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Abstract of thesis entitled
“An assessment of reproductive development of the male Indo-Pacific
bottlenose dolphin, Tursiops aduncus, in captivity”
submitted by Queeny Wing Han Yuen
for the degree of Doctor of Philosophy
at the Hong Kong Polytechnic University in May 2007
ABSTRACT
The reproductive parameters, testis size, serum testosterone levels and ejaculate
traits, of five male Tursiops aduncus (M1 – M5) were monitored for periods of
between three and five years, to investigate reproductive development and
determine at what age the onset of sexual maturity occurs. The testosterone
level of a sixth male was also investigated. Of the five main study subjects, four
males (M2 – M5), were born in captivity and therefore of known age. Weekly
ultrasonographic appearance and measurements of the testes were compared to
monthly serum testosterone levels to evaluate the reproductive development and
status of each individual. This study defined the onset of sexual maturity as the
first release of sperm in the semen, which signifies the onset and establishment
of
spermatogenesis.
Weekly
ejaculate
traits,
in
conjunction
with
ultrasonographic and testosterone data, were used to monitor the reproductive
development of the recently matured individuals and investigate their
reproductive potential, or ‘effectiveness’. Sperm from recently matured males
ii
was cryopreserved, thawed and examined for any difference in freezability
compared to those of the older mature males.
The age of onset of spermatogenesis ranged from 6y 7m to 7y 4m. The onset of
sexual maturity in two males occurred before the age of 7yr, which is younger
than previously reported. The changes in ultrasonographic appearance of the
testis during sexual maturation were consistent with one previous report in that
the testicular echopattern became more echogenic throughout the organ. The
testes also changed shape from cylindrical to being ‘cigar-shaped’, or expanded
at the caudal end. The cranial and caudal aspects of the epididymis became more
readily visualised 4 – 5 months prior to onset of spermatogenesis. Testis size
increased rapidly during a 4-month period preceding onset. In M3 and M4, testis
size at onset was 17 – 18cm in length and 170 – 190cm3 in volume. In M2 – M4,
serum testosterone level at onset varied widely, from 1.7 – 24ng/ml, therefore,
assignment of a particular level to mark sexual maturity was not possible.
Endocrine profiles proximal to onset suggest a threshold level of 3ng/ml may be
useful to identify recently matured males. Overall sperm density from one
collection session during the first year after onset ranged from 0 – 713 x 106/ml
and the highest density recorded in a single ejaculate was 1,475 x 106/ml. After
onset, spermatogenesis continued to increase in efficiency, and azoospermia or
extremely low sperm density became progressively rare.
Data of the
reproductive parameters of M5 was not consistent with the other subjects,
although the onset of sexual maturity occurred within the same age range.
Further monitoring is being conducted on this individual.
iii
Ejaculates of recently matured males (1st post-onset year) were of good quality
and generally comparable in sperm density (> 200 – 500 x 106/ml), motility (>
80%) and viability (> 90%) to those of the oldest male (M1, 19+yr). Ejaculates
collected as early as one month after onset tolerated cryopreservation well. Postthaw results of these ejaculates were comparable to ejaculates collected from M1,
a fully mature, proven sire. Semen taken from M2 during the 3rd post-onset year,
at the age of 9y 7m, was cryopreserved, used for AI in one female and produced
a healthy calf.
Testis size and testosterone levels showed seasonal changes, but sperm density
did not. Testis size and testosterone levels tended to be higher between March
and August, (spring and summer in Hong Kong), and lower between October
and February (autumn and winter). Although, overall density was lower in
January and February, ejaculates of high density (> 500 – 600 x106/ml) were
often found.
Data strongly suggest testicular activities were affected by illness. Testis size
and testosterone levels both decreased during episodes of illness. There was also
evidence of decline in semen parameters, sperm density motility and viability
during illness.
Results indicate that social structure also may impact reproductive development
and status, however further investigation of this was beyond the scope of the
present study.
iv
This study has demonstrated the combined use of ultrasonography of the testes,
serum testosterone and ejaculate traits to accurately evaluate reproductive
development and identify sexual maturation in the bottlenose dolphin, Tursiops
aduncus.
Results have also contributed towards improving Ocean Park’s
controlled breeding programme. Although the sample size of this study was
small, it is the largest and longest term study of its type reported to date. It is,
however, important to note that marked individual differences were found and
further questions about reproductive development, particularly as affected by
social structure and illness, were raised. Continued monitoring of this group and
further research on more individuals are required in order to better understand
the complex reproductive physiology of male dolphins.
v
PRESENTATIONS
ORIGINATING
FROM
THE
PRESENT STUDY
Conference Presentations
1. Yuen QWH, Brook FM, Kinoshita R.
Ultrasonographic assessment of
testicular growth and sexual maturity in the Indo-Pacific bottlenose dolphin
(Tursiops aduncus). Proceedings of the International Association for Aquatic
and Animal Medicine Conference 2004: 69-70 (abstract).
2. Yuen QWH, Brook FM, Ying MTC, Kinoshita R. Semen Collection and
Characterisation Indo-Pacific bottlenose dolphins (Tursiops aduncus).
Proceedings of the International Association for Aquatic and Animal
Medicine Conference 2005: 259-261 (abstract).
3. Yuen QWH, Brook FM, Ying MTC, Kinoshita R. Longitudinal monitoring
of body size, testis size, serum testosterone level, semen quality and postthaw motility to assess sexual maturity in the Indo-Pacific bottlenose dolphin
(Tursiops aduncus) in captivity. In: Olson D (ed), Dolphin Neonatal and
Reproduction Symposium. Indianapolis 2005; 24 -27.
vi
4. Yuen QWH, Brook FM, Ying MTC, Kinoshita R.
Some evidence of
seasonality in reproductive parameters of male Indo-Pacific bottlenose
dolphins (Tursiops aduncus). Proceedings of the International Association
for Aquatic and Animal Medicine Conference 2006: 37-43 (abstract).
5. Yuen QWH, Brook FM, Ying MTC, Kinoshita R. Longitudinal monitoring
of sexual maturity in captive male Tursiops aduncus. Proceedings of the
International Association for Aquatic and Animal Medicine Conference 2007:
158-159 (abstract).
vii
ACKNOWLEDGEMENTS
My thanks to the Marine Mammal Training and Curatorial Team at Ocean Park
for their hard work and dedication which has facilitated the smooth running of
this five-year project.
I particularly thank the dolphin trainers from Ocean
Theatre for their diligence in assisting me with the collection of poolside
samples.
I thank the Veterinary and Clinical Laboratory teams at Ocean Park. Thanks to
Dr. Nat Rourke and Dr. Crista Rayner for their early encouragement. I am very
grateful to Dr. Derek Spielman, Dr. Paolo Martelli, Dr. Nathalie Mauroo and Dr.
Nimal Fernando for their support, intellectual advice, stimulating discussions
and good humour. Thanks to Ms. Hui Suk Wai and all the technicians in the
Clinical Laboratory for their generous help and support throughout the research
process.
This project is not a stand-alone entity. Rather, it represents part of ongoing
research that has involved over 15 years of collaboration with Ocean Park in
developing techniques in controlled captive breeding. I admire and am deeply
indebted to the foresightedness, steadfastness and dedication of two individuals
who initiated this work: the late Dr. Derek Chow and Dr. Reimi Kinoshita.
viii
This research would not have been possible without the recognition and longterm commitment of the Management of Ocean Park. Here, I would like to
acknowledge Ms. Suzanne Gendron.
Dr. Fiona Brook, my supervisor, mentor and friend. I feel truly privileged to be
her student, she was a great source of inspiration and encouragement for me. I
thank her for patiently discussing issues and problems with me, instilling into me
the real value of research, giving me academic and professional advice, and
sharing her wisdom about life with me.
I thank Dr. Michael Ying, my supervisor, for his kindness and patience in
providing academic support and technical instructions.
My dear brother Jason, Carman, Harriet, Reimi, Shirley, Lily, Paul, Amy, Sveta
and Vera - my heartfelt thanks to them all for being there for me.
Finally, I owe deep gratitude to my parents - particularly to my mother for her
firm belief in me. I thank them for their support and honouring this belief by
giving me an education that has changed the course of my life. Thank you.
ix
CONTENTS
Certificate of Originality ..................................................................................... i
Abstract................................................................................................................ ii
Acknowledgements .......................................................................................... viii
Contents..... ...........................................................................................................x
List of Figures.....................................................................................................xx
List of abbreviations ...................................................................................... xxiv
Chapter 1 INTRODUCTION ...........................................................................1
1.1 The bottlenose dolphin, Tursiops.................................................................1
1.2 Status in captivity and captive management................................................3
1.2.1 Breeding................................................................................................5
1.3 Assessment of reproductive status in male Tursiops ...................................6
1.4 Assisted reproductive technologies in Tursiops ..........................................9
1.5 Need for research .......................................................................................10
Chapter 2 Literature Review The Reproductive Anatomy, Physiology and
Methods of Assessing Reproductive Status in Bottlenose Dolphins..............13
2.1 The reproductive tract of the male dolphin................................................13
2.1.1 Testis and epididymis .........................................................................13
2.1.2 The prostate gland...............................................................................16
2.1.3 The penis.............................................................................................17
2.1.4 Testicular Asymmetry.........................................................................19
2.2 Functions of the mammalian testis ............................................................20
2.2.1 Steriodogenesis ...................................................................................20
x
2.2.2 Spermatogenesis .................................................................................22
2.3 The mammalian spermatozoon..................................................................26
2.3.1 Structure..............................................................................................26
2.3.2 Spermatozoa of cetaceans ...................................................................30
2.4 Ultrasonography in reproductive medicine................................................31
2.4.1 Ultrasonographic examination of the testes in human........................32
2.4.2 Ultrasonography in assessing male reproductive status in domestic
species ..........................................................................................................33
2.4.3 Ultrasonography in non-domestic species ..........................................36
2.4.4 Safety ..................................................................................................42
2.5 Sexual maturity ..........................................................................................43
2.5.1 Overview on puberty and sexual maturity..........................................43
2.5.2 Endocrine activities and testicular development during sexual
maturity in bulls ...........................................................................................44
2.5.3 Sexual maturity in male dolphins .......................................................46
2.6 Seasonality .................................................................................................70
2.6.1 Testicular and hormonal changes in seasonal mammals ....................71
2.6.2 Seasonality in delphinids ....................................................................73
2.6.3 Seasonality in Tursiops.......................................................................77
2.7 Assisted reproductive techniques (ART) in dolphins ................................79
2.7.1 AI in dolphins .....................................................................................80
2.7.2 Semen collection.................................................................................82
2.7.3 Semen evaluation ................................................................................85
2.7.4 Semen cryopreservation......................................................................90
2.8 Captive breeding of bottlenose dolphins ...................................................99
xi
2.8.1 Controlled Breeding..........................................................................100
Chapter 3
3.1 Aim ..........................................................................................................103
3.2 Objectives ................................................................................................103
3.3 Material and method ................................................................................104
3.3.1 Subjects .............................................................................................104
3.3.2 Animal training .................................................................................106
3.3.3 Body weight ......................................................................................106
3.3.4 Body length and girth .......................................................................107
3.3.5 Equipment and protocol for ultrasonographic assessment of testes .107
3.3.6 Blood sample for testosterone evaluation .........................................110
3.3.7 Protocol for semen collection ...........................................................111
3.3.8 Semen analysis..................................................................................113
3.3.9 Semen cryopreservation....................................................................118
3.3.10 Semen thawing................................................................................121
3.3.11 Inter-and intra-operator tests...........................................................122
Chapter 4 Results...........................................................................................124
4.1 Study schedule / duration.........................................................................124
4.2 Subject demographic information............................................................125
4.3 Ultrasonographic evaluation of the testes ................................................126
4.3.1 Ultrasonographic appearance of the testes and epididymes .............126
4.3.2 Ultrasonographic assessment of testis size .......................................132
4.3.3 Correlation between testis measurements.........................................148
4.4 Correlation between body size and testis size .........................................150
4.5 Serum Testosterone level.........................................................................151
xii
4.5.1 M1.....................................................................................................152
4.5.2 M2.....................................................................................................153
4.5.3 M3.....................................................................................................154
4.5.4 M4.....................................................................................................159
4.5.5 M5.....................................................................................................160
4.5.6 M6.....................................................................................................161
4.6 Semen collection and sperm density........................................................166
4.6.1 M1.....................................................................................................166
4.6.2 M2.....................................................................................................170
4.6.3 M3.....................................................................................................172
4.6.4 M4.....................................................................................................176
4.6.5 M5.....................................................................................................180
4.7 Comparisons between testis size, serum testosterone levels and sperm
density............................................................................................................182
4.7.1 Comparison between serum testosterone and testis size ..................184
4.8 Comparison between serum testosterone and sperm density...................190
4.8.1 Comparison between testis size and sperm density ..........................194
4.9 Seasonality ...............................................................................................199
4.9.1 Testis volume....................................................................................200
4.9.2 Serum testosterone level ...................................................................209
4.9.3 Sperm density ...................................................................................219
4.10 Semen and ejaculate characterisation ....................................................226
4.10.1 Before the onset of spermatogenesis ..............................................226
4.10.2 After onset of spermatogenesis .......................................................229
4.10.3 Correlation between ejaculate parameters ......................................238
xiii
4.11 Cryopreservation....................................................................................241
4.11.1 Characteristics of ejaculates before cryopreservation ....................241
4.11.2 Characteristics of ejaculates after cryopreservation and thawing...242
Chapter 5 Discussion .....................................................................................247
5.1 Ultrasonographic examination of testes...................................................248
5.1.1 Ultrasonographic appearance of the testes........................................249
5.1.2 Testis size..........................................................................................253
5.1.3 Testicular asymmetry........................................................................259
5.2 Serum Testosterone level.........................................................................260
5.2.1 Serum Testosterone levels during sexual maturation .......................263
5.3 Semen collection and sperm density........................................................267
5.3.1 Semen collection and sperm density during sexual maturation........271
5.4 Age and Body length at sexual maturity..................................................276
5.5 Correlation between testis size, serum testosterone level and sperm density
.......................................................................................................................278
5.6 Seasonality ...............................................................................................281
5.6.1 Seasonal changes before and after the onset of sexual maturity.......287
5.7 Characteristics of Tursoips aduncus ejaculates .......................................289
5.8 Testes size, ejaculate characteristics and mating system .........................294
5.9 Reproductive ‘effectiveness’ and captive breeding .................................295
5.10 Other factors affecting reproduction and reproductive development ....297
Conclusions.......................................................................................................300
Summary and Recommendations...................................................................305
References .......................................................................................................311
xiv
Appendix 1........................................................................................................ A1
Appendix 2........................................................................................................ A3
Appendix 3........................................................................................................ A6
Appendix 4........................................................................................................ A7
Appendix 5........................................................................................................ A9
Appendix 6...................................................................................................... A22
Appendix 7...................................................................................................... A38
Appendix 8...................................................................................................... A55
Appendix 9...................................................................................................... A57
Appendix 10.................................................................................................... A71
Appendix 11.................................................................................................... A73
xv
List of Tables
Table 2.1:
Ultrasonographic measurement and appearance of testes of
T.aduncus of different reproductive status ..................................40
Table 2.2:
Summary of testis size and histology to assess reproductive status
in Tursiops ...................................................................................48
Table 2.3:
Summary of testis size and histology to assess reproductive status
in other delphinids .......................................................................50
Table 2.4:
Summary of T level used as an indicator of maturity in Tursiops
.....................................................................................................55
Table 2.5:
Summary of T level used as an indicator of maturity in other
delphinids.....................................................................................57
Table 2.6:
Testis changes monitored by B-mode ultrasonography during
sexual maturation and serum testosterone levels and sperm
density in a male T. aduncus from August 1990 - April 1992.....63
Table 2.7:
Percentage of final body length (BL) and age reached at sexual
maturity in delphinids ..................................................................67
Table 2.8:
Scale for assessing canine spermatozoa motility status...............87
Table 3.1:
Subjects and age.........................................................................105
Table 3.2:
Age, body weight, body length and body girth of subjects at the
beginning of the study (October 2002) ......................................106
Table 4.1:
Duration of data collection ........................................................124
xvi
Table 4.2:
Age, body weight, body length and body girth of subjects at
beginning (October 2002) and end of the study ........................125
Table 4.3:
Testis measurements recorded for M1 – M5 at the beginning the
study...........................................................................................133
Table 4.4:
Range of testis measurements recorded for M1 during study ...133
Table 4.5:
Annual range of testis measurements for M1 ............................136
Table 4.6:
Table 4.7:
Table 4.8:
Table 4.9:
Table 4.10:
Table 4.11:
Table 4.12:
Table 4.13:
Table 4.14:
Correlation between TL and TC in all the subjects ...................149
Table 4.15:
Correlation between body size and testis size ...........................151
Table 4.16:
Range of T level (ng/ml) recorded for M1 ................................152
Table 4.17:
Table 4.18:
Table 4.19:
Table 4.20:
Table 4.21:
Table 4.22:
Range of number of ejaculates and ToV per collection session
recorded in M1...........................................................................167
Table 4.23:
Range of OvD and HiEjD recorded in M1 ................................169
xvii
Table 4.24:
recorded in M2...........................................................................170
Table 4.25:
Table 4.26:
recorded in M3...........................................................................173
Table 4.27:
Table 4.28:
recorded in M4...........................................................................178
Table 4.29:
Table 4.30:
recorded in M5...........................................................................180
Table 4.31:
Table 4.32:
Correlation between T level and testis size ...............................184
Table 4.33:
Correlation between T level and sperm density.........................191
Table 4.34:
Correlation between testis size and sperm density ....................195
Table 4.35:
Monthly and overall study mean values (± SD) of TV, T level
size and OvD during 2002 – 2006 in M1...................................201
Table 4.36:
Monthly and overall study mean values (± SD) of TV, T level and
OvD during 2002 – 2005 in M2.................................................203
Table 4.37:
Monthly and overall study mean values (± SD) of TV, T level and
OvD during 2002 – 2006 in M3.................................................204
Table 4.38:
Monthly and overall study mean values (±SD) of TV, T level and
OvD during 2003 – 2006 in M4.................................................206
Table 4.39:
Monthly and overall study mean values (± SD) of TV and T level
during 2003 – 2006 in M5 .........................................................208
xviii
Table 4.40:
Monthly and overall study mean values (± SD) of T level during
2002 – 2006 in M6.....................................................................218
Table 4.41:
Semen collection and characteristics before the onset of
spermatogenesis (values shown are mean ±SD)........................227
Table 4.42:
Age at onset of spermatogenesis in M1 – M5 ...........................229
Table 4.43:
Semen collection and characteristics after onset of
spermatogenesis (values shown are mean ±SD)........................231
Table 4.44:
Characteristics of successive ejaculates.....................................235
Table 4.45:
Inter-correlations and correlation coefficients, r, of ejaculate
parameters ..................................................................................239
Table 4.46:
Characteristics of raw ejaculates................................................243
Table 4.47:
Overall ejaculate scores after cryopreservation and at 30 minutes
after thawing and percentages over raw scores .........................244
xix
List of Figures
Figure 2.1:
Diagrammatic representation of the testis and epididymis in a
dolphin .........................................................................................15
Figure 3.1:
Presentation of Tursiops aduncus for ultrasonographic
examination of testes at poolside ...............................................108
Figure 3.2:
Diagrams to illustrate testis measurements................................110
Figure 3.3:
Semen collection in Tursiops aduncus at poolside....................113
Figure 4.1:
Ultrasonographic image of right testis in LS – M1 ...................127
Figure 4.2:
Caudal portion of the epididymis of left testis in LS – M1 .......128
Figure 4.3:
Ultrasonographic image of left testis in LS – M3......................129
Figure 4.4:
Ultrasonographic image of left testis in LS – M4......................130
Figure 4.5:
Caudal aspect of right testis in LS – M4....................................131
Figure 4.6:
Ultrasonographic image of right testis in LS – M5 ...................132
Figure 4.7:
Weekly measurements of testis volume – M1 ...........................135
Figure 4.8:
Figure 4.9:
Figure 4.10:
Figure 4.11:
Figure 4.12:
Monthly serum testosterone level – M1 ....................................156
Figure 4.13:
Figure 4.14:
Figure 4.15:
xx
Figure 4.16:
Figure 4.17:
Figure 4.18:
Weekly overall sperm density – M1 ..........................................168
Figure 4.19:
Figure 4.20:
Figure 4.21:
Figure 4.22:
Correlation between TV and T level – M2 ................................185
Figure 4.23:
TV and T level – M2..................................................................185
Figure 4.24:
Figure 4.25:
Correlation between T level and TV – M4 ................................187
Figure 4.26:
TV and T level – M3..................................................................188
Figure 4.27:
TV and T level – M4..................................................................188
Figure 4.28:
Figure 4.29:
V and T level – M1 ....................................................................189
Figure 4.30:
T level and TV – M5..................................................................190
Figure 4.31:
Correlation between T level and OvD in M2 ............................191
Figure 4.32:
T level and OvD – M2 ...............................................................192
Figure 4.33:
T level and OvD – M3 ...............................................................193
Figure 4.34:
Correlation between T level and OvD in M1 ............................193
Figure 4.35:
T level and OvD – M1 ...............................................................194
Figure 4.36:
Correlation between TL and OvD in M4...................................196
Figure 4.37:
TL and OvD – M4 .....................................................................197
Figure 4.38:
TL and OvD – M2 .....................................................................197
Figure 4.39:
TL and OvD – M3 .....................................................................198
Figure 4.40:
TV and OvD – M1 .....................................................................198
xxi
Figure 4.41:
Mean monthly TV in M1, M2 & M3 (2002-2006)....................201
Figure 4.42:
Frequency of testis volume of individual months the annual mean
of each year (2002 – 2006) in M1..............................................202
Figure 4.43:
Frequency of testis volume of individual months above annual
mean of each year (2002 – 2005) in M2 ....................................204
Figure 4.44:
Frequency of testis volume of individual months above the
annual mean of each year (2002 – 2006) in M3 ........................205
Figure 4.45:
Mean monthly TV, 2003 – 2006 in M4 – M5 ...........................207
Figure 4.46:
Frequency of testis volume of individual months above the annual
Figure 4.47:
Frequency of testis volume of individual months above the annual
Figure 4.48:
Mean monthly T level, 2002 – 2006 in M1 and M6..................211
Figure 4.49:
Frequency of T level of individual months above the annual mean
of each year (2002 – 2006) in M1..............................................211
Figure 4.50:
Mean monthly serum T level, 2002 – 2006 in M3 and M4 .......212
Figure 4.51:
of each year (2002 – 2005) in M2.............................................212
Figure 4.52:
of each year (2002 –2006) in M3...............................................213
Figure 4.53:
of each year (2002 – 2006) in M4..............................................215
Figure 4.54:
Mean monthly T level, 2002 – 2006 in M5 ...............................216
Figure 4.55:
of each year (2002 – 2006) in M5..............................................216
xxii
Figure 4.56:
of each year (2002 – 2006) in M6..............................................218
Figure 4.57:
Mean monthly OvD, 2002 – 2006 in M1 – M3 .........................219
Figure 4.58:
Frequency of overall session density of individual months above
the annual mean of each year (2002 – 2006) in M1 ..................220
Figure 4.59:
Figure 4.60:
the annual mean of each year (2002 –2006) in M3 ...................222
Figure 4.61:
Mean monthly OvD, 2005 – 2006 in M4...................................223
Figure 4.62:
Figure 4.63:
% raw TM 30 minutes after thawing .........................................245
Figure 4.64:
% raw PM 30 minutes after thawing .........................................245
xxiii
List of abbreviations
AI
artificial Insemination
ART
assisted Reproductive Techniques
ATP
adenosine triphosphate
BG
body girth
BL
body length
BW
body weight
cAMP
cyclic adenosine monophosphate
CASA
computer-assisted semen analyzer
CCM
canine capacitation medium
CITES
Convention on International Trade in Endangered Species of
Wild Fauna and Flora
CNS
central nervous system
CS
cross-sectional view
CSG
Cetacean Specialist Group
DEN
sperm density
E1
1st ejaculate in a collection series
E2
2nd ejaculate in a collection series
E3-n
3rd to the last ejaculate in a collection series
EjC
ejaculate sperm count or sperm count of a single ejaculate, x106
EjD
ejaculate sperm density or density of a single ejaculate, x106/ml
EjV
ejaculate volume or volume of a singe ejaculate, ml
xxiv
ELFA
enzyme immunoassay method with a final fluorescent detection
by an automated VIDAS system
ET
embryo transfer
FM
forward motility, %
FSH
Follicular stimulation hormone
GH
growth hormone
GIFT
gamete intro-fallopian transfer
GnRH
gonadotrophin releasing hormone
HiEjD
sperm density of the most dense ejaculate in a collection series
ICC
intra-Class Correlation Coefficient
ICSI
intracytoplasmic sperm injection
IUCN
International Union for Conservation of Nature and Natural
Resources
IVF
in vitro fertilization
KR
kinetic Rating
L
left side
LH
luteinizing horomone
LS
longitudinal view
MMPA
Marine Mammal Protection Act
MS
motility score
OvD
overall sperm density of a collection session, x106/ml
PBS
phosphate-buffered saline
PM
progressive motility, %
PVA
poly vinyl alcohol
R
right side
xxv
RFM
rate of forward motility
RIA
radioimmunoassay
RPM
rate of progressive motility
SEA
seminiferous epithelia area
SMI
sperm motility index
SOP
speed of progression
SQA
sperm quality analyzer
TC
testis circumference
TD
testis depth
TL
testis length
T level
serum testosterone level
TM
total motility, %
ToC
total sperm count of a collection session, x106
ToV
total volume of a collection session, ml
TSH
tyhyroid-stimulating hormone
TW
testis width
TV
testis volume
TYB
N-tris (hydroxymethyl) methyl-2-aminoehane sulfonic acid
(TES)-TRIS yolk buffer
WHO
World Health Organsiation
VIA
viability, %
ZIFT
zygote intro-fallopian transfer
xxvi
Chapter 1
INTRODUCTION
1.1 The bottlenose dolphin, Tursiops
There are two species of bottlenose dolphins, the common bottlenose dolphin,
Tursiops truncatus and the Indo-Pacific bottlenose dolphin, Tursiops aduncus.
As a genus, Tursiops is widespread, occurring in most of the world’s warm
temperate to tropical seas (Wells and Scott, 2002). T. truncatus is considered the
more widely ranging pelagic species, whereas T. aduncus is limited to Indian
and Western Pacific Oceans, inhabiting more coastal waters. Near-shore coastal
water is more accessible from land and so the T. aduncus species is more
vulnerable to habitat degradation and exploitation, including capture for display,
which may cause long-term decline in populations that are already small and
localised (Brownell et al., 2003).
Concerns about over-exploitation in the wild have led to various international
policies and agreements being made to protect bottlenose dolphins and other
cetacea. International trading of bottlenose dolphins is closely monitored by the
Convention on International Trade in Endangered Species of Wild Fauna and
Flora (CITES), under the Appendix II classification.
Species in this
classification may not necessarily be facing immediate threat of extinction, like
the baiji (Lipotes vexillifer) which is classified in Appendix I, but may become
1
so unless trade is subjected to strict regulations. In the U.S.A., the Marine
Mammal Protection Act (MMPA), established in 1972, imposed a moratorium in
both the taking and importing of marine mammals. Notable exceptions to this
moratorium are taking for purposes of scientific research, public display,
photography for education or commercial purposes, or enhancing the survival or
recovery of a species or stock. Display facilities in the U.S.A., however, have
long observed a self-imposed moratorium. Bottlenose dolphins are included in
the International Union for Conservation of Nature and Natural Resources
(IUCN) Red List for Cetaceans under the Criteria of ‘Data Deficient’ (Reeves et
al., 2003). This means this species is poorly known on a global basis due to
taxonomic difficulties. The 2002 – 2010 Conservation Action Plan for the
World’s Cetaceans (Reeves et al., 2003) includes live-capture for display in
oceanaria and / or research as a threat to cetaceans. This report further equates
taking for such purposes with ‘incidental or deliberate killing, as animals
brought into captivity, or sometimes even killed during capture operations, are
no longer available to help maintain their natural population’. It also states that
‘dolphins should not be captured or removed from a wild population unless that
specific population has been assessed and it has been determined that a certain
amount of culling can be allowed without reducing the populations’ long-term
viability or compromising its role in the ecosystem’.
2
1.2 Status in captivity and captive management
Bottlenose dolphins have been maintained in captivity from as early as the 15th
century in Europe and the first oceanarium, Marine Studios in Florida, was
established in 1947 (Couquiaud, 2005a). These animals are not only charismatic
to the greater public but they also arouse vast scientific interest among
academicians, medical and husbandry caretakers. Early research conducted on
dolphins in captivity consisted of behavioural observations of social activities,
mating and calving (McBride and Hebb, 1948; McBride and Kritzler, 1951;
Tavolga and Essapian, 1957). Continual development of different research areas
and the knowledge amassed have been essential in maintaining the health and
welfare of these animals. The welfare of captive individuals bears relevance to
conservation in that dolphins are long-lived and reproductively active, thus better
survivorship reduces the demand for replacements from the wild (Fisher and
Reeves, 2005). Conservation by means of captive breeding to replace removal
from the wild and to generate knowledge through research, were the main
themes of the opening remarks at the first bottlenose dolphin breeding meeting,
‘Breeding Dolphins: Present, Status, Suggestions for the Future’, in 1975. After
this benchmark event, another such meeting did not follow until, ‘The Bottlenose
Dolphin Reproduction Workshop’ in 1999. In 2005, a meeting entitled ‘Dolphin
Neonatal and Reproduction Symposium’ was held. Delegates from facilities
around the world attended workshops, reflecting genuine concerted efforts to
achieve better management of captive dolphin groups on a global basis. Topics
discussed at these meetings were multidisciplinary and ranged from captive
group demographic and genetic analysis to advances in innovative reproductive
3
techniques. Experts from the zoological and domestic animal fields were also
invited, as useful information can be gained from their successes in reproductive
technologies in both captive and wild population management.
Captive management of bottlenose dolphins is made challenging by the
complexity of social interactions in the wild.
Wild Tursiops populations
function in a fission-fusion manner, in which individuals associate in small
groups, with compositions that change on a daily, or even hourly, basis (Connor
et al., 2000a). Male reproduction is limited by access to mates (Trivers, 1972;
Wrangham, 1980). In the wild, male dolphins cooperate and compete within
strategic social groups, or alliances, to gain mating opportunities with receptive
females (Connor et al., 2000b). Hierarchies have also been described in captive
male dolphins (McBride and Hebb, 1948; Tavolga, 1966). Social groups may
become unstable when males are maturing, as they need to establish their
positions within the hierarchy (Caldwell and Caldwell, 1972; Samuels and
Gifford, 1997; Waples and Gales, 2002). In captivity, conflict between males can
result in physical injuries and increased stress levels. Chronic stress has been
proposed to predispose an individual to infection due to suppressed immune
function (St. Aubin and Dierauf, 2001) and to inhibit reproduction (Sapolsky,
1985; St. Aubin and Dierauf, 2001). Also, when breeding is not controlled, only
the dominant males may succeed in copulating with females in the presence of
younger, subordinate males (McBride and Hebb, 1948; Tavolga, 1966). As a
result, calves have been found to be sired by the dominant animals only (Asper
et al., 1992; Duffield et al., 1999). Over-representation of a few individuals can
lead to narrowing of the gene pool and a possible increase of inbreeding in the
future. Therefore, captive management not only needs to maintain an optimal
4
social environment, but also plan breeding strategically to achieve appropriate
sex and age group compositions in the enclosure space available (Andrews,
1999).
1.2.1 Breeding
An indication of increasing success in captive breeding is the rise in the
proportion of captive-born animals from 6% in 1977 to 44% in 1999 in North
America (Andrews, 1999) and about 12% in 1991 to 35.4% in 1998 in Europe
(Hartmann, 2000). However, besides the number of calves born, there are other
aspects in breeding management to consider for a captive colony to be selfsustainable. Maintenance of genetic vigour and avoidance of inbreeding are of
utmost importance. The genetic materials of founder animals are extremely
valuable and should be utilised and retained in the captive gene pool for as long
as possible. Controlling breeding, by actively selecting the sire and dam, and
varying breeding pairs, will allow all sexually mature animals in the group to
reproduce, and thus reduce inbreeding in both the short and long term. The
reproductive lifespan of an animal may be maximised by allowing it to
reproduce as early as possible. Breeding records in the past have shown the
following concerns: loss of animals without adequate representation; few
females in a group; unknown paternity; prolonged periods without births, and
animals of reproductive age not having an opportunity to breed.
A controlled breeding programme for T. aduncus that were captured from waters
off Taiwan and Indonesia (Reeves et al., 1994) was developed in Ocean Park,
5
Hong Kong in 1993 (Kinoshita et al., 2005). This breeding programme included
strictly enforced husbandry protocols, separation of males and females, and
long-term systematic investigations of their reproductive conditions using
ultrasonography and endocrinology. The controlled unassisted breeding (i.e.,
natural copulation) strategy in this programme, resulted in 9 calves born out of
11 procedures (Brook and Kinoshita, 2005).
The result of such a high
conception rate (91%) is strong evidence that knowledge and routine monitoring
of the reproductive states of both male and female and accurate prediction of
ovulation are essential elements in a breeding management model. The use of
artificial insemination (AI), with cooled liquid stored, or cryopreserved, semen,
subsequently introduced into Ocean Park’s breeding programme, resulted in
lower conceptions rates of 28.6% and 40%, respectively (Kinoshita et al., 2005).
There is an obvious need to improve the success of this technique. A benefit of
the successful controlled unassisted breeding efforts, besides contribution to a
genetically varied captive group, is provision of much needed known-aged
individuals for continual research. While AI and semen cryopreservation
techniques are being perfected for routine use, breeding should continue, in a
controlled manner and based on sound reproductive knowledge of the individual,
to curtail further loss of genetic material (Brook and Kinoshita, 2005).
1.3 Assessment of reproductive status in male Tursiops
Fundamental science is the foundation on which to build better management
(Wildt, 1999a).
Thorough understanding of basic reproductive biology is
6
required if traditional passive breeding practices are to be replaced by actively
controlled programmes. The latter requires separation of males and females and
the time at which a designated pair is put together for copulation is determined
based on their reproductive states and genetics. Advantages of such controlled
breeding conditions include prevention of undesirable impregnation of young,
physically immature females and mating between closely related animals, or
those with unfavourable breeding records. Planned pregnancies also allow close
monitoring of gestation and better perinatal management, to optimise calf
survival. Since the inception of controlled breeding, defining the female dolphin
estrous cycle has been a major priority and much research has been conducted on
the subject, whilst investigation of the male has been limited. Being able to
follow ovarian activities and predict ovulation accurately in individual animals
form the necessary basis for controlled breeding strategies and successful births
have been reported as a result (Brook and Kinoshita, 2005).
Assessment of the reproductive conditions of both male and female dolphins is
imperative for a systematically planned breeding programme. (Robeck et al.,
1994; Robeck, 1999). It is essential to be able to identify when an animal
reaches maturity so that it may be included in breeding plans. To date, the age at
which male dolphins reach maturity is still uncertain - studies have been few and
methods for assessing age and reproductive conditions vary. Physical maturity is
perhaps better defined as adult (Laws, 1956), asymptotic (Sergeant, 1984) or
maximum (Ross, 1977; Cockcroft and Ross, 1990) body length reached. The
ability to produce sperm may be more accurately conveyed by reproductive
(Schroeder, 1990b) or sexual maturity (Brook, 1997).
Historically, sexual
maturity in Tursiops has been investigated based on testes size and histological
7
examinations for the presence of spermatozoa in dead animal specimens
(Harrison et al., 1969b; Harrison and Ridgway, 1971; Caldwell and Caldwell,
1972; Harrison et al., 1972; Harrison and Fanning, 1973; Sergeant et al., 1973;
Ross, 1977; Cockcroft and Ross, 1990). These studies proposed that sexual
maturity is attained after 11 years of age in Tursiops. Endocrine studies on
testosterone levels in live dolphins have also classified males less than 11 years
of age as sexually immature (Kirby, 1990; Schroeder, 1990a). Brook (Brook,
1997) carried out longitudinal monitoring of reproductive conditions in live T.
aduncus and found sexual maturity was attained in one subject at an estimated
age of 7 – 8 years. Due to the exact age being unknown, further study was
recommended. Terminology used in association with male dolphin sexual
maturity, such as reproductive effectiveness (Cornell et al., 1987), competence
(Asper et al., 1992; Dudley, 1999) and efficiency (Schroeder and Keller, 1989;
Schroeder, 1990b), is not clear and requires further investigation and explanation.
Ultrasonography in human reproductive medicine is routinely used for imaging
the testes (Eisenberg and Lewin, 2000) and monitoring ovarian activities in
artificial reproductive technologies (ART) (Nugent et al., 2000). It has also been
developed for applications in assisted reproductive procedures in wildlife species,
including the gorilla (Gorilla gorilla gorilla) (Pope et al., 1997), elephant
(Loxodonta africana and Elephas maximus) (Hidebrandt et al., 2000;
Hildebrandt et al., 2003) and giant panda (Ailuropoda melanoleuca)
(Hildebrandt et al., 2003).
Brook (1997) was the first to describe
ultrasonographic techniques to systematically monitor the testes and ovaries of
Tursiops and demonstrated successful applications in controlled unassisted and
assisted breeding of Tursiops aduncus (Brook and Kinoshita, 2004; Brook and
8
Kinoshita, 2005).
These techniques have subsequently been transferred to
monitoring ovarian activities of other cetacean species, such as Chinese white
dolphin (Sousa Chinensis) (Brook et al., 2004), killer whale (Orcinus orca)
(Robeck et al., 2004b) and T. truncatus (Robeck et al., 2005a). The use of
ultrasonography to gain understanding of the anatomy of the testes, their changes
during maturation and application in ART has also been recognised in nondomestic species (Hildebrandt et al., 2000).
1.4 Assisted reproductive technologies in Tursiops
Assisted reproductive technologies (ART), such as AI, may be used to enhance
controlled breeding by increasing propagation efficiency and improving genetic
management (Robeck, 1999; Wildt, 1989). These technologies, however, should
not be considered as ‘quick fix’ alternatives for failures in natural breeding due
to poor understanding and husbandry management (Wildt, 1989).
Basic
understanding of the reproductive anatomy and physiology of the species
concerned is required for the development of ART (Robeck, 1999; Wildt, 1999a).
In addition to accurate ovulation prediction and identification of the site in the
female reproductive tract at which to deposit semen, AI also requires reliable
collection of semen that is of high quality. Once the onset of spermatogenesis is
established, little is known in Tursiops about subsequent sperm outputs and if
spermatogenesis is an on-going process. Seasonality in sperm density has only
been reported in one adult T. truncatus (Schroeder and Keller, 1989). Brook
(1997) reported intermittent short periods of reduced sperm density in T.
9
aduncus and suggested further study to investigate the significance of this
finding. The semen of adult Tursiops is considered to be high in quality and has
been successfully cryopreserved (Durrant et al., 1999a; Robeck and O'Brien,
2004a). Methodologies for assessing different sperm parameters, however, have
not been reported in full detail and motility evaluation is subjective. Also, the
characteristics and quality of semen of recently matured dolphins have not been
studied. Such data is necessary for planning breeding with these young males, to
ensure they are genetically represented as early as possible, through natural
mating or assisted techniques.
1.5 Need for research
Since Brook (1997), no further long-term investigation has carried out on the
reproductive physiology of male Tursiops. More information can be gained
from similar ultrasonography-based studies of known age individuals. Areas of
the male dolphin reproductive physiology identified by Brook (1997) that
require further research included:
i.
age at sexual maturity
ii.
changes in testis size and appearance, and serum testosterone levels on
approach to sexual maturity
iii.
testis size and appearance at sexual maturity
iv.
testosterone level at sexual maturity
v.
spermatogenesis and progress after onset
vi.
azoospermia or very low sperm densities
10
vii.
reproductive cycle / seasonality
viii.
reproductive effectiveness
ix.
factors that may affect reproductive development, such as illness and
social influence
Other areas include semen characteristics, which may differ between sub-species,
and the differences and changes before and after onset.
Given that research should be initiated ‘on novel species at such time when the
extant populations, both captive and free ranging, are still demographically
stable and genetically viable to ensure adequate time and material availability to
develop the necessary protocols’ (Loskutoff, 1999), research on Tursiops is both
necessary and timely.
Although bottlenose dolphins are not an endangered
species at present, they are considered threatened and their future is not assured,
as it continues to be affected by habitat degradation and human exploitation.
Further, the existing Tursiops captive stock may potentially serve as a model in
wildlife conservation in two ways.
Firstly, management of wild Tursiops
populations may benefit from scientific and management knowledge gained in
captive research efforts. Secondly, assisted reproductive technologies proven
successful in a captive species may serve as a basis for further research to
develop techniques specific to an endangered cetacean species.
This study was a longitudinal investigation of more than four years to investigate
the reproductive physiology of the male bottlenose dolphin, T. aduncus. The
main aim of the study was to establish at what age sexual maturity (i.e., the onset
of spermatogenesis) occurs in four known-aged dolphins born in Ocean Park,
Hong Kong.
To date, the age at which male bottlenose dolphins become
11
sexually mature is not known. Ultrasonography was used to monitor testicular
changes during sexual maturation. Ejaculate traits in sexually immature, recently
mature, and fully mature males were investigated in detail. A semen
cryopreservation protocol, proven effective in bringing about births of live
calves, was used to investigate the freezability of the semen of recently sexually
mature dolphins, and to compare these to a fully mature male. Data collected
were also used to evaluate any seasonal patterns in testis size and testosterone
levels in sexually immature and mature animals. Understanding and information
gained in this study will assist controlled breeding strategies. Early knowledge
of a male dolphin’s maturity status and semen characteristics allows prompt
inclusion in breeding plans for natural mating or assisted breeding, and optimal
collection of semen for cryo-storage, should a seasonal pattern exist.
12
Chapter 2
LITERATURE REVIEW
The Reproductive Anatomy, Physiology and Methods of
Assessing Reproductive Status in Bottlenose Dolphins
2.1 The reproductive tract of the male dolphin
The male mammalian reproductive tract consists of two testes, two epididymes,
each with its ductus deferens, and various accessory glands (Setchell and Brooks,
1988). There are species differences in the range of accessory glands present in
the reproductive tract, with most males having a prostate, bulbourethral
(Cowper’s) glands and seminal vesicles. However the prostate is the only
accessory gland in cetaceans (Setchell and Brooks, 1988)
2.1.1 Testis and epididymis
The testes are paired, elongated, cylindrical structures situated within the
abdominal cavity in cetaceans (Harrison, 1969a; Green, 1977; Harrison et al.,
1997) (Figures 2.1 and 2.2). They lie caudal to the kidneys and each is attached
to the abdominal cavity by a mesorchium (Meek, 1918; Matthews, 1950;
13
Harrison, 1969a; Green, 1977). The dorsal surface of the testis abuts the
intestinal coils (Hepburn and Waterston, 1902). On gross physical examination,
the testes of dolphins are large (Meek, 1918; Matthews, 1950; Harrison, 1969a)
and encapsulated in a tunica albuginea (Green, 1977). The tunica albuginea is a
tough fibrous covering that consists of three layers, the outermost layer of the
tunica vaginalis, then the tunica albuginea proper and the inner most tunica
vasculosa, lining the parenchyma (Setchell and Brooks, 1988). On removal of
the tunica albuginea, the surface of the organ is lobular in appearance, similar to
other mammalian testes, but to a greater degree (Ping, 1926; Green, 1977). The
mediastinum testis is reportedly poorly developed, with small longitudinal
strands from which smaller tubules radiate at intervals within the parenchyma
(Matthews, 1950). The caudal end of the testes is more rounded, with a narrow
gubernaculum attached and the cranial end tapers to a more pointed process
where the caput epididymis lies (Harrison, 1969a).
The epididymis is well developed and also elongated, lying on the mesial aspect
of the mesorchium along the entire length of the testes (Matthews, 1950; Green,
1977) (Figure 2.1). It is loosely attached to the testis by fibrous connective
tissue (Matthews, 1950; Slijper, 1966). The cranial end, or the caput epididymis,
is a prominent triangular cap (Matthews, 1950).
With no morphologically
distinct corpus, the cauda epididymis immediately follows the caput (Matthews,
1950) and is triangular in cross-section (Harrison, 1969a).
The caudal
epididymis is a very convoluted tubule and adjacent loops are separated by
fibrous connective tissue, with blood vessels scattered within (Matthews, 1950).
The tubule is lined by ciliated columnar epithelial cells to facilitate movement of
14
luminal contents. Spermatozoa have been found in abundance in the lumen of
the convoluted tubule (Meek, 1918; Matthews, 1950; Ross, 1977).
vm
e
vd
ce
t
ub
ce
e
t
ub
vd
vm
-
caput epididymis
epididymis
testis
urinary bladder
vas deferens
vasal mass
Figure 2.1: Diagrammatic representation of the testis and epididymis in a
dolphin (adapted from Matthews 1950, Harrison, 1969a and Brook
1997)
The vas deferens connects the end of the cauda epididymis at the caudal pole of
the testis and has been seen to form distinct convoluted masses along its course
(Matthews, 1950). These ‘vasal masses’ (Brook, 1997) are separated by short
lengths of less convoluted vas deferens. The left and right ejaculatory ducts are
close to each other and enter the urethral crest through orifices on the seminal
colliculus. Large quantities of spermatozoa have been found throughout the vas
15
deferens in the epididymis of sexually mature animals (Meek, 1918; Matthews,
1950; Harrison, 1969a).
The position of the testes within the abdominal cavity and their juxtaposition to
large locomotor muscles, the hypaxialis lumborum and rectus abdominus,
suggest they are exposed to core body temperature, which may have a
detrimental effect on spermatogenesis. In other animal species, the exteriorly
positioned scrotum helps maintain the testes at up to 7oC below that of the core
body temperature of 36-380C, so that spermatogenesis proceeds uninterrupted
(Harrison, 1949; Waites, 1970; Williams-Ashman, 1988). In bottlenose dolphins,
there is a vascular countercurrent heat exchanger around the testes. This is
formed by a venous plexus returning cooled blood from the surfaces of the
dorsal fin and tail flukes to the testicular artery supplying the testes via a
spermatic arterial plexus (Rommel et al., 1992).
Studies that measured
temperature in the colon near to this vascular network in a sexually mature
dolphin reported cooling effects of 0.5 – 1.3oC compared to temperatures
recorded at other regions in the colon (Rommel et al., 1994). These results
suggest the temperature of the testis is regulated by the exchanger through
cooling of the arterial blood that supplies it, to prevent possible detrimental
effects on spermatogenesis.
2.1.2 The prostate gland
The dolphin prostate gland is reported to be a large and active secretory organ in
adults (Hepburn and Waterston, 1902; Matthews, 1950; Harrison, 1969a). The
16
prostate surrounds part of the urethra and has an outer fibrous capsule under
which is a powerful muscle layer, the compressor prostatae muscle (Matthews,
1950; Harrison, 1969a). The ventral (or pre-urethral) part lies on the inner curve
of the urethra and the dorsal (or post-urethral) part lies outside of the curve
(Matthews, 1950; Harrison, 1969a; Green, 1977). In longitudinal section, both
parts of the prostate appear conical in shape and in transverse section the entire
organ has a circular margin (Matthews, 1950). The prostate is composed of
lobules of closely packed racemose glands lined by columnar cells, which empty
into the urethra via prostatic ducts and sinuses at a region around the urethral
crest (Matthews, 1950; Slijper, 1966).
2.1.3 The penis
The penis lies coiled in the preputial sac, concealed beneath the genital slit
(Harrison, 1969a). The genital slit is an invagination of the abdominal skin,
where the skin folds to cover the distal portion of the penis (Slijper, 1966).
Owing to the curved shape of the penis only a small degree of stretching is
required for protrusion (Slijper, 1966).
The distal part of the penis tapers
smoothly to the tip and is called the pars intrapraeputialis, or terminal cone
(Slijper, 1966). The terminal cone may be likened to the glans penis in other
mammalian species, except in cetaceans there is no comparable swelling (Slijper,
1966). There is no os penis in any cetacean species (Slijper, 1966; Harrison,
1969a; Green, 1977).
17
The cetacean penis is of the fibroelastic type (Slijper, 1966).
There is no
lengthening or thickening in the fibroelastic penis, only stretching of the curve
on protrusion (Slijper, 1966). Rigidity in the fibroelastic penis is attributed to the
properties of the tunica albuginea, the tough fibrous sheath that surrounds the
corpus cavernosum, and the networks of collagen and elastic fiber in the
trabeculae within the corpus. There is only a small degree of blood filling of the
cavernous spaces in the corpus cavernosum. Coitus executed by a fibroelastic
penis is typically rapid (Slijper, 1966).
There are powerful and extensive muscles and ligaments associated with the
penis to facilitate coitus and ejaculation. The erector penis muscle arises from
the whole length of the pelvic bone and inserts into the corpus cavernosum of the
penis (Meek, 1918). A strong double elastic retractor muscle extends from the
rectum to the base of the preputial sac to withdraw the penis into the sac (Meek,
1918; Harrison, 1969a).
The overall anatomy of the male dolphin reproductive tract appears to be suited
to produce and deliver a large amount of seminal fluid in a short time. The large
testis size is capable of producing large quantities of spermatozoa (Matthews,
1950). Similarly, the large size of the prostate also suggests large amounts of
fluids may be produced.
The extensively convoluted nature of both the
epididymis and vas deferens allows storage of large amounts of spermatozoa, to
provide for an immediate supply as demanded by a fibroelastic coitus.
18
2.1.4 Testicular Asymmetry
Asymmetry in the testes has been noted in some cetaceans. During the 19511952 whaling season, the left testes were found to be distinctly heavier than the
right testes in Physeter macrocephalus (sperm whale) (Arvy, 1977). The mass of
the right testes was found to be consistently greater than the left in 44 Stenella
attenuata (spotted dolphin), however such asymmetry was not found to be
significant based on statistical analysis (Kasuya et al., 1974). In a study of 133
mature male Lagenorhynchus obscurus (dusky dolphin) no significance
differences were found in testicular mass or length (Van Waerebeek and Read,
1994). A more recent ultrasonographic study reported the right testicular length
and volume were greater than the left in 7 Tursiops aduncus (Brook, 1997).
Differences in testes mass have also been found in adult males of other animal
species, some showing consistent asymmetry and others inconsistent bilateral
asymmetry (Yu, 1998).
In humans, testicular asymmetry is well documented. (O'Rahilly, 1986; Lenz et
al., 1993). Differences in venous drainage have been proposed as an explanation
for such asymmetry in humans, the right spermatic vein joining the inferior vena
cava while the left joins the left renal vein (Hooker, 1970). Asymmetry may
also be explained by asymmetric innervation causing differences in endocrine
sensitivity and functional captivity between the left and the right organs
(Gerendai and Halasz, 1997).
The significance of testicular asymmetry is not known.
Measurement of
testicular size in more subjects is required to determine if there is significant
19
morphologic asymmetry in dolphins. Possible differences in vascular supply of
the countercurrent heat exchanger for the left and right testes in the bottlenose
dolphin have been proposed (Brook, 1997). To investigate this hypothesis,
colour Doppler ultrasonography may be useful in providing further information
on gonadal vasculature and blood flow (Brook, 1997).
2.2 Functions of the mammalian testis
The mammalian testes have two major functions, they are to produce steroid
hormones, androgens (Setchell and Brooks, 1988) and spermatozoa through a
sequence of highly organised cytological events in spermatogenesis (De Krester
and Kerr, 1988). Functionally, the testes can be divided into the interstitium,
which contain Leydig cells for androgen formation and secretion, and the
seminiferous tubules, which contain Sertoli cells to support spermatogenesis
(Bardin et al., 1988; De Krester and Kerr, 1988; Setchell and Brooks, 1988).
2.2.1 Steriodogenesis
Testosterone is a potent hormone. It is the major circulating androgen and almost
exclusively of testicular origin (Coffey, 1988; Hall, 1988). The concentration of
this hormone is very high within the testes, some 75 times higher than in the
peripheral circulation. In the testes, synthesis and secretion of testosterone are
primarily carried out by Leydig cells under of the regulation of luteinizing
20
horomone (LH), secreted in pulses by the anterior lobe of the pituitary
(adenohypophysis) (Hall, 1988; Johnson and Everitt, 2000a; Johnson and Everitt,
2000b).
LH is secreted in a pulsatile manner under the influence of
gonadotrophin releasing hormone (GnRH) which is also released in pulses from
the hypothalamus. LH binds to specific surfaces receptors on the Leydig cells
and accelerates steriodogenesis through raising the level of intracellular cyclic
adenosine monophosphate (cAMP) and the rates of subsequent protein reactions.
Testosterone is synthesised from cholesterol, which is stored as lipid droplets in
the cytoplasm of the Leydig cells, through a series of enzymatic reactions. The
product hormone is then secreted and passed into the seminiferous tubules to
bind with androgen receptors within the Sertoli cells. The effects of testosterone
include stimulation and maintenance of protein secretion by the Sertoli cells to
support spermatogenesis. Sertoli cells may also stimulate Leydig cell steroid
production by secretion of inhibin, which exerts a negative feedback control over
follicular stimulating hormone (FSH) secretion by the anterior pituitary.
In humans, there are marked changes in secretory patterns and levels of
gonadotropins and androgens during the process of acquiring sexual maturity
and adulthood (Coffey, 1988; Turek and Van Cauter, 1988; Johnson and Everitt,
2000c). At onset of puberty, LH levels fluctuate diurnally. Elevated secretion,
due to increased pulse frequency and magnitude, occurs at night and testosterone
levels are subsequently raised (Wu et al., 1993). During puberty, the levels of
these hormones also increase during the day but not as much as at night. Once
an adult baseline level is attained, LH no longer shows marked diurnal changes,
but testosterone continues the diurnal rhythm with levels being elevated during
the night.
Circulating testosterone levels vary widely during the course of the
21
day and this may reflect the pulsatile manner of its release as well as the diurnal
changes. Therefore it is important to take into account the time of day when
samples are taken for testosterone evaluation. As males age, the nocturnal rise in
testosterone stops and levels gradually decline in older age.
2.2.2 Spermatogenesis
Spermatogenesis in many mammals is a continuous process throughout the
reproductive lifespan of the male and takes place inside the seminiferous tubules
(De Krester and Kerr, 1988). Extensive folding of the seminiferous tubule loops
gives rise to the lobular appearance described in the testes (De Krester and Kerr,
1988). In order to support continual daily production of spermatozoa, precursor
cells must undergo self replication and since each cell must be in the haploid
state, chromosome number must be halved for restoration of the diploid state on
fertilization.
Therefore, spermatogenesis consists of stages of mitotic
proliferation and meiotic division, before a final stage of cytodifferentiation, or
spermiogenesis, which transforms the round cell into the complex structure of
the spermatozoon (De Krester and Kerr, 1988; Setchell and Brooks, 1988;
Johnson and Everitt, 2000a).
A mature spermatozoon emerges from many stages of precursor cells (De
Krester and Kerr, 1988; De Krester, 2000; Johnson and Everitt, 2000a). The
most basic precursor cells are the prospermatogonial cells which are the
quiescent germ cells of the immature testes. At puberty these cells undergo
rounds of mitosis at the basal compartment of the seminiferous tubules to form
22
spermatogonial stem cells.
These cells continue to replicate to form A1
spermatogonial cells which mark the beginning of spermatogenesis. The number
of rounds of mitotic division these cells further undergo to form spermatogonia
type B is species specific. These germ cells enter the first meiotic stage to form
resting primary spermatocytes of different subclasses, leptotene, zygotene,
pachytene and diplotene, to represent stages of the long prophase. While nuclear
divisions (karyokinesis) occur completely during mitosis, cytoplasmic divisions
(cytokinesis) are incomplete, so the spermatocytes are linked to each other by
thin cytoplasmic bridges to form a syncytium. Cells are not freed from the
syncytium until spermatozoa are formed. As the primary spermatocytes undergo
meiotic divisions, they migrate towards the lumen of the seminiferous tubues.
After the first meiotic division, secondary spermatocytes are formed and a
further division then forms spermatids and signifies the completion of
chromosomal reductions.
A spermatid undergoes elaborate cytomplasmic
remodeling; it elongates to assume the structure of a spermatozoon. The release
of fully formed spermatozoa from the syncytium into the lumen of the
seminiferous tubules is called spermiation. These cells are then washed along
the tubules in a fluid secreted by the Sertoli cells, to the epididymis for
acquisition of motility and fertilization capacity, storage and transport (Robaire
and Hermo, 1988). In humans, spermatogonia do not undergo the spermatogenic
process with spermatozoa being released into the seminferous tubule lumen until
the age of 11 – 13 years. Prior to this, at 7 – 9 years of age, gonocytes or
prospermatogonia self-replicate to form spermatogonia, which reside at the basal
compartment in numbers equal to those of Sertoli cells.
23
2.2.2.1 The Sertoli cell
The migratory course and timing of the development of the different precursor
cells inside the seminiferous tubules are highly organized by the non-germinal
Sertoli cells (Bardin et al., 1988; Johnson and Everitt, 2000a). In cross-section,
all the germ cells at a particular point on the circumference of a seminiferous
tubule should be similar. Such high degree of co-ordination is possible because
morphologically the Sertoli cells extend from the basement membrane of the
seminiferous tubule to the lumen and adjacent cells around the tubule are
connected via cytoplamsic junctional contacts. These contacts effectively form a
physical barrier, dividing the tubule into the basal compartment, containing the
spermatogonia and the adluminal compartment, containing the spermatocytes
and spermatids. The continuous cytoplasmic network encircling the base of the
Sertoli cells allows communication between adjacent cells so that the
developmental progression of the associated spermatogenic cells, which are also
bound in a syncytium, from the base to the lumen, can be synchronized. The
time required for completion of spermatogenesis is constant in a given species,
but differs between species. To ensure a continuous flow of spermatozoa, rather
than a pulsatile release due to synchronisation, initiation of spermatogenesis
along the seminiferous tubules occurs randomly.
One other important function of the Sertoli cells is to create a unique intratubular
micro-environment for developing germ cells by active secretion of fluid and
proteins and maintenance of the physical barrier which serves as a blood-testis
barrier (Bardin et al., 1988; De Krester, 2000; Johnson and Everitt, 2000a).
Secretory proteins of the Sertoli cells, such as androgen binding protein and
24
glycoproteins, are constituents of the intratubular fluid and exert a hydrostatic
and diffusional gradient that is maintained by the blood testis barrier. The main
component of this barrier is the specialized inter-Sertoli-cells junctions, thus
movement of molecules, such as proteins and ions, across the peritubular wall is
restricted. The barrier also prevents spermatozoa from leaking into the
interstitium where they will it elicit an immune response because they are
antigens. Failure of the blood-testis barrier may therefore cause subfertility, as
spermatozoa are being destroyed by the body’s own immune system.
The
barrier’s selectivity in passage of molecules into the tubular lumen protects the
developing germ cells from mutagenic molecules.
Besides androgen requirement, spermatogenesis cannot commence until Sertoli
cells have developed their functional capabilities. Prepubertally, Sertoli cells are
immature and continue to undergo proliferation until the adult morphology and
population are established during sexual maturation (Bardin et al., 1988; De
Krester, 2000; Johnson and Everitt, 2000a). Before such time, the blood testis
barrier is absent. The fluid and proteins that contribute to the unique intratubular
microenvironment required by spermatogenesis are also absent due to lack of
production and secretion. FSH has been found to have an influence on the
proliferative phase of Sertoli cells and its action is sustained through the
prepubertal period for fluid production and initiation of spermatogenesis (Bardin
et al., 1988). Also, the high degree of spatial and temporal organization would
not be possible without the adult structural features. The potential of the adult
testes, in size and sperm output, therefore, depends on the proliferation and
development of Sertoli cells in the immature testes.
25
2.3 The mammalian spermatozoon
The mammalian spermatozoon is very specialised in structure and function. All
mammalian spermatozoa have two main components, the head and the tail (or
flagellum), but differences in size, morphology of the head and length and
composition of the flagellum occur between species (Eddy, 1988).
Both
components are surrounded by a plasma membrane that delineates regional
domains, which differ in composition and function.
2.3.1 Structure
2.3.1.1 The head
The head is subdivided into the anterior acrosome, or the acrosome cap, the
equatorial segment and the postacrosomal region.
In addition, there is an
anterior band between the anterior acrosome and equatorial segment and a
serrated band between the equatorial segment and the postacrosomal region.
Further, there is a posterior ring of the post-acrosomal region that forms a tight
seal between the cytoplasmic compartments of the head and tail. The head
contains the haploid nucleus for syngamy. Prior this event, acrosome reaction
occurs, whereby fusion and vesiculation of the acrosomal membrane and plasma
membrane occur and acrosomal enzymes are released to digest a path through
the cumulus cells and zona pellucida of the oocyte (Yanagimachi, 1988). After
acrosome reaction, a region above the post-acrosomal region becomes capable of
fusion with the plasma membrane (oolemma) of the oocyte (Yanagimachi, 1988).
26
2.3.1.2 The tail
The tail consists of a centrally placed axoneme, which is a ‘9 + 2’ complex of
microtubules surrounded by dense fibers and extends the entire length of the
flagellum. The microtubules contain a type of protein called dynein that is
responsible for generating the motive force of the flagellum (Turner, 2003). The
anterior domain of the tail contains the connecting piece (neck) and the mid
piece. The latter consists of mitochondria wrapped in a tight helix around the
dense fibers and is surrounded by a mitochrondrial sheath. The mitochondria
produce adenosine triphosphate (ATP) as an energy source for cellular metabolic
activities and flagella movement (Turner, 2003). The mid piece is separated
from the posterior tail by an annulus, which is a dense fibrous ring around the
axonemal complex. The posterior tail is surrounded by a fibrous sheath and is
further subdivided into the principal piece and the end piece. The function of the
flagellum is to bring about motility for spermatoza to reach the perivitelline
space of the oocyte. Basically, a force must first be generated from the base by
bending to form waves and these waves are then propagated along the length to
the tip of the flagellum.
Biochemical changes in the components of the
flagellum during epididymal maturation change the motility pattern from slow
beating and a circular motion to generally vigourous beating, rapid and forward
movement.
Different species show different patterns of sperm motility,
however many, including dolphins, show a hyperactivated motility pattern at
specific locations in the female reproductive tract during capacitation, before
acrosomal reaction (Yanagimachi, 1988).
Hyperactivated motility is
characterised by very vigorous, whiplash-like beatings of the flagella and
intermittent linear movements. Hyperactivation not only enables spermatozoal
27
migration between localized regions in the female reproductive tract but also
provides powerful thrusting to mechanically drive though the zona pellucida.
2.3.1.3 The sperm plasma membrane
The structure of the sperm membrane is complex, some knowledge of its natural
state is useful to help understand the detrimental effects of procedures in
cryopreservation, such as cooling and thawing, that cause loss of membrane
integrity and function (Hammerstdet et al., 1990). The sperm plasma membrane
conforms to the basic fluid mosaic model of biological membranes in which
proteins are non-covalently associated with a bilayer of lipids forming the matrix
of the membrane (Yanagimachi, 1988; Hammerstdet et al., 1990; Parks and
Graham, 1992; Watson, 1995). However, the organization and composition of
the proteins and lipids differ between the delineated regions and domains of the
sperm surface in order for each to carry out its specialised function (Eddy, 1988;
Parks and Graham, 1992). The sperm membrane contains mostly phospholipids,
followed by sterols, glycolipids and free fatty acids (Eddy, 1988) and their
precise composition is important in maintaining the functional domains.
Movement of molecules is restricted by a combination of mechanisms such as
lipid-lipid interactions, lipid-protein interactions and protein barriers (Parks and
Graham, 1992). The lipid compositions of the two layers, an extracellular facing
leaflet and the cytoplasmic facing leaflet, in the bilayer configuration are
asymmetrical, and interactions occur between the leaflets. Some lipids may also
be arranged in hexagonal aggregates. However, in order to maintain the bilayer
dominant form, non-bilayer lipids are prevented from aggregating by
28
interactions with proteins. Since the lipids and proteins of the membrane are not
covalently linked, their association may be altered by changes in temperature.
For example during cooling, different lipids undergo physical phase transition,
from the liquid-crystalline to the gel phase, at different temperatures.
The
nonlayer lipids, maintained in the bilayer through protein interaction, undergo
phase transition at a higher temperature than bilayer lipids, forming gel
aggregates and thus leave the bilayer. The presence of sterols may inhibit this
transition to some extent (Holt, 1997). Upon warming, these non-bilayer lipids
may not be able to regain their association with the proteins and original state
within the bilayer.
As a result, protein function may be altered and the
membrane destabilised or disrupted, which may lead to loss of function of the
domain concerned and render the spermatozoon unable to fertilise an oocyte.
Different species have different sperm membrane make up and lipid
compositions, and thermotropic phase transition may take place at different
temperatures (Holt, 1997; Medeiros et al., 2002).
Therefore, effect of
temperature changes and sensitivity to membrane disruption differ among
species. For instance, the composition of low protein and intermediate sterol
content of the rooster sperm membrane is thought to attribute to greater
resilience to physical damage at reduced temperatures. Whereas the higher
protein and lower sterol content of the boar sperm membrane is extremely
susceptible to irreversible membrane changes due to thermotropic phase
transition (Parks and Graham, 1992). In addition to the boar, the sperm of bulls
rams and stallions are very vulnerable, whereas, the sperm of humans and rabbits
are, like the rooster, more resistant (Medeiros et al., 2002).
29
2.3.2 Spermatozoa of cetaceans
The overall anatomy of the spermatozoa of cetaceans has been found to be
similar to that of other eutherian mammals (Fleming et al., 1981; Miller et al.,
2002), however differences in spermatozoal total length, size and morphology of
the head have been reported among cetacean species. In agreement with the
findings in terrestrial mammals, the length of cetacean spermatozoa generally
correlated negatively with body size (Cummins and Woodall, 1985; Kita et al.,
2001). The larger whales Berardius bairdii (Baird’s beak whale) and
Balaenoptera edeni (Bryde’s whale), had smaller sized spermatozoa. The size of
spermatozoa in cetaceans is suggested to be related to reproductive strategy (Kita
et al., 2001) as found in terrestrial species (Cummins and Woodall, 1985).
Ultrastructures of the head, midpiece and principle piece in the spermatozoa of
11 species of cetaceans have been identified using scanning and transmission
electron microscopy (Kita et al., 2001; Miller et al., 2001). Sperm heads of the
Orcinus orca (killer whale) and Delphinapterus leucas (beluga whale) were
found to be wider than other species, and are almost square and paddle-shaped.
Such unique head morphology may also be explained by differences in
reproductive strategy and capacitatation mechanism (Kita et al., 2001). Further,
the heads of these whales have reniform acrosomal regions and lack the vertical
postacrosomal ridges found in the Lagenorhynchus obliquidens (Pacific whitesided dolphin) and Tursiops truncatus (Atlantic bottlenose dolphin) sperm
(Fleming et al., 1981). Instead, there are cytoplasmic bands and regular electron
dense structures in the anterior acrosome and postacrosomal region that may
contribute to sperm-oocyte fusion and / or post fusion events (Miller et al., 2001),
30
as suggested for the vertical postacrosomal ridges of bottlenose dolphin sperm
(Fleming et al., 1981).
2.4 Ultrasonography in reproductive medicine
The use of ultrasonography for diagnostic purposes in human medicine was first
presented by an Austrian neurologist/ psychiatrist, Karl Dussik, in 1942 (Levi,
1997). Clinical use in obstetrics and gynaecology followed later, along with
examination of lesions of the breast (Wild and Reid, 1954) and in 1958 with
various normal and pathological conditions of the abdomen and pelvis identified
in 100 patients (Donald et al., 1958). Since then, obstetrical examinations have
relied heavily on ultrasonography to monitor gestational progress, including
embryonic and fetal development and malformation (Brent et al., 1991). As the
technology became increasingly advanced and more accessible, ultrasonography
became an efficient and relatively cost-effective imaging modality.
It is
routinely used in the evaluation of human female reproductive organs and for
monitoring functional changes during spontaneous and induced ovarian cycles.
Further, ultrasonography presents images in real-time, it has become the
preferred method for monitoring follicular growth and ovulation and its use has
made significant contributions to the advancement of assisted reproductive
technologies (ART) (Nugent et al., 2000).
31
2.4.1 Ultrasonographic examination of the testes in human
Ultrasonography of the human testes was introduced in the 1970s and has since
become the diagnostic modality of choice for testicular abnormalities (Behre et
al., 1989; Eisenberg and Lewin, 2000; Sidhu, 2006).
2.4.1.1 Assessment of testis size
Pubertal increase in testes size is largely due to the proliferation of the Sertoli
cells in the seminiferous tubules, therefore accurate measurement of the testes
size can be used to monitor sexual maturation (Rundle and Sylvester, 1962;
Takihara et al., 1983). Furthermore, since the bulk of the testis is made up of
seminiferous tubules, the size of the organ should be indicative of the status of
spermatogenesis (Takihara et al., 1987; Lenz et al., 1993). Various studies have
found testicular volumes obtained by ultrasonographic technique are less
variable and more accurate compared to the more traditional palpation methods
or using calipers, volumetric models and orchidometers (Rivkees et al., 1987;
Behre et al., 1989; Fuse et al., 1990; Lenz et al., 1993; Paltiel et al., 2002).
Ultrasonography was adopted as the gold standard in measuring testicular
volume in one study, when found the orchidometer was too insensitive to
volume differentials to monitor growth impairment (Diamond et al., 2000).
Several formulas have been used to derive testicular volume from measurements
taken at different planes, however, Lambert’s empirical formula for an ellipsoid
(Volume cm3 = length x width x depth x 0.71) (Lambert, 1951) has been found
to provide the most accurate estimate, thus it is recommended for routine clinical
32
use in conjunction with ultrasonography (Paltiel et al., 2002). Ultrasonographic
study has found that human testes increased in volume up to the age of 20 years
and no further changes were found up to the age of 59 years (Lenz et al., 1993).
This study also found positive correlation between testicular volume and sperm
count and percent of morphologically normal spermatozoa (Arai et al., 1998).
2.4.1.2 Assessment of testis appearance
In addition to size, the ultrasonographic appearance of the testicular parenchyma
may also be used to evaluate maturation in humans. The parenchyma of the
normal human testes is of homogenous reflectivity (Sidhu, 2006). Another study
found the prepubertal testis was of a low-level echogenicity and belonged to the
age group 0.5 to 9 years (Hamm and Fobbe, 1995). Postpubertal testes were of
medium level echogenicity and belonged to the age group of 16+ years. The
change in testicular echogenicity during puberty may be explained by
maturational developments of the seminiferous tubules; increase in length and
luminal diameter and thickening of the basement membrane, and increase in
germ cell number and the presence of spermatozoa.
2.4.2 Ultrasonography in assessing male reproductive status in domestic
species
The use of ultrasonography in veterinary medicine began in 1970 to detect
pregnancies in pigs (Pechman and Eilts, 1987).
33
2.4.2.1 Evaluation of testicular development
In males, B-mode ultrasonography was first used to determine normal
ultrasonographic appearance and measurements of the testes.
Subsequent
studies then investigated changes during maturation and relationships with
reproductive stages. Such studies have been conducted in dogs (Eilts et al.,
1993), bulls (Pechman and Eilts, 1987; Cartee et al., 1989; Chandolia et al., 1997;
Arteaga et al., 2005), boars (Cartee et al., 1986), and goats and sheep (Ahmad et
al., 1991). The ultrasonographic appearances of the testes of these species are
reported to be similar, i.e., homogenous with low to medium echogenicity. In
goats, changes in the echogenicity of the testes and epididymis were noticeable
from 12 weeks of age onwards and adult ultrasonographic appearance was
attained by 18 weeks, after which time no further changes took place (Ahmad et
al., 1991). A study on young bulls showed ultrasonographic measurements of
testis diameter increased from < 2cm at 2 weeks of age to > 5 cm at 46 weeks
when sexual maturity was reached (Chandolia et al., 1997). Changes in testis
echopattern of these bulls were quantitatively measured as pixel (picture
elements) values. These values increased in stages of age 2 – 6 weeks, 6 – 16
weeks, followed by 28 weeks onwards. Peak pixel values were finally reached at
44 weeks. Further, ultrasonographic changes found in this study were supported
by endocrine profiles of LH, FSH and testosterone and were speculated to be due
to cellular development of the maturing testes and production of different germ
cells, including spermatozoa.
34
2.4.2.2 Evaluation of testicular function
In domestic species, direct or indirect (scrotal width or circumference)
measurements of testis size are recognised to reflect sperm output, semen quality
or breeding soundness. Manual methods to measure testis size are cumbersome
and consist of the use of flexible cloths, calipers or tapes (Willet and J.I., 1955;
Hahn et al., 1969; Gebauer et al., 1974; Palasz et al., 1994; Pant et al., 2003).
Some of these studies were conducted on young bulls from 18 months old;
authors noted testis growth correlated significantly with semen traits, such as
ejaculate volume, sperm density and count (Palasz et al., 1994; Brito et al., 2002;
Pant et al., 2003). An ultrasonographic study conducted on mature bulls found
that,
although
ultrasonographic
testis
diameter
correlated
well
with
measurements taken manually, they did not correlate with semen parameters
(spermatozoa motility and morphology) (Cartee et al., 1989). Another
ultrasonographic study also reported a lack of correlation between testis diameter
and semen traits (volume, sperm density and number of cells per ejaculate) in
mature bulls (Cartee et al., 1986). Based on semen traits, this study classified
boars as satisfactory, questionable and unsatisfactory breeders. It is unfortunate
that correlation analysis between testis size and semen traits was not conducted
based on this classification as it may have yielded some interesting results. The
study’s small sample size, n = 14, may have prevented such analysis. In addition,
this study also reported young boars at 9 months old produced semen with traits
comparable to older 15-month old individuals. Another study on bull testes
reported declines in ultrasonographic echopattern, in pixel values, and semen
quality (motility and morphology) after testicular temperature was raised by
scrotal insulation (Arteaga et al., 2005). Both parameters generally increased
35
some time after removal of insulation. The authors suggested delay in recovery
may be explained by the time required for the seminiferous tubules to respond to
adverse stimulus and transport of spermatozoa from the tubules to the
epididymis.
responses
Therefore, studies that only measure same day, or same week,
may
fail
to
demonstrate
correlations
between
testicular
ultrasonographic evaluation and semen quality.
2.4.3 Ultrasonography in non-domestic species
Despite routine and diverse applications in human and mainstream veterinary
medicine, the use of ultrasonography in zoo and non-domestic animal species
has been limited. Ultrasonographic examination of these species presents a host
of practical problems due to size, need for restraint, positioning, demeanor and
excitability of the subjects.
Some of these problems may be overcome by
operant training, so that the animal co-operates in the examination procedure and
thereby manual restraint and possible associated stress may be eliminated.
2.4.3.1 Ultrasonography in development of assisted reproductive
technologies (ART)
Ultrasonography provides much needed information for better understanding of
the reproductive anatomy and physiology, which is essential to manage breeding
of wildlife species in captivity. Such information is species-specific and this
must be remembered in investigations in order for it to be useful for the
36
development of ART.
In conjunction with endocrine monitoring, B-mode
ultrasonography has been relied on to characterise the ovarian cycle and predict
ovulation in Indo-Pacific humpback dolphins (Sousa Chinensis) (Brook et al.,
2004), bottlenose dolphins (Tursiops aduncus and truncatus) (Brook, 1997;
Robeck et al., 1998; Brook, 2001; Robeck et al., 2005a), killer whales (Orcinus
orca) (Robeck et al., 2004b) and the giant panda (Ailuropoda melanoleuca)
(Hildebrandt et al., 2003). It has also been crucial in the visualisation of the
reproductive tract for artificial insemination in elephants (Loxodonta maximus)
(Brown et al., 2004) and oocyte retrieval in lowland gorillas (Gorilla gorilla
gorilla) (Hatasaka et al., 1997).
Ultrasonographic evaluation for male reproductive activity has been less widely
applied. Currently, in the zoological setting, semen analysis is the primary tool
used in determining the fertility of an individual. However, with the exception
of trained individuals in a few species, semen can only be collected by
electroejaculation, which requires manual or mechanical restraint and anesthesia.
Due to the risks associated with restraint and anesthesia, semen collection can
not be carried out on a regularly repeated basis, thus a complete picture of the
reproductive capacity of an individual can not be obtained. Furthermore, low
semen volume and sperm count have been reported in samples collected by
electroejaculation in primates (Pope et al., 1997). In species that have testes
within the abdomen, such as the orders Cetacea, Sirenia and Proboscidea,
ultrasonography offers the only means of visualising these organs.
