SEMEN QUALITY AND EGG HATCHABILITY IN LOCAL TURKEY

1
SEMEN QUALITY AND EGG HATCHABILITY IN LOCAL TURKEY FED
DIETS CONTAINING MORINGA OLEIFERA AND GONGRONEMA
LATIFOLIUM LEAF MEAL.
BY
YUSUF, MERCY
PG/MSc./12/61409
DEPARTMENT OF ANIMAL SCIENCE
FACULTY OF AGRICULTURE
UNIVERSITY OF NIGERIA, NSUKKA
NOVEMBER, 2014
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TITLE PAGE
SEMEN QUALITY AND EGG HATCHABILITY IN LOCAL TURKEY FED DIETS
CONTAINING MORINGA OLEIFERA AND GONGRONEMA LATIFOLIUM LEAF
MEAL.
BY
YUSUF, MERCY
PG/MSc./12/61409
AN M.Sc RESEARCH THESIS PRESENTED TO THE DEPARTMENT OF ANIMAL
SCIENCE, UNIVERSITY OF NIGERIA NSUKKA, NSUKKA IN PARTIAL
FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MASTER OF
SCIENCE DEGREE (M.Sc) IN REPRODUCTIVE PHYSIOLOGY
SUPERVISOR: PROF. S.O.C. UGWU
NOVEMBER, 2014
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CERTIFICATION
This is to certify that this research work was carried out by Yusuf, Mercy with the
Registration number PG/M.Sc/12/61409, post-graduate student of the Department of Animal
Science, faculty of Agriculture, University of Nigeria Nsukka, in partial fulfillment of the
requirement for the award of Master of Science Degree in Reproductive Physiology. This
work is original and has not been submitted in part or full for any other degree or higher
degree in this or any other university.
--------------------------------Professor S.O.C Ugwu
(Supervisor)
------------------------------Date
---------------------------------Professor A.G. Ezegwe
(Head of Department)
------------------------------Date
-----------------------------External Examiner
-------------------------Date
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DEDICATION
This work is dedicated to God almighty, the only one true God for his faithfulness and
enabling grace that carried me through the course of this study, may his name be praise. I also
dedicate this work to my Mum Mrs. Jael Yusuf, and my entire Sibling. I love you all.
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ACKNOWLEDGEMENTS
I would like to thank my supervisor, Prof. S.O.C Ugwu for accepting me as a Master student and
for offering unreserved assistance throughout my studies. I especially appreciate his commitment
to mentor me, his constructive criticism of my manuscripts and speedy feedback, which helped
me complete my studies on time. Despite being busy, he has always found time to read my
manuscripts, for which I am very thankful. A special thanks goes to the former Head of the
Department Dr. A. E. Onyimoyi, for the warm welcome I received when I first came to the
Department and for giving me the opportunity to pursue my carrier in reproductive physiology, I
am forever grateful.
Thanks are conveyed to Head of the Department Animal Science and a professor of Reproductive
physiology, Prof. A. G. Ezekwe for all the knowledge, discussions and motivation you are my
best. I am grateful to Dr. N.S. Machebe and Dr. C. C. Ogbu for their interest in my research,
positive attitude, technical support and encouragement were especially valuable. I also wish to
acknowledge Dr. A.O. Ani, and Dr. Mrs. H. Foleng, for valuable words of encouragement,
technical support, for knowing they are always there for me.
My thanks also extend to Mr. Igbokwe, I. E and Mr. Okorie, C. A, for their tremendous
assistance, being concerned and always there when I needed help, you made things easy for me
and this work is a reality, thanks. I owe a very big Thanks to my Godfather for helping source
Utazi without cost in far Kogi state, such a kind gesture, may God reward you and raise up
helpers for you. Thanks are also due to my friends; Mr. Joshua I. Eze, Norah, Mr. Bright, A., and
Mr. Solomon, for their friendship, support and help. I also wish to thank the present and former
Ph.D. and M.Sc. students in Animal Science, I may not called all by names, Oyelion Chika,
Dozie, Mathew, Alafuro Akandi, Pascal, Tari, and the list goes on and on for their friendly chats,
for spending time together, pleasantries, it was all refreshing, for which I am grateful. I am also
grateful to my landlord for creating a pleasant environment that made life a bit easy for me.
I am grateful to the Departmental administration and to the various divisions and sections of the
Departmental for their unreserved help facilitating the successful completion of this research.
Special thanks go to the Departmental Lab Technicians for their wonderful help during the
laboratory analysis. The academic and support staff at the Department of Animal Sciences, and
all the farm staff involved in the study are acknowledged for their support. I owe especial thanks
to Farm Manager Mr. Chime, Samuel for the assistance and encouragement, it really helps.
I wish to express my deepest love and gratitude to my mum Mrs. Jael Yusuf, My Siblings;
Timothy, Hannatu, Alice, Ezekiel and Barnabas for their steadfast love, sacrifice, financial
support, encouragement and for sustaining me with their prayers for my success, I thank you all
for believing in me.
Finally, I thank the Almighty God.
Yusuf, Mercy
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TABLE OF CONTENT
Title
Certification
Dedication
Acknowledgement
Table of content
List of Tables
List of Figures
Abstract
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Chapter One
1.0 Introduction
1.1 Background of the study
1.2 Problem statement
1.3 Objectives of the study
1.4 Justification of the study
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Chapter Two
2.0.0 Literature Review
2.1.0 Origin and Distribution of Turkey
2.1.2 Description of Local Turkey
2.1.3 Turkey Production in Nigeria
2.2.1 Body Weight and sexual maturity
2.2.2 Body Weight and Semen Quality
2.3.1 Physiology of semen production
2.3.2 Physiology of Avian Sperm
2.3.3 Lipid Peroxidation of Semen
2.3.4 Metabolic Aspect of Antioxidant Defense.
2.4.0 Enhancing Reproductive Efficiency of Turkey
2.5.0 Origin and Distribution of Moringa Oleifera
2.5.1 Nutritional Properties of Moringa Oleifera
2.5.2 Amino acid Content of Moringa Oleifera leaves
2.5.3 Therapeutic Properties of Moringa Oleifera
2.5.4 Effect of Moringa Oleifera on Reproduction in Male
2.6.0 Origin and Distribution of Gongronema latifolium
2.6.1 Nutritional Properties of Gongronema latifolium
2.6.2 Therapeutic Properties of Gongronema Latifolium
2.6.3 Effects of Gongronema latifolium on Reproduction of Male Animal
2.7.0 Artificial Insemination
2.7.1 Artificial Insemination in Turkey
2.7.2 Semen Collection
2.7.3 Semen Quality Evaluation
2.7.3.1 Semen Colour
2.7.3.2 Volume of ejaculate
2.7.3.3 Motility Evaluation
2.7.3.4 Motility Evaluation Technique
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2.7.3.5
2.7.3.6
2.7.3.7
2.8.0
2.8.1
2.8.2
2.8.3
2.8.4
2.8.5
2.8.6
2.9.0
2.9.1
2.9.2
2.9.3
2.9.4
2.9.5
2.9.6
2.9.7
2.9.8
2.9.1
2.10.1
2.10.2
2.10.3
2.10.4
2.10.5
2.11.0
2.11.1
2.11.2
2.11.3
2.11.4
Morphology Semen Evaluation
Morphology Assessments Technique
Sperm Concentration
The Biochemistry of Semen
Determination of Fructose Concentration
Fructose as a Constituent of Seminal Plasma.
Importance of Fructose Test in Evaluation of Fertility
Evaluation of Seminal Chemical Elements on Fertility
Sodium and Potassium Concentration in Semen
Sodium and Potassium Effects on Semen Quality and Fertility
Factors affecting poultry semen
Ambient Temperature
Micro Bacterial Contamination
Photoperiod
Nutrition
Age Factor
Oxidative stress
Frequency of Ejaculation
Breed/species variation
Semen Collection Technique
Artificial insemination
Site, Depths and Time of Insemination
Fertilizing Capacity of the Sperm Cell in vitro
Duration of Fertile Period in Turkey Hen
Evaluation of Fertility and Hatchability
Factors influence Fertility
Age Factor
Body weight of the Hen
Nutrition
Stress
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CHAPTER THREE
MATERIALS AND METHODS
3.1. Location and Duration of the study
3.2. Plan of the Study
3.3. EXPERIMENTAL MATERIALS
3.3.1. Materials and Processing
3.3.2 Procurement and Management of Experimental Animals
3.3.3. Training of Toms for Semen Collection
3.4 Data Collection
3.4.1 The Effect of M. oleifera and G. latifolium on Body Weight:
3.4.2. Semen collection
3.5 Semen Evaluation
3. 5.1 Semen Colour
3.5.2 Semen volume
3.5.3 Motility Evaluation
3.5.4 Sperm Concentration
3.5.5 Dead and Live /Normal and Abnormal Spermatozoa
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3.5.6
3.5.7
3.6
3.6.1
3.6.2
3.9
3.9.1
Sperm Morphological Evaluation
Biochemical Analysis
Fertility trial (Phase 1V: Fertility and Hatchability)
Artificial insemination
Egg collection, storage and hatchability
Experimental Design
Statistical analysis
Chapter Four
RESULTS AND DISCUSSION
4.1
Effects of M. oleifera and G. latifolium on Body Weight (kg)
4.1.1 Effects of M. oleifera supplementation on Semen Colour and Volume
4.1.2 Effects of Moringa oleifera on Progressive Motility
4.1.3 Effects of Moringa oleifera on Sperm concentration
4.1.4 Effects of Moringa oleifera on Sperm Viability (Live/Dead)
4.1.5 Effects of Moringa oleifera on Sperm Morphology
4.2.1 Effects of Gongronema latifolium on Semen Colour and Volume
4.2.2 Effects of Gongronema latifolium on Progressive Motility
4.2.3 Effects of Gongronema latifolium on Sperm Concentration
4.2.4 Effects of Gongronema latifolium on Sperm Viability (Live/Dead Ratio)
4.3.1 Effects of Gongronema latifolium on Sperm Morphology
4.3.2 Combined Effects of M. oleifera and G. latifolium on Semen colour and Volume
4.3.3 Combined Effects of M. oleifera and G. latifolium on Progressive Motility
4.3.3 Combined Effects of M. oleifera and G. latifolium on Sperm Concentration
4.3.4 Combined Effects of M. oleifera and G. latifolium on Sperm Viability
4.4.1 Combined Effects of M. oleifera and G. latifolium on Sperm Morphology
4.4.2 Effects of M. oleifera and G. latifolium inclusion on Percent Fertility of toms Semen
4.4.3 Effects of M. oleifera and G. latifolium on Percent Dead -in- Shell Embryos
4.5.1 Effects of M. oleifera and G. latifolium on Percentage Hatched Eggs
4.5.2 Combined Effects of M. oleifera and G. latifolium on Percent Dead-in -Shell Embryos
4.5.3 Combined Effects of M. oleifera and G. latifolium on Percent Egg Hatchability
4.6.1 Effect of M. oleifera and G. latifolium on Fructose Concentration in Toms Semen
4.6.2 Cations Concentration in Tom Semen fed varying levels of M. oleifera and G.latifolium
4.7.1 Effects of M. oleifera and G. latifolium on Fructose Composition of Turkey Toms
Semen
4.8.1 Associations between semen quality parameters and body weight of treated tom
CHAPTER FIVE
5.0. 0 Summary and Recommendation
5.1. 0 Summary
5.2.0 Recommendations
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LIST OF TABLES
Table 1: Vitamin and mineral content of Moringa Oleifera leaf
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Table 2: Phytochemicals/Vitamins composition of Gongronema Latifolium
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Table3: Seminal characteristics of Domestic animals
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Table 4: Species Differences in Chemical Composition of Seminal Plasma
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Table 5: Composition of the Experimental Diets M. oleifera (MO): G. latifolium (GL)
Table 6: Treatments combination of Factorial Experimental Arrangement
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Table7: Treatment Arrangement
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Table 8: The Effects M. oleifera on Semen Characteristics of Turkey Toms
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Table 9: The Effects of G. latifolium on Semen Characteristics of Turkey Toms
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Table 10 Combined Effects of M. oleifera and G. latifolium on Semen quality Traits of Toms.
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Table 11: Effects of M. oleifera and G. latifolium on Fertility and Hatchability of Turkey Eggs
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Table 12: Combined Effect of M. oleifera and G. latifolium on Fertility and Egg
Hatchability of Turkey Tom’s semen
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Table 13: Chemical Composition of Semen 0f Turkey supplemented with M. oleifera
or G. latifolium
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Table 14: Chemical Composition Turkey Semen Fed Combined level of M. oleifera and
G. latifolium
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Table 15: Measures of Association Between Body Weight and Semen Characteristics
of Tom
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LIST OF FIGURES
Figure 1: Semen collection, evaluation and insemination
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Figure 2: Slide preparation
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Figure 3: Effect of treatments on body weights of Turkeys across weeks
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Figure 4: Viability and morphological examination (Stained spermatozoa)
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Figure 5: Hatched live poult and dead in-shell embryos
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x
ABSTRACT
The experiment was conducted to determine semen quality, fertility, egg hatchability and some
biochemical parameters in Nigerian local turkey toms fed diets containing Moringa oleifera (MO),
Gongronema latifolium (GL) leaf meals and their combinations. A total of 72 Nigerian local turkeys
comprising of 54 males and 18 females were used for the study. The males were randomly divided
into 9 treatment groups, each treatment was replicated 3 times with 2 toms per replicate. The
treatment diets were given only to the toms, starting from three month of age through the
experimental period. The experimental animals were fed and given water properly, twice a day
without restriction. All the management practices were carried out to the best of ability. The males in
all the treatment groups were weighed weekly to determine their daily and weekly body weight gain.
At 26 weeks of age, toms were trained for semen collection, and 32 weeks of age, semen was collected
using abdominal massages. Samples were analyzed for colour, volume, progressive motility, sperm
concentration, viability and sperm morphology. Fresh semen sample were also collected per
treatment in vials’ stored in ice block and analyzed for fructose, Na and K. A total of 18 hens were
randomly shared 2 per treatment corresponding to the 9 treatments. Pooled Semen from each
treatment was used to inseminate the hens twice a week at the beginning of egg lay and once a week
subsequently. A total of 225 eggs were collected and incubated in weekly batches, analyzed for
fertility and hatchability. The result revealed that M. oleifera and G. latifolium leaf meals had
significant (P<0.05) effects on the semen quality parameters measured. M. oleifera fed tom at 3kg
yielded the best result: ejaculate volume 0.58ml, motility= 92.93%, Conc.= 4.82(x10/ml 9), live
sperm= 94.13%, normal sperm 91.38% and corresponding lower values for percentage dead and
abnormal sperm. While, G. latifolium treated toms had a lower value for their semen quality
parameters when compared with the control group. Interaction effects of M. oleifera and G. latifolium
leaf meals were significantly (P<0.05) different. Compared with control semen quality traits were
higher at 3kgMO+1.5kgGL inclusion, lower at 1.5kgMO + 1.5kgGL, and significantly reduced semen
quality of toms fed 1.5kgMO+ 3kgGL diets. Similarly, the percent fertile eggs, and percent hatched
eggs were greatly improved at 3kgMO diets with corresponding decrease in percent infertile eggs and
dead in shell embryos than the control. However, toms fed diet at 1.5kgGL and 3kgGL had their
values for these parameters being severely reduced. Hatchability values increased to 88.39% and
83.33% at 3kg MO+1.5kgGL and 3kg MO+3kgGL respectively with a decrease in percent dead- inshell embryos to 16.99% and 19.12% respectively. Seminal fructose concentration (mg/100ml) was
significantly (P<0.05) increased (5.86+2.76) at 3kgGL when compared with the control, but M.
oleifera had a negligible increase in fructose concentration. However, M. oleifera fed toms (3kg) had
a significant(P<0.05) increase in concentration of Na and K (0.39 and 0.35) respectively. These
result suggest that improved fertility, eggs hatchability and reduction in percent embryo mortality can
be achieved using M. oleifera at 3kg/100kg diet and combination of M. oleifera+G. latifolium at rate
of 3kgMO+1.5kgGL, but treatment with G.latifolium at the rate of 1.5kg, 3kg and combination at rate
of 1.5kgMO +3kgGL caused reduced fertility in local Nigerian turkey.
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CHAPTER ONE
INTRODUCTION
1.1.
Background of the study
In Nigeria, poultry industry is once again experiencing growth due to the current regime’s
effort at encouraging investments in the industry through several economic and agricultural
policies and reforms including removal of import duties on agricultural products (Fasina et
al., 2007). Notwithstanding, the current trend in growth within the industry it is still
experiencing challenges as some species of poultry are left out. For instance, turkey
production has not been as successful as chicken production in Nigeria. Its production is
largely at the small holder level. This has been attributed to high cost of feed, inconsistencies
in feeding program as well as lack of information on its nutritional requirements (Ojewola et
al., 2002). Also, reproductive problems experienced under natural mating conditions, low
fertility and poor hatchability as a result of poor quality semen due to oxidative stress
amongst other factors (Bucak et al., 2010) militate against turkey production in Nigeria. This
situation is also evident from the FAO report (FAOStat, 2011), which shows that the
population of local turkeys in Nigeria is only about 1.05 million, being the smallest when
compared with other poultry species. It is important to come to terms with the fact that
advancement in the industry depends on the use of birds with high reproductive rate, adoption
of better mating methods, use of high quality semen in insemination as well as good nutrition.
According to Donoghue and Donoghue (1997), avian spermatozoa are rich in polyunsaturated
fatty acids (PUFA) which makes them vulnerable to lipid peroxidation especially during invitro manipulation. In particular fatty acids are the most vulnerable to lipid peroxidation.
Generally, some features of avian semen have also been found to put it under pressure of
oxidative stress. For instance, there is limitation in antioxidant recycling, because of very low
activity or even absences of hexose mono-phosphate shunt in avian spermatozoa (Sexton,
1974). Also, the low production of NADPH (the coemzymes for glutathione reductase) has
been implicated as a factor in reducing fertility of avian sperm. There are also observations
that leukocyte contamination of the semen is responsible for increased generation of free
radicals which affect the performance of turkey sperm (Halliwed and Gutteridge, 1999).
Furthermore, the activity of antioxidant enzymes in turkey spermatozoa is also lower
compared to that of chicken and this makes turkey sperm more vulnerable to the problem of
peroxidation (Aitken, 1999). Worthy of note, is the fact that turkey spermatozoa are very
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dependent on oxidative metabolism to maintain optimal ATP level needed for sperm
metabolism (Wishart, 1982). Therefore, any damage resulting from these discrepancies may
leads to alteration of the membrane irreversibly, thereby affecting sperm function and
fertilizing ability. In effect, antioxidant protection is thus absolutely vital for maintaining the
fertility of turkey spermatozoa.
Studies have revealed how the reproductive efficiency of male breeder can be affected by a
variety of factors such as breeding methods, environment (daily photoperiod, temperature
housing, and nutrition) and frequency of semen collection and technique of artificial
insemination (AI), especially in turkey (Sexton, 1983 and Lake, 1984). In addition, these
authors have stressed the importance of evaluating the semen prior to insemination to
improve the reproductive efficiency. Antioxidants have been reported to be efficient in
diminishing lipid oxidation in avian spermatozoa which is a major factor in reduction of
fertility. Worthy of note is the fact that natural antioxidant has the ability to increase the
antioxidant capacity of the seminal plasma and reduce the risk of certain deleterious free
radicals on sperm fertilizing ability (Chanda and Dave, 2009). Dawson et al. (1990) reported
that the antioxidant properties of ascorbic acid are essential in maintaining the membrane and
the genetic integrity of sperm cells by preventing oxidative damage to the sperm DNA. Also,
studies have shown that antioxidants especially those of plant origin such as Moringa oleifera
and Gogronema Latifolium have greater application potential for therapeutic and reproductive
uses.
Moringa Oleifera plant in the family of Moringacea is native to India, naturalized in tropic
and sub-tropical areas of the world (Price, 2002). It is widely distributed and cultivated in the
northern part of Nigeria and it is called Zogale in Hausa. The plant is characterized as fast
growing and drought resistant with an average height of 12 meters at maturity (Fuglie, 2001).
All parts of the moringa tree is said to have beneficial properties. Nutritional analyses by
Gopalan et al. (1989) and Fuglie (2001) indicate that Moringa leaves contain a wealth of
essential amino acids, vitamins and minerals with higher values in their dried form than in its
fresh form, except for vitamin C which is high in its fresh leaves. Fuglie (1999) also reported
some specific plant pigments with demonstrated anti oxidant properties such as carotinoids,
lutein, alpha-carotine, beta-carotine, xanthins and chlorophyll. Other phytochemicals
contained in moringa which have powerful antioxidant ability include kaempferol, queretin,
rutin, kaffeoylquinic acids, vitamins A, C and E, some valuable micro nutrients such as
selenium and zinc are also found in the leaves of Moringa.
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Recently, scientists have put more interest on the role of this plant in improving male
reproduction efficiency. Cabacungan (2008) reported that a steady diet of moringa fruit boost
the sperm count of male thus, improving their chances of fertilizing an egg. Interestingly,
Serrano M. R (2008) reported an increase in sperm count in male mice when 1%
concentration of moringa ethanolic leaf extract was administered. Cajuday and Pocsidio,
(2010) also observed that mice administered with high and medium dose of the plant extract
had enhanced spermatogenesis. This evidence was supported by increase in testicular and
epididymal weights as was confirmed in the report of Gonzales (2001).