37
2.4.3.2 Ultrasonographic anatomy of male dolphin reproductive tract
Stone (1990) reported that ultrasonography can demonstrate the dolphin testes
and gave a brief description of the echopattern as fairly homogenous, of
medium-level reflectivity like that of humans (Stone, 1990). Subsequent work
by Brook (1997) and Brook et al. (2000) provided detailed descriptions of the
examination
technique
and
ultrasonographic
appearance
of the
male
reproductive tract, and the application of ultrasonographic monitoring in
controlled breeding in captivity (Brook, 1997; Brook et al., 2000).
Such
information forms a foundation for further systematic and long-term
investigations on male dolphin reproductive physiology. In addition to baseline
profiling, ultrasonographic monitoring may help to determine when sexual
maturity occurs, identify possible seasonal changes and detect reproductive
pathologies.
The following ultrasonographic anatomical description of the male dolphin
reproductive tract is based on Brook’s work on Tursiops aduncus (Brook, 1997;
Brook et al., 2000). Consistent with anatomical descriptions in the literature
(Meek, 1918; Matthews, 1950; Harrison, 1969a; Green, 1977), the testes are
elongated and cylindrical in shape and lie longitudinally in the abdomen,
immediately caudoventral to the kidneys, in proximity to two large muscle
groups (hypaxialis lumborum muscle and rectus abdominus muscle), intestines
and urinary bladder.
The testicular parenchyma is well demarcated by a
hyperechoic border of the tunica albuginea. The mediastinum is visualised as a
central, hyperechoic, linear structure extending the full length of the testis in
longitudinal section. In cross-section, the testes are round and the mediastinum
38
extends dorso-medially from the centre and does not appear to extend across the
entire width of testis. When a testis is large enough, its dorsal surface abuts the
hypaxialis lumborum muscle and the ventral surface of the rectus abdominus
muscle. The echogenicity of these muscle groups is similar in all animals,
regardless of age or size, therefore can serve as a reference to assess differences
in testicular echogenicity between animals.
2.4.3.2.1 Assessment of male dolphin reproductive status
A grading system (given in Table 2.1) was proposed to characterise testes of
dolphins of different reproductive status, based on morphology, size and
echopattern. Testicular volume was derived by Lambert’s empirical formula for
an ellipsoid, using length measurement taken in longitudinal section and width
and depth measurements taken in cross-section (Lambert, 1951). The increase in
parenchymal echogenicity observed as an animal gained sexually maturity was
hypothesised to be due the increasing diameter of the seminiferous tubules of
maturing testes. As the lumen dilates, the tubule walls separate, presenting
increased interfaces for the ultrasound beam to reflect and so more echoes, or
stronger signals, are returned, as indicated by an increase in ‘brightness’. Also
the presence of free spermatozoa may increase echogenicity. Testicular
appearance was helpful in distinguishing between animals that were sexually
mature or immature.
The anatomy of the epididymis as visualised by ultrasonography is again
consistent with the literature, in that it is an elongated structure laying on the
39
dorso-lateral surface along the entire length of the testes (Matthews, 1950; Green,
1977).
The younger the subject, the harder it is to clearly visualise the
epididymis in its entirety. The head of the epididymis is a triangular structure,
Table 2.1:
Ultrasonographic measurement and appearance of testes of
T.aduncus of different reproductive status Brook (1997; 2000)
Testis ultrasonographic appearance
Status
TL
(cm)
TV
(cm3)
Mature
14.0
–
2
2.8
151.2
–
531.8
Sub-adult
/ maturing
8.8
–
10.0
26.6
–
45.7
II
Elongated
Consistently
cylindrical
Immature
4.4
–
6.4
4.0
–
10.3
III
Elongated
Consistently
cylindrical
Grade shape / contour
I
Elongated
Cylindrical,
expanded at
distal portion
*parenchyma
Homogeneous
‘speckled’
echopattern of
mid to high level
intensity
Hyperechoic
Lobulation,
particularly in
elderly male
Boarder well
defined
Homogeneous
echopattern
Less echogenic
Poorly
differentiated
Hypoechoic
mediastinum
Linear &
well defined
Hyperechoic
With
extensions of
linear
hyperechoic
structure
Linear &
well defined
Hyperechoic
Linear &
well defined
Hyperechoic
*Echogenicity of the testis parenchyma is judged in comparison with hypaxialis
lumborum muscle, above the dorsal surface of the testis.
resembling a cap over the cranial end of the testis and is isochoeic or
hyperechoic compared with the testicular parenchyma. In cross-section, the
body of the epididymis is triangular, with one side attached to the surface of the
testes, and it shows the same echogenicity.
40
The distal two-thirds of the
epididymis is more hypoechoic and the tubular and convoluted appearance of the
structure becomes more discernable towards the distal aspect. In older, fully
mature dolphins, the caudal epididymis is reported to be large, extending some
1.5 – 3.5cm beyond the caudal end of the testis. The tubules here are hypoechoic
and may be up to 7mm in diameter. A post-ejaculation decrease in diameter of
up to 2mm, measured ultrasonographically, led to the speculation that these
tubules act as a reservoir for seminal fluids, as well as spermatozoa, since the
dolphin reproductive tract lacks seminal vesicles. Brook (1997) stated it was not
possible to ultrasonographically differentiate the vas deferens from the
epididymis in any animal. The presence of a ‘vasal mass’, a rounded mass of
tubules, was suggested to be useful to identify an animal that had been sexually
mature for some years.
2.4.3.3 The application of ultrasonography in captive dolphin breeding
The significance of ultrasonographic monitoring of the reproductive status of
both male and female dolphins has been well demonstrated in a captive
controlled breeding programme (Brook, 1997; Brook and Kinoshita, 2005).
Information on testicular activities gained from ultrasonographic examination
facilitates selection for a sexually mature male for natural mating or collection of
semen for AI. Accurate prediction of ovulation by ultrasonography greatly
facilitates better planning and management, allowing timely union between a
selected male and the cycling female. The application of ultrasonography in
captive breeding of bottlenose dolphins is further reviewed in Section 2.8.1.
41
2.4.4 Safety
At diagnostic exposures, ultrasound of mammalian tissues has been considered
to be safe (Brent et al., 1991).
At present there is no direct evidence of
biological effects of ultrasound at low exposures used clinically in developing
embryos or fetuses, or at cellular level. The effect of ultrasound on testes appears
to depend on the intensity and/or duration of exposure. No detrimental effect on
testicular development, sperm production and semen quality was found in bulls
after a 3-minute exposure of low acoustic intensity (Coulter and Bailey, 1988).
A study on rat testes after a 10-minute exposure at a higher acoustic intensity of
1 Watt/cm2 found testicular temperature raised to 39oC and disruption of
spermatogenesis due to loss of cellular integrity in developing germ cells,
spermatids and spermatocytes (Dumontier et al., 1977). However, such effects
are reversible, as the stem cells, spermatogonia, and the key supporting nongerm cells, Sertoli and Leydig cells, were not affected. The individual operator,
however, should also appreciate the principles and concepts of the biophysics
and bioeffects of ultrasound and exercise prudent practice.
The physical effects of ultrasound of concern are heating and cavitation (Holland,
1998). As ultrasound enters tissues it is propagated and energy is lost through
attenuation due to absorption, scattering and reflection (Merritt, 2005).
In
absorption, acoustic energy is converted into heat. The rate at which temperature
is raised inside the tissue depends on factors such as spatial focusing or intensity
(mW/cm2), ultrasound frequency (MHz), exposure duration and tissue type. The
risk of heating is increased at higher intensities and longer exposures. Therefore,
an operator should avoid stationing the transducer over a particular region of the
42
body for a prolonged period and duration of examination should be restricted to
the time required to ascertain the necessary findings.
2.5 Sexual maturity
2.5.1 Overview on puberty and sexual maturity
It is difficult to arrive at a consensus for defining puberty and the period this
developmental process spans within a species. This is because puberty involves
a cascade of physiological, morphological and behavioural changes that lead to
fertility of an individual. Therefore, puberty is temporal and progressive, and the
time required to complete the process varies between species. Sexual maturity is
one of the developmental landmarks of puberty. In humans and primates, it
pertains to the first menses (menarche) in females or first release of spermatozoa
(spermarche) in males, and developmental stages of secondary sexual
characteristics, such as breast, genitalia and pubic hair growth (Plant, 1988;
Marson et al., 1991). The age and size at maturity of an individual depends on
body and environmental conditions (Plaistow et al., 2004).
Testis development during sexual maturity is governed by neuroendocrine
control along the hypothalamicpituitary- gonadal axis (Amann, 1983; Plant,
1988; Styne, 1994; Ojeda et al., 2006). At the onset of puberty, activation of the
gonads results from a rise in the pulsatile secretion of gonadotropins, LH and
43
FSH, in both frequency and amplitude from the anterior pituitary (Plant, 1988).
Pulsatile LH secretion is driven by pulsatile secretion of GnRH by the
hypothalamus (Plant, 1988; Styne, 1994). Little is know about the stimulation of
the release of GnRH during puberty, or reduction during juvenile pause or
prepubertal hiatus, except a control mechanism resides in the central nervous
system, in the form of a pulse generator in the hypothalamus (Plant, 1988; Styne,
1994; Ojeda et al., 2006).
2.5.2 Endocrine activities and testicular development during sexual
maturity in bulls
A review by Amann (1983), based on multiple reports of studies on bulls,
presents a speculative sequence of endocrine and cellular events, initiated by the
increase of LH, that lead to sexual maturity. Prior to 8 weeks of age, LH levels
were very low (< 0.3ng/ml) and mean testosterone level was 0.39ng/ml in calves
(Amann and Walker, 1983). From week 11 – 13, LH increased significantly to >
2ng/ml and remained at higher levels until week 20 (Amann and Walker, 1983).
During this period, Leydig cells were found to differentiate and mature to adult
form and function. Synthesis and secretion of testosterone by the Leydig cells
was stimulated by the presence of LH. Testosterone levels were initially low
and began to rise by about week 16 (Amann and Walker, 1983). By week 18,
there was a marked increase in intratesticular testosterone concentration and
such increase is postulated to be responsible for the development of immature
Sertoli cells and their proliferation (Amann and Walker, 1983).
44
Also by this
time, testosterone reached levels detectable in the blood and continued to
increase until week 28 (Amann and Walker, 1983). Since testosterone exerts a
negative feedback control on the hypothalamus and anterior pituitary (Plant,
1988; Styne, 1994), this may explain the decrease in LH levels (< 1ng/ml)
observed in the presence of continual testosterone increase (Amann and Walker,
1983). By week 24, inside the seminiferious tubules under the influence of
testosterone, differentiation of gonocytes into prespermatogonia is maximal
(Curtis and Amann, 1981).
By week 28, Sertoli cells reached the adult
complement and the cytoplasmic junctional contacts between adjacent cells were
established in order to provide the necessary environment for spermatogenesis to
proceed. By week 32, the onset of spermatogenesis is evidenced by significant
increase in the number of primary and secondary spermatocytes and formation of
spermatids (Curtis and Amann, 1981). During the period between weeks 24 and
32, increases in seminferious tubules diameter (from 127 ± 2 to 208 ± 10µm)
and length (from 1510 ± 124 to 2010 ±128 m/testis) resulted in a rapid three-fold
increase in testis weight, from 42 ± 3 to 117 ± 10g (left testis only) (Curtis and
Amann, 1981). From the time when spermatozoa first appear in the lumen of the
tubules at week 32, i.e., the establishment of spermatogenesis, the efficiency of
sperm production gradually increased. A bull is said to have reached sexual
maturity or puberty when ejaculates produced contain at least 50 x 106 sperm and
≥ 10 % progressively motile at the earliest age of 39 weeks old (Wolf et al.,
1965).
45
2.5.3 Sexual maturity in male dolphins
For male dolphins the question of what constitutes sexual maturity is not as
straight forward as for females, where sexual maturity is identified when the
animal has ovulated for the first time (Perrin and Reilly, 1984). It is difficult to
be certain exactly when a male dolphin has reached sexual maturity (Kasuya and
Marsh, 1984; Bryden and Harrison, 1986a). The process is complex and many
studies have used different criteria based on gross and histological examinations
of excised testes of dead animals.
Studies on live dolphins are few and
historically testosterone level in the peripheral blood was the only clinical
indicator for maturity. More recently, techniques for semen collection under
conditioned behaviour have become routine and ultrasonography is available for
visualization of the intra-abdominal testes. However, there is only one
comprehensive and long-term longitudinal study that investigated the
reproductive development of male dolphins using ultrasonographic measurement
of testes, sperm density and serum testosterone level evaluation (Brook, 1997).
The age and size at which sexual maturity is attained in bottlenose dolphins are
still not clear. This, in part, is due to inconsistencies between studies in the
reproductive parameters used to correlate to age and size and in defining what
constitutes sexual maturity. The age of dolphins from the wild is unknown and
evaluation of dentine growth layer groups (GLG) of the teeth can only provide
an estimate. Morphometric measurement, such as total body length, is easy to
obtain in stranded or captive animals, but single measurements are not useful in
assessing reproductive status. Body lengths of sexually mature dolphins have
been found to vary widely, rendering it an unreliable parameter to predict sexual
46
maturity.
Coupled with ultrasonographic technique and semen collection,
longitudinal studies on known aged individuals born in captivity will provide
information on the reproductive development and growth at specific ages in
dolphins.
This information is essential to improve captive population
management and for the development of ART.
2.5.3.1 Excised testes: Testis size and histology
Most of the studies of excised dolphin testes combine testis size with histological
examinations for seminiferous diameter and the presence of spermatozoa to
assess reproductive status. Criteria for defining sexual maturity varied in
complexity; some simply noting spermatozoa in the lumen of the seminiferous
tubules (Harrison et al., 1972; Harrison and Fanning, 1973; Kasuya et al., 1974;
Ross, 1977) others the presence of different developmental germ cell types
(spermatogonia, spermatocytes, primary and secondary, and spermatids) and
spermatozoa (Hirose and Nishiwaki, 1971; Harrison and Ridgway, 1971;
Miyazaki, 1977; Collet and Saint Girons, 1984; Miyazaki, 1984; Hohn and
Chivers, 1985; Chavez Lisambart, 1998; Rosas et al., 2002).
Such
inconsistencies make direct comparison between studies listed in Tables 2.2 and
2.3 difficult.
47
Table 2.2:
Summary of testis size and histology to assess reproductive
status in Tursiops
Study &
N
Species
&
origin
Testis
length
(cm)
Ross
1977
N=13
T. truncatus
S. Africa
T. aduncus
S. Africa
-
C
42 – 52
C
45 – 55
T. truncatus
S. Africa
Harrision
et al. 1973
N=8
Sergeant
et al. 1973
N=31
Harrision
et al. 1972
(incl.
Harrison et
al. 1971 &
1969b)
a
STD
(µm)
C
<100
*
Cockcroft
et al.
1990
N=53
Testis
weight
(g)
T. truncatus
S. Australia
T. truncatus
Queensland
T. truncatus
N.E.
Florida
T. truncatus
Florida &
California
T. truncatus
Brisbane
T. truncatus
Japan
T. truncatus
Hawaii
T. gilli
California
n/s
C
310 –
1160
C≤
18
C
655 – 805
C
1270–
1730
C≤
31
C
15 – 98
n/s
≤ 33
≤150
-
C
5 - 60
-
C
80
-
C
907
C
20
C
14 – 18
C
18
C
20
-
BL
(cm)
Status
(n)
No mature ST
>9
225
I
50% mature
ST
9 – 12
n/s
P
75-100%
mature ST
14.5
240
SM
100 –
196
239 –
249
I
(4)
MR
(2)
224 –
2 54
M
(4)
261 –
272
162 –
201
I
(2)
I
(2)
202
M
(1)
171 –
235
I
(5)
Few sperm in
ST or Ep
9 – 11
Sperm in ST
& / or Ep
10 – 17
47
No sperm
4 – 11
No sperm in
Ep
ST w/ lumen
Sperm in ST
& Ep
-
25 – 80
200
C
11 – 39
C/E
10 – 15
C/E
100 – 200
C/E
300 – 700
C
6 – 20
Age
by
GLG
(Y)
189 – 243
C
1077
-
Histology
of the
testes
<7
n/s
-
30 – 100
ST no lumen
No sperm in
Ep
-
-
120
ST w/ lumen
w/ few sperm
-
80
C
22 - 35
70
C
1126
ST w/ some
mitotic
activity
7 – 15
n/s
MR
≥12
245
SM
98 –
260
I
(9)
259
260
-
-
-
-
-
-
-
Sperm in Ep
& vas
-
-
-
Maturity was also based on fusion of epiphyses mid-thoracic vertebrae
N
Number of animals in study
n
Number of animals in status
n/s
Not specified
Testes length and weight
C = Combined testes
E = Either right or left testis
ST
Seminiferous tubules
STD
ST Diameter
Histology of the testes
Gonia = Spermatogonia
Cytes = Spermatocytes
1o = Primary
2o = Secondary
Tids = Spermatids
Ep = Epididymis Age
GLG = Growth Layer Groups
BL
Body Length
Status IM = Immature
P = Puberal or Pubertal
MR = Maturing
M = Mature
SM = Sexually Mature
IA = Inactive
48
IM
?
(1)
MR or
Sick
(1)
260 –
277
IA
(2)
210 –
225
IM
(3)
IM
(1)
IM
(1)
M
(2)
225
276
303 –
333
A common finding between dolphin species is rapid increase in testis size before
the onset of sexual maturity. A study on Tursiops truncatus, showed that large
fluctuations in testes weight began from 10 years of age (Sergeant et al., 1973).
In this study, immature testes weighed between 10 – 15g, maturing testes
weighed 100 – 200g and mature testes weighted 300 – 700g from 12 years and
over (Table 2.2). Another study on wild T. truncatus, reported rapid increase in
testes weight began at the age of 10 years, when combined testes weighed less
than 100g (Cockcroft and Ross, 1990) (Table 2.2). Based on seminiferous
tubule development, the combined weight of mature testes in this study was
310g or above. A large increase in combined testes weight from < 80g to almost
400g between the age of 8 to 12 years was reported in Delphinus delphis
(Common dolphin) and mature testes weighed 350g or above (Hui, 1979) (Table
2.3). In Stenella attenuata (spotted dolphin) accompanied by rapid increase in
seminiferous tubule diameter, testes rapidly increased in weight from less than
100g to over 1000g between 7 and 13 years of age (Perrin et al., 1976) (Table
2.3). The adult testes weight in this study was 500g or above. Miyazaki (1977)
observed rapid increase in testes weight during puberty when a testis weighed
between 6.8g to 15.4g in Stenella coeruleoalba (striped dolphin) (Table 2.3).
Single testis weight continued to increase, accompanied by rapid increase in
seminiferous tubule diameter, up to 70g when a fully mature state was
considered reached based on presence of spermatozoa in all the seminiferous
tubules examined.
49
Table 2.3:
Summary of testis size and histology to assess reproductive
status in other delphinids
Worker
Year
Harrison
et al.
1969b
N = unk
Spp &
Origin
Testis
Length
(cm)
Delphinus
Delphis
S.
California
-
Stenella
graffmani
& longirostris
Mexico
Harrison
et al. 1969b
N = unk
Delphinus
bairdi
Harrison
et al. 1971
N = 16
Sotalia
fluviatilis
S. America
n=4
Kasuya
et al. 1974
N = 44
Perrin
et al. 1976
N =115?
-
Lagenorhychus
obliquidens
S. California
Hirose
et al. 1971
N = 117
C/E
38
Stenella
caeruleoalba
Japan
Stenella
attenunata
Pacific ocean
Japan S. coast
Stenella
attenunata
N.E tropical
Pacific
-
Testis
Weight
( g)
C/E
380 – 550
C/E
950 – 3200
C
6-20
C
250 – 500
C
1020– 1850
C/E
30 – 60
C/E
200 – 640
Stenella
Coeruleoalba
Pacific coast
Japan
Histology
-
ST no lumen
Early
activity
No sperm
abundant
sperm
-
C
> 3000
-
-
-
-
179 – 186
?
187 – 198
SM
165 – 175
IM
175 – 180
?
Sperm in Ep
180 – 214
SM
No sperm
163 – 184
IM
170 – 194
?
200
SM
> 190
M
123 – 133
IA
(2)
148
A
(1)
-
-
Early
activity
Ep no sperm
Active (no
detail)
Active
w/ sperm
-
-
-
Inactive
(no detail)
158
E
69
114
ST w/ lumen
Sperm in Ep
-
ST no lumen
w/ gonia
ST w/ gonia,
1o & 2o cytes
All germ cell
type
ST w/ only
gonia, some
cytes
-
60 –
70
n/s
n/s
Sperm in
peripheral
section of
testis
Sperm in
center
section of
testes
63
-
E
200 – 300
Rapid
↑
n/s
E
500 – 2000
up to
170
From no copious
sperm in Ep
33 –
65
M44
34 –
104
M 55
45 –
2 14
M
131
ST
w/ gonia
Only
ST
w/ gonia &
cytes
ST w/ sperm
(No. ST w/
sperm out of
20)
50
IM
200
49
E
80 – 225
(15.5)
177
ST no sperm
E
7.7
E
9.3
E
142
E
7 – 15
Status
(n)
160
-
C
447
-
BL
Cm
-
C
20 – 50
-
Age by
GLG
Y/G
-
C/E
> 1100
<7
Miyazaki
1977
N = 348
STD
(µm)
165 – 215
190 – 220
> 220
IM
(53)
MR
(22)
M
(28)
> 220
M&R
(9)
Y
<2
< 160
IM
Y
2–8
160 – 187
P
Mean 197
(max)
< 220
SM
Y
6.5 –
14
G
7 – 12
G
10 – 14
mean
12
Y
2.5 –
11.5
Y
2.5 –
14.5
Y
6.5 –
13.5
(9)
155 – 170
180 – 240
mean 195
P
SM
IM
(70)
n/s
P
(58)
219
(210)
M
(220)
Table 2.3:
Worker
Year
*Hui 1979
N = 34
Sergeant
et al. 1980
Best
et al. 1984
N = 25
Spp &
Origin
Delphinus
delphis
S.California
Lagenorhychus
acutus
N.W. Atlantic
Sotalia
fluviatilis
S. America
Testis
Length
(cm)
Testis
Weight
(g)
-
C
≥ 350
-
-
E
15
Collet
et al. 1984
N = 26
Delphinus
delphis
E. N. Atlantic
Cont’d
STD
(µm)
-
E
1 - 15
E
40
E
180 - 370
C
8-148
C
1275 2110
C
56 - 393
Sperm in Ep
-
Miyazaki
1984
N = 350
33 - 65
34 - 79
ST w/ gonia
& cytes
40 - 60
E
30-40
50
100 250
E
≤ 100
19 - 86
(64)
E
100 - 400
51 - 201
(136)
E
≥ 400
133 298
(199)
Globicephala
macrorhynchus
Stenella
coeruleoalba
Pacific coast
Japan
-
-
45 - 214
30 - 69
(46)
Hohn 1985
N = 269
Stenella
attenunata
E.N. tropical
Pacific
-
-
BW Kg
40 - 47
BW Kg
36 - 42
ST marrow
w/ gonia
only. Ep
poorly
developed
ST small
lumen. w/
gonia &
cytes
Large Ep
lumen
All germ cell
types
Reduced Ep
lumen
ST w/ gona
& cytes only
No sperm in
Ep
0% mature
ST
< 50% 99% Mature
ST
Sperm in
Ep:
Scanty copious.
100% mature
ST w/ cytes,
tids or sperm
ST w/ gonia
only
100-150
Pacific coast
Japan
BL
(cm)
G
7-12
175 - 190
G
126 - 210
G
233
G
upto 22
244 - 251
BW Kg
10 - 35
E
70 - 800
Kasuya
et al. 1984
N = 51
Spermatogen
esis
assumed
Age by
GLG
Y/G
-
E
5-20
E
15 - 34
Histology
52 - 135
(77)
93 - 276
(169)
51
ST w/ sperm
(No. ST w/
sperm out of
20)
ST w/ no
lumen &
gonia only
ST w/ small
lumen
gonia, cytes
& rare tids
ST w/ sperm
83 - 138
Status
(n)
MR &M
IM
(16)
IN
(1)
M
(4)
IM
(6-8)
144 - 152
A
(3)
140 - 147
IA
(3)
117 - 187
IM
(4)
92 - 194
PR
(2)
206 - 223
SM & R
(11)
Y
<9
< 409
IM
Y
7 - 15
(16)
141 - 455
(414)
MR
-
-
Y
> 15 46
(17)
Y
≤ 14.5
Y
2.5 4.5
Y
≥ 6.5 17
(9)
Y
11
Y
17
Y
22
394 - 525
(422)
M
approx
< 235
IM
approx
185 - 199
P
approx
208 239
M
154 - 188
(175)
IM
(61)
174 - 204
(186)
P
(27)
175 - 231
(200)
M
(181)
Table 2.3:
Worker
Year
Van
Waerbeek
et al. 1994
N = 133
Spp & Origin
Lagenorhychus
obscurus
coastal Peru
Testis
Length
(cm)
Testis
Weight
(g)
E
≤ 24
E
12.3 –
58.5
E
≤ 310
Cont’d
STD
(µm)
Lagenorhychus
obscurus
Peruvian
waters
-
Age by
GLG
Y/G
BL
(cm)
No seminal
fluid in Ep
-
C
≤ 9730
Seminal
fluid in Ep
36 - 70
Chavez
Lisambart
1998
N = 48
Histology
75 121
-
110 263
ST w/ no
lumen &
gonia only
ST w/ small
lumen
gonia, cytes
& rare tids
ST w/ sperm
IM
(38)
-
Y
1 – 4.3
Y
3.5 –
4.5
Y
4.5 –
24.8
(11)
n/s
M
(125)
140 – 195
IM
(18)
178 – 183
P
(2)
173 – 199
(190)
M
(28)
Sousa chinesis
Jefferson
E
Hong Kong
G
207
19.8 –
2000
5 – 8.5
23.2
N=2
Rosas
ST w/ all
Stenella
G
Mean
C
244
163
germ cell
Coeruleoalba
2002
8
12
70
types
S.E.
Brazil
N=1
*maturity also based on robustness = BL/BW and flipper bones (epiphyses and diaphyses) fusion
N
Number of animals in study
n
Number of animals in status
n/s
Not specified
Testes length and weight
C = Combined testes
E = Either right or left testis
ST
Seminiferous tubules
STD
ST Diameter
Histology of the testes
Gonia = Spermatogonia
Cytes = Spermatocytes
1o = Primary
2o = Secondary
Status
(n)
-
SM
(1)
Tids = Spermatids
Ep = Epididymis Age
BL
Body Length
Status IM = Immature
P = Puberal or Pubertal
MR = Maturing
M = Mature
SM = Sexually Mature
IA = Inactive
2.5.3.1.1 Resting phase in spermatogenesis
Some researchers
investigated
detailed
cellular characteristics
of the
seminiferous tubules to differentiate between animals that fulfilled some criteria
for sexual maturity but differed in histological appearance of the testes (Harrison
52
and Ridgway, 1971; Hirose and Nishiwaki, 1971; Collet and Saint Girons, 1984)
(Table 2.3). Some animals showed elongation of the epididymes, large lumens,
and a higher proportion of Sertoli cells to spermatogonia but an absence of
further developmental germ cell types beyond spermatocytes. This was
interpreted as a sign of arrested or abortive spermatogenesis and these animals
were reported to be mature but in a ‘resting’ phase (Hirose and Nishiwaki, 1971;
Collet and Saint Girons, 1984). A resting phase is associated with male
reproductive cycles and seasonality by many workers. However, it must be
cautioned that there may be overlaps in the different criteria used to assess
sexual maturity, therefore, the resting animals observed may in fact be immature
animals with larger body and / or testes size. Collet and St. Grions (1984) did
not find with certainty spermatozoa in any of the specimens, suggesting the
possibly that none of the specimens classified as resting were sexually mature.
Also this study further explained that, since the subjects were stranded animals,
they were not representative of the normal population and pathologies found in
them may have disrupted spermatogenesis.
2.5.3.2 In vivo studies
2.5.3.2.1 Testosterone level
Serum testosterone level (T level) monitored on a regular basis over a period of
time has been used as an indicator of the reproductive status of male dolphins.
Current information on the T level in dolphins are mostly based on Tursiops
53
since they are the most common captive species and can be readily trained for
blood collection (Tables 2.4). It is difficult to deduce the accepted T level at
different maturity stages when testosterone alone is used to assess reproductive
status.
There is substantial overlap in results between Kirby (1990) and
Schroeder (1990a; 1990b), which rely only on testosterone levels to indicate
sexual maturity.
The T level assigned for immature subjects by Schroeder
(1990a; 1990b) were found in pubescent subjects by Kirby (1990). In addition,
there is a big gap in T level between pubescent and sexually mature animals
given by Kirby (1990), therefore, should the levels of an animal fall in this gap,
assignment of maturity status to this animal would be uncertain based on T level
evaluation alone.
Again caution must be exercised when interpreting and
comparing the results of these studies, because methodologies for testosterone
evaluation assays, blood collection and the duration of the investigative period
differ.
54
Table 2.4: Summary of T level used as an indicator of maturity in Tursiops
Worker
Year
Spp &
Origin
T level
ng/ml
Semen Analysis
<1
Tursiops
aduncus
Taiwan &
Indonesia
*Brook
1997
N=8
0.3 – 6.6
No sperm in
semen
1.6 – 19.6
density:
0 - 237 x106 /ml
Volume: < 64ml
0–3
3–5
Schroeder
1990 a& b
** N = 21
Schroeder
et al. 1983
N=1
***Kirby
1982 and
Kirby
et al. 1979
Tursiops
truncatus
5 – 54
Tursiops
truncatus
3 – 55
0.26 ±
0.19
1.76 ±
2.44
Tursiops
truncatus
density:
800 – 1300 x
106/ml
count:
9
0-54596 x 10
(1332 ejac.)
count:
0 - 63x109
volume: < 40ml
% motility:
90 - 100%
RFM 4 - 5
(179 ejac.)
-
14.1 ±
12.6
Tursiops
truncatus
BL
(cm)
Status
(n)
< 6+
192 –
202
J / IM
(2)
AD /
MR (1)
6–
7+
> 7+
Tursiops
truncatus
Florida /
Mississippi
208 –
232
10 –
13
-
RM
(1)
M
< 11
J/
IM (16)
11 –
13
PU
(7)
-
7
19
0.4 – 3.9
-
US, BL
-
SM?
-
SM
<
242
IM
(2)
Sick?
(1)
260
-
-
A/
SM (15)
1.3 – 5.2
Other
criteria
used
M
(4)
PR /
IM
PU /
MR
-
0.1 – 1.7
Harrison
et al.
1971
207
1–
10
10 –
14
> 13
– 15
< 0.8
Judd
et al.
1977
N=2
Age
(Y)
GLG
ST
histol.
TW
ST
histol.
4.9 – 24.0
≥ 10
sired
*Age, estimates only, assessment was based on body size, growth patterns and length of time in
captivity.
** Includes Schroeder (Schroeder and Keller, 1989)
*** cited in Kirby (Kirby, 1990)
Status
J = Juvenile = IM = Immature
PR = Prepubescent = IM = Immature
PU = Pubescent = MR = Maturing
AD= Adolescent = MR = Maturing
A = Adult = SM = Sexually Mature
RM = Reproductively Mature
260 –
290
M
(2)
Ejac. = Ejaculate samples
ST = Seminiferous tubule
BL = Body Length
US = Ultrasonogphic examination of the testes
Semen Analysis:
RPM = Rate of progressive motility scale 0 – 5, 0
no movement, 5 fastest
55
A study combining ultrasonographic assessment of testis development and
semen analysis with testosterone evaluation, proposed a baseline level of <
1ng/ml for sexually immature male dolphins (Brook, 1997). Again, there is
substantial overlap in concentration levels between the maturing and mature
status. In this study, semen was collected on a weekly basis and sexual maturity
is defined as when spermatozoa first appeared in an ejaculate and T level at this
stage were found to be above 1ng/ml. Further monitoring of multiple parameters
on more subjects is required to confirm the levels of testosterone at different
stages in T. aduncus and to investigate if there are associations between T level
in the blood with testis size and sperm output.
A study on Orcinus orca (killer whale) subjectively estimated onset of puberty
based on T level of six captive subjects (Robeck and Monfort, 2005b) (Table
2.5). The study also included four wild caught mature subjects. The onset of
puberty was defined as the first year that T level elevated (P < 0.05) above
1ng/ml and sexual maturity as the first year following pubertal onset for which
medium levels exceeded (P < 0.05) those of the year of pubertal onset and every
sample was above 1ng/ml. The T level of four of the captive born subjects
exhibited transitions from juvenile status, < 0.4ng/ml, to pubertal status, >
1ng/ml at ages between 7 – 9 years old. The mean T level at sexual maturity
was 6.0 ± 3.3ng/ml and was attained at 10 – 14 years (mean 11.6 ± 2.1 years) in
3 subjects. To date, this study presents the largest number of known aged
subjects and the most comprehensive information on testosterone levels during
sexual maturation in any dolphin species, however, further study of more captive
born subjects is required to consolidate findings.
56
Marked fluctuations in T level in mature dolphins are reported. These have been
attributed to seasonal changes, health and social status of the animals. All of
these have been found to affect testosterone levels in other mammalian species.
Table 2.5:
Summary of T level used as an indicator of maturity in other
delphinids
Worker
Year
Robeck
et
al.
2005
N = 10
Spp &
Origin
Testosterone
ng/ml
Testis
tissue
Serum
0.1 ± 0.2
2.9 ± 3.2
-
Orcinus orca
5.6 ± 2.9
0.3 – 0.4
Kita
et al.
1999
N = 78
Globicephala
macrorhynchus
0.8 – 3.5
Taiji, Japan
1.5 –
2 2.0
Wells
1984
N=2
Stenella
longirostris
Hawaii
Age
(Y)
BL
(cm)
Status
(n)
-
1–7
8 – 12
496 ±
18
J
P (4)
≥ 13
548 ±
2
SM
Count:
51 x 108
Density:
634 x 106/ml
14.4 –
154.9
42.0 –
264.2
33.0 –
287.1
<1–
> 60
Semen
Analysis
-
Status
J = Juvenile = IM = Immature
PR = Prepubescent = IM = Immature
PU = Pubescent = MR = Maturing
AD= Adolescent = MR = Maturing
A = Adult = SM = Sexually Mature
RM = Reproductively Mature
Ejac. = Ejaculate samples
ST = Seminiferous tubule
57
-
-
-
-
9 – 16
-
IM
(10 –
12)
MR
(7 – 9)
M
(19 –
56)
172 –
183
Other
criteria
used
Dorsal
fin
height
&
width
ST
histol.
(Kasuya
et al
1984)
GLG
2.5.3.2.1.1 Seasonal changes in testosterone level
In T. truncatus, seasonal changes in T level have been described as levels
elevated above baseline levels or the mean level for mature individuals.
Seasonal elevations greater than 10ng/ml from baseline levels of 2 – 5ng/ml
have been reported (Kirby, 1990). In another study, a mean level of 8.8ng/ml
was found in three mature males, and levels in one 15 year old male were
consistently above this level during a six-month period between January and
June (Brook, 1997), Schroeder (1990a;1990b) and Schroeder and Keller (1989;
1990c) reported that peak T level occurred in June and July, 2 – 6 weeks before
peak sperm density levels in September – October.
In Orcinus orca, seasonal changes in T level have been found in pubertal and
sexually mature animals (Robeck and Monfort, 2005b).
Pubertal animals
exhibited higher levels from March to July (mean 4.2 ± 3.4ng/ml) and low levels
in months between December and February. In sexually mature animals, higher
levels were in March to June (mean 7.2 ± 3.3ng/ml) and low levels were
between September and December. These results indicate there are individual
differences and individual variations from year to year, therefore, when
analysing levels on the basis of a group, period of peak activities may be
prolonged and season pattern less pronounced. Further study is required to
investigate seasonal changes in T level in sexually immature dolphins.
58
2.5.3.2.1.2 Effect of health status on testosterone level
Fluctuations may also be due to the health status of the animal. The T level of a
breeding male T. truncatus had fallen below 1ng/ml 3 weeks before its death
(Kirby, 1990). In a pubescent male T. aduncus, testosterone levels were found to
decrease during illness from above 1ng/ml to 0.08 – 0.9ng/ml (Brook, 1997).
Illness is one of the many consequences that can be linked to chronic stress (St.
Aubin, 2001). In marine mammals, stress is thought to impair reproductive
function because of an elevated release of glucocorticoid which suppresses the
release of GnRH from the hypothalamus. Consequently the release of LH and
FSH from the anterior lobe of the pituitary is also inhibited, which disrupts
function of the testes, including steroidogenesis, thus decreasing levels of
testosterone (St. Aubin, 2001).
2.5.3.2.1.3 Effect of social status on testosterone level
Social status and dominant versus submissive interactions between animals have
also been suggested to play a role in affecting reproductive activities and
gonadal functions (Robeck et al., 1994; Brook, 1997; Boyd et al., 1999). Suzuki
(Suzuki et al., 2003) found a significant difference in T level between an older
adult male killer whale of 14 years old and a younger sub-adult male of 11 years
old. The study suggested the low T level found in the sub-adult may reflect the
‘immatureness’ of the individual or his low social status, as he was often
attacked by two larger males and a female. In terrestrial mammals, socially low
ranking male baboons were found to be prone to stress and stress induced
59
suppressed testosterone secretion (Sapolsky, 1983). Social status has also been
suggested to have some relevance in seasonal changes in T level since marked
and consistent seasonal changes were exhibited only by the dominant male
bottlenose dolphin in Brook (1997). However, this male was the only healthy
and fully mature male in the study group. Data from more healthy and mature
males will help gain better understanding on what constitutes normal
testosterone patterns and the influence of social status.
2.5.3.2.2 Ultrasonographic assessment of testis size
Brook (1997; 2000) used real-time B-mode ultrasonography, in conjunction with
testosterone evaluation and semen analysis, to assess the reproductive status of
male bottlenose dolphins.
Since ultrasonography is non-invasive, it can be
carried out repeatedly under cooperative behaviour. Brook’s studies yielded
important longitudinal data on changes in testis morphology and size, which was
not available previously for live dolphins.
A grading system, from I to III, based on size, morphology and echopattern (See
Table 2.1), was proposed to characterise testes of dolphins of different
reproductive status.
The differences in testis length and volume observed
between each reproductive status were distinct. The smaller immature testes
were graded as Grade III and the larger adult testes, Grade I. Testis size should
not be relied on alone for assessing reproductive status, further examination of
morphology and echopattern should also be carried out. The shapes of both
immature, Grade III, and sub-adult, Grade II, testes were consistently cylindrical
60
throughout the length of the organ, whereas there was an expansion at the distal
portion of the adult testes, Grade I. The echopattern of the adult testes was
homogenous, of mid to high level intensity, with a lobular echo-texture, which
was found to be more prominent in older subjects.
The mediastinum was
hyperechoic with linear hyperechoic extensions, which were again more
prominent in older subjects. Echogenicity was reduced in sub-adult and juvenile
testes, the mediastinum, although of equal echogenecity as that in mature testis,
lack linear extensions. The echo-pattern of the parenchyma of the immature
testis was different, being markedly hypoechoic in relation to the neighbouring
muscles and poorly differentiated. An anatomical feature of the dolphin testis
noted in ultrasonography is rounded protrusions adjacent to the caudate
epidiymidis, speculated to be part of the vas deferens. These protrusions were
referred to as vasal mass and since they were only found in the testes of older
animals, their presence is suggested to be useful in differentiating animals that
have recently matured or have been mature for several years.
One T. aduncus reached sexual maturity during the study conducted by Brook
(1997).
Table 2.6 gives details of the changes in testis size during sexual
maturation and summarises changes of other accompanying reproductive
parameters. This subject was estimated to be about 6 years old at the beginning
of the study, and his testes were graded between grades II – III. A year later,
spermatozoa were first found in his ejaculate samples and signified the
establishment of spermatogenesis and onset of sexual maturity. During this
period prior to onset, the testis size of this subject almost doubled in length and
increased 5 fold in volume. Such rapid increase in testis size has been widely
reported in histological studies of other dolphin species such as the Delphinus
61
delphis (Hui, 1979; Collet and Saint Girons, 1984) Stenella coeruleoabla
(Miyazaki, 1977) and Stenella attenuata (Perrin et al., 1976). Accompanying
changes in morphology and echopattern observed brought about an upgrade of
the testes to between grade II – I. By the end of the study, his testes were
classed as Grade I. Also prior to onset, from weeks 27 to 31, testis size reduced
notably and the animal was diagnosed as suffering from a bacterial infection.
Testis size resumed growth after the animal recovered from the illness. The
study further reported irregular fluctuations in testes size during the remainder of
the study period but found that testis length did not fall between 14cm after the
onset of sexual maturity. Since the findings reviewed here were only based on a
single individual, investigations on more known aged animals will help confirm
the growth pattern of the testes during sexual maturation and effect of illness on
testis size. Longer-term investigation will yield more information to help assess
testis size fluctuations, particularly in the context of seasonal changes.
62
Table 2.6:
Testis changes monitored by B-mode ultrasonography during
sexual maturation and serum testosterone levels and sperm
density in a male T. aduncus from August 1990 - April 1992
(Brook 1997)
Study
Wk
(Mo)
1
(Aug)
Est.
Age
6+
7
Right Testis
TL
TV
(cm)
( cm3)
Left Testis
TL
TV
(cm)
(cm3)
10
45.7
9.3
42.9
10
49.7
9.9
61.2
Testes
Sonographic
Appearance
hypoechoic
Grade II -III
T level
ng/ml
Status
Comment
0.32
J
Body length 202cm
0.54
8/9
(Oct)
13.5
121.8
13.5
113.5
3.4
13
16.00
195.4
15.5
180.3
3.5
27
(Jan)
11.2
81.4
10.3
65.7
0.08
31
(Mar)
9.7
59.9
9.5
45.5
0.9
35
10.0
29.8
9.8
32.1
1.1
39
14.5
86.5
13.3
102.0
2.8
- echogenicity
similar to Grade I
- less obvious
lobulation than
mature testis
- Grade II-I
40
15.9
41
15.2
199.5
14.8
184.9
5.5
45
(Jun)
17.8
297.0
18.9
301.9
2.5
17.3
243.0
16.7
234.1
6.6
51
(Jul)
7+
14.9
-T levels 1st time
rose above 1 ng/ml
- rapid testis
growth
max. increase,
6cm, since wk 7
-rapid testis length
reduction
- fell ill
- illness
-recovery from
illness
- testis length & T
level continue to
increase
rapid testis growth
& maturation
occurred
MR
T level 1st time rose
above 5ng/ml
-Max. testis
volume
-Testis fluctuate
thereafter, not fall
< 14cm
Highest T level
54
55/56
(Aug)
7+
57-87
88
(Apr)
89
16.3
180.5
16.0
229.6
3.0
≈14.0 –
18.2
≈154.5
– 297.2
≈14.5 –
17.0
≈ 181.2
– 298.1
≈
1.6 – 6.3
Nr.
8+
Grade I
18.3
17
288.7
SM
onset of
spermatogenesis
10-13 wks after T
level > 5ng/ml
-sperm in ejaculate
- Body length
207cm
intermittent
azoospermic
periods
Max. sperm
density, 237.2 x
106/ml
Max. testis length
63
2.5.3.2.3 Semen density
The establishment of spermatogenesis, hence sexual maturity, is indicated by the
first appearance of spermatozoa in an ejaculate sample (Brook and Kinoshita,
2005). To date, there is no extensive information on the ejaculate characteristics
of sexually immature dolphins. Brook (1997) is the only study that investigated
the establishment of spermatogeneisis in bottlenose dolphins and found in one
subject that this event took place at an estimated age of 7+ years (Tables 2.4 and
2.6). The study further reported sperm density of this dolphin ranged from 0 to
237.2 x 106/ml during a 32-week post onset period and episodes of azoopermia
that lasted up to 48 days. A fully mature male in the same study produced sperm
density levels that ranged from 0 to 139.4 x106/ml. Another longitudinal study
of a 15 year-old T. truncatus also reported a minimum density level of zero but a
much higher maximum density level of 1587 x 106/ml (Schroeder and Keller,
1989). Further study on more dolphins is required to add to Brook (1997)
findings at establishment of spermatogenesis. Monitoring must also be longer
after establishment to investigate in detail changes in ejaculate characteristics
and how much time is required for sperm density of recently mature dolphins to
reach full adult capacity.
2.5.3.3 Body length at sexual maturity
Body length is a simple parameter to attain and easier to assess consistently,
provided the method used is accurately performed. Knowledge of maximum
body length or average adult size is part of understanding of a species life history.
64
This parameter is useful in wild population management, as any changes may
provide some indication of the effect of exploitation (Perrin and Reilly, 1984).
Longitudinal monitoring of body length is important in gauging growth
trajectories and measurements of adult length, or asymptotic length, are essential
in growth models, such as Gompertz (Read et al., 1993; Clark et al., 2000) and
Bertalanffy (Cockcroft and Ross, 1990) which are useful tools in managing
captive and wild populations.
Variations in methodologies and unclear criterion to assess reproductive status in
male dolphins make the body length at sexual maturity difficult to determine
(Perrin and Henderson, 1984a). This is evidenced by the substantial overlaps in
body lengths between animals of different maturity (Hirose and Nishiwaki, 1971;
Collet and Saint Girons, 1984; Hohn, 1985; Chavez Lisambart, 1998) (Table 2.2)
and render body length a poor indicator of sexual maturity. Based on rapid testis
growth prior to onset of sexual maturity found in two separate studies, in
Delphinus delphus and T. aduncus, a lack in corresponding significant increase
in body length led both authors to conclude body length on its own can not be
used to accurately assess reproductive development and predict sexual maturity
in male dolphins (Hui, 1979; Brook, 1997).
The percentage of final body length reached at sexual maturity has been found to
be quite constant in female marine mammals (Laws, 1956) and thus useful as an
indicator of an animal’s proximity to sexual maturity. In cetaceans, the average
percentage final body length reached at sexual maturity was found to be 85.1%
in females (Laws, 1956). Such percentage is less constant in males, Law (1956)
suggests this is due to greater variation in growth rates, but a lack of consensus
65
in consistency in assessing male sexual maturity among workers may also play a
role.
Table 2.7 summarises the percentage of final body length at sexual maturity
deduced from literature on various species of male delphinids. The average for
this percentage is 93% for male delphinids which is higher than the average for
female cetaceans, 85.1% (Laws, 1956). Sexual dimorphism found in Orcinus
Orca, T. truncatus and Stenella attenuata is suggested to be caused by a longer
period of pubertal growth in males than in females (Kasuya et al., 1974; Read et
al., 1993; Clark et al., 2000). There are a number of factors that may contribute
to explaining why the percentage of final body length at sexual maturity in Table
2.7 is wide ranging, 83 – 99%. First, as stated earlier, the criteria used to assess
male sexual maturity are not consistent between studies. The life histories of
species such as Orcinus orca and Lagenorhynchus obscurus (dusky dolphin) can
be presumed to be very different, and the difference in final body size attained is
considerable. Some body lengths listed are obtained by calculation or deduction
from growth models or curves as opposed to observed average measurements.
Over estimation of body length derived from growth curves has been reported in
a study of T. truncatus in the wild (Cockcroft and Ross, 1990).
66
Table 2.7:
Percentage of final body length (BL) and age reached at
sexual maturity in delphinids
Worker &
Year
Spp &
Origin
a
Robeck et al.
2006 & Clark
et al. 2000
Chavez
Lisambart
1998
N = 48
a
Brook
1997
N=1
Cockcroft
et al. 1990
N = 53
Miyazaki 1997
N = 1752
Kasuya
et al. 1984
Orcinus Orca
captive born
N. America
Lagenorhychus
obscurus
Peruvian waters
Tursiops aduncus
Taiwan &
Indonesia
Tursiops truncatus
S. Africa
Stenella
Coeruleoalba
Pacific coast
Japan
Globicephala
macrorhynchus
Japan
Stenella
attenunata N.E
tropical Pacific
Stenella
attenunata Pacific
ocean, Japan
Sexual maturity
A)
GLG age
BL
Year
b
548
11.6
d
180
c
207
4.3
7-8
14.5
240
8.7
219
15.8
414
B)
BL
Final
GLG age
Year
657.2
d
d
191.7
6.5
A/B
%
83
94
220
-
94
243
-
99
238
473.5
14
-
Perrin
12
et al. 1976
200
195
N = 115
Kasuya
10.3
12.5
et al. 1974
d
197
203.3
N =195
a
Sergeant
Tursiops truncatus
12
et al. 1973
270
N.E Florida
245
N = 31
a
Captive subjects
b
Known age
c
Age estimated by body size, growth pattern and length of time in captivity
d
Measurements deduced from growth equations / models / curves
92
89
98
97
91
Although most of the studies are based on wild animals, two are based on
captive subjects.
Brook (1997) noted the percentage of final body length
reached at sexual maturity in captive T. aduncus (Table 2.7) is higher than that
found in T. truncatus by Sergeant (Sergeant et al., 1973) and suggested the effect
of captivity as possible a cause.
Improved and consistently high plane of
67
nutrition and decreased level of energy expenditure, as found in captive
conditions, may elevate growth, consequently captive animals attained larger
body size at sexual maturity (Laws, 1956; Sergeant et al., 1973; Read et al., 1993;
Robeck and Monfort, 2005b). More research based on a clearer definition of
male sexual maturity is required to further investigate usefulness of the
percentage of final body length in predicting sexual maturity. The use of captive
known aged subjects will yield better understanding on growth and effect of
captivity on body size at sexual maturity.
2.5.3.4 Age at sexual maturity
In T. truncatus the age, by GLG count, at sexually maturity is reported to be
above 10 years (Sergeant et al., 1973; Ross, 1977; Cockcroft and Ross, 1990)
thus, it is difficult to predict the onset of sexual maturity based on age alone.
The age at sexual maturity reported in one captive T. aduncus was 7 – 8 years,
which is much younger than the age reported for T. truncatus in the wild (Brook,
1997) (Tables 2.4 and 2.6). Again, this may be explained by improved growth
rate in captive conditions, thus animals reach the body size threshold for sexual
maturity to occur at an earlier age. This finding was based on 1 captive animal
only, and its estimated age was not obtained by count of GLG of the teeth.
Further study on known aged subjects is required to investigate the effect of
captivity on age at sexual maturity and how consistent age is as an indicator.
68
2.5.3.4.1 Aging by growth layer group (GLG) count
GLGs are groups of incremental dentinal and cemental growth layers in cyclic
repetitions seen in longitudinal thin sections of the teeth (Bryden, 1986b). Since
the deposition of dentine is progressive with age, up to 11 layer groups, and
cement continues to accumulate thereafter (Kasuya et al., 1974), counting of the
number of growth layers can provide an estimation of an animal’s age. This
method of age determination has been used in many cetacean studies, including
studies shown in Tables 2.2 and 2.3 where reproductive development and growth
of an individual are corresponded or related to a certain age or age range. Age
determined by GLG is not without uncertainty due to technical differences
between workers and multiple teeth are required.
The latter may pose a
challenge in field or stranding situations. A study suggested that dentinal and
cemental GLGs are deposited annually based on calibration on the teeth of a
known-aged captive born dolphin bottlenose dolphin (Cockcroft and Ross, 1990).
However, this subject only lived for 7 years and other workers have suggested to
take into considerations the rate at which GLGs are deposited may change
during different stages of an animal’s lifespan (Perrin et al., 1976). Further, age
determination through GLG can not be conducted on teeth that have been worn
to the gum. Tooth wear has been suggested to be related to very old age (Perrin
et al., 1976). When the pulp cavity is completely filled, GLG can not be counted
accurately and partial occlusion may lead to underestimation of GLG, hence age
(Perrin et al., 1976). Poor calcification of secondary dentine has been reported
in dolphin teeth with 12 – 16 dentinal growth layers and accurate counting was
not possible (Kasuya et al., 1974; Miyazaki, 1977). GLG counts are difficult to
carry out and some studies report counting being conducted by more than one
69
technician to ensure reliability (Cockcroft and Ross, 1990; Jefferson, 2000).
Such finding further highlights the need to carry out studies on known-aged
animals where possible.
2.6 Seasonality
Natural selection ensures that reproduction occurs in harmony with existing
environmental conditions (Bronson, 1988).
Reproductive cycle in a given
species is geared towards optimizing annual seasonal changes in environmental
conditions to maximize survival of the young (Boyd et al., 1999). Seasonal
patterns not only differ between species and populations of the same species, but
also differ between sexes, as each faces its own unique set of environmental
challenges (Bronson, 1988). The extent of seasonal effects may vary and species
may be classified on a continuum of facultative-obligate seasonal breeders (Asa,
1996).
Species that dwell in an environment where favourable conditions are
predictable and invariant from year to year are likely to be obligate breeders.
However, when environmental conditions are unpredictable, breeding may be
facultative, seasonal changes may be subtle and males may remain
reproductively capable throughout the year, to be in a constant state of readiness
for opportunistic breeding. Successful reproduction and survival of offspring is
dependent on the female, therefore the male reproductive cycle should coincide
with her. While production of spermatozoa is not energetically costly per se,
physical and behavioural changes associated with obtaining matings, such as
development of coat and colour, body musculature and weapons for display or
70
combat, establishment of social dominance through aggression, demarcation of
territory and mate searching and guarding, are very demanding in resources. In
manatees, males must travel long distances to search for females which are only
estrous during warm months. Therefore, reduction in reproductive activities
during non estrous periods will allow an individual to recuperate, live longer and
sire more offspring (Boyd et al., 1999).
2.6.1 Testicular and hormonal changes in seasonal mammals
Marked seasonal changes found in highly seasonal mammalian species include
structural and functional regression and redevelopment under the influence of
gonadotrophins (Lincoln, 1989).
Changes in the size and weight of the testes
reflect histological changes within the seminiferous tubules, including luminal
size, Sertoli cell size, germ cell population, and the number of germ cells
completing spermatogenesis. In regression, the tubules shrink because of the
involution of the basement membrane and occlusion of the lumen. While cell
number remains the same, Sertoli cells also undergo reduction in size due to
reduced cytoplasmic volume and nucleus. The tight junctions between adjacent
cells may also lose integrity and failure of the blood-testis barrier may lead to
infertility.
Germ cells of all types (spermatogonia, spermatocytes and
spermatids) at specific locations within the tubules are generally low in number
during regression due to reduced efficiencies in mitotic and meiotic divisions.
The number of spermatozoa produced is thus reduced also. In more pronounced
seasonal species, there may be a complete absence of spermatozoa in tubules that
71
are severely reduced in size with completely occluded lumens. Abnormalities in
germ cells and degenerate cell fragments may be found within the tubules.
Occurrence of immature sperm cells, spermatozoa with structural abnormality
and reduced motility in the epididymis is higher during testicular regression
(Lincoln, 1989). There appears to be a minimal weight requirement for
spermatogenesis to occur. A general rule has been proposed that if the weight of
the testes drops below 30% of the seasonal maximum, spermatogenesis can be
expected to be completely disrupted (Lincoln, 1989).
Reduction in pulse frequency and magnitude of LH and FSH secretions by the
anterior pituitary reduce the secretion of testosterone by the Leydig cells. Such
decline in gonadotrophins is controlled by the GnRH generator in reducing
GnRH secretion from the hypothalamic neurons or increased sensitivity of the
hypothalamus to the inhibitory or negative feedback effect of gonadal steroids,
such as testosterone.
In very seasonal species, testosterone level may become
very low or undetectable during periods of testicular regression.
Seasonal
changes of spermatogenesis and steroidogenesis may not necessarily parallel
each other. Testosterone levels may fluctuate in the absence of obvious changes
in spermatogenesis (Lincoln, 1989). In addition, secretions of prolactin, thyroidstimulating hormone (TSH) and growth hormone (GH) are also inhibited during
low seasons (Bronson, 1988).
Prolactin and GH are involved in increasing
sensitivity of the gonads to LH by redevelopment of LH receptors.
72
2.6.1.1 Seasonal puberty
The regressed state of the testis is comparable to an immature testis and the
events in redevelopment, resumption of testis size and activity, are comparable
to that of puberty (Lincoln, 1989).
Complete reversal of the events described
for regression takes place during redevelopment. Testis size measurement can be
used to infer tubules activities and resumption of spermatogenesis or
augmentation of the process.
Counting of the number of spermatozoa in the
epididymis or ejaculate can directly monitor increased efficiency in
spermatogenesis.
A delay of up to several weeks for spermatozoa to be
produced and fill the epididymis has been observed, since during regression the
epididiymis is completely devoid of spermatozoa if spermatogenesis had ceased
completely. Blood sampling for hormonal assays is also useful in monitoring
seasonal patterns in secretion. However, due the episodic or pulsatile nature of
the secretion of the hormones concerned, a sampling frequency that can capture
a cyclic pattern is essential and study duration must be long. In addition, peak
endocrine secretion may not be coincident with peak testis size or
spermatogenesis.
2.6.2 Seasonality in delphinids
There are many species of dolphins and their habitat distribution covers a wide
range of environmental conditions, therefore their reproductive strategies vary.
In wild caught or stranded animals, male reproductive cycles have been
investigated using indicators such as testis size and histological examination of
73
the seminiferous tubules, and presence or absence of sperm in the epididymis.
Longitudinal monitoring of testosterone level in peripheral blood of captive
dolphins has been routinely conducted and may be used to study seasonal
changes.
A large study, consisted of 852 animals and over 100 sexually mature testis
samples, found reproduction in Lagenorhynchus obscures (dusky dolphin), in
Peru highly seasonal (Van Waerebeek and Read, 1994). Male reproductive
activities were found to be in synchrony with the female reproductive activies.
Maximum testis weight and copious seminal fluid in the epididymis was found
in September and October. Testis weight decreased in November but seminal
fluid was in the epididymis year-round, suggesting complete cessation of
spermatogenesis was unlikely. The tubule diameter obtained in another study of
Lagenorhynchus obscures in Peruvian waters was reported to not correspond
well with the data on testis weight and birth. Tubule diameters were found to be
largest in January and March and smallest in June and July (Chavez Lisambart,
1998).
This study consisted of only 28 animals and it lacked samples for
September and October. Tubule diameter increased from June to August and
data interpolation between August and November depicted a downward trend.
Such data presentation is misleading, as tubule diameter may continue to
increase after August before falling in November.
Further, lower tubule
diameter in November should be expected as testis weight was found decreased
during this month in the earlier study.
The Stenella species, attenuata (spotted dolphin), coeruleoalba (striped dolphin)
and longirostris (spinner dolphin), have been shown to be seasonal. The mean
74
weight of the testis of mature Stenella attenuata has been found to correlate with
the diameter of the follicles in females in the same school (Kasuya et al., 1974).
It is speculated that since large follicles ovulate readily, therefore males present
within the school should be at peak reproductive potential. Also in this study,
seasonal decline in spermatogenesis was suggested where spermatozoa were not
found in smears of testes that were above the weight at puberty. Full regression
was not found in a study of the northern and southern offshore stocks of Stenella
attenuata, in eastern tropical Pacific Ocean, as spermatids were observed in
mature testes (n = 181) throughout the year (Hohn and Chivers, 1985). The
mean diameter of tubule lumens in this population was greatest in April, the
incidence of specimens with spermatozoa peaked in May and maximum testis
weights were found in July and August. The study suggested testis size is not a
good single indicator of reproductive seasonality for Stenella attenuata as peak
spermatozoa levels coincided with peak calving season when testes weights were
relatively low. Year round calving found in the northern offshore stock indicates
that not all males exhibit the same seasonal pattern and some remain
reproductive throughout the year. A resting stage was assigned to male Stenella
coeruleoalba (n = 9) which have attained sexual maturity age (determined by
dental growth layer group) and body length but their testes weights were less
than that of the mature individuals and the seminiferous tubules were not
composed of all stages of spermatogenetic cells (Hirose and Nishiwaki, 1971).
It is suggested that the resting stage is indicative of a male reproductive cycle.
Based on samples collected only from October to December, a later study
deduced a seasonal change in reproductive activity in another group of Stenella
coeruleoalba (Miyazaki, 1977).
Testis weight, tubule size and incidence of
75
mature animals with spermatozoa found in all the tubules examined decreased
from the October to December during 1968 – 1973. The finding that peak male
reproductive activity in October did not coincide with the main mating seasons
of this dolphin population suggests it may occur during other months of the year
which were not sampled. For Stenella longirostris, heavier testes were found
from February to August and they reduced during September through January
for the northern white-belly population (Perrin and Henderson, 1984a). The
testis weight of the eastern stock of the same species was found to be less than
that of white-belly form and peak weights occurred between March and June.
2.6.2.1 Resting phase in testicular activities
In Delphinus delphis (common dolphin), a resting phase for mature testes is also
described. Three individuals stranded on the Atlantic coasts of France during
December and January were in a state of complete inactivity, with marked
diminished tubule diameter, Sertolian syncytium and germ cell types (Collet and
Saint Girons, 1984). However, stranded animals are not representative of a
healthy population since they often present systemic pathologies which may
disrupt spermatogenesis, resulting in the inactivity described. The sample size of
this study was small (n = 26), samples sparsely covered the calendar year and
not every month was presented. Also, the study reported that spermatozoa were
not found in any of the samples. The assignment of sexually mature status is thus
questionable and the reproductive activity in this population of common
dolphins cannot be fully understood. An earlier study on the same species did
not recognize a resting phase. Large testes were found throughout the year and
76
indicated that there was no seasonal rut for the population in water of southern
California (Hui, 1979).
2.6.3 Seasonality in Tursiops
Results of studies on seasonality in Tursiops are varied and lack consensus, and
data about male cycles are scarce.
Population studies of wild and captive
dolphins generally found some degree of seasonality in reproduction, with
flexibility and diffused peaks of activities (Urian et al., 1996). Calves are born
all year round with more births concentrated in certain months, depending on the
population studied (Cornell et al., 1987; Cockcroft and Ross, 1990; Urian et al.,
1996).
There is no evidence of a seasonal pattern in testis weight, tubule
diameter and presence of spermatozoa in the epididymis (Cockcroft and Ross,
1990). Longitudinal monitoring of circulating testosterone levels are reported to
show a biphasic pattern, with increased levels in spring and fall in T. truncatus
(Harrison and Ridgway, 1971; Kirby, 1990) and spring and early summer in T.
aduncus (Brook, 1997). A two-year study based on a single mature male found
peak testosterone levels consistently occurred in July, however, lower levels did
not follow an obvious pattern (Schroeder and Keller, 1989). Sperm density
showed week to week fluctuations and very low levels occurred repeatedly
between November and January (Schroeder and Keller, 1989). Episodes of
azoospermia, or extremely low sperm density, were exhibited by two T. aduncus,
however, their occurrence did not follow any pattern (Brook, 1997). Since
azoospermia was found in a recently sexually mature animal, further study is
77
required to investigate changes in sperm density immediately after the onset of
spermatogenesis. The only longitudinal data available to date on testis size did
not find marked seasonal increase, but testes of T. aduncus showed a tendency to
be larger during spring and summer (Brook, 1997). Seasonal changes were not
found in the echo pattern of testicular parenchyma (Brook, 1997).
There are other factors in addition to environment stimuli to consider in studying
reproductive patterns. Stress caused in social situations, particularly with social
subordination, can exert a negative effect on reproductive activities
superimposed on a breeding season.
In captive situations, the limited
environment and logistics of animal group management may result in social
instability and stress. Prompt inhibition of LH secretion in male mice has been
found to follow defeats in fighting episodes (Bronson, 1988). Testosterone
levels differed between high and low ranking baboons during periods of overt
aggression (Sapolsky, 1983). In one study of T. aduncus, seasonal tendency in
testis size and testosterone level was more evident in the dominant male
compared to the subdominant or younger males (Brook, 1997). Longitudinal
studies aimed to investigate seasonality in reproductive activities must span
multiple years in order to account for individual variations and shifting of peak
and low activity levels from year to year (Hirose and Nishiwaki, 1971; Van
Waerebeek and Read, 1994; Brook, 1997; Kirby, 1990).
78
2.7 Assisted reproductive techniques (ART) in dolphins
ART encompasses techniques such as artificial insemination (AI), embryo
transfer (ET), in vitro fertilisation (IVF), gamete intro-fallopian transfer (GIFT),
zygote intro-fallopian transfer (ZIFT) and intracytoplasmic sperm injection
(ICSI) (Go, 2000; Wildt, 1999a). These techniques involve retrieval, storage and
transferring of genetic materials across distances and time, therefore, limitations
in conventional captive breeding strategies can be circumvented. Before ART
can be developed in any species, basic understanding of the reproductive process
is required as each species is specialised in their adaptations to reproduce
(Robeck et al., 1994; Wildt, 1999a; Wildt and Wemmer, 1999b).
The success of controlled unassisted breeding based on scientific data and well
defined husbandry and management protocols has been demonstrated in
Tursiops aduncus (Brook and Kinoshita, 2005). However, limitations inherent
in breeding programmes of small captive groups that solely rely on natural
breeding prevent such programmes from reaching the goal of preserving genetic
diversity and population self-sustainability. Physical space required to maintain
a sizeable group for genetic viability, animal incompatibility, limited resources
and risks of animal transport for inter-institutional breeding exchange and
associated social group disruption are some limitations of natural breeding
(Lasley et al., 1994; Robeck et al., 1994; Wildt, 1999a; Wildt and Wemmer,
1999b). Integration of appropriate assisted reproductive techniques (ART) can
help overcome these limitations.
79
ART should not be viewed as a ‘quick-fix’ alternative to inadequacies in
husbandry and management (Wildt, 1989) or a solution to reproductive
abnormality in wildlife species (Robeck et al., 1994). ART is most effective
when applied to animals that are highly fertile with a good reproductive history
(Lasley and Anderson, 1991; Robeck et al., 1994). Use of ART in certain types
of infertility may exacerbate problems of limited space and genetic variation in
captivity by reproducing more subfertile animals (Lasley and Anderson, 1991).
Techniques such as AI should be used to enhance the reproductive efficiency
achieved by conventional breeding, not to replace it (Robeck et al., 1994).
Besides exchange of genetic materials between isolated groups, genetic
management can be further enhanced by strategic infusion of genetic materials
stored from earlier generations into future generations (Wildt, 1989; Wildt,
1999a). In addition, genetic materials may be exchanged between captive and
free-ranging individuals, to introduce genetic vigor to both populations (Wildt,
1989; Wildt, 1999a).
Once an assisted technique is developed and proven
successful in a non-endangered, more easily accessible species, some degree of
extrapolation may be carried out so that transfer of the technique to a related
endangered species in which limited time and individuals can be afforded for
research can be quicker (Loskutoff, 1998).
2.7.1 AI in dolphins
Comparatively basic understanding of the reproductive physiology required for
the development of AI means that it is a feasible option in some cetaceans.
80
Information obtained in recent years and on-going research ( Brook et al., 1991;
Robeck et al., 1993; Robeck, 1996; Brook, 1997; Robeck et al., 1998; Brook,
1999; Robeck et al., 2000; Brook, 2001) has led to the development and
successful application of AI in T. aduncus (Kinoshita et al., 2004; Brook and
Kinoshita, 2005), T. truncatus (Robeck et al., 2005a), Orcinus orca (killer whale)
(Robeck et al., 2004b) and Lagenorhynchus obliquidens (Pacific white-sided
dolphin) (Robeck et al., 2003).
AI entails deposition of semen directly into the female reproductive tract. For
this technique to be useful, understanding of both the male and female
reproductive tract and physiology is essential so that semen can be collected
safely and reliably, normal ejaculates traits characterised, and insemination
conducted at a time and site that optimises chances of fertilisation. Fresh, cool
liquid-stored, or cryopreserved semen may be used for insemination. Much
research has been conducted to characterise and monitor the bottlenose dolphin
estrus cycle by endocrine methods (Schroeder, 1985; Schroeder, 1990a; Robeck
et al., 2001a; Robeck et al., 2005a) and ultrasonographic visualisation of the
ovary (Brook, 1997; Brook, 2001; Robeck et al., 2001a; Brook and Kinoshita,
2005; Robeck et al., 2005a). Some of these studies also included attempts to use
exogenous hormones to induce or control estrus and ovulation, to enhance the
reproductive potential of mature females by reducing periods of anestrus
(Robeck et al., 2001a; Robeck et al., 2005a). Accurate prediction of ovulation is
essential in deciding the optimal time to carry out insemination, so that fewer
insemination attempts are required prior to ovulation and valuable semen is not
wasted (Robeck et al., 2005a). Insemination techniques were developed from
studying the female reproductive tract of dead specimens (Robeck et al., 1994).
81
Specific sites within the female reproductive tract where semen have been
deposited and successfully brought about pregnancies include the uterine body
and the uterine horn ipsilateral to the ovary with the preovulatory follicle
(Kinoshita et al., 2004; Robeck et al., 2005a; O'Brien and Robeck, 2006). In
male dolphins, the fundamental reproductive anatomy and physiology is not
fully understood and little research has been conducted towards development of
short-term storage and cryopreservation of semen.
2.7.2 Semen collection
The method of sample collection must also be taken into account for semen
analysis and evaluation standardisation. The materials from which collection
containers are made have been found to affect sperm motility (Balerna et al.,
1985).
This study found polyetheylene- and polyurethane-based plastic
containers depressed motility and recommended the use of high quality
polypropylene or polystyrene containers because of their biocompatible
properties and safety in case of accidental breakage.
During collection,
contaminates such as urine in semen samples affect pH and sperm function.
Motility of human sperm decreases in proportion to the amount of urine present
and contact time (Kim and Kim, 1998). Another study that investigated the
effect of a pH range, 5.25 – 9.75, on human semen samples, reported a mean
sample pH of 7.65 and found spermatozoa generally tolerated alkaline conditions
better than acidic conditions (Makler et al., 1981). This study also showed
82
immotility induced by acidification may be reversed by neutralisation, however,
such reversibility was not achieved with alkalinised samples.
Early attempts to collect semen from dolphins used electroejaculation (Hill and
Gilmartin, 1977; Seager et al., 1981). The degree of manual restraint required
for this procedure may affect the quality of the semen collected and the stress
induced rendered the technique inappropriate for routine application. Semen
collection under trained behaviour was subsequently developed. Keller (1986)
and Wu (1996) were first to describe the training steps, which included voluntary
extension of the penis and ejaculation into a collection vessel upon tactile
stimulus. Seawater contamination during poolside semen collection must be
avoided as immediate cessation of progressive motility regardless of sperm
density is reported (Schroeder et al., 1983).
Urination may occur during
voluntary semen collection. The effect of urine contamination on dolphin
spermatozoa has not been fully reported to date. The current pH value of semen
reported for bottlenose dolphins, T. truncatus, is about 7.7 (Robeck and O'Brien,
2004a).
As a result of trained behaviour, semen samples can be collected frequently to
gain some understanding of spermatogenesis and possible cyclicity, to develop a
standardised semen evaluation protocol, to assess outcomes of experiments on
different processing conditions and the effect of cryopreservation.
83
2.7.2.1 Frequency of semen collection
Frequency of collection has been shown to affect semen characteristics in bulls
and stallions. Increased collection frequency resulted in decreased ejaculate
volume, sperm density and count (Cunningham et al., 1967; Magistrini et al.,
1987). Motility was found to increase slightly in a 5-collection day per week
schedule, compared to a 3-collection day per week schedule (Magistrini et al.,
1987). On a weekly basis, higher numbers of spermatozoa were yielded in a 6collection per week schedule (6 collection days per week or ejaculation twice in
succession on 3 collection days) compared to a once a week collection schedule
(Pickett et al., 1975; Almquist, 1982). The high collection frequency of 6
collections per week from puberty did not have a detrimental effect on the
growth and reproductive development of bulls (Almquist, 1982). In stallions,
when ejaculates were collected in succession (up to 5 times) the number of
spermatozoa per ejaculate decreased (Pickett et al., 1975; Squires et al., 1979;
Pickett et al., 1985). Differences in motility between the first two ejaculates was
slight (Pickett et al., 1985) or statistically insignificant (Pickett et al., 1975).
Such significant inter-animal variations have been reported in these semen
collection frequency studies (Cunningham et al., 1967; Pickett et al., 1975;
Pickett et al., 1985; Magistrini et al., 1987) that investigators must be mindful
that semen and breeding potential must be evaluated strictly on an individual
basis.
The potential effect of frequency of semen collection on dolphin semen
characteristics and sperm quality has not been investigated.
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2.7.3 Semen evaluation
Routine evaluation of ejaculate includes sperm motility, viability, density,
volume and pH. Accurate semen analysis is essential as semen quality is often
used to indicate fertility potential, disorder and conception rates in human and
animals (Aitken, 1989; Durrant, 1990; Auger et al., 2000; Andrade-Rocha, 2003;
Palmer and Barth, 2003; Hoflack et al., 2005). Standardisation in evaluation is
useful particularly for comparison purposes: to compare seminal parameters
between animals of different age, or the outcome under different experimental
conditions when determining an optimum cryopreservation protocol for a
specific species.
2.7.3.1 Sperm motility
Manual assessment of sperm motility is subjective (Auger et al., 2000; Howard
et al., 1984) and is conducted at 20oC for human samples (WHO, 1999) or body
temperature (35 – 38oC) of the species concerned (Seager and Platz, 1977;
Howard et al., 1984; Robeck and O'Brien, 2004a).
Motility is basically
determined by counting the numbers of motile and immotile spermatozoa in
randomly selected microscopic fields (Mortimer, 1994). The number of motile
spermatozoa counted is converted into a percentage to give total motility (TM).
Only spermatozoa that are free from clumping should be counted (Mortimer,
1994). World Health Organisation (WHO) guidelines further classify motile
spermatozoa into a) rapid progressive, b) slow or sluggish progressive and c)
85
non-progressive (WHO, 1999). The sum of Grades a) and b) provides the
percentage of forward or progressive motility (PFM or PPM).
Assessment of dolphin spermatozoa motility is based on the manual methods
described above and thus also subjective (Durrant, 1990; Robeck and O'Brien,
2004a; O'Brien and Robeck, 2006).
Progressively motile spermatozoa are
categorised or ranked based on a scale that was originally developed for
assessing canine spermatozoa (Seager and Platz, 1977) (Table 2.7). This scale is
adapted to denote the rate of movement exhibited by progressively motile
spermatozoa and is referred to as kinetic rating (KR), or rate of progressive
motility (RPM), or speed of progression (SOP). Here, 5 indicates the most rapid
forward or progressive movement and 0 no forward or progressive movement
(Durrant et al., 1999a; Robeck et al., 2001a; Robeck and O'Brien, 2004a;
O'Brien and Robeck, 2006).
Further, motility parameters may be
mathematically combined to provide a single index for evaluation and
comparison between motility results from different cryopreservation conditions.
Durrant (1999a) used motility score (MS) which was derived by motility x SOP.
Robeck and O’Brien (2004a), Robeck et al. (2004b) and O’Brien and Robeck
(2006) devised sperm motility indices (SMI) for dolphin spermatozoa, derived
by TM x PPM x KR and PM x KR, respectively.
It is often assumed that the most rapid and linear form of motility is associated
with increased fertility. However, it has been shown that slow or sluggish linear
or nonlinear motility is significantly related to conception rate (Dunphy et al.,
1989). The longevity of rapidly motile spermatozoa may be compromised by
faster depletion of their energy source, intracellular ATP (Dunphy et al., 1989).
86
Table 2.8:
Scale for assessing canine spermatozoa motility status
(Seager, 1977)
Category / Description of sperm movement
Rank
All dead
0
Slight side-to-side movement, no progressive progression
1
Rapid side-to-side movement, no progressive progression
2
Rapid side-to-side movement, occasional progressive
3
progression
Slow, steady progressive progression
4
Rapid, steady progressive progression
5
2.7.3.2 Sperm viability
Spermatozoal viability, or vitality, is a quantitative parameter. Supravital stains
can be used to distinguish dead, or non viable, sperm cells based on the concept
that dead cells sustain plasma membrane damage that allows uptake of stains
(Mortimer, 1994; WHO, 1999). Stain solution, eosin-nigrosin, has been used to
assess the viability of the spermatozoa of many species (Bjorndahl et al., 2003),
including that of dolphins (Durrant et al., 1999a; Robeck and O'Brien, 2004a).