On the other hand, Gongronema latifolium of the family asclepiadaceae is a tropical
rainforest plant primarily used as spice and vegetable and in traditional folk medicine. It is
commonly called Utazi by the Igbo tribe in South Eastern of Nigeria. (Ugochuku et al., 2003;
Ugochuku and Babady, 2002). Phytochemical screening of the ethanolic extract of the plant
shows that the root contains poly-phenol in abundance, Alkanoids, glycosides and reducing
sugars in moderate amounts (Antai et al., 2009). Other chemicals such as B-sistosterol,
lupenylester, pregnane ester and essential oil were found in the plant extracts as reported by
Ekundayo (1980). Atawodi, (2005) also reported the antioxidant potentials of the plant,
which was confirmed by the report of Nwanjo et al. (2006). In addition, the plant is suggested
to be able to mop up reactive oxygen species in the system. According to Ugochuku and
Babady (2002); and Ogundipe et al. (2003) ethanolic and aqueous extracts of the plant had
hypoglycemic, hypolipidermic and antioxidant properties.
Evaluation of biochemical constituents of semen is an important criterion for assessing male
fertility. Biochemical constituents of seminal plasma are said to play a role as sperm
metabolites, nutrition of ejaculated sperm and provision of protection to spermatozoa against
proteinase inhibitors, which help in sperm capacitation and local immunosuppression (Pesch
et al., 2005). Therefore, ensuring that the various major biochemical constituents of semen
are available in there right proportions is an indication of semen quality.
1.2 Problem Statement
Turkey is one species of poultry that are bred exclusively by artificial insemination due to the
differences in body weight between the male and female. Most times the male weighs twice
more than the female, consequently the larger body sizes (weight) of the male accounts for
poor mating ability. Thus tom often spend more time preparing to mate with the female,
hence the female might lose interest or is weakened due to the long time spent by the male
and this results in ineffective mating. Secondly, studies have revealed that turkey toms are
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naturally clumsy during natural mating and as such when semen is released little or nothing is
discharged into the vulva of the hen turkey as a result of the tom’s awkward mating skills and
thus much of the semen is wasted. Unfortunately, the full potentials of artificial insemination
technique in Nigeria have not been fully utilized and improvement in turkey production is
limited by fewer numbers of experts in artificial insemination technique, particularly those
working on local turkey production.
Furthermore, avian semen has been found to be rich in PUFA which put them under pressure
of oxidative stress (Donoghue and Donoghue, 1997). The activity of antioxidant enzymes in
turkey spermatozoa is lower compared to that of chicken and this makes turkey sperm more
vulnerable to peroxidation (Aitken et al., 1999). In effect, an unsatisfactory egg fertility and
hatchability due to the low quality semen of turkey constitutes a major problem for turkey
breeders. Also, during hatching and early postnatal period, the accumulation of PUFAproducing tissue makes it vulnerable to peroxidation. In poultry, vitamin E (combine with
selenium) provides protection against lipid peroxidation especially in turkey semen, this
vitamins is effectively transferred both from the paternal and maternal system to the egg yolk
and further to the developing embryo (Surai, 2002).
The biochemical constituents such as fructose, potassium and sodium of semen are important
for sperm fertility and deviation from normal values or proportion of these biochemical
components in seminal plasma may result to low or male infertility (Cevik et al., 2007).
Handler and Bulos(1965) reported that fructose serves as fuel supply for sperm cells, and in
the absence of fructose infertility would occur. In general, deviation from normal values of
biochemical components in seminal plasma is correlated with male infertility (Cevik et al.,
2007). The present study was designed to determine the effects of M. oleifera and G.
latifolium leaf meals in turkey toms’ diets on semen quality, biochemical parameters and egg
hatchability of local turkeys.
1.3 Objectives of the Study
The main objective of the study is to determine Semen Quality and Egg Hatchability in
Local Turkey Fed Diets Containing Moringa Oleifera and Gongronema Latifolium Leaf
Meals.
Specifically this study seeks to:
i.
Determine the effects of Moringa oleifera and Gongronema latifolium on daily body
weight of turkey toms.
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ii.
Evaluate the effects of Moringa oleifera and Gongronema latifolium on semen quality
parameters.
iii.
Determine the fertilizing capacity of the spermatozoa obtained from the treated toms
on hatchability of eggs laid by inseminated hens.
iv.
Determine the concentration of some biochemical constituents of turkey semen and
correlate them with some notable semen quality parameters.
v.
Correlate body weight of the treated toms with their semen quality parameters.
1.4 Justification
The greatest achievement of every producer in the poultry industry is to maintain breeder
males capable of producing viable spermatozoa that can fertilize eggs which will hatch with
minimum mortality. However, the numbers of hatched eggs is dependent on the quality and
quantity of the spermatozoa, thereby determining the profitability of the production.
Unfortunately, reproductive efficiency in turkey has been compromised due to emphasis on
high body weight during genetic improvement. Artificial insemination therefore, has become
the most effective and widely used techniques in turkey production and for causing
reproductive improvement in most farm animals. The success of artificial insemination is
directly dependent on the quality of semen output and appropriate handling procedures to
sustain the fertilizing potential of the spermatozoa. In Nigeria, the turkey industry has not yet
utilized the high reproductive potentials offered by artificial insemination, as a major tool to
improve and optimize the genetic potential of the local breeds and eliminate the reproductive
challenges in turkey production.
The practice of using drugs or hormones to enhance reproductive efficiency in poultry has
been questioned in many areas because of their cumulative negative health effects in the
animal as well as their products (meat and egg) meant for human consumption. Alternative
measures are now being recommended for improving reproduction in farm animals through
the application of organic extracts of plant components (leaf, seeds, stem and root)
administered through feed or water. Most recently, some available underutilized plants have
been proved to have nutritional, medicinal and therapeutic properties which can improve
semen quality, fertility and even hatchability of the turkey eggs. Moringa oleifera and
Gongronema latifolium contain nutrients, vitamins, minerals, some beneficial phytochemicals
and antioxidants which are known to stimulate growth and improve reproductive efficiency in
humans and animals. The plants have been used as natural feed additives and have generally
6
been proven to be effective and non-toxic when consumed by humans and animals. It is
against this background that this research was conducted.
7
CHAPTER TWO
LITERATURE REVIEW
2.1 Origin and Distribution of Turkey
Turkey is of family Meleagrididea, genus Meleagris, and specie Meleagris gallopavo The
modern domesticated turkey is descended from one of six subspecies of wild turkey,
Meleagris gallopavo (Michael, 2008), ancient MesoAmericans domesticated this subspecies.
The Aztecs (Mexican Spanish guajolote, from Nahuatl huexolotl) domesticated the turkey
and used it as a major source of protein (meat and eggs), and also employed its feathers
extensively for decorative purposes, as did many other Mesoamerican culture.
Turkeys were taken to Europe by the Spanish, who had found them as a favorite domesticated
animal among the Aztecs and other Mesoamerican peoples. After being introduced to Europe,
many distinct turkey breeds were developed (examples include Spanish Black, Royal Palm).
In the early 20th century, many advances were made in the breeding of turkeys, resulting in
varieties such as the Beltsville Small White etc (Crowe et al., 2006).
2.1.2 Description of Local Turkey
Local strains are available, but most species are not indigenous. For example; The guinea
fowl (Numididae) originated in West Africa; the Muscovy duck (Cairina moschata) in South
America; pigeons (Columba livea) in Europe and turkeys (Meleagrididae) in Latin America.
Turkeys (Meleagrididae) birds being native to Latin America, however, the local breeds of
turkeys kept by rural producers in the tropics usually have black feathers, as distinct from the
white-feathered breeds that are raised intensively, brown color also exists and has numerous
pale barring and mottling of the feathers especially of the tail, primaries, secondary and wing
coverts; a metallic sheen of the plumage usually accompany the black and brown color
phases (Mallia, 1998). Body weight ranges from 7 to 8 kg in males and from 4 to 5 kg in
hens. They have good meat conformation, produce about 90 eggs per year and have medium
to good hatchability. They are more susceptible to disease than either chicken or ducks.
In general, there are three colour varieties of local turkey in Nigerian they are; white, black
and lavender. Adult males have a naked, heavily bumpy head that is normally bright red in
colour but turns to white overlaid with bright blue when the birds are excited. Other
distinguishing features of the common turkey are a long red fleshy ornament (called a snood)
that grows from the forehead over the bill; a fleshy wattle growing from the throat; a tuft of
coarse, black hair like feathers (known as a beard) projecting from the breast; and more or
8
less prominent leg spurs. The male turkey, or gobbler, or tom, may be 130 cm long and weigh
10 kg, though average weight is less (Encyclopedia Britannica, 2010). Female turkeys, or
hens, generally weigh only half as much as the males and have less warty heads than do the
males. Domesticated strains of the common turkey, developed for their fine-tasting flesh,
may be much heavier.
2.1.3 Turkey Production in Nigeria
Turkey rearing is very popular in many parts of the world, but its production is not popular in
Nigeria. Turkey production has largely remained at the small holder level in Nigeria,
primarily because of the management problem often encounter to lack of incentives by
Government (Udokainyang, 2001). Mbanasor and Sampson (2004) stated that, there is
obviously lack of information on specific requirements for turkey production in Nigeria,
which may be attributed to low level of research. They further identified high cost of feeds,
diseases incidence and, high costs of other inputs such as housing, drugs, feed as farmer’s
pressing problems. While, low fertility and poor hatchability as a result of poor quality semen
due to seminal oxidative stress (Bucak et al., 2010), as the most militating factors against
turkey production in Nigeria. Recently, turkey breeding is gaining importance in Nigeria as
local turkeys are said to possess some innate resistance to certain local diseases in addition to
adaptability to prevailing climatic conditions (Zahraddeen et al., 2005).
2.2.1 Body Weight and sexual maturity
It is fairly well established that body weight is an important criterion for adequate early
production. There is still insufficient evidence regarding optimum body structure and
composition, this parameter is useful as another monitoring tool. According to Foote (1978),
major development of the reproductive system takes place between birth and puberty.
Consequently, the young male must be fed and managed to achieve good growth and
minimize disease and other undesirable stresses. In production, of great importance is
nutrition (Reid, 1960). Good nutrition promotes rapid growth, early sexual activity and the
possibility of collecting semen at an early age (Foote, 1969) in various farm animals. It seems
as though early maturing birds achieve a threshold level of body mass and commence
production when the minimum physiological age is reached, while late maturing birds at the
same age do not have the body mass required for production (Flipse and Almquist, 1961).
The first stage of sexual maturity is noted by the appearance of physical characteristics, such
as comb development etc. With these characteristics, the bird is at the point of beginning the
9
move from a juvenile to an adult stage. But, at this initial stage of the onset of sexual
development, what they do not show is the correlated effects that occur in other traits as a
result of selection based on bodyweight (www.hybridturkeys.com 2014).
2.2.2 Body Weight and Semen Quality
Omeje and Marine (1990) reported that genotype differences affected body size and semen
characteristics of cocks, except the pH value. Total testicular weight is approximately 1% of
the total body weight, depending on the breed of bird (Sturkie and Opel, 1976). Ramamurthy,
et al. (1989) observed significant positive correlation between body weight and seminal
volume, pH, and abnormal spermatozoa rate, whereas motility, spermatozoon concentration
and live spermatozoon rate are negatively correlated with body weight in poultry. On the
contrary, Soller et al. (1965b) reported a negative correlation between sperm motility and rate
of gain. Marini and Goodman (1969) observed that in a male line selected for increased body
weight, sperm motility was decreased. In the same line, a negative correlation was found
between growth rate and mating activity (Rappaport and Soller, 1966). In a series of four
natural mating trials and one artificial insemination trial, body weight was poorly correlated
to fertility (r = -0.39 to 0.09) in the cock (Wilson et al., 1979). This negative correlation
between fertility and body weight has also been seen in turkeys (Berg and Shoffner, 1954). In
a similar study, Edens et al. (1973) found decreased metabolic activity in sperm cells of a
high weight line, which agrees with the decreased sperm motility and lower mitochondrial
mass of sperm cells seen in a strain of chickens exhibiting high feed intake (Morrison et al.,
1997).
2.3.1 Physiology of Turkey Reproduction
In turkeys, sperm production in the testes starts when the toms are about 150-250 days old.
Spermatogenesis occurs in the seminiferous epithelium and is the process where stem cells
produce diploid spermatogonia that undergo mitosis and then meiosis to divide into haploid
spermatocytes.
The
spermatocytes
then
undergo
meiosis
to
form
spermatids.
Spermatogenesis is ultimately controlled by neurons (Sharp and Gow, 1983), and depends on
testosterone, follicle stimulating hormone (FSH), and the activity of Sertoli cells (Sharpe
1994). The transformation of spermatids into sperm cells is referred to as spermiogenesis and
takes place during 8-10 morphological steps (Gunawardana, 1977; Tiba et al., 1993) in the
seminiferous epithelium. Spermiogenesis includes the formation of an acrosome and
10
axoneme, loss of cytoplasm and nuclear condensation of the cell (Oliva and Mezquita, 1986;
Sprando and Russell, 1988).
A few toms may produce traces of semen at five months of age, but first production of
amounts sufficient for satisfactory fertilization occurs somewhat later. The majority of a flock
is not usually in adequate semen production until about eight months of age (Lorenz et al.,
1959). These sperm, after going through several stages that take about a month to complete,
finally leave the testes and travels down the ducts termed the vasa deferentia. The release of
fully formed sperm cells from the seminiferous epithelium into the lumen of the seminiferous
tubules is known as spermiation. Cells are suspended in fluid secreted by the Sertoli cells.
Passage through the seminiferous tubules depends on hydrostatic pressure of the fluid and
contraction of myoepithelial cells (Rothwell and Tingari, 1973). Sperm cells are immotile at
spermiation (Ashizawa and Sano, 1990). Sperm acquire the potential for motility as they pass
through the excurrent ducts (Ashizawa and Sano, 1990), and mature in these ducts and are
stored there for only a short time. The posterior parts of the vasa deferentia are thickened by
increased musculature, and they are expanded into "bulbous ducts" just before they terminate
in narrow papillae, which pour semen onto the surface of the phallus. This organ contains
erectile tissue, but the process of erection differs from the mammalian penis, the tissue
becomes engorged with lymph instead of blood (Almquist and Barber, 1974). The nonintromittent organ of galliforms (turkey, chicken and quail) consists of folds and bulges that
make contact with the female’s cloaca at mating. From an anatomical perspective, there are
considerable differences between the non-intromittent organs of the chicken and turkey
(Bakst and Dymond 2013).
2.3.2 Physiology of Avian Sperm
Thurston and Hess (1987) described avian spermatozoa as elongated, flagellated cells that
can be divided into the head, midpiece, and tail. The chicken spermatozoa were described as
vermiform cells 0.5-0.7μm wide and approximately 90μm long. Fine structural changes
during transit through excurrent ducts have also been observed in rooster sperm (Tingari,
1972). An in-depth structural assessment of the head, neck and midpiece of sperm from
White Leghorns using a transmission electron microscope indicated that the midpiece
contains approximately 30 mitochondria (Bakst and Howarth, 1977). Mitochondrial swelling
result to degenerative sperm (Lake et al., 1968) and significant positive correlations exist
among mitochondrial status, midpiece integrity and fertilizing capacity of fresh semen (Xia et
al., 1988). However, the major integral component of poultry spermatozoa membrane that are
11
involved in a series of biochemical and functional changes ultimately required for sperm
maturation, the acrosome reaction and fertilization, is the lipid (Brèque et al., 2003). It is
known that poultry sperm contain a high proportion of polyunsaturated fatty acids (PUFAs)
in the plasma membrane (Ravie and Lake, 1985; Surai et al., 1998). Phospholipids in
Chicken and turkey spermatozoa are enriched mainly with n-6 PUFAs, including arachidonic
(20:4n-6) and docosatetraenoic (22:4n-6) acids. But turkey’s spermatozoa are characterized
by the lowest degree of lipid unsaturation and the longest fertile period in the sperm storage
tubules (SST). The major phospholipids class of turkey sperm is phosphatidylcholine,
comprising up to 39 % of the total phospholipids content. It has been shown that the
phospholipid content of turkey spermatozoa decrease by 30 % during 24 hours storage at
4 °C, with most of the loss (20 %) occurring between 1 and 4 hours (Douard et al., 2003).
Inclusion of typical antioxidants such as Vitamin E in the extender has not proven to be
effective in preventing lipid peroxidation of turkey sperm during semen storage (Long and
Kramer 2003; Douard et al., 2004). Inclusion of organic antioxidant in diet of poultry may
provide great deal of protection and improved fertilizing capacity of sperm.
2.3.3 Lipid Peroxidation of Semen
The fact that the avian spermatozoa plasma membrane contains high levels of
polyunsaturated fatty acids (PUFAs) means that turkey spermatozoa are particularly
vulnerable to the deleterious effects of lipid peroxidation (Douard et al., 2003, 2004, 2005;
Zaniboni and Cerolini, 2009), and the generation of free radicals in the body belong to its low
antioxidant capacity leading to oxidative stress which has been implicated in the aetiology of
several phatological condition such as lipid peroxidation, protein oxidation, DNA damage
and cellular degeneration related to so many disease conditions (Ames et al., 1993). Chicken
and turkey spermatozoa contain high amounts of polyunsaturated fatty acyl groups (Ravie
and Lake, 1985) and spontaneous peroxidation occurs during in vitro semen storage in both
species (Fujihara and Koga, 1984; Cecil and Bakst, 1993). The indication of high
concentrations of polyunsaturated fatty acids (PUFAs) and age-dependent changes in the
PUFA composition of turkey sperm membranes (Douard et al., 2003) and, the presence of
reactive oxygen species (ROS), trigger a chain of chemical reactions called lipid peroxidation
that affect spermatozoa membrane functions and DNA integrity (Aitken, 1995). Normal byproducts of oxidative metabolism form free radicals of O2 and H2O2, which induce the
formation of lipid peroxides that are extremely toxic to sperm (Wishart, 1984). In turkey
semen, malonaldehyde MAL which is a by-product formed during peroxidation increased
12
with length of in vitro storage (Cecil and Bakst, 1993). Most importantly, lipid peroxidation
is a major contributor to the lower fertility rates associated with stored turkey semen (Long
and Kramer, 2003).
In generation there are three major features of semen that put them under pressure of
oxidative stress:
-Limitation in antioxidant recycling, because of very low activity or even absence of hexose
monophosphate shunt in avian spermatozoa (Sexton, 1974), the production of NADPH, the
coenzymes for glutathione reductase, is also limited. This means that recycling in the chain of
vitamin C- GSH as the primary defence preventing conversion of superoxide radical to more
powerful radical (OH+) would be of great importance for spermatozoa survival.
-Sperm storage within oviductal sperm storage tubule (SST) at a body temperature of 41˚c
can be considered the risk factor for lipid peroxidation and antioxidant role of the SST has
been proposed.
-Its also suggested that spermatozoa cannot carry out extensive biosynthetic repair of sperm
function. For instance, when damaging alteration occurs to the sperm membrane, it
irreversible alters sperm function and the antioxidant protection is thus absolutely vital for
maintain the fertility ability of spermation.
In avian semen, natural antioxidants (such as vitamin E, vitamin C, selenium and carotenoids)
associated with antioxidant enzymes protect spermatozoa against oxidative damage by
inhibiting, scavenging or suppressing the formation of ROS (Breque et al., 2003; Surai et al.,
2006). However, these antioxidants are usually present in insufficient amounts to counteract
the lipid peroxidation that occurs during ex-situ semen storage (Douard et al., 2004; Zaniboni
and Cerolini, 2009).
2.3.4 Metabolic Aspect of Antioxidant Defense
Free radicals and other reactive oxygen species such as hydroxyl (OH-), superoxides (O2),
Nitrogen oxide (NO2-) etc. and non free radicals like hydrogen peroxide and single oxygen
are constantly formed in the body during normal cellular metabolism,
during energy
production in the mitochondria electron transfer chain, phagocytosis, arachidonic
metabolism, ovulation, fertilization etc. (Halliwed and Gutteridge, 2007), they can also be
produced from external source such as food, drugs, smoke and other pollutant from the
environment (Miller and Britigan 1997). It is widely accepted that superoxide radicals’
formation is usually the result of electron leakage from the mitochondrial electron transfer
chain due to uncoupled oxidative phosphorylation (Halliwed and Gutteridge, 1991). There
13
are also observations that leukocyte contamination of the semen is responsible for increase
generation of free radicals. However, if semen contamination is minimal, metabolic
differences between the species especially in terms of mitochondrial oxidative
phosphorylation activity (Halliwed and Gutteridge, 1991) would determine differences in the
rate of formation of supraoxide radicals, probably stress factors, responsible for uncoupling
of oxidation and phosphorylation in mitochondria, could stimulate electron leakage and
superoxide radical formation.
Turkey spermatozoa are very dependent on oxidative metabolism to maintain optimal ATP
level (Wishart, 1982). In this respect, the lower unsaturation of turkey sperm lipid could be an
advantage in term of prevention of lipid peroxidation. However, activities of antioxidant
enzymes in turkey spermatozoa are also lower compared to chicken (Aitken et al., 1989).
Also the ration of polyunsaturation fatty acid to antioxidation in the spermatozoa is a very
important determination of their survival in vitro, which could also be a factor in survival of
spermatozoa in the oviduct. A unique feature of avian reproduction is spermatozoa storage
within oviduct sperm storage tubule (SST) (Bakst et al., 1994) for several weeks. Therefore,
maintain of membrane stability and prevention lipid peroxidation during this high
temperature (41oC) sperm storage could be an important strategy for avian species.
2.4.1 Enhancing Reproductive Efficiency of Turkey
The current trend in growth and expansion in the scope of turkey production in Nigeria to
keep pace with the rising demand of turkey have necessitated conscious effort to identify and
analyze ways to enhance turkey production among farmers in order to bridge the production
deficit gap between chicken and turkey. Modern strains of commercial turkeys and meat type
chickens have been primarily selected on the basis of growth rate, feed conversion and meat
yield. Unfortunately, this has engendered a series of negative effects on reproductive
performance, including early but generally limited persistence of sexual maturity and
declining egg fertility (Brillard, 2004). The study of Douard et al. (2003) has also
demonstrated that membrane susceptibility to lipid peroxidation is higher in fresh or stored
(48hrs) ejaculate collected from older birds compared to young turkey breeder males.
In order to counteract these problems militating against the turkey industry in Nigeria,
approaches have been proposed by several researchers. The turkey industry has to take
advantage of the potentials offered by artificial insemination, a strategic tool to select male
and female lines, and also to optimize the production of breeder male. Indeed, artificial
insemination in breeder turkeys has replaced natural mating for over 50 years in the
14
developed world. As it virtually suppresses sexual behaviour constraints in the selection of
male and female lines, facilitates high reproductive performance, and ultimately allows
permanent optimization of the genetic potential from the best sires. Furthermore, the addition
of antioxidants in the diluents during in vitro storage and also dietary supplementation of
antioxidant in breeder flocks is being proposed by Jean-Pierre (2007). Surai (1999) described
the use of antioxidant supplements such as tocopherol or organic selenium in turkey feeds to
improve animal health and spermatozoa quality (Neuman et al., 2002b; Zaniboni et al., 2006;
Dimitrov et al., 2007; Zaniboni and Cerolini, 2009; Slowinska et al., 2011). This is supported
by the report of Breque et al. (2003) who stated that the use of antioxidants is therefore,
important to protect spermatozoa from lipid peroxidation and maintain their membrane
integrity, motility and fertilizing ability during storage. In avian semen, natural antioxidants
(such as vitamin E, vitamin C, selenium and carotenoids) associate with antioxidant enzymes
to protect spermatozoa against oxidative damage by inhibiting, scavenging or suppressing the
formation of ROS (Surai et al., 2006).
Surai (1999) further elaborated that there are about three levels of antioxidant defense in the
cell. Thus, first level of defense: prevention of radical formation, using superoxide dismutase
(SOD) and catalase metal binding protein Se-GSH-Px. Second defense: the prevention of
chain formation, by propagation GSH Uric Acid, vitamin A, E C, carottenoid, Se- GSH-Px,
and lastly, repair damaged molecules carried out by lipase and proteasess etc. Kitanov et al.
(2003); Semerdjiev et al. (2008) stated that, plants can produce phytochemicals with sexenhancing potency and with the ability to stimulate high reproductive potential in animals.
Their result was supported by report of Machebe et al. (2013), who reported a marked
improvement in fertility and hatchability of turkey hen egg fed Okra seed and pumpkin seed
extract. Several studies have confirmed that in vitro supplementation of diluents with vitamin
E favors maintenance of sperm motility and viability, a prerequisite to in vivo sperm storage
in the oviduct (Douard et al., 2004).
2.5.0 Origin and Distribution of Moringa Oleifera
Moringa Oleifera plant family of Moringacea is native to India, naturalized in tropic and subtropical area around the world (Price, 2002). Moringa is best known of three species of the
genus Moringa (Kristin, 2000). The Romans, Greeks and Egyptians extract edible oil from
the seeds and it for perfume and skin lotion. According to the Fuglie (2000) in the 19th
century, plantations of Moringa in the West Indies exported the oil to Europe for perfume and
15
lubricants for machinery. People in the Indian sub-continent have long used Moringa pods for
food the edible leaves are eaten through West Africa and in parts of Asia. Price (2002) stated
that Moringa grows best in dry, sandy soil, it tolerates poor soil including coastal areas. It is a
fast-growing drought resistant plant, with average weight of dry matters at maturity (Fuglie.
2002). Many part of the plant are edible including the immature and mature seed pods,
leaves, oil pressed from the mature seeds and the root (FAO, 1999; Rajangam et al., 2001)
and are reported to contains a profile of important nutrient with medical evidence for its
nutritional, therapeutic, and prophylactic properties.
2.5.1 Nutritional Properties of Moringa Oleifera
The nutritional properties of Moringa Olefeira are so many and so well known that there is
little or no doubt about its substantial health benefits (Fahey, 2005). A very large number of
reports have been published on the nutritional properties of Moringa, in both scientific
journals and medical literature. The leaves are the most nutritious part of the plant, being a
significant source of vitamins, minerals and amino acid. Nutritional value per 100g (3.5Oz):
Energy 92cal and 205cal. Carbohydrate 12.5g and 38.2g,; Dietary fiber 0.90g and 19.2mg;
Fat 1.70g and 2.3mg; Protein 6.70g and 27.1g; Water 78.66g in fresh and dried leaves
respectively.
Table 1: Vitamin and Mineral Content of Moringa Oleifera Leaves
Vit./Minerals
Fresh leaves
Carotene (vit. A) 6.78 mg
Thiamine(B1)
0.06 mg
Riboflavin(B2)
0.05 mg
Niacin(B3)
0.8 mg
Vitamin C
220 mg
Calcium
440 mg
Copper
0.07g
Iron
0.85mg
Magnesium
42mg
Phosphorus
70mg
Potassium
259mg
Zinc
0.16mg
Source:
Gopalan et al. (1989)
Dried leaves
18.9 mg
2.64 mg
20.5 mg
8.2 mg
17.3 mg
2,003 mg
0.57mg
28.2mg
368mg
204mg
1,324mg
3.29mg
Fuglie (2002)
2.5.2. Amino acid Content of Moringa Oleifera leaves (per 100g of edible portion)
Arginine 406.6mg, 1325mg. Histidine 149.8mg, 613mg. Isoluecine 299.2 mg, 825 mg.
Lysine 492.2 mg, 1,950 mg. Lysine 342 mg, 1,325 mg. .Methionine 117.7 mg, 350 mg.
16
Phenylalanine 310.3 mg, 1,388 mg. Threonine 117.7 mg, 1,188 mg. Tryptophan 107 mg, 425
mg. Valine 374.5 mg, 1,063 mg in fresh and dried leaves respectively. (Gopalan et al., 1989;
Fuglie, 2002).
2.5.3 Therapeutic Properties of Moringa Oleifera
Aside the wealth of mineral, vitamins and amino acid content of this plant, scientists
observed that M. Oleifera contains unique compounds with enact mechanism that help in
purging, preventing and even reversing damages caused by toxins. Phytochemicals are, in
strictest sense of the word chemicals produced by plants commonly thought to have an
impact on health, flavor, texture, smell, or colour of plants, but are not required by humans as
essential nutrients (Fahey, 2005). An examination of the phytochemicals contents of M.
Oleifera, showed that this plant is rich in compound containing the simple sugar such as,
rhamnose, higher amount of polyphenols (an antioxidant compound) and it is rich in a fairly
unique compound called glucosinolate and isothiocyanates (Bennett et al., 2003 and Fahey et
al., 2001). Specific component of Moringa that have hypotensive, anticancer, and
antibacterial activities include 4- (4-O-acetyl-α-L-rhamnopyranosyloxy)benzyl isothiocyanate
(Abram et al., 1993), 4-α-L-rhamnopyranosyloxy)benzyl isothiocyanate (Abuye et al., 1999),
niazimicin (Akhtar and Ahmad, 1995), pterygospermin (Anderson et al.,1986), benzyl
isothiocyanate (Anwar and Bhanger, 2003) and 4-α-L-rhamnopyranosyloxy)benzyl
glucosinolate (Asres, 1995). Other known common recognized phytochemicals such as
carotenoid (β-carotene or pro-vitamin A) (Fuglie, 1999).
M. Oleifera contains Silymarin a flavonoid found with enact mechanism that help purge the
liver of hepa toxins and even reverse the damage that they cause. It also exert protective
effects on hepatic marker enzymes, lipid peroxidation, and enhance it recovery (Pari and
Kumal 2003; Ashok and Pari 2007). Chung et al. (2002) reported an anti-fungal activity of
Moringa
against
dermatophytes
such
as
Ttrychophyton
rubrum,
Trichophyton,
Mentagrophytes etc. it has been shown to lower serum cholesterol, decrease lipid profile of
liver, heart and Oarta and also increase the excretion of feacal cholesterol. Fuglie (1999)
reported some specific plant pigment found in Moringa with demonstrated potent antioxidant
properties such as carotenoids-luitein, alpha-carotene and beta-carotene, xanthins and
chlorophyll. Others known powerful antioxidant includes kaemferol, quercetin, rutin and
caffeuylquinic acids, vitamins C, E and A and essential micronutrient with antioxidant
activity, such as selenium and zinc. Carotenoids are also known as an important component
of the antioxidant defense system of avian spermatozoa (Surai et al., 2006) which also play
17
important roles in endocrine and immune systems, appearing as pigments in the feathers, skin
and eggs of birds (Sirri et al., 2007).
2.5.4 Effect of Moringa Oleifera on Reproduction in Male Animal
Research in to the reproductive action of M. Oleifera, shows that it enhances male sexual
function including libido, and improves sperm quality and anti-erectile dysfunction among
many others. Thawatchai et al. (2012) reported an increase in mounting number and
enhanced intromission in rats treated with M. Oleifera extract after subjecting them under
stress. On further investigation of the mechanism of action, it was revealed that Moringa
extract possess Monoamine Oxidase type B inhibitor (MAOᴃI). Dopamine is mainly
mobilized by MAOᴃ, the activity of MAOᴃ is used to reflect the availability of dopamine,
which plays a crucial role in regulation of male sexual function in many aspects including
motivation, reinforcement, motor response to sexual stimuli and male genital reflex
(Dominguez and Hull, 2005). Serrano and Pocsidio (2008) observed an increase in sperm
count in male mice when 1% of Moringa extract was administered subcutaneously for two
weeks. A work done by Bureau of plant industry (BPI) in USA showed that a steady diet of
Moringa fruit boosts the sperm count of men, thus improve their chances of fertilizing an egg
(Cabacunga, 2008).
2.6.0 Origin and Distribution of Gongronema latifolium
Gongronema latifolium Benth, is a leafy vegetable belonging to the order Apocynales, family
Asclepiadaceae, and sub-family Asclepiadciopeae (Nielsen, 1965). It is abundantly available
in virgin forests in many parts of sub Saharan Africa and some parts of China (Nielsen, 1965;
Ying and Ping-tao, 1997). It is commonly called “Utazi” in Southeastern Nigeria, and
“Arokeke” in Southwestern Nigeria (Ugochukwu et al., 2003). G. latifolium is a common
forest climber with hollow stems and broadly ovate leaves that are widely cordate at the base.
It grows in the forests of Southeastern Nigeria (Akpan, 2004), and is of West African origin
(Nielsen, 1965). The crop can be propagated by seeds and stem cuttings (Agbo and Obi
2006). There are difference species with varying morphology, morphological variation such
as flower colour, leaf sizes could be caused by variations in chromosome numbers, and sizes
and shapes. The morphological characters of one of the four clones indicated that polyploidy
in the plant resulted in higher leaf size and its inability to flower. The non-flowering nature of
the clone is not a disadvantage per se, as the plant can be mass propagated by vegetative
cuttings.
18
2.6.2 Nutritional Properties of Gongronema latifolium
The nutritional and medicinal importance of G. latifolium cannot be overemphasized. It is a
good source of vitamins, minerals and proteins (Okafor, 2005). Crude protein 26.99%, Crude
fibre 31.86%, Ether extracts 10.30, Ash 6.79, Carbohydrate 15.5, Moisture 8.56 and Nitrogen
free extract 24.18. The levels of vitamins A, C, E and β-carotene in the crop species are
relatively high, measuring 40.82mg/100g, 15 mg/100g, 3.71mg/100g and 6.80 mg/100g
respectively (Machebe et al., 2011). The crop has been identified to be nutritionally high in
iron, zinc, vitamins, protein and amino acids (Agbo et al., 2009). It is used as a leafy
vegetable and spice in south eastern Nigeria (Agbo et al., 2005). Glew et al. (1997);
Akwaowo et al. (2000) and Ajayi et al.(2006) suggested that consumption of 100g (DM) of
G. latifolium leaves may be capable of providing 27g of protein which satisfies recommended
daily allowance of protein for children. These also represent a potentially rich source of
some, but not all of the essential amino acid that is necessary for humans. A child consuming
100g of G. latifolium would be ingesting approximately 6.07g of fatty acid which translates
into 54.6kcal of energy. Lipid content of the leave contain modest useful amount of the
essential fatty acid, linoleic acid 31.1%(Glew et al., 2004). Edible triglycerides, such as those
in olive oil, have cardioprotective effect.
The phytochemical/vitamin composition of G. latifolium are presented in table 2.
Table 2 Phytochemicals/Vitamins composition of G. latifolium
Vitanins/Phytochemical
Composition (mg/100g)
Beta carotene
6.80
Vitamin E
3.71
Vitamin C
15.0
Vitamin A
40.82
Sources: Machebe et al. (2011)
2.6.2 Therapeutic Properties of Gongronema Latifolium
The plant plays a vital role in the treatment and prevention of varied health related problems
including liver diseases, diabetes mellitus, high blood pressure, loss of appetite, dysentery,
stomach pains, worm infections, cough and malaria fever (Agbo et al., 2005; Okafor, 2005).
Medicinal importance of the plant is further elaborated by the presence of five bioactive
compounds including Alkaloid 9.10%, Phenol 2.23mg/100g, Tannin 2.54%, Phytate
6.5mg/100g, Cyanogenic glycoside 0.02 mg/100g, and others includes Lycopen 5.16
19
mg/100g, Moisture 10.2% in the dry leaves (Machebe et al., 2011). These are suggested to
proffer varied pharmacological effects (Gamaniel and Akah 1996) in the crop species which
are relatively substantial. Cyanogenic glycosides were present at a low level, while
haemagglutinin, known to agglutinate erythrocytes and leucocytes are absent. Similarly, the
leaf extracts have been shown to possess anti-oxidative properties and are being utilized in
management of diabetes mellitus and other tropical diseases (Agbo et al., 2005; Ugochukwu
et al., 2003). Other reports show the present of saponins, and flavonoids which had been
shown to possess antioxidant and antimicrobial properties (Morebise and Fafunso 1998;
Hernandenz et al., 2000).
2.6.3 Effects of Gongronema latifolium on Reproduction of Male Animal
Some plants have been reported to possess antifertility properties due to the nature of their
phytochemical constituents. Such plant will usually result in reducing the sperm count,
motility, viability and cause visible alteration of sperm morphology. Such impairment of
male fertility has been reported in herbs that have antimalarial activity. According to
McGarvey et al. (2001); Weber et al. (2001) and Pastuszewska et al. (2006), plants with high
alkaloid contents were observed to be responsible for increased serum concentration of
estradiol and prolactin. This has the capacity to inhibit gonadothrophin action of the testis and
subsequent impairment of male fertility. This findings were confirmed by others researches
who reported significant reduction (P < 0.05) in the sperm count, sperm viability, sperm
motility and weight of testis with increased percentage of sperm head abnormality, especially
at higher doses of P. biglobosa and G. latifolium extracts (Ikpeme et al., 2012);
Alstoniabounai and Azaridichta indica (Oze et al., 2007 and Raji et al., 2003). This reduction
suggests that these spice plants are efficacious in disrupting spermatogenic processes and
pathways. This probably suggests that those leaf extracts might impair fertility if caution is
not exercised by consumer. G. latifolium and ocimum gratissimum have been associated with
high antimalaria properties, rich in alkanoids and glycosides, plant chemicals associated with
antimalaria and antifertility potential. Ugonna, (2003) suggested in his findings that
prolonged treatment with G. latifolium could significantly alter the fertility potential of the
male animal.
2.7.0 Artificial Insemination
The current increasing number of farmers in Nigeria showing interest in artificial
insemination technique is a reflection that the farmers are willing to adopt modern
20
technologies to improve their poultry species (especially turkeys). Artificial insemination
(A.I) is a vital tool for rapid improvement of infertility in Turkey by allowing maximum use
of the best toms on numerous hens. According to Zahraddeen et al. (2011) the benefits of this
technology are however derived when it is available to the farmers and are effectively utilized
by them. Unfortunately this technology is at its infancy in Nigeria especially among Turkey
farmers.
Artificial insemination (AI) in the broad sense, is a technological process involving semen
collection, processing, evaluation and artificially deposition of a quality semen into the
female genital to fertilize the ova (ovum) thereby by-passing natural mating (McDonald,
2003). AI was the first great biotechnology applied to improve reproduction and genetics of
farm animals; it has an enormous impact worldwide in many species particularly in dairy
cattle (Foote, 2002). In poultry, it was first applied by the chicken industry, and later had its
greater impact in turkey production (Burkey, 1984). Many of the principles and procedure
used where adopted from cattle (Wilson, 1978)
The basic procedures for semen collection and AI were established in the 1930s as reviewed
by Lake, (1995). A non-invasive method of semen collection from roosters ‘‘abdominal
massage method’’ as described by Burrows and Quinn (1937). For turkeys, the technique is
adapted by massaging the area around the cloaca before milking the semen (Lake and Stewart
1978a).
2.7.1 Artificial Insemination in Turkey
The primary objective of AI is to deposit optimum number of normal motile spermatozoa in
the female reproductive tract so that they can reach the Oocyte at the most favourable time
ensuring sperm capacitation and fertilization of the ova (Bearden et al., 2004). Artificial
insemination has been widely applied to poultry. Turkeys are the only commercial livestock
species completely dependent upon artificial insemination (AI) for fertile egg production
(Juliet and Bakst, 2008). AI is used extensively with freshly collected semen. It is used 100%
for turkey breeding because mating is difficult. AI in turkey was originally implemented in
order to control disease such as Mycoplasma meleagridis. It has, since then continued as a
means of ensuring high level of fertility (95% or more) when perform by skilled staff
(www.Hybridturkey.com.2009). Wilson et al. (1979) reported that fertility from natural
mating was poorly correlated with the physical characteristic of the male. Further indication
shows that with AI sperm concentration, motility, percentage live spermatozoa were
significantly correlated with fertility.
21
2.7.2. Semen Collection
Semen collection, processing, and AI have been reviewed by Sexton (1979) and Lake (1986)
and more recently by Donoghue and Wishart (2000). The pioneers in the poultry field were
Burrows and Quinn (1937), who developed the method of abdominal massage and pressure to
collect semen. In 1937 Burrows and Quinn described a non-invasive method, the ‘‘abdominal
massage method’’, for collection of semen from roosters. The technique involves restraining
the male, and gently stroking the back of the bird from behind the wings towards the tail with
firm rapid strokes. The male responds with tumescence erection of the phallus, at which time
the handler gently squeezes the cloaca expressing semen through the external papillae of the
ducti deferentis collecting the semen into a container. For turkeys, the technique is adapted by
massaging the area around the cloaca before milking the semen (Lake and Stewart, 1978a, b).
Adaptations are also made for species such as waterfowl which have penis-like copulatory
appendages (Bramwell, 2014). The proximity of the cloaca increases the likelihood of
obtaining semen contaminated with faeces, urates, and bacteria that are detrimental to semen
quality.
2.7.3 Semen Quality Evaluation
The assessment of semen defines what constitutes normal fertile semen, and that criteria can
be applied in the appraisal of the quality of ejaculated semen (Cooper, 1977). Gee (1995)
stated that semen quality characteristics of poultry birds gives an excellent indicator of their
reproductive potential and has been reported to be a major determinant of fertility and
subsequent hatchability of eggs (Peters et al., 2004). Fertility and hatchability on the other
hand are the major determinant of profitability in the hatchery enterprise. In the light of the
preceding statement, Mann (1954) earlier stated that, in spite of the wealth of information
gained by past and present students of semen, there is as yet no single seminal characteristics
known, which alone could serve as the means of judging 'male fertility'. The best criterion of
the fertilizing capacity of spermatozoon is of course, the actual ability to fertilize the ovum”.
More traditional semen evaluation procedures include determination of semen volume, color,
concentration, motility, viability and morphology of spermatozoa have been developed over
the years. Many of these assessments correlate with the fertilizing capacity of spermatozoa
when fresh semen is evaluated (Wishart, 1995a).
2.7.3 1 Semen Colour
Good quality semen has a thick consistency and a pearly or creamy white colour (Bearden et
al., 2004). Semen with a reduced concentration of spermatozoa is grayish in color and watery
22
in appearance and concentration of spermatozoa is very low.. Deep yellow buff colored
semen usually has a high content of defective and/or under developed spermatozoa, which if
used will result in lower fertility. If semen looks visually normal in color and consistency,
you can be relatively sure that spermatozoa concentration and motility of semen are
satisfactory enough to assure high fertility. However, when in doubt, discard it
(www.Hybridturkey.com. 2007). The use of semen that is discoloured, watery, or
contaminated by fecal material, urates, or blood will lead to lowered fertility particularly if
the semen is subjected to short-term or long-term storage. Bearden et al. (2004) suggested
that all contaminated samples should not be use but discarded.
2.7.3.2 Volume of ejaculate
Semen volume is a physical parameter, evaluated immediately after ejaculate collection
(Ilinca et al., 2008). Poultry semen is viscous and highly concentrated, containing 6 (roosters)
to 12 (toms) billion spermatozoa/ml. The semen of the domestic fowl according to Hafez
(1978) varies from a dense opaque suspension to a watery fluid with a relatively high density.