A 2003 study showed that variations in mixing time between sample and stain
solution, from 30 – 300 seconds did not affect the final percentage of dead
spermatozoa in human semen (Bjorndahl et al., 2003). This study also cautioned
that prolonged storage of stains in a humid environment before examination
might result in false high percentages of dead spermatozoa. Spermatozoa are
killed during the initial preparatory process of staining and drying, condensed
water vapour in a humid environment could reconstitute the dried stain which
may then enter any unstained cells (Bjorndahl et al., 2003). Such findings
87
warrant prompt examination of slides after preparation. A study on boar semen
recommended the mixing time between sample and stain solution to be strictly
30 seconds and the smear to be dried quickly on a warm plate, as opposed to at
room temperature (Tamuli and Watson, 1994). Pre-warming of stain solution to
30oC has been shown to result in lower percentages of dead spermatozoa in bull
semen by reducing the detrimental effects of sudden temperature difference
(Hancock, 1951).
2.7.3.3 Sperm density
Sperm density is also a quantitative parameter. The method most commonly
used to determine sperm density in human semen is haemocytometry (Mortimer,
1994; Swan and Elkin, 1999; WHO, 1999). WHO guidelines entail application
of different dilution factors depending on the density of the sample as estimated
during motility assessment. The higher the density of the semen sample, the
higher the dilution factor used. However, effect of different dilution factors on
the accuracy of counting has not been investigated. Volumes of diluent and
sample used are below 1ml, therefore, extreme care must be exercised in making
such micro-volumetric dilutions (Mortimer, 1994), and correct use of
micropipettes is essential. Sperm density of dolphin semen is in general higher
than that of humans; normal density in humans is between 20 and 250 x 106/ml
(Andrade-Rocha, 2003). Sperm densities reported for Tursiops truncatus include
a mean of 373 x 106/ml, which is high (Robeck and O'Brien, 2004a), and
maximum values of ≥ 1,500 x 106/ml (Hill and Gilmartin, 1977; Schroeder and
88
Keller, 1989; Briggs et al., 1995). Details of methods for the determination of
dolphin sperm density have not been reported, thus, it is not certain if the
application of WHO guidelines for dilution of dolphin ejaculates is appropriate.
2.7.3.4 Semen analysis by automated system
Automatic semen analysis systems, computer-assisted semen analyser (CASA)
and sperm quality analyser (SQA), eliminate the subjective nature of manual
assessment and provide quantitative measures of spermatozoa motion and speed.
CASA is expensive and to fully benefit from the advantage of high precision, it
requires expertise in sample preparation and parameter settings (WHO, 1999).
SQA is considered a more practical and inexpensive device (Hoflack et al., 2005)
and it provides a sperm motility index (SMI) based on density, progressive
motility and percentage of normal spermatozoa through electro-optical methods
(Bartoov et al., 1991). SMI has been found to correlate well with manual
assessment in human semen (Bartoov et al., 1991; Fuse et al., 2005). A bull
semen analysis study found only moderate correlation between SMI and results
of manual assessment, even at an optimum sperm density of 50 x 106/ml and
SMI of frozen-thawed semen was not useful in predicting non-return rate
(Hoflack et al., 2005). Inaccuracies in SMI have been found in species that have
high density semen (Hoflack et al., 2005). Spermatozoa in highly dense samples
can not move freely, collision between cells occurs and velocity of progressive
motility is reduced (Hoflack et al., 2005). Lower SMI in dense samples may
also be due to a lack of penetration of light adequate for optical examination
(Hoflack et al., 2005).
89
The use of CASA to evaluate dolphin spermatozoa has not been reported.
2.7.4 Semen cryopreservation
2.7.4.1 Basic principles
Cryopreservation refers to storage temperatures well below 0oC; -79oC when
using solid carbon dioxide, -140 -160oC in liquid nitrogen vapour and -196oC
when submerged in liquid nitrogen (Watson, 1995).
The purpose of the
procedure is to completely halt metabolic activity so that the life span of the
spermatozoa can be extended (Medeiros et al., 2002).
Some basic
understanding of cryobiology and the effects of cooling spermatozoa from body
to subzero temperatures is helpful in devising experiments to establish optimal
conditions for cryopreserving semen of any species. A cryopreservation
procedure includes steps such as extension, cooling, addition of cryoprotectant,
packaging, storage and thawing; at each step, spermatozoa are subjected to stress
and can lose their ability to function normally (Watson, 1995). A major cause
of stress to sperm cells during cryopreservation relates to the formation and
dissolution of ice, both extra- and intra-cellularly (Watson, 1995; Watson, 2001).
90
2.7.4.1.1 Ice formation
As the temperature of a solution containing spermatozoa lowers to below its
freezing point, water gradually crystallises out as ice and the solution becomes
more concentrated. An osmotic gradient is created and the cells are said to be
exposed to solution effects (Watson, 1995). In the presence of a hypertonic
solution, water leaves the cells through osmosis. Severe dehydration causes cell
shrinkage, which may lead to injury such as irreversible membrane collapse
(Medeiros et al., 2002). Also the presence of extra-cellular ice in a constrained
frozen environment exerts mechanical stress on the cells (Medeiros et al., 2002).
Cell damage caused by solution effects may be reduced by increasing freezing
rates, thus reducing the length of exposure of the cells to extreme hypertonic
conditions. However, if freezing rate is too fast, there is insufficient time for
water to leave the cells and intracellular ice formation occurs, which is fatal to
the sperm cell (Watson, 1995; Woods et al., 2004). Therefore, cell viability
when plotted against cooling rates is represented by a U-shape curve (Medeiros
et al., 2002; Watson, 1995; Woods et al., 2004). The optimum freezing rate is
one that best balances the damaging effects of the two phenomena, solution
effects and intracellular ice formation, to gain maximum cell survival. Optimum
cooling rates vary between species. Spermatozoa survival is also dependent on
thawing and the optimal warming rate is dependent on the cooling rate (Woods
et al., 2004). Generally, fast warming rates are preferred, because slow rates
may encourage recrystallisation of any thermodynamically unstable small ice
crystals left in the cells from freezing, leading to fatal damage (Holt, 2000a;
Woods et al., 2004).
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2.7.4.1.2 Cryoprotectant
The use of a cryoprotectant is to avoid formation of intracellular ice and to
restrict solution effects (Medeiros et al., 2002).
The most commonly used
cryoprotectant is glycerol, which is a permeating cryoprotectant and acts at both
the extra- and intracellular level (Medeiros et al., 2002). Its presence in the
solution stimulates osmosis, or cell dehydration, so that less water molecules are
available for possible ice formation (Medeiros et al., 2002). The colligative
properties, including depression of freezing point and the consequent lowering of
salt concentration of the solution, help to reduce the harmful solution effects
(Holt, 2000a).
Upon permeating the cell membrane, the stress induced by
osmosis is decreased, because its presence within the cells replaces water
molecules required for maintaining cellular volume and interactions (Medeiros et
al., 2002). Glycerol is cytotoxic to spermatozoa (Holt, 2000a; Watson, 2001;
Medeiros et al., 2002). It is used in concentrations between 4 – 10% (v/v)
depending on the sensitivity of the spermatozoa of different species (Watson,
2001). Its presence inside the cell may also exert transitory osmotic effects and
cause cell volume fluctuations that may lead to cell damage (Watson, 2001).
The toxic and damaging effects of glycerol must be taken into account when
devising a cryopreservation protocol.
The influence of glycerol may be
minimised by usage at a working concentration as low as possible, addition and
removal in a stepwise fashion (Holt, 2000b; Watson, 2001) or withholding
addition until temperature reaches near below that of freezing point.
Components in cryodiluents, such as egg yolk and sugars (e.g., lactose) also
have cryoprotecting properties (Holt, 2000a). Sugars increase the amount of
unfrozen water at any given temperature or reduce the salt concentration of the
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solution, thus reducing solution effect injury to cells (Holt, 2000a).
The
lipoprotein fraction, particularly phospholipids, of egg yolk, provides
cryoprotection at the level of the cell plasma membrane lipid phase transition
(Parks and Graham, 1992; Holt, 2000a).
2.7.4.1.3 Cryopackaging
The method in which spermatozoa is packaged for freezing and storage is
generally dictated by the practical requirements of AI (Holt, 2000a). The straw
(0.25 and 0.5ml) packaging method is often preferred over the pellet method for
a number of reasons. After thawing, straws can be fitted onto an inseminating
pipette for direct insemination (Holt, 2000a), without the need to transfer the
content between receptacles and in doing so risking contamination. However,
the disadvantage of this direct approach is the cryoprotectant is not removed
before insemination (Holt, 2000a). For clear identification and easy access, each
straw can be marked on the exterior prior to loading (Watson, 2001). Given
each straw is sealed properly, with either a ‘poly vinyl alcohol’ (PVA) plug or
ball bearing, leakage should not occur. Contact of straw content with the liquid
nitrogen it is stored in is prevented and possible contamination avoided (Holt,
2000a). Freezing rates for straws can be controlled, either by suspension in the
vapour phase above liquid nitrogen or an automated programmable freezer (Holt,
2000a). The volume of pellets is usually between 50 and 200µl (Holt, 2000a;
Watson, 2001). Preparation of pellets can not be conducted in a sterile manner,
as samples are required to be in direct contact with dry ice for formation of
frozen spheres (Holt, 2000a) and their packaging into cryo-vials, again, involves
93
direct contact with liquid nitrogen.
Upon contact with dry ice, freezing
commences and the rate at which it proceeds can not be controlled. However,
the thawing process of pellets in a pre-warmed medium does allow the removal
or reduction of the amount of cryoprotectant present (Holt, 2000a).
2.7.4.2 Dolphin semen cryopreservation
Studies on the effect of cryopreservation of bottlenose dolphin spermatozoa are
few. The method first reported was based on one successful in cryopreserving
canine spermatozoa (Seager et al., 1975; Fleming et al., 1981). A homemade
egg yolk and sugar based diluent, containing 4% (v/v) glycerol as cryoprotectant,
was used to extend the raw semen. The extended sample was cryopackaged in
pellet form and transferred into liquid nitrogen (-176oC). Thawing took place
after a 10-day cryopreservation period, using different thawing media under
37oC incubation, in either pure air or in 5% CO2 air. The highest post-thaw
percentage of progressive motile cells was 80% at 30-minute and 6 – 8 hour post
thaw in canine capacitation medium (CCM) and 5% CO2 air compared to 85%
before freezing.
Schroeder (1990a) subsequently presented a more detailed
report of a cryopreservation technique, again, based on the canine spermatozoa
protocol (Seager et al., 1975).
However the glycerol concentration was
increased from 4% to 6%. Post thaw motility after a storage time of 3 to 5 years
was consistently 60% or above and grade 5 motility (5 being the best on a
subjective scale of 1 – 5). The thawing medium used was saline.
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Durrant (1999) was first to report a series of experiments to investigate the
conditions listed below based on samples from two adult T. truncatus:
1. Cooling or no cooling
2. addition of cryoprotectant before or after cooling to attained a final
concentration of 4% glycerol
3. cryopacking in pellet (250µl) or vial (0.5ml)
4. Medium for thawing
5. Thaw sample incubation time
6. Thaw sample temperature incubation
7. centrifugation of thawed sample
8. removal of supernatant (containing cryoprotectant) and resuspension of thaw
samples
9. addition of caffeine to thaw sample
In order to account for the differences in motility between the ejaculates used in
these experiments, post-thaw motility in this study was represented by the
percentage of initial motility score (MS).
The most effective combination of
conditions in cryopreservation was with 30 minutes of cooling to 4oC, addition
of cryoprotectant after cooling, a medium freeze rate of 12.8oC/min and
packaged in vials. An increase in post-thaw motility was found and maintained
for up to 90 minutes post-thaw with samples that were thawed in phosphatebuffered saline (PBS), followed by post-thaw treatments of centrifugation to
remove cryprotectant, resuspension in a N-tris (hydoxymethyl) methyl-2aminoethane sulfonic acid (TES)-TIS yolk buffer (TYB) incubation at 4oC. The
addition of caffeine to stimulate motility in spermatozoa of other species, such as
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the ram (Maxwell et al., 1995), produced mixed results in dolphin spermatozoa,
thus, warrant further investigation.
Robeck and O’Brien (2004a) designed a comprehensive set of experiments not
only to determine optimal conditions for dolphin sperm cryopreservation and
thaw, but also to address practical implications of liquid storage, cryopackaging
and prefreezing, in preparation for further development in sperm sex sorting.
The semen donors of this study were three proven sires aged between 14 and 34
years. Sperm density of all the samples was standardised to approximately 400 x
106/ml through centrifugation for concentration or addition of excess seminal
plasma for dilution. Such standardised raw density gave rise to a standardised
prefreezing density of approximately 100 x 106/ml, achieved after addition of
cryodiluents.
The experiments used different cryodiluent compositions,
comprising the egg yolk and lactose mix, Androhep Enduraguard, Equex STM
or N-tris (hydroxymethyl) methyl-2-aminoehane sulfonic acid (TES)-TRIS yolk
buffer (TYB) . Cryoprotectant used for all the experiments was glycerol at a
final concentration of 3% and was added after cooling to 5oC. Freezing was
carried out in stages, the rates used in each experiment may be viewed overall as
slow, medium and fast. Only the straw form was used for cryopackaging, as it
can be prepared in a more sterile manner, and is less prone to pathogenic
contamination than pellet form (Holt, 2000a; Watson, 2001). Different straw
volumes (small 0.25ml and large 0.5ml), were investigated since one may be
preferential depending on storage requirement and end usage.
Semen was
thawed at either slow, medium or fast rate. Based on SMI at 6 hours post-thaw,
the optimum treatment combinations were a) cryodiluent TYB, fast freezing rate,
in large straw and medium thawing rate or b) the same cryodiluent at the same
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freezing rate, but small straw and fast thawing rate.
Significant individual
variations were found in two of the three cryopresevation methods investigated.
Further experiments in the same study (Robeck and O'Brien, 2004a) produced
results that are of significant relevance to the development of sperm-sorting
technique in T. truncatus. Post-thaw motility was well maintained after storage
of 24 hours at 4oC in TYB. In practice, this means once a sample is cooled in
correct media composition there is time for transportation to another facility
where the sample may be used for AI, or to a laboratory for further processing
such as sperm-sorting and/or cryopresevation.
Using the optimal combination
of treatments found, samples were cryopreserved at prefreezing density of 100 x
106/ml (standard) and 20 x 106/ml (low), achieved by centrifugation and resuspension with appropriate amount of TYB. Decreased motility was observed
with the low prefreezing density. This finding suggests high dilution rates have
a detrimental effect and similar findings were cited from studies of ram (Graham,
1994) and boar (Pursel et al., 1973) spermatozoa.
Removal of protective
proteins in seminal plasma during dilution was proposed to be the cause of the
detrimental effect observed. In addition, this finding has important implications
in sperm-sorting as, due to limitations in sorting speed, low sperm densities (10
– 20 x 106/ml), are normally used (Garner, 2006). The findings show further
research is required to investigate the potential beneficial effects of seminal
plasma addition during freezing and / or after thawing to low density samples.
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2.7.4.2.1 Spermatozoa sex sorting
The difference in DNA content between X- and Y- chromosome bearing
spermatozoa is the basic principle on which flow cytometric sorting was
developed to separate the two populations of spermatozoa (Garner, 2006). Sexsorted spermatozoa are used in conjunction with assisted techniques such as AI,
close to the time of predicted ovulation, or IVF with subsequent ET (Maxwell et
al., 2004). Sperm sex sorting is an expensive technology and cells are subjected
to many damaging effects, therefore, treatments in sample preparation, storage,
cryopreservation and thaw must be carefully researched and optimised to attain
maximal cell survival.
This technology has been implemented in domestic
species such as cattle, swine, sheep and horses (Garner, 2006). Differences in
sorting efficiency exist between species due to differences in sperm head
morphology and DNA content difference between the X- and Y-chromosome
carrying sperm (Garner, 2006). Individual variations within a species are also
expected due to differences in susceptibilities of the gametes to the stress and
insults imposed by preparatory procedures, such as dilution, staining, media
composition changes, in addition to laser exposure and elevated pressure of the
sorting process itself (Garner, 2006).
Experiments based on results of previous cryopreservation work have been
carried out to establish methodologies for dolphin spermatozoa sex sorting
before and after cryopreservation (O'Brien and Robeck, 2006). Results of these
experiments showed that seminal plasma supplementation did not have any
effect on sperm characteristics and when conventional straw freezing was
compared to directional freezing in 2ml vials, the latter was better for sorted and
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non-sorted dolphin spermatozoa.
The study suggests more insemination trials
are required, particularly to determine the minimum AI dose, which is currently
believed to be 270 x 106 progressively motile spermatozoa (Robeck et al., 2005a)
and 150 x 106 progressively motile spermatozoa (O'Brien and Robeck, 2006),
and associated pregnancy rate. If properly managed, AI with sex sorted semen
will allow a more stringent management strategy through controlling the sex
ratio of captive groups, whilst improving genetic diversity.
2.8 Captive breeding of bottlenose dolphins
Propagation of T. truncatus has been the most successful of all cetacean species
maintained in captivity (Ridgway, 1995). In North America, 44% of the captive
population was captive born in 1999 (Andrews, 1999). In Europe, the percentage
of captive-born dolphins reached 35.4% in 1998 (Hartmann, 2000). The
proportion of calves that survived to two years was 67.3% in North America
(Joseph et al., 1999).
Improved breeding success in recent years has been
attributed to improved husbandry and health management (Cornell et al., 1987;
Joseph et al., 1999; Kinoshita et al., 1999). In the past, breeding occurred in
large pools of several adult females with usually just one adult male (Schroeder,
1990a; Asper et al., 1992; Ridgway, 1995). Competition between females, and
aggression between males and toward mother and calf, led to high calf mortality
(McBride and Hebb, 1948; Essapian, 1963). Segregation of pregnant females in
advance of parturition and mothers and calves being maintained in designated
maternity pools, with or without compatible females, improved calf survival
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(Asper et al., 1992; Kinoshita et al., 1999; Couquiaud, 2005b). However, this
did not address the long term genetic consequences of only one or a few males
siring calves. There is increasing pressure for facilities to maintain their current
captive groups in a sustainable manner, as taking from the wild is no longer an
option in most countries. Sustainability in insolated captive groups can only be
achieved through well managed breeding programmes that focus on preserving
genetic variability, rather than only the number of animals produced (Bainbridge
and Jabbour, 1998).
2.8.1 Controlled Breeding
Central to a managed breeding programme is breeding by controlled access.
This can be achieved by separation of mature females and males. Selection of a
breeding pair and when they are placed together for natural copulation, or when
is semen collected for AI, should be based on knowledge of their past and
current reproductive conditions. Therefore, methods to reliably monitor the
reproductive activities of each individual must be developed. Ultrasonography
has been used to detect folliculogenesis, predict ovulation accurately and thereby
control natural breeding (Brook, 2001; Brook, 1997; Brook and Kinoshita, 2005).
In these studies, each female was placed with a selected mature male 12 to 24
hours before ovulation, copulation was observed and ovulation confirmed by
ultrasonographic examination, the animals were then returned to their respective
social groups. The success rate of this controlled breeding strategy was high,
with 10 births and nine live calves resulting from 11 procedures.
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Pair selection in controlled breeding can overcome undesirable social and
genetic consequences encountered in the mixed-pool situation. Assignment of a
single male encourages successful copulation and ensures known paternity. In
the presence of more than one male, paternity is unknown, and aggression and
competition may prevent mating from happening at all (McBride and Hebb,
1948). Since the dominant male is more likely secure matings (Asper et al.,
1992), this again leads to the problem of calves born to only the dominant males.
In 1999, it was reported that only 58.6% of the males in captivity have sired
calves in North America (Tavolga, 1966; Duffield et al., 1999).
Over
presentation of the genes of a few males in the gene pool will increase
inbreeding in the future. The adverse genetic consequence of inbreeding is loss
of genetic diversity, in which the individuals tend towards being homozygous,
i.e. carrying two copies of similar allelic genes at the same loci (Duffield and
Amos, 2001). Inbreeding reduces survivorship and reproductive fitness of the
offspring (Duffield and Amos, 2001), which in turns leads to further genetic loss
as they are less likely to reproduce successfully. Changing the combination of
breeding pairs reduces inbreeding (Brook and Kinoshita, 2005). Active selection
of the sire ensures that the genetic material of every sexually mature male,
particularly that of any unrepresented founders, is retained in the genetic pool.
Exclusion of males with poor breeding records ensures that resources are not
wasted. Further, knowledge of when a young male has reached sexual maturity
not only prompts separation from females to prevent undesirable pregnancies,
but also identifies the earliest possible time to allow a young individual to breed
so that its genes are insured, in case of unforeseen loss.
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The use of ART enhances controlled breeding by allowing exchange of genetic
materials, thus increasing vigor, across physical distances and generation time.
Successful application of AI, using non-sorted cooled liquid-stored and
cryopreserved spermatozoa, has been reported in Tursiops (Kinoshita et al., 2004;
Robeck et al., 2005a). Further, one female T. truncatus calf has been born as a
result of AI using sorted sperm (O'Brien and Robeck, 2006). The ability to
predetermine the sex of the offspring contributes towards achieving social
groupings that mimic those seen in the wild (O'Brien et al., 1999). A carefully
matched male to female ratio will minimise social conflicts between males,
particularly when the size of the captive group continues to grow and enclosure
space becomes more limited (O'Brien et al., 1999). Socially induced stress is
also reduced, the thereby the health and welfare of dolphins in captivity
improved.
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Chapter 3
3.1 Aim
The primary aim of the study was to determine at what age sexual maturity
occurred in a group of male Tursiops aduncus in captivity by monitoring the
reproductive physiological changes, including testes size, testosterone level and
sperm density, during the maturation process.
Monitoring was continued after the attainment of sexual maturity to further
investigate male reproductive seasonality.
Semen samples from recently sexually mature subjects were cryopreserved to
investigate the ability to withstand freezing.
3.2 Objectives
The specific objectives of this study were to:
1. determine changes in size and appearance of the testis during sexual
maturation using ultrasonography
2. evaluate changes in serum testosterone levels during sexual maturation
3. identify the onset of spermatogenesis by examining semen samples
4. investigate any association between reproductive parameters
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5. determine any seasonal or cyclic changes in the reproductive parameters
6. characterise ejaculates of Tursiops aduncus
7. investigate the effect of cryopreservation on semen from subjects of
different ages and number of post-onset years
3.3 Material and method
3.3.1 Subjects
The main subjects of this study were five male Tursiops aduncus owned by
Ocean Park Corporation (Table 3.1).
The exact ages of the four youngest
subjects were known because they were born at Ocean Park under an existing
controlled breeding programme. The oldest subject was caught in Indonesian
waters. His age was estimated based on his physical size and appearance on
arrival at Ocean Park in 1987.
The subjects are maintained in a formation of interconnected, concrete semicovered pools in a facility called Ocean Theatre. This facility is a show arena
with a spectator stand of 3,500 seats.
(See Appendix 1 for photograph and
schematic layout of this facility and pool dimensions). The subjects take part in
a minimum of two show demonstrations and one close encounter session daily.
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Table 3.1:
Study
subject
ID
M1
Given name
at Ocean
Park
Molly
M2
Subjects and age
1984 (aest.)
Age at beginning
of study
(October 2002)
19+ y
Popeye
2nd May 1994
8y 5m
M3
To To
19th July 1995
7y 3m
M4
Perky
17th Sept 1998
4y 1m
M5
Leo
1st May 1999
3y 5m
Date of Birth
a
Age estimated in 1989 based on body size, growth pattern and length of time in captivity
(Brook, 1997)
The usual socialisation pattern was the two older males (M1 and M6) being
paired together and the younger males also paired according to their age, or
sometimes placed together as a group of four.
However, due to show
requirements and animal loss, groupings were subject to changes from time to
time. For most of the duration of the study, the subjects were maintained in one
facility and females were maintained in a facility some distance away. When
females were present in the same facility, the males were maintained in separate
pools, allowing only restricted visual and auditory contact through solid metal
gates that divide the pools. Tactile contact was absent, unless a male was paired
with a female for controlled breeding purposes.
Diet consisted of capelin (Mallotus villosus), herring (Clupea harengus pallasi),
sardine (Clipeidae sardinops sagax) and squid (Loligo vulgaris) imported frozen
from U.S.A. The amount and composition of food intake were formulated by
Park veterinarians according to individual requirements.
105
3.3.2 Animal training
All measurements and samples were taken from the subjects using cooperative
behaviours. The behaviours required in this study were achieved by operant
conditioning and use of positive reinforcements. Briefly, a subject’s response or
presentation of a behaviour upon a specific cue was reinforced with food, verbal
praise or touching.
3.3.3 Body weight
Body weights were obtained by subjects sliding out of the water on to a scale
platform. Subjects were weighed weekly, as per Ocean Park’s medical care
protocol. Weighing was carried out in the morning during the first feed. Table
3.2 shows the subject weights at the beginning of the study.
Table 3.2:
Age, body weight, body length and body girth of
subjects at the beginning of the study (October 2002)
19+y
Body weight
(kg)
125.8
Body length
(cm)
217.0
Body girth
(cm)
126.0
M2
8y 5m
129.6
225.0
129.0
M3
7y 3m
122.6
223.5
120.5
M4
4 y 1m
104.5
210.0
117.5
M5
3y 5m
87.2
190.0
113.0
M6
19+y
141.4
232.0
122.0
Subject
Age
M1
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3.3.4 Body length and girth
Body measurements were taken in shallow water. Body length was obtained
with the subject floating at the surface in ventral presentation (‘belly up’). A
flexible measuring tape was placed along the ventral surface, from the tip of the
rostrum to the notch between the flukes. Body girth was obtained with the
subject floating in the resting position and the measuring tape was passed around
the section of body in front of the dorsal fin where girth is greatest. Following
Ocean Park’s medical protocol, body measurements were taken once a year for
subjects over 12 years old and once every 6 months for subjects less than 12
years old. Table 3.2 shows the body lengths and girth measurements of the
subjects at the beginning of the study.
3.3.5 Equipment and protocol for ultrasonographic assessment of testes
A portable Aloka ultrasound unit (SSD 900, Co., Ltd., Japan), in conjunction
with a 3.5MHz linear array transducer, was used for ultrasonographic
examination of the testes. The unit was made suitable for outdoor poolside use
by fitting of a splash-proof, custom-made plastic cover. All examinations were
recorded on thermal-printing paper using a Sony Echocopier (Sony Products Ltd.,
Japan) and subsequently scanned (CanoScan D1250U2, Canon Inc., Japan) for
storage in electronic format.
The protocol for ultrasonographic examination of dolphin testes in this study
was developed by Brook (1997). Examination was carried out at poolside with
107
the subject cooperatively stationed in lateral recumbency in the water. The body
alignment was such that each animal was in a straight line and relaxed, with the
ventrum close to and parallel with the side of the pool (Figure 3.1). Subject
stationed at the poolside for ultrasonographic examination.
To support the
animal and minimize movement, one trainer placed his legs in the water under
the caudal section of the subject’s body, near the peduncle. A second trainer was
sometimes present at the head for further support and provision of positive
interaction with the subject during ultrasonographic examination. All
measurements were taken when the subjects were apnoeic to minimise error due
to movement.
Figure 3.1: Presentation of Tursiops aduncus for ultrasonographic
examination of testes at poolside
For longitudinal measurements (LS), the transducer was placed on the subject’s
flank at a level above the genital slit, parallel to the long axis of the body. To
108
locate the testes, the transducer was moved away from the genital slit towards
the dorsal aspect.
Once the organ was located, the transducer was moved
towards the flukes to locate the caudal end of organ; this point was marked by
the index finger tip of the animal trainer. The transducer was then moved
towards the head to locate the cranial end of the organ and this point also marked.
The distance between the two points was measured with a flexible ruler and
represented the length of the organ (Figure 3.2). The echopattern of the testicular
parenchyma was also assessed and recorded in this view.
For cross-sectional measurements (CS), the transducer was rotated 90o anticlockwise at the widest part of the testes identified in LS. In this orientation, the
transducer was moved along the organ to ensure the greatest dimensions were
captured (Figure 3.2), whilst also surveying the echopattern of the testicular
parenchyma. Dimensions in this view were measured using the ultrasound unit’s
built-in electronic callipers function in the ‘freeze-frame’ mode.
Ultrasonographic measurements of each testis were:
1. Length (TL), cm
2. Depth or dorsoventral diameter (TD), cm
3. Width or lateral diameter (TW), cm
4. Circumference (TC), cm
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2
3
4
1
Figure 3.2: Diagrams to illustrate testis measurements
(1 = length in LS 2 = the widest part to obtain TD, TW
and TC in CS, 4 = circumference in CS, denoted by
dashed line)
The volume of each testis was determined using Lambert’s formula for an
ellipsoid: Testis Volume (TV), cm3 = length x width x depth x 0.71.
Ultrasonographic examination of the testes was carried out weekly, before semen
collection and on the same day as monthly blood sample collection.
3.3.6 Blood sample for testosterone evaluation
Blood samples were taken at poolside upon voluntary presentation of flukes for
venipucture with a 21G butterfly needle set (JMS Singapore Pte., Ltd.) attached
to a 10ml syringe.
Blood was collected following Ocean Park’s medical
protocol – i.e. once a month in the morning during the first feed. Blood was
110
collected for serum testosterone level evaluation on the same day as
ultrasonographic examination and semen collection.
After collection, the sample was left to stand for 30 minutes to clot in a plain
tube (Thrombin 5NIH units, BD Vacutainer® Systems, U.K.) and serum was
obtained by centrifugation at 4500 rpm for 10 minutes. Testosterone level was
evaluated using a commercially available enzyme immunoassay kit (VIDAS
Testosterone), coupled with an automated VIDAS analyser (bioMérieux sa,
France). Specifications of this test kit system and assay protocol are given in
Appendix 2.
3.3.7 Protocol for semen collection
In this study, the sequence of training steps for semen collection by manual
manipulation was based on the method described by Keller (1986) and Wu
(1996). Briefly, a hand signal, specific to semen collection, was used for a
subject to station upright at the water’s edge, facing the trainer. This signal
subsequently became a brief moving target that led the subject into the ventralup position. Tactile stimulus was initially applied at the periphery of the anal slit
to signal for exposure of the penis to its full length. In a short time, most animals
automatically extended the penis without stimulation. The tip of the penis was
placed at the neck of a collection bottle (polypropylene, wide mouth, round,
500ml or 200ml, Nalgene® Labware, Nalge Nunc International, U.S.A.) Slight
pressure was applied at the base of the penis as the signal for ejaculation. Upon
completion of the behaviour, the subject was given reinforcement and was not
111
signalled to present for another collection until the penis was fully retracted and
the genital slit closed.
In each session, ejaculate samples were collected in separate bottles in
succession until no more semen was present in spite of effort, or when
micturition occurred. On site, collection session duration and cloudiness of
ejaculates were noted on a worksheet (Appendix 3). Ejaculate samples were
incubated at 37oC (Plentikool, Igloo Product Corp., U.S.A. or Minitub, Abfullund Labortechnik GmbH & Co., KG, Germany) and were evaluated in a
laboratory within an hour of collection. Semen collection was carried out once a
week, immediately after ultrasonographic examination and on the same day as
blood sample collection.
After completion of all evaluations, ejaculate samples were discarded and the
bottles were rinsed several times and soaked in tap water for approximately 15
minutes. A plastic bottle brush was used for further cleaning followed by a final
rinse with purified Type I water (Milli-Q Synthesis, Millipore China Ltd., Hong
Kong). Bottles were then sterilised by autoclave at 133oC (M9 Ultraclave,
Midmark® Corporation, U.S.A.) and stored in a hot oven (Memmert GmbH +
Co., KG, Germany) to dry until the next use.
112
Figure 3.3:
Semen collection in Tursiops aduncus at poolside
(note the acutely tapered penis tip see Section 2.1.3 for
anatomical description)
3.3.8 Semen analysis
The protocol used was based on techniques and procedures developed and used
for evaluating dolphin semen (Fleming et al., 1981; Seager et al., 1981;
Schroeder, 1990a; Durrant et al., 1999a; Robeck and O'Brien, 2004a).
Procedures used for analysing human semen in WHO (1999) and Mortimer
(1994) were also adapted.
Individual ejaculates were evaluated for the following parameters:
1. pH
2. Volume, ml
3. Total motility (TM), %
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4. Progressive motility (PM), %
5. Rate of progressive motility (RPM, ranked on a subjective scale 0 - 5,
where 0 denotes no progressive movement and 5 denotes most rapid
progressive movement)
6. Viability (VIA), %
7. Density, 106/ml and count 106/ml
On arrival at the laboratory, ejaculate samples continued to be maintained in
37oC for evaluation. All slides and cover slips (22mm x 22mm) were polished
and pre-warmed on a hot plate (Dri-Bath, Barnstead / Thermolyne, U.S.A.) set at
37oC. A 37oC warm stage (TH 60-6 75x50x1.4mm with 6mm aperture, Linkam
Scientific Instruments, U.K.) was mounted on to a microscope (BX50, Olympus
Optical Co., Ltd., Japan) for motility evaluations and was removed for
evaluations of the other parameters which did not require spermatozoa to be
alive.
3.3.8.1 pH
For determination of pH, 4-patch pH test strips at operating range 5.0 – 10.0
(Neutralit, Merck & Co., Inc., Germany) were used. The strips were fully wetted
with raw ejaculate samples and pH was read after 30 seconds.
114
3.3.8.2 Volume
For determination of ejaculate volume (EjV), graduated glass 10ml and 50ml
cylinders were used. Smaller volumes were measured using 3.5ml disposable
pipettes (Copan Italia S.p.a., Italy).
Volume of each ejaculate was added together to determine the total semen
volume (ToV) for a collection session.
3.3.8.3 Motility
For motility evaluation, ejaculate samples were evaluated in the natural state and
when diluted. For dilution, a modified sperm washing medium with bovine
serum albumin 5.0mg/ml (Irvine Scientific, U.S.A.) was used. Firstly, 5µl of
natural sample was delivered on to a slide using a micropipette (P20,
Pipetman®P, Gilson Inc., U.S.A.). For diluted preparation, 5µl of sample was
delivered on to 5µl of washing medium. Cover slips were placed over the
preparations and they were left to stand for 30 seconds before evaluation.
A slide preparation was surveyed first at 100x total magnification (i.e., 10x
objective and 10x ocular lenses) bright field, followed by detailed evaluation at
400x total magnification.
In a given field, motile cells were noted to
subjectively determine total motility (TM) in percentage. Within the motile cells
population, those that were free and showed forward movement were counted
and converted into percentage for progressive motility (PM). The rate at which
these cells forwardly progressed (RPM) was ranked on a scale of 0 - 5, where 0
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denotes no progressive movement and 5 denotes most rapid progressive
movement. Evaluations were based on at least 200 cells. Several microscopic
fields were examined for samples of low density.
Remaining at 400x total magnification and from the same wet slide preparation,
sperm clumping (spermatozoa stuck together in non-specific manners) was noted.
Also, density of the sample was estimated from the number of spermatozoa per
bright field. This estimate was used to decide on the dilution factor to be applied
to raw samples for determining sperm density.
3.3.8.4 Viability
For viability evaluation, raw samples were mixed with eosin-nigrosin stain
(Sigma Chemical Co., U.S.A.) in equal volumes. 5µl of sample was pipetted on
to 5µl of stain solution on a warm slide over the hot plate. The mixture was
gently aspirated and expelled three times for thorough mixing and then left to
stand for 1.5 minutes.
A smear was made by placing a cover slip at
approximately 45o angle in front and interfacing the periphery of the droplet mix.
Sliding of the cover slip at an angle in one smooth forward motion spread the
mixture along length of the slide. The slide was left on the hot plate for the
smear to dry as soon as possible
A smear was surveyed first at 100x total magnification bright field, followed by
cell differentiation (Diffcount, Modulus Data Systems, U.S.A.) at 1000x total
magnification under oil immersion. Sperm heads that were stained bright pink
116
were counted as dead cells and cells with heads that were white (unstained) were
live, or viable. Cells that showed evidence of stain uptake at the neck region
were also classified live. Evaluation was based on at least 200 cells and the
number of viable cells was converted into a percentage.
3.3.8.5 Sperm density and count
For density evaluation, spermatozoa were initially killed by freezing at -70oC
(Revco, Thermo Electron Corp., U.S.A.) for approximately 15 minutes. Thawed
samples were diluted with phosphate-buffered saline (Dulbecco `A' tablets,
Oxoid Ltd., U.K.) according to factors determined during motility evaluation.
Dilution factors used were 0 (no dilution), 1:5, 1:10, 1:20, 1:50 and 1:200.
Dilution was carried out in micro-volumes (i.e., < 1ml) in 1.5ml Eppendorf tubes
(Sarstdet Aktlengesellschaft & Co., Germany) using pipettes of operating ranges
2 – 20µl, 20 – 200µl and 200 – 1000µl (P20, P200 and P1000, respectively).
Neubaur-improved haemocytometers (Paul Marienfeld Gmbk & Co., KG,
Germany) were used for manual counting to determine sperm density. Before
use, haemocytometers and cover slips were polished with lint-free paper. The
cover slips were pressed firmly onto the wetted sides of the haemocytometers
until Newton’s rings were observed between the glass surfaces. Thoroughly
mixed diluted samples were then charged, through capillary action, into both
sides of the haemocytometers. The haemocytometers were left to stand for 10
minutes in a humidified chamber to prevent samples from drying out.
117
A charged haemocytometer was surveyed first at 100x total magnification bright
field, followed by counting at 400x total magnification. Of the 25 large squares
a haemocytometer grid contains, 5 were counted with the ‘L’ rule applied (i.e.,
cells that fell on the triple-ruling on the left side of the square were counted,
whilst those on the right were not counted). These squares are those situated in
the middle and all four corners of the grid. Both sides of the haemocytometer
were counted and the mean count was used for conversion into density in x
106/ml for an ejaculate (EjD).
The density and volume of each ejaculate were multiplied together to determine
sperm count (x106) of that ejaculate sample (EjC). Total sperm count (ToC) for
an individual in a collection session was the summation of all ejaculate counts
(EjC). Division of the total sperm count (ToC) by the total volume (ToV) of all
the ejaculates produced the overall density (x 106/ml) the collection session
(OvD).
3.3.9 Semen cryopreservation
The protocol used in cryopreserving dolphin semen in this study was based on
those described by Schroeder (1990a) and Robeck et al. (2001a) with some
modifications made. This protocol was successful in cryopreserving semen that
resulted in the births of live calves from artificial insemination (Brook and
Kinoshita, 2004; Kinoshita et al., 2004). The major steps in cryopreserving
dolphin semen were extension, cooling, equilibration with cryo-protectant, cryopackaging and storage. At each step, 0.3 – 0.5ml of samples was taken for
118
motility and viability evaluations following the protocol given in Section 3.3.8.
Each sample at each step was evaluated three times and the mean was used for
statistical analysis.
3.3.9.1 Extension
The extender used in the protocol was prepared in-house based on Seager (1969)
and Schroeder (1990a}. This extender contained 11% (v/v) lactose (Riedel-de
Haën, Seelze GmbH, Germany), 20% egg yolk, 1000IU of penicillin (G/ml)
(Irvine Scientific, U.S.A.) and 1.25mg of streptomycin sulphate per ml (Gibco
BRL, Life Technologies, U.S.A.). Detail of extender preparation is given in
Appendix 4. Two extender batches of 400ml were made in one day and frozen
at -70oC in aliquots of 5 – 10ml. When required, aliquots were thawed at room
temperature.
Within an hour of collection, selected ejaculate samples were extended with prewarmed extender in the incubator at 37oC. Equal volume of extender was added
to the sample drop by drop using 3.5ml disposable pipettes, with gentle swirling
between each drop to mix.
3.3.9.2 Cooling
Cooling of the extended sample to 4oC was carried out in a bench-top
refrigerator (Engel Portable Refrigerator, Sawafuji electric Co., Ltd., Japan).
119
The tube containing the extended sample was transferred from the incubator to a
water bath (beaker with 200ml of water) of 37oC and the entire set up was placed
in the refrigerator.
A thermocouple probe (Digi-Sense®, DualogR™, Cole
Palmer Instrument Co., U.S.A.) was placed in the water bath to monitor
temperature. Temperature changes were recorded every 15 minutes.
3.3.9.3 Addition of cryo-protectant and equilibration
Equilibration of the extended sample with cryoprotectant was carried out when
the temperature of the water bath reached 4oC.
In advance of this step,
cryoprotectant, glyercol (Sigma-Aldrich Inc., U.S.A.), was added to another tube
of extender to bring about a concentration of 12% (v/v). The glycerolated
extender was mixed thoroughly and placed in the refrigerator to cool to 4oC. An
equal volume of the glycerolated extender was added to the sample using 3.5ml
disposable pipettes at a rate of 1drop per 5 seconds, whilst the sample remained
submerged in the water bath. Gentle swirling was applied in between each drop.
The final mixture contained 6% glycerol and was left to equilibrate at 4oC for 2
hours.
3.3.9.4 Cryo-packaging and storage
For cryo-packaging in pellet form, blocks (100 x 165 x 35mm) of solid carbon
dioxide or dry ice, acquired from a local commercial source, were used. A
heated tailored-made block of round metal studs (17mm in diameter) was
120
pressed firmly on to the blocks of dry ice to make depressions in which pellets
were formed. At the end of the 2-hour equilibration period, in addition to
motility and viability evaluations, some of the sample was used to determine prefreezing density. The bulk of the remaining sample was placed in a 0oC ice bath.
Using pre-cooled sterile 3.5ml disposable pipettes, samples were dropped
vertically, 3cm above the dry ice blocks into the depressions and pellets of
approximately 40-50µl in volume were formed. The pellets were left to stand
for 3 minutes and then transferred in to a flask (Thermoflask, Thermolyn, U.S.A.)
of liquid nitrogen by tilting the dry ice blocks over the flask.
For storage, the pellets were transferred into 2ml cryogenic vials (Nalgene®
Labware, Nalge Nunc International, U.S.A.) using a plastic spoon with a long
handle under liquid nitrogen. Each sample was stored in duplicate vials. The
vials were affixed to cryocanes (Nalgene® Labware, Nalge Nunc International,
U.S.A.) which were slowly lowered into a liquid nitrogen tank (MVEXC 34/18,
Chart Bio-Medical Division, U.S.A.) with a low level alarm (QLA100 Quantum
Production, U.K.). A colour coded inventory system was devised to label the
samples and record their locations inside the liquid nitrogen tank.
3.3.10 Semen thawing
Semen thawing was carried out after 2 weeks of cryogenic storage. The steps in
thawing were removal from storage, thawing at 37oC, washing by centrifugation
and re-suspension. To investigate the effect of centrifugation and re-suspension,
aliquots of approximately 100µl of thawed samples were taken for each step for
121
evaluations.
Post-thaw semen evaluations included motility and viability
evaluations, following the protocol given in Sections 3.3.8.3 and 3.3.8.4.
Evaluations were carried out at 0, 30 and 60 minutes post-thaw, at 37oC. Three
aliquots were thawed for each sample and the mean was used for analysis.
For thawing, 6 – 7 pellets (approximately 0.3ml in total volume) were
transferred into a 1.5ml Eppendorf tube containing 0.9ml of pre-warmed
washing medium (bovine serum albumin). The tube was placed in a 37oC flow
water bath (Tempette® Junior TE-8J, Techne Inc., U.S.A.) for 1 minute to thaw.
The thawed sample was centrifuged at 800g (5415D, Eppendorf-Natheler-Hlnz,
GmBH, Germany) for 10 minutes. Approximately 900µl of supernatant was
removed and the sediment was mixed by agitation (Maxi Mix II, Thermolyne
Corp., U.S.A.). The sample was re-suspended with equal volume of pre-warmed
washing medium and incubated at 37oC.
3.3.11 Inter-and intra-operator tests
Throughout the study, all ultrasonographic examinations, semen evaluations and
cryopreservations were carried out by one operator / technician, the author.
Intra- and inter-operator tests were carried out to investigate the repeatability and
reproducibility of the methods and protocol used.
Repeatability and reproducibility were good for ultrasonographic examination of
the testes. Intra-operator measurement variability was lower with a repeatability
coefficient of <1cm for nearly all the LS and CS measurements. Limits of
122
agreement between operators were good and were mostly close to or with in ±
1cm. The limits of agreement were larger for circumference measurements, this
was likely caused by slight animal movements under the influence of pool water
current.
Measurement variability within an operator and between operators were higher
in semen analysis. Repeatability coefficients for motility analyses of over 50%
were larger than those of viability. Reproducibility was better by comparison;
limit of agreement was greatest for percentage progressive motility (PM), which
was 7.17 ± 38.43%. For reproducibility, the two operators assessed a slide
sample one immediately after the other, whereas for repeatability, a duplicate
slide preparation was made for assessment some time after the first, as decided
by the non-observer.
Therefore, the greater intra-operator measurement
variability was likely to be due to inherent differences between the slide
preparations, which can be due to incomplete mixing of the sample, or time
induced differences, particularly if a sample was very dense. Measurement
variability in motility analysis can be expected due to the subjective nature of the
assessment of this parameter. Inter- and intra-operator variability for viability
were both low, < 5%, and assessment of this parameter was not subjective.
Repeatability of sperm density assessment was good, the lower reproducibility
found may reflect differences between the operators in sample preparation.
The results and evaluations of the repeatability and reproducibility tests of
ultrasonographic measurement of the testes and semen analysis are presented in
detail in Appendices 5 and 6.
123
Chapter 4
RESULTS
4.1 Study schedule / duration
th
The study began on 9 October 2002, (week 1), when collection of most data
began (Table 4.1).
Table 4.1: Duration of data collection
Data type
Weekly
ultrasonography and
semen analysis
Weekly
ultrasonography
Weekly semen
analysis
Monthly
ultrasonography
Monthly
ultrasonography and
semen analysis
Monthly testosterone
Weekly body weight
Start of collection
month
study
/ year
week
End of collection
month
study
/ year
week
Oct / 2002
1
M1, *M2,
M3
Feb /2003
18
*M4
Jun / 2003
35
May / 2003
32
Oct / 2005
159
Jul / 2006
197
Jul / 2006
Jun /
2006
Aug /
2006
197
Dec /
2006
4
Dec /
2006
Oct / 2002
Oct / 2002
1
Jul / 2002
-
*data collection ceased due to death of the subject
124
196
M5
*M4
M5
Sept /
2006
Dec /
2006
Jul /
2006
Body length
Subjects
Jan /
2007
205
M1, M3,
*M4
222
M5
222
209
222
201
223
M1, *M2,
M3, *M4,
M5, M6
M1, *M2,
M3, *M4
M5
M1, *M2,
M3, *M4,
M6
M5
4.2 Subject demographic information
A total of 5 males were included in the study; data from one other male (M6)
was collected opportunistically for use in some analyses. Details of the age, body
length (BL), body girth (BG) and body weight (BW) of subjects at the start and
end of the study are given in Table 4.2.
Measurements of BL, BG and BW and of M2, M3, M4 and M5, show these
younger subjects were still growing.
Both M1 and M6 were of the same
estimated age and had reached sexual and physical maturity before the beginning
of the study. M2 was also sexually mature (first release of spermatozoa in 2001).
M2 – M5 were both physically and sexually immature.
Table 4.2: Age, body weight, body length and body girth of subjects at
beginning (October 2002) and end of the study
Age
y /m
Start End
n
Body length
(BL) (cm)
Start
End
Body girth
(BG) (cm)
Start
End
n
M1
19+
23+
9
217.0
219.0
126.0
123.0
194
125.8
125.2
M2
8/5
11
6
225.0
233.0
129.0
126.0
129
129.8
146.1
M3
7/3
11/1
10
223.5
234.0
120.5
119.0
195
122.6
133.0
M4
4/1
7/10
10
210.0
222.0
117.5
119.0
182
104.5
119.3
M5
3/5
7/7
10
190.0
210.0
113.0
114.0
194
87.2
105.5
M6
19+
23+
10
232.0
232.0
122.0
123.0
194
141.4
138.1
n = number of body length and girth measurements taken
nm = number of body mass measurements taken
125
m
Body mass
(BW) (kg)
Start
End
4.3 Ultrasonographic evaluation of the testes
The testes of all the subjects were visualised using the transabdominal
ultrasonographic technique described. Characteristics of the ultrasonographic
appearance, or echopattern, were assessed according to Brook (1997) and Brook
et al. (2000), to differentiate between individuals of different reproductive status.
Progressive changes in echopattern and texture within an individual were also
noted during sexual maturation.
4.3.1 Ultrasonographic appearance of the testes and epididymes
4.3.1.1 M1
At the start of the study this animal was fully mature and a proven sire. The
testes were well demarcated by a hyperechoic border. On longitudinal section,
the contour of the testes showed expansion from the caudal aspect to the midsection and then tapered towards the cranial end (Figure 4.1). Echopattern of the
testicular parenchyma was homogenous, speckled, of medium to high intensity
and remained unchanged throughout the study period. Echogenicity was
isoechoic to the adjacent hypaxialis lumborum muscle.
The testicular
mediastinum was visualised as a hyperechoic line that extended the entire length
of the parenchyma. Lobulation was also seen within the parenchyma. Based on
these factors, testes in M1 were classified as Grade I (Brook, 1997; Brook et al.,
2000).
126
EpCa
rr
EpCr
Testis
caudal aspect
cranial aspect
Figure 4.1: Ultrasonographic image of right testis in LS – M1
The epididymes were also readily visualised in M1 (Figure 4.1). The head, or
cranial portion, of the epididymis (EpCr) was triangular in shape and extended
from the cranial border of the testes. The echopattern was the same level of
echogenicity as the testicular parenchyma. The body of the epididymis extended
along the dorso-lateral surface of the testis, and the caudal portion (EpCa), or tail,
was large and irregularly shaped.
(Figure 4.1) The caudal portion of the
epididymis was also relatively echolucent due to the tubules here being of larger
diameter (Figure 4.2)
127
Figure 4.2: Caudal portion of the epididymis of left testis in LS – M1
4.3.1.2 M2
M2 was born in May 1994 and first produced / released spermatozoa in
September 2001, so was sexually mature at the beginning of the present study.
The ultrasonographic appearance of the testes of M2 was similar to that of M1
and also classified as Grade I.
4.3.1.3 M3
M3 was born in July 1995 and was not sexually mature at the beginning of study.
The onset of spermatogenesis in this subject occurred during October 2002, the
1st month of the study.
The contour of the testis showed a more cylindrical structure than M1 and M2,
with just slight expansion in width from the mid-section to the caudal aspect.
The echopattern of the testicular parenchyma was slightly hypoechoic when
128
compared to the hypaxialis lumborum muscle. The mediastinum was again a
prominent, hyperechoic, linear structure. Testes were classified as Grade II – I.
As the study progressed, changes in the ultrasonographic appearance of M3’s
testes were noticeable. Expansion at the midsection became more marked and
the echopattern of the testicular parenchyma increased to levels similar to those
in M1 and M2. Lobulation of the parenchyma also increased. The caudal
portion of the epididymis became more differentiated and the round hypoechoic
structures became more numerous and easier to discern. By the end of the
present study, testes in M3 were Grade I (Figure 4.3).
cranial aspect
caudal aspect
Figure 4.3: Ultrasonographic image of left testis in LS – M3
4.3.1.4 M4
M4 was born in September 1998 and was sexually immature at the beginning of
the study.
129
The testes were cylindrical with well demarcated margins (Figure 4.4). The
echopattern of the parenchyma was homogenous and hypoechoic compared to
hypaxialis lumborum muscle group. The appearance of the mediastinum was the
same. Visualisation of the epididymis was not possible at this stage. Testes in
M4 were classified as Grade III.
Figure 4.4: Ultrasonographic image of left testis in LS – M4
As the study progressed the ultrasonographic appearance of M4’s testes changed
markedly. Although the shape of the testes remained consistent and cylindrical,
without notable expansion in width, parenchymal echogenicity increased until it
was isoechoic to the hypaxialis lumborum muscle group. Lobulation of the
parenchyma gradually became discernable. The cranial and caudal portions of
the epididymis became apparent. The caudal portion of the epididymis was less
echolucent compared that of M1, M2 and M3, however, round hypoechoic
structures were beginning to be discernable about 8 months after the onset of
spermatogenesis occurred. By that time testes in M4 were Grade I (Figure 4.5).
130
Figure 4.5: Caudal aspect of right testis in LS – M4
4.3.1.5 M5
M5 was born in May 1999 and was sexually immature at the beginning of the
study.
The shape of testes and appearance of the mediastinum were similar to those of
M4 at the beginning of the study.
The testicular parenchyma was poorly
differentiated and hyopechoic. Testes in M5 were classified Grade III.
During the study period, the appearance of the testes showed little change. The
echogenicity of the testicular parenchyma increased slightly, but remained
hypoechoic compared to the hypaxialis lumborum muscle group. Visualisation
of the epididymis was still not possible by the end of the study. Testes in M5
remained Grade III (Figure 4.6).
131
Figure 4.6: Ultrasonographic image of right testis in LS – M5
4.3.2 Ultrasonographic assessment of testis size
Testis size was assessed to determine annual growth in subjects that had not
reached full or physical maturity. Measurements were also used to compare with
serum testosterone levels and sperm output after the onset of spermatogenesis to
evaluate overall progress in testicular activities.
At the beginning of the study, testis size appeared to be related to age, with the
oldest male having the largest testes and the youngest, the smallest testes (Table
4.3).
During the study, mean testis size increased year by year in all subjects except
M1. Fluctuations in testis size were recorded in all males and differences in
testis size were found between individuals of similar ages.
132
Table 4.3: Testis measurements recorded for M1 – M5 at the beginning the
study
M1
Age
y/m
19+
RL
(cm)
21.6
LL
(cm)
21.0
RV
LV
3
(cm ) (cm3)
588.1 544.8
RC
(cm)
19.5
LC
(cm)
19.1
M2
8/5
17.5
17.2
179.4 267.8
11.9
14.8
M3
7/3
18.0
16.5
179.4 159.9
11.8
11.7
M4
4/1
7.1
7.9
14.6
14.4
5.3
5.0
M5
3/5
6.2
6.0
7.4
8.3
4.1
4.4
Subject
R = right side, L = left side
L = length, V = volume, C = circumference
Significant testicular asymmetry was found in four subjects, and testis length
(TL) alone could not be used to assess testis size. In M2, both testes were of
similar length, but the left testis volume (TV) was nearly 100cm3, or > 50%,
larger than the right (Table 4.3). TV measurements were more accurate in
indicating testis size and therefore used in further analyses.
4.3.2.1 M1
The overall range of testis measurements recorded for M1 is given in Table 4.4.
Table 4.4: Range of testis measurements recorded for M1 during study
Measurement
n
Min
Max
Mean
SD
Right length (cm)
178
18.3
22.6
19.9
0.7
Left length (cm)
178
16.7
21.6
19.5
0.8
Right volume (cm3)
178
317.6
604.9
466.3
58.8
Left volume (cm3)
178
323.7
593.7
465.1
54.6
Right circumference (cm)
178
15.6
20.1
18.0
0.9
Left circumference (cm)
178
15.7
20.4
18.2
0.8
133
No significant difference (P > 0.05) was found in TV measurements between the
left and right testes. Significant differences (P < 0.05), however, were found in
TL and circumference (TC) measurements.
The right TL measurements
recorded were larger than those of the left side, whereas the left TC
measurements recorded were larger than those of the right.
TV measurements recorded during the study, from October 2002 to August 2006,
are shown in Figure 4.7. Measurements fluctuated throughout the study but did
not fall below 300cm3, or rise above 610cm3. Variations about the mean values
were small compared to that of other subjects (Table 4.4).
Measurements
increased from February to reach peaks in March to May, and then decreased
until January / February the following year. However, decreases in measurement
were interrupted by small increases in December 2004 and again in December
2005.
After the lowest volume measurements were reached, subsequent
increases were rapid and uninterrupted. The most rapid increase was observed
from February to April 2005, when the right TV increased from 393.7cm3 to a
peak measurement of 555.9cm3, an increase of over 70% in 2 months. In the
same year, TV began to decrease from May until January 2006.
TL and TC measurements generally followed the same rise and fall pattern as
TV. TL measurements did not fall below 16cm and circumference measurements
did not fall below 15cm, or rise above 21cm. The similar pattern demonstrated
by TC measurement indicates that, if there is no significant asymmetry, the use
of this parameter is acceptable for assessing testis size when measurements
required to calculate TV, such as TL, cannot be obtained.
134
Figure
4.7:
Weekly
measurements
of
testis
135
volume
–
M1
Table 4.5: Annual range of testis measurements for M1
*Mean length (cm)
Study year
(age - y/m)
2003
(20+)
2004
(21+)
2005
(22+)
2006
(23+)
*Mean volume (cm3)
n
Min
Max
Mean ± SD
Min
Max
Mean ± SD
50
18.5
21.6
20.2 ± 0.7
419.3
585.8
505.3 ± 40.9
50
17.6
21.1
19.4 ± 0.8
345.6
557.3
453.3 ± 58.2
46
18.8
20.1
19.7 ± 0.4
396.1
540.2
467.3 ± 39.1
33
18.3
20.2
19.3 ± 0.5
322.6
499.1
425.9 ± 42.6
n = number of ultrasonographic measurements taken
*mean of right and left testis measurements
Table 4.5 shows the range of TV and TL measurements recorded on an annual
basis. Variations in annual mean values were small compared to those found in
other subjects.
Minimum, maximum and mean values differed from year to
year but did not follow any trend. M1 was a fully mature male and the testes had
reached their maximum size. In the absence of growth, variation found about the
annual mean values suggests there were other factors that may have influenced
testis size.
All testis measurements recorded for M1 are shown in Appendix 7.
4.3.2.2 M2
Significant differences (P < 0.05) were found in TL, TC and TV measurements
between the left and right testes.
The left testis measurements recorded were
consistently larger than those of the right.
136
Measurement
N
Min
Max
Mean
SD
Right length (cm)
128
13.4
24.1
17.7
2.3
Left length (cm)
129
14.0
25.0
18.2
2.3
Right volume (cm3)
127
62.3
585.6
208.4
105.6
Left volume (cm3)
128
83.6
712.8
251.1
118.9
129
8.0
18.5
12.3
2.2
129
9.0
19.8
13.4
2.2
TV measurements recorded during the study, from October 2002 to May 2005,
are shown in Figure 4.8. Measurements fluctuated markedly throughout the
study period, and the largest measurements recorded were about 9 times greater
than the smallest (Table 4.7). The smallest measurements (62 – 83cm3) recorded
were smaller than those recorded for M3 and M4, although these animals were
younger. The largest measurement recorded (712.8cm3) was the largest in all
subjects. Variations about the mean values were also the largest. Measurements
increased from February to reach peaks in April to June and then decreased until
January the following year.
Increases and decreases in measurement were
sometimes rapid, for example in 2004, overall TV increased by about 100cm3
during March and then rapidly decreased from June to August. In August, M2
presented clinical signs of illness that warranted both oral and intravenous
antibiotics. TV began to increase again in January the following year, about a
month after M2 was clinically well. These results may indicate some effect of
illness on testis size.
137
TL and TC measurements generally followed the same rise and fall pattern as
TV. TL measurements did not fall below 13.4cm and TC measurements did not
fall below 8cm.
Study year
(age - y/m)
2003
(9/5)
2004
(10/5)
2005
(11)
*Mean length (cm)
n
*Mean volume (cm3)
Min
Max
Mean ± SD
Min
Max
Mean ± SD
50
13.9
20.2
17.2 ± 2.0
73.1
337.0
198.1 ± 81.2
50
14.7
21.9
18.4 ± 2.2
112.7 445.7 246.2 ± 107.7
29
16.2
24.6
18.5 ± 2.5
139.5 649.2 254.4 ± 146.8
basis. Variations in annual mean values were the largest found in all the subjects.
Minimum, maximum and mean values increased consistently from year to year,
indicating that, although this subject was sexually mature by 8y of age, the testes
had not reached full adult size.
Further, annual increments in testis
measurements were small compared to the large annual fluctuations. For
example from 2003 to 2004 mean value for TV increased by only 48.1cm3
whereas the annual SD value was 107.7 cm3. This finding suggests there were
other factors influencing testis size in M2 in addition to growth.
138
4.3.2.3 M3
Measurement
n
Min
Max
Mean
SD
Right length (cm)
181
15.5
24.1
19.0
2.0
Left length (cm)
181
15.9
23.7
18.8
2.0
Right volume (cm3)
181
116.0
565.9
243.5
104.2
Left volume (cm3)
181
109.2
542.9
224.6
94.5
181
10.1
18.3
13.0
2.0
181
9.7
18.0
12.6
1.8
between the left and right testes. The right testis measurements recorded were
larger than those of the left.
M3 reached sexual maturity 2 weeks after the commencement of the study in
October, the mean left and right testis measurements were 17.8cm and 183.1cm3
and body size measurements were 225cm and 123.1kg.
139
Figure 4.8: Weekly measurements of testis volume – M2
140
TV measurements recorded during the study, from October 2002 to August 2006,
are shown in Figure 4.9.As seen in M2, measurements fluctuated throughout the
study period, however, changes were less marked and occurred at different times.
Variations about the mean values overall were also large. In the 1st study year,
measurements showed a biphasic pattern, increasing from January to a peak in
March, then decreasing until May. Then another peak was reached in July before
decreasing to September. From March to May 2004 TV underwent a notable
increase of > 170cm3, to > 300cm3, after which TV generally remained at higher
measurements than found in the 1st year and did not fall below 140cm3.
Subsequent fluctuations followed a less marked biphasic pattern.
In 2004
measurements increased to form a peak in May followed by another a larger
peak in September. In 2005, measurements increased to form a peak in March,
followed by another a larger peak in June. The rapid fluctuations in TV during
this year may have been influenced by illness, M3 showed general weight loss
and was presented with clinical signs in February – June 2005. In 2006, from
January to April TV showed the most rapid increase of about 400cm3 and from
April to June TV decreased by about 350cm3. Decline in blood parameters
began in February that year, illness continued beyond the study period.
141
Study year
(age - y/m)
2003
(8/3)
2004
(9/3)
2005
(10/3)
2006
(11/1)
*Mean length (cm)
*Mean volume (cm3)
n
Min
Max
Mean ± SD
Min
Max
Mean ± SD
52
15.8
18.7
17.3 ± 0.7
112.3
216.3
165.2 ± 27.6
49
16.2
21.4
18.4 ± 1.7
124.3
360.0
215.4 ± 75.1
47
17.2
23.9
20.4 ± 1.7
150.4
483.0
294.8 ± 82.2
33
17.0
23.7
19.8 ± 2.2
150.0
554.4 283.8 ± 136.3
between the left and right testes. The left TV and TC measurements recorded
were larger than those of the right side, however the right length measurements
recorded were larger than those of the left side.
Table 4.9 shows the range of TV and TL recorded on an annual basis. Variations
in annual mean values were large.
Minimum, maximum and mean values
increased from year to year, indicating, though sexually mature, from 7 – 11y of
age the testes of M3 were still growing. Annual increment or growth was
greatest between 2004 and 2005, with an increase of 79.4cm3 in mean TV and
2cm in mean TL, compared to 50.2cm3 in TV and 1cm in TL the previous year.
Despite incomplete data representation in 2006, continual growth is suggested by
the larger maximum TV attained.
142
Figure 4.9: Weekly measurements of testis volume
– M3
143
4.3.2.4 M4
Measurement
n
Min
Max
Mean
SD
Right length (cm)
151
7.1
20.0
13.1
4.1
Left length (cm)
152
7.4
20.0
12.9
4.0
Right volume (cm3)
150
10.6
282.2
87.6
73.8
Left volume (cm3)
151
11.1
274.1
91.0
75.0
150
4.4
14.3
8.4
2.7
151
4.5
14.0
8.7
2.7
TV measurements recorded during the study, from February 2003 to July 2006,
are shown in Figure 4.10. Measurements fluctuated throughout the study, but
again in a different pattern from other subjects. Variations about the mean
values were not as large as in M2 and M3.
During the first 2 years,
measurements showed relatively small changes. TV then began to increase
rapidly from January to May 2005, when the right testis was 228cm3 and left
testis was 217cm3. During this period, the onset of spermatogenesis took place
in April 2005; the mean left and right testis measurements were 17.1cm and
179cm3 and body size measurements were 225cm and 121.7kg.
144
TL and TC measurements generally followed the same pattern as TV. After
onset of spermatogenesis, TL measurements did not fall below 15cm and TC
measurements did not fall below 9cm.
Study year
(age - y/m)
2003
(5/5)
2004
(6/5)
2005
(7/5)
2006
(7/10)
*Mean length (cm)
*Mean volume (cm3)
n
Min
Max
Mean ± SD
Min
Max
Mean ± SD
44
7.1
8.9
8.2 ± 0.4
10.6
28.0
18.3 ± 5.1
52
9.1
14.7
11.1 ± 1.9
27.4
84.7
42.0 ± 11.6
43
14.8
18.9
17.0 ± 1.0
95.8
228.1 158.5 ± 40.3
19
16.8
20.0
18.4 ± 1.0
120.1
267.0 194.5 ± 44.2
Table 4.11 shows the range of TV and TL measurements recorded each year.
Minimum, maximum and mean values increased from year to year, again
indicating normal growth. Annual increment in growth was greatest between
2004 and 2005, during which time the onset of sexual maturity occurred. There
were increases of 116.5cm3 in mean TV and 6.9cm in mean TL, compared to
increases of 23.7cm3 in TV and 2.9cm in TL between 2003 and 2004.
4.3.2.5 M5
145
Measurement
n
Min
Max
Mean
SD
Right length (cm)
126
5.6
8.3
7.0
0.7
Left length (cm)
126
5.4
8.2
6.9
0.7
Right volume (cm3)
124
5.0
18.0
10.3
3.2
Left volume (cm3)
125
5.1
17.4
10.5
3.0
124
1.4
5.7
4.4
0.6
124
3.6
5.5
4.5
0.5
No significant difference (P > 0.05) was found in TV between the left and right
testes.
Significant differences (P < 0.05), however, were found in TL and TC
measurements. The right TL measurements recorded were larger than those of
the left side, whereas the left TC measurements recorded were larger than those
of the right.
TV measurements recorded during the study, from June 2003 to December 2006,
are shown in Figure 4.11. Variations about the mean values overall were small.
Measurements showed a general upward trend during the study period without
showing any obvious pattern in fluctuation.
TL and TC measurements generally followed the same upward trend as TV.
146
147
Study year
(age - y/m)
2003
(4/1)
2004
(5/1)
2005
(6/1)
2006
(7/7)
*Mean length (cm)
*Mean volume (cm3)
n
Min
Max
Mean ± SD
Min
Max
Mean ± SD
44
5.8
6.8
6.2 ± 0.2
5.6
8.8
7.3 ± 0.8
31
5.6
7.6
6.9 ± 0.4
6.5
14.0
10.0 ± 1.6
43
7.0
8.2
7.6 ± 0.3
9.4
16.1
12.6 ± 1.8
11
7.7
8.1
7.9 ± 0.2
12.7
16.1
15.1 ± 1.0
basis. Variations in annual mean values were larger after the 1st study year.
Minimum, maximum and mean values gradually increased from year to year,
with annual increments being very similar. Unlike other males, there was no
significant testis growth in M5 before the first release of spermatozoa took place,
in February 2006. At sexual maturity, the mean left and right testis
measurements were 7.8cm and 11.1cm3 and body size measurements were
200cm and 101.6kg.
4.3.3 Correlation between testis measurements
Pearson product-moment correlation coefficient, r, was used to examine the
strength of associations between TL and TC. Analysis was conducted for each
subject individually. Analysis for TV was not conducted since it was derived
from TL measurement, therefore, not an independent variable.
148
149
Table 4.14: Correlation between TL and TC in all the subjects
M1
M2
M3
M4
M5
n
178
129
181
151
124
r
0.71
0.97
0.96
0.94
0.83
r2
0.50
0.90
0.92
0.88
0.64
P
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
n = number of paired data
Correlations between TL and TC varied in strength, r = 0.5 – 0.9 show a
moderate to excellent degree of relationship and statistical significance (P < 0.01)
in all the subjects (Table 4.14). The strongest correlations were found in M2,
M3 and M4. Given such strong correlation between the two parameters, it is
recommended that TC measurement may be used in place of TL to ascertain
testis size and to compare measurements between animals.
4.4 Correlation between body size and testis size
Spearman rank correlation coefficient, rs, was applied to obtain the strength of
associations between body size parameters (BL and BG), and testis size
parameters (TL and TC). Data from all subjects plus M6 were used.
When all the subjects were tested together (N = 6), correlation coefficients, rs,
between BL and TL and between BG and TL indicated only moderate
association (Table 4.15).
150
Table 4.15: Correlation between body size and testis size
Testis length
6
4
Testis circumference
6
4
N
Body
0.55
0.95
length
Body
0.52
0.40
girth
N = number of subjects in analysis
0.23
0.95
0.52
0.40
However, when data from M2, M3, M4 and M5 were considered separately from
fully grown males (N = 4), associations between BL and TL and between BL
and TC were excellent (Table 4.14). Results show that BL is a better indicator
of testis size than age in animals that have not reached full physical maturity.
The small number of subjects in these tests must be taken into consideration
when interpreting the association between body and testis size.
4.5 Serum Testosterone level
Blood samples were collected once a month from all subjects for evaluation of
testosterone levels in the serum. Testosterone levels varied throughout the study
period in all subjects and consistent annual increases were determined in subjects
that had not reached full maturity.
Individual differences were found in testosterone profiles, even between
individuals of similar ages or during similar post-onset periods. Results suggest
that serum testosterone level cannot be relied on alone to accurately assess the
reproductive status of an individual.
151
4.5.1 M1
Testosterone levels (T level) during the study period ranged from 11.3 –
78.4ng/ml, with an overall mean of 44.5 ± 18.8ng/ml (Table 4.16), the highest
recorded T level in all subjects.
Table 4.16: Range of T level (ng/ml) recorded for M1
2003
Age (y / m)
2004
2005
2006
Overall
20+ y
12
12
12
12
48
Min
11.3
17.8
27.7
12.4
11.3
Max
70.2
73.8
78.4
64.2
78.4
Mean
40.0
45.0
50.7
42.6
44.5
SD
20.4
21.6
17.7
15.5
18.8
N
n = number of evaluations
Figure 4.12 shows M1’s T level fluctuated greatly throughout the study period,
from October 2002 to September 2006. Lowest levels were consistently found
from November to January, with higher levels recorded between April and
August. Peak levels in each month were often separated by an interim decline.
The annual minimum, maximum and mean T level were similar (Table 4.16).
These results indicate this subject had reached full reproductive capacity.
All T level recorded for M1 are shown in Appendix 8.
152
4.5.2 M2
T level during the study period ranged from 0.2 – 21.8ng/ml, with an overall
mean of 3.7 ± 4.2ng/ml (Table 4.17). M2 became sexually mature in 2001 and T
level was maintained above 3ng/ml for most of the study.
Figure 4.13 shows T level fluctuated throughout the study period (data collected
from October 2002 to April 2005). Fluctuations were not as large as seen in M1.
Lower levels generally occurred between September and February. T level
consistently began to rise above the mean in February every year. Higher levels
were found between April and June.
2003
Age (y / m)
2004
2005
Overall
9y 5m
N
12
12
7
31
Min
0.2
0.9
0.9
0.2
Max
7.9
8.6
21.8
21.8
Mean
3.0
3.5
5.4
3.7
SD
2.9
2.5
7.6
4.2
The annual mean, minimum and maximum T level of M2 increased gradually
from year to year during the study (Table 4.17). These results indicate that,
although sexually mature, this male had not reached full reproductive status by
153
the age of 11 years. However, this animal was chronically ill, dying in May
2005, so data must be interpreted with caution.
The overall testosterone profile of M2 was lower than that of M1. This may be
due to age, poorer health status, or possibly the fact that M1 was the more
dominant male in the group.
4.5.3 M3
mean of 4.7 ± 9.1ng/ml (Table 4.18).
Figure 4.14 shows M3’s T level fluctuated throughout the study period (data
collected October 2002 to September 2006). After the onset of spermatogenesis
at the beginning of the study levels fluctuated, but remained below 10ng/ml
during the first 3 years. The largest fluctuations occurred during the last year,
when T level reached a peak of 64.2ng/ml in March 2006.
The annual mean and maximum T level of M3 increased from year to year
during the study (Table 4.18). Results indicate that, although sexually mature,
this male had not reached full reproductive capacity by the end of the study
period, when he was 11y 3m of age.
154
2003
Age (y / m)
2004
2005
2006
Overall
8y 3m
N
12
12
12
12
48
Min
1.2
0.9
0.5
0.2
0.2
Max
6.3
5.6
9.4
64.2
64.2
Mean
2.7
3.0
4.6
8.4
4.7
SD
1.4
1.6
2.9
17.8
9.1
Despite being younger, T level were generally higher than in M2 and there was a
more dramatic rise in annual mean levels over the study period. This may be due
to the poorer health status of M2.
155
Figure 4.12: Monthly serum testosterone level – M1
156
157
158
4.5.4 M4
mean of 6.7 ± 9.8ng/ml (Table 4.19).
2003
Age (y/m)
2004
2005
2006
Overall
5y1m
N
11
12
12
10
45
Min
0.1
0.4
0.8
1.7
0.10
Max
4.9
6.0
49.0
26.2
49.0
Mean
0.8
2.2
14.9
8.8
6.7
SD
1.4
2.0
14.2
7.4
9.8
Figure 4.15 shows M4’s T level fluctuated greatly after the onset of
spermatogenesis. T level in the first study year were mostly below 1ng/ml.
Levels in the following year were mostly above 1ng/ml. Fluctuations in the
following year were first above 3ng/ml, then above 5ng/ml. Levels fell below
1ng/ml again in November and December 2004, and then sharply increased to
24ng/ml in April 2005, when onset of spermatogenesis occurred.
Levels
continued to rise to reach a peak of 49ng/ml in June the same year. Levels then
fell sharply to below 3ng/ml for the reminder of the year, which may be due to
illness. After the onset of spermatogenesis, T level did not fall below 1ng/ml in
this subject.
159
The annual mean, minimum and maximum T level of M4 increased from year to
year until 2005 (Table 4.19). Data collected in 2006 were few due to the sudden
death of the subject in July. Results indicate that, although sexually mature, this
male had not reached full reproductive capacity by the age of 7y 10m.
Despite being younger, the testosterone profile of M4 was generally higher than
those seen in M2 and M3.
4.5.5 M5
T level in this male ranged from 0.1 – 1.4ng/ml, with an overall mean of 0.3 ±
0.3ng/ml (Table 4.20), the lowest recorded in all the subjects.
2003
Age (y/m)
2004
2005
2006
Overall
4y5m
N
10
12
12
12
46
Min
0.1
0.1
0.1
0.1
0.1
Max
0.4
0.3
0.8
1.4
1.4
Mean
0.2
0.1
0.3
0.5
0.3
SD
0.1
0.1
0.2
0.5
0.3
160
Figure 4.16 shows M5’s T level fluctuated comparatively little throughout the
study period, from October 2002 to September 2006. Although T level were
very low (< 1ng/ml) the onset of spermatogenesis occurred in February 2006,
when the subject was 6y 9m of age.
T level mostly remained below 1ng/ml for the remainder of the study period.
The testosterone profile of M5 was much lower than those seen in other males.
All T level recorded for M5 are shown in Appendix 8.5.
4.5.6 M6
T level in this male ranged from 1.6 – 57.8ng/ml, with an overall mean of 22.8 ±
16.5ng/ml (Table 4.21), notably lower than similarly aged M1. T level recorded
in this subject did not fall below 1ng/ml.
2003
Age (y / m)
2004
2005
2006
Overall
20+ y
n
11
12
12
12
47
Min
8.6
3.2
4.3
1.6
1.6
Max
57.8
32.0
56.2
28.6
57.8
Mean
34.7
19.9
26.8
10.9
22.8
SD
18.5
9.4
18.1
9.4
16.5
161
Figure 4.17 shows M6’s T level fluctuated greatly throughout the study period.
Lowest levels were consistently found in November, December or January, with
higher levels recorded between April and August. These results were similar to
the pattern seen in M1, although overall levels were lower.
The annual minimum, maximum and mean T level were similar between 2002
and 2005 (Table 4.21). These results indicate this subject had attained full
reproductive capacity. A decline in T level was observed during 2005 – 2006
when this subject was ill.