He further stated that the differences in volumes and sperm concentration of the domestic
fowl semen depends largely on the relative contribution of the various reproductive glands,
the number of spermatozoa that could be obtained from a breed/strain and the extent to which
the genetic potentials can be exploited.
Ejaculate volume and sperm concentration are dependent on strains, frequency of collection
and age of the male (Bakst and Cecil, 1992; Kelson et al., 1996; Vanwambeke, 1996).
Differences among species in the numbers of sperm ejaculated reflect differences in sperm
production per gm of testis, testis size, sperm resorption (within the epididymis, for example)
and other sperm losses.
2.7.3.3 Motility Evaluation
Motility of spermatozoa has always been considered a primary requirement to fertilize eggs.
Motility is known to be an important characteristic in predicting the fertilizing potential of an
ejaculate (Gadea, 2005), but even this relationship occasionally fails to give a true picture, as
motile spermatozoa are by no means always fertile. Although the spermatozoa are brought to
the fertilization site mainly by uterine contractions (Langendijk et al., 2002), sperm motility
is required for penetration of the zona pellucida. Therefore, several methods have been used
for motility assessment. The simplest way to evaluate sperm motility is by estimating the
number of motile spermatozoa under a light microscope or using phase contrast microscopy.
23
This method is subjective since it depends on the interpretation by an individual (Vyt et al.,
2004b). It is however a cheap method and facilitates evaluation of high number of samples
throughout which makes it popular in commercial AI- centres.
There have been conflicting reports concerning the relationship between sperm motility and
fertility. Most researchers have found a significant correlation between sperm motility and
fertility (Kummerer et al., 1972; Wishart and Palmer, 1986), while Wall and Boone (1973)
found no correlation between them. The fertility of turkey spermatozoa has been reported to
be positively correlated with sperm motility measured by subjective ‘scoring’ method
(Donoghue, 1998; Bearden et al., 2004). Sperm motility is a primary determinant of male
fitness (Fronman et al., 1999). Bowling et al (2003) reported that sperm motility is
independent of testis size as measured by ultrasound and also showed that males
characterized by high sperm motility may weigh less than low sperm motility counterpart.
2.7.3.4 Motility Evaluation Technique
The scoring method of motility evaluation involves placing a small drop of sodium citrate
solution on a clean glass slide warmed to 38oC. This drop should be of such size that placing
a coverslip upon it (below) will allow it to occupy all the space under the coverslip, but not
spill out beyond it. With a clean glass rod, a very small dab of whole semen is drop on the
buffer and a coverslip is placed on the slide. The prepared slide is then examined under low
magnification (microscope). If the sperm are close together so as to make motility estimate
difficult, a second slide may be prepared. Motility is rated on a basis of 0 to 100% where 0
represents an estimated 0-5% progressively motile sperm, 1 represents 5-15%, etc (Bearden
et al., 2004).
2.7.3.5 Morphology Semen Evaluation
The microscopic appearance of spermatozoa can give information on morphological
abnormalities, cell membrane integrity and the acrosome. Microscopic estimate of sperm
motility tend to vary great between different laboratories. Some of this variability is due to
the method of estimation and the individual evaluators. For this reason evaluation of semen
quality tend to emphasize sperm morphological assessment rather than motility (Hudson,
1972; Morrow, 1980). An ejaculate normally contains some morphologically abnormal
spermatozoa (Foote, 1968). Morphological abnormalities give an indication of aberrations in
the spermatogenesis. Some malformations compromise the function of the cells and cannot
be compensated for, therefore leading to culling of the male animal. Abnormal shape of the
head which carries the genetic material or abnormalities of the mitochondrial sheet which is
24
important for the function of the flagella, are therefore called primary abnormalities.
Remainders of cytoplasm, proximal or distal droplets, and small tail abnormalities are called
secondary abnormalities and can be compensated for by the semen dose (Donadeu, 2004).
Additionally, morphological anomalies (e.g. coiled tails) acquired by inappropriate handling
of semen are called tertiary abnormalities. The normalcy of the ejaculate, however, depends
on the percentage of spermatozoa that possess primary and secondary abnormalities and most
importantly, estimation of normalcy of the acrosome because of its obvious role in
fertilization (Saacked, 1978).
The morphological structure of turkey semen is quite different from that of mammals. The
turkey spermatozoon is long, cylindrical and sharp at both ends. Like other species, the
spermatozoon is composed of the acrosome, head, mid-piece and tail. It is 0.5 µm at its
widest point. The acrosome is 2 µm, the head is 13 µm, the mid-piece is 4 µm and the tail is
85 µm long (Etches, 1996). It is very important to know the proportion of defective
(abnormal) spermatozoa in a semen sample to determine fertility. Etches (1996) further
classified the morphological defect types of semen assessed in vitro as follows: Neck bending
,(mid-piece bending).; Mid-piece damage.; Acrosome damage.; Bending, Swelling, Knotting
or rounding; Whole head swelling; and Tail defects. Serhat et al. (2002) reported
abnormalities of Acrosome 41%, mid-piece 25% as leading abnormalities, which was
attributed to the connection point between the head and mid-piece of poultry semen is very
sensitive to external factors (Maeda et al., 1986 and Tsukunaga, 1987) and large or swollen
head and knotted head, scoring 11 ± 0.11%. Researchers with similar results suggested that
mid-piece abnormalities were due to the sensitivity of this region, as it deteriorates quicker
than other regions and the tail’s movement cause bending (Maeda, et al., 1986, Yamane et
al., 2002). Lastly, Tail defects were 22 ± 0.18%, and the most frequent types were folding,
bending and knotting of the tail. However, Tsukunaga, (1987) stated that poultry semen could
swell in seminal plasma after ejaculation and it was difficult to determine the abnormal
swelling rate. Again, researchers claim that tail defects in poultry semen are secondarily
occurring mechanical defects that cannot be considered as true (primary) defects (Tsukunaga,
1987).
2.7.3.6 Morphology Assessments Technique
Morphology can be assessed by staining techniques that do not require highly qualified
personnel (Shipley, 1999). Although several stains can be used, staining spermatozoa of farm
animals for morphological examination is usually combined with membrane integrity
25
assessment using a dye that is excluded by live cells, such as eosin. Therefore, besides being
helpful for assessing sperm morphology, the eosin-nigrosin stain can be used to discriminate
between live and damaged cells. This staining technique is widely used and is considered a
simple and reliable technique that is easy to apply and its outcome correlates with fertility
(Tsakmakidis et al., 2010). Normal morphology is correlated with fertility (Alm et al., 2006;
Xu. et al., 1998) and should therefore be performed routinely.
2.7.3.7 Sperm Concentration
The knowledge of sperm concentration not only provides a basis for calculating the number
of sperm cell per insemination dose, but also serves as a measure of semen quality
(Christensen, 1981; Bearden et al., 2004) because lower sperm concentration could be an
indication of problem (Hafez, 1985). Sperm concentration can be estimated by a packed cell
volume method, or spermatocrite. Semen is drawn into a microcapillary tube and one end of
the tube is sealed. The sperm cells are centrifuged and the percent packed cells are measured
using a microcapillary reader (Maeza and Buss, 1976). Optical density is another indirect
method to estimate sperm concentration. Bilgili and Renden (1984) found 550nm to be the
optimum wavelength to estimate concentration in a spectrophotometer. Direct sperm cell
counts can be made by use of a hemocytometer (Allen and Champion, 1955). Different glass
chambers are described to count cells in a known volume. Haemocytometers, such as the
Neubauer, Thoma and Bürker chamber are reusable glass chambers with fixed volume used
for counting immobilized spermatozoa in grit. Other reusable glass chambers as the Mackler
chamber are used for assessing concentration as well as motility (Tomlinson et al., 2001).
Haemocytometers are considered as the standard method for determining sperm
concentration and have a lower coefficient of variation than disposable chambers
(Christensen et al., 2005; Tomlinson et al., 2001).
As with motility, there are conflicting reports of the relationship of concentration to fertility.
The majority of reports have found no relationship between the two (McCartney, 1956;
Cooper and Rowell, 1957), while other researchers have seen positive correlations between
sperm concentration and fertility (McDaniel and Craig, 1959; Kammerrer et al., 1972).
Sperm concentration is influenced by breed, nutrition, season and even method of ejaculation
(Butswat et al., 2001). Ejaculates containing less than 500 million cells per ml have been
associated with low fertility rate (Bearden et al. 2004).
26
Table 3: Seminal Characteristics of Domestic Animals
Domestic
Volume Sperm Conc. Total
Animal
(ml)
(x109/ml)
sperm(x109)
Bovine, dairy
6
1.2
7
Bovine beef
4
1.0
4
Ovine
1
3.0
3
Caprine
0.8
2.4
2
Porcine
225
0.2
45
Equine
60
0.15
9
Canine
5
0.3
1.5
Chicken
0.5
3.5
1.8
Turkey
0.5
7.0
3.5
Turkey(local) 0.5
2.8
3.5
Source: Gomen (1977); Noirault and Brillard (1990)
Sperm
Motility (%)
70
65
75
80
60
70
80
85
85
84
Normal
sperm (%)
89
80
90
70
60
70
80
80
90
90
Semen collection
weeks (no)
4
4
20
20
3
3
3
3
3
3
2.8.1 The Biochemistry of Semen
On the basis of the preceding semen quality evaluation, a modern fertility examination must
be considered incomplete with the mere biophysical examination of the ejaculate. However
biochemical analysis of the semen is necessary in order to exclude anything that may lead to
fertility problems. Therefore, apart from conventional method of semen analysis involving
estimation of mass motility, sperm concentration and morphology of sperms; assessment of
some other biochemical constituents has been suggested (Argov et al., 2007; Turba et al.,
2007; Brinsko et al., 2007; Meseguer et al., 2004).
According to Mann (1945)” Whole semen' as ejaculated, generally appears as a viscous,
creamy, slightly yellowish or greyish fluid, and consists of spermatozoa or' sperm', suspended
in the fluid medium, called seminal plasma; its composition depends in the first place, on the
proportion of sperm and plasma, and is further determined by the size, storage capacity, and
secretory output of several different organs which comprise the male reproductive tract”. The
seminal plasma is a composite mixture of fluids secreted by accessory organs and certain
other glands located in the wall of the urethral canal, which provides the medium and vehicle
for spermatozoa survival, and it volume and composition varies according to species, is
determined by the size, storage capacity and secretory output of different organs of the male
reproductive tract (Mann, 1964; Gundogan, 2006). There is little seminal plasma in bird
semen and even among some of the mammals, but on the whole, the higher mammals,
including man, produce relatively dilute semen with a considerable proportion of seminal
plasma.
27
The following mammalian species have been found to contain fructose in their semen: man,
bull, ram, boar, stallion, goat, opossum rabbit, guinea-pig, rat, mouse, hamster (Mann, 1949)
and, among the lower animals. There are, however, considerable quantitative differences
between the various species. In the bull and goat, for example, the concentration of fructose
in semen sometimes reaches a level of 1000 mg/l00 ml., but in the boar and stallion it seldom
exceeds 50 mg/l00 ml., the cock semen has no fructose or a negligible amount only, but it
contains a certain amount (20-100 mg/l00 ml.) of anthrone reactive material of which a
variable fraction disappears on oxidation with glucose oxidase and must therefore, be
identical with glucose (Mann and Hancock, 1952). Rabbit semen, contains occasionally an
appreciable admixture of glucose in addition to fructose (Mann and Parsons, 1950).
Biochemical estimates of seminal plasma are used for semen evaluation as seminal plasma
play its role in sperm metabolites, nutrition of ejaculated sperms and provides protection to
spermatozoa against proteinase inhibitors, which help in sperm capacitation and local
immunosuppression (Pesch et al., 2005). Seminal plasma comprises of ions (Na+, K+, Zn+,
Ca++, Mg++, C++); energy sources (fructose, sorbitol, glycerylphosphocholine); organic
compounds (citric acid, amino acids, peptides, low and high molecular weight proteins, lipid,
hormones, cytokines); and nitrogenous components such as ammonia, urea, uric acid and
creatinine. Reducing substances such as ascorbic acids and hypotaurine also exist in the
seminal plasma of ruminants. Evaluation of these biochemical factors is an important
criterion for assessing male fertility. Deviation from normal values of these biochemical
components in seminal plasma is correlated with male infertility (Cevk et al., 2007).
2.8.1 Determination of Fructose Concentration
The identification of the seminal sugar as fructose by Mann, (1946a) opened a new chapter in
fertility research in many animals including man (Kimmig, 1959; Schirren, 1955, 1961). It
was shown that in several species fructose is secreted either by the seminal vesicles or by
functionally related organs (Mann, 1946c; 1947; l948a, b). This made it possible to use the
chemical assay of fructose in semen as an indicator of the relative contribution made by the
seminal vesicles towards the make- up of the whole semen.
In a research conducted by Mann and his associate to test not only fructose in differennt
animals (stallion, bull, ram, rabbit, guinea-pig, rat), but also to establish a relationship
between the fructose content in semen and the nature of certain endocrine anomalies. Thus
Mann & Parsons (1947) introduced the so-called fructose test, which was further developed
by Mann, Lutwak-Mann & Price (1948), as well as Mann & Parsons (1950). The fructose
28
analysis of semen carried out by these researchers including Harvey (1961); Davis and
McCune (1950) was based on calorimetrical analysis of the colour reaction between fructose
and resorcinol (Schirren, 1956).
2.8.2 Fructose as a Constituent of Seminal Plasma
In the seminal plasma of several species, including bull, ram, rabbit, boar and man, fructose
accounts for practically the whole of the yeast fermentable reducing sugar. Little glucogen, if
any, is present in the seminal plasma; this was shown by applying a method which makes use
of mould glucose oxidase which oxidizes glucose quantitatively but leave fructose untouched.
The level of fructose in seminal plasma varies from one species to another and even within
the same species there are individual differences. The highest values were observed in bull
where the concentration sometimes exceeds 1g. fructose /100ml seminal plasma. The main
function of fructose in semen is to supply the spermatozoa with readily glycolyzable material.
On storage, the content of seminal fructose falls progressively and lactic acid accumulates.
Normally spermatozoa utilize fructose, as this is the chief sugar available in seminal plasma,
but their enzymic equipment enables them to metabolize efficiently glucose and mannose. In
man, fructose concentration consider as a 'normal concentration' by a number of authors
(Harvey, 1951; Schirren, 1955, 1961) is between 1200 and 4500ug/ml. Harvey,
(1948) reported that there is about 2-5mg of fructose per ml seminal fluid and it appears to be
higher in men who are more fertile. All lower figures for concentration must be considered
pathological; this is to say, such concentrations normally result in infertility. Fructose is also
likely involved in protein complexes, particularly in coagulated semen (Montagnon et al.,
1982). Haendler (1965) showed that fructose is present as a fuel supply for sperm cells, and
without fructose infertility would result. Break down of fructose via fructolysis (for energy
consumption) may result in lactic acid production. Hence, the addition of fructose will not
greatly change the metabolic rate, but will extend the life span of the sperm. Excessive
dilutions suppress sperm motility and the metabolic rate of the sperm (Nishiyama, 1961;
Bearden et al., 2004).
2.8.3 Importance of Fructose Test in Evaluation of Fertility
Redenz (1933) has shown that bull spermatozoa contain glycolyse glucose, fructose, and
mannose to lactic acid, and that the presence of these sugars, but not that of sucrose, lactose,
or glycogen, is beneficial to sperm motility. His findings were confirmed by others and it has
since become an established fact that the metabolism of spermatozoa in several mammalian
species including man, ram and bull, is predominantly of a glycolytic character (Ross, Miller
29
and Kurzrok, 1941; Salisbury, 1946).The activating influence of fructose on motility of
spermatozoa is shown by a previously immotile spermatozoa obtained directly from the
epididymis of a bull, ram, or boar, resemble washed ejaculated sperm in that they are
incapable of survival under purely anaerobic conditions. While in the epididymis, the
spermatozoa have no access to fructose and are immotile; the onset of motility coincides with
their passage along the male genital tract and contact with the seminal plasma.
A more positive relationship can be shown between the fructose content of seminal plasma
and age. Nowakowski & Schmidt (1959) reported similar experiments, which led to the same
conclusion. On the other hand, several authors found out that absolute fructose concentration
was inversely proportional to the number of spermatozoa Schirren (1963). Stossier (1960)
found notable lower percentages of fructose with increasing numbers of spermatozoa. Harvey
(1951) had assumed that the fructose in human ejaculate would be at least partially
consumed, most likely just before ejaculation. The proven relationships between fructose
content of seminal plasma and testosterone production have been previously reported by
Mann's (1946) in animal. Landau & Loughead (1951) verified these results on human beings
for the first time with four patients (eunuchs), on a large basis by Nowakowski & Schirren
(1956). Landau & Loughead (1951), reported a decreased in fructose concentration in men
with symptoms of androgen deficiency, a return of the fructose concentration to normal in
these cases was attained by treatment with testosterone, so that here again a relationship
probably existed between the fructose concentration and the testosterone concentration. At
this point, (Mann, 1945) reference should be made to the fact that lower fructose
concentration always suggested a decrease testosterone production by the Leydig cells, when
there was no sign of inflammation of the prostate region or seminal vesicles. Low fructose in
semen is characteristic of ejaculatory duct obstruction, bilateral congenital absence of the vas
deference, partial retrograde ejaculation and androgen deficiency (WHO, 2010).
30
Table 4: Species Differences in Chemical Composition of Seminal plasma
Species
Fructose Conc.
Sodium Conc.
References
347mg/100m
Potassium
Conc.
71.4mg/100ml
Bull
361mg
Ram
Buck
435mg
295.7mg
103mg/100ml.
-
71mg/100ml.
-
Oramus et al.(1980); Mann (1954)
Roca et al. (1993)
Boar
Stallion
14.7 mg
15mg/100ml
587 mg/100ml.
68mg/100ml
197mg/100ml.
62mg/100ml
Oramus et al.(1980); Mann (1954)
Mann. (1954)
Rooster
4 mg/l00ml
3.96μg/ml
2.88μg/ml
Mann (1954); Mass´anyi et al.(2008)
Turkey
3.2 - 8.0mg/100ml
3.14μg/ml
3.42μg/ml
Gamal and Rizik (1972); Mass´anyi et al. (2008)
Man
250mg/100ml
250mg/100ml
89mg/100ml
Mann and Schirren, (1955)
Oramus et al.(1980);Igboeli and Rakha (1971)
2.8.4 Evaluation of Seminal Chemical Elements on Fertility
Mammalian seminal plasma and spermatozoa are known to contain a broad variety of macroand micro-elements (Marzec-Wróblewska et al., 2012). These chemical elements represent a
vital ecophysiological component for the preservation and fertilization capacity of
spermatozoa. Some of them are essential for proper sperm cell functions (e.g., sodium, Na;
potassium, K; calcium, Ca; magnesium, Mg); others are required in relatively narrow limits
(e.g., zinc, Zn; copper, Cu; manganese, Mn; cobalt, Co; selenium, Se; iron, Fe) (Massányi et
al., 2003; Massányi et al., 2004). The sperm cell contains potassium (K+) as a major cation,
whereas sodium (Na++) is the principal cation in the seminal plasma. Potassium is a natural
metabolic inhibitor and by increasing the cellular concentration, it increases the ratio of
potassium to sodium which again reduces the metabolic activity of the sperm. The influence
of major biologically active inorganic components on spermatozoa viability parameters has
been studied in animals as well as in humans (Massányi et al., 2003; Massányi et al., 2004;
Eghbali et al., 2008; Atig et al., 2012; Peter et al., 2008; Sørensen, et al., 1999). Positive
effects on the sperm cell motility, morphology, and concentration were reported particularly
for Zn, Mg, Se, and Ca (Eghbali et al., 2008; Atig et al., 2012; Sørensen, et al., 1999). Fe,
Cu, and their compounds are essential metal cofactors for a variety of bioactive molecules;
however, disturbances in their regulative absorption mechanism with subsequent aberrant
concentrations may have a negative impact on the sperm viability and morphology (Massányi
et al., 2003, 2004; Roychoudhury et al., 2008).
31
2.8.5. Sodium and Potassium Concentration in Semen
In a research conducted to determine the level of some major biochemical constituents in
seminal plasma of Lohi rams. Tariq et al. (2013) reported Na and K, in seminal plasma as
222.90 mg/dl, 48.21 ppm. Tariq et al.(2013) reported the concentration of chemicals in
seminal plasma of bovine as; Na 179.44mg/ dl, K 25.97mg/dl, Fe 4.15mg/dl, Cu 2.39 mg/ dl,
Mg7.65mg/ dl, Zn 23.59 mg/ dl. While, Cragle et al. (1958) reported that potassium is more
concentrated within the sperm cells of bovine semen than in seminal plasma. But the sodium
and calcium are more concentrated in the seminal plasma than within the sperm cells. Igboeli
and Rakha (1971) used flame photometer for potassium and sodium concentration and atomic
absorption spectrophotometer for magnesium and calcium in whole semen and seminal
plasma. Sodium concentration (mg/100 ml) in seminal plasma and whole semen and the
fraction of pre ejaculate was 320, 347 and 335, respectively and in the same order potassium
was 69.4, 71.4 and 152; Ca was 34.0, 35.3 and 4.1 and Mg++ was 8.8, 8.3 and 5.7.
Mass´anyi et al. (2008) reported that the concentration of cadmium in rooster is 9.06 and in
turkey 4.10μg/ml. In zinc 5.25μg/ml in rooster and 3.70μ g/ml in turkey were detected.