162
Figure 4.15: : Monthly serum testosterone level – M4
163
Figure 4.16: : Monthly serum testosterone level – M5
164
165
4.6 Semen collection and sperm density
Semen was collected using voluntary behaviours and all subjects co-operated
well. Multiple ejaculates were collected in succession until the male did not
present again, or until no further ejaculate was available. The duration of a
collection session ranged from two minutes to over one hour. Subjects M3, M4
and M5 were able to deliver semen prior the onset of spermatogenesis.
Sperm density data presented here includes overall session sperm density (OvD)
and highest ejaculate density (HiEjD) per session.
The former (OvD) was
derived by division of total sperm count (ToC) by the total semen volume (ToV)
of a session, the latter (HiEjD) was the density of the most ‘concentrated’
ejaculate collected in a series.
Sperm density was found to fluctuate erratically from week to week. Sperm
density profiles were found to be different between individuals when they were
of similar ages or similar periods after onset of spermatogenesis.
4.6.1 M1
4.6.1.1 Ejaculate number and volume
In a single session M1 (20 – 23+y) produced 1 – 28 ejaculates, with ToV of 4.9
– 221.7ml.
The first ejaculate was usually of much larger volume than
subsequent ejaculates. The number of ejaculates per session increased from 2003
166
to 2005 (Table 4.22). This increase may have been due to better / more rigorous
collection attempts as the study progressed, or to a ‘learning effect’ in this male.
Table 4.22: Range of number of ejaculates and ToV per collection session
recorded in M1
Study
year
No. of ejaculates
Total volume (ml)
n
Min
Max Mean
SD
Min
Max
Mean
SD
Overall
182
1
28
9.8
5.8
4.9
221.7
92.7
42.3
2003
50
1
12
4.8
2.4
5.2
121.3
64.8
26.4
2004
52
2
19
9.2
3.9
4.9
167.2
89.0
35.6
2005
47
1
28
14.0
6.8
39.5
221.7
117.6
48.2
2006
33
4
23
12.1
4.6
25.4
192.0
105.6
37.1
n = number of collection sessions
ToC ranged from 0 – 70,260.4 x 106. There was only 1 session, in April 2003, in
which no spermatozoa were found in the semen of M1. OvD ranged from 0 –
980.3 x 106/ml and the mean OvD for the duration of the study was 241.0 ±
162.0 x 106/ml (Table 4.23). The highest HiEjD recorded in M1 was 3045 x
106/ml, in June 2005. The mean HiEjD was 853.6 ± 572.3 x 106/ml (Table 4.23).
The 1st ejaculate was usually the lowest in density, likely due to the larger
volume. This will be discussed further in Section 4.9 on semen and ejaculate
characteristics.
167
Figure 4.18: Weekly overall sperm density – M1
168
Table 4.23: Range of OvD and HiEjD recorded in M1
Study
year
Overall sperm density
( x 106/ml)
n
Highest ejaculate sperm
density ( x 106/ml)
Min
Max
Mean
SD
Min
Max
Mean
SD
Overall
182
0
980.3
241.0
162.0
0.3
3045.0
853.6
572.3
2003
50
0
594.8
182.9
151.2
0.3
2650.0
753.1
667.7
2004
52
46.0
751.9
262.3
134.5
201.3
2210.0
908.7
481.4
2005
47
77.8
796.3
290.0
143.1
125.0
3045.0
980.1
562.1
2006
33
0.7
980.3
225.6
213.0
3.6
2345.0
736.2
540.4
OvD recorded during the study, from October 2002 to June 2006, are shown in
Figure 4.18. OvD fluctuated on a weekly basis, from mostly > 100 x 106/ml year
round and did not exhibit continuous periods of very low levels, (i.e., < 10 x
106/ml).
The annual mean, minimum and maximum OvD and HiEjD of M1 increased
from 2003 to 2005 (Table 4.23). This is likely due to the increasing number of
ejaculates collected. These results are explained earlier in Section 4.6.1.1.
All results of total volume, overall sperm density and total count recorded for
M1 are shown in Appendix 9.
169
4.6.2 M2
The onset of spermatogenesis in this male took place in September 2001 when
the animal was 7y 4m. At the beginning of the present study, spermatogenesis
had been established for just over a year.
In a single session M2 (9 – 11y) produced 1 – 19 ejaculates with ToVof 4.1 –
178.9ml (Table 4.24). The number of ejaculates per session increased from 2003
to 2005 (Table 4.24). This increase, again, may have been due to improved
collection technique as the study progressed, or to a ‘learning effect’ in this male,
but may also be due to increased efficiency in semen production.
recorded in M2
Study year
(post –
onset year)
No. of ejaculates
Total volume (ml)
n
Min
Max
Mean
SD
Min
Max
Mean
SD
Overall
130
1.0
19.0
6.1
4.2
4.1
178.9
50.1
30.5
2003 (2nd)
49
1.0
10.0
3.6
1.7
4.1
107.4
38.7
25.0
2004 (3rd)
50
1.0
18.0
6.7
3.8
7.1
179.0
57.7
36.9
2005 (4th)
31
3.0
19.0
9.3
5.1
19.7
98.2
55.8
21.2
170
ToC ranged from 0 – 44,284.0 x106.
There was only 1 session, in November
2002, in which no spermatozoa were found. OvD ranged from 0 – 1,692.4
x106/ml, with a mean OvD of 112 ± 186.3 x 106/ml (Table 4.25). The highest
HiEjD recorded in M2 was 2,450 x 106/ml, in October 2002. The mean HiEjD
was 440 ± 473.8 x 106/ml (Table 4.25).
Study year
(post –
onset year)
Overall
2003
(2nd)
2004
(3rd)
2005
(4th)
( x 106/ml)
n
density ( x 106/ml)
Min
Max
Mean
SD
Min
Max
Mean
SD
130
0
1692.4
112.0
186.3
0.1
2450.0
440.0
473.8
49
0
1692.4
98.0
246.2
0.1
2450.0
335.5
493.1
50
0.1
633.4
132.6
161.3
0.2
2030.0
556.6
528.9
31
0.1
423.3
100.7
92.3
0.1
995.0
414.0
276.4
OvD recorded from October 2002 to May 2005, are shown in Figure 4.19. OvD
fluctuated from week to week.
OvD was very low (< 10 x 106/ml) for
consecutive months from November 2002 to March 2003 (2nd post-onset year)
and from February to May 2004 (3rd post-onset year). After that, OvD fluctuated
mostly above 100 x 106/ml for the reminder of the study. Fluctuations in HiEjD
were greater and followed a similar pattern to OvD. HiEjD was also very low (<
10 x 106/ml) for consecutive months from December to March during 2002 –
171
2003 and March to May during 2003 – 2004. After that HiEjD fluctuated mostly
from above 200 x 106/ml for the reminder of the study period.
The annual mean, minimum and maximum OvD and HiEjD of M2 increased
from 2002-2004 (Table 4.25). Data from 2004 – 2005 were incomplete due to
death of the subject. Results suggest M2 had not reached full reproductive
capacity at the time of death.
The sperm density profile of M2 was generally lower than in M1.
4.6.3 M3
The onset of spermatogenesis in this male took place in October 2002, when the
animal was 7y 3m.
In a single session M3 (8 – 11y) produced 0 – 14 ejaculates with ToV of 0 –
60.5ml (Table 4.26). A lack of semen (aspermia) was found in 4 collection
sessions; 1 in September 2003, the 1st post-onset year, 2 in March 2004 and 1 in
January 2006, the 4th post onset year. The number of ejaculates and ToV per
session increased from 2003 to 2005, 1st – 3rd post-onset years (Table 4.26).
172
recorded in M3
Study year
(post –
onset year)
No. of ejaculates
Total volume (ml)
n
Min
Max
Mean
SD
Min
Max
Mean
SD
Overall
186
0
14.0
3.4
2.4
0
60.5
18.9
13.2
2003 (1st)
50
0
5.0
2.1
1.1
0
44.0
15.6
12.2
2004 (2nd)
50
0
10.0
2.7
2.3
0
56.5
17.5
15.1
2005 (3rd)
53
1.0
14.0
4.5
2.5
4.2
60.1
23.1
12.0
2006 (4th)
33
0
11.0
4.1
2.6
0
58.4
19.4
12.3
ToC ranged from 0 – 81,922 x106. No spermatozoa were found in 30 out of a
total 183 (16.3%) sessions (excluding aspermic sessions); 18 in the 1st post-onset
year, 11 in the 2nd and 1 in the 3rd.
The occurrence of azoospermia did not
follow any seasonal pattern. OvD ranged from 0 – 2,493.8 x106/ml and the
mean OvD for the duration of the study was 209 ± 438.4 x 106/ml (Table 4.27).
The highest HiEjD recorded in M3 was 5,170 x 106/ml, in August 2005, 3rd postonset year. The mean HiEjD was 662.1 ± 1,044.8 x 106/ml (Table 4.27).
173
174
Study year
(post –
onset year)
Overall
2003
(1st)
2004
(2nd)
2005
(3rd)
2006
(4th)
n
( x 106/ml)
Min Max Mean SD
density ( x 106/ml)
Min
Max Mean
SD
183
0
2493.8
209.0
438.4
0.03
5170.0
662.1
1044.8
48
0
55.0
2.6
8.8
< 0.03
155.0
15.4
35.5
49
0
670.0
72.2
156.5
0.1
1325.0
270.3
340.4
53
0
2493.8
380.1
653.7
0.1
5170.0
920.9
1366.2
33
0
1369.8
437.8
408.6
0.1
4060.0
1086.3
987.4
OvD recorded from October 2002 to June 2006, are shown in Figure 4.20. After
the onset of spermatogenesis in October 2002, M3’s OvD was very low (0 to <
10 x 106/ml) until December 2003. From December 2003 to February 2004,
OvD sporadically increased from < 10 x 106/ml up to 500 x 106/ml. OvD
dropped again and then remained at 0 – < 10 x 106/ml until June 2004, when it
began to fluctuate above 10 x 106/ml on a more consistent basis. A shorter
continuous period of very low OvD (< 10 x 106/ml) occurred from February to
April 2005, during the 3rd post-onset year.
After that, OvD fluctuated
dramatically from week to week. The highest OvD recorded (2,493.8 x 106/ml)
was in July 2005. For the remainder of the study, OvD fluctuated mostly from
above 100 x 106/ml, except January – March 2006, when densities sometimes
fell below 10 x 106/ml. Fluctuations in HiEjD followed a similar pattern to OvD.
From the 3rd post-onset year, HiEjD
fluctuated around 300 x 106/ml. The HiEjD of 5,170 x 106/ml recorded during
the 3rd post-onset year was also the highest single density recorded in all subjects.
175
The annual mean overall OvD and HiEjD of M3 increased consistently during
the study (Table 4.27).
This suggests M3 had not reached full reproductive
capacity. Efficiency in spermatogenesis was unlikely to have reached full adult
capacity when the study ended in June 2006.
The sperm profile of M3 became higher than that of M2 from the 3rd post-onset
year.
4.6.4 M4
The onset of spermatogenesis in this male took place on 19th April 2005 when
the animal was 6y 7m.
In a single session M4 (6 – 8y) produced 1 – 11 ejaculates with ToV of 0.7 –
84.2ml (Table 4.28). The number of ejaculates and ToV per session increased
after the onset of sexual maturity (Table 4.28).
176
177
recorded in M4
Study year
(post –
onset year)
No. of ejaculates
Total volume (ml)
n
Min
Max
Mean
SD
Min
Max
Mean
SD
9
1.0
5.0
3.1
1.6
0.5
11.8
5.2
4.2
Overall
55
1.0
11.0
5.4
2.2
0.7
84.2
26.0
17.3
2005 (1st)
46
1.0
11.0
5.3
2.3
0.7
84.2
24.6
18.0
2006 (2nd)
9
3.0
8.0
6.0
1.6
16.6
50.2
33.4
10.7
2004
(Pre-onset)
ToC ranged from < 0.03 – 24,090.5 x106. After onset, spermatozoa were found
in every ejaculate. OvD ranged from < 0.03 – 963.6 x106/ml. The mean OvD
for the duration of the study was 117.8 ± 212.4 x 106/ml (Table 4.29). The
highest HiEjD recorded in M4 was 1,475 x 106/ml, in April 2005, 1st post-onset
year. The mean HiEjD was 273.7 ± 384.9 x 106/ml (Table 4.29).
Study year
(post –
onset year)
n
Overall
2005
(1st)
2006
(2nd)
( x 106/ml)
density ( x 106/ml)
Min
Max
Mean
SD
Min
Max
Mean
SD
55
< 0.03
963.6
117.8
212.4
< 0.03
1475.0
273.7
384.9
46
< 0.03
712.7
62.4
151.6
< 0.03
1475.0
162.9
291.7
9
124.0
963.6
400.9
259.9
211.3
1260.0
791.0
352.4
178
OvD recorded from October 2002 to July 2006, are shown in Figure 4.21. After
onset of spermatogenesis in April 2005, M4’s OvD was very low (< 10 x 106/ml)
until February 2006, 10 months post-onset. Then OvD began to increase to > 10
x 106/ml. Large fluctuations occurred from March 2006 onwards and a peak of
963.6 x 106/ml was reached in June the same year. OvD fluctuated mostly from
> 100 x 106/ml for the remainder of the study period. Fluctuations in HiEjD
followed a slightly different pattern to OvD. HiEjD was low but it sporadically
fluctuated above 100 x 106/ml during the 10-month post-onset period while OvD
was more consistently low (< 10 x 106/ml). HiEjD first reached above 200 x
106/ml within 5 months of the onset of spermatogenesis. This was notably more
rapid than observed in M3 after over a year of onset.
Large fluctuations
occurred from March 2006 and a peak of 1,475 x 106/ml was reached in April
the same year. HiEjD fluctuated mostly from > 200 x 106/ml for the reminder of
the study period.
The annual mean overall OvD and HiEjD of M4 increased during the study
(Table 4.29), suggesting this male had not reached full reproductive capacity
when the study ended in July 2006.
It is difficult to compare the sperm density profile of M4 with that of other
young males as the post-onset monitoring period was relatively short. However,
the profile of the 1st post-onset year was higher than that of M3 of the same
period.
179
4.6.5 M5
The onset of spermatogenesis in this male took place on 6th February 2006, when
the animal was 6y 9m, very similar to M4.
In a single session M5 (6 – 8y) produced 0 – 5 ejaculates with ToV of 0 – 4.4ml
(Table 4.30). Aspermia was found in 4 collection sessions prior the onset of
spermatogenesis. The mean number of ejaculates and ToV per session increased
from 2005 to 2006 (Table 4.30). This increase is likely due to increased
efficiency in semen production after the onset of sexual maturity.
recorded in M5
Study year
(post –
onset year)
2005
(Pre-onset)
2006 (1st)
No. of ejaculates
Total volume (ml)
n
Min
Max
Mean
SD
Min
Max
Mean
SD
13
0
3.0
1.5
1.3
0
4.4
0.7
1.2
26
1.0
5.0
2.8
1.4
< 0.1
2.8
0.9
0.9
180
Spermatozoa were found in the semen of M5 only after centrifugation and in 5
collection sessions only. ToC was very low < 0.03, with OvD also low. (Table
4.31).
Study year
(post –
onset year)
n
2006
(1st)
26
density ( x 106/ml)
( x 106/ml)
Min
Max
Mean
SD
Min
Max
Mean
SD
0
< 0.03
-
-
-
< 0.03
-
-
After the onset of spermatogenesis, M5’s sperm output was significantly lower
than levels observed in M3 and M4.
M5 is shown in Appendix 9.
Spermatozoa were often not detectable on initial wet-mount preparations of
ejaculates that were of extremely low counts (< 0.03 x 106) or density (< 0.03 x
106/ml). Such ejaculates were found occasionally in all the subjects, but more
frequently in M3 (Section 4.6.3.2), M4 (Section 4.6.4.2) and M5 (Section
4.6.5.2), and they were often the first ejaculates collected in each session. The
differences in characteristics between the 1st, 2nd and other ejaculates collected in
a series are presented in detail in Section 4.10. Examination of sediments of
181
centrifuged samples increased the likelihood of detecting spermatozoa in these
ejaculates, however, it is possible that sparse spermatozoa may have been
overlooked due to technical error, as well as a true absence. Therefore, when
examining semen of sexually immature subjects for the first release of
spermatozoa, extreme care is required to avoid misinterpretation of reproductive
status. The vigilance of the weekly collection regime of this study, in
conjunction with multiple collections in a session, plus centrifugation, ensured
the first release of spermatozoa was pinpointed with an accuracy of at least one
week.
4.7 Comparisons between testis size, serum testosterone levels and
sperm density
Testis size were compared with serum testosterone levels and sperm density to
determine any relationships. For these comparisons, only data collected on the
same day were used for analysis. Analysis was conducted for each subject
individually. Pearson product-moment correlation coefficient, r, was obtained to
examine the strength of associations between the different parameters.
182
183
4.7.1 Comparison between serum testosterone and testis size
Testis length (TL) and volume (TV) measurements were compared with serum
testosterone level (T level)
Table 4.32:
Correlation between T level and testis size
Testis length
Testis volume
n
r
P value
r
P value
M1 41
0.41
< 0.01
0.46
< 0.01
M2 30
0.82
< 0.01
0.89
< 0.01
M3 43
0.57
< 0.01
0.67
< 0.01
M4 28
0.61
< 0.05
0.71
< 0.01
M5 22
0.40
> 0.05
0.40
> 0.05
Correlation between testis size and T level varied in strength between subjects,
with r values ranging from 0.4 – 0.9 (Table 4.32), which suggest a fair to very
good relationship. Association was stronger between TV and T level in M1 –
M4. Statistical significance (P < 0.05 and P < 0.01) was found in all the r values,
except those of M5.
The strongest association was found in M2 (TL r = 0.82, TV r = 0.89,
Figure 4.22). Figure 4.23 shows TV and T level over time. TV and T level
increased and decreased concurrently, with both parameters being higher during
the summer months, March to July, and lower during the winter months,
184
November to January (Figure 4.23). The pattern in which these parameters
varied are further assessed in Section 4.9.
25
y = 0.0358x - 4.6494
2
R = 0.7831
20
15
T level
(ng/ml)
10
5
0
0
50
100
150
200
250
300
350
400
450
500
550
600
650
TV (cm3)
Figure 4.22: Correlation between TV and T level – M2
(Solid markers represent TV and T level recorded in March to
July .Dotted line denotes overall mean T level)
650
30
Testis volume
T level
600
550
25
500
450
20
400
TV 350
(cm3) 300
15
250
10
200
150
5
100
50
Month of study
Figure 4.23: TV and T level – M2
185
5
5
M
ar
-0
Ja
n0
4
4
N
ov
-0
4
Se
p0
Ju
l -0
4
4
M
ay
-0
4
M
ar
-0
Ja
n0
3
3
N
ov
-0
Se
p0
3
Ju
l -0
3
M
ay
-0
3
0
M
ar
-0
Ja
n0
3
0
T level
(ng/ml)
Associations between testis size and T level were similar in M3 and M4, they
ranged from moderate to good (TL r = 0.6, TV r = 0.7, Figures Figure 4.24 and
Figure 4.25).
A similar pattern of concurrent fluctuations was seen in M3
(Figure 4.26), but was less well defined than in M2. Parameters were higher
between March and August, and lower between September and January (Figure
4.26). These results are further assessed in Section 4.9.
In M4, both testis size and T levels increased concurrently and markedly during
the four months prior to onset of spermatogenesis, from January 2004 to April
2005 (Figure 4.27).
Subsequently, the parameters also showed concurrent
variations. As in M2 and M3, TV and T level also showed a tendency to be
higher in March to August (Figure 4.27). This pattern will be further reviewed
in Section 4.9.
70
y = 0.0597x - 9.4525
2
R = 0.4534
60
50
40
T level
(ng/ml)
30
20
10
0
0
50
100
150
200
250
300
350
400
450
500
550
600
650
TV (cm3)
August. Dotted line denotes overall mean T level)
186
55
y = 0.1135x - 2.882
R2 = 0.5053
50
45
40
35
T level 30
(ng/ml) 25
20
15
10
5
0
0
50
100
150
200
250
300
350
400
TV (cm3)
Figure 4.25: Correlation between T level and TV – M4
(Solid markers represent TV and T level recorded in March to August.
Dotted line denotes overall mean T level)
Associations between testis size and T level in M1 were fair to moderate (testis
length r = 0.41, testis volume r = 0.46, Figure 4.28).
Fluctuations of these
parameters were often concurrent (Figure 4.29). There was a tendency for both
TV and T level to be higher between March and August (Figure 4.29). These
findings are further assessed in Section 4.9.
Associations in M5 were fair (both TL and TV r = 0.4) with an overall upward
trend in both parameters (Figure 4.30). Further monitoring is required to study
the relationship between testis size and T level in this subject.
187
650
600
90
Testis volume
T level
80
550
70
500
450
60
400
50
350
TV
300
(cm3)
T level
40 (ng/ml)
250
30
200
150
20
100
10
50
0
D
ec
-0
Fe 2
b03
Ap
r-0
3
Ju
n03
Au
g03
O
ct
-0
D 3
ec
-0
3
Fe
b04
A
pr
-0
4
Ju
n04
A
ug
-0
4
O
ct
-0
D 4
ec
-0
Fe 4
b0
A 5
pr
-0
5
Ju
n05
A
ug
-0
5
O
ct
-0
D 5
ec
-0
Fe 5
b0
A 6
pr
-0
6
Ju
n06
0
Month of study
650
600
70
Testis volume
T level
60
550
500
50
450
400
40
350
TV
300
(cm3)
30
250
200
20
150
100
10
50
Month of study
188
6
Ju
n0
Fe
b06
A
pr
-0
6
5
A
ug
-0
5
O
ct
-0
5
D
ec
-0
5
Ju
n0
Fe
b05
Ap
r-0
5
4
A
ug
-0
4
O
ct
-0
4
D
ec
-0
4
Ju
n0
Fe
b04
A
pr
-0
4
0
O
ct
-0
3
D
ec
-0
3
A
ug
-0
3
0
T level
(ng/ml)
90
80
y = 0.1616x - 26.687
R2 = 0.2079
70
60
50
T level
(ng/ml) 40
30
20
10
0
150
200
250
300
350
400
450
500
550
600
650
3
TV (cm )
650
600
90
Testis volume
T level
80
550
70
500
450
60
400
TV 350
(cm3)
50
300
40
250
30
200
150
20
100
10
50
0
D
ec
-0
2
Fe
b03
Ap
r-0
3
Ju
n03
A
ug
-0
3
O
ct
-0
3
D
ec
-0
3
Fe
b04
A
pr
-0
4
Ju
n04
A
ug
-0
4
Oc
t-0
4
D
ec
-0
4
Fe
b05
A
pr
-0
5
Ju
n05
A
ug
-0
5
Oc
t-0
5
D
ec
-0
5
Fe
b06
A
pr
-0
6
Ju
n06
0
Month of study
189
T level
(ng/ml)
30
4.0
Testis volume
T level
3.5
25
3.0
20
2.5
TV 15
(cm3)
2.0 T level
(ng/ml)
1.5
10
1.0
5
0.5
0.0
A
ug
-0
3
O
ct
-0
3
De
c03
Fe
b04
A
pr
-0
4
Ju
n04
A
ug
-0
4
O
ct
-0
4
D
ec
-0
4
Fe
b05
A
pr
-0
5
Ju
n05
Au
g05
O
ct
-0
5
D
ec
-0
5
Fe
b06
A
pr
-0
6
Ju
n06
A
ug
-0
6
Oc
t -0
6
0
Month of study
Figure 4.30: T level and TV – M5
4.8 Comparison between serum testosterone and sperm density
Serum testosterone level was compared with overall session sperm density (OvD)
(derived by division of total sperm count by total semen volume) and highest
ejaculate density (HiEjD – density of the most concentrated ejaculate in the
collection series). Analysis was not conducted on M5 due to extremely low
density semen samples.
Correlation between T levels and sperm density varied in strength and direction
between subjects, | r | values ranged from 0.01 to 0.4 (Table 4.33) which suggest
non-existent to, at best, fair relationships. Association was stronger between T
level and OvD in all subjects except M1.
190
Table 4.33:
Correlation between T level and sperm density
Overall density
(OvD)
n
Highest ejac. Density
(HiEjD)
r
P value
r
P value
M1
39
0.01
> 0.05
0.07
> 0.05
M2
30
0.38
<0.05
0.16
> 0.05
M3
41
-0.11
> 0.05
-0.09
> 0.05
M4
13
-0.33*
> 0.05
-0.18*
> 0.05
*
rs Spearman rank correlation coefficient
The strongest association was found in M2, (OvD r = 0.38, Figure 4.31) and this
was statistically significant (P < 0.05). This is illustrated by Figure 4.32,
showing T level and OvD over time.
T level and OvD showed some
fluctuations that were concurrent, however, OvD often fluctuated independently
of T level.
25
y = 0.0151x + 2.4223
2
R = 0.1424
20
15
T level
(ng/ml)
10
5
0
0
100
200
300
400
500
OvD (x106/ml)
Figure 4.31: Correlation between T level and OvD in M2
(Solid markers represent T level and OvD recorded in March to
July. Dotted line denotes overall mean T level)
191
600
600
30
Sperm density
T level
500
25
400
20
300
15
OvD
(x106/ml)
T level
(ng/ml)
5
4
5
M
ar
-0
Ja
n0
4
N
ov
-0
4
4
Se
p0
Ju
l -0
4
M
ay
-0
4
M
ar
-0
Ja
n0
N
ov
-0
Se
p0
Ju
l -0
M
ay
-0
M
ar
-0
Ja
n0
3
0
3
0
3
5
3
100
3
10
3
200
Month of study
Figure 4.32: T level and OvD – M2
T level was inversely associated with sperm density in the youngest subjects
(M3 and M4). M3’s OvD remained very low, or zero, during the 1st post-onset
year, between January and October 2003, while T level fluctuated. (Figure 4.33).
Conversely, between May 2005 and January 2006, OvD markedly increased and
fluctuated, while T level decreased and remained at a relatively constant low
level. The post-onset monitoring period was too short in M4 to investigate any
relationship between T level and sperm density.
Association was the weakest in M1 (r < 0.1, Figure 4.34). Both Figures 4.34 and
4.35 show OvD fluctuated independently of T level.
192
2400
2200
90
Sperm density
T level
80
2000
70
1800
1600
60
1400
50
OvD 1200
(x106/ml)
T level
40 (ng/ml)
1000
800
30
600
20
400
10
200
0
Ja
n0
M 3
ar
-0
M 3
ay
-0
3
Ju
l -0
3
Se
p03
No
v03
Ja
n04
M
ar
-0
M 4
ay
-0
4
Ju
l -0
4
Se
p04
N
ov
-0
4
Ja
n05
M
ar
-0
M 5
ay
-0
5
Ju
l -0
5
Se
p05
No
v05
Ja
n06
M
ar
-0
M 6
ay
-0
6
0
Month of study
90
y = 0.0013x + 49.266
R2 = 1E-04
80
70
60
T
50
level
(ng/ml)
40
30
20
10
0
0
100
200
300
400
500
600
700
800
OvD (x106/ml)
Figure 4.34: Correlation between T level and OvD in M1
(Solid markers represent T level and OvD recorded in March to
193
900
1200
90
Sperm density
T level
80
1000
70
800
60
50
T level
(ng/ml)
OvD 600
(x106/ml)
40
400
30
20
200
10
0
M
Ja
n0
3
ar
-0
3
M
ay
-0
3
Ju
l -0
3
Se
p03
N
ov
-0
3
Ja
n04
M
ar
-0
4
M
ay
-0
4
Ju
l -0
4
Se
p04
N
ov
-0
4
Ja
n05
M
ar
-0
5
M
ay
-0
5
Ju
l -0
5
Se
p05
No
v05
Ja
n06
M
ar
-0
6
M
ay
-0
6
0
Month of study
4.8.1 Comparison between testis size and sperm density
Testis length and volume was compared with overall session density and highest
ejaculate sperm density. Analysis was not conducted on M5.
Correlation between testis size and sperm density varied widely, with | r | values
ranging from no relationship (0.01) to a moderate relationship (0.6) (Table 4.34).
Association between testis size and sperm density was statistically significant (P
< 0.05 and P < 0.01) in all subjects except M1.
194
Table 4.34: Correlation between testis size and sperm density
Testis length
Testis volume
r
P value
r
P value
0.09
> 0.05
0.11
> 0.05
-0.01
> 0.05
0.04
> 0.05
0.35
< 0.01
0.35
< 0.01
0.25
< 0.01
0.24
< 0.01
0.24
< 0.01
0.20
< 0.01
0.21
< 0.05
0.18
< 0.05
0.58
< 0.01
0.38
< 0.01
0.57
< 0.01
0.39
< 0.01
n
M1
OvD
HiEjD
M2
OvD
HiEjD
M3
126
OvD
HiEjD
M4
176
151
OvD
HiEjD
52
The strongest association was found in M4 (TL r = 0.6, Figure 4.36, TV r = 0.4).
This association is also shown in Figure 4.37. Associations in M2 and M3,
ranging between r = 0.2 – 0.4, were only fair (Figures 4.38 and 4.39).
Association was the weakest in M1 (r < 0.2). In this male, an association was
better reflected in TV (Figure 4.40).
Comparisons between the reproductive parameters investigated were sound,
since they were based on data collected on the same day. Generally, the stronger
associations between the parameters were found in the youngest subjects, M2 –
M5. This is likely to be due the growing status of these subjects as shown in the
increase in testis size over as the study year progressed (Sections 4.3.2),
compared to M1, a fully mature male. Any degree of association between
195
parameters in M1 may be interpreted as inherent biological / functional
relationships.
1200
y = 124x - 2067
R2 = 0.3415
1000
800
OvD
(x106/ml)
600
400
200
0
12
14
16
18
20
22
TL (cm)
Figure 4.36: Correlation between TL and OvD in M4
196
24
24
22
1200
Testis length
Sperm density
20
1000
18
16
800
14
TL
(cm)
12
600
10
8
OvD
(x106/ml)
400
6
4
200
2
Ju
n06
A
pr
-0
6
M
ay
-0
6
06
ar
-0
6
M
Fe
b-
Ja
n06
-0
5
N
ov
-0
5
D
ec
-0
5
O
ct
Se
p05
A
ug
-0
5
-0
5
Ju
l
Ju
n05
0
A
pr
-0
5
M
ay
-0
5
0
Month of study
Figure 4.37: TL and OvD – M4
26
24
2000
Testis length
Sperm density
1800
22
1600
20
18
1400
16
1200
TL 14
(cm)
1000
12
10
800
8
600
6
400
4
200
2
Month of study
197
Fe
b05
Ap
r-0
5
4
D
ec
-0
4
A
ug
-0
4
O
ct
-0
4
Ju
n0
Fe
b04
Ap
r-0
4
3
D
ec
-0
Ju
n0
3
A
ug
-0
3
O
ct
-0
3
0
Fe
b03
A
pr
-0
3
Oc
t -0
2
D
ec
-0
2
0
OvD
(x106/ml)
26
24
3500
Testis length
Sperm density
3000
22
20
2500
18
16
2000
TL 14
(cm) 12
OvD
(x106/ml)
1500
10
8
1000
6
4
500
2
0
O
ct
-0
D 2
ec
-0
2
Fe
b03
A
pr
-0
3
Ju
n03
Au
g03
Oc
t -0
D 3
ec
-0
3
Fe
b0
Ap 4
r-0
4
Ju
n04
A
ug
-0
4
O
ct
-0
4
De
c04
Fe
b0
A 5
pr
-0
5
Ju
n05
A
ug
-0
5
O
ct
-0
D 5
ec
-0
5
Fe
b06
A
pr
-0
6
Ju
n06
0
Month of study
650
600
1200
Testis volume
Sperm density
550
1000
500
450
800
OvD
(x106/ml)
TV 400
(cm3) 350
600
300
250
400
200
150
200
100
50
0
O
ct
-0
De 2
c02
Fe
b0
Ap 3
r-0
3
Ju
n03
A
ug
-0
3
O
ct
-0
D 3
ec
-0
3
Fe
b04
Ap
r-0
4
Ju
n04
A
ug
-0
4
O
ct
-0
4
D
ec
-0
4
Fe
b05
A
pr
-0
5
Ju
n05
A
ug
-0
5
Oc
t -0
5
D
ec
-0
5
Fe
b06
A
pr
-0
6
Ju
n06
0
Month of study
Figure 4.40: TV and OvD – M1
198
All the associations between parameters found in M2 were of statistical
significance (P < 0.05 or < 0.01) and this subject exhibited the strongest
associations in 2 out of the 3 comparisons investigated. Data of M2 showed
marked seasonal changes, and changes in the parameters parallelled each other.
The topic of seasonal change is further investigated in Section 4.9 and individual
differences observed in testicular activities are discussed in detail in Section 5.
For all the subjects, the strongest associations found were between testosterone
level and testis volume. These results demonstrate the usefulness of
ultrasonography to monitor and assess reproductive activities in dolphins.
4.9 Seasonality
Fluctuations in serum testosterone levels, testes volume and sperm density were
evaluated to determine any seasonal pattern. For this comparison, serum
testosterone data from M6 were also included. The mean values of each month
were plotted to assess any overall pattern during the study. The annual mean
values (determined in Sections 4.3 for testis volume, 4.5 for serum testosterone
level and 4.6 for sperm density), were used to assess data from each separate
study year; a frequency plot for each month with values above the annual mean
was used to determine if there was a seasonal pattern on a yearly basis. All
analyses were conducted for each subject individually.
199
4.9.1 Testis volume
Testis sizes were measured by ultrasonography on a weekly basis and since testis
volume (TV) measurements were most accurate in indicating testis size (see
Section 4.3.2), this was used to examine seasonal patterns.
4.9.1.1 M1
The overall mean TV recorded for M1 was 466.4 ± 53.7cm3. TV showed a
seasonal pattern, with higher monthly mean measurements (> 460cm3) from
March to August and lower monthly mean measurements from September to
February (Table 4.35 and Figure 4.41). Annual peak measurements and
measurements above the annual mean were most frequently recorded from
March to August and in November (Figure 4.42).
4.9.1.2 M2
The overall mean TV recorded for M2 was 229.6 ± 111.1cm3. This subject
showed the most marked seasonal pattern in TV, with higher monthly mean
measurements (> 220cm3) between March and August, and lower monthly mean
measurements between October and February (Table 4.36 and Figure 4.41).
Annual peak measurements and measurements above the annual mean also were
most frequently recorded from March to August (Figure 4.43). Measurements
recorded between November and February were always below the annual mean
TV: 198.1cm3 in 2003, 246.2cm3 in 2004 and 254.4cm3 in 2005.
200
Table 4.35: Monthly and overall study mean values (±
± SD) of TV, T level
size and OvD during 2002 – 2006 in M1
Testis volume
Serum T level
n
cm3
n
ng/ml
n
x 106 /ml
Jan
17
404.63 ± 11.9
4
22.78 ± 7.77
17
193.52 ± 38.99
Feb
16
445.53 ± 9.76
4
35.30 ± 17.32
16
144.32 ± 22.36
Mar
17
485.65 ± 12.48
4
52.59 ± 21.90
17
219.9 ± 26.13
Apr
14
506.75 ± 8.4
4
50.70 ± 12.65
14
363.9 ± 62.69
May
16
510.19 ± 10.3
4
59.39 ± 19.28
16
326.2 ± 39.6
Jun
15
482.74 ± 9.09
4
53.82 ± 19.65
15
310.18 ± 49.66
Jul
12
488.52 ± 8.25
4
45.30 ± 14.78
13
253.56 ± 57.94
Aug
14
471.05 ± 10.68
4
46.83 ± 17.13
13
269.62 ± 31.3
Sep
10
455.11 ± 1.94
4
40.61 ± 13.54
12
244.82 ± 24.95
Oct
18
450.38 ± 12.66
4
28.70 ± 9.53
16
249.87 ± 23.10
Nov
16
452.95 ± 15.93
4
33.51 ± 16.93
17
165.09 ± 36.25
Dec
14
453.89 ± 16.27
4
18.41 ± 8.08
15
200.90 ± 42.86
Overall
466.4 ± 53.7
44.5 ± 18.8
241.0 ± 162.0
M1
M2
M3
600
550
500
450
400
350
TV
(cm3)
300
250
200
150
100
50
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Month
Figure 4.41: Mean monthly TV in M1, M2 & M3 (2002-2006)
201
Dec
Year 4
Year 3
Year 2
Year 1
15
14
13
12
11
Freq.
above
annual
mean
10
9
8
7
6
5
4
3
2
1
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Month
Figure 4.42: Frequency of testis volume of individual months above the
annual mean of each year (2002 – 2006) in M1
4.9.1.3 M3
The overall mean TV recorded for M3 was 234.1 ± 98.8cm3. TV showed a
pattern similar to M1 and M2, with higher monthly mean measurements (>
230cm3) between March and August, and lower monthly mean measurements
between September and February (Table 4.37 and Figure 4.41). Annual peak
measurements and measurements above the annual mean were most frequently
recorded from March to August. This pattern was not as apparent during the 1st
study year, probably due to physical growth of the testes. Overall there were
variations from year to year in the pattern found (Figure 4.44). Measurements
recorded in December and January were always below the annual mean TV:
165.2cm3 in 2003, 215.4cm3 in 2004, 294.8cm3 in 2005 and 283.8cm3 in 2006.
202
Results of the last study year should be interpreted with caution as data
collection for that year was incomplete.
Table 4.36:
Monthly and overall study mean values (±
and OvD during 2002 – 2005 in M2
Testis volume
Overall sperm
density
Serum T level
n
cm3
n
ng/ml
n
x 106 /ml
Jan
13
122.44 ± 8.5
3
0.92 ± 0.88
13
47.25 ± 11.55
Feb
12
141.931 ± 2.68
3
1.60 ± 1.53
12
43.24 ± 13.3
Mar
13
223.18 ± 16.33
3
3.82 ± 2.85
13
17.74 ± 9.58
Apr
9
341.36 ± 44.1
3
9.11 ± 8.91
11
121.16 ± 37.47
May
12
399.49 ± 34.86
2
5.30 ± 2.34
11
140.51 ± 31.57
Jun
8
364.13 ± 21.05
2
7.96 ± 0.55
8
198.42 ± 84.15
Jul
9
321.54 ± 12.42
2
4.44 ± 2.93
9
169.14 ± 60.15
Aug
9
256.98 ± 8.28
2
4.74 ± 2.76
9
98.26 ± 31.62
Sep
8
205.88 ± 6.4
2
1.60 ± 0.83
8
148.55 ± 30.56
Oct
12
186.47 ± 7.98
3
1.18 ± 0.66
13
219.55 ± 127.3
Nov
12
155.66 ± 7.81
3
0.78 ± 0.42
11
79.12 ± 18.88
Dec
11
132.37 ± 9.42
3
1.56 ± 1.76
11
127.70 ± 51.13
Overall
229.6 ± 111.1
3.7 ± 4.2
203
112.0 ± 186.3
15
14
Year 3
Year 2
Year 1
13
12
11
10
Freq.
above
annual
mean
9
8
7
6
5
4
3
2
1
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Month
Figure 4.43: Frequency of testis volume of individual months above annual
mean of each year (2002 – 2005) in M2
Table 4.37:
Testis volume
Overall sperm
density
Serum T level
n
cm3
n
ng/ml
n
x 106 /ml
Jan
17
166.59 ± 9.17
4
1.75 ± 1.47
13
243.08 ± 98.8
Feb
16
209.68 ± 18.38
4
3.42 ± 3.60
11
118.28 ± 64.79
Mar
16
297.99 ± 34.99
4
15.60 ± 27.30
12
26.84 ± 14.37
Apr
15
294.77 ± 38.26
4
3.11 ± 1.62
11
220.62 ± 131.18
May
16
268.56 ± 22.13
4
3.82 ± 2.90
10
146.11 ± 71.12
Jun
16
276.32 ± 28.99
4
4.38 ± 3.06
16
227.18 ± 110.6
Jul
12
285.58 ± 24.19
4
1.54 ± 0.89
10
702.90 ± 294.75
Aug
13
237.98 ± 13.82
4
5.23 ± 2.56
12
599.63 ± 233.52
Sep
12
217.87 ± 29.27
4
2.33 ± 1.83
9
304.55 ± 122.01
Oct
18
197.27 ± 16.09
4
3.04 ± 2.61
11
291.84 ± 109.48
Nov
16
192.89 ± 9.05
4
2.97 ± 1.58
10
334.55 ± 118.29
Dec
14
178.48 ± 12.15
4
1.84 ± 1.40
10
242.83 ± 140.44
Overall
234.1 ± 98.8
4.7 ± 9.1
204
209.0 ± 438.4
Dec
15
Year 4
Year 3
Year 2
Year 1
14
13
12
11
10
Freq.
above
annaul
mean
9
8
7
6
5
4
3
2
1
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Month
Figure 4.44:
Frequency of testis volume of individual months above
annual mean of each year (2002 – 2006) in M3
4.9.1.4 M4
The overall mean TV recorded for M4 was 86.3 ± 74.2cm3. TV in this subject
showed an overall pattern similar to M1, M2 and M3, with higher monthly mean
measurements (> 80cm3) from March to August, and lower monthly mean
measurements from September to February (Table 4.38 and Figure 4.45).
However, when data from each study year were reviewed separately, there was
variation between years. For the first two years of the study, measurements
showed a tendency to be higher from September to January (Figure 4.46). A
seasonal pattern, similar to those in M1 – M3, emerged during the last two study
205
Dec
years, with larger TV frequently recorded above the annual mean measurements
(228.1cm3 in 2005, 267.0cm3 in 2006) in March to August (Figure 4..46).
Data from this subject must be interpreted with caution since not all the months
for the last study year were represented.
Table 4.38:
±SD) of TV, T level
Testis volume
Serum T level
n
cm3
n
ng/ml
n
x 106 /ml
Jan
13
59.05 ± 9.19
4
1.16 ± 1.11
4
10.30 ± 1.60
Feb
14
75.81 ± 3.48
4
4.94 ± 4.32
4
23.56 ± 7.70
Mar
15
101.93 ± 20.63
4
10.11± 13.53
4
192.2 ± 109.39
Apr
15
113.57 ± 27.48
4
7.27 ± 9.94
5
355.09 ± 136.92
May
14
121.23 ± 25.88
4
7.28 ± 8.98
6
219.10 ± 109.16
Jun
15
108.39 ± 20.25
4
12.24 ± 21.01
8
252.64 ± 112.88
Jul
14
88.27 ± 24.03
4
7.38 ± 9.11
4
37.38 ± 29.16
Aug
13
81.01 ± 23.03
3
4.74 ± 7.30
4
21.00 ± 11.04
Sep
8
69.27 ± 17.5
3
4.38 ± 4.98
2
0.50 ± 0.45
Oct
14
69.11 ± 4.13
4
1.16 ± 1.27
4
25.72 ± 10.42
Nov
12
65.87 ± 13.94
3
2.73 ± 1.59
4
4.39 ± 3.25
Dec
9
52.87 ± 12.89
4
0.72 ± 0.68
2
54.96 ± 18.71
Overall
86.3 ± 74.2
6.7 ± 9.8
206
117.8 ± 212.4
160
M4
M5
140
120
100
TV 80
3
(cm )
60
40
20
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Month
Figure 4.45: Mean monthly TV, 2003 – 2006 in M4 – M5
Year 4
Year 3
Year 2
Year 1
15
14
13
12
11
Freq.
above
annual
mean
10
9
8
7
6
5
4
3
2
1
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Month
207
Dec
4.9.1.5 M5
The overall mean TV recorded for M5 was 10.4 ± 3.0cm3. TV in this subject did
not show an annual pattern (Table 4.39 and Figures 4.45 and 4.47). These results
were expected, since the testes of this subject were still growing.
± SD) of TV
and T level during 2003 – 2006 in M5
Testis volume
Serum T level
n
cm3
n
ng/ml
Jan
12
10.35 ± 0.85
4
0.13 ± 0.06
Feb
10
9.38 ± 0.63
4
0.16 ± 0.09
Mar
13
10.09 ± 0.77
4
0.28 ± 0.28
Apr
10
10.86 ± 1.00
4
0.44 ± 0.49
May
9
11.75 ± 0.98
4
0.29 ± 0.29
Jun
11
11.84 ± 1.00
4
0.24 ± 0.18
Jul
11
10.72 ± 0.91
4
0.21 ± 0.20
Aug
10
10.23 ± 1.02
4
0.42 ± 0.38
Sep
5
10.45 ± 1.14
4
0.37 ± 0.55
Oct
12
10.56 ± 1.00
4
0.13 ± 0.05
Nov
13
9.81 ± 0.93
4
0.17 ± 0.11
Dec
9
9.73 ± 0.9
4
0.14 ± 0.06
Overall
10.4 ± 3.0
208
0.3 ± 0.3
15
Year 4
Year 3
Year 2
Year 1
14
13
12
11
10
Freq.
above
annual
mean
9
8
7
6
5
4
3
2
1
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Month
Testis volume overall did not show a well-defined seasonal pattern and did reach
measurements above annual means at any time of the year. There was some
tendency for testes to be bigger from March to August, which are the spring and
summer months, and smaller from November to February, which are the late
autumn and winter months in Hong Kong.
4.9.2 Serum testosterone level
Blood was collected for serum testosterone levels (T level) on a monthly basis
and these data were used to determine changes that might show a seasonal
pattern. Blood was also collected monthly in M6, therefore the testosterone
level of this subject was also included for analysis.
209
Dec
4.9.2.1 M1
The overall mean serum T level recorded for M1 was 44.5 ± 18.8ng/ml. T level
showed a seasonal pattern with higher monthly mean levels (> 44ng/ml) between
March and August and lower monthly mean levels between September and
February (Table 4.35 and Figure 4.48). Annual peak testosterone levels and
levels above the annual mean also were most frequently recorded from March to
August (Figure 4.49). Levels recorded in October, December and January were
always below the annual mean levels: 40.0ng/ml in 2003, 45.0ng/ml in 2004,
50.7ng/ml in 2005 and 42.6ng/ml in 2006.
4.9.2.2 M2
The overall mean T level recorded for M2 was 3.7 ± 4.2ng/ml. T level showed a
prominent seasonal pattern, similar to that of TV, in this subject, with higher
monthly mean levels (> 3ng/ml) between March and August, and lower monthly
mean levels between September and February (Table 4.36 and Figure 4.50).
Annual peak testosterone levels and levels above the annual mean were most
frequently recorded from March to August (Figure 4.51).
Levels recorded
between November and February were always below the annual mean levels:
3.0ng/ml in 2003, 3.5ng/ml in 2004 and 5.4ng/ml in 2005.
210
M1
M6
90
80
70
60
T level
(ng/ml)
50
40
30
20
10
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Month
Figure 4.48: Mean monthly T level, 2002 – 2006 in M1 and M6
6
Year 4
Year 3
Year 2
Year 1
5
4
Freq.
above
3
annual
mean
2
1
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Month
Figure 4.49: Frequency of T level of individual months above annual mean
of each year (2002 – 2006) in M1
211
45
M2
M3
M4
40
35
30
T level 25
(ng/ml)
20
15
10
5
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Month
Figure 4.50: Mean monthly serum T level, 2002 – 2006 in M3 and M4
Year 3
Year 2
Year 1
6
5
4
Freq.
above
annual 3
mean
2
1
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Month
212
4.9.2.3 M3
The overall mean T level recorded for M3 was 4.7 ± 9.1ng/ml. T level in this
subject showed a less well defined pattern with higher monthly mean levels (>
3ng/ml) between February and August, except July, and lower monthly mean
levels between September and January, except October (Table 4.37 and Figure
4.50). Annual peak testosterone levels, and levels above the annual mean, were
most frequently recorded from March to August, except July and in November
(Figure 4.52). Year-to-year variations in testosterone did not follow a particular
pattern, as observed in the TV, which may be attributable to growth, or
maturation. Levels recorded between December and January were always below
the annual mean levels: 2.7ng/ml in 2003, 3.0ng/ml in 2004, 4.6ng/ml in 2005
and 8.4ng/ml in 2006.
6
Year 4
Year 3
Year 2
Year 1
5
4
Freq.
above
3
annual
mean
2
1
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Month
of each year (2002 –2006) in M3
213
Dec
4.9.2.4 M4
The overall mean T level recorded for M4 was 6.7 ± 9.8ng/ml. This level is high
due to the dramatic increase shortly before and after onset (see Section 4.6.4)
and therefore cannot be used for comparison for all the study years. T level in
this subject showed a similar pattern to M3, with higher monthly mean levels (>
4ng/ml) between March and September, and lower monthly mean levels between
October and January (Table 4.38 and Figure 4.50). Annual peak T level and
levels above the annual mean were most frequently recorded from February to
July (Figure 4.53). Again there were year-to-year variations, which may be
attributable to growth or maturation. The tendency for levels to be higher from
March to August was more apparent during the last two years of the study.
Levels recorded from October and January, except November, were always
below the annual mean levels: 0.8ng/ml in 2003, 2.2ng/ml in 2004, 14.9ng/ml in
2005 and 8.8ng/ml in 2006.
214
Year 4
Year 3
Year 2
Year 1
6
5
4
Freq.
above
annual 3
mean
2
1
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Month
4.9.2.5 M5
The overall mean T level recorded for M4 was 0.3 ± 0.3ng/ml. Although T level
was very low in this subject, this pattern was similar to all other males, with
higher monthly mean levels (> 0.2ng/ml) between March to September and
lower monthly mean levels between October and February (Table 5.39 and
Figure 4.54). Levels above the annual mean were most frequently recorded from
March to August (Figure 4.55). Levels recorded between October and February
were always below the annual mean levels: 0.2ng/ml in 2003, 0.1ng/ml in 2004,
0.3ng/ml in 2005 and 0.5ng/ml in 2006.
This consistent seasonal tendency was unexpected because of M5’s growth
status and the year-to-year variations found in M3 and M4. Therefore, findings
215
here may be incidental and specific to this individual. Further monitoring is
required.
1.6
1.4
1.2
1.0
T level
(ng/ml) 0.8
0.6
0.4
0.2
0.0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Month
Figure 4.54: Mean monthly T level, 2002 – 2006 in M5
Year 4
Year 3
Year 2
Year 1
6
5
4
Freq.
above
annual 3
mean
2
1
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Month
216
Dec
4.9.2.6 M6
The overall mean T level recorded for M6 was 22.8 ± 16.5ng/ml. T level in this
subject showed a similar seasonal pattern to M1, with higher monthly mean
levels (> 22ng/ml) in March to September, except July, and lower monthly mean
levels between October to February (Table 4.40 and Figure 4.48). The lower
monthly mean levels in June (22.9ng/ml) and July (19.6ng/ml) were likely
affected by low levels recorded in June and July 2006 (3.0ng/ml and 1.6ng/ml
respectively) when M6 was ill. Annual peak T level and levels above the annual
mean also were most frequently recorded from March to September (Figure
4.56). Levels recorded in November and January were always below the annual
mean levels: 34.7ng/ml in 2003, 19.7ng/ml in 2004, 26.8ng/ml in 2005 and
10.9ng/ml in 2006. Data from 2006 should be interpreted with caution as the
subject was ill for the latter half of that year.
Serum testosterone level showed a seasonal pattern that was similar to testis
volume. There was a tendency for testosterone level to be higher from March to
August / September and lower for a slightly longer duration, from September /
October to February, all through the autumn and winter period.
217
± SD) of
T level during 2002 – 2006 in M6
n
Serum T level (ng/ml)
Jan
4
8.78 ± 3.73
Feb
4
17.58 ± 6.66
Mar
4
29.32 ± 14.15
Apr
4
34.84 ± 19.29
May
4
34.64 ± 20.14
Jun
4
22.86 ± 17.82
Jul
4
19.61 ± 19.02
Aug
4
24.26 ± 18.42
Sep
4
23.59 ± 15.46
Oct
4
16.90 ± 10.10
Nov
4
14.38 ± 9.22
Dec
4
14.37 ± 10.80
Overall
22.8 ± 16.5
6
Year 4
Year 3
Year 2
Year 1
5
4
Freq.
above
annual 3
mean
2
1
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Month
218
Dec
4.9.3 Sperm density
Semen was collected on a weekly basis and multiple ejaculates were collected in
a succession in each session.
The overall sperm density (OvD) was used to
determine if changes showed a seasonal pattern in sperm output. OvD of a
session was derived by division of total sperm counts (ToC) by total semen
volume (ToV) of all the ejaculates collected in a series.
4.9.3.1 M1
The overall mean OvD for M1 was 241.0 ± 162.0 x 106/ml. Monthly mean OvD
were higher (> 240 x106/ml) from April to October and lower between
November and February (Table 4.35 and Figure 4.57). Annual peak OvD or
OvD above the annual mean were recorded in every month of the year, but less
often in February (Figure 4.58).
1000
M1
M2
M3
900
800
700
600
OvD
500
6
(x10 /ml)
400
300
200
100
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Month
Figure 4.57: Mean monthly OvD, 2002 – 2006 in M1 – M3
219
Nov
Dec
15
Year 4
Year 3
Year 2
Year 1
14
13
12
11
10
Freq.
above
annual
mean
9
8
7
6
5
4
3
2
1
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Month
Figure 4.58: Frequency of overall session density of individual months
above annual mean of each year (2002 – 2006) in M1
4.9.3.2 M2
were higher (> 110 x106/ml) from April to October, (except in August and
December) and lower between January and February, and again in November
(Table 4.36 and Figure 4.57). Annual peak OvD or OvD above the annual mean
were recorded in every month of the year, though less often in January, February,
March and August (Figure 4.59).
220
Dec
8
Year 3
Year 2
Year 1
7
6
Freq. 5
above
annual 4
mean
3
2
1
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Month
4.9.3.3 M3
were higher (> 209 x 106/ml) from April to December, (excluding May) and
lower between January and March (Table 4.37 and 4.56). Monthly mean OvD
were notably higher, (600 – 700 x 106/ml), in July and August. Annual peak
OvD, or OvD above the annual mean, were recorded in every month of the year,
except March, less often in May (Figure 4.60).
221
Dec
Year 4
Year 3
Year 2
Year 1
8
7
6
5
Freq.
above
annual 4
mean
3
2
1
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Month
above annual mean o f each year (2002 –2006) in M3
4.9.3.4 M4
were higher (> 117 x 106/ml) from March to June and lower between July and
February (Table 4.38 and Figure 4.61). Annual peak OvD or OvD above the
annual mean were recorded from March to June, and in December (Figure 4.62).
OvD above the annual mean have not occurred in the remaining months of the
year. The results of this subject must be interpreted with caution, as the postonset monitoring period was short (< 2 years) and because of the animal’s death
in July, not all the months for the last study year were represented.
Overall, sperm density did not show the seasonal pattern that was found in testis
size and serum testosterone level.
Sperm densities were maintained at higher
222
Dec
levels (above the annual mean) while the other reproductive parameters were at
low levels. Sperm densities above the annual mean were seen at any time of the
year and varied from year to year. There were also some variations between
individuals in the timing of increased sperm densities. These were concentrated
around April and May in M1 and M2, but occurred in August and September in
M3. Density was overall lower in February, but also in March in the younger
subjects, M2 and M3.
550
500
450
400
350
OvD 300
(x106/ml) 250
200
150
100
50
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Month
Figure 4.61: Mean monthly OvD, 2005 – 2006 in M4
223
Oct
Nov
Dec
8
Year 4
Year 3
7
6
Freq. 5
above
annual 4
mean
3
2
1
0
1
2
3
4
5
6
7
8
9
10
11
12
Month
The variations (SD) in monthly mean values of testis volume, testosterone level
and sperm density were nearly always smaller than the SD about the annual
mean values for all the subjects (see Tables 4.35 – 4.40 of this section and
Tables 4.5, 4.7, 4.9, 4.11, and 4.13 in Section 4.3.2 for testis volume, Tables
4.16 – 4.21 in Section 4.5 for testosterone level and Tables 4.25, 1.27 and 4.29 in
Section 4.6 for sperm density). This indicates large fluctuations between years.
These fluctuations may be due to climate differences between seasons and years,
age-related growth / maturational changes, illness or other causes.
Serum testosterone levels showed a slightly more circumscribed seasonal pattern
than testis volume. In M1, M2, M3 and M6, annual changes in testis volume
were of a similar pattern to changes in serum testosterone levels. This was
expected, as association between the two parameters was good (see Section 4.7.1
224
on Comparison between testosterone level and testis size). A seasonal pattern
was not so clear in younger, recently matured males, M4 and M5, in which
annual changes are more likely attributable to growth and maturation.
There was no clear seasonal pattern in sperm density, which fluctuated
erratically from week to week (See Section 4.6 on sperm density). There was
only a tendency to be lower in February, and also in March in the younger
subjects M2 and M3. In M1 – M3, although sperm densities did occasionally
reach above the annual mean, there was also an overall tendency for it to be
lower in March, which marked the beginning of the ‘high’ season for testis
volume and testosterone.
A seasonal pattern was most obvious in M2, where the ‘peak season’ for both
testis size and testosterone levels was from March to August, (spring to mid
summer), and the ‘low season’ was from September to February, (autumn to
winter).
Interpretation of seasonal patterns must be made with caution, as subjects were
few in this study. Data sets for some years were incomplete, or small, due to
difficulties in data collection, or loss of subjects, and a large degree of individual
difference was found.
225
4.10 Semen and ejaculate characterisation
Semen was collected to evaluate individual / species characteristics by assessing
ejaculate volume, pH, and density, and sperm motility and viability. Each subject
presented for semen collection until no more semen was present in spite of effort,
or when micturition occurred (see Section 3.3.7 on Protocol for semen
collection).
Multiple ejaculates were collected from each subject during a
collection session. Significant differences were found between successive
ejaculates from a single collection series in M1 – M4. Characteristics of the
ejaculates were also notably different between subjects. Differences were also
found between subjects of similar age before and after the onset of
spermatogenesis. There were inevitable differences in the number of collection
sessions and number of ejaculates collected for each subject, therefore, data are
interpreted accordingly.
4.10.1 Before the onset of spermatogenesis
At the beginning of the study M3, M4 and M5 were azoospermic, i.e., the onset
of spermatogenesis had not occurred in these subjects.
4.10.1.1 M3
M3 was aged 7y 3m when semen collection began. The semen of this subject
was monitored for 2 collection sessions, over a period of 2 weeks, before the
226
onset of spermatogenesis. The number of ejaculates per session and ejaculate
volume were small (Table 4.41). The ejaculate pH (> 9) was higher than those
recorded in other sexually immature subjects.
Table 4.41: Semen collection and characteristics before the onset of
spermatogenesis (values shown are mean ±SD)
Parameter
M3
M4
M5
Age (y/m)
7/3
4/8 - 6/7
6/5 - 6/9
Collection session characteristics
Number of sessions (ns)
2
9
13
Session duration (min)
4.0 ± 2.8
5.9 ± 2.8
6.8 ± 6.6
2.0 ± 0
3.1 ± 1.6
1.5 ± 1.3
4.0 ± 2.1
5.2 ± 4.2
0.8 ± 1.3
3
27
18
volume (ml)
2.7 ± 0.3
1.7 ± 2.3
0.6 ± 1.0
pH
9.3 ± 0.4
9.0 ± 0.3
8.5 ± 0 (ne=2)
No. of ejaculates / session
Semen characteristics
Overall volume (ml)
Ejaculate characteristics
Number for ejaculates (ne)
4.10.1.2 M4
was monitored for 9 collection sessions, over a period of 23 months, before the
227
onset of spermatogenesis. Weekly semen collection was not always possible
during the pre-onset period as access to this subject was sometimes prevented by
procedures related to animal husbandry and relocation. M4 produced a higher
number of ejaculates per session and larger ejaculate volumes than the older M3
(Table 4.41).
4.10.1.3 M5
was monitored for 13 collection sessions, over a period of 4 months, before the
onset of spermatogenesis. The delay in commencing semen collection and lower
collection frequency during the pre-onset period were due again to access, which
was sometimes prevented by procedures related to animal husbandry and
relocation. M5 produced the lowest number of ejaculates per session, and lower
ejaculate volume and pH than older subjects M3 and M4 (Table 4.41).
M3 – M5 cooperated well for semen collection at young ages, youngest 4y 8m,
prior to onset of sexual maturity, and successfully delivered semen in multiple
ejaculations. The number of ejaculates that were collected in a session was
small and ejaculates were of low volume. Mean ejaculate pH > 9 of pre-onset
semen was higher than semen containing spermatozoa.
228
4.10.2 After onset of spermatogenesis
Spermatogenesis was established in M1 and M2 when the study began. The
onset of spermatogenesis took place in M1 in August 1991 at an estimated age of
7 – 8y (Brook, 1997).
The onset of spermatogenesis occurred in M2 in
September 2001 at age of 7y 4m (unpublished data).
As the study progressed, the first release of spermatozoa in M3, M4 and M5 was
identified; the month and exact age of the first release of spermatozoa (i.e., onset
of spermatogenesis) in the ejaculates of these subjects are shown in Table 4.42.
Table 4.42: Age at onset of spermatogenesis in M1 – M5
Subject
Month of Onset
Age
M1
August 1991
7 – 8+ y
M2
September 2001
7y 4m
M3
October 2002
7y 3m
M4
April 2005
6y 7m
M5
February 2006
6y 9m
4.10.2.1 Session semen volume, sperm count and sperm density
Total session volume (ToV) was the sum of the volume of all ejaculates
collected in a single session. Total count (ToC) was the sum of the count of all
ejaculates in a collection session. Overall sperm density (OvD) of a session was
derived by division of total ToC by ToV. Two mean values were obtained for
ToV, ToC and OvD, one derived by inclusion of the first ejaculate in the
229
collection series (E1) and the other derived by exclusion of this ejaculate. A
paired-samples ‘T’ test was applied to determine any significant differences
between the 2 paired values.
Significant differences (P < 0.05) were found in ToV, ToC and OvD between
values derived by inclusion and exclusion of E1 (Table 4.43) in 4 out of 5
subjects. ToV and ToC were higher when E1 was included, whereas OvD was
lower. Although exclusion of E1 yielded higher OvD, the OvD values presented
in Section 4.6 were derived from inclusion of E1. Inclusion of E1 ensured that all
semen and spermatozoa produced by an individual were accounted for.
Although E1 is generally of much lower density, in a single collection session an
individual subject may produce only 1 ejaculate, therefore exclusion of E1will
not provide accurate data with which to fully evaluate testicular function.
4.10.2.1.1 M1
M1 was aged over 19y when semen collection began for this study, so
spermatogenesis had been ongoing for more than 12 years. In general, M1
produced the highest number of ejaculates per session, and the highest ToV, and
ToC (Table 4.43). OvD derived by exclusion of E1 was slightly lower than that
of the younger subject M3.
4.10.2.1.2 M2
M2 was 8y 5m when semen collection began for this study, so spermatogenesis
had been ongoing in this subject for more than 1 year. M2 produced a higher
number of ejaculates per session, and higher ToV and ToC than younger subjects
230
(Table 4.43), but lower than M1. Both OvD values (derived by inclusion and
exclusion of E1) were lower than those of M3 and M4. However, OvD derived
by exclusion of E1 was higher than that of M4, but still lower than M3.
Table 4.43: Semen collection and characteristics after onset of
spermatogenesis (values shown are mean ±SD)
Parameter
Post-onset duration
(y)
M1
M2
M3
M4
st
M5
2nd - 4th
1st - 4th
1¼
182
130
184
55
39
19.0
±13.0
9.8
±5.8
12.6
±10.0
6.1
±4.2
9.7
±7.6
3.4
±2.4
10.3
±5.6
5.4
±2.2
7.3
±3.3
2.5
±1.4
26.0
±17.3
3053.9
±5650.2
117.8
±212.4
1.5
±2.5
12th -15th
1st
Collection session characteristics
Number of sessions
Session duration
(min)
No. of ejaculates /
session
Overall semen characteristics – including all ejaculates
Total volume (ToV)
(ml)
Total count (ToC)
(x106)
Overall density (OvD)
(x106/ml)
92.7
±42.3
22654.5
±17480.3
241.0
±162.0
50.1
±30.5
5577.0
±8248.1
112.0
±186.3
18.9
±13.2
5007.3
±12743.0
209.0
±438.4
<0.1
<0.1
Overall semen characteristics - excluding 1st ejaculate (E1)
Total volume (ToV)
(ml)
Total count (ToC)
(x106)
Overall density (OvD)
(x106/ml)
49.0
±35.1
17087.6
±13610.5
371.9
±267.4
17.1
±18.4
3982.8
±6071.6
217.1
±267.7
4.9
±6.4
2125.4
±5464.8
375.6
±769.1
9.1
±13.5
1284.5
±2337.5
127.0
±216.2
0.6
±2.1
<0.1
<0.1
Bold typeface within the same column denotes mean values are significantly different (P < 0.05)
for a given parameter between values derived by inclusion and exclusion of E1
231
4.10.2.1.3 M3
M3 produced a higher OvD than M2, M4 and M5 (Table 4.43). Ejaculate
number per session and ToV were lower than those of the younger M4, but ToC
was higher.
4.10.2.1.4 M4
M4 generally produced a lower number of ejaculates per session, and lower ToV,
ToC and OvD than older males (Table 4.43). OvD derived by inclusion of E1
was slightly higher than that of M2. The number of ejaculates per session and
ToV were higher than in M3.
Similar to M1 – M3, significant differences (P < 0.05) were found in ToV and
ToC between values derived by inclusion and exclusion of E1 in M4 (Table 4.43).
However, no significant difference (P < 0.05) was found between OvD derived
by inclusion and exclusion of E1 and the values were comparable. This finding
indicates that E1 may be of comparable density to those of other ejaculates in a
collection series in younger / more recently mature males.
4.10.2.1.5 M5
M5 produced the lowest number of ejaculates per session, and the lowest ToV,
ToC and OvD (Table 4.43). No significant differences (P < 0.05) were found in
the all parameters between values derived by inclusion and exclusion of E1.
232
Overall, semen characteristics improved (higher in ToV, ToC and OvD) with age,
or the number of years after onset of spermatogenesis. This is to be expected as
testes of younger subjects (M2 – M5) have yet to reach adult size and functional
capacity.
The OvD values presented here indicate semen and sperm output
fluctuated largely during the study period, further supporting the finding that
sperm density fluctuated erratically from week to week (Section 4.6). Although
results to date indicate the 1st ejaculate was mostly lower in density, this was not
always the case; E1 can be of comparable density to the rest of the ejaculates in a
collection series and therefore should not be discarded at the first instance.
4.10.2.2 Ejaculate characteristics
Ejaculates were collected and analysed individually. A total of 2,917 ejaculates
were collected during the study and the number of ejaculates collected from each
subject ranged from 67 – 1,336 (Table 4.44). For statistical analysis, data from
the 1st (E1) and 2nd ejaculates (E2) were treated separately and data for the 3rd and
remaining ejaculates were pooled together as one group (E3-n). ANOVA test was
applied to determine any significant differences between E1, E2 and E3-n.
The quality of the ejaculates of M1 to M4 was generally good; total sperm
motility (TM) and viability (VIA) were mostly > 80% and rate of progressive
motility (RPM) > 3 (Table 4.44). Mean value for pH of all the ejaculate groups
was > 8.1, which was lower than the mean pH of ejaculates produced before the
onset of spermatogenesis (Table 4.41).
233
4.10.2.2.1 M1
M1 produced the highest mean ejaculate volume (EjV) (44.8 ± 18.0ml),
ejaculate density (EjD) (579.5 ± 507.7 x106/ml) and ejaculate count (EjC)
(6900.0 ± 15478.2x106) (Table 4.44). Other ejaculate parameters, (mean TM,
PM, VIA and EjD) of E1 were generally poorer compared to the ejaculates of the
younger subjects M2 – M4. The PM of all the ejaculates was notably lower than
in M2 – M4.
Significant differences (P < 0.05) in EjV, PM, VIA, EjD and EjC were found
between E1, E2 and E3-n (Table 4.44). The mean EjV of E1 was higher, but it was
lower in EjD than E2 and E3-n. Due to EjV being the highest in E1, EjC was also
highest in this ejaculate. All sperm characteristics (TM, PM, RPM and VIA) of
E1 were poorer than those of E2 and E3-n. Mean EjV, EjD, EjC, PM and VIA of
E2 were significantly different (P < 0.05) from E3-n. Ejaculate quality of E2 was
better (higher EjD, EjC, motility and VIA) than E1 and E3-n.
4.10.2.2.2 M2
M2 produced higher EjV than the younger subjects M3 – M5 (Table 4.44).
Despite the high EjV in E1, EjD and EjC were low (73.1 ± 239.6 x106/ml and
1,618.2 ± 3,655.6 x106). EjD and EjC of E2 and E3-n were higher than those of
M4, but lower than M3. Sperm characteristics were generally similar to M3 and
M4, though mean TM and VIA were slightly higher.
234
Table 4.44: Characteristics of successive ejaculates
(values shown are mean ±SD)
E1
E2
E3-n
M1
ne
143 – 181
170 – 178
929 – 977
EjV (ml)
44.8 ± 18.0
11.5 ± 8.0
7.0 ± 4.7
e
pH
8.5 ± 0.4 (ne = 294)
8.5 ± 0.6 (n = 111)
8.4 ± 0.2
TM (%)
89.3 ± 12.8
87.8 ± 12.0
82.6 ± 18.2
PM (%)
33.4 ± 35.9
65.1 ± 36.5
55.3 ± 37.3
a
RPM
4.1 ± 1.3
3.9 ± 1.4
2.8 ± 1.8
VIA (%)
88.8 ± 8.3
92.3 ± 6.1
90.6 ± 8.3
EjD (x106/ml)
151.0 ± 208.9
579.5 ± 507.7
356.6 ± 360.7
EjC (x106)
6900.0 ± 15478.2
5546.2 ± 6879.4
2294.2 ± 2491.4
M2
ne
100 – 129
103 – 115
348 – 365
EjV (ml)
4.7 ± 3.8
4.1 ± 2.7
36.6 ± 21.3
pH
8.4 ± 0.5 (ne = 144)
8.4 ± 0.2
8.5 ± 0.4 (ne = 82)
TM (%)
87.1 ± 14.6
86.4 ± 17.0
82.9 ± 25.5
PM (%)
63.7 ± 37.5
71.2 ± 33.5
41.4 ± 36.8
a
RPM
3.7 ± 1.7
3.9 ± 1.5
3.1 ± 1.9
VIA (%)
90.2 ± 9.8
89.7 ± 12.1
84.2 ± 13.4
EjD (x106/ml)
73.1 ± 239.6
344.9 ± 419.3
232.4 ± 264.4
EjC (x106)
1618.2 ± 3655.6
1722.3 ± 2857.1
965.9 ± 1494.7
M3
ne
80 – 183
96 – 147
157 – 230
EjV (ml)
2.8 ± 3.5
2.0 ± 1.6
15.1 ± 10.9
e
pH
8.4 ± 0.4
8.5 ± 0.6 (n = 92)
8.5 ± 0.5 (ne = 90)
TM (%)
86.9 ± 16.9
80.5 ± 28.6
84.2 ± 22.7
PM (%)
63.0 ± 40.2
63.1 ± 38.6
67.0 ± 37.3
a
RPM
3.5 ± 1.6
3.4 ± 1.9
3.6 ± 1.8
VIA (%)
84.4 ± 17.2
86.4 ± 17.7
86.5 ± 16.7
EjD (x106/ml)
499.0 ± 953.7
426.0 ± 765.9
167.6 ± 380.7
6
EjC (x10 )
1822.2 ± 3854.5
780.2 ± 1954.5
4167.3 ± 9057.0
M4
ne
34 – 55
38 – 51
106 – 133
EjV (ml)
3.5 ± 7.5
2.0 ± 1.5
21.8 ± 17.9
pH
8.3 ± 0.1
8.2 ± 0.3 (ne = 21)
8.1 ± 0.3 (ne = 36)
TM (%)
85.5 ± 9.7
81.2 ± 17.0
79.6 ± 19.3
PM (%)
58.6 ± 37.3
63.6 ± 38.5
63.1 ± 35.7
a
RFM
3.0 ± 1.8
3.2 ± 1.8
3.3 ± 1.7
VIA (%)
82.7 ± 14.7
82.0 ± 19.0
78.8 ± 15.8
EjD (x106/ml)
119.2 ± 239.2
114.5 ± 196.9
219.6 ± 374.6
EjC (x106)
781.5 ± 1546.9
254.7 ± 545.0
1822.9 ± 3390.9
M5
ne
37
24
6
EjV (ml)
0.9 ± 1.2
0.5 ± 1.4
0.8 ± 1.6
pH
8.4 ± 0.4 (ne = 15)
8.6 ± 0.8 (ne = 7)
6
EjD (x10 /ml)
< 0.03
< 0.03
0
EjC (x106)
< 0.03
< 0.03
0
a
Rate ranked on a subjective scale of 0 – 5; 0 = no progressive movement and 5 = most rapid
progressive movement
Bold typeface with in the same row denotes mean value are significant different (P < 0.05) for a
given parameter among E1, E2 and E3-n within a subject.
235
E1 was significantly different (P < 0.05) from E2 and E3-n in all the ejaculate
parameters, except pH and TM (Table 4.44). E1, E2 and E3-n were significantly
differently (P < 0.05) from each other in EjD. Ejaculate quality of E1 was poorer
overall (lower EjD, PM, RPM and VIA) than E2 and E3-n. E2 and E3-n were
similar in sperm characteristics (TM, PM, RPM and VIA).
4.10.2.2.3 M3
M3 produced lower EjV than older subject M2 and younger subject M4 (Table
4.44).
However, the notably higher EjD resulted in higher EjC. Sperm
characteristics were generally better than those of M4.
E1 was significantly different (P < 0.05) from E2 and E3-n in EjV, EjD and EjC
(Table 4.44). No significant difference was found between E1, E2 and E3-n in pH
or sperm characteristics. Also, no significant difference was found between E2
and E3-n in all the ejaculate characteristics.
4.10.2.2.4 M4
M4 mostly produced lower mean EjV, EjD and EjC than M1 – M3 (Table 4.44).
Mean PM of E2 was comparable to those of older subjects. Ejaculate and sperm
characteristics of M4 were overall slightly poorer than M1 – M3. The smaller
number of ejaculates collected in this subject may affect these results.
236
E1 was significantly different (P < 0.05) from E2 and E3-n in EjV and EjC (Table
4.44). E2 was significantly different (P < 0.05) from E1 and E3-n in EjD. No
significant difference was found between E1, E2 and E3-n in ejaculate pH and
sperm characteristics.
4.10.2.2.5 M5
M5 produced the lowest mean EjV (Table 4.44). The number of spermatozoa
produced by this subject was extremely low (< 0.03 x 106), and motility and VIA
analyses were not possible. Spermatozoa were found only after centrifugation
and they were not motile. It was not certain if sperm motility was affected by
centrifugation.
Again, ejaculate characteristics overall improved (higher in density, motility and
viability) with the number of years after onset of spermatogenesis. However,
ejaculate quality of the youngest subject M4 was still considered on the whole
good and an individual ejaculate can be of very high density (> 600 x106 / ml)
and motility (> 90%). Such high quality ejaculates warrant consideration of a
young, recently matured dolphin to be selected for breeding, either naturally or
via semen collection for AI.
Significant differences in sperm characteristics (motility and viability) between
the 1st, 2nd and remaining successive ejaculates were not found in the younger
subjects, M3 and M4. This may be due to age or inherent differences between
237
individuals. Further investigation on more subjects is required to confirm such
finding.
The significant differences (P < 0.05) in volume and sperm density between the
1st, 2nd and subsequent ejaculates in the collection series highlight the importance
of collection of multiple ejaculates in a session in order to gain a true picture of
sperm production in an individual. This will also guide appropriate collection of
samples for artificial insemination or cryopreservation. In this study, it was
found that the 1st ejaculate did not accurately reflect spermatogenesis, as it was
significantly lower in density regardless of age, or years after onset. The large
volume and low relative density of the 1st ejaculate renders it undesirable for use
in artificial insemination, as further processing is required to concentrate larger
volumes to acquire a better insemination dose in a smaller volume, and to reduce
space required for cryo-storage, as this is expensive. These results show that
several, serial ejaculates should be collected in a single session to ensure better
sample size and density for cryopreservation.
All results of ejaculate volume, sperm density and count of E1 and E2 of M1 –
M5 are included in Appendix 9.
4.10.3 Correlation between ejaculate parameters
Ejaculate data of subjects M1 – M4 were pooled together and Pearson productmoment correlation coefficient, r, was obtained to examine the strength of
associations between ejaculate parameters, EjV, EjD, TM, VIA, PM and RPM.