Higher concentration of copper was found in rooster semen (6.79 μ g/ml) in comparison with
turkey semen (4.29 μg/ml). The level of sodium (3.96μg/ml; 3.14μ g/ml) and potassium
(2.88μg/ml; 3.42μg/ml) was very similar in both species. Correlation analysis detected high
positive correlation between cadmium and zinc (r = 0.701) in rooster and between sodium
and potassium (r = 0.899) in turkey semen. In man, seminal plasma sodium and potassium
have commonly been found to be in the range 110-120mM (Na) and 20-30 mM (K)
respectively (Huggins et al., 1942; Skandhan & Mazumdar, 1981).
2.8.6 Sodium and Potassium Effects on Semen Quality and Fertility
Gür and Demirci (2000) detected a positive impact of Na on all spermatozoa vitality
characteristics assuming that Na is crucial for proper physicochemical properties of semen.
This agrees with the result of Tariq et al. (2013), who further concluded that the seminal Na
is indispensable for a suitable antioxidant milieu and activity. On the other hand, negative
associations between the K concentration, motility, and progressive motility. This also agrees
with Gür and Demirci (2000) as well as Sheth and Rao (1962), proposing that oxygen uptake,
glycolysis, and fructolysis could be inhibited by K and indicating that this element may
adversely affect spermatozoa activity. Moreover, negative correlations together with high
concentrations of K in the Mo groups confirmed the suggestions of Ford (2001), and Griveau
32
et al. (1994) that at low pH the K+ ion pairs with the superoxide causing a significant increase
in lipid peroxidation and free radicals formation, which are inversely correlated with sperm
motility and antioxidant status, especially with superoxide dismutase and GSH, which are
directly responsible for superoxide scavenging (Tvrdá et al., 2011). López et al. (2013)
reported that a moderate associations were found between Na (r = -0.428), K (r = 0.354), and
Se (r = 0.354) with progressive motility and, concluded that, several biochemical components
of seminal plasma were related to semen quality in AI boars. Tariq et al. (2013) reported that
there is significant negative correlation between biochemical constituent (Na, Mg, Cu) and
sperm characteristics (ejaculated volume, mass activity, motility percentage, sperm
concentration and spermatozoa abnormalities). On the contrary, Tariq et al. (2013) reported
that Na, Fe, Cu, Mg, and Zn were positively correlated with the motility and antioxidant
parameters. Inversely, K exhibited the positive associations with malondialdehyde.
2.9.0 Factors affecting poultry semen quality
Semen quality could be affected by age, lighting, season, body weight, and diet (Sexton,
1986; 1987) as well as semen collector. Factors affecting the quality and quantity of sperm
could be a hormonal system, feed, temperature and season, frequency of ejaculation, libido,
physical factors, age and disease.
2.9.1 Ambient Temperature
The sperm membrane is susceptible to changes in temperature, and this may affect the
movement of the sperm, causing deterioration in quality and fertilizing capacity. Therefore
care must be taken to maintain the required temperatures (Senger, 2003). A decrease in
ambient temperature after collection of semen decreases the activity (motility) of the sperm
and semen should also not be exposed to the sun (Anderson, 2001). The ambient temperature
is never constant and higher temperatures can increase the metabolism of the sperm cell,
while cooler temperatures reduce the metabolic rate and slows down the sperm movement
(Hafez and Hafez, 2000). The amount of good quality semen is decreased by hyperthermia
associated with high ambient temperatures with high relative humidity or fever.
2.9.2 Micro Bacterial Contamination
Semen collection in farm animal species is not a sterile procedure, and some degree of
contamination with bacteria cannot be avoided (Varner et al., 1998; Althouse et al.,
2000; Aurich and Spergser, 2007; Bielanski, 2007; Yániz et al., 2010).Virtually all semen
samples are contaminated at the time of collection (Almond and Poolperm, 1990). Poultry
33
semen becomes heavily contaminated with bacteria as it issues from the papillae on the wall
of the cloaca during collection. Sexton et al. (1980) reported that turkey semen collected by
artificial ejaculation contains on the average 1300 x 106 bacteria/ml. Microorganisms have a
deleterious effect on sperm function, both directly by altering the structure of the sperm, by
affecting its motility (Depuydt et al., 1998) or by provoking a premature acrosome reaction
(Kohn et al., 1998), and indirectly stimulating the production of antibodies that can be
directed against the sperm glycocalyx complex (Kurpisz and Alexander, 1995). Some reports
indicate that metabolic products such as endotoxins from some bacteria appear to have
detrimental effects on the survival of sperm (Almond and Poolperm, 1990). Thus, semen
quality and the quantity of viable sperm cells may be reduced with bacterial contamination.
Some reports indicated that metabolic products, such as endotoxins from some bacteria and
fungi appear to have detrimental effects on the survival of sperm. Several micro-organisms
identified in poultry semen include Staphylococcus albus, Staphylococcus aureus,
Escherichia coli, Proteus spp., Hemolytic streptococci spp., Diphtheroid bacilli and Bacillus
spp. (Sexton et al., 1980). Enterobacter spp. (Ngu et al., 2014).
2.9.3 Photoperiod
Time of the day for the collection of semen also affects the quality and quantity of semen.
Semen production has been noted to be higher when collected in morning and evening when
the environment is cooler (Mass´anyi et al., 2008). According to Bearden et al. (2004), the
lighting in the laboratory can suppress the metabolic rate, motility, and fertilizing capacity of
the sperm. A greater effect was observed when semen was in contact with oxygen (O2). The
enzyme catalase will prevent the harmful effect of light or photoperiod. This demonstrates
that light causes a photo-chemical reaction in the semen that result in the production of
hydrogen peroxide.
2.9.4 Nutrition
Boars on the high plane of nutrition produced a larger ejaculate with more spermatozoa but
there was no difference in sperm motility, sperm concentration or morphology (Dutt and
Barnhart, 1959). Ejaculate volume, sperm density, and fertility ability of toms can be affected
by restricted feed intake (Etches, 1996).
2.9.5 Age Factor
34
In poultry species, semen quality parameters such as volume, concentration and motility
change negatively with increasing age of the male, leading to a progressive decline in fertility
(Thatohatsi, 2009). In the volume evaluation, attention must be paid to the age of the male
because the quantity of the collected semen in growing animal increases with age. In
cockerels, it has been shown that increasing age negatively affects the biochemical
parameters of semen (Hafez and Hafez, 2000). Similarly, Kotlowska et al.(2005) reported
changes in semen quantity and quality to be related to increasing age in cockerels.
2.9.6 Oxidative Stress
Oxidative stress is a condition associated with an increased rate of cellular damage induced
by oxygen and oxygen derived oxidants commonly known as ROS (Sikka et al., 1995).
Among various causes, one of the most important factors contributing to poor quality semen
has been reported to be oxidative stress (Bucak et al., 2010). Oxidative stress (OS) has been
attributed to affect the fertility status and physiology of spermatozoa (Agarwal, et al., 2008).
The term oxidative stress is generally applied when oxidants outnumber antioxidants (du
Plessis et al., 2008, Desai et al., 2010), the imbalance between the production of reactive
oxygen species (ROS) and a biological systems ability to readily detoxify the reactive
intermediates or easily repair the resulting damage (Agarwal et al., 2003). All cellular
components including lipids, proteins, nucleic acids and sugars are potential targets of
oxidative stress (Agarwal et al., 2008). Oxidative damage in proteins ranges from specific
amino acid modifications and peptide breakage to loss of enzyme activity (Stadtman and
Levine, 2003). The production of ROS by sperm is a normal physiological process, but an
imbalance between ROS generation and scavenging activity is detrimental to the sperm and
associated with male infertility (Sharma and Agarwal, 1996). Excess of free radicals
generation frequently involves an error in spermiogenesis resulting in the release of
spermatozoa from the germinal epithelium exhibiting abnormally high levels of cytoplasmic
retention (Sanocka and Kurpisz, 2004). The peroxides are generally associated with
decreased sperm functions and viability (Aitken et al., 1989).
2.9.7 Frequency of Ejaculation
The frequency of ejaculation and the period of semen collection had an impact on semen
quality. Long abstinence periods (Pascual, 1993) and successive ejaculations (Ollero et al.,
1994) have been associated with membrane alterations of spermatozoa. Sperm volume and
concentration in semen samples decreased gradually with increase in ejaculation frequency
35
(Ollero et al., 1996; Kaya et al., 2002), the sperm motility did not change considerably. In a
study by Ollero et al. (1996), the maximum proportion of viable cells was obtained in the
second ejaculate after an abstinence period of 3 days. The authors concluded that the use of
the second and/or a mixture of second and third ejaculates would improve the results in
artificial insemination. Kaya et al. (2002) further elaborate that the increased in semen
collection frequency may have an effect on sperm quality and the composition of the seminal
plasma (Kaya et al., 2002), although it remains to be determined whether this has an impact
on field fertility.
2.9.8
Breed/species variation
Semen quality traits are indicated to vary according to breeds (Machebe and Ezekwe, 2000).
An investigation of thirteen commercial breeder lines revealed a significant interaction
between strain and duration of fertility (Fiser and Chambers, 1981). Nwachukwu et al. (2006)
reported that naked neck and frizzled genotypes produced higher ejaculates than the normal
feathered breeds of cockerels. Similarly, Zahraddeen et al. (2005) reported higher semen
volume and other seminal characteristics for exotic white Nicholas toms than the local breed
of toms. In contrast, no effects of duration of fertility were found in five commercial lines of
broiler breeders (Kirby et al., 1998).
2.10.0 Semen Collection Technique
Effective harvest of semen involves obtaining the maximum number of sperm of highest
possible quality in each ejaculate. This involves proper semen collection procedures used on
males that are sexually stimulated and prepared. The initial quality of semen is determined by
the male and cannot be improved even with superior handling and processing methods.
Semen quality can be lowered, by improper collection and processing techniques. Semen
collection is a complex procedure involving coordinated efforts between the animal handler
and the collector (Bearden et al., 2004).
2.10.1 Artificial insemination
The technique currently used for AI in poultry was developed in the 1930s and involves
applying pressure to the hen’s abdomen and everting the vaginal orifice through the cloaca
(Quinn and Burrows, 1937). This procedure is also referred to as cracking, venting or
everting the hen. Semen is deposited 2–4 cm into the vaginal orifice concurrently with the
release of pressure on the hen’s abdomen. Insemination is accomplished with straws, syringes
or plastic tubes. Bakst et al. (1994) avian spermatozoa are normally inseminated into the
36
lower vagina from where, even with untreated spermatozoa, only 1–2% is able to reach and
enter the SST at the uterovaginal junction, where they are subsequently stored for days or
weeks before fertilization. A significant feature of the reproductive physiology of the hen is
her ability to store fertile spermatozoa for long periods of time. Sperm storage tubules (SST),
which are structures found in the distal half of a the oviduct of all avian species studied to
date, sequester and store spermatozoa which are slowly released over time to insure an
adequate population of spermatozoa at the site of fertilization (Bakst, 1993). In large scale
commercial operations, automated semen dispensers using individual straws loaded with a set
AI dose are commonly used. Industry standard for insemination dose in chickens and turkeys
is 100 and 200 million spermatozoa per insemination, respectively (Etches, 1996). Older
hens, however, require either duplicate inseminations, or more than 250 million sperm per
week to maintain fertility (Brillard and McDaniel, 1986). With artificial insemination, the
quality of the spermatozoa is a more limiting factor for fertility than the number inseminated
(Wishart and Palmer, 1986), and furthermore, sperm quality is more likely to determine
fertility than oviduct selection (Froman et al., 1999). Therefore, a low (or minimal) dose of
spermatozoa can be used in order to differentiate between sperm quality of males.
2.10.2 Site, Depths and Time of Insemination
Variations among such artificial insemination techniques as depth of insemination and time
of insemination can influence the rate of fertility (Judd, 2001). Bakst et al. (1994) avian
spermatozoa are normally inseminated into the lower vagina from where, even with untreated
spermatozoa, only 1–2% is able to reach and enter the SST at the uterovaginal junction,
where they are subsequently stored for days or weeks before fertilization. Lorenz (1959)
recommended deep semen deposition whereas Rooney et al. (1966) found no fertility
differences when inseminating hens at 1.25cm or 5cm depth. Ogasawara et al. (1968)
reported optimal fertility with insemination less than 5cm in which the spermatozoa were
placed close to the sperm storage gland. Also, Biellier et al.,(1961) found that deep
insemination, (8cm) of Broad Breasted Bronze hen produced better fertility compared to
2.5cm depth of insemination. However, Wentworth et al., (1975) inseminated three different
lines of turkeys (Large White Hybrid, Bronze and Large White inbred) and showed
significantly greater fertility at 2cm depth of insemination compared with 7cm depth of
insemination. Unfortunately, there was no consistency in superior fertility with shallow depth
of insemination. Wentworth et al., (1975) stated that the depth of insemination did not affect
the duration of fertility in Bronze hens, but Large White had a longer duration of fertility
37
following shallow insemination. According to Bakst and Brillard, (1995) turkey hens are
generally inseminated before they begin to lay, usually 14 to 17 days after increased lighting
for stimulation of egg production. On the contrary, chickens, AI is usually initiated when
15% to 20% of the hens are in egg production. In turkeys, Brillard and Bakst (1990)
demonstrated that sperm numbers in the SST of hens inseminated before the onset of lay was
twice that of hens inseminated at the beginning of egg production. Turkey hens inseminated
before the onset of egg production can produce fertilized eggs up to 16 weeks after
insemination (Christensen and Bagley, 1989). The precise mechanisms supporting prolonged
sperm storage in the SST are unknown but are thought to include reversible suppression of
respiration and motility of spermatozoa as well as stabilization of the plasma membrane and
maintenance of the acrosome (Bakst, 1993).
2.10.3 Fertilizing Capacity of the Sperm Cell in vitro
The measure of a successful AI program is sustained hen fertility. McDaniel (1995);
Donoghue, (1999); Mellor (2001) shown that the necessity of male selection based on semen
fertilizing ability.
The ability of spermatozoa to penetrate cervical mucus has been
considered to be a potentially important because this attribute might be useful I predicting
fertilizing ability. As reviewed by Bakst et al. (1994), the behaviour or fate of sperm within
the hen's vagina constitutes a critical determinant of fertility in the domestic fowl. Motile
sperm ascend the vagina and enter specialized sperm storage tubules (SST), which are located
at the juncture of the vagina and shell gland. Sperm remain within the SST for a period of
days to weeks. If the oviduct is patent upon their release, sperm pass rapidly up the oviduct,
presumably by antiperistalsis, to the infundibulum, which is the site of fertilization in the hen.
sperm within a sperm storage tubule are always oriented with their acrosomes toward the
blind end of the tubule and their long axes parallel to the long axis of the tubule (Bakst, et al.,
1994), sperm metabolize fatty acids, and the large lipid droplets located within the apical
cytoplasm of SST epithelial cells (Bakst, et al., 1994), appear to be a likely source of
exogenous fatty acids. The SST is blind-end tubules. If SST epithelial cells secrete a fluid
into a tubule's lumen, then a current would be generated within the lumen. Froman et al.
(1999) hypothesize that sperm reside within the SST by actively maintaining their position
against a current. In such a case, low intracellular ATP content would result in the egress of
viable sperm from the SST.
In general, sperm motility describes the ability of sperm to move properly towards an egg or
the quality of the sperm, which is a reason in successful fertilization (Quill and Garbers,
38
2002). By measuring the reduction of resazurin, Cooper and Rowell (1957) found it may be
possible to identify males with low fertilizing capacity, which could be due to the significant
association found between reduction time of methylene blue and motility (McDaniel and
Craig, 1962). Following capacitation spermatozoon can bind to the zona pellucida of the egg
and undergo the acrosome reaction (Yanagimachi, 1994) so far. Therefore, assessing the
portion of a sperm population that is motile is possibly the most widely-used measure of
accessing semen quality (Farrell et al., 1998). According to Love (2011), reduced percentage
of in vitro fertilization rate and acrosomal reaction are due to inseminating the oocyte by the
spermatozoa containing lower HYP motility spermatozoa in stallion. On the contrary Wise et
al. (2003) stated that de novo spermatozoa motion kinematics (motility and morphology) is
not always correlate to its fertility. This result might be due to considering the different
breeds of animal at different climatic condition.
The predictive value of the sperm mobility assay was attributed (Froman and Feltmann,
1998) to its simulating a critical step for internal fertilization in the hen: the net movement of
a sperm population against resistance. Consequently, this postulate constituted an alternative
to the hypothesis that the net movement of motile sperm within the vagina is affected by
selection exerted by the oviduct, in particular, an immunological barrier (Steele and Wishart
1992).
2.10.4 Duration of Fertile Period in Turkey Hen
Females in avian species share with other females (reptiles, hymenoptera) the ability to store
spermatozoa for prolonged periods in specialized structures of the oviduct called sperm
storage tubules located in the uterovaginal junction and in the infundibulum. Upon selection
and storage, sperm are progressively released from the storage sites and then transported to
the infundibulum, the site of fertilization of the oocyte (Bakst et al., 1994). Depending on
species and individuals, avian females may therefore lay several fertile eggs after a single
mating or insemination, thus defining the so-called ‘duration of fertile period’, which is the
number of days during which a given female lays fertile eggs after a single deposition of
sperm in the oviduct. In the turkey, duration of the fertile period may reach up to 8-10 weeks,
but the chances of each egg being fertilized progressively decline as time after semen
deposition increases (Lorenz, 1950; McCartney, 1951)
With AI programs, it is often desirable to determine the fertility status of a flock before the
next weekly insemination. There are several options available: breaking-out fresh eggs and
examining the GD to differentiate a fertilized from an unfertilized or early dead embryo;
39
setting normal but culled eggs (checked, hairline cracked, or dirty eggs) in a spare incubator
for 24-36 hr before breaking-out counting sperm in the outer PL; and counting sperm holes in
the inner PL. The above procedures are reviewed in Bakst and Long (2010). Sustained
fertility in the avian female depends on its ability to store adequate viable spermatozoa in
sperm storage tubules and to supply the infundibulum with sufficient numbers of sperm to
fertilize a succession of ova. Only the morphologically normal spermatozoa are capable of
ascending through the vagina of the hen to the region where the sperm storage tubules are
located (Bakst et al., 1994).
2.10.5 Evaluation of Fertility and Hatchability
While candling-fertility is useful, there is an eight or more day lag between the last AI and
candling-fertility determination, which overlaps with the next insemination (hen insemination
is generally at 7-day intervals). Fertility can be assessed at the hatchery before or after eggs
hatch. By candling, one can assess flock fertility as well as other sources of hatch failure such
as eggs set upside down, cracked eggs and embryonic mortality (Mauldin, 2002). Candling
can be performed quickly using a table (mass) candler or more slowly (yet more accurately)
using a spot candler. Egg breakout is the process where eggs that have been candled and
deemed not viable are removed from the incubator and opened to assess fertility and
embryonic development (Mauldin, 2002). Embryonic mortality is often classified as early
dead, mid-dead or late dead (Wilson, 1995). Early dead embryos occur during the first week
of incubation and are characterized by a blood ring or network of blood vessels. The embryo
may also adhere to the side of the egg. Embryos that die during the second week of
incubation are characterized by the presence of a hard beak and an egg tooth. Dead embryos
that are fully covered in feathers occur during the third week of incubation (Wilson, 1995).
Candling eggs and performing a breakout analysis is an estimate of flock fertility.
2.11.0 Factors influence Fertility
Several factors influence fertility after AI. Semen quality can be affected by age of the tom or
rooster, lighting schedule, season, body weight, and diet (Sexton, 1986, 1987) as well as the
semen collector. Timing of AI is important and is usually performed in the late afternoon to
minimize the number of hens with hard-shelled eggs in the shell gland.
In addition to the above problems with the ‘scale’ of fertility, we have the problem of the
inherent variation in the hen’s response to insemination, in terms of the proportion of fertile
eggs that she subsequently lays. This is, in turn, a function of the number of spermatozoa
40
which she retains in her oviduct and transfers to the egg at fertilization and is true not only
with respect to individual hens, but with respect to different inseminations made into the
same hen within a period of a few days (Wishart et al., 1992). Some reporters indicated that
metabolic products, such as endotoxins from some bacteria and fungi appear to have
detrimental effects on the survival of sperm. Watson (1990) observed that not only pathogen
but, other microflora can have adverse effects on the fertility of semen by the production of
toxins and by utilization of metabolic substrates. There is also a direct influence of bacteria
on fertilization (conception) especially if the number of bacteria reaching the site of
fertilization in the oviduct results in the step-wise decrease in sperm counts during transit to
the oviduct. Regardless of whether or not bacterial contamination reduces semen quality,
interferes with fertilization or causes uterine infection.
2.11.1 Age Factor
A sigmoidal decline in fertility is expected over a period of 2 to 21 days post insemination
(Kirby and Froman, 1990; Kirby et al., 1998) with a faster decline seen in older birds. The
quicker decline with age suggests poorer semen quality of males (Bramwell et al., 1996).
However, no difference in duration of fertility was found between flocks at 39 and 59 weeks
of age (Fiser and Chambers, 1981), which would indicate that the duration of fertility is not
related to age. With advancement in age the reproductive performance of hen start declining,
with marked increase in body weight, egg weight, decrease in egg number, and a drastic
decline in hatchability, embryonic mortality and increased number of cull chick (Durape,
2007). Infertility in older hens has been attributed to less receptors sites on the ovum for
sperm to bind and penetrate prior to fertilization (Keith, 2009).