238
Analysis for ejaculate EjC was not conducted since it was derived from volume
and density and so not an independent variable.
Table 4.45: Inter-correlations and correlation coefficients, r, of ejaculate
parameters
ne
≈ 2250
VOL
DEN
TM
EjV
EjD
TM
VIA
PM
RPM
1
-0.16**
0.01
-0.03
-0.19**
-0.09**
1
0.09**
0.31**
0.36**
0.21**
1
0.53**
0.37**
0.45**
1
0.42**
0.46**
1
0.53**
VIA
PM
1
RFM
**Correlation is significant P < 0.01
ne ≈ approximate number of paired ejaculate data
Correlations between ejaculate parameters varied in strength, with r = 1 – 0.5
(Table 4.45) which suggests little or no relationship, to a fair or moderate
relationship. Statistical significance (P < 0.01) was found where | r | (0.09).
EjV was inversely associated with EjD and the progressive motility parameters
(PM and RPM), i.e., the greater the EjV, the lower the EjD and the lower the
sperm motility, which was expected. However, the strength of such associations
was not marked, as ejaculates may occasionally be high in both volume and
density.
The associations of density with TM, PM and RPM were positive but not strong.
Sperm motility of an ejaculate increased with density to a certain point. When
239
density reached vey high levels (> 1500 x 106 /ml) spermatozoa became so
densely packed that motility was prevented, despite a 1:1 dilution (ejaculate
sample : diluent) on evaluation. Motility was affected at all levels ranging from
sluggish progressive movement (RPM < 1), to no progressive movement (RPM
= 0 and PM = 0), to a complete lack of tail movement (TM = 0).
EjD correlated more strongly with VIA (r = 0.3) than with TM (r = 0.09). In
continuation from the explanation given above, spermatozoa that were immotile
in ejaculates that reached extremely high densities, could be viable, thus, they
were accounted for in VIA, but not in TM.
The correlation between TM and VIA was the strongest, r = 0.53, which, again,
was expected. VIA accounts for spermatozoa that are motile in TM evaluation.
The association was not 100% because VIA also accounts for viable
spermatozoa that may not be motile in TM evaluation.
Associations among the motility parameters, i.e., TM, PM and RPM, were
moderate r = 0.4 – 0.5, again expected, since microscopic evaluations of these
parameters were not completely independent of each other.
Stronger
associations, r = > 0.5 (P < 0.01), were found between these parameters in the
younger subjects, M2 – M4. Lower associations in M1 (r = 0.2 – 0.4) may be
explained by the tendency of this subject to have ejaculates of higher density
(Section 4.9.2.2, Table 4.44); densely packed spermatozoa may exhibit tail
motility, but not in a progressive manner. Inherent individual differences in
semen composition and spermatozoa characteristics should also be taken into
consideration.
240
The significant positive correlations (P < 0.01) between EjV, EjD and
spermatozoa
characteristics
confirm
the
significant
differences
found,
particularly between E1 and E2 in M1 and M2 (Table 4.44). The importance of
successive ejaculate collection is, thus, further reinforced.
Inter-correlations derived by Pearson product-moment analysis for individual
subjects are included in Appendix 10.
4.11 Cryopreservation
Ejaculates from M1, M2, M3 and M4 were cryopreserved and thawed after a
period of approximately 2 weeks (14.8 ± 0.5 day) following the procedures
described in Methodology (Section 3.3.9).
Criteria used to decide which
samples were selected for cryopreservation were i. ejaculate raw sperm density,
ii. motility and iii. viability. In a series of ejaculates collected from one subject,
ejaculates nearest to 400 x 106/ml were selected, so that sperm density
approaches 100 x 106/ml after 2 stages of 1:1 dilution, as detailed in the protocol
(Sections 3.3.9.1 and 3.3.9.3). This follows the standardised pre-freezing density
used in Robeck (2004a).
4.11.1 Characteristics of ejaculates before cryopreservation
A total of fifty-two ejaculates were cryopreserved. These samples were collected
between March 2005 and June 2006, every month during this period was
represented, except September and December 2005.
241
Details of cryopreserved ejaculates are shown in Table 4.46. The ejaculate in the
collection series most frequently selected for cryopreservation was the second
ejaculate (E2). Only two ejaculates from M2 were cryopreserved prior to death.
Overall, raw ejaculates had good total motility (TM) (> 87%), progressive
motility (PM) (> 86%), rate of progressive motility (RPM) (> 4) and viability
(VIA) (>91%).
Kruskal-Wallis (non-parametric) test was applied, no significant difference (P >
0.05) was found between subjects in raw ejaculate PM, RPM and VIA, however
there were significant differences (P < 0.05) in ejaculate TM and DEN. The
greatest difference in mean TM was between M1 and M3, the mean TM of M1
being the lowest of all the subjects (Table 4.46). M3 had the greatest mean DEN.
4.11.2 Characteristics of ejaculates after cryopreservation and thawing
Overall, ejaculate TM and VIA decreased after cryopreservation (Table 4.47). At
30-minutes post-thaw, TM was > 42%, PM was > 60%, RPM was > 3 and VIA
> 55%. Paired-samples ‘T’ test was applied and significant difference (P < 0.05)
was found between raw / pre-freezing and post-thaw ejaculates in all the
parameters, indicating they were affected by cryopreservation and / or thawing.
242
Table 4.46: Characteristics of raw ejaculates
M1
M2
M3
M4
14 – 15y
2.5 – 3.5y
2.5 –
3.5y
2 – 14m
ne
19
2
17
14
Ejac. no. in series
*E2
*E2, *E6
*E2
*E2
692.0
± 248.1
4384.1
± 2601.2
87.9
± 10.6
91.0
± 4.3
4.6
± 0.3
91.5
± 6.5
178.6
± 98.8
377.2
± 159.6
2488.7
± 2024.2
92.0
±0
87.7
± 8.5
4.2
± 0.2
94.3
± 1.4
79.6
± 41.8
988.5
± 791.6
6629.1
± 13438
93.3
± 2.1
91.4
± 5.2
4.6
± 0.4
94.7
± 3.2
277.3
± 238.1
408.5
± 350.6
1377.6
± 1270.7
89.6
± 3.3
86.5
± 9.2
4.4
± 0.4
91.2
± 5.5
94.1
± 94.6
Post-onset duration
Density (DEN)
(x 106/ml)
Count (CNT)
(x106)
Total motility (TM)
(%)
Progressive motility
(PM) (%)
b
Rate of progressive
motility (RPM)
Viability (VIA)
(%)
Pre-freezing DEN
(x 106/ml)
ne = number of ejaculates cryopreserved
* = mode
b
Rate ranked on a subjective scale of 0-5, where 0 denotes no progressive movement and 5
denotes most rapid progressive movement
For each subject, post-thaw parameters were expressed as % raw, values to
correct for variations in raw ejaculate characteristics (Table 4.47). Figures 4.63
and 4.64 show the % raw TM and % raw PM for individual ejaculates of all the
subjects 30 minutes after thawing. Mean % raw for TM and PM were generally
> 50% and > 60%, respectively. When the Kruskal-Wallis test was applied,
significant difference (P < 0.05) was found between subjects in post-thaw scores
and % raw of all the ejaculate parameters, except VIA. The highest mean % raw
for all the post-thaw parameters were those of M3 (62 – 85%), followed by M2
243
and M4 with similar results (Table 4.47). Despite having the most samples, the
% raw in M1 was the lowest in all parameters except VIA.
Table 4.47: Overall ejaculate scores after cryopreservation and at 30
minutes after thawing and percentages over raw scores
n
e
Total
motility
(TM)
%
Progressive
motility
(PM)
%
% raw
% raw
a
Rate of
progressive
motility
(RPM)
Viability
(VIA)
rate
% raw
%
% raw
M1
M2
M3
M4
19
2
17
14
42.5
± 13.8
47.5
± 13.5
61.0
± 12.2
66.9
± 12.8
3.1
± 0.7
68.4
± 16.6
55.8
± 8.3
60.8
± 7.2
48.6
± 8.1
52.8
± 8.8
65.9
± 11.9
76.2
± 21.0
3.4
± 0.8
81.7
± 23.0
57.3
± 9.8
60.6
± 9.5
58.4
± 9.6
62.7
± 10.4
78.2
± 10.9
85.8
± 13.2
3.9
± 0.6
85.7
± 15.0
61.6
± 8.7
65.0
± 8.6
47.3
± 9.3
52.9
± 10.6
65.5
± 13.8
76.8
± 20.7
3.2
± 1.0
72.1
± 25.0
56.6
± 7.4
62.0
± 6.6
ne = number of ejaculates cryopreserved and thawed
a
Rate ranked on a subjective scale of 0-5, where 0 denotes no progressive movement and 5
denotes most rapid progressive movement
244
100
M1
M2
M3
M4
90
80
70
60
% raw
TM 50
40
30
20
10
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Sample
Figure 4.63: % raw TM 30 minutes after thawing
100
M1
M2
M3
M4
90
80
70
% raw 60
PM
50
40
30
20
10
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Sample
Figure 4.64: % raw PM 30 minutes after thawing
Results indicate that semen of younger dolphins (e.g., M3 and M4), is suitable
for cryopreservation. Further, provided density is good (≥ 400 x106/ml), semen
245
19
can be successfully cryopreserved as early as two months after the onset of
spermatogenesis. However, it must be noted that the number of subjects and
ejaculate samples in this study were small. Also, factors such as variations in
ejaculate sperm density and slight differences in sample processing conditions
must be taken into account when interpreting data.
All results of characteristics of raw ejaculates and ejaculates 30 minutes after
thawing for M1 – M4 are included in Appendix 11.
246
Chapter 5
DISCUSSION
The aim of this study was to determine the age of sexual maturity in Tursiops
aduncus born in captivity, and therefore of known age. The study defined the
onset of sexual maturity by the onset of spermatogenesis, which is indicated by
the release of spermatozoa in the semen. Ultrasonography was used to assess the
size and appearance of the testes on a weekly basis and compared these with
monthly serum testosterone levels.
Semen was collected weekly, under
voluntary behaviour, for detection of spermatozoa using bright-field microscopy.
The age, measurements of the reproductive parameters and body size during
onset were then evaluated.
Weekly ultrasonographic examination of the testes, monthly serum testosterone
evaluation and weekly semen collection continued after the onset of
spermatogenesis to further monitor post-onset progress and investigate possible
seasonal changes in testicular activities in recently matured subjects. Two more
males, M1 and M2, which had been sexually mature for 2 – 11 years at the start
of the study, were also investigated.
In order to fully and accurately evaluate testicular function and characterise the
semen of T. aduncus, ejaculates from each subject in this study were collected in
succession to constitute a series and each was also evaluated individually.
247
Information on the semen characteristics of Tursiops is scarce, with few data
from adult Tursiops truncatus (Atlantic bottlenose dolphin) being reported in
any detail (Schroeder and Keller, 1989; Robeck and O'Brien, 2004a) . To date,
there is no information about the ejaculate characteristics of recently matured
dolphins, or of the differences between successive ejaculates. This information is
important to properly manage captive breeding and / or semen collection for
cryopreservation.
Ejaculates collected from as early as the first month after onset of
spermatogenesis were cryopreserved to investigate the ability of spermatozoa of
recently mature individuals to withstand freezing.
Post-thaw spermatozoa
characteristics were compared to the cryopreserved ejaculates collected from
older subjects that had been sexually mature for some years.
5.1 Ultrasonographic examination of testes
All the subjects of the present study cooperated well with the ultrasonographic
procedure whilst remaining in the water at the poolside.
The necessary
behaviour had been established in the older subjects (M1, M2 and M3) for some
years prior to commencement of the present study, and no problems were
experienced. At the beginning of the study, the behaviour was not well
established in the younger subjects M4 and M5, and there was some delay in
commencing ultrasonographic data collection in these dolphins. The delay in
M5 was longer because access to this subject was restricted as he was initially
248
placed in a female / nursery group and then, during weaning, other husbandry
related procedures to monitor the health of this individual took precedence.
Overall, the animals responded well to the ultrasonographic sessions, unlike
previous reports (Stone, 1990; Brook, 1997). Most of the time they were keen
and prompt to present when given the cue, and the position held was stable for as
long as required. With increasing experience of the author, an examination
session for each animal required only 5 minutes. With current training standards,
removal of an animal from the water for routine ultrasonographic examination of
the testes is no longer justified. However, if there is a clinical need
ultrasonographic examination may be conducted on land under manual restraint,
as detailed in Stone (1990) and Brook (1997).
A difficulty encountered while conducting B-mode ultrasonography outdoors
was visualisation of the image on the monitor screen.
When sunlight was
intense it was very difficult to discern subtle differences in gray-scale.
Positioning of equipment, shading and in-built functions of image / display
optimisations were of some help. Generally, more patience and longer scanning
time can overcome such difficulties.
Head mounted video display glasses may
improve image visualisation but would require some time for acclimatisation by
the operator, who also needs to be able to see the animal while scanning.
5.1.1 Ultrasonographic appearance of the testes
The testes were readily visualised and the ultrasonographic appearances during
different reproductive stages were consistent with previous reports (Brook, 1997;
249
Brook et al., 2000). At the beginning of the study, the testes of M1 (age 19+y)
and M2 (age 8y 5m), were of mature status, or Grade I. They were ‘cigarshaped’, with expansion towards the caudal aspect. The echopattern of the
parenchyma was speckled and homogenous, of mid to high level intensity, with
a prominent lobular echo-texture. Overall echogenicity was isoechoic to the
adjacent hypaxialis lumborum muscle. The testes of M4 (age 4y 5m) and M5
(age 4y 1m) were of the immature status, or Grade III. They were consistent in
shape, of low level echogenicity and lacked the lobular appearance.
The
appearance of the testes of M3 (age 7y 3m), was intermediate and between
Grades II and I. There was slight expansion towards the caudal aspect, the
parenchyma was speckled and homogenous, but slightly hypoechoic when
compared to the hypaxialis lumborum muscle. To the best of my knowledge,
there are no further reports of the ultrasonographic appearance of the testes of
Tursiops.
5.1.1.1 Testis appearance during sexual maturation
The onset of spermatogenesis in M3 took place two weeks after the present study
began. At the beginning of the study, the testes of M3, (aged 7y 3m), were in
transition, between grades II and I. Although size measurements of 17 – 18cm
and 160 – 170cm3 were comparable to those of Grade I testes described (Brook,
1997; Brook et al., 2000), the parenchyma was comparatively hypoechoic. As
the study progressed, parenchymal echogenicity and lobulation increased and the
testes reached the Grade I status soon after onset, with maximum measurements
of 24cm and 554.4cm3 at the age of 10y 9m.
250
The onset of spermatogenesis occurred in M4 during the study period and the
changes in testis appearance and size that took place were consistent with one
previously reported system of grading dolphin testes (Brook et al., 2000). At the
beginning, the testes of this subject were Grade III, the parenchyma was
markedly hypoechoic and measurements were 7 – 8cm in length and 10 – 16cm3
in volume. Testes were Grade II by March 2004, remaining that way until
January 2005 (age 5y 6m to 7y 4m). Parenchymal echogenicity and testis size
increased but they were still less echogenic than the hypaxialis lumborum. Size
increased to 13.5cm in length and 55cm3 in volume. During February 2005,
lobulation of the parenchyma began to be discernable. Means of both the left and
right testis measurements were 14.9cm and 103.1cm3. In April 2005 (age 6y
7m), the first release of spermatozoa took place in M4. By this time, the testes
had reached Grade I, ie. parenchymal echogenicity had markedly increased,
echo-texture was very ‘speckled’, and mean measurements were 17.2cm and
178.0cm3. Lobulation of the parenchyma continued to grow more marked and
by the end of the study, maximum measurements recorded were 20cm and
267cm3, at the age of 7y 7m.
The onset of spermatogenesis also took place in M5 during the study, but the
appearance (and size) of the testes did not conform to the changes that took place
in M2 and M3, nor those previously reported (Brook, 1997; Brook et al., 2000).
The first release of spermatozoa, at age 6y 9m, occurred in the absence of
notable changes in testis appearance.
By the end of the study period,
echogenicity of the testicular parenchyma had increased slightly, but was still
hypoechoic compared to the hypaxialis lumborum and the testes remained at
Grade III, at age 7y 8m, in both appearance and size.
251
The changes in the ultrasonographic appearance in the testes of M4 may reflect
anatomical observations of maturing testes. Brook (1997) and Brook et al. (2000)
suggested increased echogenicity in maturing testes was likely due to dilation of
seminiferous tubules, which results in a larger number of interfaces for reflection
of ultrasound, plus the presence of spermatozoa, which also increases testicular
echogenicity. Increase in seminiferous tubule diameter has been reported in the
testes of maturing Stenella attenuata (spotted dolphin) (Perrin et al., 1976)
Stenella coeruleoalba (striped dolphin) (Miyazaki, 1977; Miyazaki, 1984) and
Delphinus delphis (common dolphin) (Hui, 1979). Mature tubules were found to
have lumens and contain spermatozoa. Studies in terrestrial mammals, (primates,
bulls, rams, rats and guinea pigs), found that testicular growth is a result of
increasing seminiferous tubule diameter. Increase in tubule diameter was partly
associated with proliferation and maturation of Sertoli cells to attain adult and
cell population, so that spermatogenesis can take place in a spatially and
temporally co-ordinated and efficient manner (Curtis and Amann, 1981; Amann,
1983; Bardin et al., 1988; De Krester and Kerr, 1988; De Krester, 2000).
Comparison to the hypaxialis lumborum to assess the overall parenchymal
echogenicity is simple and useful in the evaluation of reproductive status in
dolphin testes, but it is subjective. Computer-assisted ultrasonographic image
analysis, such as pixel value or intensity, used in beef bull studies (Chandolia et
al., 1997; Arteaga et al., 2005), yield quantitative data that may improve the
accuracy in assessing changes in testicular tissue, particularly during transitions
between grades.
252
In addition to changes in echopattern, the study found the emergence of the
epididymis in the ultrasonographic image to be a useful marker in recognising
sexual maturity.
Visualisation of the epididymis was not possible at the
beginning in M4 and M5. Visualisation of the epididymis of M4’s testes began
in December 2004, and by January 2005 it was readily visualised. However, the
difference between the cranial and caudal aspects was not discernable before
onset of spermatogenesis.
At 8 months post-onset, the echopatterns of the
cranial and caudal aspect of the epididymis began to differ in both M3 and M4.
By 9 months post-onset, the caudal aspect was appreciably echolucent compared
to the cranial aspect. Visualisation of the epididymis of M5 was still not possible
by the end of the study. There is little information available about changes or
appearance of the epididymis in dolphins (Meek, 1918; Matthews, 1950;
Harrison, 1969a; Brook and Kinoshita, 2005) and further investigation is
required to confirm the usefulness of ultrasonographic observations to indicate
an animal’s proximity to sexual maturity.
5.1.2 Testis size
The testes of sexually mature T. aduncus were large. M1 was a proven sire and
at the beginning of the study was over 19 years old. He had the largest testes, at
19.8cm in length and 510.0cm3 in volume. The testes of this subject were fully
mature and growth was not expected or found. The mean testis length was 19.8
± 0.7cm and mean volume was 466.4 ± 53.7cm3. Annual fluctuations were small
compared to younger males. Ross (1977) reported testis lengths of 24 – 28cm in
253
sexually mature T. aduncus, from South African waters. These measurements
are larger than those found in this study. This is likely to be due to differences in
body size between different populations. The body length of mature animals in
the South African population was reported to be > 240cm. The average body
length of the mature animals in this study (from Indonesia and Taiwan) was <
220cm.
Despite being sexually mature at the beginning of the study, comparison
between annual data showed that the testes of M2 gradually increased in size
between the ages of 8y 5m and 11y. At the beginning of the study, the testes of
M2 were the second largest of the group, being 17.3cm and 223.6 cm3. During
the study period, the testes of this subject grew; the exact increase was difficult
to determine as testis size fluctuated greatly within each study year and therefore
annual mean measurements were determined for comparison. The mean size
during the last year was 18.5 ± 2.5cm and 254.4 ± 146.8cm3, i.e., smaller than
the testes of M1. The testis size of this subject fluctuated to the extent that the
annual maximum volume reached was > 4 times greater than the minimum.
During the last study year, M2 reached a maximum testis size (24.6cm,
649.2cm3) that was greater than the maximum reached in M1 (21.6cm,
585.8cm3) . M2, with a body length of 232cm, was a much bigger animal than
M1 and this may be why testes were larger. The present study found a moderate
correlation (r = 0.6) between body length and testis length and, a stronger
correlation (r = 0.8) was reported in an analysis based on a larger number of
sexually mature males (Brook, 1997). The large fluctuations found may be due
to growth, and / or other factors, such as illness, seasonal changes or social
254
interactions. The possible influence of these factors on testicular function will
be discussed further below.
5.1.2.1 Testis size during sexual maturation
During the present study, the onset of spermatogenesis was documented in three
males (M3, M4 and M5). Annual means and ranges show that the testes of these
subjects continued to grow between 4y and 11y of age, and data suggest that
testicular growth may continue beyond 11 years old. The pattern in which the
testis size changes took place differed between subjects.
From the age of 6y 3m, testis volume in M4 increased more than four fold
during the four months before onset, from 40cm3 to 179cm3 recorded at the time
of onset. Testis length increased from 12.5cm to 17cm. Serum testosterone
levels also increased rapidly during this period. A rapid increase in testis size
during puberty has been reported in studies of several species of wild dolphins; T.
truncatus (Sergeant et al., 1973; Hui, 1979; Cockcroft and Ross, 1990), Stenella
attenuata (Perrin et al., 1976) and Stenella coeruleoalba (Miyazaki, 1977),
Globicephala macrorhynchus (short-finned pilot whale) (Kasuya and Marsh,
1984) and in one captive Tursiops aduncus undergoing sexual maturity (Brook,
1997). In the latter, testis size increased by 100% in length and 750% in volume
over a period of 10 months before onset, measurements reached at the time of
onset were 16.2cm and 205.1cm3. Rapid increase in testis size is likely to be due
to rapid in increase in the seminiferous tubule diameter (Perrin et al., 1976;
Miyazaki, 1977) and length, as found in pubertal bulls (Curtis and Amann, 1981).
255
A rapid increase in testes size was not recorded in M3, probably because the
onset of spermatogenesis occurred only two weeks after the commencement of
the study. Testis size at this time was 17.8cm and 183.1cm3. From the age of 4y
1m to 6y 9m, testis size in M5 increased slowly from 6.2cm and 7.4cm3 to 7.8cm
and 11.1cm3 at the time of onset of spermatogenesis. No rapid enlargement took
place in the testes of this subject. A rapid increase in testis size may be useful
for predicting sexual maturity in dolphins but results of this study show there are
individual differences and this may not always occur before the onset of
spermatogenesis.
The testis measurements reached at sexual maturity in M3 and M4 were similar
to each other and were comparable with measurements reported in Brook (1997).
However, the testis size of M5 at sexual maturity was significantly smaller than
M3 and M4, and the male (M1) reported in Brook (1977). Ross reported a
length measurement of > 20cm for maturing testes in South African Turisops
aduncus. At the end of the study, M5 was sexually mature, but testes were much
shorter. This male was also smaller, with a body length of 210cm. The testicular
echopattern lacked parenchymal echnogenicity and lobulation seen in the
sexually mature testes of other males. The sparse spermatozoa found in the
semen of M5 may be due to a few mature tubules that can function to produce
spermatozoa. Histological examination of testicular tissue of Stenella attenuata
suggested maturation of seminiferous tubules may occur at different times, with
those at the periphery of the organ producing spermatozoa first, followed by
those at the core (Kasuya et al., 1974). In two other detailed histological studies
of the testes of Globicephala macrorhynchus and Tursiops truncatus sexual
maturity status was subdivided into stages (early maturing, late maturing or stage
256
2 and mature or stage 2/3) based on the percentage of mature tubules found
(Kasuya and Marsh, 1984; Cockcroft and Ross, 1990). These classifications take
into account that not all seminiferous tubules reach maturity at the same time and
full maturity (i.e., 100% mature tubules) can be more accurately identified.
Similar studies in the future should investigate if a correlation can be made
between testicular echopattern and histology for more precise identification of
testis maturity in live animals.
It remains uncertain why the testes of M5 were significantly smaller at sexual
maturity. It may be due to individual differences, or possibly the study’s earlier
and more vigilant monitoring of testis size and semen has led to detection of
spermatozoa earlier, when testes size are smaller. Such individual difference is
significant and casts some doubt on the use of ultrasonographic testis
measurements alone to predict sexual maturity. It is important to monitor other
parameters, particularly semen, at the same time to ensure correct identification
of sexual maturity. The number of male T. aduncus studied whilst undergoing
sexual maturity remains small, (N = 4). Monitoring of more animals is required
to further evaluate testis size at sexual maturity and to gain more information to
better understand differences between individuals.
After onset of spermatogenesis, testes of M3 and M4 continued to increase and,
for both subjects, the peak size recorded was very similar, at approximately
210cm3 in volume. The post-onset time taken to reach peak measurements was
also similar, at about a month. The testis size of both subjects subsequently
decreased. The testes of M3 became smaller (< 130cm3) until February the
following year, whereas those of M4 were larger (176 – 228cm3) until August.
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There may be a seasonal component to the differences in testis size changes
between these two subjects. M3 reached sexual maturity in October, in autumn,
when testis size showed a tendency to be lower until February. M4 reached
sexual maturity in April, when testis size showed a tendency to be higher until
August. The speculation that the season during which sexual maturity is reached
may influence testis size immediately after onset is based on two individuals
only, it is proposed with caution until more observations can be made on more
animals.
After onset, M4’s testis volume decreased to 176cm3 in June. Medical records at
that time show M4 presented with clinical symptoms of elevated white blood
cell count and intermittent low grade pyrexia, and this may have caused the
decrease in testis size. Symptoms abated in July and testis then returned to peak
size.
Brook (1997) reported testis size increase in one pubertal male was
interrupted by an episode of illness prior to sexual maturity and further
suggested this may have prolonged the maturation process.
Harrison and
Ridgway (1971) reported the testes of a wild captured T. truncatus, which died
of a bacterial infection, were abnormal, while the testis on one side was grossly
enlarged and infected, the other was of normal size but had no spermatozoa.
Collet and St Girons (1984) questioned the reliability of results of stranded dead
subjects in being representative of a population, since pathologies in stranded
dolphins may have affected testicular function. Studies on captured or stranded
animals have no history of the medical conditions of these animals, therefore,
assignment of reproductive status based on testis size alone must be made with
caution.
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Rapid and large-scale changes in testis size were found in all recently mature
subjects M2 – M4.
This may a normal pattern for testes that are undergoing
growth to attain adult size and function. Illness may have also influenced these
changes, as all subjects had episodes of illness during the study period. Also,
social or environmental stress, may have influenced testicular development in
these subjects. Research on more individuals is necessary to further investigate
testicular development in young dolphins and to gain better understanding of the
effect of illness and / or stress.
5.1.3 Testicular asymmetry
Testicular asymmetry has been reported in some cetacean species, such as the
sperm whale, Physeter macrocephalus (sperm whale) (Arvy, 1977) Stenella
attenuata (Kasuya et al., 1974) and the T. aduncus (Brook, 1997). In a study of
three T. aduncus, Brook (1997) reported the right testis was bigger than the left
in all three males.
In the present study, statistically significant testicular
asymmetry was found in all subjects. In M2 and M3 there was significant
asymmetry in all measurements (length, volume and circumference). In M3 the
right testis was bigger, however, in contrast to Brook (1997), the left testis in M2
was consistently larger than the right. In M1, the right testis was longer but
smaller in circumference than the left, and volumes were not significantly
different.
Possible differences in vascular supply of the left and right testes in the
bottlenose dolphin have been proposed as a possible cause of testicular
259
asymmetry (Brook, 1997).
Colour Doppler ultrasonography of the dolphin
testes may be useful in providing information on gonadal vasculature and blood
flow. This will help to further understand the overall anatomy of dolphin testes,
the growth of these organs during the maturation process, and may explain the
individual differences.
5.2 Serum Testosterone level
Prior to the introduction of ultrasonographic examination of the testes, serum
testosterone level was relied on to assess reproductive status in captive dolphins
(Judd and Ridgway, 1977; Kirby, 1990; Schroeder, 1990a). A baseline level of
1 – 3ng/ml is generally accepted for healthy, sexually mature male dolphins
(Kirby, 1990; Schroeder, 1990a; Robeck et al., 1994; Robeck et al., 2001a;
Robeck and Monfort, 2005b). However, whether the level of testosterone in the
peripheral blood reflects the intra- or peri-testicular environment is uncertain. In
Globicephala macrorhynchus, testosterone levels in the testes, determined by
extraction from homogenised testis tissue, were more up to 180 times higher
than serum levels (Kita et al., 1999). Also, it is not certain if the methods used
to evaluate testosterone level in different studies are comparable, nor if the
different commercial test kits used may yield different results. Nonetheless,
when testosterone level is monitored regularly over a long period of time and on
an individual basis, the profile gained is useful in indirect assessment of
reproductive condition.
260
In this study, the mean serum testosterone levels of two fully mature males of
similar age (19+y), M1 and M6, were 44.5 ± 18.8ng/ml and 22.8 ± 16.5ng/ml
respectively.
These values were higher than the mean value of 8.8ng/ml
reported previously for two sexually mature T. aduncus aged 15 – 20+y (Brook,
1997). These differences may be due to normally fluctuating levels differing
between individuals, or that data from the present study was more
comprehensive and longer term, thus recording more normal range values. The
test kit used for evaluating serum testosterone level in the present study was an
enzyme immunoassay method with a final fluorescent detection (ELFA) by an
automated VIDAS system, which differed from the kit used in Brook (1997).
The ELFA kit had lower cross-reactivity levels with other testosterone
derivatives / steroidal compounds than the conventional radioimmunoassay (RIA)
kit used by Brook (1997). The maximum level recorded (in M1) was 78.4ng/ml,
which is again higher than the 19.6ng/ml reported in Brook (1997). Most
previous studies also reported lower levels in sexually mature T. truncatus;
24ng/ml in Harrison and Ridgway (1971), 54ng/ml at age 19y in Schroeder and
Keller (1983) and Schroeder (1990a) and 70ng/ml at age 23y in Kirby (1990). It
is likely that these differences in levels between the present study and others are
due to differences between individuals, differences in duration of monitoring
period, frequency and time of blood sampling, and method used for hormone
evaluation. Alternatively, these values may represent a normal range in sexually
mature Tursiops, and the differences may be caused by the differences in time
when blood samples were collected among the studies.
Nonetheless, such
uncertainties indicate that, despite testosterone being the most studied
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reproductive parameter in male Tursiops, there is still much to learn about basic
physiology in these animals, and further research is needed in this area.
In the present study, fluctuations were found in monthly serum testosterone
levels which did not appear to have any association with times when females
were present in the same holding facility. Brook (1997) also found no effect of
the presence of females. In a mixed group of captive Stenella longirostris
(spinner dolphin), testosterone levels exhibited a seasonal pattern, but did not
show an obvious relationship with male – female pairing duration (Wells, 1984).
In contrast, a study on wild white rhinoceros (Cerathotherium simum simum)
found males that accompany a receptive female had higher faecal androgen
metabolite concentrations than those which roamed alone, even when pairing
occurred outside of the mating season (Kretzschmar et al., 2004). It was not
within the remit of this study to determine the effect of the presence of females
on testosterone level, or its significance in dolphins, but this may require further
investigation.
The lowest testosterone level recorded for M1 was 11.3ng/ml (mean 44.5 ±
18.8ng/ml) and for M6 was < 3ng/ml (mean 22. 8 ± 16.5ng/ml). These low
levels were recorded during episodes of illness. Clinical signs of illness included
elevated white blood cells, abnormalities in other blood parameters, intermittent
pyrexia and unusual hepatic appearance on ultrasonography. M6 was ill for
more than 6 months and the testosterone profile for that year was altered, with an
annual mean level (10.9ng/ml) that was substantially lower than those of other
years (34.7ng/ml, 19.9ng/ml and 26.8ng/ml). An adverse effect of illness on
testosterone levels in dolphins has been reported (Brook, 1997; Kirby, 1990). In
262
both of these reports, testosterone levels in the affected males decreased to <
1ng/ml. These findings highlight the importance of longitudinal monitoring of
testosterone levels to assess the reproductive status of an individual. A marked /
sustained decrease in testosterone levels may also be a useful indicator of illness
in male dolphins.
M1 showed consistently higher testosterone levels than other subjects. There
may be several factors affecting this. M1 was 19+y, had been fully mature for
more than 14 years and had the largest testes. The testes are a prime source of
testosterone, so this may simply be a reflection of size. However, M1 was also
the dominant animal in this ‘bachelor’ group, which consisted of the more
recently matured subjects (M2 – M5) and another fully mature male (M6). In a
study of wild mountain gorillas (Gorilla gorilla beringei), a tendency for
dominant males to have higher testosterone levels than subordinate males was
found (Robbins and Czekala, 1997).
In wild baboons (Papio anubis),
testosterone recovered to elevated levels quicker after a stressful event in
dominant males than in low ranking males (Sapolsky, 1983). The effect of social
status on reproductive function in Tursiops was beyond the scope of this study
but further research is needed to investigate this.
5.2.1 Serum Testosterone levels during sexual maturation
Prior to the onset of spermatogenesis, testosterone levels increased in most males.
Levels in M4 increased rapidly from < 1ng/ml in December 2004 to > 24.1ng/ml
by April 2005, when onset occurred. Levels in M4 occasionally rose > 3ng/ml
263
in the 15 months before a more dramatic increase was seen. If testosterone level
was used alone, M4 may have been misinterpreted as being sexually mature
from the age of 5 years.
For the purpose of this study, archival data of
testosterone levels from M3 from June 2001 were reviewed to determine if there
was any similar pre-onset pattern. Levels in M3 rose from 0.5ng/ml to 1.7ng/ml
over a 6-month period before onset in October 2002. There was insufficient data
to determine if this occurred in M2 prior to onset. Levels in M5 did not show
any increase. Brook (1997) documented an increase in the testosterone level in
M1 (‘Molly’) from < 1ng/ml to 6.6ng/ml over a 5-month period prior to onset.
Results show that the onset of spermatogenesis in T. aduncus can also occur at
lower testosterone levels. A less rapid increase in testosterone levels in Orca
orcinus (killer whale) was found in a study in which three males reached sexual
maturity (Robeck and Monfort, 2005b). Mean level prior to sexual maturity (at 7
– 9y) was 0.7 ± 0.7ng/ml; sexual maturity was reached 2 – 7 years (4.3 ± 2.5
years) later, with a mean testosterone level of 6.0 ± 3.3ng/ml. Differences
between species are expected and there may also be an effect of season on
differences between individuals in the present study, which will be discussed
further. To date, the present study is the only detailed, longitudinal investigation
of changes in serum testosterone during sexual maturation in T. aduncus. The
individual differences found warrant research on more animals to determine how
often the rapid increase occurs prior to onset of spermatogenesis and whether it
is a useful sign for predicting sexual maturity in dolphins.
Testosterone levels recorded closest to when sperm was first detected were
2.1ng/ml in M2, 1.7ng/ml in M3, and 0.1ng/ml in M5 in the present study, and
3ng/ml in M1 (Brook, 1997). These levels are generally lower than the 3ng/ml
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commonly reported for sexually mature T. truncatus (Harrison and Ridgway,
1971; Kirby, 1990; Schroeder, 1990a). The 24.1ng/ml recorded in M4 at onset
was much higher and there may be a seasonal component to explain this, which
will be discussed further. In a study of Globicephala macrorhynchus, it was
found that spermatozoa were produced at a minimum serum testosterone level of
0.82ng/ml (Kita et al., 1999), but it was also found that levels within the testis
were much higher than in serum. In another study on testis tissue of adult mice,
spermatogenesis was maintained when intra-testicular testosterone level was
reduced by a factor of 30, from 0.39 ± 0.04ng/ml to 0.01 ± 0.001ng/ml
(Cunningham and Huckins, 1979). Based on present results and findings from
other species, it is possible the onset of spermatogenesis in dolphins can take
place when testosterone levels in the serum are low. The present study’s early
and vigilant monitoring may have led to prompt detection of spermatozoa at an
earlier age. Also, levels reported here at onset must be viewed with caution, as
they were from blood samples taken closest to the day when spermatozoa were
first detected and therefore may not accurately reflect the circulating level on the
actual day. Further, given diurnal changes in testosterone and the pulsating
manner of its release found in humans, livestock and laboratory species (Pelletier
et al., 1981; Plant, 1988; Turek and Van Cauter, 1988), monthly evaluation is
probably not adequate to fully document the changes in testosterone level of a
dolphin undergoing sexual maturation.
Like testis size, factors such as illness and stress may affect serum testosterone
levels. Stress may be socially induced, particularly in young male dolphins,
which are often submissive, with low social ranking within a group (McBride
and Hebb, 1948; Wells, 1984; Samuels and Gifford, 1997; Waples and Gales,
265
2002; Suzuki et al., 2003). Testosterone levels in M2 and M3 often fell to <
1ng/ml. M2 presented clinical signs that warranted administration of both oral
and intravenous antibiotics when his serum testosterone level fell to < 1ng/ml
during August – December 2004.
M3 showed persistent weight loss and
sporadic clinical symptoms in 2004 – 2006, which may explain why its postonset monthly profile was lower that that of M4. Levels in M4 also sharply fell
from 49ng/ml in June 2005 when he presented with persistent pyrexia, however
post-onset levels in this male never fell below 1ng/ml.
Results further
demonstrate the unstable nature of serum testosterone and warn against the use
of this parameter alone in determining sexual maturity in Tursiops. Although
illness is the most apparent cause for these changes, effect of social interactions
within a group can not be excluded. The effect of social status on testosterone
level / testicular functions was beyond the scope of this study, and further
research is required.
The season during which M4 reached sexual maturity may explain why its
testosterone levels (and testis size) at onset and profile after onset were much
higher than those of M3. At onset M4’s level was 24.1ng/ml and further rose to
reach 49.0ng/ml two months later. M4 reached sexual maturity in April during
spring when serum testosterone level showed a seasonal tendency to be higher
until August (summer) in this captive group.
Whereas, M3 reached sexual
maturity in October, or autumn, when testosterone level showed a tendency to be
lower until the end of winter in February. The influence of season on hormonal
profile has been observed in female lambs (Karsch and Foster, 1981; Foster,
1988). When lambs reached the expected age of puberty outside of the breeding
season, the hormone changes associated with ovulation (i.e., increase in
266
oestradiol and LH surge), were delayed until the following breeding season. It
was proposed that remaining sensitivity of the neuroendocrine axis (responsible
for the secretions of GnRH, LH and FSH) to gonadal steroids due to a lack of
maturity combined with the environment effects of season might explain the
delay.
Any seasonal influence on the testosterone secretion in maturing or
recently matured T. aduncus is speculative at this time and based on two
individuals only. Further investigation on more individuals is required.
5.3 Semen collection and sperm density
This study followed a strict semen collection protocol – i.e. ejaculates were
collected individually, in succession, until no more semen was present in spite of
effort, or when micturition occurred. Only two other studies reported the use of
a similar collection protocol to investigate testicular activities in Tursiops
(Schroeder and Keller, 1989; Brook, 1997). Criteria used for cessation of a
collection session in these studies were not specified, except for an ‘adequate
number of spermatozoa’ being collected for investigative purposes, such as
development of semen cryopreservation methods (Robeck and O'Brien, 2004a).
In the latter study, maximums of four ejaculates were collected in one session.
In the present study, multiple ejaculates were obtained from each subject during
a collection session. Since ejaculates collected in a series differed in volume and
density, an overall density (OvD) was obtained mathematically to evaluate
267
efficiency in spermatogenesis and for comparisons between individuals. OvD
was derived by summation of the total ejaculate sperm count (EjC) divided by
the total ejaculate volume (EjV). This study also examined the densities of the
most concentrated ejaculates in a series (HiEjD) and demonstrated that T.
aduncus are capable of producing very dense ejaculates after recent onset of
spermatogenesis and on a year-round basis. This information is important when
planning
controlled
breeding
programmes
or
semen
collection
for
cryopreservation.
A large number of ejaculates and total semen volume can be collected from T.
aduncus. In M1 – M4 (ages 7 – 23y), the maximum number of ejaculates
collected successively was 11 – 28 and ToV ranged from 60 – 222ml. The
oldest subject (M1) produced the greatest number of ejaculates and ToV. This is
probably because the testes of this individual were the largest, but may also be
due to its age, as the process of sexual maturity has been proposed to take
several years (Cornell et al., 1987; Cockcroft and Ross, 1990; Schroeder, 1990a).
These amounts are similar to one report of a 19-year T. truncatus, which
produced up to 12 ejaculates per session and 60ml ToV (Schroeder and Keller,
1989). Brook (1997) reported a maximum of only five ejaculates per session,
but similar ToV of 77.5ml in three T. aduncus, aged 7 – 15+y, but these results
may have been affected by poorer collection techniques and variations in early
protocols. Many factors may affect ejaculate production in dolphins and
differences may be due to simple individual variation, differing age or testis size,
cooperation of the animal, or the stringency of the collection protocol.
The
large ejaculate number and ToV found in the present study resulted in very high
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total sperm counts (ToC), of 24,090 – 81,922 x 106. This range is comparable to
the 63,000 x 106 reported by Schroeder and Keller (1989).
In M1 – M4, mean and maximum OvD were 241 – 112 x 106/ml and 964 – 2494
x 106/ml, respectively.
The mean OvD of M1 (241 x 106/ml) and M3 (209 x
106/ml) are comparable to values reported in earlier studies (224.7 x 106/ml –
Schroeder and Keller, 1989; 237.2 x 106/ml – Brook, 1997), despite the
differences in age, the number of ejaculates per session and ToV. Schroeder and
Keller (1989) also reported a maximum OvD of 1,587 x 106/ml, which is lower
than the maximum values recorded in M2 (1692.4 x106/ml) and M3 (2493.8 x
x106/ml) in this study, though both were much younger. The densities of the
most concentrated ejaculates were 1475 – 5170 x 106/ml. The densest ejaculate
was produced by M3 in August during the 3rd post-onset year.
Another study
also reported sexually mature T. truncatus produced densities as high as 3,000 x
106/ml (Briggs et al., 1995). Data from the few studies reported indicate sperm
densities in Tursiops do not appear to differ considerably between subspecies,
and that even very young, recently matured males may produce very dense
ejaculates.
A complete absence of spermatozoa or extremely low densities were
occasionally found in the present study and this is consistent with previous
reports (Schroeder and Keller, 1989; Brook, 1997), however, this may not apply
to all males. A complete absence of spermatozoa only occurred once in M1 and
Brook (1997) reported azoospermia never occurred in one fully mature (20+y) T.
aduncus. Care must be taken to accurately define true azoospermia, as a failure
to detect sperm on microscopic examination is also possible. The present study
269
found that the 1st ejaculate in a collection series was typically of low density,
therefore if only the 1st ejaculate is collected and evaluated, misinterpretation of
overall density may occur. The very rare occurrence of azoospermia suggests
that a ‘resting’ phase in the context of a male reproductive or testicular cycle, as
described in other dolphin species (Hirose and Nishiwaki, 1971; Collet and Saint
Girons, 1984) does not exist in T. aduncus. This study did not find sperm density
to show any obvious seasonal pattern and it is still not clear why extremely low
densities can occur in fully mature dolphins. It appears, however, that this
phenomenon is short lived and further collection of ejaculates soon afterwards
may provide better data / samples for controlled breeding or research
programmes.
A decline in semen parameters was recorded in M1 during an illness in 2005
(June – August). M1 presented with persistent clinical signs of elevated white
blood cell count and pyrexia, treated with both oral and intravenous antibiotics.
During June and July, the number of ejaculates collected per session decreased
from 15 to 1 – 6 and OvD decreased from > 300 x 106/ml to 150 – 125 x106/ml.
It was also noted that total motility (TM) decreased from > 70% to < 40% and
viability (VIA) decreased from ≥ 90% to < 82%. Semen and ejaculate
characteristics returned to normal as the animal’s condition improved. Kirby
(1990) and Brook (1997) both postulated a decline in testicular size and function
as consequences of illness, however, to the best of the author’s knowledge, there
is no other report on the effect of illness on sperm density and other semen
parameters in dolphins. Further investigation of this phenomenon is needed.
270
5.3.1 Semen collection and sperm density during sexual maturation
Results show that sexually immature T. aduncus are capable of delivering semen
under cooperative behaviour long before sexual maturity occurs. The number of
ejaculates and volume collected were smaller when subjects were immature.
This is likely to be due to the smaller size of the testes and accessories glands,
such as the epididymis and prostate, which are responsible for the production
and storage of seminal fluid.
This study found the onset of spermatogenesis, (i.e., sexual maturity), may occur
before the age of 7 years, which is younger than previously reported for T.
aduncus (Brook, 1997). The age of sexual maturity in T. truncatus is generally
reported as ≥ 10 years (Harrison and Ridgway, 1971; Kirby, 1990; Schroeder,
1990a) with ‘reproductive effectiveness’ being reached some years later. Sexual
maturity in M2 – M5 occurred within a 9-month range (6y 7m – 7y 4m). The
age at sexual maturity was very similar between M2 (7y 4m) and M3 (7y 3m).
M2 reached sexual maturity prior to the start of the study, in September, 2001.
As semen collection was not conducted regularly at that time, it is possible that
spermatozoa may have been present in the semen of this dolphin earlier.
Spermatozoa were first detected in M3 on 23rd October 2002, only two weeks
after commencement of the study. Prior to the study, semen was last collected
from M3 on 22nd June 2002, but no spermatozoa were found. The age at sexual
maturity of M4 and M5, was also similar, 6y 7m and 6y 9m, respectively; these
ages at sexual maturity are the youngest reported for Tursiops (Sergeant et al.,
1973; Ross, 1977; Cockcroft and Ross, 1990; Kirby, 1990; Schroeder, 1990a;
Brook, 1997).
It may be because the present study used more careful and
271
rigorous monitoring at younger ages, so that the earlier presence of fewer
spermatozoa was not missed. The methodology used to detect spermatozoa was
stringent and included the use of centrifugation and systematic microscopic
screening of multiple wet-mount slide preparations. There is no evidence that
commencement of semen collection at a young age induces precocity, or
‘hastens’ the maturation process. The number of subjects in the present study
remains small, and given the individual differences found, research on more
males is required to confirm the age / age range at which onset of
spermatogenesis occurs in T. aduncus and to further investigate if early semen
collection has any physiological effect in bringing about sexual maturity at a
younger age.
Results show that once onset of spermatogenesis has taken place in T. aduncus,
there is continuous progress in sperm output, with periods of azoospermia, or
extremely low densities immediately following onset. In M3 and M4, a lack of
spermatozoa or extremely low density (< 0.03 x 106/ml) was found up to 1 year
and 7 months post-onset. After this time, occurrence of these conditions was rare,
as indicated by the increase in annual mean sperm density. Azoospermia did not
last for more than three weeks in M3 and was found only occasionally in M4.
Similarly, Brook (1997) reported periods of azoospermia following sexual
maturity in one male lasted 9 – 48 days. Azoospermia or extremely low sperm
densities in young, recently mature males appears to be normal, and histological
studies also indicate that maturation (i.e., to contain sperm) of seminiferous
tubules throughout the testis is progressive (Kasuya et al., 1974; Kasuya and
Marsh, 1984). In Globicephala macrorhynchus, it takes over 2 years to be reach
100% mature tubules from < 50% mature tubules in early maturing stage.
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In
bulls, efficiency of sperm production increases gradually from first release of
sperm, at age 32 weeks, until 1 year of age, when it reaches that of a fully mature
individual (Curtis and Amann, 1981). Consequently, some livestock bulls are
classified under different stages of sexual development after first release of
sperm based on the number of sperm an ejaculate contains. Bulls are classified
to be at puberty when an ejaculate containing ≥ 50 x 106 sperm (of ≥ 10%
motility) is first collected (Amann, 1983; Wolf et al., 1965) and at full sexual
maturity when two ejaculates of ≥ 500 x 106 sperm (with at least one of ≥ 50%
motility) are collected consecutively (Tatman et al., 2004).
It is not clear why episodes of azoospermia were longer and more frequent in
M3 than M4.
As a result, the mean OvD for the 1st post-onset year was
substantially lower in M3 (2.6 ± 8.8 x 106/ml) than in M4 (62.4 ± 15.6 x 106/ml).
This may be due to the smaller testis size (and lower testosterone levels) in M3
and the possible influence of the season during which maturity was reached. In
bulls, calves born in autumn took longer to release the first sperm and reach the
sperm count required for sexual maturity status (i.e., ≥ 500 x 106 sperm /
ejaculate), than those born in spring (Tatman et al., 2004). Also, the size of the
testes of the bulls born in autumn was larger at first sperm and sexual maturity,
as they had a longer time to develop. This indicates that other factors influence
onset and efficiency of spermatogenesis in addition to testis size. Again, the
speculation that season may have an effect on testicular development in dolphins
is made based on two individuals only, and further study is recommended.
Since the pulsatile release of LH and FSH from the anterior pituitary plays a
pivotal rule in male reproductive development (Plant, 1988) longitudinal
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monitoring of these hormones may provide further understanding of the sexual
maturation process in Tursiops and may help to explain why differences occur
between individuals. To date, there is one basic report on LH and FSH levels in
T. truncatus, which found that serum LH level was significantly higher in adult
dolphins than in sexually immature animals (Schneyer and Odell, 1984).
However, this was a cross sectional study, lacking longitudinal monitoring
which yields data that are more useful for understanding developmental
processes. Also, the study group contained both captive and wild animals of
mixed gender, the method of age / reproductive status assignment of the animals
was not clear and the RIA tests used were developed for human serum.
Although M5 was a similar age to M4 at onset, both body size and testis size
were smaller than those reported (Brook, 1997) and those recorded for M2 – M4.
Only scarce spermatozoa were found in the semen of this subject during the 10month post-onset period monitored. The differences exhibited by M5 in testis
size, testosterone level and body size at onset indicate future studies should
include monitoring of semen, as well as the other parameters, in order to
accurately assess the reproductive status of an individual. Ultrasonography of
testes and semen collection from M5 is being continued to gain further
understanding of reproductive development in this subject.
After onset, semen was not delivered in three collection sessions by M3, despite
two attempts at ejaculation during each session. Husbandry records for these
sessions note the arrival and removal of females in adjacent pools. This
coincided with unstable blood parameters and weight loss noted in veterinary
records. It is not certain if aspermia in this subject was a result of loss of
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cooperative behaviour due to the presence of females, or due to illness.
Schroeder and Keller (1989) also reported a lack of ejaculate in one T. truncatus,
but no explanation was given for this.
The study maintained a strict collection frequency of once a week to avoid any
differences varying collection frequency may cause. Increased collection
frequency has resulted in decreased ejaculate volume, sperm density and count
in bulls and stallions (Cunningham et al., 1967; Magistrini et al., 1987). Motility
was found to increase slightly when collection frequency was increased from 3collection days to 5-collection days per week in stallions (Magistrini et al., 1987).
The present study noted a possible ‘learning effect’ in M1 and M2, as the
number of ejaculates collected per session notably increased from the 1st year of
the study. These males had been sexually mature for some years so it is not
thought likely the increased number was due to increased production in the
physiological sense, but rather that the animals had undergone ‘acclimatisation’
to the successive ejaculate collection protocol. It may also be that the handling
of animals improved and sessions were not ended arbitrarily, as this tended to
happen at the beginning of the study. There was no evidence that the present
protocol had any effect on spermatogenesis or behaviour.
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5.4 Age and Body length at sexual maturity
Body length is often taken into consideration when using other parameters, such
as testis size and histology, to assess reproductive status in wild dolphins (Hirose
and Nishiwaki, 1971; Sergeant et al., 1973; Hui, 1979; Collet and Saint Girons,
1984; Perrin and Henderson, 1984a). However, substantial overlaps in body
length in animals of different maturity status, and individual differences, would
suggest this parameter is of limited use as an indicator of sexual maturity and
should not be relied on alone. Ages of animals in these studies were estimated
by growth layer group (GLG) count, results of which are subject to uncertainty
due to technical differences between workers and difficulties differentiating
multiple layers. This method too is of limited use if the parameters (e.g., body
length) it is compared with are not reliable. In the present study, M2, M3 and
M4 all reached sexual maturity at a larger body size but at younger ages than
previously reported for T. aduncus (Brook, 1997), however, the exact age of the
male (M1) in the latter study was unknown and this must be taken into
consideration when comparing it with known aged subjects. M2 was the largest
and oldest male to reach sexual maturity in this group, at 226cm long and 7y 4m.
This male was not regularly monitored at the time the onset of spermatogenesis
was documented and it may be that this occurred earlier and at a smaller body
size. Its larger body size may also be because his dam was a T. aduncus from
Taiwan, and was larger than the Indonesian animals. This again alerts
investigators to use parameters appropriate to the population they are studying.
M3 and M4 both had body lengths of 225cm at the time of onset, when M3 was
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7y 3m, whilst M4 was 8 months younger, at 6y 7m. M5 was only 200cm long at
sexual maturity, but was of a similar age (6y 9m) to the other males. The study
has shown that sexual maturity may occur before 7 years of age, but that body
size at sexual maturity varies in Tursiops. Research on more individuals is
required to gain more data and better understanding.
Law (1956) proposed that sexual maturity occurs in male delphinids at 93% of
the final body length. Similarly, a percentage of 91% is reported in Tursiops
truncatus (Sergeant et al., 1973). Authors of both studies suggested growth was
more rapid in captive animals due to improved nutrition. One T. aduncus (M1 in
the present study) reached sexual maturity at 94% of final body length (Brook,
1997). This percentage is difficult to deduce in the captive born males of the
present study. Data indicate that M2 had not reached full maturity when it died,
aged 11 years. M4 was younger and also died before growth was completed.
Therefore no conclusion can be made about percentage body size at onset in
these males. Both M3 and M5 were physically immature at the end of the present
study and continued monitoring is required to determine the percentage of final
body length at sexual maturity. If this percentage can be better defined, it may
become a useful measure for predicting sexual maturity in male dolphins of the
same species and origin. Further research is required to investigate the effect of
captivity on growth and reproductive development, but this can only be valid if
comparisons are made with wild counterparts of similar origin.
Final body size is reached some years after sexual maturity and indicates an
animal has reached physical maturity. Since male dolphins in the wild compete
to gain access to females for mating, success is more likely when body size is
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larger. It has been reported that prior to physical maturity, males may not be
considered as reproductively ‘effective’ in the social context (Connor et al.,
2000b), but these males are physiologically, or sexually, mature, i.e. producing
sperm. In captive situations when breeding is selectively controlled, usually
only one male is allowed access to a female, therefore the difference between
sexual and social maturity is not significant and captive males may be
considered to be reproductively effective once they are sexually mature.
5.5 Correlation between testis size, serum testosterone level and
sperm density
Comparisons between parameters were made only with data that were collected
on the same day. Associations of varying degrees were found between testis size,
serum testosterone level and sperm density, but these associations differed
between individuals. It is difficult to interpret any relationship between two
parameters based simply on comparison of numerical values, as the reproductive
physiology in male dolphins is complex. Any association found may not indicate
a direct causal relationship, or a lack in association may not necessarily mean a
relationship does not exist. There are other factors to be considered. In captivity,
it has been postulated that social factors may influence reproductive activities
and development in male dolphins (Brook, 1997; Robeck et al., 2001a).
Evaluation of such factors was beyond the scope of this study, therefore, the
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following discussion focuses on the differences found between subjects due to
age and reproductive status, as opposed to the functional / physiological aspects
of the reproductive parameters.
There was a considerable difference between the older M1 and younger males,
M2 – M4 in the strengths of association between all the parameters.
M1
exhibited the lowest correlations in all comparisons, (r = 0 – 0.5), while
correlations in M2 – M4 were r = 0.1 – 0.9. It may be that M1 was fully grown
and fully mature, whereas M2 – M4 were still growing, as evidenced by body
and testis size. Age related growth changes in these subjects may have had an
overall effect and resulted in the stronger associations found. However, Brook
(1997) found correlations between testis length and testosterone levels in one
growing male and one fully mature male did not differ. The sample size in this
study was very small and results may not be reliable.
Associations were weakest between sperm density and other parameters. In M1,
associations between testosterone level – sperm density and testis volume –
sperm density were almost non-existent (r = 0 – 0.1), whereas moderate
association was found between testis volume and testosterone level (r = 0.4 – 5,
P < 0.05). In M2 – M4, although associations between testosterone level –
sperm density and testis volume – sperm density were better than in M1 (| r | =
0.1 – 0.6), they were still weaker than the association between testis volume and
testosterone level, where r > 0.6 (P < 0.05). These results indicate that, although
changes in testis size and testosterone may follow a similar pattern, changes in
sperm density do not (see Section 5.6 of this chapter). Brook (1997) also found
weaker associations with sperm density. In analyses that combined data from
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three subjects, Brook (1997) reported r = 0.2 (P > 0.05) for correlation between
testosterone level – sperm density and r = 0.5 (P < 0.05) for correlation between
testis size – sperm density. Correlations between testis size and testosterone
were significant and slightly higher, r = 0.5 – 0.6, but the author advised that
comparisons should be interpreted with caution, as sperm density data were few
and blood samples were not always collected on the same day. No association
between testosterone level and sperm density was also reported in Orcinus orca
(Robeck and Monfort, 2005b).
Results show testosterone level and sperm density in T. aduncus often fluctuate
independently. Changes in androgenic activity of the testes, in the absence of
obvious changes in spermatogenic activity, have been described in some
terrestrial mammals (Lincoln, 1981) and it is known that spermatogenesis can be
maintained at low intra-testicular concentrations in sexually mature rats
(Cunningham and Huckins, 1979). Therefore spermatogenesis can proceed
without any interruption, even with a large decline in serum testosterone level.
Also, given the pulsatile manner in which testosterone is secreted (Turek and
Van Cauter, 1988), evaluation on a monthly basis may not be adequate for
comparisons with sperm density, a parameter which also fluctuated widely. The
individual differences found warrant further investigation of the association
between testosterone level and sperm density in dolphins.
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5.6 Seasonality
Tursiops can be fertile throughout the year, as evidenced by ovulations and / or
conceptions occurring in every month of the year (Harrison and Ridgway, 1971;
Cornell et al., 1987; Wells et al., 1987; Cockcroft and Ross, 1990; Asper et al.,
1992; Urian et al., 1996; Brook, 1997; Duffield et al., 1999; Wells, 1999). In
captive settings, increased breeding activity during certain months of the year
may not necessarily demonstrate biological seasonality, since husbandry
management may preclude male and female association, irrespective of gonadal
activity (Cornell et al., 1987; Asper et al., 1992; Brook, 1997). To date, no
distinct seasonal pattern of reproduction has been found in captive Tursiops
(Brook, 1997; Robeck et al., 2001a).
Longitudinal data on male Tursiops
reproductive seasonality are sparse and are mostly based on serum testosterone
levels (Harrison and Ridgway, 1971; Schroeder and Keller, 1989; Kirby, 1990;
Brook, 1997). A capture-release study on wild T. truncatus presented some
evidence that showed larger testes size (length and diameter) occurred in
summer (Wells, 1999). Details of the ultrasonographic procedure for obtaining
testis size were not given and winter captures appear to lack representation. To
date, there are two studies that investigated seasonal changes in testicular
activities that included evaluation of sperm density (Schroeder and Keller, 1989;
Brook, 1997) and only Brook (1997) also investigated testis size. Therefore,
there remains a need for more research to determine if a seasonal pattern exists
in the reproductive physiology of male Tursiops.
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This study was carried out in a dolphin facility situated in Hong Kong. Hong
Kong, with a total area of 1,104 km2, is located in the Northern hemisphere, on
the south eastern coast of China, slightly below the Tropic of Cancer (22o15’N
latitude and 114o10’E longitude). Hong Kong has a sub-tropical climate with
distinct seasons, although weather for this region for most of the year is
temperate. Spring in Hong Kong is March – mid-May, with temperatures of 18
– 27oC; summer is late May – mid September, with temperatures of 26 – 33oC;
autumn is late September – early December, with temperatures of 18 – 28oC and
winter is mid-December – February, with temperatures of 14 – 20oC (The Hong
Kong Observatory). M2 – M4 were born in Hong Kong, the sires and dams
were from Taiwanese and Indonesian waters. M1 was from Indonesian waters
and had been in Ocean Park for over 15 years.
The data from this study were analysed in two ways to determine if the changes
in testis size, serum testosterone levels and sperm density showed any seasonal
pattern. First, all data were pooled and the mean value of each month was used
for comparison between different months. Secondly, data of each year were
considered separately; the mean value for each year was determined, to reduce
the impact of year-to-year differences due to other factors, such as normal
growth in younger males, or social changes. Frequencies of months with levels
above the annual mean were used to assess seasonal patterns.
In M1 and M2, there was a tendency for testis size to be larger from March to
August (i.e., spring and mid summer), this period coincides with when ovulation
is more common (March – July) in this group (Brook, 1997). Testis size was
smaller from November to February (i.e., mid autumn and winter). Reports of
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seasonal changes in sexually mature testes are conflicting. In a study of Stenella
attenuata in Japan, Kasuya et al. (1974) suggested changes in mature testis
weight were indicative of seasonality because this correlated with the peak
diameter of ovarian follicles in July. Based on a lack of spermatozoa found in
testes that satisfied the weight criterion for maturity, this study also suggested a
decline in spermatogenesis occurred, however, it may also be that the onset of
spermatogenesis may not have taken place in these testes. In a later study of
Stenella, Hohn and Chivers (1985) found testis size changes did not affect
spermatogenesis and concluded testis size was not a good indicator of
reproductive seasonality.
Reports on testis size changes in Tursiops are few. Cockcroft (1990) found no
evidence of a seasonal pattern in testis weight or presence of spermatozoa in the
epididymis in T. truncatus off the coast of Southern Africa. Only Brook (1997)
studied testis size on a long-term basis and reported the testes size of a fully
mature, wild-born male (15+y), in Hong Kong showed a seasonal pattern. Testis
length was larger from January to August (i.e., mid winter to mid summer) and
smaller from September to December (i.e., autumn to winter), with a small rise
in between in November. This testis size increase occurred earlier than found in
the present study.
The study period in Brook (1997) was less than two
consecutive years and a minimum of two consecutive years is recommended to
characterise changes that occur on an annual basis (Turek and Van Cauter, 1988).
In the present study, annual change was assessed using a mean derived from
each year’s data to control for any major inconsistencies between each year due
to factors other than season (e.g., illness and social re-grouping because of
animal relocation or loss). Brook (1997) used an overall mean to assess a 20-
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month data set, which may not be adequate in detecting subtle changes in one
year if large changes occurred in another. Also individual differences between
males and differences within the same animal from year to year (Hohn and
Chivers, 1985; Kirby, 1990) can not be precluded. The subject in Brook (1997)
was a wild born male and had been in captivity for only four years. All the
subjects in the present study were captive born, except M1, which had been at
Ocean Park since 1987.
It has been postulated that captivity may affect
reproduction in Tursiops (Laws, 1956; Sergeant et al., 1973; Bryden and
Harrison, 1986a; Robeck et al., 2001a), but there is no empirical evidence for
this and information remains too limited to determine if this is true. Results of
the present study show that, although testis size in T. aduncus showed some
seasonal changes, these were not the dramatic seasonal ‘rut’ changes reported in
more typically seasonal species, such as Phocoena phocoena (habour porpoise)
(Read and Hohn, 1995; Neimanis et al., 2000). In this species, testes reached
maximum mass in June and July, then underwent regression and cessation of
spermatogenesis.
The present study did not find any seasonal decline in
spermatogenesis and changes in testis size can not be used to interpret
seasonality in T. aduncus.
Serum testosterone levels in the present study showed a vague pattern similar to
testis size. In M1, M2 and M6 there was a tendency for levels to be higher
between March and August / September, and lower between September /
October and February. Changes in testosterone level are also reported in T.
truncatus. Harrison and Ridgway (1971) reported testosterone levels of an adult
male were highest in September and October in one year, but in April and May
in the following year. Schroeder and Keller (1989) reported testosterone levels in
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a 19-year male peaked in June and declined in July / August. Kirby (1990)
concluded seasonal fluctuations in testosterone in dolphins were biphasic, with
increased activity in ‘spring and fall’, but that males remained capable of siring
calves all year round. Like the present study, Brook (1997) found testosterone
level changes followed a similar pattern to testis size in males in Hong Kong,
however levels increased earlier and decreased earlier than in the present study.
The present study found no decline in spermatogenesis despite changes in
testosterone levels and male dolphins can impregnate females whether
testosterone level is increasing or decreasing (Kirby, 1990). Serum testosterone
level, like testis size, thus can not be used to interpret seasonality in T. aduncus.
Since M1 was a fully mature and healthy male, the seasonal pattern found in its
testis size and serum testosterone level may be considered the ‘norm’ (Brook,
1997). Interestingly, in the present study, the most distinct seasonal pattern was
seen in M2, a chronically ill male that had not reached full maturity. This
subject eventually succumbed to a bacterial infection, and died in 2005 at 11
years of age. The maximum testicular volume during spring/summer, from May
to June was > 4 times larger than the minimum during winter (January).
Changes in testosterone level were also large and followed a distinct pattern,
with peak levels (> 7ng/ml) in spring and summer (May and June) and lowest
levels, (< 1ng/ml) during autumn and winter (November and December). Some
declines paralleled episodes of illness such as one in August – September 2004,
during this time testis volume rapidly decreased by > 150cm3 and serum
testosterone levels fell < 3ng/ml.
However, an obvious temporal association
between testicular decline and illness was not always discernable. The overall
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picture suggests that health and growth had some influence on the pattern seen in
M2.
Sperm density fluctuated erratically from week to week in all subjects and no
patterns were seen. Although there was a tendency for densities to be lower in
winter (February in M1; January – March in M2; March in M3) ejaculates of
high density (> 600 x 106/ml) were still found, so high overall densities may
occur at any time of year in T. aduncus. Results show changes in testis size and
testosterone level had no obvious effect on spermatogenesis or the reproductive
‘effectiveness’ of an individual. This is consistent with the results of a long-term
study on two adult Orcinus orca (Robeck and Monfort, 2005b), which reported
no pattern in sperm density, despite apparent seasonal changes in testosterone
level.
Spermatogenesis is not energetically costly and female Tursiops can
ovulate at any time of year (Brook, 1997; Brook, 2001). For these reasons it
makes biological sense that spermatogenesis is maintained year-round.
Periods of 2 – 8 weeks have been reported between high serum testosterone
levels and peak sperm density in male T. truncatus (Schroeder and Keller, 1989;
Schroeder, 1990a; Schroeder, 1990b). Robeck et al. (1994) suggested this delay
might be due to an inhibitory effect of high serum testosterone on
spermatogenesis, as observed in some terrestrial species. However, Kita and
Kashiwagi (1999) showed that large amounts of germ cells and spermatozoa can
be found at high intra-testicular testosterone levels. The present study did not
find a consistent temporal association between the two parameters. Likewise, no
association between these parameters was found in two adult Orcinus orca
(Robeck and Monfort, 2005b). Any delay between peak testosterone levels and
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peak sperm density is more likely due to time required for recruitment of germ
cells, completion of spermatogenesis and release into the epididymis for further
maturation and storage (Robeck et al., 1994) and this may vary. It must be noted,
however, that the number of animals studied to date remains small and further
investigation is required to determine if there is a real association between these
parameters
Results show sperm density in T. aduncus is not seasonal and there is no
reduction in spermatogenesis. There is an overall tendency for testis size and
testosterone levels to decrease during winter, but this does not affect
reproductive effectiveness of an individual and 25% of calves born at Ocean
Park were conceived during this season.
5.6.1 Seasonal changes before and after the onset of sexual maturity
It is not certain if the reproductive system of sexually immature male dolphins
undergo seasonal changes. It was difficult to assess seasonal changes in the
sexually immature subjects because of rapid growth and maturational changes
that took place before onset of spermatogenesis. However analysis of each
year’s data separately helped to overcome this difficulty and to investigate
seasonal patterns. It has been speculated earlier that the season during which
sexual maturity is reached may have an influence on testis size, testosterone
level and sperm density. M4 reached sexual maturity during spring, testis size
and testosterone level, at sexual maturity and immediately afterwards, were
higher than those of M3, which reached maturity during autumn.
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In M4,
seasonal tendencies in testis size and testosterone level similar to those in older
mature subjects became apparent during the year of onset (aged 6 – 7 years) but
a pattern was not clear and the monitoring period after onset was short. There
was no clear pattern in M3 during a four year period after onset. Similarly
Brook (1997) did not find any seasonal change in the testes of ‘Molly’ before or
immediately after onset, at age 6 – 8 years.
There was some evidence to suggest that testosterone may begin to show a
seasonal tendency before onset of spermatogenesis, but this was only found in
one male. Study of more sexually immature individuals is required to confirm
this finding. Testosterone levels of ‘pubertal’ male Orcinus Orca were found to
show a seasonal pattern similar to sexually mature individuals, with higher levels
(4.2 ± 3.4ng/ml) in March to July (Robeck and Monfort, 2005b). However,
semen was not collected from these individuals, therefore, it is not certain they
were sexually immature.
This study is the first to investigate and describe seasonal changes before and
immediately after the onset of sexual maturity in T. aduncus. However, data
were based on a small sample size and findings should be interpreted with
caution.
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5.7 Characteristics of Tursoips aduncus ejaculates
In order to accurately characterise semen of Tursiops, ejaculates collected in
succession were evaluated individually.
Ejaculates were evaluated for pH,
volume, total motility (TM), progressive motility (PM), rate of progressive
motility (RPM), viability (VIA), density and count. In order to avoid the effect
of temperature change, ejaculates were incubated at 37oC until evaluation, all
glass slides were warmed before use and microscopic examination for motility
was carried out on a warm-stage at 37oC.
Ejaculated semen of T. aduncus remains in the liquid / aqueous state and does
not coagulate after standing for any length of time. The absence of coagulum
formation may be explained by the lack of the seminal vesicles and coagulating
glands in cetaceans (Slijper, 1966; Harrison, 1969a). These glands contribute the
necessary chemical constituents for semen coagulum formation in humans and
other terrestrial mammals (Coffey, 1988; Setchell and Brooks, 1988).
This study showed that, with suitable training, sexually immature T. aduncus are
capable of delivering semen with no detrimental effect on behaviour.
The
number of ejaculates and volume collected were less when subjects were
sexually immature. The pH of pre-onset semen (> 9) was higher than the pH of
semen that contained spermatozoa. Such difference may be due to a lack of
maturation in the secretory and absorptive functions of the epididymis, resulting
in differences in ionic compositions in the seminal fluid that may affect pH.
Semen that does not contain spermatozoa lacks the post ejaculation changes
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caused by metabolic activity of spermatozoa in the conversion of fructose to
lactic acid, which again may alter pH (Setchell and Brooks, 1988).
In M1 – M4, significant differences (P < 0.05) were found between the 1st and
2nd ejaculate in volume, density and sperm count. Typically, the 1st ejaculate in a
collection series had the largest volume; mean values were 15.2 – 44.8ml and the
maximum recorded was 99.5ml in M2. Overall, density of the 1st ejaculate was
significantly lower than the other ejaculates in the series, in which mean values
were 73 – 168 x106/ml. However, this ejaculate may occasionally be dense, with
densities as high as > 900 x 106/ml found in M1 – M4. Therefore, the 1st
ejaculate should not be discarded until all ejaculates have been collected and
examined under the microscope.
The density of the 2nd ejaculate was
significantly higher (P < 0.05) than that of the 1st and in general was the highest
of all ejaculates in a series. The mean values of this ejaculate were 220 – 580 x
106/ml and the maximum was 5170 x106/ml (see Section 5.3 of this chapter). To
date, there are a number of reports on various semen characteristics in T.
truncatus (Durrant et al., 1999a; Yoshioka et al., 1999; Robeck and O'Brien,
2004a; Robeck et al., 2005a; O'Brien and Robeck, 2006). The present study is
the first to note the 1st ejaculate as being significantly different to the subsequent
ejaculates in a collection series in T. aduncus. This finding may be due to
different collection protocols, or may represent differences between species;
further research on the semen of more dolphin species is required to confirm this.
Spermatozoa characteristics of ejaculates of T. aduncus were comparable to
those reported for T. truncatus (Durrant et al., 1999a; Robeck and O'Brien,
2004a). They were good even when collected from as early as the first month
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after onset; TM and VIA >90%, PM >80% and RPM > 4. It is said that possible
abnormalities associated with recent onset are likely to be due to a lack of
spermatozoal maturation in the epididymis, including an absence of progressive
motility and retained cytoplasmic droplets (Robaire and Hermo, 1988); the
present study did not find any obvious abnormalities in spermatozoa related to
recent onset in T. aduncus. Further tests, such as staining for morphology and
acrosome examination under phase-contrast and fluorescence microscopy should
confirm this.
There was some evidence that the spermatozoa characteristics of the 1st ejaculate
were poorer than subsequent ejaculates in a collection series.
Significant
differences in motility and viability between the 1st and 2nd ejaculates were found
in M1 and M2, but not in M3 or M4. This may represent differences between
individuals and / or age. Since the volume of the 1st ejaculate was largest in M1
and M2, the resultant low sperm densities may be responsible for the lower
sperm motility and viability found. A study of a cross-sectional sample of 250
men reported a positive moderate – good correlation (r = 0.4 – 0.55) between
sperm density and motility (Agarwal et al., 2003). The correlations found
between these parameters in the present study were lower (r < 0.4).
Analyses
of bull and human semen using a sperm motility index (SMI), derived by
automated optical methods, found that indices were lower at low densities, or
low percentages of motile cells (Bartoov et al., 1991; Hoflack et al., 2005).
These findings further suggest motility is inherently lower when density is low.
It was also noted that variability in SMI was higher (i.e., lower repeatability) at
low densities (≤ 22 x 106/ml), and also at ‘extremely’ high densities (≥ 1,135 x
106/ml) (Hoflack et al., 2005); repeatability results in the present study also show
291
this. Further, SMI decreased faster over time as density increased (Hoflack et al.,
2005) and these studies reported a significant linear relationship between SMI
and density up to < 40 – 50 x 106/ml. At densities 50 – 250 x 106/ml, SMI did
not differ significantly, but from 250 – 1,000 x 106/ml, SMI sharply decreased
(Hoflack et al., 2005). The authors suggested the condensed state of sperm cells
at high concentrations resulted in collisions in movement, thus reducing
progressive motility.
The ejaculates of dolphins are usually much more
concentrated than those of humans or bulls. In the present study, the weak
associations found between density and motility (r = 0.01 – 0.4) may be
explained by reduced motility (TM) due to time and reduced progressive motility
(PM and RFM) because of high density, at times (at densities > 1,500 x 106/ml)
to the extent that no progressive movement was possible, (i.e., both PM and
RFM were zero). A 1:1 dilution ratio of neat sample to diluent helped to
improve motility and increasing this ratio may facilitate more accurate
evaluation of extremely high density samples (> 2,000 x 106/ml).
The differences between the 1st and 2nd ejaculates have important implications
for assessing testicular function and semen collection for cryopreservation. To
fully evaluate reproductive condition of an individual Tursiops, multiple
ejaculates must be collected, as collection of the 1st ejaculate alone does not
reflect the full reproductive potential of an individual. Ejaculates should be
collected in separate containers, so that subsequent ejaculates in the collection
series are not ‘diluted’.
Successive ejaculate collection and evaluation of
ejaculates individually allow selection of the optimum sample for AI or
cryopreservation. The 1st ejaculate is usually less desirable because of the large
volume and relatively low density. Further processing is required to
292
‘concentrate’ the sample to acquire an optimal insemination dose in a restricted
volume and to make good use of cryo-tank space, as this is expensive.
Laboratory processing of raw ejaculates is demanding on both time and
resources and can usually be avoided by collection of more ejaculates. The
effect on spermatozoa of centrifugation and addition of seminal plasma to
‘concentrate’ or ‘dilute’ ejaculates has been investigated in T. truncatus
(Robeck and O'Brien, 2004a; Robeck et al., 2005a; O'Brien and Robeck, 2006)
but not in T. aduncus. While collection should be conducted separately, the
study strongly recommends retaining the 1st ejaculate until a collection session is
completed and all ejaculates in the series evaluated. This safeguards against an
individual not producing further ejaculates and provides a backup if subsequent
ejaculates are lost – e.g. contaminated by urine or pool water.