2.11.2 Body weight of the Hen
Hen body weight has great effect on the overall flock fertility. Excess body weight, as well as
lighter hens may have decrease fertility (Keith, 2009) this hens have a reduced ability to store
viable sperm cells for long time, and cannot internally store as many total sperm cells,
fertility will be complicated. Goerzen (1996) found a negative correlation between hen
weight and duration of fertility.
2.11.3 Nutrition
Birds, like other farm animals need adequate nutrition to carry out reproductive function, and
also invest some nutrient in egg production (Dzoma, 2010). Egg size an indication of
41
maternal investment, is also a good predictor of hatchability as well as chicks survival at one
month of age (Bonato et al., 2009). Starvation, deficiencies of some macro and micro nutrient
can adversely affect fertility, hatchability, and chick survival (Dzoma, 2010).
2.11.4 Stress
Stress factors have been noted to affect fertility in breeder flock (Thatohatsi, 2009). Stress of
all kinds are said to compromise virtually every system of the body, producing stress
chemicals which in turn diminish the function of organs and glands (Durape, 2007). Any
degree of stress can have a negative effect on fertilization rate (Donoghue and Washart, 2000;
Obidi et al., 2008). Thus, rough handling of hens during capture, prior to insemination and
dropping the hen hard after insemination have been found to cause infertility in breeders,
hens must be handling with care during and after insemination, otherwise, semen may be
regurgitated from the vagina.
42
CHAPTER THREE
MATERIALS AND METHODS
3.2. Location and Duration of the study
The research was carried out at the poultry unit of the Department of Animal Science
Teaching and Research farm, University of Nigeria, Nsukka. Nsukka lies in the derived
savannah region, and located on latitude 7o 24’E and longitude 6o 25’N (Offomata. 1975),
with an altitude of 447m above the sea level (Breinholt et al., 1981). The climate is a typical
humid tropic, with a relative humidity range of 56.01 – 103.83%. The annual rainfall ranging
from 986-2098mm, with the rainy season around April-October and dry season is between
November-March, while the natural day length for Nsukka is between 12 – 13 hours (Iyang,
1978). The average diurnal minimum temperature ranges from 22o-24.7oC while the average
maximum temperature ranges from 33 – 37oC (Energy center, UNN. 2008). The experiment
lasted for twenty four (24) weeks.
3.2. Plan of the Study
The experiment was conducted in four phases. Phase I: measurement of weekly body weight.
Phase II: Semen Collection and Evaluation, Phase III: Biochemical parameters and Phase IV:
Fertility and Hatchability assessment.
3.3. EXPERIMENTAL MATERIALS
3.3.1. Materials and Processing
Moringa oleifera and Gongronema latifolium were used as the treatments, other materials
that formed part of the experimental diet include: maize, wheat offal, soya bean meal, palm
kernel cake. Fish meal, salt, bone meal and Micro nutrient (vitamin and mineral premix).
Moringa oleifera leaves was harvested from house hold farms, gardens, and fences in Billiri
Gombe state the northern part of Nigeria. It was handpicked then shade dried in an open air
room at room temperature for 5 days. Gongronema latifolium was purchased from local
famers in Orba local market and in town in Kogi state. The plants were handpicked and
shade dry for 7 days to dry. The leaves from both plants was then grounded to powder
separately and stored in clean jute bags until usage.
43
Table 5: Composition of the Experimental Diets M. oleifera (MO): G. latifolium (GL)
Ingredient
Treatments
Maize
Wheat offal
Soya bean meal
Palmkernel cake
Fish meal
M. oleifera
G. latifolium
Bone meal
Limestone
Lysine
Methionine
Vitamin/premix
Salt
Total
1
49
12
18
13
3
2
2
0.25
0.25
0.25
0.25
100
Proximate
Crude Protein (%)
Energy (Kcal/Kg ME)
Crude Fibre (%)
2
48
11
17
14
3.5
1.5
2
2
0.25
0.25
0.25
0.25
100
3
48
11
16
14
3
3
2
2
0.25
0.25
0.25
0.25
100
%/ Composition (100kg)
4
5
6
48
48
48
11
12
11.5
16
16
15.5
15
13
13.5
3.5
3
3.5
1.5
1.5
3
1.5
2
2
2
2
2
2
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
100
100
100
7
48
11
14.5
13.5
3.5
3
1.5
2
2
0.25
0.25
0.25
0.25
100
8
48
11.5
14.5
13.5
3
1.5
3
2
2
0.25
0.25
0.25
0.25
100
9
48
11
14
13
3
3
3
2
2
0.25
0.25
0.25
0.25
100
Dry matter
18.6
2,890.03
6.23%
3.3.2 Procurement and Management of Experimental Animals
A total number of 72 healthy growing local turkeys of about six weeks of age, 54 males and
18 females were purchased from a reputable farm in Ekwulobia, Anambra state. The birds
were quarantine and allowed to acclimatize in Departmental farm for two weeks. They were
given commercial feed and water ad libitum throughout the acclimatization period, after
which they were transfer to the experimental pens. At the eleventh weeks the treatments
commences. The birds were randomly assigned to nine (9) experimental diets; each of the 9
treatments consists of six (6) males, three replications and two toms (2) per replicate with two
(2) females per treatments. The first treatment received a basal diet i.e without any of the
treatments (control), treatments 2 and 3 contained 1.5 and 3kg levels of M. oleifera leaves
diet respectively. Treatments 4 and 5 received G. latifolium diet at the same levels as M
oleifera, while the last four groups (6,7,8 & 9) were given four levels of the bi-herbal diet;
1.5kg M.O +1.5kg G.L, 3kg M.O + 1.5kg G.L, 1.5kg M.O +3kg G.L, and 3kg MO + 3kg
G.L respectively. The effects of these herbs at various levels were later analyzed for semen
quality, fertility and hatchability and biochemical parameters of turkey tom semen.
44
Table 6: Treatments combination of Factorial Experimental Arrangement
Factor B
Gongronema
Latifolium (GL )
Dose (Levels)
b0 (0) GL
b1 (1.5kg) GL
Factor A Moringa oleifera (MO )
a0 (0)MO
a1 (1.5kg) MO
0GL+0MG
1.5MO+0GL
0MO+1.5GL
1.5MO+1.5GL
a2 (3kg) MO
3MO+0GL
3MO+1.5GL
b2 (3kg) GL
0MO+3GL
3MO+3GL
1.5MO+3GL
The groups of turkey toms were reared separately from the female in well-ventilated
constructed pens according to treatment and fed diet containing 19% crude protein at the
start, which was subsequently changed to the required crude protein. The composition of the
breeder diet is presented in Table 4. Water was provided ad libitum and routine vaccinations
were carried out.
Table7: Treatment Arrangement
Treatments
T1=
T2=
T3=
T4=
T5=
combinations
Treatments
Basal Diets (control)
1.5kgMO/100kg diet
3kgMO/100kg diet
1.5kgGL/100kg diet
3kgGL/100kg diet
T6=
T7=
T8=
T9=
combinations
1.5kg +1.5kgGL/100kg diet
3kgMO+ 1.5kgGL/100kg diet
1.5kgMO +3kgGL/100kg diet
3kgMO +3kgGL/100kg diet
3.3.3. Training of Toms for Semen Collection
At the age of twenty six weeks (26) all the tom from each treatment group were trained each
for semen collection using the abdominal massage technique as described by Burrows and
Quinn (1937) and to check for each treatment age at puberty. The training was done twice a
week for three weeks. By twenty nine (29) weeks, all the toms were ready for semen
collection (www.hybridturkey.com, 2007).
3.4 Data Collection
3.4.1 The Effect of M. oleifera and G. latifolium on Body Weight:
Tom’s body weight was measured and recorded as initial body weight. Thereafter,
individually toms were weighed and recorded on weekly bases, in the morning before feeding
through twenty four weeks of age.
45
3.4.2. Semen collection
The goal of semen collection is to obtain the maximum volume of clean, high quality semen
with the minimal amount of handling (Bakst and Dymond, 2013). Semen was collected using
the abdominal massage technique as described by Burrows and Quinn (1937); Bakst and
Long, (2010), which involves massaging the cloacal region to achieve phallic tumescence.
This is followed by a ‘cloacal stroke’, a squeezing of the region surrounding the sides of the
cloaca to express the semen as described by Kalamah et al. (2002). Little additional semen
was expressed after two cloacal strokes. With each collection of the Ejaculates, the semen
was examination visually and a subsequent immediate microscopic evaluate (motility) on an
individual bases per male, per treatment. To minimize the chances of faecal contamination of
the semen as recommended by Kalamah et al. (2002), feed were removed late afternoon of a
day before semen collection. Semen was collected a minimum of twice per week, with two
days rest between next milking.
Figure 1: Semen collection, evaluation and insemination
3.5 Semen Evaluation
3. 5.1 Semen Colour
Fresh semen was assessed visually on collection and score as outlined by Bearden et al.
(2004), 1 (Good quality turkey semen) = viscous and creamy- white; 2 (Samples with low
concentration) = watery or less opaque and 3 = Pink/Yellow appearance. Records were taken
within 30 minutes of collection.
3.5.2 Semen volume
The Semen volume from each of the treated toms were measured with the use of a calibrated
collection test tube graduated in ml. and then the volume was read off and recorded in ml.
46
3.5.3 Motility Evaluation
A small drop of normal saline solution was placed with the aid of a micropipette on clean
glass slide warmed at 38oC. With a clean glass rod, a very small dab of whole semen was
drop on the buffer, a coverslip was placed upon the drop, allowing it to occupy all the space
under the coverslip, but not flood out beyond it.
It was then placed on a microscope for examination with low magnification (microscope). A
magnification of x 400 was used. Several fields were examined and an estimate to the nearest
0 to 20% of motile sperm was made. The motility determination was carried out by taking
into consideration subjective measurements based on my judgment.
3.5.4
Sperm Concentration
The sperm concentration was measured using the direct cell count method. Here,
hemocytometer (Improved Neubauer counting chamber) was used for counting sperm cells as
described by Baker et al., (1985). It consists of specially designed slides that contains two
counting chambers and two dilution pipettes. The area of the counting chambers is
0.0025mm² and a depth of 0.1 mm. the square is sub-divided into 25 smaller squares. The
sperm cells were diluted by drawing 0.05ml semen with a tuberculin syringe and dropping it
in a 10ml beaker containing a mixture of 0.2ml Ethyl Alcohol solution and 0.75ml (2.9%)
sodium citrate. The diluted sample was thoroughly mixed and loaded into the counting
chamber. Before usage the counting chamber and the coverglass of the hemocytometer were
thoroughly cleaned, using tissue paper, it was soaked in a petridish with base covered with
fitter paper and little water added for an hour to enhance visibility of individual sperm cells
within the counting chamber. Thereafter, it was dry cleaned with tissue paper before use. The
coverglass was placed on the flat horizontal surface and a firm pressure was used to slide the
coverglass into position on the counting chamber till a rainbow effect (Newton’s rings) is
obtained on both sides of the counting chamber (this was attained when the coverglass stuck
firmly to the counting chamber and refused to fall-off even on complete inversion of the
counting chamber). I drop of the diluted semen was then placed on one end of the chambers
holding the capillary tube at an angle of 45o and also on the other end, this was allowed to
settle for 20mins. The loaded hemocytometer was then placed on the stage of the phase
contrast microscope and the count of the number of cells in a specified area was obtained at
magnification of x20 then x40 and finally at x100 for clear sight. Because the cells were
distributed randomly across the entire area of the counting chamber with some of them lying
on the ruled lines, it was necessary to adopt a standard counting technique for including or
47
excluding cells which lie on the ruled lines of the Improved Neubauer counting chamber
(accepting cells at the boundary of the top and right boundaries and excluding those at the left
and bottom boundaries as they will enter into the nearest counting chambers as cell counts).
When the cells were settled out of suspension, the number lying on 5 of the 0.04mm2 squares
were counted. For the final result, the concentration of sperm per volume was found using the
formula:
Formula: Sperm Concentration = N x DF X 106
AxD
Where N is the number of cells,
1
= 20
0.05
(0.05ml sperm + 0.2ml ethyl alcohol + 0.75 ml Nacl) = 0.05 + 0.2 + 0.75 = 1ml and Sperm
cells alone = 0.05ml.
A is the area of chamber counted = 0.002mm2 and
D the depth of chamber = 0.1mm. (Baker et al.,1985).
Total sperm count = sperm concentration x total volume of ejaculate (x109) (Hafez, 1985).
DF the dilution factor =
3.5.5
Dead and Live /Normal and Abnormal Spermatozoa
Percentage live/dead spermatozoa and percentage normal/abnormal sperm cells were
determined using the staining technique of Bakst and Cecil. (1997). The histological smears
were made within 20 min after semen collection. The stains were prepared exactly according
to the methods described by Bakst and Cecil. (1997). A mixture of Eosine/Nigrosine was
used as dye/stain, a drop at warm temperature (38oC) was placed near one end of a glass slide
on slide warmer (38oC). Using a plastic dropper, a small amount (1 drop) of semen was drop,
with a glass rod it was stirred in 2 or 3 circular movements of the rod. With a second slide,
the dye-semen drop at an angle (Figure 1) was spread in a thin layer. A smear was made by
placing a warm slide over the first and spreading the mixture evenly between the two slides.
The two slides were separated by pulling the ends in. Thereafter, on microscopic
examination, the slides were respectively placed on the stage of a phase contrast microscope
and observed at 100 magnifications.
Normal and abnormal morphology was observed,
stained spermatozoa were considered dead and damage, while live and normal spermatounstained ones were considered alive for every 100 counted sperm per slide and the average
count of each type determined. The live and dead sperm cells were recorded in percentage.
48
3.5.6 Sperm Morphological Evaluation
Using the same slide prepared for
fo the Dead-Alive
Alive evaluation, sperm cells were counted in
100 under "light dry" magnification and recorded the number of sperm that have abnormal
and abnormal morphologies expressed in percentages.
Figure 2 slide preparation
3.5.7 Biochemical Analysis
The semen of toms was collected using method described above. The semen sample was
collected in a label small test tube and was analyzed using Colorimetric methods as described
by Lindner and Mann (1964). While, sodium and potassium were estimated by the atomic
flame photometer described by David (1960).
3.6 Fertility trial (Phase 1V: Fertility and Hatchability)
Fertility trial was carried out using pooled semen from the turkey toms. Semen collected from
each tom were pooled on treatment basis and used to inseminate two hens per treatment, the
hens were inseminated a dose of 0.25ml twice before the onset of egg production thereafter
once weekly. Insemination was done late afternoon after egg lay. Eighteen hens in all were
inseminated.
3.6.1 Artificial insemination
The goal of AI is to produce a succession of fertilized eggs between successive
inseminations. Birds do not have an estrous cycle that synchronizes copulation with
ovulation. Therefore,
e, alternatively, at twenty weeks of age the hens started squatting
displaying signs of approaching puberty. H
Hens were inseminated with semen according to
treatment, two times a week before the onset of egg production and once weekly thereafter.
At the twenty two weeks the hens stated laying (Froman et al.,
., 2011; Bakst, 2011).
49
Fertility test was performed using eighteen (18) hens divided into two per treatment
correspond to the nine (9) treatment. T 1 hens were inseminated with pooled semen from T 1
toms while T 2 females were inseminated with pooled semen from T 2 toms and the same for
all the nine treatment. The females were insemination by “venting” as described by Hafez
(1985). Venting was done by applying pressure to the left side of the abdomen around the
vent thereby causing the cloaca to evert and the oviduct to protrude so that a syringe was
inserted into the oviduct and the appropriate amount of semen delivered.
Semen was pooled from donor males in 10ml beakers and maintained warm on an improvised
incubation kit throughout the insemination process. All syringes were equally maintained
warm on the incubation Kit. Freshly collected undiluted pooled semen was drawn with a 1ml
syringe and deposited into the vagina of the female at the depth of 2cm within less than 5
minutes of semen collection. As the semen was expelled into the vagina, pressure around the
vent was released which assisted the hen in retaining sperm in the vagina or the oviduct.
Rough treatment of hens was avoided during capture before insemination and each hen was
released gently after insemination to prevent semen regurgitating from the vagina which may
result in lowered fertility as described by Macpherson et al. (1977). Inseminating the hens
was done repeatedly after every one week with 0.25ml of undiluted semen.
Prior to semen collection and artificial insemination, the toms and hens were kept off feed
and water for six hours to minimize contamination of semen with faeces and urates
(www.hybridturkeys.com, 2010). Other precautions ensured during the insemination process
were as follows:
•
The exteriorized vents of the hens were not touched with bare hands to avoid
contamination;
•
Syringes used for insemination were only used once;
•
Contaminated semen was not used for insemination; and,
•
The vents of toms and hens were properly cleaned with cotton wool moistened with
physiological saline before semen collection and insemination.
3.6.2
Egg collection, storage and hatchability
Egg collection and storage started after the hen have started laying and have received second
insemination. Eggs collected on a daily basis were labelled using a marker on collection to
indicate each treatment. Eggs were saved within the experimental house in egg crates at room
temperature. After seven days of collection, eggs were sorted out to remove cracks, extra
50
small and large ones. Whole eggs with acceptable size were then transported to a commercial
hatchery in Nsukka and set in trays for incubation at 58-60% RH and 370C. Candling was
carried out on the 14th day of incubation. Eggs started hatching late on the twenty-seventh
day of incubation. Unhatched eggs were opened and causes identified as early or late
embryonic mortality, and classified as dead-in-shell embryos.
Fertility was calculated using the expression:
%
=
× 100
Hatchability and embryonic mortality were obtained as follows:
%
%%
ℎ "
#
=
& − ( − )ℎ
*"
$
$
× 100
+, =
-
.
. $
#
× 100
3.10 Experimental Design
3.10.1 Statistical analysis
Data obtained was analyzed using one-way analysis of variance (ANOVA) in accordance
with a completely randomized design (CRD) using computer statistical package (SPSS,
2009). Significant differences in the treatment means were separated using Duncan’s
procedure and accepted at 5 or 1% level of probability. The statistical models for the various
trials are given below;
Data collected was analyzed using 3x3 factorial in CRD. The statistical model for the design
is as follow:
/012 = 3 + 50 + 61 + 756801 + 9012
Where, A, and B represent the two factors (treatments)
Xijk = overall observation on effects of the treatments
µ = population mean or overall mean
Ai = Effects of Moringa oleifera on the ith toms
Bj = Effects of Gongronema latifolium on the jth toms
(AB)ij = effects of the M. oleifera + G. latifolium on the ith and jth toms
∑ijk = Experimental or random error.
51
CHAPTER FOUR
RESULTS AND DISCUSSION
The results of the effects of treatments on body weight, semen quality parameters, and egg
fertility and hatchability of local turkey toms fed diets supplemented with M. oleifera and G.
latifolium are presented and discuss accordingly:
7.00
6.00
T1
5.00
Body weight, kg
T2
T3
4.00
T4
T5
3.00
T6
T7
T8
2.00
T9
1.00
0.00
week
Fig. 3. Effect of treatments on body weights of Turkeys across weeks
:1 = ; , & ,, :2 = 1.5@ABC, :3 = 3kgM, T4 = 1.5kgGL, T5 = 3kgGL, T6 = 1.5kgMO +
1.5kgGL, T7 = 3kgMO + 1.5kgGL, T8 = 1.5kgMO + 3kgGL, T9 = 3kgMO + 3kgGLT1.
4.1 Effects of M. oleifera and G. latifolium on Body Weight (kg)
Figure 3 shows that M. oleifera, G. latifolium diets at various levels of inclusion had no
negative effects (P>0.05) on the daily increase in body weight of treated toms. Toms fed
supplemented diet at various levels of inclusions had statistically the same body weight
increase with toms in the control group, except at the second week of experimental treatment,
where toms fed 3kgMO had a significant (P < 0.05) increase in body weight when compared
to other treatments combinations and the control group. However, there was no indication of
weight loss in the treated toms administered with either of the herbs (M. oleifera and G.
latifolium) or their combined treatment as shown in figure 2. The result of this study concur
with the report of Ekaluo et al. (2011) who observed that all the rats treated in his study had
52
general increase in body weights (both treatment and control groups during the treatment
period). The general increase in body weights of the toms indicated that the treatment had no
adverse effect on growth and body weight of the toms. Body weight may be a good indicator
of semen volume and semen concentration in some cockerel breeds. Generally, poultry
breeds with heavier body weight have been found to have larger testes and produce more
sperm cell during spermatogenesis and thus resulting in a higher semen concentration
(Adeyemo et al., 2007).
The mean ± MSE values of semen quality evaluation of ejaculates from Toms fed diets
supplemented with Moringa oleifera and those of the control are presented in Table7 and 8.
Table 8: The Effects Moringa oleifera on Semen Characteristics of Turkey Toms
Treatment
CS
SV (ml)
PM (%)
SC(x109/ml)
LS (%)
DS (%)
NS (%)
ABS (%)
0kg
1.00
0.39a
81.31a
3.78a
87.73a
12.27c
82.33a
17.67b
1.5kgMO
1.00
0.49a
87.93b
4.11ab
89.58b
10.42b
84.90a
15.10b
3kgMO
1.00
0.58b
92.59c
4.82b
94.13c
5.87a
91.38b
8.62a
SEM
0.04 NS 0.05
1.57
0.09
0.68
0.63
0.12
Means within the same row with different superscripts are significantly different; *=P<0.05
Keys: NS= not significant CS= semen colour, SV= semen volume, PM= progressive motility, SC=
sperm concentration, LS= live sperm, DS= dead sperm, NS= normal sperm and ABS= abnormal
sperm.
0.12
4.1.1 Effects of M. oleifera supplementation on Semen Colour and Volume
The result indicates that semen colour of toms fed diets contain M. oleifera did not differ
significantly (P>0.05) with those of the control group. The ejaculates from M. oleifera treated
toms and the control group had the same white creamy colour and vicious semen, indicating
good quality semen containing a lot of spermatozoa. This result slightly contradicts the report
of Fatoba et al.(2013) who observed that semen colour of rats in control group (5.0 ml saline)
and low dose (5.0 ml) of the Moringa extract, produced white colour semen, whereas, higher
doses (10.0 ml, 15.0 ml and 20.0 ml) of the extract produced milky (good semen colour)
semen in albino rats. A typical watery whitish color is thought to be resulting from semen
oxidation due to prostate secretions (Ali, 2002).
The ejaculate volume of toms fed diet containing M. oleifera (3kg) did differs significantly
(P<0.05) with those of 1.5kgMO and the control group. The value for semen volume of Toms
fed 3kgMO diets significant increase to 0.58ml, compared with 0.49ml (1.5kgMO) and
0.37ml for toms in the control which are statistically the same. The value recorded in this
53
study for tom in the control group were higher than 0.16 and 0.17ml reported by (Oleyi et al.,
1997), 0.17ml (Zaharaddeen et al., 2005), 0.35ml (Nwachukwu et al., 2006) 0.18ml (Ngu et
al., 2014) for indigenous local toms. It is slightly lower when compared with 0.53ml, 0.44ml
and 0.36ml recorded for exotic breeds (Kotłowska et al., 2005). However, the increase in the
semen volume recorded (0.49ml and 0.58ml) in this study for treated toms could be linked
with the ability of the dietary supplement to induce sperm production. This suggest that M.
oleifera leaf meal might have enhanced the development and activity of the seminiferous
tubules and the interstitial cells of toms due to administration of significant amount of the
herb and it possible role in enhancing hormone functions. This is supported by the reported
Adaikan and Ngu (2000) who suggested that M. oleifera induce sperm production which may
be due to relative increases in testes and epididymal weights (Cajuday and Pocsidio, 2010).
The increases in testes weight signify high sperm production due to increase in number of
sperm producing cells and consequently increase in the number of enlongated spermatid in
the seminiferous tubules of treated animal (Gonzales et al., 2001). The increase in semen
volume with higher dosage of M. oleifera (3kgMO) recorded in this study agrees with the
findings of Fatoba et al. (2013) who recorded significant increase in semen volume of albino
rats treated with Moringa root extract. Upendra et al. (2000) observed the effects of herbs on
male broiler breeders, and reported significantly higher semen volume per ejaculate and other
semen quality traits in male breeder broiler fed diets supplemented with herbal (SPEMAN
forte VET) formulation consisting mainly of Argyreia speciosa, Tribulus terrestris,
Leptadenia reticulate, Crocus sativus, Anacyclus pyrethrum, Withania somnifera and
Asteracantha longifolia (Muzumdar, 1999; Nadakarni, 1993), compared to the control. Ezike
et al. (2010) observed an increase in semen volume in toms under free range, suggesting that
forage picked by toms contained different beneficial phytochemicals which may have helped
in improving semen volume and viscosity in turkey male breeder. Some phytochemicals exert
beneficial effects on gametogenic and androgenic functions of testes, it also acts as nerve
tonic, regulating neurohomornal functions, while stimulating the activity of seminiferous
tubules (Upendra et al., 2000)
4.1.2 Effects of Moringa oleifera on Progressive Motility
Percentage progressive motility of M. oleifera supplemented toms were significantly
(P<0.005) different from those of the control group. Dietary administration of 1.5kg and
3kgMO in diet of turkey toms significantly increased percent progressive sperm motility.
Noteworthy, is the dose dependent increase from 87.93% to 92.59% in sperm motility with
54
higher dose of M. oleifera 1.5kg and 3kg respectively, when compared to 81.31% for the
control groups. This result highlights the positive effects of M. oleifera in enhancing sperm
motility by providing the substrate (ATP) needed for motility. This observation is in
agreement with the report of Fatoba et al. (2013) who recorded significant higher sperm
motility in albino rats treated with increase doses of Moringa root extract. And also, the
earlier findings of Upendra et al. (2000) who studied the effect of herbal preparation
(SPEMAN forte VET) in male breeder broiler and observed a significant increase in sperm
motility when compared with the control. In a similar study Machebe et al. (2013) reported
significant increased (89.61%, 94.14%, 85.75% and 93.50%) in sperm motility of tom treated
with plants root extracts, which are all natural sources of antioxidants. The values recorded in
the treated toms were higher when compared to 75.39% and 77.15% for synthetic sources of
vitamin C and E as reported by Oleye et al. (2007). While, value recorded (71.31%) for toms
in the control group was slightly lower than 84.23% and 83.47% reported by Zaharadden et
al. (2005) and 81.27% reported by (Ngu et al., 2014) for same indigenous breeds of turkey
and 90.8% for exotic turkeys (Holsberger et al., 1998). Sperm count, motility and
morphology are key indices of male fertility as these are prime markers of testicular
spermatogenesis (Morakinyo et al., 2008). Plant materials contains great amount of beneficial
phytochemicals, anti-oxidants, vitamins and minerals which are known to increase growth
and stimulate reproduction in humans and animals (Nwangwa et al., 2007; Machebe et al.,
2011).
4.1.3 Effects of Moringa oleifera on Sperm Concentration
Mean values of sperm concentration was significantly (P <0.05) affected by the dietary
inclusions of M. oleifera in the diet of turkey toms. Table 8 shows that sperm concentration is
higher at 3kgMO (4.82 x109) than 3.78x109 for the control toms, but statistically similar to
lower level of inclusion (1.5kg MO). The mean values obtained in this study are in agreement
with those reported by Nwachukwu et al. (2006). However, the concentration of spermatozoa
in the M. oleifera treated toms was higher than 1.73±0.18x108sperm/ml reported by Ngu et
al. (2014), 9.96 – 14.19x106/ml. (Machebe et al., 2013) for Indigenous turkeys and 3.23 -4.8
billion sperm/ml semen in Indigenous cocks (Ajayi et al., 1995). Again, when compared
with sperm concentration of the exotic breeds of turkeys, the range of 6.30 to 7.02×109/ml
(Kotłowska et al., 2005) and 8.3×109/ml (Neuman et al., 2002) were quite higher than the
values recorded in this study for the indigenous breeds of turkey.
55
Ejaculates with low sperm concentration have been associated with low fertility (Bearden et
al., 2004). The higher sperm concentrations recorded in M. oleifera treated toms suggest that
testicular development and proper hormone balance were triggered by treatment with M.
oleifera leave meal. This suggestion is supported by the report of Saalu et al. (2011) which
showed that rats treated with M. oleifera leaves extract showed normal seminiferous
epithelium and high spermatozoa production. Cabacungan. (2008) showed that a steady diet
of Moringa fruit boosted the sperm count of men thus, improving the chance of fertilizing an
egg. Also Serrano (2008) reported an increase in sperm count in male mice administered with
1% concentration of Moringa ethanol extract. In the same trend, Cajuday and Pocsidio (2010)
recorded a significantly higher relative testes weight, larger diameters of seminiferous tubules
and a relatively mature body weight in all the Moringa treated Mice than the control. It
appears that the antioxidants present in M. oleifera leaves further preserved and enhanced the
process of spermatogenesis. Numerous reports suggest the elevation of varying detoxification
and antioxidant enzymes and biomarkers as a result of treatment with Moringa or
phytochemicals isolated from it (Kumar and Pari 2003).
4.1.4 Effects of Moringa oleifera on Sperm Viability (Live/Dead)
Table 8 present experimental result comparing percentage live and dead sperm cells between
groups of toms fed diets with and without M. oleifera. The result showed that percentage live
sperm were significantly higher (P < 0.05) in the treated toms (89.58±0.68% and
94.13±0.68%) at 1.5kgMO and 3kgMO respectively, compared with 87.73±0.63% in the
control group. The study indicates percent values of 12.27±0.68%, 10.42±0.68% and
5.87±0.63% for dead sperm cells in 0kg (control), 1.5kgMO and 3kgMO respectively. This
result confirm earlier findings of Fatoba et al. (2013) on Moringa root extract treated rats
having higher mass activity and sperm cell livability than the control. However, the present
study observed a persistent dose dependent but moderate increase in percent spermatozoa
viability from the toms fed diets containing 1.5kg and 3kgMO. Consequently, toms under the
control diet contained higher percentages of nonviable spermatozoa than the treated toms,
leading to the conclusion that sperm viability in turkeys is at least, partly influenced by the
amount of supplementation of natural antioxidant source. This result is in conformity with the
report of Oleye et al (1997) who recorded an increase in percentage live sperm from 65.74%
to 78.52% and a corresponding decrease in percent dead sperm cells from 13.79% to 12.46%
in the control and 125mg/kg vitamin E supplementation. Correspondingly, Vitamin C
supplementation to toms under the same study had the highest percentage live sperm 81.01%
56
and a lower value for dead sperm cells 8.73% compared with the control values of 63.25%
and 17.52% for live /dead sperm cells. Multinucleated giant cells (MCG) an indication of
degeneration were observed in testes of control breeder turkey which were absent in the
group treated with dietary supplementation of ascorbic acid (Neuman et al., 2002). Zanboni
et al. (2006) believed that the effect of vitamin E is likely due to its powerful antioxidant
activity, being able to quench oxygen radicals in fat soluble matrices. It is also likely that
vitamin E supplementation facilitated effective spermatocytogenesis while playing its
antioxidant role at the sites of sperm formation in the testis of supplemented toms.
In this study, the protective effects of Moringa leaf may be attributed to the presence of
phytoconstituents (polyphenols, tannins, anthocyanin, glycosides, thiocarbamates) that
scavenge free radicals, activate the antioxidant enzymes, and inhibit oxidases (Liu
2006, Amin 2005). The phenolics present in Moringa fruit extract are able to terminate the
radical chain reaction by converting free radicals to more stable products, in addition to the
phenolics, which could serve as antioxidants and may effectively scavenge various reactive
oxygen species and free radicals under in vivo conditions. The aqueous extract
of Moringa leaves contains certain nonphenolic, biologically active components such as
selenium, thiocarbamates, glucosinolates, and their hydrolysis products such as
glucoraphanin, isothiocyanate sulforaphane, nitriles (Faizi et al., 1994). The use of lycopene
as a dietary supplement has also been reported to improve the viability of poultry
spermatozoa and native immunity of the birds (Mangiagalli et al., 2010).
Figure 4: Viability and morphological examination (Stained spermatozoa)
4.1.5 Effects of Moringa oleifera on Sperm Morphology
Percentage normal and abnormal sperm cells of turkey toms under study were significantly
(P<0.05) different. The result highlights the positive effects of M. oleifera (3kg) on
57
percentage normal sperm (91.38%±0.12) compared with control (82.33%±0.12) which is
statistically similar to 84.90%±0.12 for 1.5kgMO. Consequently, a proportional decrease was
recorded for percent abnormal sperm to high dose of M. oleifera (8.62±0.12%), while the
control toms had the highest percent abnormal sperm cells (17.67%±0.12).
Percent
morphologically intact sperm recorded in this study were in consistent with acceptable range
of (80-90%) reported by Bearden et al. (2004) for turkey sperm.
Sperm morphology is an indicator of some disorders in spermatogenesis. According to
Anderson (2001), partial or complete degeneration of the sperm tubules may result to high
production of abnormal spermatozoa thereby reducing the proportion of normal spermatozoa.
However, increase in abnormal sperm can also be attributed to aging of spermatozoa resulting
in loss of membrane integrity following peroxidation in the vas deferens (Noirault and
Brillard, 1995). Alkan et al. (2001) attributed sperm abnormalities to it relatively long and
slender mid-piece of chicken sperm cell which makes it vulnerable to damage. Increase in
percentage abnormal sperm impairs fertility of breeder flock (Thatohatsi, 2009). Here, M.
oleifera contain fundamental antioxidant and phenolic compounds that helps in protecting the
testis against morphologic, spermatogenic and oxidative changes brought about by toxic
materials and certain antineoplastic agents (Siddhuraju and Becker, 2003; Saalu et al., 2011).
It also contains Vitamin C which is an anti-oxidant/anti-stress and has a counteracting effect
on heat stress that may exert a degenerating action on sperm production of avian species
(McDanniels et al., 2004). M. oleifera leaf extract pretreatment has been shown to shield
testes from a variety of toxic substances (Stohs and Bagchi, 1994).
Table 9 summarized the effects of G. latifolium on semen quality parameters of local toms as
presented below:
Table 9: The Effects of Gongronema latifolium on Semen Characteristics of Turkey Toms
Treatment
CS
0kg
1.00
1.5kgGL
SV (ml)
a
b
PM (%)
0.43
88.42
1.47 b
0.38ab
3kgGL
1.47b
SEM
0.04
b
SC(x109/ml)
b
LS (%)
b
DS (%)
a
NS (%)
80.89
ABS (%)
c
19.11a
3.65
90.69
9.31
71.80a
2.41a
88.91a
11.09b
76.44b
23.56b
0.28a
71.22a
2.21a
87.S57a
12.43b
75.78a
24.22b
0.05
1.57
0.09
0.68
0.63
0.12
0.12
Means within the same row with different superscripts are significantly different. *=P<0.05
Keys: NS= not significant CS=semen colour, SV=semen volume, PM=progressive motility,
SC=sperm concentration, LS=live sperm, DS=dead sperm, NS=normal sperm and ABS=abnormal
sperm.
58
4.2.1 Effects of Gongronema latifolium on Semen Colour and Volume
The result shows significant decrease (P<0.05) in the semen color and sperm volume in the
G. latifolium treated groups of toms when compared with the control group as shown in Table
8. The semen colour of the control group (1.00) were creamy white and vicious semen,
whereas, toms fed diets at low(1.5kg) and higher doses of 3kgGL produced watery, less
vicious, clear semen (1.47). The ejaculate volume produced by toms fed diets without G.
latifolium (control) had a mean value of 0.43±0.06ml which was slightly higher than 0.35ml
reported by Nwachukwu et al.(2006), and also higher than 0.18ml (Ngu et al., 2014) for
indigenous breeds of toms. The ejaculate volume recorded in the control group (0.43ml) was
higher than the accepted range of 0.25-0.35ml reported by Barsk (1990) but within range
(0.28ml) in the treated tom. The reduction in semen colour and volume in G. latifolium
treated toms evidently suggested negative effect of G. latifolium on the semen colour and
volume. This appears to be true, because colour of semen can present different shades as
influenced by different factors (Mann and Lutwak-Mann, 1981) including type of feed,
contamination with urine, breed of animal etc. These observations were in agreement with the
report of Upendra et al. (2000) who studied the effects of these herbs (SPEMAN forte VET)
in the diet of male breeder broiler and observed a significant increase in seminal fluid
viscosity when compared to the control. In support of this finding, Ugonna (2013) and
Ikpeme et al. (2012) also reported a significant reduction in semen quality in rats treated with
G. latifolium, especially at higher dose and longer duration. The reduction in some semen
quality parameters was earlier observed when plants associated with antimalaria properties
are used (Ezeonwu et al., 2013; Oze et al., 2007 and Ugonna, 2013), such plant with
antifertility properties usually result in impairment of male fertility. Chemical composition of
G.latifolium shows that it contains among others substance alkaloid up to 9.10% and
cyanogenic glycosides (poisonous gas) may be present at a low level (0.02 mg/100g). These
are chemicals linked with adverse effects on semen production. Also, Russell et al. (1981)
reported that nicotine contains 90-95% of the total alkaloids have been reported to cause
decrease in weight of reproductive organ, causes testicular degeneration, disorganization of
the testicular cytoarchitecture and decreased serum testosterone level in animal treated with
it (Oyeyipo et al., 2010).
4.2.2 Effects of Gongronema latifolium on Progressive Motility
Dietary administration of G. latifolium in the diets of toms had significant (P < 0.05) effects
on percentage progressive sperm motility when compared with the control groups. The result
59
shows that toms treated with 1.5kgGL and 3kgGL are statistically similar but different from
the control group. However, it was observed that percent progressive motility reduced
significantly from 88.42% recorded for the control to 71.80% and 71.22% at 1.5kg and 3kg
inclusions of G. latifolium respectively. Note, the control group maintained moderate motility
value of 88.42%, but even at lower dose of 1.5kgGL, G. latifolium significantly reduced
sperm motility which could be attributed to prolonged treatment. Ikpeme et al. (2012)
reported a mean value 76.82+ 0.05% for rats treated with G. latifolium, which is slightly
higher than what was obtained for this study.
Although, it appear that there is yet no report on the effect of G. latifolium on semen
characteristic in male turkey, report on male albino rats treated with G. latifolium showed that
it causes reduction in sperm motility (Ikpeme et al., 2012; Ugonna, 2013; Oyeyipo et al.
2014) as well as in other semen quality characteristics. Also Oyeyipo et al. (2014) observed
decreased sperm count, motility and normal sperm morphology of treated rats which
demonstrates that nicotine (95% Alkaloid) impair some semen qualities. According to
Oyeyipo et al. (2011) reduction in sperm motility is said to be associated with an impairment
of spermatogenesis consequent to reduction of testosterone secretion caused by the nicotine.
4.2.3 Effects of Gongronema latifolium on Sperm Concentration
There was visible reductions (P <0.05) in the sperm concentration with G. latifolium
administration. Table 9 indicates that G. latifolium had the same effects of decreasing sperm
concentration (2.21x109/ml and 2.41x109/ml) at higher and low doses respectively, when
compare with the control group which had a higher mean value of 3.65x109 /ml. The sperm
concentration value obtained in this study for the control group is reasonable when compare
with 3.91x109/ml reported for chicken (Modupe et al., 2013) but higher than 2.81 x109 /ml in
local toms and 4.66 x109 /ml for exotics turkeys as reported by (Zaharaddeen et al., 2005).
The significant reduction observed in sperm concentrations of the treated toms may be
associated with the impairment of spermatogenesis consequent to reduction of testosterone
secretion caused by G. latifolium earlier suggested by Oyeyipo et al. (2007). This also agrees
with the report on G. latifolium induced reduction in sperm concentration of rats administer
G. latifolium especially at higher doses (Ikpeme et al., 2012; Ugonna, 2013). Such adverse
effects of induced impairment of spermatogenesis on sperm are associated with plant that has
anti-malaria properties and appreciable level of alkaloids (Aydos et al., 2001; Raji et al.,
2003; Oyeyipo et al., 2011 and Ezeonwu et al., 2013).
60
4.2.4 Effects of Gongronema latifolium on Sperm Viability (Live/Dead Ratio)
The effects of G. latifolium leaves meal on sperm viability shows significant (P < 0.05)
differences in the group fed G. latifolium leaves meal (Table 9) and the control group. The
result show that G. latifolium fed toms, had a significant reductions in percent live sperm
from 90.63± 0.68% in the control group to 87.13±0.68% and 85.47±0.68% in 1.5kgGL and
3kgGL inclusions respectively. Statistically, 1.5kgGL and 3kgGL values for percent viability
are the same, but different from the control. Noteworthy, is the significantly decreased, with a
concomitant increased in the proportion of dead spermatozoa in G. latifolium treated toms,
especially at higher doses. This result is in agreement with the report of Ugonna (2013) who
described a dose dependent decrease (control, 300mg/kg GL and 500mg/kg GL) in
percentage live sperm (91±7.53%, 60±11.75% and 59±0.35%) respectively on administration
of G. latifolium leave extract to male rats, and suggested that prolonged treatment of animals
with aqueous extract of O. gratissimum or G. latifolium could significantly alter the fertility
potential of male animal. Similarly, the adverse effects of G. latifolium on sperm cells
viability compares favourably with the reports of Ikpeme et al. (2012) who reported that
Sperm viability was significantly reduced, especially at higher doses on administration of G
latifolium extract in albino rats in comparison with the control. In another study, the effects of
ethanol extract of Azadirachta indica stem bark on sperm viability causes significant dosedependent decreases in weights of the testis, epididymis and seminal vesicles but an increase
in that of the adrenal gland (Raji et al., 2003) of the treated rats.
The result of this study revealed that treatment with G latifolium causes reduction of percent
live sperm, with severe decreases as the level of the leaves meal (3kgGL) increased in the
diet of toms. This reduction in the percentage live sperm suggests that the G latifolium are
effective in disrupting spermatogenic processes and pathways by causing reproductive
endocrine malfunction in the treated male (Raji et al., 2003). Ekaluo et al. (2009) suggested
that this decrease is an indication of the increase in the rate of induced mutation on the sperm
cells during spermatogenesis.
4.2.5 Effects of Gongronema latifolium on Sperm Morphology
Table 9 compares sperm cells morphology of toms in the treated groups and the control.
Sperm cells abnormalities were significantly (P<0.05) higher in the G. latifolium treated
groups, especially at higher doses of G. latifolium diets, when compares with the control.
However, percentage normal and abnormal spermatozoa are statistically the same in 1.5kGL
and 3kgGL levels of inclusions, but different from the control. Sperm abnormalities were
61
inversely proportional (lower) to normal sperm (higher) in the control group but increased
across the treatment levels (1.5kg and 3kg) GL. This result revealed that despite its wealth of
beneficial nutrients, supplementation of G. latifolium leaves meal in the diets of turkey toms
might adversely affects sperm morphology. This was observed from the results which
showed that percentage normal sperm decreased from 80.38±0.12% in the control group to
78.37±0.12% and 75.33±0.12% in G. latifolium treated groups.