Ejaculate characteristics described in this study are for semen collected under
trained behaviour. Although unlikely, it is not known if these differ from the
characteristics of semen deposited into the female reproductive tract by natural
copulation. However, the successful application of AI in T. aduncus (Kinoshita
et al., 2004; Brook and Kinoshita, 2005), suggests any difference may not be
significant. Also, only the semen of T. aduncus was investigated and direct
comparison with the ejaculate characteristics of another dolphin species should
be made with caution.
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5.8 Testes size, ejaculate characteristics and mating system
The functional relationship between testis size, ratio to body size, and mating
system has been discussed for primates (Harcourt et al., 1981), cetaceans
(Landino, 1985; Payne and Bird, 1985) and some terrestrial mammals (Kenegy
and Trombulak, 1986).
A multi-male, competitive mating system is
characterised by high copulation frequencies, by one or a number of males, with
one female. This requires production of adequate sperm by each male in order to
compete with others and ensure paternity (Harcourt et al., 1981; Kenegy and
Trombulak, 1986).
Breeding in Tursiops follows this multi-male strategy
(Landino, 1985; Wells et al., 1987; Connor et al., 2000a; Mesnick and Ralls,
2002), which may explain the large testes, which are to produce large number of
ejaculates and sperm required (Matthews, 1950; Slijper, 1966; Harrison, 1969a;
Kenegy and Trombulak, 1986; Brook, 1997; Atkinson, 2002).
Further to this, coagulum formation in the semen of some species serves as a
‘copulation plug’ to prevent passage of spermatozoa by competitors (Devine,
1975; Mattan and Shephard, 1976; Dewsburry and Baumgardner, 1981). In
dolphins, however, semen does not coagulate, so other strategies are needed to
enhance the chance of successful fertilisation. Brook (1997) suggested the large
volume of semen, combined with delivery under high pressure, may serve to
displace semen deposited by competitors. The large volume of the 1st ejaculate
found in this study supports this hypothesis, in that it may serve to displace
semen from previous males. Brook (1997) also suggested the acutely tapered tip
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of the dolphin penis (Slijper, 1966) (see Figure 3.3 Section 3.3.7) allows high
pressure delivery of semen, facilitating displacement of semen already in the
vagina / pseudocervix. Physiologic characteristics of the large volume of the 1st
ejaculate also may serve to ‘prime’ the relatively long female tract for conditions
such as pH that are optimal for spermatozoa longevity and / or provide an
aqueous environment to facilitate sperm migration to the oocyte. Wild male
Tursiops are reported to sometimes ‘herd’ and ‘guard’ estrous females (Wells,
1999; Connor et al., 2000b), so repeated copulation within short intervals is
ensured. Re-insemination with smaller volumes of high density semen further
enhances success in sperm competition.
5.9 Reproductive ‘effectiveness’ and captive breeding
The present study defines sexual maturity as the onset and establishment of
spermatogenesis, denoted by the presence of spermatozoa in the semen. Once an
individual produces spermatozoa, it is physiologically capable of impregnating a
female and so is reproductively effective. Previous studies of wild cetaceans
made a distinction between sexual maturity and social maturity (Perrin et al.,
1976; Kasuya, 1984; Miyazaki, 1984), with the latter defined as the ability to
successfully compete for a female (Connor et al., 2000b).
For success in
competitive mating, maximum body size (i.e., physical maturity), is an
advantage, but usually occurs some years after sexual maturity. More recent
studies indicate that ‘social maturity’ may not be as significant as previously
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thought, even in the wild, physically immature, juvenile males have achieved
paternity through alternative strategies (Connor et al., 2000b; Krutzen et al.,
2004). For dolphins in captivity, the reported age of successful sires in
uncontrolled breeding conditions is usually around 12 years (Duffield et al.,
1999) to ≥ 16 years (Asper et al., 1992), although there are anecdotal reports of
younger males being successful (Duffield et al., 1999). Natural breeding can be
selectively controlled in captive groups, with a single male given uncontested
access to a female, resulting in pregnancy and delivery of healthy offspring
(Brook and Kinoshita, 2005).
Under these conditions, a male may be
reproductively effective as soon as sexual maturity is reached.
This study further showed that the reproductive effectiveness of recently
matured males may be enhanced by early collection and crypreservation of
ejaculates. Post-thaw results showed that ejaculates collected as soon as 2 – 14
months post-onset in T. aduncus are suitable for cryopreservation. Cryopreseved
semen, collected from M2 at 9y 7m of age, was used for AI in one female in
2003. This procedure was successful and a healthy male calf was born in 2004.
Results have made an important contribution to Ocean Park’s breeding
programme. Information on the testicular activities of young males was useful
for making breeding plans and for sire selection. For some time, two older
founder males sired all the calves born at Ocean Park. Inclusion of younger
males in a controlled breeding programme has helped to increase genetic
variation. Early cryopreservation of semen from young males also allows AI,
even after death, so maximising the available gene pool.
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5.10 Other factors affecting reproduction and reproductive
development
In the present study, changes found in testis size and testosterone levels were not
related to spermatogenesis, but these changes may be important for other reasons.
Bryden and Harrison (1986) speculated that the social status of a male dolphin
within a group might affect testes size. The authors observed that whilst the
large testes of bottlenose dolphins may be useful to promote successful
fertilisation through repeat matings in an aquatic environment, male seals which
breed with many females in a harem during one season do not have large testes.
Therefore, they concluded that size is a not a reliable indicator of function.
However, although both classed as marine mammals, seals and dolphins are
phylogenetically separate, have very different reproductive systems and breeding
strategies, and these may not be the only reasons for difference in testes size.
Dolphins in the wild form dynamic groups of specific composition and hierarchy
and they compete for mating opportunities (Wells et al., 1987; Connor et al.,
2000a; Connor et al., 2001), therefore status within the group may affect testis
size and testosterone level. In one small study of T. aduncus, Brook (1997)
found that the dominant males had the largest testes but were not necessarily the
largest animals. Similarly, the present study found that the dominant male (M1)
had the largest testes and highest testosterone level. However, this study group
consisted of only one other fully mature male and the remaining subjects were
much younger. There are anecdotal accounts of dominant male Tursiops having
297
much larger testes than subordinate males of the same age, kept in the same pool
(Brook, personal communication), but this has not been studied in detail.
Larger testes, higher testosterone levels and more rapid pubertal development are
reported in dominant adolescent macaques (Macaca mulatta) (Bercovitch, 1993)
and mandrills (Mandrillus sphinx) (Wickings and Dixson, 1992). It was not
within the remit of this study to investigate the effect of social status on
reproductive development, however findings suggest that there may be an
association.
Direct evidence of the effect of social interactions would be
difficult to isolate in this group, as husbandry and show procedures dictate that
group compositions change often. Mostly, the older, more dominant males (M1
and M6) were paired together, but they were sometimes placed with the group of
younger males. Behaviours did change and this may have caused additional
social pressure within the group, as it was not the ‘norm’. Also this is not a
typical structure in the wild, where older males tend to separate from younger,
‘bachelor’ groups (Wells et al., 1987; Connor et al., 2000b). Social instability is
a constant issue in captive male dolphin groups (McBride and Hebb, 1948;
Caldwell and Caldwell, 1972; Sweeney, 1990; Samuels and Gifford, 1997;
Waples and Gales, 2002). Some authors also believe that stress induced by
social instability renders an individual more susceptible to illness (Sweeney,
1990; St. Aubin and Dierauf, 2001; Waples and Gales, 2002) and can disrupt
reproductive functions (St. Aubin and Dierauf, 2001). More research is needed
to resolve whether dominant behaviour affects testis size / testosterone level, or
vice versa.
298
The effect of illness on testicular function has been reported (Kirby, 1990; Brook,
1997). Brook (1997) suggested illness may have delayed sexual maturity in one
pubertal T. aduncus. The present study found illness affected testis size and
testosterone levels in M2, M3 and M4, with direct declines and changes in
overall reproductive profiles. These effects are not always immediately obvious
and it is known that a dolphin may not show clinical symptoms until late in
illness (Sweeney, 1990).
The effects of social ‘stress’ and illness on
reproductive function in these animals is not documented in detail and,
surprisingly, there is little empirical research in this area, even though it may be
a direct cause of illness or early death in male dolphins.
Although both social stress and associated illness may impact reproduction and
are very important issues for the management of dolphins in captivity, very few,
controlled studies have been conducted in these areas. Further research to
investigate the relationship between reproduction and social status in male
dolphins is recommended.
299
Chapter 6
CONCLUSIONS
The present study found ultrasonography can be used to assess testicular
development in Tursiops aduncus.
The shape of the immature testes is
consistent throughout the structure and the echopattern is hypoechoic in
comparison to the hypaxialis lumborum m. At the onset of spermatogenesis, the
distal portion of the testes usually shows expansion, the ‘speckled’ appearance is
more apparent and echogenicity is comparable to that of the hypaxialis
lumborum. However, these changes were not seen in one subject, therefore such
signs may be useful to identify sexual maturity in most males but may not apply
in some.
Visualisation of the epipidymis was not reliable until a few months before sexual
maturity occurred. This may be a useful sign of imminent onset of
spermatogenesis in T. aduncus.
A rapid testis size increase a few months before onset was seen in one male in
the present study.
Rapid testis size increase may be useful in identifying
imminent sexual maturity. Testicular growth continues beyond the onset of
spermatogenesis and results indicate a period of more than four year may be
required for full adult testis size and function to be achieved in T. aduncus.
300
Testis size at onset of sexual maturity ranges from 8 – 18cm in length and 10 –
188cm3 in volume, but are mostly > 16cm and > 170cm3. Size alone can not be
relied upon to accurately identify sexual maturity.
Serum testosterone levels fluctuate widely and can not be relied on to assess
reproductive development in T. aduncus.
Serum testosterone levels differ
between subjects at sexual maturity and varies within individuals. Levels
recorded in immature males range from < 0.1 – 24.1ng/ml and in mature males
from 1.6 – 78.4ng/ml. There is a large overlap in levels, therefore testosterone
can not be used to differentiate reproductive status.
A rapid increase in testosterone coinciding with rapid increase in testis size was
recorded in one male. Again, this may be a useful indicator of imminent onset of
spermatogenesis in T. aduncus.
Testosterone levels recorded at onset of spermatogenesis in T. aduncus range
from 0.1 – 24.1ng/ml, although onset mostly occurred at levels of < 3.0ng/ml.
Monitoring of this parameter on an individual basis is still required as the
monthly profile of individual males differs markedly before and after onset.
The age at sexual maturity in T. aduncus ranges from 6y 7m – 7y 3m and body
length ranges from 200 – 226cm. Therefore, body length is not a good indicator
of sexual maturity.
The differences exhibited by one subject in this study indicate that semen
collection is required, in addition to testis size and serum testosterone level, for
correct identification of sexual maturity in young T. aduncus.
301
A weekly
collection schedule ensures more precise identification.
Centrifugation of
dolphin semen for spermatozoa detection can further ensure correct
identification of onset of spermatogenesis.
Sperm density in T. aduncus fluctuates erratically but can be maintained at high
levels year-round. Overall sperm density in T. aduncus in Hong Kong ranges
from 0 – 2494 x 106/ml and increases with age. From onset, some years are
required for spermatogenesis to reach full capacity.
Fully mature T. aduncus may exhibit rare azoospermia, or extremely low sperm
density (< 0.03 x 106/ml). This does not appear to be physiologically significant.
High density ejaculates (> 400 x 106/ml) are produced by T. aduncus as early as
the 1st month after onset of spermatogenesis, however, azoospermia or extremely
low densities (< 0.03 x 106/ml) normally occur for up to 1y 7m. There are
marked differences between individuals. Semen collection should continue
during this period to accurately characterise development on an individual basis
and for early and opportunistic cryopreservation of ejaculates of acceptable
densities.
There is an association between testis size and testosterone level in T. aduncus,
and these two parameters show seasonal changes.
Sperm density does not
correlate with testis size or testosterone level, and does not show a seasonal
pattern. There is no obvious temporal association between sperm density and
testosterone level.
302
The reproductive physiology of T. aduncus shows no clear seasonal pattern.
Although there are fluctuations in testis size and testosterone levels, these do not
show a dramatic seasonal ‘rut’ change. Male T. aduncus are reproductively
‘effective’ and capable of impregnating females in any month of the year and
25% of calves born at Ocean Park were conceived during winter.
The ejaculate quality of T. aduncus is good, even ejaculates collected as early as
the 1st month after onset. Ejaculate density is usually > 200 x 106/ml, total
motility and viability are > 90%, and progressive motility is > 80% with rates >
4.
The 1st ejaculate in T. aduncus is significantly different to remaining ejaculates
in a collection series. It is high in volume and low in density, but may be high in
sperm count. Subsequent ejaculates are lower in volume, much higher in density
and have high counts. Therefore, collection of only the 1st ejaculate does not
allow accurate evaluation of testicular function or development of an individual.
Also, for cryopreservation and AI, subsequent ejaculates may be used to
minimise sample handling and processing to attain a suitable cryo-package /
insemination dose. Ejaculate characteristics found in this study may be specific
to T. aduncus, thus, may not apply in other dolphin species.
Captive male T. aduncus can be reproductively effective as soon as they are
sexually mature. The study further shows that the reproductive effectiveness of
a recently mature male may be enhanced by collection and cryopreservation of
semen.
303
While the onset of sexual maturity may take place rapidly, full maturity is a
gradual process in male T. aduncus and may take several years.
Illness appears to affect testicular activities in T. aduncus. The effects of illness
on testis size and testosterone level may be directly associated with a decline, or
they may be indicated in the overall reproductive profile. In captivity, illness
can be a consequence of socially induced stress. Therefore, health and social
interactions must be taken into consideration when assessing reproductive
development in young bottlenose dolphins and may help to explain why
individual differences occur.
304
Chapter 7
SUMMARY AND RECOMMENDATIONS
The primary aim of this study was to determine the age at sexual maturity in
male Tursiops aduncus. Ultrasonography was used to evaluate testis appearance,
and serum testosterone levels were measured monthly. Sexual maturity was
defined by the onset of spermatogenesis, indicated by the first release of sperm
in the semen as detected on bright-field microscopy. Ejaculate traits in sexually
immature, recently mature, and fully mature males were investigated in detail. A
semen cryopreservation protocol, proven effective in bringing about births of
live calves, was used to investigate the freezability of the semen of recently
sexually mature dolphins, and results compared to a fully mature male. Data
were also used to evaluate any seasonal patterns in testis size and testosterone
levels in sexually immature and mature animals.
This study monitored one wild born and four known age male T. aduncus, for
periods of between three and five years. To date, this represents the most
extensive investigation of reproductive physiology in male Tursiops.
Understanding and information gained has improved controlled breeding
strategies at Ocean Park and added significantly to the body of knowledge about
male reproductive physiology in Tursiops.
305
A total of three subjects reached sexual maturity during the study period. The
onset of spermatogenesis occurred in two subjects before the age of seven years.
This is the first time the actual onset of spermatogenesis has been identified in
this species and results show this occurs earlier than previously reported (Brook,
1997). More data are required to confirm this.
At sexual maturity, testis appearance and size in two males were similar to
previous reports (Brook, 1997; Brook et al., 2000), however in one male the
testes remained small and hypoechoic. The sample size of the present study was
small and further research is required to determine whether the third male is
unusual or whether testis size at sexual maturity in T. aduncus can vary
markedly. Similar studies should also carefully consider the method by which
the presence of sperm is determined so that sexual maturity is precisely
identified and data between animals is compared accurately.
This study found that echopattern of the testicular parenchyma can indicate
progress in spermatogenesis after onset, but interpretation of ultrasonographic
images is subjective. It would be useful to develop a computerised technique to
quantitatively assess testicular echopatterns in dolphins. It would also be useful
to compare echopatterns with histology of specimen testes so that more precise
identification of testis maturity can be made in live dolphins.
Testosterone levels have limited usefulness in assessment of sexual maturity or
reproductive status in T. aduncus. Levels at onset of spermatogenesis were
significantly different between the three subjects (< 0.1 – 24.1ng/ml).
Such
large variations may represent inherent individuality, or reflect the pulsatile
nature of the hormone.
Thus, a few blood samples can not be used for
306
assignment of reproductive status in male T. aduncus. More research is needed
for better understanding of the basic physiology of this hormone in Tursiops,
including normal range of values and ways / patterns of fluctuation.
This study shows that spermatogenesis progressively increases in efficiency for
some time after onset. The number of years required to reach full adult capacity
in T. aduncus is not certain and requires further investigation.
Data show an expected correlation between testis size and testosterone level, but
there was no convincing correlation between sperm density and testis size, or
between sperm density and testosterone level.
The latter may be because
monthly testosterone data is inadequate for comparison. Further study of a
larger number of fully mature subjects is suggested to further investigate the
relationship between sperm density and other reproductive parameters in
Tursiops.
This study is the first to demonstrate that reproductive physiology of male T.
aduncus is not typically seasonal and that spermatogenesis is maintained year
round. So sexually mature males are capable of impregnating females in any
month of the year. This information is important for better management of
captive breeding programmes.
There were variations in reproductive parameters between individuals and within
an individual from year to year. They are likely to represent differences due to
age, stages of reproductive development, or possibly other factors. Since the
sample size was small, no conclusions can be drawn about individual variations
307
or asynchronicity of reproductive parameters in male Tursiops. Longitudinal
investigation of more individuals is required.
This is the first report of cryopreservation of sperm from recently matured T.
aduncus. Results show semen collected as soon as two months after onset of
spermatogenesis can be cryopreserved successfully. This information is
extremely important as curators can ensure preservation of genetic material at
the earliest opportunity. The number of ejaculates cryopreserved was, however,
small and the storage period relatively short.
Further study is required to
investigate the effect of longer storage times. Although this study did not find
any gross abnormalities in ‘young’ spermatozoa on bright-field examination,
future studies of detailed morphological analysis of thawed sperm would be
useful, as any abnormality can compromise fertilisation.
Based on results of the present study, stages of sexual development are identified
and the following grading system is proposed to classify the different stages:
Stage I:
First release / detection of spermatozoa;
Annual mean overall density < 10 x 106/ml;
Consecutive individual ejaculates without sperm or density < 0.03
x 106/ml;
Up to 1 year after onset
Stage II:
Annual mean overall density 10 – 80 x 106/ml;
Consecutive series of ejaculates without sperm or density < 0.03
x 106/ml;
Up to 2 years after onset
308
Stage III:
Annual mean overall density 100 - 200 x 106/ml;
Rare azoospermia or density < 0.1 x 106/ml;
From 3rd post-onset year onwards
Stage IV:
Full maturity / adult level;
Annual mean overall sperm density > 200 x 106/ml;
Rare azoospermia or density < 0.3 x 106/ml
Data are based on one fully mature and four recently matured males. Continual
monitoring of these subjects and more individuals is required to refine the
proposed staging format. Similar studies should take care to clearly define a
semen collection protocol, and to be stringent in ejaculate collection and
detection of spermatozoa, as these criteria may affect results and lead to
misinterpretations of reproductive status.
There has been much speculation that social status and patterns affect
reproductive activities in bottlenose dolphins. Social instability and aggression
between captive male Tursiops have been documented.
Over time, such
pressures may lead to stress and illness. Results of the present study indicate a
need for research in this area - to identify ‘stressors’ due to group composition,
or other interactions that may affect reproductive development. Such research is
important to improve wellbeing and enhance survival of male dolphins.
It is essential to have some method to reliably monitor the reproductive
development of male dolphins and identify when they reach sexual maturity, to
facilitate husbandry and controlled breeding.
Active selection for breeding
allows young, recently mature individuals to be represented in small / isolated
309
gene pools as early as possible. In this way, their reproductive lifespan is
maximised. Control of breeding through selection of mating pairs or assisted
reproductive techniques (ART) reduces inbreeding and optimises available
genetic materials. The development of these techniques demands understanding
of the reproductive physiology and status of both females and males. The overall
goal being to achieve a genetically healthy, self-sustaining captive population.
The author recognises the sample size in the present study was limited, however
it is the most comprehensive study of its type to date. The author does not claim
data define male Tursiops reproductive physiology, but provide further valuable
information about the species. It is hoped that these results and further questions
raised will provide a frame-work and guide for future investigations into
reproductive physiology of male Tursiops.
Tursiops are the most common
dolphin species kept in oceanaria today and so most accessible for detailed study.
Any understanding and knowledge gained from the better understanding and
management of this species may help conservation of other cetacean species,
both in captive situations and in the wild.
310
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APPENDIX 1
Study location – Ocean Theatre, Ocean Park, Hong Kong
There are six interconnected pools at one of Ocean Park cetacean facilities called
Ocean Theatre where the subjects of the study are maintained. The facility is
outdoors and semi-covered, except the main show demonstration pool. Each
pool is equipped with solid metal gates. Pool layout is shown in Figures A1.1
and A1.2. Pool dimensions are given in Table A1.1.
Figure A1.1: Picture of Ocean Theatre
A1
5
Main show
demonstration
pool
3
1
4
2
Figure A1.2: Configuration of pool layout at Ocean Theatre
Table A1.1: Dimensions of pools at Ocean Theatre
Pool
Min. horizontal
dimension (m2)
Min. depth
(m)
Surface area
(m2)
Volume
(m3)
Main
10.90
5.50
1975.00
3316.00
1
7.90
4.00
93.00
396.00
2
7.90
4.00
93.00
396.00
3
7.93
2.70
63.00
206.00
4
8.00
2.80
75.00
234.00
5
6.86
1.68
49.00
92.00
A2
APPENDIX 2
Testosterone Assay Protocol
For testosterone level evaluation a commercial test kit VIDAS Testosterone,
coupled with an automated VIDAS analyser (bioMérieux sa, France), was used.
Approximately 10ml of blood was collected by venipuncture from the dorsal
fluke vein. The blood was left to stand to clot in a plain tube and serum was
harvested by centrifugation at 4500rpm for 10 minutes. This test requires 200µl
of serum sample.
The information provided by the manufacturer of the kit is as follows.
A2.1 Principle
The assay principle of this test kit combines an enzyme immunoassay sandwich
method with a final fluorescent detection (ELFA).
A2.2 Measurement range
The measurement range of the VIDAS Testosterone kit extends between 0.1 and
13ng/ml. Samples with a concentration greater than 13ng/ml must be retested
after dilution 1:2.
The concentration of testosterone in the diluent and the
dilution factor must be taken into account to obtain the concentration in the
sample.
A3
A2.3 Detection limit
Defined as the smallest concentration of testosterone which is significantly
different from the zero concentration with a probability of 95%: 0.1ng/ml.
A2.4 Accuracy
A2.4.1 Standardisation
VIDAS testosterone test is calibrated according to the ID-GCMS technique
(Isotope Dilution – Gas Chromatography Mass Spectrometry).
A2.4.2 Dilution test
Three sera collected from male patients were diluted in a serum collected from a
female patient (0.26ng/ml) and tested singly in three series. The ratio of the
mean concentration measured over the mean expected concentration is expressed
as a mean recovery percentage (Table A2.1).
Table A2.1: Recovery concentration after dilution
Serum Dilution
Factor
1
2
3
1/1
1/2
1/4
1/8
1/1
1/2
1/4
1/8
1/1
1/2
1/4
1/8
Mean expected
concentration
(ng/ml)
9.71
4.98
2.62
1.44
11.07
5.66
2.96
1.61
8.09
4.17
2.22
1.24
Mean measured
concentration
(ng/ml)
9.71
4.98
2.37
1.19
11.07
5.60
2.51
1.26
8.09
3.95
1.81
1.02
A4
Mean recovery
percentage
(%)
100.0
100.0
90.6
82.9
100.0
98.8
84.7
78.4
100.0
94.7
81.8
82.6
A2.5 Precision
Four samples were tested in duplicates in 40 different runs (2 runs per day) on
the same VIDAS
Within-run reproducibility (intra-assay precision) and reproducibility (total
precision) were calculated according to the recommendations of NCCLS
Document EP5-T2 volume 12 number 4 (Table A2.2).
Table A2.2: Reproducibility (intra-assay and total precision)
Sample
1
2
3
4
N
80
80
80
80
Mean (ng/ml)
0.39
1.86
4.55
9.07
Within run
reproducibility
CV %
5.32
7.60
2.73
3.61
Reproducibility
CV %
11.52
11.93
7.8
5.8
A2.6 Specificity
Table A2.3: Cross-reactivity
Tested compound
Testosterone
5 α-hydrotestosterone
∆4-androstenedione
5 α-androstane -3α, 17β-diol
5-androstane- 3β, 17β-diol
19 nortestosterone (nandrolone)
11 β-hydroxytestostone
Desoxycroticosterone
Corticosterone
Progesterone
SDHA
Estradiol, Estriol, Estrone
Cross-reactivity %
100.00
0.98
0.07
0.14
0.02
6.40
0.85
<0.01
<0.01
<0.01
<0.01
<0.01
A5
A2.7 Correlation
216 serum samples were testes in parallel using the VIDAS kit and a
radioimmunoassay kit. The results were:
VIDAS = 1.060 RIA = 0.137 with a correlation deviation of 0.966
A2.8 Comparison with other tests methods
Testosterone concentrations of a sample determined using kits from different
manufacturers may vary according to the assay and calibration techniques. If a
different assay technique is used and as part of patient follow-up, the laboratory
must confirm concentration obtained with the previous technique.
A6
APPENDIX 3
Semen collection form
A7
APPENDIX 4
Extender Preparation (400ml)
The extender used in the protocol was prepared in-house and was based on the
descriptions by Seager (1969) and Schroeder (1990a).
Eggs were purchased from a fresh food market on the day of extender
preparation. They were swabbed with alcohol, then rinsed in purified water and
dried with sterile gauze. After splitting the eggshell, the yolk was transferred
carefully between the two halves to allow the albumen to separate. For further
removal of albumen, the yolk was placed on a pad of sterile gauze and rolled
slowly across the pad to allow sufficient time for absorption. An 18G needle
was used to score the surface of the yolk near the edge. The yolk, loosely
wrapped in the mat, was lifted and tilted at an angle to allow the content to
empty into a sterile container. The yolk sac adhered to the mat was discarded.
Eggs were processed in this manner, each with new sterile gauze pads, until the
volume of yolk required was achieved.
To make up the lactose solution, 44g of lactose (D(+)-Lactose, Riedel-de Haën,
Seelze GmbH, Germany) was first added to 180ml deionised distilled water
(Gibco Products, Invitrogen Corp., Hong Kong) over a hot plate with a stirrer
(Nuova II, Thermolyne Corp., U.S.A.). When a solution state was reached, more
water was added to make up a volume of 276ml. 80ml of prepared egg was
A8
added to the lactose solution. Once the temperature of the mixture reached 60oC,
it was maintained at this temperature for 60 minutes to inactivate protein
components of the yolk. The mixture was cooled to room temperature and 4ml
of antibiotic solution, containing 1.08g of penicillin G potassium (Irvine
Scientific, U.S.A.) and 2.25g of dihydrostreptomycin sulfate (Gibco BRL, Life
Technologies, U.S.A.), was added.
The entire mixture was stirred and
transferred to a homogeniser (Omi Mixer, Omi International, U.S.A.).
Homogenisation was carried out at control-setting 4.25 for 2 minutes, followed
by 30 seconds rest and then further hemogenisation at the same setting for
another 30 seconds.
The final extender volume of 400ml was divided into aliquots of 5 – 10ml and
stored frozen at -70oC (Revco, Thermo Electron Corp., U.S.A.). Prior to use, the
aliquots required were thawed at room temperature.
unused was discarded.
A9
Any thawed extender
APPENDIX 5
Ultrasonographic evaluation of dolphin testes: Repeatability and
reproducibility study
In a longitudinal study, it is important that methods used to take measurements
are consistent, so that results are reproducible and repeatable. Any differences in
size of organs detected over time may therefore be attributed to physiological
changes, such as growth.
The design of the protocol for poolside ultrasonographic examination of
dolphin testes takes into account the ease of training dolphins for cooperative
behaviours and negates the need to remove the animal from the water. However,
this does mean that measurements are prone to error due to movement. In
particular, since testes length is an indirect measurement of the distance between
two marked points on the surface of the animal’s skin, the subject must be very
still, the body straight and in a relaxed state when measurements are taken.
For the reasons given above, an inter- and intra-operator study was conducted to
assess the repeatability and reproducibility of the ultrasonographic technique
used for measuring dolphin testes dimensions. Measurements were performed by
two trained operators, one of whom was the author.
A10
A5.1 Materials and Methods
The ultrasonographic examination protocol given in Section 3.3.5 was used to
conduct longitudinal measurements to obtain testes length and cross-sectional
measurements to obtain testes circumference, depth and width. Five subjects
(M1, M2, M3, M4 and M6) aged 4 – 19 years were examined by two operators
(Operator A and Operator B), independently.
Operator A is a trained ultrasonographer by profession, with more than 16 years
of ultrasonographic imaging experience in dolphins and other animal species.
Operator B, the author, was trained by Operator A for a 2-month period. First, by
demonstration and on-site supervision followed by supervision through
reviewing of printed images.
For inter-operator investigation, each subject was examined once by each
operator. The operators took turns to examine both sides of one subject
before going onto the next. Operators did not communicate with each other until
all subjects were examined.
Due to limited animal access time, intra-operator investigation was carried
on a separate day with Operator B only. The testes of five subjects were
measured twice. Subjects were rotated each time when both sides were
examined, so as to maintain an interval of approximately 10 minutes between
repeated measurements.
A11
To reduce bias, Operator B was blinded from measurements taken while
on-site. Un-scaled plastic strips were used to record testes lengths. The
distances recorded on these strips were later read against a ruler to obtain
exact length measurements.
A piece of cardboard was placed over the
monitor screen where cross sectional measurements were displayed. These
measurements were recorded in print for review off-site.
To test intra- and inter-operator reliability, intra-class correlation coefficient,
ICC, models 2 and 3 (Portney and Watkins, 2000) in SPSS 14.0 for windows,
were used respectively. Also limits of agreement and repeatability coefficients
(Bland and Altman, 1986) were ascertained to assess rater agreement and
measurement variability.
A5.2 Results
Measurements (testis length, TL, circumference, TC, depth, TD and width TW)
taken by Operator B for intra-operator test are shown in Table A5.1. In intraoperator analysis, ICCs for all the measurements were > 0.9, which
indicates repeatability was good (Table A5.2). Most repeatability coefficients
were < 1.0cm, indicating 95% of the differences found between repeated
measurements were within 1cm which was considered acceptable (Table A5.2,
Figures A5.1 – A5.8).
A12
Table A5.1: Ultrasonographic measurements taken by Operator B
Length
cm
L
R
1st measurements
19.2
M1
17.8
M2
20.1
M3
12.8
M4
18.4
M6
2nd measurements
19.5
M1
17.2
M2
19.4
M3
12.8
M4
18.6
M6
Circumference
cm
L
R
Depth
cm
L
R
Width
cm
L
R
20.6
17.3
21
13.3
23.0
18.3
13.3
13.4
7.4
15.2
17.8
11.9
13
7.2
15.7
6.2
4.4
4.5
2.4
4.9
6.0
3.8
4.2
2.3
5.0
5.5
4.1
4
2.3
4.7
5.3
3.8
4.0
2.2
5.0
20.8
17.6
20.5
13.6
23.0
17.9
13.3
13.6
7.0
15
18.1
12.2
14.5
6.7
16.0
6.1
4.3
4.7
2.3
4.8
6.1
3.9
4.7
2.2
5.2
5.0
4.0
4.0
2.0
4.7
5.4
3.9
4.5
2.1
5.0
Table A5.2: Intra-operator analysis - Operator B
Intra-operator
analysis:
Operator B
Length
L
R
Circumference L
R
Depth
L
R
Width
L
R
ICC
Model
(3,1)
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.98
Difference between
1st & 2nd
measurements
Mean
SD
0.16
0.46
-0.06
0.34
0.16
0.26
-0.38
0.72
0.04
0.13
-0.16
0.22
0.10
0.10
-0.12
0.23
A13
Repeatability
coefficient
(2SD)
0.92
0.68
0.52
1.40
0.26
0.44
0.20
0.46
1.1
0.9
0.7
0.5
Difference
0.3
between
2 measurements 0.1
(cm)
-0.1
-0.3
-0.5
-0.7
-0.9
0
5
10
15
20
25
Mean left length measurement (cm)
Figure A5.1: Difference against mean for left TL taken by Operator B
(Dotted line denotes mean, dashed line denotes mean ±2SD)
1
0.8
0.6
0.4
Difference
0.2
between
2 measurements 0
(cm)
-0.2
-0.4
-0.6
-0.8
-1
0
5
10
15
20
25
Mean right length measurement (cm)
Figure A5.2: Difference against mean for right TL taken by Operator B
A14
1
0.8
0.6
0.4
Difference
0.2
between
2 measurements 0
(cm)
-0.2
-0.4
-0.6
-0.8
-1
0
2
4
6
8
10
12
14
16
18
20
Mean left circumference measurement (cm)
Figure A5.3: Difference against mean for left TC taken by Operator B
1.2
1
0.8
0.6
0.4
0.2
0
Difference
-0.2
between
2 measurements -0.4
(cm)
-0.6
-0.8
-1
-1.2
-1.4
-1.6
-1.8
-2
0
2
4
6
8
10
12
14
16
18
20
Mean right circumference measurement (cm)
Figure A5.4: Difference against mean for right TC taken by Operator B
A15
1
0.8
0.6
0.4
Difference
0.2
between
2 measurements 0
(cm)
-0.2
-0.4
-0.6
-0.8
-1
0
1
2
3
4
5
6
7
Mean left depth measurement (cm)
Figure A5.5: Difference against mean for left TD taken by Operator B
1
0.8
0.6
0.4
Difference
0.2
between
2 measurements 0
(cm)
-0.2
-0.4
-0.6
-0.8
-1
0
1
2
3
4
5
6
7
Mean right depth measurement (cm)
Figure A5.6: Difference against mean for right TD taken by Operator B
A16
1
0.8
0.6
0.4
Difference
0.2
between
2 measurements 0
(cm)
-0.2
-0.4
-0.6
-0.8
-1
0
1
2
3
4
5
6
Mean left width measurement (cm)
Figure A5.7: Difference against mean for left TW taken by Operator B
1
0.8
0.6
0.4
Difference
0.2
between
2 measurements 0
(cm)
-0.2
-0.4
-0.6
-0.8
-1
0
1
2
3
4
5
6
Mean right width measurement (cm)
Figure A5.8: Difference against mean for right TW taken by Operator B
A17
Measurements taken by Operators A and B for inter-operator test are shown in
Table A5.3. In inter-operator analysis, ICCs for all the measurements were
again > 0 .9, which indicates the reproducibility was good (Table A5.4). The
limits of agreement were mostly close to or within ± 1cm, which indicate good
agreement and they were comparable to repeatability coefficients (Table A5.4,
Figures A5.9 – A5.16). The limits of agreement were larger for circumference
measurements (Figures A5.11 – A5.12).
Table A5.3: Ultrasonographic measurements taken by Operators A & B
Length
Circumference Depth
cm
cm
cm
L
R
L
R
L
R
Operator A measurements
21.0 20.8 19.5
20.7 6.3 7.0
M1
20.2 19.3 15.9
14.6 5.1 4.9
M2
19.8 20.8 14.2
14.7 4.6 4.8
M3
9.5 12.4
8.4
7.1
2.8 2.3
M4
18.8 22.3 16.5
18.7 5.5 6.0
M6
Operator B measurements
20.4 20.4 17.9
18.8 5.8 6.3
M1
19.5 19.6 14.9
13.9 4.9 4.5
M2
19.2 19.9 13.4
13.8 4.4 4.5
M3
9.6 12.7
7.2
7.3
2.4 2.4
M4
18.0 22.5 17.2
17.6 5.7 5.9
M6
A18
Width
cm
L
R
6.1
5.0
4.4
2.5
5.1
6.2
4.4
4.5
2.2
5.9
5.7
4.6
4.2
2.2
5.3
5.6
4.3
4.3
2.2
5.3
Table A5.4: Inter-operator analysis
Length
L
R
Circumference L
R
Depth
L
R
Width
L
R
Difference between
ICC
model operator measurements
(2,1)
Mean
SD
0.99
0.52
0.36
0.99
0.10
0.53
0.97
0.78
0.88
0.97
0.88
0.76
0.97
0.22
0.29
0.97
0.28
0.30
0.97
0.18
0.25
0.97
0.30
0.28
Limit of
agreement
(mean ± 2SD)
0.52 ± 0.71
0.10 ± 1.07
0.78 ± 1.76
0.88 ± 1.51
0.22 ± 0.54
0.28 ± 0.61
0.18 ± 0.50
0.30 ± 0.57
1.4
1.2
1
0.8
Difference
between
0.6
2 measurements
(cm)
0.4
0.2
0
-0.2
-0.4
0
5
10
15
20
25
Mean left length measurement (cm)
Figure A5.9: Difference against mean for left TL taken by Operators A &
B (Dotted line denotes mean, dashed line denotes mean ±2SD)
A19
1.2
1
0.8
0.6
0.4
Difference
between
0.2
2 measurements
(cm)
0
-0.2
-0.4
-0.6
-0.8
-1
0
5
10
15
20
25
Mean right length measurement (cm)
Figure A5.10:
Difference against mean for right TL taken by
Operators A & B
2.6
2.4
2.2
2
1.8
1.6
1.4
Difference
1.2
between
1
2 measurements 0.8
(cm)
0.6
0.4
0.2
0
-0.2
-0.4
-0.6
-0.8
-1
0
2
4
6
8
10
12
14
16
18
20
Mean left circumference measurement (cm)
Figure A5.11: Difference against mean for left TC taken by
Operators A & B
A20
2.4
2.2
2
1.8
1.6
1.4
1.2
Difference
1
between
0.8
2 measurements
0.6
(cm)
0.4
0.2
0
-0.2
-0.4
-0.6
-0.8
-1
0
5
10
15
20
25
Mean right circumference measurement (cm)
Figure A5.12: Difference against mean for right TC taken by
Operators A & B
1
0.8
0.6
0.4
Difference
0.2
between
2 measurements 0
(cm)
-0.2
-0.4
-0.6
-0.8
-1
0
1
2
3
4
5
6
7
8
9
10
Mean left depth measurement (cm)
Figure A5.13: Difference against mean for left TD taken by
Operators A & B
A21
1
0.8
0.6
0.4
Difference
0.2
between
2 measurements 0
(cm)
-0.2
-0.4
-0.6
-0.8
-1
0
1
2
3
4
5
6
7
Mean right depth measurement (cm)
Figure A5.14: Difference against mean for right TD taken by
Operators A & B
1
0.8
0.6
0.4
Difference
0.2
between
2 measurements 0
(cm)
-0.2
-0.4
-0.6
-0.8
-1
0
1
2
3
4
5
6
7
8
9
10
Mean left width measurement (cm)
Figure A5.15: Difference against mean for left TW taken by
Operators A & B
A22
1
0.8
0.6
0.4
Difference
0.2
between
2 measurements 0
(cm)
-0.2
-0.4
-0.6
-0.8
-1
0
1
2
3
4
5
6
7
Mean right width measurement (cm)
Figure A5.16: Difference against mean for right TW taken by
Operators A & B
A5.3 Discussion and Conclusion
This study was conducted to investigate the reproducibility and repeatability of
an ultrasonographic protocol to measure dolphin testes dimensions conducted
poolside by two operators. Repeatability and reproducibility were good. The
larger limits of agreement in TC may be due to the projection of the transducer
or animal movement when measurements were recorded. TC, TD and TW were
taken at the largest cross section of the testis, the transducer was also positioned
to ensure ultrasound beam was projected perpendicular the surface of the organ.
Movement may have caused slight displacement of the transducer.
A23
In comparison to another intra-operator study (Brook, 1997) which used
the same protocol to measure dolphin TL at poolside, standard deviations in the
TL in this study was smaller. Brook (1997) also found that length measurements
taken with the subject out of the water and positioned in lateral recumbency on
the poolside deck were more accurate. However, for the purpose of this study
which required weekly measurements of the testes, removal of subjects from the
water for ultrasonographic examination under manual restraint, was considered
to be highly impractical and not warranted give repeatability was good.
In conclusion, repeatability and reproducibility of the ultrasonographic technique
for measuring dolphin testes with the animals remaining in water were both
good. However, operators must be cautious of movement caused by the water
current and ensure correct animal body presentation.
A24
APPENDIX 6
Dolphin semen analysis: Repeatability and reproducibility study
In the absence of technology such as computer assisted sperm analysis (CASA)
manual evaluation of semen, particularly sperm motility, is subjective (Keel,
1990; Robeck et al., 1994; O'Brien and Robeck, 2006) Evaluation of dolphin
semen presents further challenges because sperm motility and density are in
general much higher than the reference values given by WHO (1999) for human
semen. Therefore an intra- and inter-rater study was carried out to assess the
repeatability and reproducibility of dolphin semen evaluation.
A6.1 Materials and Methods
The protocol for dolphin semen quality evaluation protocol given in Section
3.3.8 was used to test reliability for the following parameters:
1. Total motility (TM), %
2. Progressive motility (PM), %
3. Rate of progressive motility (RPM) (ranked on a subjective scale 0 - 5,
where 0 denotes no progressive movement and 5 denotes most rapid
progressive movement)
4. Viability (VIA), %
5. Density, 106/ml
A25
6. pH
7. Volume, ml
To reduce bias, all sample IDs were masked by two technicians who did not
participate in the evaluation process (non-raters).
Samples for motility
evaluation were prepared by the non-raters. Each ejaculate sample was valuated
twice by 2 operators (Raters A and B) independently.
Rater A is a clinical laboratory technician with five years experience in handling
and evaluating dolphin semen. Rater B, the author, was trained by Rater A for a
two-month period, through demonstration and on-site supervision.
For motility (TM, RPM and RPM) evaluations, 12 ejaculate samples were
used. After preparation, mounted slides were left to stand on the warm stage for
30 seconds before evaluation. The two raters took turns in evaluating each
sample.
For VIA and density evaluation, 8 ejaculate samples were used. Sample
preparation for density evaluation was carried by the two raters. Results were
recorded on worksheets.
To test intra- and inter-rater reliability, intra-class correlation coefficient, ICC,
models 2 and 3 (Portney and Watkins, 2000) in SPSS 14.0 for windows, were
used respectively. Also limits of agreement and repeatability coefficients (Bland
and Altman, 1986) were ascertained to assess rater agreement and measurement
variability.
A26
A6.2 Results
Semen evaluation scores by Rater A for intra-operator test are shown in Table
A6.1. In intra-rater analysis, ICCs for Rater A ranged from 0.25 and 0.99 (Table
A6.2). Repeatability coefficients for Rate A were overall were large (Table A6.2,
Figure A6.1 – A6.5), except that of viability, < 5%. Repeatability was lower in
PM and RPM and higher in VIA and density.
Table A6.1: Semen evaluation scores by Rater A
Sample
1
2
3
4
5
6
7
8
9
10
11
12
Sample
13
14
15
16
17
18
19
20
TM (%)
1st
2nd
50
5
85
95
40
5
95
95
80
80
95
90
98
98
95
99
98
85
75
80
85
85
90
95
Viability
(%)
1st
2nd
93.0
93.0
92.0
91.0
97.0
97.0
98.0
98.0
97.0
95.0
98.0
94.5
94.5
95.5
94.0
94.0
PM (%)
1st
2nd
85
50
85
95
80
70
95
95
85
80
95
95
95
95
95
99
98
80
80
85
80
85
95
95
Density
(x106/ml)
1st
2nd
500
450
550
460
855
945
415
345
530
550
961
930
1655
1785
1020
985
A27
RPM
1st
4.5
4
2.5
5
3
4.5
4.5
4.5
5
4
3.5
5
2nd
0.5
4.5
2.0
5
4.5
4.5
4.5
5
3.5
4
4
5
Table 6.2:
Intra-rater
analysis:
Rater A
TM %
PM %
RPM
Viability %
Density
(x106/ml)
Intra-rater analysis – Rater A
Difference between
1st and 2nd scores
Mean
SD
6.17
16.93
3.67
12.35
0.25
1.37
0.69
1.44
8.25
73.72
ICC
model (3,1)
0.81
0.39
0.25
0.80
0.99
Repeatability
coefficient
(2SD)
33.86
24.71
2.75
2.88
147.44
45
40
35
30
25
20
Difference 15
between
10
2 scores
5
(%)
0
-5
-10
-15
-20
-25
-30
0
20
40
60
80
100
120
Mean total motility (%)
Figure A6.1: Difference against mean for TM (%) scored by Rater A
A28
30
25
20
15
Difference
between
2 scores
(%)
10
5
0
-5
-10
-15
-20
-25
0
20
40
60
80
100
120
Mean progressive motility (%)
Figure A6.2: Difference against mean for PM (%) scored by Rater A
3.5
3
2.5
2
1.5
Difference 1
between
0.5
2 scores
0
(%)
-0.5
-1
-1.5
-2
-2.5
-3
0
1
2
3
4
5
6
Mean rate of progressive motility
Figure A6.3: Difference against mean for RPM scored by Rater A
A29
2.5
2
1.5
1
Difference 0.5
between
2 scores
0
(%)
-0.5
-1
-1.5
-2
-2.5
91
92
93
94
95
96
97
98
99
Mean viability (%)
Figure A6.4: Difference against mean for VIA scored by Rater A
160
140
120
100
80
60
Difference
between
2 counts
(x106/ml)
40
20
0
-20
-40
-60
-80
-100
-120
-140
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Mean density (x106/ml)
Figure A6.5: Difference against mean for density counted by Rater A
A30
Semen evaluation scores by Rater B for intra-operator test are shown in Table
6.3. In intra-rater analysis, ICCs for Rater B ranged from 0.05 and 0.99.
Repeatability coefficients for Rate B were overall were large (Table 6.4, Figure
6.6 – 6.10), except that of viability, < 5%. Again repeatability was lower in PM
and RPM and higher in VIA and density (Table 6.4). Repeatability coefficients
for Rate A were large (Figure 6.1 – 6.5), except that of viability, < 5%.
Table A6.3: Semen evaluation scores by Rater B
Sample
1
2
3
4
5
6
7
8
9
10
11
12
Sample
13
14
15
16
17
18
19
20
TM (%)
1st
2nd
80
35
90
95
35
15
94
96
95
85
97
95
93
95
97
95
98
80
80
85
80
85
95
95
Viability
(%)
1st
2nd
93.5
94.5
95.0
94.5
95.5
95.5
98.5
97.5
97.5
94.5
96.5
98.0
96.5
96.5
93.5
94.0
PM (%)
1st
2nd
50
0
30
85
90
10
90
95
85
90
90
95
85
92
95
97
95
80
92
80
85
80
95
95
Density
(x106/ml)
1st
2nd
513
535
495
510
1160
1145
405
430
575
695
1195
1225
1475
1430
820
880
A31
RPM
1st
4.0
3.5
4.5
4.0
4.0
4.5
3.5
4.0
4.5
3.5
3.0
4.5
2nd
0
4
2.5
4.5
4.5
4.5
3.5
4.5
3.5
3.0
3.5
4.5
Table A6.4: Intra-rater analysis – Rater B
Intra-rater
analysis:
ICC
Difference between
Repeatability
model (3,1)
1st and 2nd scores
coefficient
Rater B
Mean
SD
(2SD)
TM %
0.80
4.92
14.21
28.42
PM %
0.29
7.33
32.79
65.58
RPM
0.05
0.42
1.36
2.73
VIA %
0.65
0.19
1.39
2.77
Density
0.99
-26.56
49.08
96.16
(x106/ml)
45
40
35
30
25
20
Difference
between 15
2 scores 10
(%)
5
0
-5
-10
-15
-20
-25
0
20
40
60
80
100
120
Figure 6.6:
Difference against mean for TM (%) scored by Rater B
A32
70
60
50
40
30
Difference 20
between
2 scores 10
(%)
0
-10
-20
-30
-40
-50
-60
0
20
40
60
80
100
120
Figure A6.7: Difference against mean for PM (%) scored by Rater B
3.5
3
2.5
2
1.5
Difference 1
between
0.5
2 scores
0
(%)
-0.5
-1
-1.5
-2
-2.5
-3
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Figure A6.8: Difference against mean for RPM scored by Rater B
A33
3.5
3
2.5
2
1.5
Difference 1
between
0.5
2 scores
0
(%)
-0.5
-1
-1.5
-2
-2.5
-3
93.5
94
94.5
95
95.5
96
96.5
97
97.5
98
98.5
Mean viability (%)
Figure A6.9: Difference against mean for VIA scored by Rater B
80
60
40
20
0
Difference
between -20
2 counts
(x106/ml) -40
-60
-80
-100
-120
-140
0
200
400
600
800
1000
1200
1400
1600
Figure A6.10: Difference against mean for density counted by Rater
A34
For both Raters repeatability was lower in PM and RPM and higher in VIA and
density. This was likely to be due to the more objective nature in the evaluations
of VIA and density.
In inter-rater analysis, ICCs for parameters ranged from 0.17 to 0.91 (Table
A6.5). The ICCs indicate that inter-rater reliability, or reproducibility, of the
protocol varied for the different parameters. Limits of agreement for all the
parameters, with the exception of VIA, were large, which indicates large score
variability between the raters (Table A6.5, Figures A6.11 – A6.15).
reproducibility for PM and RPM was lower and higher for TM, VIA and density.
Table A6.5: Inter-rater analysis
Interrater analysis
TM %
PM %
RPM
Viability %
Density
(x106/ml)
ICC
model (2,1)
(with 1st
score)
0.81
0.20
0.17
0.73
0.91
Difference between
rater scores
Mean
SD
Limit of
agreement
(mean ± 2SD)
-5.08
7.17
0.21
-0.38
-18.94
-5.08 ± 20.62
7.17 ± 38.43
0.21 ± 1.73
-0.38 ± 3.15
-18.94 ± 356.67
10.31
19.22
0.86
1.58
178.33
A35
20
15
10
5
0
Difference
between -5
2 scores
(%)
-10
-15
-20
-25
-30
-35
0
10
20
30
40
50
60
70
80
90
Figure A6.11: Difference against mean for TM scored by Raters A & B
50
45
40
35
30
25
20
Difference
15
between
2 scores 10
5
(%)
0
-5
-10
-15
-20
-25
-30
-35
0
10
20
30
40
50
60
70
80
90
Figure A6.12: Difference against mean for PM (%) scored by Raters A & B
A36
3
2.5
2
1.5
1
Difference
0.5
between
2 scores
0
(%)
-0.5
-1
-1.5
-2
-2.5
-3
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Figure A6.13: Difference against mean for RPM scored by Raters A & B
5
4.5
4
3.5
3
2.5
2
1.5
Difference 1
between 0.5
2 scores
0
(%)
-0.5
-1
-1.5
-2
-2.5
-3
-3.5
-4
-4.5
-5
73.5
74
74.5
75
75.5
76
76.5
77
77.5
78
78.5
79
Mean viability (%)
Figure A6.14: Difference against mean for VIA scored by Raters A & B
A37
350
300
250
200
150
100
Difference 50
between
0
2 counts
(x106/ml) -50
-100
-150
-200
-250
-300
-350
-400
0
200
400
600
800
1000
1200
1400
1600
1800
Figure A6.15: Difference against mean for density counted by Raters A &
A6.3 Discussion and Conclusion
This study was conducted to investigate the reproducibility and repeatability of a
dolphin semen quality evaluation protocol carried out by two raters. Two
statistical techniques, ICC and one based on a 95% confidence interval were use
to analysis repeated measurements.
Repeatability was comparable for each rater and was variable for the
different parameters. The parameter with the lowest repeatability was RPM
followed by PM. The reason for this finding may be the delay in time between
the first and second evaluations of each sample. Over time the percentage of
cells showing progressive motility and their rate may differ, particularly when
A38
sperm density was very high (> 1, 000 x 106/ml). In addition, it was also
possible that the motility parameters were prone to differences between
preparations of the same ejaculate samples. Repeatability was high in TM. This
parameter is less specific than PM and RPM, and was comparatively less
affected by time delay. The methods to determine VIA and density were more
objective, thus, repeatability was good.
Measurement variability was high as indicated by large 2SD values
(repeatability coefficients). For motility (TM, PM and RPM) scores, time delay
was again a possible cause, particularly when sperm density was very high.
Differences between aspirates of the same ejaculate sample were also likely,
particularly if samples are not homogenous or mixed thoroughly. When only a
few is present in one microscopic field (at x400 total magnification), because of
uneven distribution of cells on a slide preparation or low sperm density,
examination of several fields was required which may cause higher viability.
Within-rater inconsistencies must also be considered.
Reproducibility was similar to repeatability. Lower reproducibility for PM and
RPM further suggests the evaluations of these parameters were subjective.
Reproducibility for TM, VIA and density was good. Limits of agreement for the
motility parameters were better for inter-rater analysis; smaller measurement
variability was found between raters. Such findings may be due to the shorter
time delay between rater evaluations of the same preparation, compared to the
time delay in the repeated preparations of one ejaculate sample.
A39
In general, the limits of agreements for all the parameters, except VIA (< 5%),
were large. Clinically, the interval between limits of agreement may be useful in
establishing guideline values for technicians to decide whether to discard or
retain a sample for further processing or storage, so a small interval is preferable.
Since dolphin semen is generally of good quality and very high density, and that
all semen evaluations were carried out by one technician, such interval may be
regarded with less critical importance.
For density, the limit of agreement suggests large discrepancies between the
raters. Such differences were likely to be due to individual variations in sample
preparation.
Again, for data collection in the present study, all sample
preparations for density evaluation were carried out by one technician and
particular care was taken in preparing very dense samples.
In conclusion, the protocol used to evaluate the quality of dolphin semen was
repeatable and reproducible to some degree.
While evaluation of total
motility was easier for the two technicians to agree, evaluations of progressive
motility and rate were subjective. These parameters are often difficult to assess
manually, as dolphin semen is very dense and cell movement is very rapid and
may not be uniform throughout the slide preparation. Lastly, timing is a very
important factor and meticulous care must be taken in processing and preparing
dolphin semen samples for evaluation.
A40
APPENDIX 7
Ultrasonographic measurements of Tursiops aduncus testis size
Table A7.1:
Date
9-Oct-02
16-Oct-02
23-Oct-02
30-Oct-02
6-Nov-02
13-Nov-02
20-Nov-02
27-Nov-02
4-Dec-02
12-Dec-02
18-Dec-02
24-Dec-02
31-Dec-02
8-Jan-03
15-Jan-03
22-Jan-03
28-Jan-03
5-Feb-03
12-Feb-03
19-Feb-03
25-Feb-03
5-Mar-03
12-Mar-03
19-Mar-03
25-Mar-03
2-Apr-03
9-Apr-03
17-Apr-03
27-Apr-03
5-May-03
13-May-03
21-May-03
26-May-03
03-Jun-03
12-Jun-03
18-Jun-03
25-Jun-03
09-Jul-03
16-Jul-03
23-Jul-03
30-Jul-03
RTL
20.00
21.60
21.50
21.30
19.30
22.60
20.20
21.10
19.40
21.00
21.00
19.90
20.50
19.00
19.20
20.00
20.60
20.60
20.70
21.00
20.60
21.10
21.00
21.80
20.80
19.80
20.40
21.00
20.30
20.70
21.40
20.20
20.60
19.80
19.40
20.30
20.60
20.50
20.60
20.10
RTC
19.50
RTW
6.30
6.50
RTD
6.20
5.90
RTV
554.65
588.14
18.70
18.70
18.70
19.40
18.90
19.60
19.00
18.00
17.70
18.90
16.80
17.40
17.50
17.90
18.10
18.10
18.10
18.40
18.70
18.70
19.50
19.50
19.10
19.10
19.00
18.90
17.30
19.10
18.40
19.80
18.90
19.40
19.00
18.20
18.90
18.30
19.10
18.90
6.20
6.30
6.20
6.50
6.10
6.50
6.20
5.90
5.70
6.40
5.60
5.60
5.80
5.90
6.00
5.90
5.90
6.00
6.20
6.20
6.30
6.40
6.50
6.30
6.00
6.20
5.50
6.20
6.10
6.60
6.10
6.40
6.30
6.10
6.10
6.20
6.60
6.10
5.70
5.60
5.70
5.80
6.00
6.00
5.90
5.50
5.50
5.60
5.10
5.50
5.30
5.50
5.50
5.60
5.60
5.70
5.60
5.70
6.10
6.00
5.70
5.90
6.00
5.80
5.50
6.00
5.60
5.90
5.90
6.00
5.80
5.50
5.90
5.40
5.50
5.90
539.47
533.54
484.26
604.93
524.92
584.26
503.85
483.83
467.43
506.38
415.69
415.49
419.05
460.79
482.66
483.24
485.59
509.92
507.81
529.43
572.99
594.36
547.15
522.54
521.42
536.16
435.99
546.73
519.03
558.48
526.39
539.83
503.30
483.56
526.39
487.30
530.92
513.61
M1
A41
LTL
19.50
21.00
20.50
20.50
20.00
20.20
20.50
21.10
19.60
20.60
19.00
20.80
17.50
20.20
20.20
20.50
21.00
19.00
19.50
19.40
19.60
21.00
20.80
21.20
20.50
20.50
20.40
20.10
20.80
19.70
20.50
20.70
20.10
19.50
19.90
20.10
19.60
20.60
20.40
20.00
19.80
LTC
18.20
19.00
18.10
20.00
18.20
18.50
19.60
18.00
20.40
19.00
18.30
18.30
18.40
17.80
18.30
17.80
18.00
19.40
19.10
18.80
18.80
19.80
19.50
19.70
19.10
19.20
18.50
19.40
18.50
19.30
18.70
19.50
19.60
19.00
19.20
18.40
19.30
18.60
19.20
18.80
18.30
LTW
6.00
6.30
5.90
6.70
6.10
6.00
6.20
5.90
6.70
6.20
6.10
5.90
6.20
5.70
5.90
6.10
5.90
6.30
6.30
6.10
6.20
6.50
6.40
6.80
6.10
6.10
6.20
6.40
6.00
6.20
6.10
6.40
6.50
6.10
6.10
5.90
6.30
6.00
6.30
6.00
6.10
LTD
5.60
5.80
5.60
6.00
5.40
5.70
6.20
5.60
6.30
5.90
5.60
5.80
5.50
5.70
5.70
5.30
5.50
6.00
5.90
5.90
5.80
6.10
6.00
5.80
6.00
6.10
5.60
6.00
5.80
6.10
5.80
6.00
6.00
6.00
6.10
5.80
6.00
5.80
5.90
6.00
5.60
LTV
465.19
544.81
480.90
585.11
467.75
490.50
559.49
494.97
587.39
535.02
460.82
505.36
423.69
465.97
482.32
470.56
483.83
509.92
514.62
495.73
500.42
591.18
567.09
593.65
532.71
541.59
502.88
548.01
513.93
528.99
514.96
564.36
556.57
506.73
525.74
488.35
526.02
508.98
538.37
511.20
480.22
Date
04-Aug-03
13-Aug-03
20-Aug-03
27-Aug-03
01-Sep-03
10-Sep-03
17-Sep-03
22-Sep-03
01-Oct-03
08-Oct-03
15-Oct-03
22-Oct-03
27-Oct-03
05-Nov-03
12-Nov-03
19-Nov-03
26-Nov-03
03-Dec-03
10-Dec-03
31-Dec-03
06-Jan-04
12-Jan-04
19-Jan-04
26-Jan-04
02-Feb-04
09-Feb-04
16-Feb-04
23-Feb-04
01-Mar-04
08-Mar-04
15-Mar-04
22-Mar-04
29-Mar-04
12-Apr-04
19-Apr-04
27-Apr-04
03-May-04
10-May-04
17-May-04
24-May-04
31-May-04
07-Jun-04
14-Jun-04
21-Jun-04
28-Jun-04
02-Jul-04
12-Jul-04
19-Jul-04
26-Jul-04
02-Aug-04
09-Aug-04
16-Aug-04
23-Aug-04
RTL
19.50
19.60
20.00
19.60
19.40
19.90
19.40
18.40
19.20
18.50
18.90
19.00
19.10
19.40
19.20
18.50
18.50
18.40
19.50
18.40
18.70
18.70
18.40
18.70
19.30
19.40
19.30
19.00
19.00
19.30
19.30
19.40
19.70
20.20
19.90
20.50
21.00
20.40
21.20
21.10
20.20
19.60
20.00
20.20
20.10
20.60
20.10
19.50
20.50
20.40
20.40
20.00
20.50
RTC
17.60
18.30
18.10
18.90
18.60
18.80
17.80
17.60
17.20
18.10
17.00
17.00
17.30
17.40
16.80
16.90
16.60
16.90
16.40
16.40
16.70
16.30
15.90
16.50
17.10
18.00
17.60
17.10
17.40
17.20
17.60
18.40
18.20
18.70
19.10
18.30
18.20
19.00
19.30
18.50
18.60
19.20
18.10
18.70
18.20
19.30
17.90
18.70
18.80
20.10
18.10
17.80
RTW
5.80
5.90
5.90
6.10
6.30
6.10
5.80
5.90
5.60
6.20
5.60
5.60
5.70
5.90
5.60
5.70
5.40
5.70
5.20
5.30
5.50
5.30
5.10
5.50
5.60
6.00
6.00
5.70
5.90
5.60
5.90
6.00
5.90
6.20
6.50
6.20
5.90
6.50
6.50
6.10
6.10
6.50
5.90
6.30
6.10
6.70
6.10
6.30
6.50
6.30
7.00
5.80
5.90
RTD
5.40
5.80
5.60
6.00
5.50
5.80
5.50
5.30
5.40
5.30
5.20
5.20
5.40
5.20
5.10
5.70
5.10
5.00
5.20
5.20
5.10
5.10
5.00
5.00
5.20
5.50
5.20
5.10
5.20
5.40
5.50
5.70
5.70
5.70
5.70
6.20
5.70
5.60
5.80
5.60
5.80
5.70
5.60
5.60
5.50
5.60
5.30
6.00
5.40
5.60
5.70
5.60
5.40
RTV
433.63
476.21
469.17
509.33
477.27
499.88
439.39
408.51
412.23
431.62
390.76
392.83
417.41
422.59
389.33
426.76
361.74
372.32
374.37
360.04
372.42
358.88
333.13
365.12
399.03
454.54
427.53
392.15
413.87
414.38
444.66
471.07
470.38
506.85
523.48
559.49
501.42
527.22
567.46
511.75
507.42
515.59
469.17
505.99
478.79
548.77
461.38
523.34
510.88
511.00
577.91
461.22
463.72
A42
LTL
19.60
19.90
19.80
20.00
18.50
19.80
19.00
18.50
19.50
18.00
19.10
18.40
19.00
19.00
18.80
18.00
18.00
18.00
18.70
18.00
17.70
18.30
16.70
18.20
18.90
17.60
18.50
17.90
19.00
19.30
19.60
18.30
19.40
19.90
19.80
20.60
19.50
20.20
20.80
LTC
18.80
18.10
18.50
18.50
18.50
18.50
18.30
18.20
17.50
18.00
17.10
17.60
17.20
17.00
16.60
16.70
17.30
16.60
16.70
17.90
16.30
16.30
17.20
17.80
17.00
17.90
18.50
18.50
17.60
16.40
17.90
18.20
18.20
18.70
19.00
18.70
18.70
19.00
19.10
LTW
6.20
5.80
6.00
6.00
6.40
6.20
6.10
6.00
5.60
6.00
5.50
5.60
5.60
5.60
5.50
5.50
5.60
5.60
5.60
6.00
5.20
5.30
5.50
5.70
5.70
5.80
6.10
6.30
5.80
5.30
6.00
6.10
5.90
6.00
6.70
6.20
6.20
6.10
6.50
LTD
5.70
5.70
5.80
5.80
5.40
5.60
5.60
5.50
5.50
5.40
5.40
5.60
5.30
5.20
5.10
5.20
5.40
5.00
5.00
5.40
5.20
5.10
5.40
5.70
5.10
5.60
5.70
5.50
5.30
5.20
5.40
5.50
5.70
6.00
5.40
5.70
5.70
6.00
5.70
LTV
491.79
467.10
489.22
494.16
453.95
488.09
460.82
433.46
426.43
414.07
402.76
409.69
400.38
392.83
374.41
365.51
386.47
357.84
371.76
414.07
339.81
351.20
352.15
419.84
390.09
405.87
456.70
440.37
414.68
377.65
450.88
435.92
463.22
508.64
508.62
516.88
489.28
524.92
547.15
20.30
20.10
19.90
19.50
19.50
19.50
19.80
20.10
19.70
20.40
19.00
19.50
19.20
18.80
19.20
18.60
18.60
18.90
18.50
18.30
18.00
18.60
17.90
19.90
18.70
18.70
6.00
6.30
6.00
6.30
6.40
6.20
6.20
5.80
6.10
5.80
7.00
6.30
6.30
5.90
5.90
5.80
5.60
5.60
5.60
5.40
5.90
5.80
5.70
5.60
5.60
5.70
510.22
530.45
491.69
488.45
496.20
480.70
470.66
488.35
494.86
478.84
528.81
488.45
489.53
Date
30-Aug-04
06-Sep-04
13-Sep-04
20-Sep-04
27-Sep-04
04-Oct-04
11-Oct-04
18-Oct-04
25-Oct-04
28-Oct-04
09-Nov-04
16-Nov-04
22-Nov-04
29-Nov-04
06-Dec-04
13-Dec-04
20-Dec-04
30-Dec-04
03-Jan-05
10-Jan-05
17-Jan-05
24-Jan-05
31-Jan-05
07-Feb-05
15-Feb-05
21-Feb-05
28-Feb-05
07-Mar-05
14-Mar-05
21-Mar-05
29-Mar-05
04-Apr-05
18-Apr-05
25-Apr-05
02-May-05
09-May-05
23-May-05
30-May-05
06-Jun-05
20-Jun-05
27-Jun-05
04-Jul-05
11-Jul-05
18-Jul-05
25-Jul-05
02-Aug-05
08-Aug-05
15-Aug-05
22-Aug-05
19-Sep-05
26-Sep-05
03-Oct-05
17-Oct-05
RTL
20.30
20.00
20.40
20.30
19.80
20.10
19.70
19.20
20.60
19.70
20.50
19.50
20.50
20.00
20.70
19.80
20.00
20.10
20.50
19.60
19.50
19.80
19.50
19.80
19.40
19.00
19.70
19.90
20.40
20.20
20.00
19.50
20.20
20.40
21.00
20.00
19.90
20.00
19.70
19.70
19.80
19.80
19.80
19.40
19.60
19.80
20.50
20.10
19.40
18.70
19.50
19.60
19.60
RTC
19.40
18.70
18.60
18.30
17.90
18.50
17.80
17.90
17.80
18.40
18.70
18.10
19.30
19.20
18.80
17.80
18.20
18.10
17.90
17.90
17.00
17.70
17.70
17.90
16.80
18.00
18.20
18.80
18.20
18.00
18.10
19.10
19.80
19.20
18.80
19.40
18.20
19.00
18.60
18.40
18.30
18.20
17.40
17.80
17.80
18.00
17.50
17.80
17.60
16.80
17.20
16.60
17.20
RTW
7.10
6.60
6.10
6.40
5.90
6.20
5.90
6.00
6.00
6.20
6.30
6.10
6.60
6.30
6.50
5.90
5.90
5.90
5.90
6.10
5.70
5.90
6.10
6.10
5.60
6.20
6.00
6.50
6.20
5.80
5.90
6.80
6.80
6.60
6.10
6.30
5.90
6.10
6.20
6.20
6.10
5.80
5.90
6.00
5.90
6.00
5.60
5.90
6.00
5.70
5.80
5.60
5.90
RTD
5.20
5.20
5.70
5.20
5.60
5.50
5.30
5.40
5.30
5.50
5.60
5.50
5.70
5.90
5.50
5.40
5.70
5.60
5.50
5.30
5.00
5.40
5.20
5.30
5.10
5.20
5.60
5.50
5.40
5.60
5.70
5.40
5.70
5.60
5.90
6.10
5.70
6.10
5.70
5.50
5.60
5.70
5.10
5.30
5.50
5.40
5.60
5.40
5.20
4.90
5.10
5.00
5.00
RTV
532.13
487.34
503.61
479.66
464.48
486.64
437.37
441.68
465.11
476.96
513.50
464.50
547.56
527.81
525.42
447.89
477.55
471.51
472.31
449.90
394.58
447.89
439.16
454.50
393.39
434.92
469.96
505.11
484.92
465.83
477.55
508.39
555.90
535.33
536.61
545.71
475.16
528.38
494.30
476.96
480.22
464.76
423.01
438.01
451.57
455.48
456.44
454.67
429.75
370.83
409.54
389.65
410.52
A43
LTL
19.60
19.20
19.50
19.30
19.20
19.70
18.60
19.30
19.20
18.50
19.70
20.30
19.50
20.50
19.50
19.10
19.30
19.40
20.20
17.90
18.30
19.00
19.00
19.50
19.00
19.00
19.50
20.00
18.90
20.40
19.00
19.60
19.75
20.00
20.10
19.90
20.00
21.60
20.20
19.80
19.60
19.80
18.80
19.70
19.60
19.80
19.60
19.00
19.40
19.50
19.60
19.00
18.70
LTC
17.90
19.30
18.70
18.80
18.00
18.70
18.80
18.40
18.30
17.60
18.00
18.80
18.70
18.10
18.00
17.50
18.90
18.00
18.00
18.40
17.70
17.50
18.00
17.30
17.30
17.20
18.70
18.90
18.30
17.60
18.90
18.40
18.90
19.20
19.10
19.20
19.20
18.90
19.10
18.50
17.80
18.00
18.30
18.30
18.50
19.10
17.90
17.80
17.20
17.60
16.50
17.60
16.90
LTW
6.00
6.60
6.20
6.30
6.00
6.10
6.20
6.20
6.20
5.70
5.80
6.40
6.20
5.90
5.80
5.60
6.50
6.10
6.20
6.30
5.90
5.90
6.10
5.80
6.00
5.90
6.00
6.40
5.90
5.60
6.40
6.00
6.20
6.40
6.20
6.40
6.40
6.10
6.20
6.10
5.90
6.10
6.10
5.90
6.20
6.40
5.90
6.10
5.60
5.90
5.50
5.70
5.60
LTD
5.40
5.60
5.60
5.60
5.40
5.80
5.80
5.50
5.50
5.50
5.60
5.60
5.70
5.60
5.70
5.60
5.50
5.40
5.30
5.40
5.40
5.20
5.40
5.20
5.00
5.00
5.90
5.70
5.70
5.60
5.60
5.70
5.90
5.80
6.00
5.80
5.80
5.90
5.90
5.70
5.50
5.40
5.60
5.70
5.60
5.80
5.50
5.20
5.40
5.30
5.00
5.50
5.10
LTV
450.88
503.84
480.70
483.44
441.68
494.86
474.89
467.27
464.85
411.78
454.30
516.56
489.28
480.90
457.72
425.27
489.88
453.72
471.28
432.36
413.96
413.87
444.36
417.57
404.70
397.96
490.11
518.02
451.28
454.22
483.48
475.93
512.94
527.10
530.88
524.47
527.10
551.94
524.63
488.80
451.57
463.07
455.97
470.38
483.16
521.83
451.57
427.90
416.53
432.93
382.69
422.91
379.19
Date
24-Oct-05
31-Oct-05
07-Nov-05
14-Nov-05
21-Nov-05
28-Nov-05
05-Dec-05
12-Dec-05
03-Jan-06
09-Jan-06
16-Jan-06
23-Jan-06
06-Feb-06
13-Feb-06
20-Feb-06
27-Feb-06
06-Mar-06
13-Mar-06
20-Mar-06
27-Mar-06
03-Apr-06
10-Apr-06
17-Apr-06
24-Apr-06
01-May-06
22-May-06
29-May-06
05-Jun-06
12-Jun-06
19-Jun-06
26-Jun-06
28-Aug-06
RTL
19.40
19.40
19.50
19.50
19.30
19.60
19.30
19.70
18.60
19.50
18.30
18.30
18.70
19.40
19.60
20.20
19.90
20.20
20.00
20.00
19.90
19.50
19.40
19.50
20.00
20.00
19.80
19.20
19.40
18.60
19.00
19.70
RTC
16.70
16.80
15.90
17.50
16.90
17.20
17.30
17.10
17.10
16.30
15.60
15.80
16.80
17.60
17.10
17.80
18.10
18.50
17.20
18.40
18.30
18.10
18.80
18.30
18.10
17.30
17.50
18.20
18.90
16.70
17.30
17.10
RTW
5.60
5.60
5.30
5.90
5.60
5.90
5.90
5.70
5.90
5.50
5.20
5.50
5.70
5.80
5.90
5.70
6.00
6.30
5.70
6.30
6.20
6.00
6.20
5.90
6.40
6.10
5.80
6.00
6.30
5.40
5.80
5.90
RTD
5.00
5.10
4.80
5.20
5.10
5.00
5.10
5.10
5.00
4.80
4.70
4.50
5.00
5.40
5.00
5.60
5.50
5.50
5.30
5.30
5.40
5.50
5.80
5.80
5.40
4.90
5.30
5.60
5.70
5.20
5.30
4.90
RTV
385.67
393.39
352.22
424.76
391.36
410.52
412.32
406.60
389.58
365.51
317.55
321.58
378.39
431.40
410.52
457.80
466.26
496.95
428.98
474.14
473.04
456.89
495.31
473.78
490.75
424.44
432.14
458.04
494.62
370.82
414.68
404.36
A44
LTL
19.25
19.50
18.60
18.90
19.30
19.50
19.10
18.50
18.20
18.50
18.40
18.30
19.00
19.60
19.30
19.30
20.50
19.20
19.60
19.80
19.40
19.50
19.20
20.20
19.80
19.40
18.90
18.70
18.50
20.00
19.10
18.30
LTC
18.30
17.30
17.30
17.20
17.00
17.70
17.50
17.80
17.70
17.10
16.40
15.70
17.10
17.70
18.30
18.60
19.00
18.20
18.60
18.50
18.60
18.00
17.80
17.40
18.80
16.80
17.80
18.20
17.90
18.30
17.10
16.40
LTW
6.40
5.60
5.70
5.80
5.70
6.10
6.10
5.80
5.70
5.80
5.40
5.30
5.50
5.80
6.40
6.30
6.30
6.20
6.00
6.30
6.10
6.20
6.00
5.70
6.20
5.50
5.90
6.00
5.90
6.00
5.80
5.40
LTD
5.20
5.40
5.30
5.20
5.10
5.20
5.00
5.50
5.60
5.10
5.00
4.70
5.40
5.40
5.20
5.50
5.80
5.40
5.80
5.50
5.80
5.20
5.40
5.40
5.80
5.20
5.50
5.60
5.40
5.70
5.10
5.10
LTV
454.85
418.67
398.95
404.72
398.35
439.16
413.61
419.01
412.47
388.53
352.73
323.66
400.65
435.85
456.04
474.81
531.84
456.40
484.28
487.11
487.32
446.36
441.68
441.45
505.53
393.94
435.45
446.11
418.48
485.64
401.13
357.83
Table A7.2:
Date
9-Oct-02
16-Oct-02
23-Oct-02
30-Oct-02
6-Nov-02
13-Nov-02
20-Nov-02
27-Nov-02
4-Dec-02
12-Dec-02
18-Dec-02
24-Dec-02
31-Dec-02
8-Jan-03
15-Jan-03
22-Jan-03
28-Jan-03
5-Feb-03
12-Feb-03
19-Feb-03
25-Feb-03
5-Mar-03
12-Mar-03
19-Mar-03
25-Mar-03
2-Apr-03
9-Apr-03
17-Apr-03
27-Apr-03
5-May-03
13-May-03
21-May-03
27-May-03
03-Jun-03
12-Jun-03
18-Jun-03
25-Jun-03
02-Jul-03
09-Jul-03
16-Jul-03
23-Jul-03
30-Jul-03
04-Aug-03
13-Aug-03
20-Aug-03
27-Aug-03
01-Sep-03
10-Sep-03
17-Sep-03
22-Sep-03
RTL
17.50
17.60
16.80
15.30
14.50
14.50
15.20
14.20
13.90
14.00
13.50
14.00
13.40
14.70
14.20
15.40
15.10
15.00
16.20
15.60
15.50
16.40
17.50
17.60
17.60
18.60
18.40
18.90
19.60
19.50
18.70
19.40
19.50
19.60
19.60
20.10
19.60
19.70
19.50
18.60
19.20
18.10
18.70
18.20
17.60
17.30
16.50
M2
RTC
11.90
11.50
12.30
RTW RTD RTV LTL
3.80 3.80 179.42 17.20
3.70 3.60
3.90 3.90 190.06 17.20
LTC
14.80
13.80
13.90
LTW
5.10
4.60
4.40
LTD
4.30
4.20
4.40
LTV
267.81
10.70
10.60
10.00
9.96
3.50
3.40
3.20
3.30
3.20
3.30
3.10
3.00
133.83
107.76
101.92
11.50
11.50
11.50
10.80
3.80
3.70
3.80
3.50
3.50
3.60
3.50
3.40
165.25
152.26
148.26
127.58
9.10
8.70
9.30
8.90
8.00
8.70
8.90
9.00
8.40
8.90
10.10
9.80
11.20
11.30
11.40
11.70
11.90
12.50
12.80
14.80
13.60
12.90
14.20
12.60
14.40
14.20
14.10
14.10
15.00
14.20
13.90
13.70
13.40
13.40
13.90
13.00
12.30
11.70
12.20
11.50
11.60
3.00
3.10
3.20
2.90
2.60
2.90
3.00
3.00
2.70
2.90
3.30
3.20
3.70
3.70
3.70
3.90
3.90
4.00
4.10
4.90
4.40
4.30
4.60
4.20
4.60
4.60
4.60
4.50
4.90
4.60
4.50
4.60
4.30
4.30
4.50
4.20
4.00
3.80
4.10
3.70
3.80
2.80
2.50
2.70
2.80
2.50
2.70
2.60
2.80
2.60
2.80
3.10
3.10
3.50
3.50
3.60
3.60
3.70
4.00
4.00
4.50
4.20
3.90
4.40
3.90
4.60
4.50
4.40
4.50
4.60
4.40
4.30
4.10
4.20
4.30
4.30
4.10
3.90
3.70
3.70
3.60
3.60
90.65
78.14
85.27
80.71
62.30
77.83
74.21
87.67
70.78
88.78
109.68
105.65
148.95
143.43
146.59
163.48
179.29
199.94
204.93
291.19
241.42
225.04
281.66
226.78
280.94
285.12
280.22
281.80
313.67
288.85
269.27
263.79
250.04
244.18
263.78
221.29
207.12
181.68
189.56
163.61
160.26
10.60
10.30
9.90
9.70
9.00
9.40
9.70
9.60
9.00
10.00
10.40
10.70
11.70
12.00
12.80
13.40
12.90
13.70
13.50
13.50
14.70
14.90
15.50
16.00
15.70
15.80
14.50
15.70
15.90
14.90
15.10
15.10
14.10
14.90
14.10
13.90
13.00
13.50
12.90
13.00
12.60
3.50
3.30
3.20
3.10
3.00
3.10
3.30
3.10
2.90
3.20
3.50
3.60
3.80
3.80
4.10
4.50
4.10
4.40
4.30
4.40
4.80
5.00
5.00
5.20
5.30
5.10
4.70
5.00
5.10
4.90
4.90
4.80
4.70
4.80
4.50
4.50
4.50
4.40
4.20
4.20
4.00
3.30
3.30
3.00
3.10
2.70
2.90
2.90
3.00
2.90
3.10
3.10
3.20
3.70
3.80
4.00
4.00
4.10
4.30
4.30
4.20
4.50
4.50
4.90
4.90
4.70
5.00
4.60
4.90
5.00
4.60
4.70
4.70
4.30
4.70
4.50
4.40
3.70
4.20
4.00
4.10
4.00
118.91
111.34
97.47
98.25
83.96
90.64
97.84
93.76
83.60
101.42
124.03
131.69
161.72
178.39
203.77
227.48
206.48
248.51
244.18
230.93
295.98
319.50
346.16
365.43
353.72
372.96
300.86
344.42
360.29
323.27
320.49
318.75
279.81
313.94
267.42
265.70
222.24
242.73
212.32
216.40
191.98
17.50
16.10
15.70
15.10
15.70
14.50
14.40
14.30
14.40
14.60
14.20
14.40
14.20
14.00
14.40
16.10
16.10
16.20
17.40
17.50
17.80
17.30
18.50
18.60
17.60
19.30
20.00
19.90
20.20
20.00
20.60
19.60
19.80
19.90
20.20
19.60
19.90
19.50
19.60
18.60
18.90
18.80
18.50
17.80
17.70
16.90
A45
236.42
Date
01-Oct-03
08-Oct-03
15-Oct-03
22-Oct-03
27-Oct-03
05-Nov-03
12-Nov-03
19-Nov-03
26-Nov-03
03-Dec-03
10-Dec-03
31-Dec-03
06-Jan-04
12-Jan-04
19-Jan-04
26-Jan-04
02-Feb-04
09-Feb-04
16-Feb-04
23-Feb-04
01-Mar-04
08-Mar-04
15-Mar-04
22-Mar-04
29-Mar-04
12-Apr-04
19-Apr-04
27-May-04
03-May-04
10-May-04
17-May-04
24-May-04
31-May-04
07-Jun-04
14-Jun-04
21-Jun-04
28-Jun-04
02-Jul-04
12-Jul-04
19-Jul-04
26-Jul-04
02-Aug-04
09-Aug-04
16-Aug-04
23-Aug-04
30-Aug-04
06-Sep-04
13-Sep-04
20-Sep-04
27-Sep-04
04-Oct-04
11-Oct-04
18-Oct-04
RTL
17.00
16.00
16.20
16.00
16.50
16.10
16.50
16.00
15.90
15.80
16.50
15.50
15.20
14.70
14.70
15.30
16.60
15.50
15.20
15.50
16.50
17.00
17.70
18.80
18.90
20.40
20.50
21.20
22.00
21.20
21.50
21.60
21.40
20.50
20.60
21.40
21.00
21.70
19.90
19.90
19.70
19.60
19.30
19.40
18.20
18.30
18.00
17.80
17.30
19.10
17.60
17.40
17.40
RTC
11.10
10.90
10.80
10.90
11.10
11.00
10.00
10.30
10.10
10.90
10.80
9.70
9.90
9.80
10.00
9.70
9.40
10.00
9.90
10.70
10.70
11.00
12.30
14.50
13.50
14.30
15.90
15.60
15.40
15.40
15.80
15.50
15.20
16.30
16.50
16.30
15.70
15.30
15.30
14.50
14.20
13.90
13.90
13.00
12.90
12.50
12.60
12.30
12.00
13.50
12.00
11.80
11.80
RTW RTD RTV LTL
3.60 3.50 152.08 17.60
3.60 3.40 139.05 16.50
3.60 3.30 136.64 16.10
3.60 3.40 139.05 16.70
3.60 3.50 147.61 16.80
3.60 3.40 139.92 16.70
3.40 3.00 119.49 16.80
3.40 3.10 119.73 17.00
3.20 3.20 115.60 16.70
3.50 3.40 133.49 16.70
3.70 3.20 138.71 17.00
3.20 2.90 102.13 16.50
3.30 3.00 106.84 17.00
3.10 3.10 100.30 15.50
3.20 3.20 106.87 14.70
3.10 3.10 104.39 15.20
3.00 3.00 106.07 15.70
3.40 3.00 112.25 15.60
3.30 3.00 106.84 15.70
3.50 3.30 127.11 17.00
3.40 3.40 135.43 16.90
3.60 3.40 147.74 17.30
4.00 3.80 191.02 17.60
4.60 4.60 282.44 18.40
4.40 4.20 247.98 19.40
4.60 4.50 299.82 21.00
5.10 4.90 363.73 21.00
5.00 4.90 368.77 21.50
5.00 4.80 374.88 21.50
4.90 4.90 361.40 21.30
5.20 4.90 388.95 21.60
5.10 4.80 375.43 21.50
4.90 4.80 357.36 22.40
5.20 5.20 393.57 21.00
5.40 5.10 402.80 21.50
5.20 5.20 410.85 21.20
5.00 5.00 372.75 20.90
5.10 4.70 369.31 20.20
5.00 4.80 339.10 20.10
4.80 4.40 298.40 20.40
4.70 4.40 289.25 19.90
4.50 4.30 269.27 19.50
4.50 4.30 265.15 19.40
4.20 4.10 237.19 19.50
4.30 3.90 216.70 18.80
4.00 4.00 207.89 19.20
4.00 4.00 204.48 19.10
3.90 3.90 192.22 18.60
4.00 3.60 176.88 18.50
4.30 4.30 250.74 18.00
3.90 3.80 185.19 18.60
3.90 3.50 168.63 18.00
3.80 3.70 173.70 18.40
A46
LTC
12.60
11.70
11.90
11.80
11.80
11.70
12.10
11.40
11.20
11.60
11.90
11.50
11.20
11.60
11.80
10.60
10.90
11.70
10.80
11.70
13.00
13.10
13.60
11.90
14.70
16.30
16.50
15.70
16.70
17.00
17.00
16.20
16.40
16.30
17.40
17.80
16.10
16.50
16.40
15.30
16.20
14.90
14.50
14.40
14.50
13.40
13.80
13.50
12.90
12.00
13.00
13.40
12.90
LTW
4.30
3.90
3.80
3.80
3.90
3.90
3.90
3.70
3.60
3.70
4.00
3.70
3.60
3.80
3.90
3.40
3.60
3.80
3.50
3.80
4.20
4.20
4.40
3.80
4.80
5.40
5.30
5.40
5.60
5.50
5.50
5.40
5.50
5.40
5.70
5.70
5.30
5.30
5.30
5.00
5.30
4.90
4.60
4.70
4.80
4.30
4.50
4.50
4.20
3.90
4.40
4.40
4.20
LTD
3.80
3.50
3.70
3.70
3.60
3.60
3.80
3.50
3.60
3.70
3.60
3.60
3.50
3.60
3.60
3.30
3.40
3.70
3.40
3.60
4.10
4.10
4.20
3.80
4.60
4.90
5.10
4.60
5.00
5.30
5.40
4.90
4.90
5.00
5.40
5.60
5.00
5.20
5.20
4.70
5.00
4.60
4.60
4.40
4.40
4.30
4.30
4.10
4.00
3.70
3.80
4.10
4.00
LTV
204.18
159.91
160.72
166.71
167.47
166.47
176.77
156.31
153.67
162.32
173.81
156.04
152.08
150.55
146.54
121.09
136.44
155.73
132.65
165.12
206.62
211.51
230.93
188.64
304.13
394.52
403.02
379.18
427.42
440.84
455.48
403.91
428.61
402.57
469.86
480.46
393.23
395.27
393.31
340.37
374.42
312.07
291.46
286.31
281.91
252.06
262.41
243.65
220.67
184.42
220.80
230.55
219.48
Date
25-Oct-04
28-Oct-04
09-Nov-04
16-Nov-04
22-Nov-04
29-Nov-04
06-Dec-04
13-Dec-04
20-Dec-04
30-Dec-04
03-Jan-05
10-Jan-05
17-Jan-05
24-Jan-05
31-Jan-05
07-Feb-05
15-Feb-05
21-Feb-05
28-Feb-05
07-Mar-05
14-Mar-05
21-Mar-05
29-Mar-05
04-Apr-05
18-Apr-05
25-Apr-05
02-May-05
09-May-05
RTL
17.30
17.10
16.70
16.80
16.80
16.60
16.70
16.20
16.40
17.00
15.60
16.00
15.80
16.00
16.60
17.30
16.50
17.50
17.70
18.30
19.10
19.50
20.10
20.70
22.50
23.70
23.70
24.10
RTC
11.90
11.60
11.80
11.80
11.30
11.70
10.60
10.90
10.80
9.90
11.10
10.40
11.10
11.20
10.20
11.00
11.80
12.00
12.10
12.70
13.50
13.30
14.70
14.60
16.50
17.80
18.50
18.20
RTW RTD RTV LTL
3.80 3.80 177.37 17.80
3.80 3.60 166.09 18.00
3.90 3.70 171.10 18.40
3.90 3.60 167.47 17.50
3.70 3.50 154.47 17.40
3.80 3.60 161.23 17.90
3.70 3.10 136.00 16.80
3.50 3.40 136.87 17.00
3.50 3.40 138.56 17.00
3.20 3.20 123.60 16.90
3.70 3.40 139.34 16.80
3.50 3.20 127.23 16.50
3.80 3.30 140.67 16.50
3.90 3.20 141.77 16.50
3.30 3.20 124.46 17.00
3.60 3.40 150.34 17.90
4.00 3.50 164.01 17.10
4.00 3.70 183.89 17.20
3.90 3.80 186.24 18.00
4.10 4.00 213.09 18.70
4.30 4.30 250.74 19.60
4.30 4.20 250.04 20.50
4.90 4.50 314.68 20.20
4.70 4.60 317.75 21.30
5.30 5.20 440.27 23.30
5.80 5.60 546.54 24.20
6.00 5.80 585.58 24.90
5.90 5.60 565.35 25.00
A47
LTC
13.30
14.10
13.40
13.50
12.40
11.90
12.70
12.10
12.40
12.60
11.50
11.40
12.00
11.90
12.70
12.90
11.70
13.40
14.20
13.70
14.50
15.00
16.20
16.20
18.70
18.60
19.80
19.20
LTW
4.40
4.70
4.40
4.50
4.00
3.80
4.10
3.90
4.00
4.00
3.70
3.70
4.30
3.90
4.10
4.20
3.70
4.30
4.80
4.40
4.80
4.80
5.20
5.20
6.40
6.00
6.40
6.30
LTD
4.10
4.30
4.10
4.10
3.80
3.70
4.00
3.80
3.80
4.00
3.70
3.50
3.30
3.70
4.10
4.00
3.70
4.20
4.30
4.30
4.40
4.70
5.10
5.00
5.50
5.80
6.30
5.90
LTV
227.99
258.28
235.67
229.24
187.78
178.69
195.62
178.88
183.46
191.98
163.29
151.71
166.24
169.05
202.90
213.51
166.21
220.55
263.78
251.20
293.91
328.36
380.35
393.20
582.31
597.93
712.82
659.77
Table A7.3:
Date
9-Oct-02
16-Oct-02
23-Oct-02
30-Oct-02
6-Nov-02
13-Nov-02
20-Nov-02
27-Nov-02
4-Dec-02
12-Dec-02
18-Dec-02
24-Dec-02
31-Dec-02
8-Jan-03
15-Jan-03
22-Jan-03
28-Jan-03
5-Feb-03
12-Feb-03
19-Feb-03
25-Feb-03
5-Mar-03
12-Mar-03
19-Mar-03
25-Mar-03
2-Apr-03
9-Apr-03
17-Apr-03
27-Apr-03
5-May-03
13-May-03
21-May-03
26-May-03
03-Jun-03
12-Jun-03
18-Jun-03
25-Jun-03
02-Jul-03
09-Jul-03
16-Jul-03
23-Jul-03
30-Jul-03
04-Aug-03
13-Aug-03
20-Aug-03
27-Aug-03
1-Sep-03
10-Sep-03
17-Sep-03
22-Sep-03
RTL
18.00
17.30
17.50
16.70
18.00
18.30
17.50
17.80
17.20
17.20
16.20
17.20
16.50
16.60
16.70
16.90
17.30
17.30
17.70
17.60
18.70
18.60
18.70
18.30
18.10
18.00
17.90
17.10
17.50
17.50
16.90
17.00
16.90
17.10
16.70
17.00
17.40
18.20
17.90
18.50
18.50
18.50
18.20
17.30
17.00
17.10
16.50
16.50
16.30
15.50
RTC
11.80
12.30
12.30
10.70
12.30
12.30
11.50
12.50
11.00
10.60
10.50
10.90
11.00
10.30
10.20
11.00
11.10
10.20
11.40
12.30
12.30
12.50
12.80
12.30
12.10
11.40
11.70
11.10
11.00
11.50
11.50
11.50
11.50
11.40
11.60
11.90
12.00
12.90
12.20
12.80
12.80
12.80
12.40
12.40
11.90
10.80
10.90
10.60
10.60
10.10
RTW
3.90
4.10
4.20
3.70
4.10
4.10
3.90
4.20
3.60
3.50
3.40
3.60
3.60
3.30
3.40
3.60
3.50
3.30
3.70
4.00
4.00
4.10
4.20
3.90
3.90
3.90
3.80
3.60
3.60
4.00
3.70
3.80
3.70
3.70
3.70
3.90
4.00
4.20
3.90
4.20
4.10
4.10
4.10
4.10
3.90
3.50
3.50
3.50
3.40
3.40
RTD
3.60
3.70
3.60
3.10
3.70
3.70
3.40
3.80
3.40
3.30
3.20
3.30
3.40
3.30
3.10
3.40
3.50
3.20
3.60
3.80
3.90
3.90
4.00
3.90
3.90
3.40
3.60
3.50
3.40
3.30
3.60
3.50
3.60
3.60
3.70
3.70
3.70
4.00
3.90
4.00
4.00
4.00
3.80
3.80
3.70
3.40
3.40
3.20
3.40
3.10
M3
RTV
179.43
186.33
187.87
136.00
193.87
197.10
164.76
201.70
149.47
141.05
125.14
145.08
143.39
128.35
124.97
146.87
150.47
129.71
167.39
189.94
207.12
211.16
223.05
197.62
195.46
169.46
173.86
152.98
152.08
164.01
159.83
160.53
159.83
161.72
162.32
174.17
182.84
217.09
193.30
220.67
215.41
215.41
201.32
191.37
174.17
144.48
139.41
131.21
133.78
115.99
A48
LTL
16.50
16.50
18.00
18.40
18.50
19.00
17.60
17.50
17.60
17.40
16.90
17.30
16.70
16.50
16.60
16.50
16.50
17.60
18.10
17.20
18.00
18.20
18.50
18.50
17.50
17.60
17.60
17.20
17.30
16.40
16.40
16.40
16.80
15.90
16.60
16.80
17.10
17.50
17.60
18.40
18.50
18.40
17.80
17.40
17.00
17.10
16.50
16.60
16.50
16.00
LTC
11.70
12.10
11.80
11.70
12.40
12.60
12.30
12.60
11.40
11.60
11.00
11.20
10.90
9.90
10.40
10.80
11.00
10.90
11.00
12.00
11.80
12.30
12.60
12.00
12.00
11.70
11.20
11.20
11.30
10.30
11.00
10.70
10.90
11.20
11.50
11.20
11.50
12.20
12.00
12.40
12.20
12.30
12.00
12.20
11.40
10.90
10.30
10.40
10.70
9.70
LTW
3.90
4.30
4.10
3.90
4.20
4.50
4.20
4.30
3.80
4.00
3.70
3.90
3.70
3.20
3.40
3.70
3.50
3.50
3.60
3.90
3.80
4.00
4.20
3.90
3.90
3.80
3.70
3.70
3.60
3.30
3.60
3.40
3.70
3.90
3.90
3.70
4.00
4.00
3.90
4.10
4.00
3.90
3.90
4.00
3.80
3.70
3.40
3.50
3.60
3.10
LTD
3.50
3.40
3.40
3.50
3.70
3.50
3.60
3.80
3.40
3.40
3.30
3.20
3.20
3.10
3.20
3.20
3.50
3.40
3.40
3.70
3.80
3.90
3.80
3.80
3.80
3.70
3.40
3.50
3.60
3.20
3.40
3.40
3.30
3.20
3.40
3.40
3.40
3.80
3.80
3.70
3.70
3.90
3.70
3.80
3.50
3.20
3.10
3.10
3.20
3.10
LTV
159.91
171.27
178.15
178.32
204.12
212.47
188.94
203.02
161.45
168.01
146.51
153.29
140.39
116.21
128.23
138.71
143.51
148.70
157.30
176.22
184.54
201.58
209.63
194.66
184.14
175.69
157.20
158.15
159.19
122.96
142.52
134.60
145.64
140.89
156.28
150.05
165.12
188.86
185.19
198.18
194.40
198.70
182.37
187.78
160.53
143.75
123.48
127.88
134.96
109.17
Date
1-Oct-03
8-Oct-03
15-Oct-03
22-Oct-03
27-Oct-03
5-Nov-03
12-Nov-03
19-Nov-03
26-Nov-03
3-Dec-03
10-Dec-03
31-Dec-03
6-Jan-04
12-Jan-04
19-Jan-04
26-Jan-04
2-Feb-04
9-Feb-04
16-Feb-04
23-Feb-04
1-Mar-04
8-Mar-04
15-Mar-04
22-Mar-04
29-Mar-03
12-Apr-04
19-Apr-04
27-Apr-04
3-May-04
10-May-04
17-May-04
24-May-04
31-May-04
7-Jun-04
14-Jun-04
21-Jun-04
28-Jun-04
2-Jul-04
12-Jul-04
19-Jul-04
26-Jul-04
2-Aug-04
9-Aug-04
16-Aug-04
23-Aug-04
30-Aug-04
6-Sep-04
13-Sep-04
20-Sep-04
27-Sep-04
4-Oct-04
11-Oct-04
18-Oct-04
RTL
16.50
16.00
16.30
16.40
17.00
17.30
17.20
17.00
17.10
17.00
17.20
16.90
17.30
16.80
16.70
16.40
16.90
17.20
16.50
17.00
16.80
16.00
16.60
17.20
17.40
17.90
18.60
18.80
19.00
18.70
19.50
19.20
20.60
20.60
21.50
21.00
20.00
19.70
20.40
19.50
20.40
19.90
19.80
20.00
20.00
20.30
20.50
20.30
21.50
21.40
20.80
20.80
20.20
RTC
10.50
10.50
10.40
11.10
11.20
12.20
11.50
11.70
11.10
10.90
11.10
11.30
10.80
11.20
11.30
11.00
10.50
10.90
10.10
11.30
11.20
10.90
11.30
11.50
11.20
11.60
12.20
12.90
13.10
13.60
13.70
13.90
14.60
14.60
14.30
13.70
14.50
14.50
14.70
13.90
14.00
13.80
13.90
13.90
14.00
14.50
15.20
14.50
15.80
15.70
16.20
14.60
14.60
RTW
3.40
3.50
3.40
3.60
3.60
4.10
3.80
3.90
3.50
3.50
3.50
3.80
3.50
3.60
3.70
3.50
3.50
3.50
3.30
3.60
3.70
3.50
3.60
3.80
3.60
3.80
3.90
4.30
4.20
4.50
4.40
4.70
4.80
4.90
4.80
4.40
4.90
4.70
5.10
4.40
4.50
4.50
4.50
4.60
4.60
4.80
5.00
4.70
5.20
5.10
5.40
4.90
4.90
RTD
3.30
3.20
3.20
3.50
3.60
3.70
3.50
3.60
3.50
3.40
3.50
3.40
3.30
3.50
3.60
3.50
3.20
3.40
3.10
3.60
3.40
3.40
3.60
3.60
3.60
3.60
3.80
4.00
4.10
4.20
4.30
4.20
4.50
4.40
4.30
4.30
4.40
4.50
4.20
4.40
4.40
4.30
4.40
4.20
4.30
4.40
4.70
4.50
4.80
4.90
4.90
4.40
4.40
RTV
131.44
127.23
125.91
146.71
156.43
186.33
162.42
169.46
148.73
143.63
149.60
155.03
141.87
150.29
157.94
142.64
134.39
145.32
119.84
156.43
150.05
135.18
152.75
167.06
160.11
173.86
195.71
229.59
232.30
250.94
261.95
269.10
315.92
315.34
315.07
282.10
306.15
295.83
310.25
268.04
286.78
273.40
278.35
274.34
280.88
304.40
342.04
304.83
381.01
379.70
390.76
318.40
309.21
A49
LTL
16.70
15.90
16.50
17.00
17.10
17.10
17.30
17.50
17.00
17.10
17.00
16.70
16.80
16.40
16.50
16.00
17.10
16.00
16.20
16.90
16.00
16.80
16.80
16.80
16.90
17.80
18.40
18.60
18.80
18.20
18.70
19.80
19.80
20.00
20.20
20.40
20.50
19.20
19.60
19.50
19.20
19.20
19.40
20.00
20.10
20.00
20.60
21.80
20.80
21.30
20.40
19.80
20.00
LTC
10.40
10.00
11.20
10.60
11.20
11.20
10.90
11.20
11.20
11.00
10.70
10.80
10.50
11.00
10.90
10.60
10.80
10.30
10.60
10.50
10.40
11.30
10.90
10.80
11.10
11.50
11.30
12.20
12.70
12.80
13.50
14.50
14.60
14.30
14.30
13.40
13.20
13.60
13.60
13.70
13.20
13.40
13.70
13.30
13.60
13.60
14.70
14.90
15.10
14.50
14.80
14.10
13.90
LTW
3.60
3.40
3.90
3.20
3.90
3.60
3.50
3.80
3.70
3.60
3.60
3.60
3.50
3.70
3.60
3.50
3.50
3.30
3.50
3.50
3.40
3.70
3.50
3.60
3.50
3.70
3.60
4.00
4.10
4.20
4.40
5.00
4.70
4.90
4.80
4.50
4.30
4.50
4.40
4.60
4.20
4.40
4.50
4.30
4.50
4.40
4.90
4.70
5.10
4.80
4.80
4.70
4.70
LTD
3.00
3.00
3.20
3.20
3.20
3.50
3.40
3.30
3.50
3.40
3.20
3.30
3.20
3.30
3.36
3.30
3.30
3.20
3.20
3.20
3.20
3.50
3.40
3.30
3.50
3.60
3.50
3.70
4.00
4.00
4.20
4.20
4.60
4.30
4.30
4.00
4.10
4.10
4.20
4.10
4.20
4.20
4.20
4.20
4.20
4.30
4.50
4.70
4.50
4.40
4.70
4.30
4.20
LTV
128.06
115.15
146.20
123.60
151.52
152.98
146.17
155.81
156.31
148.61
139.05
140.86
133.59
142.17
141.70
131.21
140.23
119.96
128.82
134.39
123.60
154.47
141.94
141.70
146.99
168.34
164.61
195.45
218.91
217.09
245.36
295.22
303.93
299.19
296.02
260.71
256.60
251.51
257.17
261.12
240.47
251.92
260.33
256.45
269.72
268.66
322.50
341.91
338.93
319.40
326.76
284.11
280.31
Date
25-Oct-04
28-Oct-04
9-Nov-04
16-Nov-04
22-Nov-04
29-Nov-04
6-Dec-04
13-Dec-04
20-Dec-04
30-Dec-04
3-Jan-05
10-Jan-05
17-Jan-05
24-Jan-05
31-Jan-05
7-Feb-05
15-Feb-05
21-Feb-05
28-Feb-05
7-Mar-05
14-Mar-05
21-Mar-05
29-Mar-05
4-Apr-05
18-Apr-05
25-Apr-05
2-May-05
9-May-05
23-May-05
30-May-05
6-Jun-05
13-Jun-05
20-Jun-05
27-Jun-05
4-Jul-05
11-Jul-05
18-Jul-05
25-Jul-05
2-Aug-05
8-Aug-05
15-Aug-05
22-Aug-05
19-Sep-05
26-Sep-05
3-Oct-05
17-Oct-05
24-Oct-05
31-Oct-05
7-Nov-05
14-Nov-05
21-Nov-05
28-Nov-05
5-Dec-05
RTL
21.00
20.50
20.50
19.20
19.30
19.80
19.70
20.20
20.00
19.90
19.40
18.80
19.00
18.80
19.30
18.70
18.50
19.60
20.00
21.50
22.20
22.10
22.80
21.10
21.40
21.60
22.40
21.70
22.40
22.50
22.30
23.30
23.50
24.10
22.90
22.50
22.70
21.10
20.00
19.60
18.70
18.00
17.30
17.60
17.80
17.70
18.30
18.20
18.30
17.80
18.10
18.20
18.00
RTC
13.00
14.00
13.30
13.60
13.40
13.70
13.50
13.40
13.60
13.50
13.60
13.50
12.70
12.90
12.40
13.10
13.20
14.30
13.80
14.70
15.20
16.10
16.00
15.90
16.40
15.80
15.50
15.60
15.70
16.00
15.80
18.10
16.50
16.80
16.80
15.40
15.50
14.60
14.30
13.10
12.50
13.10
11.90
11.20
11.40
12.50
12.40
11.90
11.10
11.80
12.70
11.90
12.00
RTW
4.20
4.70
4.30
4.60
4.40
4.50
4.50
4.40
4.40
4.40
4.40
4.60
4.40
4.20
4.00
4.20
4.30
4.70
4.60
4.90
4.90
5.40
5.10
5.30
5.50
5.10
5.10
5.00
5.10
5.20
5.10
6.20
5.30
5.40
5.40
5.10
5.00
4.80
4.70
4.20
4.10
4.30
3.90
3.70
3.70
4.10
4.20
3.90
3.60
3.90
4.10
3.90
3.80
RTD
4.00
4.20
4.10
4.00
4.10
4.30
4.10
4.10
4.20
4.20
4.20
4.00
3.70
4.10
3.90
4.10
4.10
4.40
4.20
4.40
4.70
4.90
5.10
4.80
4.90
4.90
4.80
4.90
4.90
5.00
4.90
5.20
5.20
5.30
5.30
4.70
4.80
4.40
4.40
4.10
3.90
4.00
3.70
3.40
3.60
3.80
3.70
3.70
3.50
3.60
3.90
3.70
3.80
RTV
250.49
287.32
256.60
250.83
247.20
272.02
258.06
258.73
262.42
261.10
254.54
245.60
219.62
229.85
213.77
228.63
231.57
287.78
274.34
329.11
363.00
415.18
421.05
381.12
409.48
383.25
389.33
377.47
397.44
415.35
395.67
533.35
459.84
489.72
465.33
382.92
386.81
316.40
293.66
239.63
212.30
219.82
177.24
157.20
168.34
195.79
201.91
186.46
163.71
177.44
205.49
186.46
184.54
A50
LTL
20.10
19.50
19.40
19.20
18.50
19.60
19.20
19.40
19.70
19.40
18.90
18.20
18.50
18.40
19.20
18.90
19.40
19.30
19.80
20.50
21.00
21.20
21.90
21.40
21.30
21.50
22.40
22.40
21.80
21.50
22.30
22.20
22.50
23.70
23.10
22.60
22.40
21.20
20.00
19.50
18.50
18.10
17.00
17.50
18.40
17.50
19.50
18.00
17.70
17.90
18.00
18.30
17.70
LTC
13.40
13.30
13.40
13.00
12.90
12.80
12.90
12.80
12.80
13.30
13.20
12.70
12.40
12.30
12.00
12.50
12.60
13.10
13.40
14.20
14.80
15.10
14.70
14.60
15.00
14.60
13.40
14.40
15.50
14.90
15.30
16.40
16.40
16.60
16.00
16.40
14.70
14.50
13.20
12.60
12.00
12.40
10.80
10.80
11.20
11.80
11.00
11.20
10.70
10.80
11.80
11.10
11.20
LTW
4.50
4.40
4.40
4.30
4.40
4.30
4.30
4.30
4.30
4.50
4.50
4.30
4.20
4.20
4.00
4.30
4.20
4.30
4.30
4.80
4.80
4.90
4.90
4.90
5.00
4.80
4.40
4.70
5.20
4.90
5.00
5.60
5.50
5.50
5.30
5.50
5.00
4.80
4.40
4.10
4.00
4.10
3.60
3.40
3.80
4.10
3.80
3.80
3.80
3.70
4.00
3.70
3.80
LTD
4.00
4.10
4.20
4.00
3.80
3.90
3.90
3.90
3.80
3.90
3.90
3.70
3.70
3.60
3.60
3.70
3.80
4.00
4.20
4.30
4.60
4.70
4.50
4.40
4.60
4.40
4.10
4.40
4.70
4.60
4.80
4.90
4.90
5.10
4.90
4.90
4.30
4.40
4.00
3.90
3.70
3.80
3.30
3.40
3.30
3.40
3.20
3.30
3.00
3.10
3.50
3.30
3.30
LTV
256.88
249.76
254.54
234.47
219.62
233.37
228.61
230.99
228.55
241.73
235.50
205.59
204.12
197.53
196.30
213.50
219.83
235.69
253.89
300.42
329.21
346.65
342.86
327.58
347.83
322.40
286.91
328.89
378.28
344.07
379.99
432.51
430.53
472.00
425.93
432.44
341.94
317.90
249.92
221.38
194.40
200.22
143.39
143.63
163.82
173.20
168.36
160.26
143.26
145.77
178.92
158.64
157.59
Date
12-Dec-05
3-Jan-06
9-Jan-06
16-Jan-06
23-Jan-06
6-Feb-06
13-Feb-06
20-Feb-06
27-Feb-06
6-Mar-06
13-Mar-06
20-Mar-06
27-Mar-06
3-Apr-06
10-Apr-06
17-Apr-06
24-Apr-06
1-May-06
22-May-06
29-May-06
5-Jun-06
12-Jun-06
19-Jun-06
26-Jun-06
28-Aug-06
RTL
18.30
17.80
17.70
17.30
17.25
18.80
20.30
20.60
22.10
23.40
22.70
23.00
23.40
23.70
23.60
23.10
22.20
22.00
20.00
19.50
18.60
18.30
18.60
18.50
21.00
RTC
11.60
11.60
11.40
11.30
11.60
12.40
13.70
15.00
16.10
17.40
16.90
17.00
18.30
18.20
18.10
16.00
16.90
15.60
15.20
13.90
12.70
12.40
12.40
12.20
15.60
RTW
3.80
3.70
3.80
3.60
3.80
4.10
4.60
5.00
5.30
5.90
5.40
5.50
6.00
5.90
5.80
5.40
5.50
5.10
5.00
4.50
4.20
4.00
4.00
4.10
5.20
RTD
3.60
3.70
3.50
3.60
3.60
3.80
4.10
4.50
5.00
5.20
5.30
5.30
5.60
5.70
5.80
4.80
5.30
4.80
4.70
4.30
3.80
3.90
3.80
3.60
4.70
RTV
177.74
173.01
167.14
159.19
167.55
207.96
271.83
329.09
415.81
509.72
461.27
476.02
558.23
565.89
563.67
425.11
459.46
382.38
333.70
267.90
210.77
202.69
200.73
193.87
364.40
A51
LTL
17.60
16.80
17.20
17.20
16.70
18.70
19.80
20.60
22.20
22.40
22.70
22.50
23.00
23.60
23.50
22.80
22.00
21.80
20.20
19.00
18.60
18.20
17.70
18.80
20.50
LTC
11.20
11.20
10.80
10.80
11.10
12.30
13.50
13.80
15.00
16.80
16.90
17.10
17.60
18.00
17.40
16.80
16.00
16.00
13.50
13.10
12.60
12.50
12.40
12.50
15.10
LTW
3.80
3.80
3.50
3.60
3.70
4.00
4.60
4.60
4.90
5.50
5.40
5.90
5.80
6.00
5.60
5.60
5.40
5.40
4.30
4.40
4.00
4.20
4.00
4.10
5.00
LTD
3.30
3.30
3.30
3.20
3.30
3.80
3.90
4.20
4.70
5.10
5.40
5.00
5.40
5.40
5.50
5.10
4.80
4.80
4.20
3.90
4.00
3.70
3.90
3.80
4.60
LTV
156.70
149.58
141.05
140.68
144.77
201.81
252.20
282.57
363.00
446.11
469.97
471.26
511.46
542.89
513.90
462.33
404.87
401.19
259.02
231.49
211.30
200.81
196.05
207.96
334.77
Table A7.4:
Date
5-Feb-03
12-Feb-03
19-Feb-03
25-Feb-03
5-Mar-03
12-Mar-03
19-Mar-03
25-Mar-03
2-Apr-03
9-Apr-03
17-Apr-03
27-Apr-03
5-May-03
13-May-03
3-Jun-03
12-Jun-03
18-Jun-03
25-Jun-03
2-Jul-03
9-Jul-03
16-Jul-03
23-Jul-03
30-Jul-03
04-Aug-03
13-Aug-03
20-Aug-03
27-Aug-03
01-Sep-03
22-Sep-03
01-Oct-03
08-Oct-03
15-Oct-03
22-Oct-03
27-Oct-03
05-Nov-03
12-Nov-03
19-Nov-03
26-Nov-03
03-Dec-03
10-Dec-03
31-Dec-03
06-Jan-04
12-Jan-04
19-Jan-04
26-Jan-04
02-Feb-04
14-Feb-04
16-Feb-04
23-Feb-04
01-Mar-04
RTL
7.10
7.80
7.90
7.90
7.80
7.10
8.00
RTC
5.30
5.00
RTW
1.70
1.60
RTD
1.70
1.50
4.90
1.60
1.50
4.60
4.80
4.50
1.50
1.60
1.60
1.50
1.50
1.20
4.70
4.40
1.50
1.40
1.50
1.40
M4
RTV
14.57
13.29
LTL
7.90
LTC
5.00
5.30
LTW
1.60
1.70
LTD
1.60
1.60
LTV
14.36
7.60
7.70
4.80
1.60
1.40
12.25
8.00
4.50
1.40
1.40
11.13
7.40
7.70
7.60
8.10
7.90
8.10
8.30
8.20
7.90
8.00
7.90
8.00
8.20
7.70
8.20
8.60
8.00
8.30
8.10
8.40
8.40
8.50
8.50
8.80
8.50
8.70
8.90
8.70
8.70
8.80
8.30
9.00
8.80
8.20
9.00
9.10
9.20
9.00
4.90
5.50
5.10
5.30
5.10
1.60
1.80
1.60
1.70
1.70
1.50
1.70
1.60
1.70
1.50
12.61
16.73
13.81
16.62
14.30
5.10
5.20
5.00
5.70
5.10
5.00
5.40
5.50
5.00
5.70
6.00
6.10
6.80
6.60
6.20
6.40
5.70
6.50
6.20
6.30
6.60
5.90
6.20
6.40
6.50
6.00
6.50
6.10
6.80
6.90
6.60
7.40
1.60
1.70
1.60
1.90
1.70
1.70
1.70
1.80
1.60
1.90
2.00
1.90
2.20
2.20
2.00
2.10
1.90
2.20
2.00
2.10
2.20
1.90
2.00
2.10
2.20
1.90
2.10
2.00
2.30
2.30
2.10
2.40
1.60
1.60
1.50
1.80
1.50
1.50
1.70
1.70
1.50
1.80
1.80
1.90
2.10
2.00
1.90
2.00
1.70
1.90
1.90
1.90
2.10
1.90
2.00
2.00
2.00
1.80
2.00
1.80
2.10
2.10
2.10
2.30
15.09
15.84
13.46
19.43
14.30
14.48
16.83
16.73
13.97
20.88
20.45
21.27
26.57
26.24
22.66
25.35
19.49
26.12
22.93
24.65
29.19
22.30
24.71
26.24
25.93
21.85
26.24
20.96
30.86
31.21
28.81
35.27
12.62
13.46
10.63
11.34
11.13
7.60
5.10
1.60
1.60
13.81
8.30
8.40
8.50
8.30
8.20
8.30
8.20
8.30
8.10
8.10
8.40
8.10
8.00
8.50
8.50
8.20
8.40
8.70
8.60
8.40
8.80
8.60
8.50
8.60
8.70
8.50
8.80
9.00
8.70
9.50
9.20
9.10
9.30
4.80
5.40
5.10
5.40
5.00
5.00
4.90
5.30
4.40
5.50
5.50
6.50
6.00
6.00
6.30
6.30
6.30
6.10
5.90
6.20
6.60
6.20
6.20
5.70
6.30
6.20
6.30
6.20
6.10
6.00
6.50
7.20
1.60
1.80
1.70
1.80
1.70
1.60
1.60
1.70
1.50
1.80
1.80
2.10
2.00
2.00
2.00
2.20
2.10
1.90
1.90
2.00
2.20
2.00
2.00
1.90
2.10
2.10
2.00
2.20
2.00
1.90
2.20
2.40
1.50
1.70
1.60
1.70
1.50
1.60
1.50
1.70
1.40
1.80
1.70
2.00
1.80
1.90
2.00
1.80
1.90
1.90
1.80
1.90
2.00
1.90
1.90
1.80
1.90
1.90
2.00
1.80
1.90
1.90
1.90
2.20
14.31
18.47
16.03
17.82
15.03
14.90
14.14
16.62
12.08
19.32
17.60
23.86
21.73
22.93
23.29
23.62
24.65
22.04
20.40
23.74
26.87
22.93
23.20
21.13
24.08
24.93
25.56
24.46
25.63
23.58
27.01
34.86
A52
Date
08-Mar-04
15-Mar-04
22-Mar-04
29-Mar-04
12-Apr-04
19-Apr-04
27-Apr-04
03-May-04
10-May-04
17-May-04
24-May-04
31-May-04
07-Jun-04
14-Jun-04
21-Jun-04
28-Jun-04
02-Jul-04
12-Jul-04
19-Jul-04
26-Jul-04
02-Aug-04
09-Aug-04
16-Aug-04
23-Aug-04
30-Aug-04
06-Sep-04
13-Sep-04
20-Sep-04
27-Sep-04
04-Oct-04
11-Oct-04
18-Oct-04
25-Oct-04
28-Oct-04
09-Nov-04
16-Nov-04
22-Nov-04
29-Nov-04
06-Dec-04
13-Dec-04
20-Dec-04
30-Dec-04
03-Jan-05
10-Jan-05
17-Jan-05
24-Jan-05
31-Jan-05
07-Feb-05
15-Feb-05
21-Feb-05
28-Feb-05
07-Mar-05
14-Mar-05
RTL
9.50
9.70
9.30
8.80
9.40
9.30
9.30
9.30
9.40
9.60
9.50
9.50
9.70
9.50
9.60
9.60
9.40
9.70
9.70
9.80
9.60
9.60
13.20
9.70
9.60
13.10
12.80
13.50
13.30
13.10
13.60
13.20
13.30
12.90
13.00
12.40
13.90
12.50
13.30
13.40
12.80
12.50
12.50
12.40
13.40
13.60
14.10
15.00
14.90
15.10
15.70
16.40
16.00
RTC
6.60
6.70
6.70
6.60
6.50
6.80
6.60
6.80
6.90
7.20
7.40
6.70
7.60
7.30
7.10
7.60
7.40
6.90
7.20
7.90
7.10
7.30
7.20
7.10
6.90
7.60
8.10
7.60
7.70
7.80
7.40
7.30
7.20
6.80
7.20
6.70
6.50
6.80
6.60
6.40
6.40
6.30
6.60
6.80
6.90
7.90
8.30
8.70
9.40
10.00
10.10
10.20
10.70
RTW
2.20
2.40
2.20
2.20
2.30
2.20
2.30
2.20
2.30
2.50
2.40
2.20
2.50
2.40
2.40
2.50
2.40
2.30
2.30
2.70
2.30
2.50
2.40
2.30
2.20
2.40
2.90
2.50
2.70
2.60
2.50
2.40
2.30
2.20
2.30
2.30
2.10
2.20
2.20
2.10
2.10
2.00
2.30
2.20
2.30
2.60
2.70
2.80
3.00
3.20
3.20
3.30
3.40
RTD
2.00
1.90
2.00
2.00
1.90
2.20
1.90
2.20
2.00
2.10
2.30
2.00
2.30
2.20
2.10
2.30
2.30
2.10
2.20
2.30
2.20
2.20
2.20
2.20
2.20
2.40
2.30
2.30
2.20
2.40
2.30
2.30
2.20
2.10
2.30
2.00
2.00
2.20
2.00
1.90
2.00
2.00
1.90
2.10
2.10
2.40
2.60
2.70
3.00
3.20
3.20
3.20
3.40
RTV
29.68
31.40
29.05
27.49
29.17
31.96
28.86
31.96
30.70
35.78
37.23
29.68
39.60
35.61
34.35
39.19
36.84
33.26
34.85
43.21
34.49
37.49
49.48
34.85
32.99
53.57
60.62
55.11
56.09
58.04
55.52
51.73
47.78
42.31
48.83
40.50
41.45
42.96
41.55
37.96
38.17
35.50
38.78
40.67
45.95
60.25
70.28
80.51
95.21
109.78
114.15
122.96
131.32
A53
LTL
9.00
9.20
9.60
9.40
9.20
9.50
9.30
9.40
9.40
9.30
9.50
9.60
9.10
9.60
9.70
9.40
9.60
9.50
9.50
9.40
9.50
9.60
12.90
9.60
9.50
12.80
12.80
13.80
13.10
13.00
13.40
13.40
12.80
13.00
12.60
13.40
13.30
12.70
12.50
12.50
12.60
12.60
12.50
12.50
12.90
12.60
13.40
14.40
14.70
14.70
15.00
15.20
15.80
LTC
7.30
6.70
6.90
7.20
6.30
6.90
7.30
7.10
7.40
6.80
7.40
7.80
7.70
7.50
7.60
7.80
7.80
7.20
7.10
7.60
7.30
7.70
7.20
7.90
7.10
7.90
8.00
7.30
7.90
7.40
8.10
7.60
7.40
7.20
7.40
6.50
7.00
7.30
6.90
6.90
6.50
7.30
6.80
6.80
7.80
8.00
8.50
9.30
10.20
10.10
10.40
11.10
11.10
LTW
2.50
2.10
2.40
2.30
2.10
2.30
2.40
2.30
2.50
2.20
2.60
2.60
2.50
2.70
2.50
2.70
2.60
2.40
2.60
2.70
2.40
2.70
2.50
2.70
2.50
2.80
2.70
2.40
2.70
2.40
2.80
2.60
2.40
2.50
2.50
2.20
2.30
2.40
2.20
2.30
2.10
2.40
2.20
2.20
2.70
2.70
2.90
3.10
3.40
3.20
3.40
3.70
3.70
LTD
2.10
2.10
2.00
2.20
1.90
2.00
2.30
2.20
2.20
2.10
2.10
2.30
2.40
2.10
2.30
2.30
2.40
2.20
1.90
2.20
2.20
2.20
2.40
2.30
2.10
2.20
2.40
2.20
2.30
2.30
2.30
2.30
2.30
2.10
2.20
1.90
2.20
2.30
2.20
2.20
2.10
2.20
2.10
2.10
2.30
2.40
2.50
2.80
3.20
3.20
3.20
3.40
3.40
LTV
33.55
28.81
32.72
33.77
26.06
31.03
36.45
33.77
36.71
30.51
36.83
40.76
38.77
38.65
39.60
41.45
42.53
35.61
33.32
39.64
35.61
40.49
54.95
42.33
35.41
55.98
58.89
51.73
57.76
50.95
61.27
56.89
50.17
48.46
49.20
39.77
47.78
49.77
42.96
44.91
39.45
47.23
41.00
41.00
56.88
57.97
68.98
88.74
113.55
106.87
115.87
135.76
141.12
Date
21-Mar-05
29-Mar-05
04-Apr-05
18-Apr-05
25-Apr-05
02-May-05
09-May-05
23-May-05
30-May-05
06-Jun-05
13-Jun-05
20-Jun-05
27-Jun-05
04-Jul-05
11-Jul-05
18-Jul-05
25-Jul-05
02-Aug-05
08-Aug-05
15-Aug-05
22-Aug-05
19-Sep-05
26-Sep-05
03-Oct-05
17-Oct-05
24-Oct-05
31-Oct-05
07-Nov-05
14-Nov-05
22-Nov-05
28-Nov-05
05-Dec-05
12-Dec-05
03-Jan-06
09-Jan-06
16-Jan-06
23-Jan-06
06-Feb-06
13-Feb-06
20-Feb-06
27-Feb-06
06-Mar-06
13-Mar-06
20-Mar-06
27-Mar-06
03-Apr-06
10-Apr-06
17-Apr-06
24-Apr-06
01-May-06
22-May-06
29-May-06
05-Jun-06
RTL
15.90
16.30
17.50
17.30
17.60
18.00
18.00
17.90
17.40
17.60
17.60
17.70
17.80
18.00
18.00
18.20
18.50
19.30
18.30
18.40
17.20
17.10
16.70
16.20
17.50
17.30
17.50
16.30
17.40
17.20
16.50
16.30
16.50
15.60
15.90
16.40
16.30
16.40
16.50
17.10
17.40
17.80
17.80
19.30
18.90
19.50
20.00
19.20
19.80
19.10
19.00
19.30
18.60
RTC
11.40
12.10
11.00
11.60
12.40
12.10
13.20
12.70
13.10
12.00
11.70
12.10
12.30
12.50
12.70
13.00
13.20
12.90
12.20
12.60
10.80
10.80
11.40
10.20
11.20
10.60
10.30
10.10
9.60
11.00
10.30
9.30
9.00
9.40
9.60
9.20
9.80
9.50
9.90
10.70
10.70
12.60
11.90
12.40
12.40
12.30
14.30
13.60
12.80
12.70
12.70
11.50
RTW
3.70
3.90
3.50
3.70
4.00
3.90
4.30
4.10
4.20
3.90
3.80
4.10
4.10
4.00
4.10
4.20
4.20
4.20
4.00
4.10
4.00
3.50
3.50
3.70
3.40
3.80
3.60
3.60
3.30
3.20
3.70
3.40
3.10
3.10
3.20
3.40
3.00
3.10
3.10
3.20
3.60
3.40
4.10
3.90
4.10
4.10
4.20
4.60
4.30
4.10
4.30
4.10
3.70
RTD
3.60
3.70
3.50
3.70
3.90
3.80
4.10
4.00
4.10
3.70
3.70
3.60
3.70
4.00
4.00
4.10
4.10
4.10
3.80
3.90
3.80
3.40
3.40
3.50
3.10
3.40
3.10
2.90
3.10
2.90
3.30
3.20
2.80
2.60
2.80
2.80
2.90
3.10
3.00
3.10
3.30
3.40
3.80
3.70
3.80
3.80
3.70
4.50
4.30
4.00
3.80
3.90
3.60
RTV
150.37
167.00
152.21
168.15
194.94
189.40
225.31
208.43
212.74
180.32
175.69
185.49
191.72
204.48
209.59
222.52
226.18
235.97
197.49
208.89
185.62
144.48
141.10
148.95
130.96
158.70
138.66
120.82
126.38
113.33
143.04
125.91
101.69
89.27
101.15
110.85
100.69
111.90
108.95
120.44
146.77
146.10
196.90
197.73
209.07
215.71
220.67
282.18
259.93
222.40
220.43
219.11
175.90
A54
LTL
15.50
15.50
17.00
16.75
17.00
18.00
17.00
17.80
17.00
17.00
17.20
17.00
17.90
17.40
17.80
18.60
18.40
18.50
18.10
18.20
17.80
16.90
16.60
16.30
17.50
16.60
17.40
16.80
17.50
17.10
16.30
16.40
16.10
15.70
16.30
16.70
16.20
15.80
17.00
17.60
17.40
18.00
17.75
18.50
18.40
19.40
20.00
19.30
19.50
18.50
19.50
18.60
18.60
LTC
11.70
11.70
11.60
12.60
12.70
12.10
13.30
13.40
12.50
12.20
11.90
12.90
11.50
12.80
13.40
12.90
12.70
12.90
11.90
11.80
11.60
11.00
11.00
12.20
10.60
11.10
10.50
11.00
10.20
9.90
10.90
10.80
9.70
9.60
9.70
9.60
9.50
10.00
10.30
10.50
11.40
11.40
12.60
12.70
13.40
14.00
13.20
13.00
14.00
14.00
12.10
12.30
12.50
LTW
3.70
3.80
3.90
4.20
4.10
4.00
4.30
4.40
4.10
3.90
3.80
4.20
3.80
4.10
4.32
4.20
4.10
4.30
3.90
3.80
3.70
3.70
3.70
4.10
3.50
3.60
3.50
3.80
3.50
3.20
3.70
3.60
3.40
3.40
3.30
3.10
3.10
3.40
3.40
3.40
3.80
3.60
4.10
4.30
4.50
4.50
4.20
4.10
4.50
4.60
3.90
4.00
4.00
LTD
3.70
3.70
3.50
3.80
4.00
3.70
4.20
4.10
3.80
3.90
3.80
4.00
3.50
4.00
4.20
4.00
4.00
3.90
3.70
3.70
3.70
3.30
3.40
3.60
3.20
3.50
3.20
3.20
3.00
3.10
3.20
3.30
2.80
2.70
2.90
2.70
2.90
2.90
3.20
3.30
3.40
3.60
4.00
3.80
4.00
4.40
4.20
4.10
4.40
4.30
3.80
3.90
4.00
LTV
150.66
154.73
164.76
189.80
197.95
189.14
217.98
227.99
188.05
183.58
176.34
202.78
169.03
202.61
229.30
221.86
214.25
220.27
185.44
181.68
173.01
146.51
148.27
170.82
139.16
148.50
138.36
145.04
130.46
120.44
137.02
138.33
108.82
102.33
110.75
99.24
103.40
110.61
131.32
140.21
159.61
165.63
206.68
214.63
235.15
272.73
250.49
230.35
274.13
259.81
205.18
206.01
211.30
Date
12-Jun-06
19-Jun-06
26-Jun-06
03-Jul-06
RTL
18.50
18.20
17.10
17.60
RTC
11.10
11.60
11.30
10.50
RTW
3.60
3.80
3.60
3.40
RTD
3.50
3.60
3.60
3.30
RTV
165.50
176.77
157.35
140.21
Table A7.5:
Date
3-Jun-03
12-Jun-03
18-Jun-03
25-Jun-03
2-Jul-03
9-Jul-03
16-Jul-03
23-Jul-03
30-Jul-03
4-Aug-03
13-Aug-03
20-Aug-03
27-Aug-03
1-Sep-03
22-Sep-03
1-Oct-03
8-Oct-03
15-Oct-03
22-Oct-03
27-Oct-03
05-Nov-03
12-Nov-03
19-Nov-03
26-Nov-03
03-Dec-03
10-Dec-03
31-Dec-03
06-Jan-04
12-Jan-04
19-Jan-04
26-Jan-04
14-Feb-04
16-Feb-04
23-Feb-04
01-Mar-04
08-Mar-04
15-Mar-04
22-Mar-04
29-Mar-04
12-Apr-04
19-Apr-04
RTL
6.20
6.10
6.10
6.00
6.40
6.10
5.90
6.20
6.00
6.20
6.40
6.30
5.70
6.20
6.20
6.60
6.30
6.10
6.10
6.20
6.20
6.30
6.70
6.40
6.10
6.70
6.40
6.20
6.50
6.40
6.20
6.10
6.20
6.00
6.20
6.50
6.50
7.10
6.10
RTC
4.10
4.20
3.80
RTW
1.30
1.40
1.30
RTD
1.30
1.30
1.20
RTV
7.44
7.88
6.76
4.20
3.90
1.50
1.30
1.10
1.20
7.50
6.76
3.90
4.00
3.80
3.80
3.80
4.40
4.20
3.90
4.00
4.10
4.10
3.70
3.80
4.10
3.20
3.90
4.00
4.00
4.20
3.60
4.10
4.30
3.70
3.90
4.10
3.60
3.60
3.90
4.40
4.20
4.00
4.00
1.30
1.30
1.30
1.30
1.20
1.50
1.40
1.30
1.30
1.30
1.40
1.20
1.30
1.40
1.10
1.30
1.30
1.30
1.30
1.20
1.30
1.60
1.20
1.30
1.40
1.30
1.20
1.20
1.50
1.50
1.30
1.30
1.20
1.20
1.20
1.10
1.20
1.30
1.30
1.20
1.30
1.30
1.10
1.20
1.10
1.20
1.00
1.20
1.20
1.20
1.30
1.10
1.30
1.20
1.10
1.20
1.20
1.00
1.10
1.20
1.30
1.20
1.20
1.20
6.87
6.65
6.87
6.50
6.44
7.89
8.01
6.87
7.92
7.56
6.67
6.24
6.29
7.40
4.92
7.42
7.09
6.76
8.04
6.00
7.44
8.86
6.00
6.87
7.28
5.72
5.62
6.34
9.00
8.31
7.86
6.76
LTL
18.50
17.60
17.10
17.30
LTC
11.20
11.80
11.30
10.60
LTW
3.70
3.80
3.60
3.60
LTD
3.50
3.70
3.60
3.20
LTV
170.10
175.69
157.35
141.50
LTL
LTC
LTW
LTD
LTV
6.00
6.00
6.30
6.10
5.80
6.20
6.10
6.10
6.10
6.20
6.10
5.40
6.10
6.00
6.10
6.10
5.90
6.20
5.90
6.20
6.10
6.20
6.10
6.00
6.50
6.10
6.30
6.30
6.00
6.10
6.10
6.20
6.10
6.30
6.20
6.20
6.10
6.50
6.10
4.40
1.50
1.30
8.31
4.30
4.00
4.60
3.90
3.70
3.70
4.50
4.40
3.90
4.50
4.50
4.40
3.60
4.20
4.30
4.40
3.60
3.90
4.20
3.80
4.00
3.90
4.00
4.20
4.10
4.50
3.80
3.90
4.30
4.00
4.00
3.80
3.90
4.30
4.20
3.80
4.50
1.40
1.30
1.50
1.30
1.20
1.20
1.50
1.50
1.20
1.50
1.50
1.40
1.20
1.40
1.40
1.40
1.10
1.20
1.40
1.20
1.30
1.30
1.30
1.50
1.40
1.50
1.30
1.30
1.50
1.30
1.30
1.20
1.30
1.50
1.40
1.20
1.60
1.30
1.20
1.40
1.10
1.20
1.10
1.40
1.30
1.20
1.30
1.40
1.40
1.10
1.30
1.40
1.40
1.10
1.20
1.30
2.00
1.20
1.10
1.20
1.20
1.20
1.40
1.10
1.10
1.20
1.20
1.30
1.20
1.20
1.30
1.30
1.20
1.30
8.14
6.76
8.65
6.29
6.24
5.72
9.10
8.58
6.24
7.48
9.10
8.35
5.72
7.88
8.21
8.63
5.07
6.34
7.88
10.56
6.76
6.09
7.20
7.80
7.51
9.39
6.09
6.19
7.80
6.87
7.32
6.44
6.87
8.58
7.88
6.65
9.01
M5
A55
Date
27-Apr-04
12-May-04
19-May-04
14-Jul-04
21-Jul-04
28-Jul-04
04-Aug-04
29-Oct-04
09-Nov-04
16-Nov-04
22-Nov-04
29-Nov-04
06-Dec-04
13-Dec-04
30-Dec-04
03-Jan-05
10-Jan-05
17-Jan-05
24-Jan-05
07-Feb-05
15-Feb-05
21-Feb-05
07-Mar-05
14-Mar-05
21-Mar-05
29-Mar-05
04-Apr-05
18-Apr-05
25-Apr-05
02-May-05
09-May-05
23-May-05
30-May-05
06-Jun-05
13-Jun-05
20-Jun-05
27-Jun-05
04-Jul-05
11-Jul-05
18-Jul-05
25-Jul-05
02-Aug-05
08-Aug-05
15-Aug-05
22-Aug-05
19-Sep-05
26-Sep-05
03-Oct-05
17-Oct-05
24-Oct-05
31-Oct-05
07-Nov-05
14-Nov-05
RTL
6.50
6.80
6.30
6.40
6.60
7.00
7.10
5.60
7.00
6.60
7.10
6.50
6.80
6.90
6.90
6.60
7.10
7.00
6.80
7.30
6.70
6.80
7.80
7.20
7.50
7.10
7.10
7.20
7.10
7.50
7.40
7.30
7.00
7.50
7.70
7.20
7.50
7.10
7.20
7.30
7.10
7.40
7.30
7.50
7.10
7.60
7.20
7.60
7.40
7.70
7.70
7.55
8.10
RTC
4.20
4.20
4.00
4.30
4.80
4.10
4.40
3.60
4.00
4.10
4.10
4.00
4.50
4.60
4.20
4.20
4.10
4.20
4.30
4.60
4.10
4.30
4.50
4.30
5.30
4.90
4.30
4.50
4.60
4.60
4.80
4.50
4.10
4.90
4.80
4.80
4.30
4.30
4.90
4.40
4.50
5.30
4.10
4.50
4.70
4.80
5.00
4.90
5.00
5.00
4.40
4.50
5.10
RTW
1.20
1.30
1.30
1.50
1.70
1.40
1.50
1.20
1.30
1.40
1.40
1.40
1.60
1.60
1.30
1.30
1.30
1.40
1.40
1.50
1.40
1.40
1.50
1.50
1.80
1.70
1.40
1.50
1.50
1.60
1.60
1.50
1.40
1.60
1.60
1.60
1.40
1.40
1.60
1.40
1.60
1.70
1.30
1.60
1.50
1.60
1.70
1.60
1.70
1.60
1.50
1.50
1.70
RTD
1.50
1.40
1.20
1.30
1.30
1.20
1.30
1.10
1.20
1.20
1.20
1.10
1.30
1.30
1.30
1.30
1.30
1.30
1.30
1.40
1.30
1.40
1.40
1.30
1.60
1.40
1.30
1.40
1.40
1.40
1.50
1.40
1.20
1.50
1.50
1.40
1.30
1.40
1.50
1.30
1.30
1.70
1.30
1.30
1.50
1.40
1.50
1.50
1.50
1.60
1.30
1.30
1.60
RTV
8.31
8.79
6.98
8.86
10.36
8.35
9.83
5.25
7.75
7.87
8.47
7.11
10.04
10.19
8.28
7.92
8.52
9.05
8.79
10.88
8.66
9.46
11.63
9.97
15.34
12.00
9.17
10.74
10.59
11.93
12.61
10.88
8.35
12.78
13.12
11.45
9.69
9.88
12.27
9.43
10.49
15.18
8.76
11.08
11.34
12.09
13.04
12.95
13.40
14.00
10.66
10.45
15.64
A56
LTL
5.70
6.70
6.70
6.40
6.80
6.70
6.60
5.60
6.00
6.50
6.70
6.70
7.20
7.10
6.90
7.00
6.70
6.80
6.90
6.90
7.10
6.80
6.30
7.10
7.40
7.00
6.50
6.80
7.20
7.30
7.10
7.00
7.10
7.60
7.90
6.90
6.50
6.90
7.20
7.10
7.00
7.60
7.20
7.30
7.00
7.70
7.20
7.50
7.40
7.30
7.30
7.20
7.90
LTC
4.00
4.40
4.20
5.10
4.50
4.90
4.40
3.90
4.20
4.60
4.30
4.20
4.60
4.60
4.30
4.30
4.30
4.80
5.00
4.10
4.70
4.20
4.50
4.90
4.40
4.30
4.30
4.30
4.80
4.60
4.80
4.10
4.90
4.50
4.90
4.30
4.30
4.80
4.40
4.80
4.90
4.70
4.90
4.90
4.70
4.50
4.95
5.10
4.90
4.90
5.00
5.10
LTW
1.20
1.30
1.50
1.40
1.90
1.60
1.60
1.40
1.20
1.40
1.50
1.40
1.40
1.60
1.50
1.40
1.50
1.40
1.60
1.60
1.30
1.50
1.40
1.40
1.60
1.40
1.40
1.50
1.40
1.60
1.50
1.60
1.30
1.70
1.50
1.60
1.50
1.50
1.60
1.40
1.60
1.60
1.50
1.60
1.60
1.60
1.50
1.70
1.70
1.60
1.60
1.60
1.70
LTD
1.40
1.40
1.30
1.30
1.30
1.30
1.60
1.40
1.20
1.30
1.40
1.30
1.30
1.30
1.40
1.40
1.30
1.40
1.50
1.50
1.30
1.50
1.30
1.40
1.50
1.40
1.30
1.30
1.30
1.50
1.40
1.50
1.30
1.40
1.40
1.60
1.30
1.30
1.40
1.40
1.50
1.50
1.40
1.50
1.50
1.40
1.40
1.45
1.60
1.50
1.50
1.60
1.60
LTV
6.80
8.66
9.28
8.27
11.93
9.89
12.00
7.79
6.13
8.40
9.99
8.66
9.30
10.49
10.29
9.74
9.28
9.46
11.76
11.76
8.52
10.86
8.14
9.88
12.61
9.74
8.40
9.41
9.30
12.44
10.59
11.93
8.52
12.84
11.78
12.54
9.00
9.55
11.45
9.88
11.93
12.95
10.74
12.44
11.93
12.25
10.74
13.13
14.29
12.44
12.44
13.09
15.26
Date
21-Nov-05
28-Nov-05
05-Dec-05
12-Dec-05
03-Jan-06
09-Jan-06
16-Jan-06
23-Jan-06
06-Feb-06
13-Feb-06
20-Feb-06
27-Feb-06
06-Mar-06
13-Mar-06
20-Mar-06
27-Mar-06
03-Apr-06
10-Apr-06
17-Apr-06
24-Apr-06
01-May-06
22-May-06
29-May-06
05-Jun-06
12-Jun-06
19-Jun-06
26-Jun-06
03-Jul-06
31-Jul-06
28-Aug-06
03-Sep-06
09-Oct-06
23-Oct-06
27-Nov-06
28-Dec-06
RTL
7.90
8.00
8.00
7.50
7.80
7.90
7.90
7.80
7.75
7.90
7.30
7.50
7.90
7.50
7.60
7.70
7.75
7.70
7.80
8.15
8.30
8.00
7.70
7.70
7.70
7.80
8.00
7.90
8.20
7.80
7.80
8.00
8.10
7.90
8.20
RTC
4.60
5.10
4.60
4.60
5.00
5.70
4.80
4.90
4.60
4.50
4.50
4.40
4.60
5.20
4.40
4.70
4.80
5.20
5.10
5.10
4.60
5.00
5.20
5.60
5.20
4.70
5.20
5.30
5.50
5.60
4.80
5.10
5.10
5.00
5.10
RTW
1.50
1.60
1.60
1.50
1.80
2.00
1.60
1.70
1.30
1.40
1.60
1.50
1.50
1.70
1.40
1.50
1.60
1.70
1.80
1.70
1.50
1.60
1.70
1.90
1.70
1.50
1.70
1.70
1.80
1.80
1.60
1.60
1.70
1.70
1.60
RTD
1.40
1.60
1.30
1.40
1.40
1.60
1.50
1.40
1.40
1.40
1.30
1.30
1.40
1.60
1.40
1.50
1.50
1.60
1.40
1.60
1.40
1.50
1.60
1.70
1.60
1.50
1.60
1.60
1.70
1.70
1.50
1.60
1.60
1.50
1.60
RTV
11.78
14.54
11.81
11.18
13.96
17.95
13.46
13.18
10.01
10.99
10.78
10.38
11.78
14.48
10.58
12.30
13.21
14.87
13.96
15.74
12.38
13.63
14.87
17.66
14.87
12.46
15.45
15.26
17.82
16.95
13.29
14.54
15.64
14.30
14.90
A57
LTL
7.50
7.55
7.70
7.70
7.70
7.80
7.95
7.90
7.70
7.50
7.10
7.30
7.60
7.60
7.60
7.60
7.70
7.50
7.40
8.00
8.00
7.80
8.00
7.80
7.70
7.80
7.95
7.70
7.90
7.70
7.60
8.20
8.00
7.90
7.80
LTC
4.80
4.80
4.70
4.40
4.80
4.70
4.90
5.20
4.70
4.00
4.30
5.00
4.80
4.70
4.70
5.30
5.20
4.80
4.80
5.40
5.20
5.30
5.40
5.10
5.40
5.30
5.10
5.50
5.00
5.20
4.80
5.20
5.20
5.20
5.20
LTW
1.60
1.60
1.50
1.40
1.70
1.60
1.60
1.70
1.60
1.30
1.50
1.60
1.60
1.50
1.50
1.70
1.70
1.70
1.70
1.70
1.70
1.80
1.80
1.70
1.90
1.70
1.70
1.90
1.60
1.70
1.50
1.70
1.80
1.70
1.70
LTD
1.50
1.50
1.40
1.40
1.40
1.40
1.50
1.60
1.40
1.30
1.30
1.60
1.50
1.50
1.50
1.60
1.60
1.40
1.40
1.70
1.70
1.60
1.70
1.50
1.60
1.60
1.60
1.60
1.60
1.60
1.50
1.60
1.50
1.60
1.60
LTV
12.78
12.87
11.48
10.72
13.01
12.41
13.55
15.26
12.25
9.00
9.83
13.27
12.95
12.14
12.14
14.68
14.87
12.67
12.50
16.42
16.42
15.95
17.38
14.12
16.62
15.06
15.35
16.62
14.36
14.87
12.14
15.84
15.34
15.26
15.06
APPENDIX 8
Serum testosterone level in Tursiops aduncus
Table A8.1:
M1
T level
Oct-02
Nov-02
Dec-02
Jan-03
Feb-03
Mar-03
Apr-03
May-03
Jun-03
Jul-03
Aug-03
Sep-03
Oct-03
Nov-03
Dec-03
Jan-04
Feb-04
Mar-04
Apr-04
May-04
Jun-04
Jul-04
Aug-04
Sep-04
Oct-04
Nov-04
Dec-04
Jan-05
Feb-05
Mar-05
Apr-05
May-05
Jun-05
Jul-05
Aug-05
Sep-05
Oct-05
Nov-05
Dec-05
Jan-06
Feb-06
Mar-06
Apr-06
May-06
Jun-06
Jul-06
Aug-06
Sep-06
M1 – M3
M2
T level
24.00
22.95
11.34
17.12
32.73
32.52
69.21
70.24
52.08
64.48
46.16
35.52
20.22
26.52
17.84
18.88
31.00
60.84
47.70
61.50
73.38
43.92
73.76
64.64
44.40
58.80
31.62
27.72
41.88
78.32
55.60
78.40
68.04
54.00
33.36
35.88
30.60
42.24
12.42
34.02
59.22
64.20
45.78
59.10
52.68
25.85
50.04
35.40
Oct-02
Nov-02
Dec-02
Jan-03
Feb-03
Mar-03
Apr-03
May-03
Jun-03
Jul-03
Aug-03
Sep-03
Oct-03
Nov-03
Dec-03
Jan-04
Feb-04
Mar-04
Apr-04
May-04
Jun-04
Jul-04
Aug-04
Sep-04
Oct-04
Nov-04
Dec-04
Jan-05
Feb-05
Mar-05
Apr-05
M3
T level
0.42
0.3
0.19
0.27
1.03
3.56
5.8
7.89
7.68
3.26
4.71
1.45
1.56
0.94
3.55
1.95
1.58
4.5
7.58
4.67
8.59
2.28
2
2.49
1.57
1.1
0.94
1.36
3.7
7.06
21.84
A58
Sep-02
Oct-02
Nov-02
Dec-02
Jan-03
Feb-03
Mar-03
Apr-03
May-03
Jun-03
Jul-03
Aug-03
Sep-03
Oct-03
Nov-03
Dec-03
Jan-04
Feb-04
Mar-04
Apr-04
May-04
Jun-04
Jul-04
Aug-04
Sep-04
Oct-04
Nov-04
Dec-04
Jan-05
Feb-05
Mar-05
Apr-05
May-05
Jun-05
Jul-05
Aug-05
Sep-05
Oct-05
Nov-05
Dec-05
Jan-06
Feb-06
Mar-06
Apr-06
May-06
Jun-06
Jul-06
Aug-06
Sep-06
0.73
1.71
3.40
1.46
1.19
1.47
3.91
2.72
2.46
2.78
2.20
6.27
2.37
0.94
2.23
2.26
1.28
1.80
1.43
3.84
3.99
5.26
2.72
5.55
4.92
5.42
5.04
3.99
1.74
3.94
7.59
4.15
8.69
9.36
0.86
3.56
0.46
0.82
0.77
0.24
4.21
9.44
64.20
4.40
2.80
1.90
0.62
8.73
3.16
Table A8.2:
M4
T level
Oct-02
Dec-02
Jan-03
Feb-03
Mar-03
Apr-03
May-03
Jun-03
Jul-03
Aug-03
Sep-03
Oct-03
Nov-03
Dec-03
Jan-04
Feb-04
Mar-04
Apr-04
May-04
Jun-04
Jul-04
Aug-04
Sep-04
Oct-04
Nov-04
Dec-04
Jan-05
Feb-05
Mar-05
Apr-05
May-05
Jun-05
Jul-05
Aug-05
Sep-05
Oct-05
Nov-05
Dec-05
Jan-06
Feb-06
Mar-06
Apr-06
May-06
Jun-06
Jul-06
M4 – M6
M5
T level
0.14
0.10
0.22
0.26
0.14
0.34
0.99
0.27
0.78
0.41
4.89
0.39
3.76
0.36
0.63
6.02
0.42
3.48
5.24
1.28
1.83
2.08
1.33
1.16
0.90
0.75
2.52
7.33
23.64
24.06
22.34
49.00
19.92
15.64
11.20
2.94
3.54
1.65
2.19
10.31
26.16
8.28
7.72
10.56
14.26
Oct-02
Dec-02
Jan-03
Feb-03
Mar-03
Apr-03
May-03
Jun-03
Jul-03
Aug-03
Sep-03
Oct-03
Nov-03
Dec-03
Jan-04
Feb-04
Mar-04
Apr-04
May-04
Jun-04
Jul-04
Aug-04
Sep-04
Oct-04
Nov-04
Dec-04
Jan-05
Feb-05
Mar-05
Apr-05
May-05
Jun-05
Jul-05
Aug-05
Sep-05
Oct-05
Nov-05
Dec-05
Jan-06
Feb-06
Mar-06
Apr-06
May-06
Jun-06
Jul-06
Aug-06
Sep-06
Oct-06
Nov-06
Dec-06
M6
T level
0.1
0.1
0.1
0.31
0.1
0.1
0.1
0.19
0.38
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.31
0.25
0.19
0.1
0.23
0.1
0.17
0.11
0.24
0.24
0.15
0.15
0.38
0.8
0.26
0.56
0.3
0.18
0.2
0.13
0.1
0.1
0.3
0.76
1.28
0.22
0.54
0.1
1.08
1.35
0.1
0.34
0.14
A59
Oct-02
Nov-02
Dec-02
Jan-03
Feb-03
Mar-03
Apr-03
May-03
Jun-03
Jul-03
Aug-03
Sep-03
Oct-03
Nov-03
Dec-03
Jan-04
Feb-04
Mar-04
Apr-04
May-04
Jun-04
Jul-04
Aug-04
Sep-04
Oct-04
Nov-04
Dec-04
Jan-05
Feb-05
Mar-05
Apr-05
May-05
Jun-05
Jul-05
Aug-05
Sep-05
Oct-05
Nov-05
Dec-05
Jan-06
Feb-06
Mar-06
Apr-06
May-06
Jun-06
Jul-06
Aug-06
Sep-06
11.16
26.10
8.56
13.74
24.18
57.78
54.56
50.64
50.32
46.16
38.24
17.82
5.10
25.16
3.24
14.12
13.24
20.76
32.04
23.70
24.48
30.54
28.26
34.02
22.26
21.36
12.60
18.96
51.90
53.82
56.16
24.24
11.34
4.32
11.00
9.16
8.44
1.84
7.68
28.64
27.08
18.00
15.76
3.08
1.56
5.89
3.83
APPENDIX 9
Ejaculate (E1 and E2) characteristics and overall semen volume,
sperm count and density in Tursiops aduncus
Table A9.1:
E1 EjV
Date
09-Oct-02 31.50
16-Oct-02
2.50
23-Oct-02
6.00
30-Oct-02 25.00
06-Nov-02 19.00
13-Nov-02 29.70
20-Nov-02 64.50
27-Nov-02 4.90
04-Dec-02 37.50
12-Dec-02 47.50
18-Dec-02 57.50
24-Dec-02 49.50
08-Jan-03
13.00
15-Jan-03
41.50
22-Jan-03
54.50
28-Jan-03
74.50
05-Feb-03 70.00
12-Feb-03 35.50
19-Feb-03 72.50
25-Feb-03 43.30
05-Mar-03 49.50
12-Mar-03 57.33
19-Mar-03 35.30
25-Mar-03 39.00
02-Apr-03 61.50
09-Apr-03 41.80
14-Apr-03 16.10
22-Apr-03 46.50
27-Apr-03 57.30
05-May-03 50.50
13-May-03 47.00
21-May-03 33.50
26-May-03 50.50
03-Jun-03
4.40
12-Jun-03 16.10
18-Jun-03 36.00
25-Jun-03 74.00
02-Jul-03
58.10
09-Jul-03
39.00
E1 EjD
192.00
0.07
140.50
0.88
145.50
0.95
0.03
1.35
0.00
10.50
155.00
1.58
8.50
5.00
10.00
0.13
75.00
2.63
0.10
231.25
91.25
135.00
141.00
0.00
3.95
75.00
70.00
52.50
116.25
76.50
0.00
216.25
195.00
49.00
0.03
49.13
E1 EjC
M1
E2 EjV E2 EjD
6048.00 11.00
15.50
0.43
15.00
3512.50 8.50
16.63
4.50
4321.35 12.00
61.28 11.00
0.12
16.50
50.63 12.50
0.00
14.00
603.00 8.50
8.50
2015.00 9.30
65.36 12.50
5.50
633.25 7.50
350.00 16.50
355.00 4.30
9.06
22.50
3247.50 12.20
129.94 11.00
5.73
8.40
8163.13 10.00
3558.75 1.80
8302.50 11.90
5893.80 5.70
0.00
14.90
183.68 5.00
4297.50 12.50
3535.00 4.00
2467.50 7.50
3894.38 8.20
3863.25 5.80
0.00
37.50
3481.63 7.20
7020.00 9.40
3626.00 11.50
1.45
1915.88 5.80
555.00
50.75
365.00
480.00
650.00
20.50
15.00
17.50
41.50
156.25
165.00
30.00
110.00
100.00
1.70
1565.00
590.00
4.85
0.40
200.00
235.00
190.00
605.00
1565.00
845.00
1745.00
0.00
70.00
680.00
935.00
64.50
945.00
1550.00
47.00
405.00
1272.50
1007.50
E2 EjC
49.50
44.05
27.30
34.40
27.30
41.70
84.00
26.64
59.00
61.50
66.00
69.51
35.30
54.00
59.94
90.00
87.69
45.60
105.29
86.25
60.50
82.20
66.90
18.60
89.00
47.50
31.00
60.90
85.60
65.60
60.20
79.60
113.40
46.20
39.60
5.19
90.00
58.10
667.50 3871.50 74.88
A60
6105.00
786.63
5475.00
4080.00
2925.00
246.00
165.00
288.75
518.75
2187.50
1402.50
255.00
1023.00
1250.00
9.35
11737.50
9735.00
2085.50
9.00
2440.00
2585.00
1596.00
6050.00
2817.00
10055.50
9946.50
0.00
350.00
8500.00
3740.00
483.75
7749.00
8890.00
1762.50
2916.00
11961.50
11586.25
ToV
OvD
ToC
HiEjD
394.71
103.19
283.64
242.56
111.32
109.53
10.18
14.88
13.85
35.57
30.39
3.95
148.67
24.36
0.16
246.79
123.56
128.68
23.35
195.43
44.88
64.32
347.11
442.40
294.22
333.48
0.00
40.46
322.44
159.91
49.50
246.91
308.67
85.85
594.84
448.39
181.71
0.03
200.96
19538.00
4545.56
7743.48
8344.00
3039.00
4567.35
855.28
396.38
816.88
2187.50
2005.50
274.55
5248.00
1315.36
9.35
22210.75
10835.00
5868.00
2458.56
16856.13
2714.94
5286.73
23221.88
8227.25
26185.50
15840.30
0.00
2464.18
27600.50
10490.00
2979.89
19654.25
35003.40
3966.25
23555.63
2327.15
16354.13
1.45
15047.63
1055.00
167.75
365.00
835.00
650.00
145.50
74.00
20.50
41.50
156.25
165.00
30.00
170.00
100.00
1.70
1565.00
625.00
715.00
275.00
685.00
235.00
261.25
605.00
1565.00
2650.00
1745.00
225.00
1240.00
935.00
64.50
945.00
1550.00
512.50
1265.00
1272.50
1007.50
0.03
1380.00
E1 EjV E1 EjD
E1 EjC E2 EjV E2 EjD E2 EjC
ToV
OvD
ToC
HiEjD
Date
16-Jul-03
70.00
35.75 2502.50 15.20 617.50 9386.00 91.40 219.95 20103.50 1325.00
23-Jul-03
33.70
35.50 1196.35 7.40 172.50 1276.50 52.30 175.59 9183.35 667.50
30-Jul-03
53.90
31.63 1704.59 7.00 125.00 875.00 69.55 60.03 4175.36 219.00
04-Aug-03 11.00
3.00
33.00 12.00 114.00 1368.00 41.30 165.96 6854.00 430.00
13-Aug-03 45.75 281.25 12867.19 8.45 905.00 7647.25 85.26 464.84 39632.81 905.00
20-Aug-03 67.20
13.50
907.20 13.20 610.00 8052.00 121.30 193.16 23429.98 2620.00
27-Aug-03 9.80
242.50 2376.50 7.50 512.50 3843.75 60.00 431.77 25906.38 605.00
01-Sep-03 34.50
0.03
0.86
17.00 247.50 4207.50 104.30 273.62 28538.11 1160.00
10-Sep-03 45.50
20.25
921.38 17.50 575.00 10062.50 93.75 273.69 25658.25 1195.00
17-Sep-03 69.10
39.00 26949.00 8.60 1475.00 12685.00 113.00 390.37 44112.30 2040.00
22-Sep-03 53.20
0.00
0.00
8.40 435.00 3654.00 76.80 85.16 6540.05 435.00
01-Oct-03 52.40
44.50 2331.80 14.90 665.00 9908.50 106.60 320.84 34201.05 1170.00
08-Oct-03 57.70
57.70
105.00 8.20 390.00 3198.00 68.60 152.45 10458.00 445.00
15-Oct-03 31.90
67.50 2153.25 10.40 600.00 6240.00 55.70 317.29 17673.25 1445.00
22-Oct-03 30.50 405.00 12352.50 27.40 115.00 3151.00 89.92 388.08 34895.88 1250.00
27-Oct-03 38.90
0.00
0.00
30.90 66.50 2054.85 103.30 113.06 11678.98 735.00
05-Nov-03 24.40 735.00 17934.00 15.40 128.75 1982.75 50.00 479.04 23951.88 735.00
12-Nov-03 25.40 257.50 6540.50 11.40 695.00 7923.00 71.65 478.77 34303.70 1455.00
19-Nov-03 62.70
58.75 3683.63 10.40 315.00 327.60 102.10 119.13 12163.28 725.00
26-Nov-03 58.80
54.50 3204.60 5.60 477.50 2674.00 133.20 178.32 23751.60 665.00
03-Dec-03 53.50 260.00 13910.00 6.40 415.00 2656.00 116.50 303.97 35412.20 1305.00
10-Dec-03 56.60 151.25 8560.75 6.80 620.00 4216.00 76.50 270.37 20683.20 870.00
17-Dec-03 32.00 390.00 12480.00 11.00 800.00 8800.00 4.90 489.80 2400.00 800.00
25-Dec-03 29.00 390.00 11310.00 9.50 720.00 6840.00 49.00 479.80 23510.00 800.00
31-Dec-03 5.50
318.75 1753.13 13.20 337.50 4455.00 18.70 331.99 6208.13 337.50
06-Jan-04
42.50
33.75 1434.38 5.90 365.00 2153.50 81.50 246.57 20095.13 1080.00
12-Jan-04
21.00
0.00
0.00
39.40 49.50 1950.30 81.70 95.55 7806.33 1365.00
19-Jan-04
48.30
43.00 2076.90 13.60 388.75 5287.00 101.60 128.34 13039.51 388.75
26-Jan-04
18.70 228.75 4300.50 4.60 132.00 607.20 31.90 187.24 5972.88 228.75
02-Feb-04 34.70
32.50 1127.75 7.50 675.00 5062.50 52.20 188.65 9847.75 675.00
09-Feb-04 46.00
55.25 2541.50 12.30 705.00 8671.50 79.70 211.86 16885.30 705.00
16-Feb-04 50.50 230.00 11615.00 16.40 775.00 12710.00 120.50 248.73 29971.53 775.00
23-Feb-04 22.40
23.50
526.40 8.90 312.50 2781.25 39.20 128.07 5020.15 670.00
01-Mar-04 45.50 125.00 568.75 5.80 330.00 1914.00 75.80 100.05 7584.05 440.00
08-Mar-04 50.00 172.50 8625.00 12.20 7.50 8601.00 122.40 298.68 36558.00 935.00
15-Mar-04 19.30 415.00 8009.50 25.00 46.50 1162.50 79.50 190.09 15112.13 415.00
22-Mar-04 35.00 197.50 6912.50 1.60 56.50
90.40
65.60 219.90 14424.98 615.00
29-Mar-04 26.00
60.00 1560.00 15.20 33.00 501.60 59.00 185.87 10966.60 915.00
12-Apr-04 34.50 266.25 9185.63 14.40 485.00 6984.00 107.50 216.85 23311.36 715.00
19-Apr-04 37.10 226.25 8393.88 3.60 1805.00 6498.00 72.90 355.84 25941.73 2095.00
27-Apr-04 50.60
72.50 3668.50 2.70 2210.00 5967.00 129.20 275.30 35568.18 2210.00
03-May-04 24.90 1070.00 26643.00 4.90 1665.00 8158.50 121.00 485.78 58779.16 1665.00
10-May-04 22.50 1695.00 38137.50 8.60 343.75 2956.25 63.80 751.91 47972.08 1695.00
17-May-04 39.40 252.50 9948.50 5.50 1265.00 6957.50 63.10 424.22 26768.00 1265.00
24-May-04 41.20
52.00 2142.40 8.60 630.00 5418.00 61.20 364.85 22329.13 965.00
31-May-04 29.30
75.00 2197.50 20.30 251.25 5100.38 96.10 150.88 14499.33 391.25
07-Jun-04 41.00
84.50 3464.50 7.50 1310.00 9825.00 86.70 277.09 24024.08 1310.00
14-Jun-04 58.00 105.00 6090.00 13.00 487.50 6337.50 93.50 290.27 27140.00 690.00
21-Jun-04 27.00 237.50 6412.50 23.30 103.75 2417.38 108.40 254.43 27579.88 445.00
28-Jun-04
7.50
0.03
0.19
26.50 0.13
3.31
141.80 108.56 15393.53 812.50
02-Jul-04
61.30
15.00
919.50 12.00 67.50 810.00 99.40 45.99 4571.83 201.25
12-Jul-04
19.30 202.50 3908.25 17.50 247.50 4331.25 105.50 273.78 28884.13 1400.00
19-Jul-04
22.50
0.00
0.00
43.00 3.00
129.00 113.00 80.23 9066.03 2195.00
A61
E1 EjV
Date
26-Jul-04
59.00
02-Aug-04 81.50
09-Aug-04 52.00
16-Aug-04 32.00
23-Aug-04 37.50
30-Aug-04 59.70
06-Sep-04 63.30
13-Sep-04 60.00
20-Sep-04 52.50
27-Sep-04 33.50
04-Oct-04 26.50
11-Oct-04 43.10
18-Oct-04 45.80
25-Oct-04 41.50
28-Oct-04 76.10
09-Nov-04 39.00
16-Nov-04 46.00
22-Nov-04 35.20
29-Nov-04 27.50
06-Dec-04 40.50
13-Dec-04 22.00
20-Dec-04 51.70
30-Dec-04 47.00
03-Jan-05
45.00
10-Jan-05
39.00
17-Jan-05
46.20
24-Jan-05
50.50
31-Jan-05
44.00
07-Feb-05 30.90
15-Feb-05
6.00
21-Feb-05 46.20
28-Feb-05 37.30
07-Mar-05 62.50
14-Mar-05 32.00
21-Mar-05 52.00
29-Mar-05 61.30
04-Apr-05 51.00
18-Apr-05 46.30
25-Apr-05 74.30
02-May-05 50.00
09-May-05 32.50
23-May-05 50.90
30-May-05 54.40
06-Jun-05 29.00
20-Jun-05 42.40
27-Jun-05 41.30
04-Jul-05
49.00
11-Jul-05
48.30
18-Jul-05
51.40
25-Jul-05
62.00
02-Aug-05 83.20
08-Aug-05 66.50
15-Aug-05 67.50
E1 EjD
E1 EjC
5.58
33.50
40.25
207.58
53.00
20.25
41.25
68.00
155.00
143.00
30.50
282.50
127.50
67.50
96.00
292.35
237.50
44.00
0.00
227.50
245.00
30.50
41.00
248.75
445.00
695.00
330.00
190.00
60.00
0.50
49.00
130.00
150.00
307.50
121.00
13.50
350.00
147.50
93.75
178.75
290.00
101.75
38.00
805.00
33.75
125.00
79.00
426.25
195.00
307.50
317.50
325.00
152.50
328.93
2730.25
2093.00
6640.00
198.75
1208.93
2611.13
4080.00
8137.50
4790.50
808.25
1217.75
5839.50
2801.25
7305.60
11407.50
10925.00
1548.80
0.00
9213.75
5390.00
1576.85
1927.00
11193.75
17355.00
32109.00
16665.00
8360.00
1854.00
3.00
2263.80
4849.00
9375.00
9840.00
6292.00
827.55
17850.00
6829.25
6965.63
8937.50
9425.00
5179.08
2067.20
23345.00
1431.00
5162.50
3871.00
20587.88
10023.00
19065.00
26416.00
14962.50
10293.75
E2 EjV E2 EjD
E2 EjC
ToV
OvD
ToC
HiEjD
12.00
21.50
21.00
29.00
13.50
9.50
17.90
5.50
13.50
4.20
2.80
10.70
9.30
14.60
10.70
6.60
9.20
8.20
44.80
10.70
21.20
13.70
5.50
5.90
13.60
14.10
16.50
9.80
10.40
8.50
17.00
10.50
6.30
23.00
5.20
2.80
10.50
6.10
18.00
7.00
24.50
15.00
9.10
9.00
11.00
980.00
340.00
595.00
220.00
151.25
560.00
257.50
945.00
560.00
725.00
930.00
545.00
640.00
1120.00
312.50
1340.00
965.00
252.50
67.00
710.00
62.50
270.00
382.50
1025.00
342.50
870.00
255.00
990.00
1050.00
360.00
235.00
375.00
1010.00
212.5
575.00
1350.00
680.00
480.00
670.00
315.00
182.50
1130.00
895.00
575.00
641.67
11760.00
7310.00
12495.00
6380.00
2041.88
5320.00
4609.25
5197.50
7560.00
3045.00
2604.00
5831.50
5952.00
16352.00
3343.75
8844.00
8878.00
2070.50
3001.60
7597.00
1325.00
3699.00
2103.75
6047.50
4658.00
12267.00
4207.50
9702.00
10920.00
3060.00
3995.00
3937.50
6363.00
4887.5
4715.00
3780.00
7140.00
2928.00
12060.00
2205.00
6296.25
16950.00
8144.50
5750.00
7058.33
9.00
8.00
5.20
14.00
13.00
7.60
10.50
1426.67
1965.00
1100.00
1545.00
227.50
1090.00
505.00
12840.03
15720.00
5720.00
21630.00
2957.50
8284.00
5302.50
136.60
163.60
167.20
97.00
73.10
100.00
33.20
122.50
156.40
75.40
48.70
71.30
180.60
122.70
142.10
121.20
201.60
105.50
221.70
94.60
51.60
107.60
66.30
119.60
92.60
133.18
88.60
168.10
88.70
39.50
125.80
54.60
177.70
100.80
105.20
141.00
183.90
193.30
214.70
134.40
174.30
150.70
122.70
43.80
53.40
41.30
58.00
75.40
83.30
115.80
141.40
134.60
145.70
283.80
171.30
352.93
304.86
129.96
112.00
210.46
266.64
320.65
279.73
164.23
327.81
221.77
301.47
240.19
320.25
177.62
128.78
313.28
369.66
152.13
81.73
77.78
343.33
519.94
466.69
324.85
293.41
263.77
210.53
229.19
205.14
277.77
271.79
289.85
162.03
382.06
190.54
235.51
237.06
296.14
339.14
328.73
796.32
158.98
125.00
288.12
738.29
387.07
542.48
330.08
291.90
226.53
38767.00
28025.25
59010.13
29573.00
9499.88
11199.75
6987.13
32663.38
50149.48
21091.48
7998.13
23372.88
40051.94
36989.78
34130.73
38814.20
35807.33
13586.18
69453.55
34969.50
7850.13
8794.20
5156.73
41061.85
48146.75
62153.18
28782.00
49321.90
23396.25
8315.75
28832.05
11200.50
49360.58
27396.48
30492.25
22846.05
70260.38
36831.25
50564.03
31861.13
51617.75
51108.33
40335.45
34879.00
8489.33
5162.50
16711.03
55666.88
32243.00
62818.93
46673.19
39289.38
33006.00
980.00
630.00
895.00
800.00
268.75
965.00
318.75
945.00
722.50
725.00
960.00
545.00
640.00
1120.00
720.00
1340.00
1330.00
252.50
1030.00
1315.00
263.75
270.00
382.50
1165.00
1605.00
870.00
420.00
990.00
1050.00
440.00
700.00
375.00
1095.00
750.00
1770.00
1350.00
1255.00
525.00
670.00
875.00
1035.00
1130.00
1135.00
1315.00
641.67
125.00
1426.67
3045.00
1100.00
1545.00
651.67
1090.00
600.00
A62
E1 EjV
Date
22-Aug-05 59.00
13-Sep-05 66.30
19-Sep-05 45.30
26-Sep-05 61.00
03-Oct-05 38.50
17-Oct-05 69.20
24-Oct-05 48.50
01-Nov-05 60.80
07-Nov-05 73.00
14-Nov-05 46.20
21-Nov-05 41.20
28-Nov-05 2.50
05-Dec-05 69.20
12-Dec-05 64.20
03-Jan-06
41.20
09-Jan-06
43.00
16-Jan-06
54.00
23-Jan-06
43.00
06-Feb-06 36.50
13-Feb-06 80.80
20-Feb-06 85.00
27-Feb-06 64.80
06-Mar-06 70.00
13-Mar-06 60.80
20-Mar-06 51.00
27-Mar-06 64.00
03-Apr-06 41.20
10-Apr-06 12.40
17-Apr-06 50.80
24-Apr-06 76.20
01-May-06 52.00
22-May-06 33.00
29-May-06 66.00
05-Jun-06 65.50
12-Jun-06 68.40
19-Jun-06 57.00
26-Jun-06 66.00
E1 EjD
E1 EjC
135.50
212.20
43.00
127.50
64.50
131.25
70.00
123.75
64.50
5.13
21.50
0.54
147.50
31.50
186.25
0.00
0.00
0.00
62.50
2.63
0.00
0.00
29.00
102.50
96.00
0.00
765.00
885.00
483.75
205.00
263.75
471.25
180.00
180.50
261.25
121.25
136.00
7994.50
14088.75
1947.90
7777.50
2483.25
9082.50
3395.00
7524.00
4708.50
236.78
885.80
1.34
10207.00
2022.30
7673.50
0.00
0.00
0.00
2281.25
212.10
0.00
0.00
2030.00
6232.00
4896.00
0.00
31518.00
10974.00
24574.50
15621.00
13715.00
15551.25
11880.00
11822.75
17869.50
6911.25
8976.00
E2 EjV E2 EjD
E2 EjC
ToV
OvD
ToC
HiEjD
3.00
8.90
11.50
7.00
10.50
7.50
8.30
8.00
4.40
2.00
3.00
57.50
3.00
9.90
9.20
19.50
14.50
6.20
7.30
7.00
2555.00
1445.00
353.75
450.00
517.50
160.00
460.00
585.00
683.33
4.63
241.25
0.04
1365.00
550.00
760.00
71.50
56.50
15.00
477.50
100.00
7665.00
1286.05
4068.13
3150.00
5433.75
1200.00
3818.00
4680.00
3006.67
9.25
723.75
2.30
4095.00
5445.00
6992.00
1394.25
819.25
93.00
3485.75
700.00
8.60
13.80
6.50
7.00
11.70
12.00
4.20
2.70
6.20
11.10
3.50
5.80
21.40
11.10
7.00
8.50
0.00
305.00
1320.00
332.50
555.00
1570.00
178.75
785.00
865.00
755.00
2345.00
329.50
765.00
610.00
810.00
830.00
0.00
4209.00
8580.00
2327.50
6493.50
18840.00
750.75
2119.50
5363.00
8380.50
8207.50
2276.50
16371.00
6771.00
5670.00
7055.00
124.40
157.30
94.00
112.50
83.90
89.70
78.17
101.90
113.15
58.40
82.20
81.10
90.00
125.20
85.50
63.00
105.50
64.10
48.20
101.43
117.81
122.00
140.20
118.20
104.90
136.00
78.80
25.40
94.00
184.20
97.10
111.40
138.10
161.90
192.00
152.50
139.30
329.80
321.83
135.51
215.92
128.21
196.74
265.87
130.73
148.17
4.23
36.93
45.47
209.96
162.89
215.62
22.35
24.20
1.84
121.32
26.68
3.51
0.66
68.30
264.63
274.83
235.78
746.38
980.25
378.44
342.86
342.08
414.09
319.36
388.33
356.89
252.32
333.72
41027.43
50624.21
12738.28
24291.13
10757.00
17647.50
20782.80
13321.00
16765.42
247.00
3035.74
3687.75
18896.50
20393.30
18435.38
1408.00
2553.30
117.68
5847.76
2706.45
413.88
80.29
9574.93
31278.75
28829.88
32066.50
58815.00
24898.25
35573.50
63155.63
33216.13
46129.25
44103.00
62870.75
68523.43
38478.94
46487.25
2555.00
1445.00
390.00
760.00
517.50
850.00
930.00
600.00
780.00
5.13
241.25
468.75
1365.00
550.00
760.00
71.50
138.75
15.00
477.50
277.50
23.50
3.63
467.50
1320.00
510.00
790.00
1570.00
1805.00
785.00
1115.00
755.00
2345.00
937.50
820.00
1060.00
995.00
945.00
A63
Table A9.2:
Date
09-Oct-02
16-Oct-02
23-Oct-02
06-Nov-02
13-Nov-02
20-Nov-02
27-Nov-02
04-Dec-02
12-Dec-02
18-Dec-02
24-Dec-02
08-Jan-03
15-Jan-03
22-Jan-03
28-Jan-03
05-Feb-03
12-Feb-03
19-Feb-03
25-Feb-03
05-Mar-03
12-Mar-03
19-Mar-03
25-Mar-03
02-Apr-03
09-Apr-03
14-Apr-03
22-Apr-03
27-Apr-03
05-May-03
13-May-03
21-May-03
27-May-03
03-Jun-03
12-Jun-03
18-Jun-03
25-Jun-03
02-Jul-03
09-Jul-03
16-Jul-03
23-Jul-03
30-Jul-03
04-Aug-03
13-Aug-03
20-Aug-03
27-Aug-03
01-Sep-03
10-Sep-03
17-Sep-03
22-Sep-03
01-Oct-03
E1 EjV
52.50
12.55
49.50
63.50
17.00
74.50
50.50
11.50
24.00
50.50
13.10
18.50
30.40
46.50
56.50
67.00
33.00
62.50
27.30
50.00
7.50
48.80
0.90
23.50
23.00
7.90
18.00
29.50
17.80
34.00
52.70
11.50
25.50
79.00
32.00
50.00
21.00
30.00
38.00
13.50
88.00
20.50
6.25
56.20
42.10
15.50
13.50
29.90
40.40
63.40
E1 EjD
33.25
2450.0
0.63
0.00
0.00
3.45
25.25
18.25
0.20
16.50
0.93
0.03
0.25
0.00
0.00
0.00
0.00
0.03
0.08
0.00
1.68
1.13
800.00
32.50
160.00
42.50
22.50
23.75
300.00
165.00
2.33
103.50
63.00
14.75
17.45
2.25
4.20
63.13
30.63
75.00
12.00
89.75
8.45
2.50
2.25
47.50
51.00
312.50
54.00
185.00
E1 EjC E2 EjV
1745.63 6.00
30747.50 6.00
30.94
3.50
0.00
0.00
0.90
257.03
0.41
1275.13 1.88
209.88
3.00
4.80
3.30
83.33
7.60
12.12
3.50
0.46
2.00
7.60
4.50
0.00
5.30
0.00
7.00
0.00
2.90
0.00
7.00
1.56
5.50
2.05
3.10
4.10
12.56
8.50
54.90
2.20
720.00 11.00
763.75
2.50
3680.00 2.50
335.75
2.70
405.00
0.90
700.63
3.40
5340.00 3.80
5610.00 3.80
122.53
3.70
1190.25 2.10
1606.50 4.60
1165.25 6.20
558.40
3.90
112.50
3.30
88.20
3.10
1893.75 3.80
1163.75 3.30
1012.50 5.10
1056.00 4.00
1839.88 1.70
52.81
4.15
140.50
6.70
94.73
1.40
736.25
3.50
418.50
4.00
9343.75 2.00
2181.60 1.25
11729.00 6.90
M2
E2 EjD
1535.0
1310.0
185.00
213.75
233.75
199.00
66.25
14.50
0.05
1.15
0.40
0.45
0.00
0.18
0.00
0.18
0.53
2.08
0.20
9.50
38.50
145.00
465.00
490.00
120.00
185.00
90.00
95.00
1400.0
445.00
281.25
478.75
382.50
295.00
32.00
88.50
730.00
3.73
18.50
592.50
88.75
3.60
206.25
451.25
25.00
138.75
1040.0
955.00
1875.0
A64
E2 EjC ToV
9210.00 58.50
7860.00 24.85
647.50 56.00
63.50
192.38 19.80
95.84 74.50
374.12 52.38
198.75 17.30
47.85 27.29
0.38
58.09
4.03
16.61
0.80
20.50
2.03
7.59
0.00
10.89
1.23
69.17
0.00
76.11
1.23
7.00
2.89
72.29
6.43
37.23
0.82
4.10
80.75
7.50
84.70 51.00
1595.00 13.90
1162.50 29.10
1225.00 25.50
324.00 14.10
166.50 25.60
306.00 32.90
361.00 17.80
5320.00 34.00
1646.50 70.40
590.63 21.50
2202.25 40.50
2371.50 107.35
1150.50 35.90
105.60 53.30
274.35 21.00
2774.00 44.50
12.29 41.30
94.35 24.60
2370.00 88.00
150.88 22.20
14.94 15.05
1381.88 98.80
631.75 42.10
87.50 19.21
555.00 43.75
2080.00 36.30
1193.75 46.10
12937.5 84.30
OvD
187.28
1692.4
22.03
0.00
14.18
3.45
31.49
79.05
1.93
1.44
0.97
0.06
0.48
0.04
0.06
0.00
0.18
0.10
0.55
0.20
1.68
2.74
121.11
91.23
192.35
67.09
100.76
30.60
300.00
165.00
59.80
193.66
118.52
139.22
47.60
4.09
4.20
190.17
28.48
71.94
12.00
89.67
5.33
57.13
2.25
43.36
157.73
315.37
151.41
452.16
ToC
10955.63
42057.00
1233.44
0.00
280.73
257.03
1649.25
1367.63
52.65
83.71
16.14
1.26
3.65
0.47
4.15
0.34
1.23
6.87
20.55
0.82
12.56
139.60
1683.45
2654.75
4905.00
946.00
2579.50
1006.63
5340.00
5610.00
4210.15
4163.58
4800.05
14945.50
1708.90
218.10
88.20
8462.35
1176.04
1769.60
1056.00
1990.75
80.19
5644.73
94.73
832.75
6900.75
11447.95
6979.85
38117.00
HiEjD
1535.0
2450.0
185.00
213.75
3.45
374.12
342.50
14.50
16.50
1.15
0.40
0.53
0.35
0.50
0.10
0.18
0.58
2.08
0.20
1.68
38.50
800.00
465.00
490.00
127.50
770.00
90.00
300.00
1400.0
445.00
655.00
478.75
602.50
295.00
32.00
4.20
890.00
30.33
161.25
12.00
89.75
52.81
366.25
2.25
47.50
320.00
1040.0
955.00
1875.0
Date
08-Oct-03
15-Oct-03
22-Oct-03
27-Oct-03
05-Nov-03
12-Nov-03
19-Nov-03
26-Nov-03
03-Dec-03
10-Dec-03
31-Dec-03
06-Jan-04
12-Jan-04
19-Jan-04
26-Jan-04
02-Feb-04
09-Feb-04
16-Feb-04
23-Feb-04
01-Mar-04
08-Mar-04
15-Mar-04
22-Mar-04
29-Mar-04
12-Apr-04
19-Apr-04
27-Apr-04
03-May-04
10-May-04
17-May-04
24-May-04
31-May-04
07-Jun-04
14-Jun-04
21-Jun-04
28-Jun-04
02-Jul-04
12-Jul-04
19-Jul-04
26-Jul-04
02-Aug-04
09-Aug-04
16-Aug-04
23-Aug-04
30-Aug-04
06-Sep-04
13-Sep-04
20-Sep-04
27-Sep-04
04-Oct-04
11-Oct-04
18-Oct-04
25-Oct-04
E1 EjV
33.70
54.00
55.50
55.40
70.50
50.90
59.70
17.80
22.00
47.60
18.50
30.50
30.50
36.00
3.70
14.50
8.30
33.30
57.30
31.00
23.00
31.60
31.00
38.80
31.90
12.40
25.20
75.80
35.90
11.70
11.00
70.50
37.50
75.00
17.00
17.90
17.50
28.50
99.50
28.00
61.20
90.00
63.50
48.50
57.70
23.00
14.10
48.50
26.50
19.50
35.70
33.90
61.40
E1 EjD
27.50
11.00
18.00
4.03
14.50
15.38
13.00
53.50
11.70
2.88
180.50
52.50
1.40
7.50
173.75
3.75
32.75
39.00
0.23
2.10
5.50
0.25
3.38
0.03
1.50
71.50
0.23
9.63
0.00
99.00
14.00
23.25
4.00
13.75
465.00
88.75
375.00
77.50
5.35
102.00
35.50
98.00
5.50
3.40
36.00
73.75
34.00
24.75
74.00
27.50
29.38
1.38
0.08
E1 EjC E2 EjV
929.50
3.90
594.00
6.40
999.00 20.40
222.99
0.40
1022.25 6.40
782.59
6.90
776.10
6.40
952.30
5.90
257.40 27.50
136.85
3.00
3339.25 1.70
1601.25 2.50
42.70
4.40
270.00
3.60
642.88 13.80
54.38
0.50
217.83
1.20
1298.70 0.70
12.89
1.40
65.10
0.50
126.50
0.20
7.90
2.50
104.63
3.00
0.97
6.10
47.85
2.80
886.60
5.00
5.67
22.10
729.58
7.40
0.00
4.70
1158.30 4.00
154.00
3.00
1639.13 7.20
150.00
7.40
1031.25 6.00
7905.00 6.00
1588.63 6.90
6562.50 4.50
2208.75 3.10
532.33
6.90
2856.00 4.30
2172.60 3.30
8820.00 13.00
349.25
6.00
1649.00 5.00
2077.20 5.50
1696.25 2.00
479.40
3.20
122.38
5.20
1961.00 3.90
536.25
2.20
1048.69 7.40
46.61
4.80
4.61
4.20
E2 EjD
505.00
355.00
272.50
490.00
342.50
73.75
290.00
410.00
3.23
425.00
290.00
545.00
89.50
257.50
17.50
141.25
93.75
250.00
3.40
0.15
1.20
0.15
30.50
0.18
1.50
1.08
13.00
522.50
0.14
161.25
24.50
907.50
38.75
462.50
1055.0
2030.0
1160.0
770.00
895.00
745.00
670.00
1350.0
377.50
285.00
332.50
308.75
365.00
58.00
1200.0
120.00
635.00
10.63
66.00
A65
E2 EjC
1969.50
2272.00
5559.00
196.00
2192.00
508.88
1856.00
2419.00
88.69
1275.00
493.00
1362.50
393.80
927.00
241.50
70.63
112.50
175.00
4.76
0.08
0.24
0.38
91.50
1.07
4.20
5.38
287.30
3866.50
0.65
645.00
73.50
6534.00
286.75
2775.00
6330.00
14007.0
5220.00
2387.00
6175.50
3203.50
2211.00
17750.0
2265.00
1425.00
1828.75
617.50
1168.00
301.60
4680.00
264.00
4699.00
51.00
277.20
ToV
37.70
54.00
98.30
63.80
78.40
82.10
94.80
36.10
59.80
7.13
32.00
38.30
42.20
51.50
25.10
14.50
15.60
51.10
62.70
31.00
26.70
31.60
42.00
44.90
43.80
17.40
50.80
75.80
50.10
18.70
11.00
109.50
44.90
178.90
27.20
66.00
34.90
45.90
142.60
56.10
68.00
137.10
129.00
110.40
80.00
41.40
17.30
77.10
43.30
56.10
43.10
65.30
61.40
OvD
76.90
11.00
115.62
33.69
105.37
88.90
81.74
220.50
69.66
582.39
232.27
107.21
30.43
41.44
75.97
3.75
90.80
67.32
0.75
2.10
4.98
0.25
4.90
0.05
8.56
51.26
6.03
9.63
23.17
107.86
14.00
166.11
9.73
126.26
633.36
508.58
541.14
214.83
100.01
359.54
119.54
323.01
127.62
78.99
80.82
185.29
95.23
58.88
181.13
108.47
133.36
15.10
0.08
ToC
2899.00
594.00
11365.00
2149.34
8261.25
7299.06
7748.78
7960.05
4165.84
4155.35
7432.75
4106.25
1284.30
2134.25
1906.75
54.38
1416.45
3439.83
46.96
65.10
132.87
7.90
205.83
2.04
375.10
891.98
306.38
729.58
1160.62
2017.05
154.00
18189.18
436.75
22587.08
17227.50
33566.38
18885.70
9860.63
14261.70
20170.13
8128.60
44284.00
16462.75
8720.50
6465.50
7671.05
1647.40
4539.48
7843.05
6085.37
5747.69
985.85
4.61
HiEjD
505.00
11.00
480.00
1175.0
530.00
505.00
517.50
635.00
820.00
1285.0
855.00
545.00
180.00
885.00
173.75
3.75
322.50
250.00
28.50
2.10
5.50
0.25
30.50
0.18
35.50
71.50
13.00
9.63
4.88
161.25
14.00
907.50
38.75
995.00
1055.0
2030.0
1170.0
770.00
895.00
1435.0
1070.0
1350.0
955.00
285.00
402.50
775.00
365.00
200.00
1200.0
625.00
635.00
73.75
0.08
Date
28-Oct-04
09-Nov-04
16-Nov-04
22-Nov-04
29-Nov-04
06-Dec-04
13-Dec-04
20-Dec-04
30-Dec-04
03-Jan-05
11-Jan-05
18-Jan-05
24-Jan-05
31-Jan-05
07-Feb-05
15-Feb-05
21-Feb-05
28-Feb-05
07-Mar-05
15-Mar-05
21-Mar-05
29-Mar-05
04-Apr-05
18-Apr-05
25-Apr-05
2-May-05
9-May-05
E1 EjV
34.50
17.50
27.00
38.50
30.40
26.50
20.70
45.80
18.20
35.30
71.00
42.50
22.50
13.00
16.90
11.10
14.10
13.20
41.00
70.20
80.00
36.60
25.40
50.00
74.30
62.80
21.00
E1 EjD
1.75
47.50
4.75
0.58
25.25
68.75
1.38
5.50
59.50
6.88
15.00
14.25
1.23
11.50
71.00
2.50
26.50
2.75
3.38
11.75
5.88
0.50
521.50
91.25
101.25
217.50
405.00
E1 EjC E2 EjV
60.38
3.00
831.25
5.00
128.25
3.00
22.14
3.20
767.60
5.70
1821.88 3.90
28.46
2.20
251.90
4.00
1082.90 4.20
242.69
2.20
1065.00 3.30
605.63
2.60
27.56
1.80
149.50
3.50
1199.90 2.80
27.75
4.40
373.65
3.90
36.30
3.40
138.38
3.00
824.85
3.20
470.00
8.00
18.30
3.00
13017.50 3.10
4562.50 6.50
7522.88 5.70
13659.00 4.00
8505.00 5.00
E2 EjD
15.00
130.00
77.50
83.00
535.00
217.50
220.00
267.50
230.00
40.00
530.00
480.00
645.00
270.00
375.00
20.00
35.00
375.00
66.00
24.00
490.00
15.50
520.00
6.50
755.00
905.00
770.00
A66
E2 EjC
45.00
650.00
232.50
2656.00
3049.50
848.25
484.00
1070.00
966.00
88.00
1749.00
1248.00
1161.00
945.00
1050.00
88.00
136.50
1275.00
198.00
76.80
3920.00
46.50
1612.00
42.25
4303.50
3620.00
3850.00
ToV
41.30
22.50
37.00
60.10
38.10
45.00
35.90
74.40
60.30
46.00
97.80
63.60
36.30
28.00
19.70
56.90
51.00
57.70
44.00
89.30
88.00
39.60
45.70
61.35
98.20
81.80
83.10
OvD
6.00
65.83
94.50
28.43
135.88
205.49
54.77
57.58
119.15
19.28
66.45
85.12
105.68
82.08
114.21
85.66
93.89
61.59
7.64
33.47
49.89
1.64
423.27
118.77
242.84
242.87
263.52
ToC
247.88
1481.25
3496.38
1708.41
5177.10
9247.25
1966.34
4284.10
7184.60
887.09
6498.73
5413.38
3836.19
2298.25
2249.90
4874.25
4788.25
3553.93
336.38
2988.61
4390.00
64.80
19343.50
7286.75
23846.63
19866.50
21898.55
HiEjD
37.50
130.00
637.00
197.50
680.00
995.00
220.00
495.00
347.50
117.50
530.00
480.00
645.00
270.00
375.00
341.25
207.50
375.00
66.00
277.50
490.00
15.50
665.00
405.00
825.00
905.00
770.00
Table A9.3:
Date
09-Oct-02
16-Oct-02
23-Oct-02
30-Oct-02
06-Nov-02
13-Nov-02
20-Nov-02
27-Nov-02
04-Dec-02
12-Dec-02
18-Dec-02
24-Dec-02
08-Jan-03
15-Jan-03
22-Jan-03
28-Jan-03
05-Feb-03
12-Feb-03
19-Feb-03
25-Feb-03
05-Mar-03
12-Mar-03
19-Mar-03
25-Mar-03
02-Apr-03
09-Apr-03
14-Apr-03
22-Apr-03
27-Apr-03
05-May-03
13-May-03
21-May-03
26-May-03
03-Jun-03
12-Jun-03
18-Jun-03
25-Jun-03
02-Jul-03
09-Jul-03
16-Jul-03
23-Jul-03
30-Jul-03
04-Aug-03
13-Aug-03
20-Aug-03
27-Aug-03
01-Sep-03
10-Sep-03
17-Sep-03
22-Sep-03
E1 EjV E1 EjD
3.00
0.00
36.00
8.50
21.80
6.20
25.00
18.00
17.50
11.50
13.70
10.50
2.40
19.50
4.10
16.00
35.00
11.60
40.00
11.30
0.00
3.60
11.50
11.00
8.10
2.90
3.70
3.60
10.10
10.00
11.00
40.00
2.90
8.50
2.60
7.80
36.50
6.00
20.00
40.00
3.70
24.50
12.50
6.50
15.00
11.60
0.00
4.85
11.40
12.30
0.00
25.50
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.01
0.00
0.00
0.00
0.00
0.00
0.00
0.00
< 0.03
0.00
0.00
0.00
0.00
0.00
0.00
0.00
< 0.03
0.00
< 0.03
0.00
0.00
0.00
< 0.03
0.00
0.00
0.00
0.00
< 0.03
0.50
< 0.03
8.75
8.75
0.00
0.00
55.00
< 0.03
E1 EjC
216.75
0.10
< 0.03
0.00
0.00
< 0.03
M3
E2 EjV E2 EjD
2.50
2.50
2.50
2.30
0.00
0.00
2.45
4.00
0.50
1.65
2.40
0.00
0.22
0.00
31.90
2.50
2.20
3.50
4.40
0.45
0.00
0.00
3.70
0.00
3.00
0.00
1.85
0.04
11.80
2.80
0.00
E2 EjC
0.00
0.00
0.00
3.53
0.00
8.11
0.00
0.00
0.18
< 0.03
24.50
0.09
< 0.03
58.80
0.03
0.01
0.00
< 0.03
< 0.03
0.00
0.05
0.25
< 0.03
< 0.03
0.22
0.11
< 0.03
< 0.03
4.00
12.00
0.00
0.00
0.00
0.28
0.00
0.00
0.77
< 0.03
< 0.03
< 0.03
6.25
< 0.03
131.25
101.50
0.00
627.00
< 0.03
1.10
0.60
0.80
1.40
0.00
0.00
0.55
0.00
0.80
1.30
1.00
4.23
0.00
0.00
3.38
0.80
0.62
0.78
1.60
`
0.00
A67
0.44
ToV
OvD
ToC
5.50
2.50
38.52
11.30
21.80
6.20
27.45
22.00
20.12
14.85
16.22
10.50
0.42
22.20
36.00
18.50
37.80
15.10
44.00
11.72
0.00
3.60
22.50
11.50
4.00
2.90
5.75
4.64
24.20
14.30
11.00
40.00
2.90
9.65
8.90
10.50
39.90
6.00
23.10
43.30
4.70
24.50
13.30
6.50
15.00
11.60
0.00
8.75
11.40
12.30
0.00
0.00
0.00
20.03
0.00
0.00
0.00
0.00
0.11
< 0.03
3.63
0.00
0.02
18.86
0.00
0.00
< 0.03
0.00
0.01
0.01
0.00
0.00
< 0.03
0.00
4.45
0.00
0.00
< 0.03
0.05
0.06
< 0.03
< 0.03
< 0.03
0.00
< 0.03
0.12
0.00
0.00
0.55
0.03
0.00
0.00
0.52
0.00
8.75
8.75
0.00
3.02
55.00
< 0.03
0.00
226.32
0.00
0.00
0.00
0.00
2.23
< 0.03
58.80
0.00
0.01
418.60
0.00
0.00
< 0.03
0.00
0.22
0.11
0.00
0.00
< 0.03
0.00
17.80
0.00
0.00
< 0.03
1.10
0.85
< 0.03
< 0.03
< 0.03
0.00
< 0.03
1.28
0.05
0.00
12.66
1.15
0.00
0.00
6.87
0.00
131.25
101.50
0.00
26.45
627.00
< 0.03
HiEjD
0.00
25.50
0.98
24.50
0.03
155.00
0.05
0.25
5.80
1.45
0.06
0.85
0.03
8.13
0.58
0.78
8.75
8.75
11.50
55.00
Date
E1 EjV E1 EjD
01-Oct-03
08-Oct-03
15-Oct-03
22-Oct-03
27-Oct-03
05-Nov-03
12-Nov-03
19-Nov-03
26-Nov-03
03-Dec-03
10-Dec-03
31-Dec-03
06-Jan-04
12-Jan-04
19-Jan-04
26-Jan-04
02-Feb-04
09-Feb-04
16-Feb-04
23-Feb-04
01-Mar-04
08-Mar-04
15-Mar-04
22-Mar-04
29-Mar-04
12-Apr-04
19-Apr-04
27-Apr-04
03-May-04
10-May-04
17-May-04
24-May-04
31-May-04
07-Jun-04
14-Jun-04
21-Jun-04
28-Jun-04
02-Jul-04
12-Jul-04
19-Jul-04
26-Jul-04
02-Aug-04
09-Aug-04
16-Aug-04
23-Aug-04
30-Aug-04
06-Sep-04
13-Sep-04
20-Sep-04
27-Sep-04
04-Oct-04
11-Oct-04
18-Oct-04
4.10
4.20
6.70
7.40
7.20
14.50
16.00
13.90
6.80
8.50
11.60
0.70
11.00
6.50
1.40
1.80
2.00
7.00
7.30
6.90
8.00
27.00
12.30
0.00
19.30
2.50
9.00
20.60
34.00
0.00
0.00
26.00
8.50
0.80
10.60
37.00
39.30
3.70
27.50
37.00
34.00
34.20
41.50
8.00
18.80
19.50
14.50
11.50
4.90
48.70
12.80
9.00
E1 EjC
< 0.03 < 0.03
0.00
0.00
0.00
< 0.03 < 0.03
< 0.03 < 0.03
< 0.03 < 0.03
0.00
670.00 4556.00
0.00
24.00 278.40
0.00
0.00
3.93
43.18
0.00
0.00
1.43
2.00
25.38
45.68
500.00 1000.00
81.50 570.50
0.00
0.00
16.50 132.00
0.00
6.50
0.00
79.95
0.00
0.00
0.28
< 0.03
< 0.03
0.00
0.00
2.48
< 0.03
< 0.03
0.25
6.50
0.50
12.00
150.00
17.00
0.00
13.25
1.23
15.75
22.50
0.78
78.75
312.50
26.25
0.15
81.50
0.35
35.50
315.00
0.00
E2 EjV E2 EjD
E2 EjC
0.90
0.27
0.00
0.60
0.16
1.70
< 0.03
< 0.03
2.50
0.50
1.25
0.80
330.00
264.00
0.70
82.50
57.75
7.00
0.10
0.30
9.50
0.00
475.00
66.50
142.50
0.60
0.70
16.25
307.50
9.75
215.25
0.50
1.30
0.00
82.50
0.00
107.25
0.00
0.00
2.60
4.40
2.80
1.70
1.00
1.00
3.00
3.20
2.80
2.30
7.00
7.00
5.20
2.90
2.60
6.50
55.25
0.40
127.20
5550.00
668.10
0.00
364.38
45.33
535.50
796.50
32.16
630.00
5875.00
511.88
8.00
937.25 7.50
1.72
1.60
1728.85 1.00
4032.00 2.10
0.00
11.20
21.50
55.90
0.00
0.00
19.50
54.60
470.00 799.00
895.00 4296.00
195.00 195.00
43.50 130.50
237.50 760.00
93.75 262.25
0.00
0.00
582.50 4077.50
48.00 336.00
38.00 197.60
735.00 2131.50
290.00 754.00
530.00 3975.00
33.50
53.60
0.50
0.50
565.00 1186.50
190.00 2128.00
A68
ToV
OvD
ToC
4.10
6.50
6.97
7.40
8.90
14.50
16.00
14.50
6.80
11.00
11.60
0.70
11.80
6.50
1.40
2.50
2.00
0.14
14.30
7.00
8.60
0.00
27.60
13.00
0.00
19.30
3.00
10.30
20.60
34.00
14.90
2.50
28.60
8.50
11.50
18.30
47.50
43.70
12.20
39.90
39.80
34.00
42.60
56.50
34.20
21.70
27.50
53.33
33.10
11.20
49.70
22.31
21.20
0.00
0.00
0.02
0.00
< 0.03
< 0.03
< 0.03
3.62
670.00
0.11
24.00
0.00
26.03
0.00
1.43
41.37
500.00
570.50
4.65
0.00
44.91
0.00
0.00
0.16
0.00
< 0.03
< 0.03
< 0.03
52.50
4556.00
1.25
278.40
0.00
307.18
0.00
2.00
103.43
1000.00
81.50
66.50
0.00
386.25
0.35
22.71
0.00
0.00
0.00
10.65
< 0.03
< 0.03
0.00
0.00
2.18
6.50
8.76
107.91
318.31
23.84
32.46
88.88
7.73
15.75
140.19
8.60
62.79
368.96
77.18
0.15
335.52
11.68
34.80
297.54
119.95
9.75
295.20
0.00
0.00
0.00
109.73
< 0.03
< 0.03
0.06
0.00
62.40
55.25
100.78
1974.70
15119.50
1041.60
396.06
3546.23
307.83
535.50
5972.00
485.90
2147.56
8006.50
2122.38
8.00
11105.88
130.79
1729.35
6638.18
2543.00
HiEjD
0.60
87.50
670.00
0.50
24.00
330.00
1.43
82.50
500.00
81.50
9.50
475.00
16.25
307.50
82.50
0.01
21.50
6.50
19.50
470.00
1040.00
195.00
154.00
792.50
93.75
15.75
582.50
48.00
395.00
735.00
595.00
0.15
1325.00
33.50
35.50
565.00
415.00
Date
E1 EjV E1 EjD
25-Oct-04
28-Oct-04
09-Nov-04
16-Nov-04
22-Nov-04
29-Nov-04
06-Dec-04
13-Dec-04
20-Dec-04
30-Dec-04
03-Jan-05
10-Jan-05
17-Jan-05
24-Jan-05
31-Jan-05
07-Feb-05
15-Feb-05
21-Feb-05
28-Feb-05
07-Mar-05
14-Mar-05
22-Mar-05
25-Mar-05
29-Mar-05
04-Apr-05
13-Apr-05
18-Apr-05
25-Apr-05
02-May-05
09-May-05
24-May-05
26-May-05
30-May-05
02-Jun-05
06-Jun-05
09-Jun-05
13-Jun-05
20-Jun-05
27-Jun-05
04-Jul-05
11-Jul-05
18-Jul-05
25-Jul-05
02-Aug-05
08-Aug-05
15-Aug-05
22-Aug-05
13-Sep-05
19-Sep-05
26-Sep-05
03-Oct-05
17-Oct-05
24-Oct-05
41.50
6.50
18.70
28.10
41.50
13.50
17.50
6.20
31.00
18.00
25.50
12.50
6.00
15.20
4.80
10.90
10.30
12.50
20.20
22.00
11.30
1.00
1.00
30.50
25.00
7.30
21.00
13.80
10.60
11.20
11.70
8.25
43.00
6.60
17.50
20.00
16.00
11.00
8.00
21.50
21.50
35.80
13.00
35.00
31.50
28.70
11.50
24.00
16.00
19.20
2.00
9.50
15.30
0.03
0.00
16.50
0.00
0.01
3.88
20.00
1.25
13.25
100.00
355.00
32.25
0.25
392.50
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
20.50
6.50
0.05
15.00
6.75
0.38
8.25
0.15
0.00
< 0.03
20.00
103.50
35.50
1915.00
1111.00
1530.00
870.00
2230.00
1533.33
1565.00
945.00
980.00
810.00
745.00
460.00
78.50
735.00
685.00
E1 EjC
1.04
0.00
308.55
0.00
0.21
52.31
350.00
7.75
410.75
1800.00
9052.50
440.63
0.15
5966.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
149.65
136.50
0.69
159.00
75.60
4.39
69.30
0.65
0.00
< 0.03
400.00
1656.00
1846.00
15320.00
23886.50
32895.00
31146.00
28990.00
53666.55
49297.50
27121.50
11270.00
19440.00
11920.00
8832.00
157.00
6982.50
10480.50
E2 EjV E2 EjD
E2 EjC
ToV
OvD
ToC
HiEjD
1.50
< 0.03
85.80
0.15
13.00
1.48
196.50
29.63
140.00
287.50
4.20
12.60
25.00
29.70
43.50
18.80
24.50
9.30
36.10
19.00
25.50
18.20
6.60
20.10
14.60
12.90
12.20
11.90
6.60
28.00
23.30
34.20
18.10
6.30
38.40
9.70
29.30
17.60
16.10
30.60
16.40
11.05
6.80
10.70
17.50
24.00
23.80
60.50
16.70
33.50
29.20
43.40
25.70
40.80
39.60
33.30
23.60
29.70
18.60
23.20
5.30
15.70
18.20
0.60
< 0.03
20.80
0.34
0.30
3.19
89.20
18.91
20.15
109.87
355.00
133.31
166.08
514.08
0.83
0.00
0.00
0.69
0.01
0.11
6.69
0.49
9.59
0.60
0.28
15.91
6.00
0.04
274.74
7.37
33.76
20.69
0.51
5.86
< 0.03
53.09
160.98
131.90
1817.99
1589.72
1626.83
1165.16
2493.75
1931.57
2068.74
1215.89
1365.11
966.03
737.26
555.12
287.88
685.29
736.68
2.54
< 0.03
519.98
10.15
13.21
60.05
2185.35
175.88
727.50
2087.50
9052.50
2426.13
1096.15
10333.00
12.15
0.00
0.00
8.21
0.05
3.00
155.95
16.59
173.55
3.76
10.85
154.36
175.81
0.74
4423.30
225.59
553.74
228.60
3.46
62.68
< 0.03
1274.23
3831.30
7980.00
30360.50
53255.50
47503.50
50568.00
64089.50
78808.05
81922.00
40489.00
32216.50
28691.00
13713.00
12878.88
1525.75
10759.00
13407.67
3.00
0.50
6.10
2.20
1.50
2.00
1.00
0.30
0.30
2.00
1.00
3.00
< 0.03
39.00
0.10
6.50
1.48
655.00
98.75
70.00
287.50
3.50
1.60
0.20
1.70
2.00
1.60
4.00
3.30
6.00
1.00
13.00
11.10
0.00
1.00
1.00
2.50
1.80
0.10
2.80
0.40
2.50
2.50
1.80
305.00 1067.50
685.00 1096.00
230.00 46.00
3.00
5.10
< 0.03 < 0.03
0.00
0.00
0.13
0.50
0.00
0.00
0.50
3.00
65.00
65.00
0.00
0.00
15.50 172.05
0.00
3.00
6.63
0.00
33.00
11.25
18.25
56.67
1.13
0.00
0.00
3.00
16.56
0.00
3.30
31.50
7.30
141.68
2.82
0.00
2.90
2.10
1.80
2.60
5.30
2.70
1.00
4.60
5.30
3.20
0.60
5.40
4.30
2.00
2.20
1.50
1.70
1.90
300.42
86.75
875.00
2230.00
3305.00
3005.00
3825.00
3510.00
4355.00
5170.00
262.50
2385.00
1955.00
790.00
1225.00
277.50
515.00
1031.17
871.21
182.18
1575.00
5798.00
17516.50
8113.50
3825.00
16146.00
23081.50
16544.00
1575.00
12879.00
8406.50
1580.00
2695.00
416.25
875.50
1960.17
A69
44.50
100.00
6.50
3.88
655.00
98.75
70.00
287.50
355.00
375.00
685.00
1110.00
4.75
3.13
0.03
0.50
65.00
5.25
15.50
0.88
2.88
20.50
9.00
0.05
1215.00
33.50
357.50
117.50
1.13
27.25
300.42
1235.00
875.00
2925.00
3305.00
3005.00
3825.00
3510.00
4355.00
5170.00
3215.00
2385.00
1955.00
790.00
1585.00
700.00
4060.00
1035.00
Date
E1 EjV E1 EjD
31-Oct-05
07-Nov-05
14-Nov-05
21-Nov-05
28-Nov-05
05-Dec-05
12-Dec-05
03-Jan-06
09-Jan-06
16-Jan-06
23-Jan-06
06-Feb-06
13-Feb-06
20-Feb-06
27-Feb-06
06-Mar-06
13-Mar-06
20-Mar-06
27-Mar-06
03-Apr-06
10-Apr-06
17-Apr-06
24-Apr-06
01-May-06
22-May-06
29-May-06
05-Jun-06
12-Jun-06
19-Jun-06
26-Jun-06
8.50
16.10
15.00
10.50
10.40
14.20
11.00
17.10
0.00
12.00
7.50
8.50
10.30
10.00
23.50
15.20
33.50
27.00
17.60
11.00
21.30
19.00
18.50
28.50
15.00
8.50
23.50
14.50
9.50
19.00
930.00
625.00
270.00
645.00
8268.00
975.00
1010.00
520.00
E1 EjC
7905.00
10062.50
4050.00
6772.50
795.00
13845.00
11110.00
8892.00
E2 EjV E2 EjD
4.80
1.00
2.70
1.80
2.00
2.20
1.50
1.80
0.00
835.00 10020.00 3.70
44.00 330.00 2.70
15.25 129.63
197.50 2034.25 0.15
3.00
30.00
2.50
0.04
0.18
2.66
2.50
39.50 1323.25 10.50
102.50 2767.50 3.50
0.00
0.00
4.50
150.00 1650.00 7.50
1195.00 25453.50 1.70
112.50 2137.50 6.00
360.00 6660.00 4.80
197.50 5628.75 3.80
330.00 4950.00 6.90
0.00
0.00
9.50
151.25 3554.38 0.80
47.50 237.50 1.30
160.00 1520.00 0.80
92.50 1757.50 4.00
1200.00
1208.33
270.00
995.00
1720.00
1999.00
1450.00
1540.00
E2 EjC
ToV
OvD
ToC
HiEjD
5760.00
1208.33
4050.00
1791.00
3440.00
4378.00
2175.00
2772.00
13.30
18.30
20.20
12.40
12.50
17.10
12.95
17.10
0.00
15.72
7.50
8.50
10.45
17.10
0.04
21.80
58.40
49.60
40.10
18.50
23.00
27.40
23.30
32.30
24.90
18.23
28.50
16.95
10.30
24.50
1027.44
659.17
349.72
696.29
942.08
1133.21
1029.15
682.10
0.00
1176.94
44.00
15.25
199.72
8.40
1.88
1.26
52.82
174.32
8.27
192.57
1369.80
119.95
707.12
341.32
682.11
98.89
207.88
418.82
210.10
186.22
13665.00
12062.83
7064.26
8634.00
11776.00
19378.00
13327.58
11664.00
0.00
18501.56
330.00
129.63
2087.13
143.68
0.08
27.56
3084.41
8646.20
331.70
3562.50
31505.50
3286.50
16476.00
11024.75
16984.50
1802.75
5924.63
7099.00
2164.00
4562.50
1200.00
1208.33
736.60
995.00
1720.00
1999.00
1450.00
1540.00
2281.67 8442.17
14.00
37.80
352.50
2.75
1.88
0.68
4.50
450.00
13.75
255.00
3560.00
37.50
2045.00
1420.00
955.00
122.50
885.00
1700.00
805.00
540.00
A70
52.88
6.88
0.08
1.69
47.25
1575.00
61.88
1912.50
6052.00
225.00
9816.00
5396.00
6589.50
1163.75
708.00
2040.00
644.00
2160.00
2281.67
44.00
15.25
352.50
52.50
0.08
7.00
280.00
585.00
31.00
255.00
3560.00
385.00
2045.00
1420.00
1815.00
532.50
1305.00
1700.00
805.00
645.00
Table A9.4: M4
Date
17-May-03
24-Aug-03
06-Dec-03
14-Feb-04
02-Apr-04
16-Jun-04
12-Aug-04
12-Oct-04
16-Feb-05
19-Apr-05
26-Apr-05
03-May-05
10-May-05
23-May-05
30-May-05
06-Jun-05
13-Jun-05
20-Jun-05
27-Jun-05
04-Jul-05
11-Jul-05
18-Jul-05
25-Jul-05
02-Aug-05
08-Aug-05
15-Aug-05
22-Aug-05
13-Sep-05
19-Sep-05
26-Sep-05
03-Oct-05
17-Oct-05
24-Oct-05
31-Oct-05
07-Nov-05
14-Nov-05
21-Nov-05
28-Nov-05
05-Dec-05
12-Dec-05
03-Jan-06
09-Jan-06
16-Jan-06
23-Jan-06
06-Feb-06
13-Feb-06
20-Feb-06
27-Feb-06
06-Mar-06
13-Mar-06
E1EjV
0.40
0.50
1.50
7.00
2.10
9.00
8.00
2.00
1.40
5.50
2.50
2.80
20.00
6.40
26.00
3.50
9.40
74.50
8.80
47.00
42.50
30.50
26.50
57.50
26.20
9.40
12.00
75.20
14.50
28.30
48.20
12.00
16.50
6.00
7.00
13.70
41.50
27.50
7.00
28.20
11.90
19.00
13.70
15.50
6.00
16.80
21.50
22.60
10.00
76.20
E1 EjD
0.00
0.00
0.00
0.00
0.00
0.00
E1 EjC
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
< 0.03
0.00
0.00
0.25
< 0.03
2.63
46.25
35.25
1.50
0.50
0.00
2.50
0.00
23.75
0.00
13.75
33.50
4.75
0.88
0.00
0.00
0.00
20.50
12.50
7.13
0.80
0.00
0.00
1.38
116.25
36.25
5.63
2.38
0.30
0.20
12.25
16.00
5.50
18.00
14.50
19.00
0.00
0.00
< 0.03
0.00
0.00
5.00
< 0.03
68.25
1618.75
331.25
111.75
4.40
0.00
106.25
0.00
629.38
0.00
360.25
314.90
57.00
65.80
0.00
24.60
206.25
42.75
5.60
0.00
0.00
37.81
813.75
1022.25
66.90
45.13
4.11
3.10
73.50
268.80
118.25
46.80
145.00
1447.80
E2EjV
1.00
E2 EjD
E2 EjC
1.00
0.60
0.90
1.10
1.00
0.20
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
1.00
0.10
1.20
2.20
3.80
3.50
3.00
0.80
3.90
2.00
2.60
54.50
0.50
2.50
3.20
2.20
1.40
2.00
3.50
0.50
0.50
0.60
2.60
1.10
0.07
0.70
1.60
0.80
1.40
3.70
7.00
1.10
3.00
3.00
2.60
2.50
1.20
0.10
2.70
0.80
6.10
< 0.03
0.05
< 0.03
1.75
0.03
78.75
74.00
270.00
9.25
3.38
0.00
39.00
0.63
3.38
7.75
73.50
24.75
1.23
2.50
0.00
0.05
0.23
0.75
9.00
36.50
0.55
5.00
0.08
7.25
127.50
48.75
24.75
0.00
65.50
110.50
170.00
44.00
106.25
93.50
120.00
52.75
< 0.03
0.01
< 0.03
3.85
0.10
275.63
222.00
216.00
36.08
6.75
0.00
214.50
0.31
8.44
24.80
161.70
34.65
2.45
8.75
0.00
0.03
0.14
1.95
9.90
2.56
0.39
8.00
0.06
10.15
471.75
341.25
27.23
A71
196.50
287.30
425.00
52.80
10.63
252.45
96.00
321.78
ToV
2.00
0.50
3.60
7.60
3.00
11.80
11.50
5.70
1.40
8.00
2.60
7.50
24.50
12.20
45.70
12.80
16.20
84.21
19.80
52.00
55.00
33.00
33.80
30.70
33.30
15.58
20.50
78.70
17.60
0.70
52.50
10.10
20.00
8.54
10.00
3.20
43.95
28.90
19.40
28.20
18.10
30.00
20.10
21.00
22.10
16.80
26.80
7.20
18.15
13.40
OvD
ToC
< 0.03
0.00
< 0.03
4.67
0.01
32.16
55.30
96.12
1.91
0.68
< 0.03
6.20
0.02
19.28
0.81
23.10
51.10
9.01
0.95
< 0.03
0.04
0.02
35.75
19.07
48.05
0.69
14.07
1.12
1.66
73.66
36.25
9.95
6.30
10.88
14.07
24.34
16.00
9.18
44.71
22.44
43.38
< 0.03
0.01
< 0.03
114.40
0.10
1469.71
707.79
1557.08
161.14
13.43
< 0.03
341.00
0.56
651.54
24.80
769.30
795.85
184.73
74.55
< 0.03
0.03
1.26
361.05
381.33
410.36
6.89
45.03
49.36
47.96
1428.93
1022.25
180.04
188.98
218.62
295.48
537.86
268.80
245.93
321.89
407.30
581.33
HiEjD
0.05
87.50
0.03
351.67
145.00
272.50
9.25
3.38
39.00
0.63
23.75
7.75
69.00
420.00
59.00
2.50
0.05
1.13
73.00
102.50
151.67
0.80
32.75
37.00
7.25
127.50
36.25
32.25
43.25
65.50
110.50
170.00
44.00
106.25
93.50
120.00
60.00
Date
20-Mar-06
27-Mar-06
03-Apr-06
10-Apr-06
17-Apr-06
24-Apr-06
01-May-06
22-May-06
29-May-06
05-Jun-06
12-Jun-06
19-Jun-06
26-Jun-06
03-Jul-06
E1EjV
7.50
26.50
13.00
10.00
9.50
18.00
42.00
16.00
21.00
15.70
13.00
13.00
19.00
24.00
E1 EjD
687.50
99.00
775.00
650.00
116.25
270.00
211.25
885.00
425.00
424.50
305.00
965.00
177.00
108.75
E1 EjC
5156.25
2623.50
10075.00
6500.00
1104.38
4860.00
8872.50
14160.00
8925.00
3807.25
3965.00
12545.00
3363.00
2610.00
E2EjV
5.00
2.00
3.50
2.80
E2 EjD
425.00
320.00
1475.00
755.00
E2 EjC
2125.00
640.00
5162.50
2114.00
5.50
5.50
6.30
5.00
0.80
1.50
5.20
2.50
6.50
855.00
5.25
675.00
820.00
925.00
935.00
1260.00
1015.00
212.50
4702.50
28.88
4252.50
4100.00
740.00
1402.50
6552.00
2537.50
1381.25
A72
ToV
17.10
38.80
25.80
15.80
9.50
30.80
49.20
34.80
29.70
32.50
16.60
25.00
31.60
50.20
OvD
496.22
206.75
712.70
607.37
116.25
339.10
185.11
627.60
465.04
263.15
387.23
963.62
253.10
124.04
ToC
8485.29
8021.88
18387.78
9596.50
1104.38
10444.25
9107.50
21840.63
13811.75
8552.45
6428.00
24090.50
7998.10
6227.00
HiEjD
687.50
900.00
1475.00
755.00
116.25
855.00
211.25
885.00
820.00
925.00
935.00
1260.00
1015.00
212.50
Table A9.5: M5
Date
13-Oct-05
24-Oct-05
01-Nov-05
07-Nov-05
14-Nov-05
21-Nov-05
28-Nov-05
05-Dec-05
12-Dec-05
03-Jan-06
09-Jan-06
16-Jan-06
23-Jan-06
06-Feb-06
13-Feb-06
20-Feb-06
27-Feb-06
06-Mar-06
13-Mar-06
20-Mar-06
27-Mar-06
03-Apr-06
10-Apr-06
17-Apr-06
24-Apr-06
01-May-06
22-May-06
29-May-06
05-Jun-06
12-Jun-06
19-Jun-06
26-Jun-06
03-Jul-06
31-Jul-06
03-Sep-06
09-Oct-06
23-Oct-06
27-Nov-06
28-Dec-06
E1EjV
E1 EjD
E1 EjC
E2EjV
E2 EjD
E2 EjC
0.03
0.03
0
0
4
1.4
0
1.3
1.5
0.2
0.5
0.05
0.2
0.35
0.1
0.3
0.02
0.1
0.3
0
0.3
0.1
0.4
6
1
2
2.5
1.5
1
0.8
1.6
0.6
0.75
0.4
0
1.1
1
0.8
0
0.03
0.4
0
0
0
0
0.1
0.1
0.05
0
0
0
0
0
0
0.02
0
0
0.2
0.1
< 0.03
0
< 0. 03
0
0.8
6.8
0.7
0.3
0.4
0.5
0.15
1
0.5
0.3
0.1
0.1
0.1
0.005
0
0
< 0.03
< 0. 03
0
0
< 0.03
< 0.03
0
0
0
0
0
0
0
0
< 0. 03
< 0. 03
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
< 0.03
0
0
0
0
0
0
0
0
< 0. 03
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
A73
ToV
OvD
ToC
0.09
0
0
0
0.03
4.7
1.4
0
1.3
1.5
0.5
0.6
0.1
0.2
0.35
0.12
0.3
0.02
0.1
0.3
0.2
0.45
0.1
0.4
6.8
11.85
2.7
2.8
1.9
1.5
0.95
2.6
1.1
1.05
0.5
0.1
1.2
1.005
0.8
0
0
0
0
0
< 0.03
0
0
0
0
0
0
< 0.03
0
0
0
0
0
< 0.03
0
0
0
< 0.03
< 0.03
0
0
0
0
< 0. 03
0
0
0
0
0
0
< 0. 03
0
0
0
0
0
< 0. 03
0
0
0
< 0. 03
< 0. 03
0
0
0
0
0
0
0
0
0
0
0
APPENDIX 10
A10.1 Correlation between ejaculate parameters
Inter-correlations between ejaculate parameters (listed see below) derived by
Pearson product-moment analysis for individual subjects, M1 – M4 (Tables
A10.1 – A10.4).
1. Volume (VOL) ml
2. Density (DEN) x106/ml
3. Total motility (TM) %
4. Viability (VIA) %
5. Progressive motility (PM) %
6. Rate of progressive motility (RPM) ranked on a scale of 0 - 5, where 0
movement
(see Sections 3.3.8.3 – 3.3.8.5 for methods of assessment)
A74
Table A10.1: Inter-correlations and correlation coefficients, r, of ejaculate
parameters – M1
ne
≈ 1300
VOL
DEN
TM
VIA
PM
RPM
VOL
1.0
DEN
-0.24**
1.0
TM
-0.12**
0.09**
1.0
VIA
-0.10*
0.25**
0.38**
1.0
PM
-0.25**
0.48**
0.22**
0.32**
1.0
RPM
-0.16**
0.24**
0.33**
0.35**
0.42**
1.0
parameters – M2
ne
≈ 550
VOL
DEN
TM
VIA
PM
RPM
VOL
1.0
DEN
-0.24**
1.0
TM
0.02
0.11*
1.0
VIA
-0.20**
0.38**
0.55**
1.0
PM
-0.29**
0.31**
0.54**
0.50**
1.0
RPM
-0.17**
0.19**
0.52**
0.42**
0.59**
1.0
parameters – M3
ne
≈ 330
VOL
DEN
TM
VIA
PM
RPM
VOL
1.0
DEN
-0.11**
1.0
TM
0.10
0.07
1.0
A75
VIA
0.02
0.38**
0.67**
1.0
PM
0.04
0.35**
0.52**
0.67**
1.0
RPM
0.03
0.25**
0.61**
0.72**
0.86**
1.0
parameters – M4
ne
≈ 200
VOL
DEN
TM
VIA
PM
RPM
**
VOL
1.0
DEN
-0.07
1.0
TM
0.14
0.20**
1.0
VIA
0.04
0.50**
0.62**
1.0
Correlation is significant P < 0.01 *Correlation is significant P < 0.05
ne ≈ approximate number of paired ejaculate data
A76
PM
-0.05
0.45**
0.51**
0.67**
1.0
RPM
0.03
0.49**
0.54**
0.71**
0.89**
1.0
APPENDIX 11
Characteristics of raw ejaculates and cryopreserved ejaculates 30
minutes after thawing
Raw and frozen-thawed were assessed for the following parameters:
1. Total motility (TM) %
2. Viability (VIA) %
3. Progressive motility (PM) %
4. Rate of progressive motility (RPM) ranked on a scale of 0 - 5, where 0
movement
5. post-thaw parameters were expressed as % raw
(see Sections 3.3.8.3 – 3.3.8.5 for methods of assessment)
A77
Table A11.1: Raw and frozen-thaw ejaculate characteristics for
M1 – M4
Date
ID
07-Mar-05
14-Mar-05
21-Mar-05
6-Jun-05
20-Jun-05
04-Jul-05
11-Jul-05
02-Aug-05
08-Aug-05
25-Oct-05
01-Nov-05
07-Nov-05
13-Feb-06
13-Mar-06
20-Mar-06
27-Mar-06
29-May-06
05-Jun-06
26-Jun-06
15-Mar-05
21-Mar-05
14-Mar-05
26-May-05
9-Jun-05
13-Jun-05
20-Jun-05
04-Jul-05
11-Jul-05
02-Aug-05
08-Aug-05
24-Oct-05
07-Nov-05
14-Nov-05
16-Jan-06
13-Feb-06
13-Mar-06
10-Apr-06
17-Apr-06
M1
M1
M1
M1
M1
M1
M1
M1
M1
M1
M1
M1
M1
M1
M1
M1
M1
M1
M1
M2
M2
M3
M3
M3
M3
M3
M3
M3
M3
M3
M3
M3
M3
M3
M3
M3
M3
M3
Raw
TM
93.0
92.0
92.0
91.3
86.7
45.0
84.3
90.3
92.0
92.0
91.3
91.0
91.3
89.7
89.3
90.3
88.3
87.3
93.0
92.0
92.0
93.3
95.3
93.0
89.7
88.7
93.0
95.0
93.0
90.3
94.3
92.3
94.3
93.3
96.3
95.0
94.0
94.3
Raw Raw Raw Thaw Thaw
PM RPM VIA TM PM
85.0 4.5 94.5 54.8 53.3
85.0 4.5 90.0 66.7 73.5
88.3 4.5 91.8 57.3 62.5
93.0 5.0 93.0 46.8 67.2
90.0 4.0 79.8 23.8 36.7
80.0 4.5 69.5 10.2 30.0
89.0 4.8 88.0 34.0 72.5
91.7 4.3 94.5 49.7 75.0
97.3 4.7 94.3 37.3 73.3
91.7 4.5 94.8 47.7 56.7
93.3 4.0 95.2 55.0 65.0
96.0 4.5 96.5 52.3 72.7
95.3 5.0 93.8 21.7 51.7
95.0 4.8 92.5 42.3 60.0
94.3 4.5 95.2 36.7 56.0
91.0 5.0 91.0 34.0 58.3
89.7 4.3 95.5 45.0 60.0
91.7 4.3 92.7 51.0 66.7
91.7 4.7 96.2 40.7 68.3
93.7 4.3 93.3 42.8 57.5
81.7 4.0 95.3 54.3 74.3
85.0 4.2 92.2 62.5 45.0
93.3 4.5 88.7 72.3 79.2
85.0 3.8 96.5 66.5 79.5
76.7 4.7 97.0 60.2 85.8
94.0 4.5 91.2 62.2 78.3
90.7 4.8 97.2 54.3 92.2
95.3 4.7 97.7 55.2 87.7
89.3 4.2 96.0 62.0 87.8
88.3 4.0 97.7 42.7 66.7
91.7 4.7 95.3 59.0 78.5
93.0 4.5 97.0 65.0 92.3
96.3 5.0 96.5 65.7 75.0
93.7 5.0 97.2 55.8 77.7
96.7 5.0 87.3 31.5 73.5
94.3 4.7 92.0 54.3 77.7
93.7 4.8 95.7 65.0 75.0
96.0 5.0 94.8 58.7 77.3
A78
Thaw
RPM
3.8
4.4
3.8
3.3
2.4
1.7
3.7
3.5
3.3
2.8
3.5
3.7
2.0
3.3
2.5
2.7
2.7
3.3
2.3
2.8
3.9
3.0
3.6
3.7
4.6
4.3
4.6
4.4
4.3
4.3
4.4
4.3
4.2
3.9
2.8
3.5
3.2
3.3
Thaw % raw % raw
VIA
VIA TM
55.0 59.0
58.2
68.0 72.5
75.6
59.3 62.3
64.6
60.2 51.3
64.7
46.7 27.5
58.5
34.5 22.6
49.6
51.2 40.3
58.1
55.8 55.0
59.1
54.5 40.6
57.8
62.0 51.8
65.4
61.5 60.2
64.6
63.3 57.5
65.6
52.8 23.7
56.3
62.2 47.2
67.2
57.2 41.0
60.1
45.5 37.6
50.0
45.8 50.9
48.0
66.7 58.4
71.9
57.7 43.7
60.0
50.3 46.6
53.9
64.2 59.1
67.3
65.5 67.0
71.1
65.0 75.9
73.3
47.7 71.5
49.4
73.0 67.1
75.3
56.3 70.1
61.8
68.0 58.4
70.0
59.0 58.1
60.4
57.2 66.7
59.5
69.0 47.2
70.6
74.7 62.5
78.3
68.5 70.4
70.6
60.5 69.6
62.7
57.3 59.8
59.0
41.7 32.7
47.7
54.3 57.2
59.1
66.8 69.1
69.9
62.2 62.2
65.6
Date
ID
30-May-05
6-Jun-05
24-Oct-05
31-Oct-05
16-Jan-06
23-Jan-06
27-Feb-06
20-Mar-06
27-Mar-06
10-Apr-06
17-Apr-06
29-May-06
05-Jun-06
26-Jun-06
M4
M4
M4
M4
M4
M4
M4
M4
M4
M4
M4
M4
M4
M4
Raw
TM
91.7
86.0
93.3
89.3
88.7
92.7
88.3
87.7
88.7
82.0
91.7
87.7
92.7
93.7
Raw Raw Raw Thaw Thaw
PM RPM VIA TM PM
61.7 3.5 95.2 61.3 80.0
78.3 4.5 86.3 56.7 69.2
87.3 4.5 81.3 46.7 48.3
78.3 4.8 89.3 43.0 53.3
87.0 4.0 87.0 44.3 35.8
83.7 4.0 86.3 42.3 50.0
93.0 4.2 87.3 42.0 60.0
91.7 4.5 97.3 59.7 77.3
95.0 5.0 97.2 46.7 71.7
89.0 4.7 95.8 44.7 82.3
83.3 4.3 85.2 25.0 70.0
95.7 4.5 95.3 49.3 71.0
92.3 4.5 96.7 44.3 71.7
95.0 4.8 96.2 56.7 76.7
A79
Thaw
RPM
4.6
2.5
2.0
2.8
2.5
2.8
1.8
4.3
4.3
2.7
1.8
4.0
4.0
4.0
Thaw % raw % raw
VIA TM
VIA
61.2 66.9
64.3
59.8 65.9
69.3
53.2 50.0
65.4
59.5 48.1
66.6
51.0 50.0
58.6
48.8 45.7
56.6
40.2 47.5
46.0
53.2 68.1
54.6
60.7 52.6
62.4
63.7 54.5
66.4
50.3 27.3
59.1
64.3 56.3
67.5
59.5 47.8
61.6
67.3 60.5
70.0

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