This finding was also recorded by Ikpeme et al. (2012) who reported that abnormal sperm
morphology was significantly increased as the concentration of both P. biglobosa and G.
latifolium extracts increased in the treated rats. In a similar study, Raji et al. (2003)
administered Azadirachta stem bark extract to male rats, and sperm morphology was
adversely affected in the Azadirachta extract treated rats which were unable to impregnate
female rats throughout the duration of the study. However, these female rats conceived and
littered physically normal litters about four weeks after cohabitation with untreated male rats.
Premature sperm and onset of multinucleated giant cells (MCG) an indication of degeneration
where observed in testes of chicken fed Neem seed kernel meal and absent in the control
(Mohan et al., 1997). According to Ikpeme et al. (2010) and Glover and Assinder (2006)
distortion in the fertility of male mammals is directly correlated with the distortion in
spermatogenesis. In addition, the observed increase in percentage of sperm head abnormality
and subsequent reduction in sperm count may have resulted from the alteration in the
epididymal environment as was reported by (Ekaluo et al., 2009). In a similar development
(Nwanjo et al., 2007), asserted that this increase in percentage abnormalities is an indication
of the increase in the rate of induced mutation on the sperm cells at the level of
spermatogenesis.
The effects of combinations of M. oleifera and G. latifolium inclusions in the diets of local
toms on the ejaculate characteristics are presented below:
62
Table 10 Combined Effects of M. oleifera and G. latifolium on Semen quality Traits of Toms.
Parameters
CS
A
1.00
B
1.00
Treatment
C
D
1.00
1.00
SV (ml)
PM (%)
0.41b
81.20b
0.33d
83.13b
0.55a
87.93a
0.30e
72.47c
0.38c
88.87a
0.08
2.12
SC (x 109/ml)
LS (%)
DS
3.33 c
89.28a
10.72b
3.29d
87.58b
12.42a
4.28 a
89.87a
10.13b
3.35c
86.93b
13.07a
3.84b
89.79a
10.21b
0.16
1.12
1.09
NS
84.10 b
82.97 b
89.00a
76.10c
84.67 b
1.21
c
b
d
a
ABS
15.90
17.03
11.00
23.90
E
1.00
SEM
0.06 NS
15.33
c
0.20
Means within the same row with different superscripts are significantly different. *=P<0.05,
NS= Not significant, MO= Moringa oleifera and GL= Gongronema latifolium
A= Control, B = 1.5kgMO+1.5kgGL, C= 3kgMO+1.5kgGL, D= 1.5kgMO+3kgGL, E
=3kgMO+3kgGL,
CS= semen colour, SV=semen volume, PM=progressive motility, SC=sperm concentration, LS=live
sperm, DS=dead sperm, NS=normal sperm and ABS=abnormal sperm.
4.3.1 Combined Effects of M. oleifera and G. latifolium on Semen colour and Volume
The result indicates that Toms fed combined diets of M. oleifera and G. latifolium at different
levels
of inclusion produced ejaculates that are statistically the same in semen colour,
meaning that the combined levels of the two herbs have no significant effect (same shade) on
the semen colour as both the treated and the control toms had same mean value of 1.00. On
the other hand, semen volume is significantly (P<0.05) different. The mean values of the
ejaculated at 3kgMO + 1.5kgGL (0.55ml) was significantly higher than the control and other
ejaculate values from the bi-herbs. Toms in the control group had an ejaculated volume
(0.41ml) far better than the values recorded from toms treated in B, D and E levels of
combination. Combined herbs at 3kgMO+1.5kgGL showed an improvement in semen
volume, when compared with the single treatment with G. latifolium and the control group.
This result revealed that combining M. oleifera and G. latifolium at 3kgMO + 1.5kgGL may
significantly improve semen volume better than control, single dose of G. latifolium and
other inclusion combinations. The improvement in semen quality observed in this study may
be due to the phytochemicals present in both plant and the possibilities of M.oleifera
suppressing induce antifertility effects on treatment with G. latifolium alone. M. oleifera is
known to exert beneficial effects on gametogenic and androgenic functions of testes, it also
acts as nerve tonic, regulating neurohomornal functions, stimulating the activity of
seminiferous tubules. Upendra et al. (2000) observed the effects of herbs in male broiler
63
breeder, and reported a significant higher semen volume in the ejaculate and other semen
qualities when diets were supplemented with herbal preparations (SPERMAN forte VET)
when compared with the control. On the contrary, reduction in semen quality was recorded
with plant associated with antimalaria properties (Ezeonwu, 2011; Oze et al., 2007), which
may have antifertility properties with a resultant impairment of male fertility.
4.3.2 Combined Effects of M. oleifera and G. latifolium on Progressive Motility
The results in Table 10 shows that toms fed combined diets of M. oleifera and G. latifolium at
B, C, D and E levels of inclusions, produced semen with significant (P<0.05) differences in
progressive motility and also different from the control group (A). Statistically, treatment C
and E are the same but different from A and B which are equally the same but different from
treatment D. The highest values were recorded in C and E which showed an improvement
due to the combined treatment when compared with the control. Treatment D had the lowest
mean value (72.47%) for progressive motility indicating a probable adverse effect of G.
latifolium and that M. oleifera inclusion at lower dose could not mask this adverse effect.
This finding revealed that supplementing turkey breeder toms with combined formulas of
these herbs with G. latifolium at higher dose may negatively affect sperm progressive
motility. It was also observed that the combinations of M. oleifera and G. latifolium with G.
latifolium at a high dose significantly showed a dose dependent reduction in sperm motility.
But the means values of 87.93% and 88.87% recorded suggested that M. oleifera might have
suppressed the deleterious properties of G. latifolium herb as evidenced in the improved
percentage motility recorded for the combined levels at higher dose of M. oleifera (3kgMO
+1.5kgGL and 3kgMO+3kgGL) when compared with the combinations at higher doses of G.
latifolium. Thus, it can be infer that M. oleifera inclusion at that level might have offer
protection against the negative effects on reproduction and oxidative stress caused by G.
latifolium, which may be attributed to the high antioxidant and androgenic properties of M.
oleifera.
This result is in consonance with the reports of Ekaluo et al. (2011); Raji et al. (2003) and
Ezeonwu et al. (2011) who pointed out that plant with higher alkaloid and glycoside content
have high toxicity and may exert antifertility effects on the treated animal. A significant
decreased in the percentage motile sperm (72.47) recorded in this study for treatment D is
lower than 76.82+ 0.05% reported by Ikpeme et al., (2012), but higher than 43+9.35%
recorded in albino rats treated with bi-herbs G. latifolium and Ocimum grassimum (Ugonna,
64
2013). The observed increase in progressive motility was in line with the reports of Aslam et
al. (2005), Amaglo et al. (2010) and Gowrishankar et al. (2010) who reported a correlation
between the flavonoids and sperm production, they also noted that Moringa inhibited 6-betahydroxylation of testosterone thereby producing an androgenic effects by enhancing sexual
drive through increased serum and testicular testosterone levels (Cajuday and Pocsidio,
2010).
4.3.3 Combined Effects of M. oleifera and G. latifolium on Sperm Concentration
The mean values for sperm concentration of toms fed diets containing combined levels of
M.oleifera and G. latifolium was significantly different (P<0.05). However, the mean value
for sperm concentration of treatment C is higher (4.28 x 109/ml) and statistically different
from, B and E which are also different, but treatment A and D were statistically the same.
Treatment C with the highest value of 4.28 x 109/ml suggest a conspicuous effect of the
higher dose of M. oleifera and lower dose of G. latifolium in enhancing sperm concentration
of the toms. Furthermore, treatment E showed a reasonable improvement compared to the
control group, suggesting that M. oleifera might have assisted in reducing the adverse effects
of G. latifolium. The sperm concentrations in the treated toms recoded in this study were
higher than 1.73±0.18x108sperm/ml reported (Ngu et al., 2014) in Indigenous turkeys but
concur with 3.23-4.8 billion sperm/ml semen in Indigenous cocks (Ajayi et al., 1995). When
compared with the sperm concentration in exotic turkeys, the range of 6.30 to 7.02
(×109sperm/ml) reported by Kotłowska et al. (2005) was quite higher than the values
obtained for the local indigenous breeds. In agreement with the findings of this study, Ikpeme
et al. (2012) reported a reduction in sperm count in rats administered a high dose of G.
latifolium extract combined with P. biglobosa which was attributed to disruption in
spermatogenic processes, and alteration in the epididymal environment as reported by
Nwanjo et al. (2007) and Ekaluo et al. (2009).
4.3.4 Combined Effects of M. oleifera and G. latifolium on Sperm Viability
Sperm viability was significantly (P<0.05) different among treatments. Percentage normal
sperm were significantly higher and statistically the same in treatment A, C and E, (89.28,
89.87 and 89.79) % respectively. While, treatments B and D values for percent normal sperm
(86.93 and 87.87) % respectively was lower and similar. When comparing percent live sperm
cells in the different treatment combinations with the value recorded for the control group, it
can be inferred from the result obtained that toms on combined treatment at
3kgMO+1.5kgGL and 3kgMO+3kgGL slightly improved percent normal sperm cell better
65
than other inclusion and the control. M. oleifera is known to exerting superior effects on the
combination, considering their respective physiologic roles as powerful anti-oxidants.
Consequently, the results of the effects of these two herbs on percentage dead sperm cells
shows that statistically, treatment A, C and E had similar treatment effect, meaning that the
levels of inclusion of these herbal combinations had the same positive effects of reducing
percent dead sperm in the treated toms equal to the control. However, treatments B and D are
statistically equal with increased percent dead spermatozoa 12.42% and 13.07% respectively.
The present results revealed that in line with documented evidence on the antioxidant potency
of these herbs, percent live spermatozoa were less improved with their combinations. Percent
live sperm and the corresponding decreased in percent dead sperm in the control group
yielded equal result as in 3kgMO+1.5kgGL and 3kgMO+3kGL supplemented turkey toms.
While, toms fed diets supplemented with 1.5kgMO+1.5kgGL and 1.5kgMO+3kgGL
combination had are significant reductions in percent live sperm and a corresponding increase
in percent dead sperm. This finding concur with the findings of Ugonna et al. (2013) who
noted that at higher dose of treatment with G. latifolium, sperm viability was not affected.
Zanboni et al. (2006) reported that the positive effect of vitamin E was likely due to its
powerful antioxidant activity, being able to quench free radicals. G. latifolium had an adverse
effect on sperm viability when used alone and at higher inclusion level (combined) in the
diets of toms. This could probably be associated with hormone imbalance and or decrease in
serum testosterone level, because high level of serum testosterone has been associated with
increased sexual, physical and mental energy, vitality and sex drive (Yamamoto et al., 1998).
Ugonna (2013) confirmed a dose dependent decrease FSH, LH and testosterone in rats treated
with G. latifolium.
4.3.5 Combined Effects of M. oleifera and G. latifolium on Sperm Morphology
Percent sperm morphology shows significant (P<0.05) deviate in the treated groups and
value obtained from the control group. Table 10 shows that treatment A, B, and E are
statistically the same and different from treatment C and D. Treatment C (3kgMO+1.5kgGL)
had a significant increase in percent normal sperm with a decrease in abnormal sperm
(11.00%) when compare with the control value (15.90%). However, combination at
1.5kgMO+3kgGL had a significant increase (23.90%) in abnormal sperm with decrease
percent normal spermatozoa (76.10%). Abnormal sperm in treatments A and E are
statistically similar but different from treatment B, C and D. Turkey toms supplemented with
combination of M. oleifera and G. latifolium in treatment had the same effects with that of the
66
control, implies that the levels of inclusion of these herbs may be too small to cause any
significant effect in improving sperm morphology, while at higher inclusion of G. latifolium
(1.5kgMO+3kgGL), produced undesirable result on sperm morphology. On the contrary,
3kgMO+1.5kgGL inclusion gave a better result.
According to Anderson (2001), partial or complete degeneration of the sperm tubules may
result to high production of abnormal spermatozoa thereby reducing the proportion of normal
spermatozoa. The significant increases in percent normal sperm with a corresponding
decrease in sperm abnormality recorded at higher inclusions of these herbs is an indication
that the combinations had positive effect on the testicular functions and subsequently reduced
spermatozoa abnormities. The result of the combined treatment revealed that M. oleifera at
3kg inclusion in the combination might have proved protection against induced abnormalities
cause by G. latifolium on the spermatozoa. This observation is supported by the report of
Saalu et al. (2011) who observed that rat’s co-treatment with M. oleifera leaves extract and
Hydroxyurea had their testis protected against the morphologic, spermatogenic and oxidative
status changes induced by Hydroxyurea. Ibukun et al., (2014), on the androgenic activities of
Zingiber officinale extract commonly called ginger Co-administer with nicotine (constitutes
90-95% total alkaloids), AZO (a herbs) clearly ameliorated nicotine-induced infertility and
maintained normal sperm function and fertility. Similar effect was observed by (Nwanjo et
al., 2007). On the contrary, Ugonna et al. (2013) observed an insignificant effect, even at
higher dose of G. latifolium on sperm viability.
Table 11 and 12 summarizes the respective mean values ± SEM of the fertility and hatchability
traits of turkey hens inseminated with semen from toms fed varying levels of M. oleifera and G.
latifolium.
Table 11: Effects of M. oleifera and G. latifolium on Fertility and Hatchability of Turkey Eggs
Parameters
Treatments
Mean ± SEM
Fertility indices
0kg
1.5kg MO
3kg MO
1.5kgGL
3kgGL
Overall Total Eggs
Percent Infertile Eggs
25
19.90+3.17c
25
12.91+0.15b
25
10.33+0.15a
25
21.98+0.25c
25
25.18+0.25d
Percent Fertile Eggs
80.10+6.89c
87.19+3.33 d
89.67+4.79e
78.02+6.76b
74.82+3.43a
Dead-in-shell Embryos (%)
19.20+5.64c
12.67+5.55a
14.33+6.00b
10.72+5.27a
23.32+5.67d
Percent Hatched Eggs
60.32+5.78b
74.52+4.91d
75.35+5.55d
65.25+5.45c
51.52+5.47a
Means within the same row with different superscripts are significantly different. *=P<0.05,
Key: MO= M. oleifera, GL= G. latifolium, 0kg = basal diet
67
4.4.1 Effects of M. oleifera and G. latifolium inclusion on Percent Fertility of toms Semen
The results show significant (P < 0.05) differences among the treatment groups. The highest
percent of infertile eggs were recorded in hens inseminated with semen from toms fed 3kg
GL diet, while eggs of hens inseminated with semen from the toms in the control group had
19.90% infertile eggs. From the result, it can be deducted that at 1.5kg and 3kg M. oleifera
supplementation in the diets of toms used to inseminate turkey hens, the number of infertile
eggs reduced significantly, revealing the effects of M. oleifera in enhancing fertility of tom
spermatozoa. On the other hand, supplementing turkey toms with G. latifolium leaves meal
seems to have adverse effect on the semen quality as revealed in Table 8 consequently, affect
the fertilizing ability of their respective semen. The semen from the control group had a lower
percent infertile eggs (19.90+0.17) when compared to 21.98+0.22 and 25.18+0.22 recorded in
the G. latifolium treated group.
Percentage fertile eggs were significantly (P < 0.05) higher in toms fed 1.5kgMO and 3kgMO
(87.19% and 89.67%) respectively. While, the values of 80.10% recorded for the control and
78.02% for 1.5kgGL are statistically similar, but different from 74.82 %( 3kgGL) which is the
lowest. Fertility values recorded in this study were higher than 58.40% recorded for local
toms as reported by (Ngu et al., 2013), 40.1% by Idi (2000) in chicken. This may be possible
because, excellent fertility can be obtained by AI in many cases than from natural mating.
However, the methods of insemination must be practiced skillfully to obtain good results
(Lake, 2011). The result is also supported by the report of Machebe et al. (2013) who
recorded 50.2%, 87.3%, 51.3% and 94.3% fertility in hens treated with basal diets (control),
okra seed extract, guava root extract; and pumpkin seed extract treated hens respectively.
The reduction in the percent infertile eggs recorded in this study in toms fed M. oleifera may
be attributed to antioxidant properties and nutritional benefits of M. oleifera leaves meal
which is able to preserved the fertilizing ability of the spermatozoa and also protect it from
oxidative damages. However, the increase in the percent of infertile eggs could be associated
with the qualities of the semen which might have cause reduction in the fertility potential of
the spermatozoa. From the findings of this study, it can be deducted that any chemical agent
or substance that can affect reproductive activity will as well affect the semen quality and
quantity thereby reduce the fertility rate of the animal. The reduction in the fertility rate of
turkey semen can be attributed anti-androgenic property of the plant or its extract. This study
affirmed to the dangers of extended administration of G. latifolium leaves meal alone to
turkey toms. On the other hand M. oleifera leaves meal showed improvement in fertility of
68
turkey semen.
This is result is supported by the report of Durape (2007) who used
phytochemicals to increase fertility from 95.1% to 97.3% in broiler breeders’ flock. Upenrda
et al. (2003) reported improved fertility and hatchability of eggs fertilized by semen of broiler
breeder giving herbal supplementation. Similarly, Narahari (2003) suggested that herbal
formulation improves fertility in male breeder. However, infertility record obtained could be
attributed to the quality of the semen used for insemination as well as the physiology of the
hen at the time of the insemination (Keith, 2008).
4.4.2 Effects of M. oleifera and G. latifolium on Percent Dead -in- Shell Embryos
Percentage dead-in-shell embryos were significantly different (P < 0.05) across the treatment
groups. The percent dead-in-shell embryos significantly reduced in eggs fertilized by semen
from tom’s treated with 1.5kgGL and 1.5kgMO which were statistically the same. While the
eggs fertilized by semen from 3kgMO had 14.33% dead- in- shell embryos which are also
fair when compared with 19.20% and 23.32% for control and 3kgGL respectively. The result
of this study shows that lower values of 12.67% 14.33% and 10.72% dead- in-shell embryos
recorded for eggs fertilized by semen from toms supplemented with 1.5kgMO, 3kgMO and
1.5kgGL respectively, contains antioxidant that might have protracted its beneficial effect
from the semen to the developing embryos and consequently reduced percent dead-in-shell
embryo. But the higher values recorded 19.20% and 23.32% dead- in-shell for the control
group and 3kgGL supplemented toms, may be due to the reduced semen quality and higher
percent dead sperm which is strongly associated with embryos sustainability. The percent
dead-in- shell embryos obtained in this study ranges from 10.72% to 23.32%, which compete
favourable with 42.75%, reported for local and 35.16% for exotic breeds of turkey (Ngu et
al., 2013). However, Machebe et al. (2013) reported similar improvement in turkey hens
treated with plants extract (Okra seed, guava root, pumpkin seed,) and lower values of
17.10% 11.14% and 20.15% in percentage dead-in-shell was recorded. G. latifolium treated
toms had slightly higher values 23.32+5.67%, the reason for the higher values may be
referred back to the earlier records of higher percent abnormal sperm recorded under G.
latifolium treated toms.
The result of this study agrees with the findings of Keith (2008) who stated that, even though
it takes a single sperm to fertilize an egg, adequate number of morphologically intact sperm is
required to ensure hatchability. Devegowda (2009) attributed embryonic mortality to when
few sperm were present to fertilize an egg and when there is a decrease in the number of
viable sperm inseminated. According to Bramwell (2002) early embryonic mortality can be
69
as a result of low sperm activity in the individual hen. Peter and Dicman (2006) reported that
if eggs are stored for more than one week after lay, there is an increase occurrence’ of early
embryonic mortality and abnormalities as a result of reduction in egg white viscosity and
degradation of albumen. Furthermore, factors such as poor egg storage, egg size, and age of
the breeder and incubators shortcoming could be the reason for higher embryonic mortality
(Peter and Dicman, 2006).
4.4.3 Effects of M. oleifera and G. latifolium on Percentage Hatched Eggs
Hatchability rate in percent was significantly higher in egg fertilized by semen from toms fed
diet 3kgMO which is statistically the same with those toms fed 1.5kgMO. Moderate values
were recorded in toms fed 1.5kgGL (65.25%) and in the control group (60.32%). However,
egg hatchability in egg lay by hens inseminated with semen from G. latifolium treated toms at
3kgGL had the lowest hatchability rate of 51.52%. Percentage hatched egg values recorded in
this study were moderately lower than the range of 95 -100(%) reported for exotic turkeys
(Keith, 2008), and higher than the range of 22±1.31- 51±2.52% reported by Machebe et al.
(2013) for herbal treated hens. The control group had percentage hatchability of 60.32%
which is higher than the value of 56.25% reported by Ngu et al. (2013) for local toms.
Fertilizing capability level of a quality sperm is usually closely related to embryo survival,
and a survived embryo will hatched. Therefore to ensure high percentage hatchability the
quality of the semen is of paramount important. Thus it can be inferred that supplementing
turkey toms diet with beneficial plant herbs with potent antioxidant can significantly improve
the percentage hatchability of turkey which is the ultimate goal of every breeder. To support
this claims, studies have shown that natural antioxidants, including vitamin E and C,
Selenium, and carotenoids (which is present in M. oleifera) plays vital roles in avian
reproduction by maintaining antioxidant defenses of the spermatozoa and embryonic tissues
(Surai et al., 2006). However, if the production of oxygen radicals exceeds the capacity of the
antioxidants to detoxify them, then the sperm can be irreversibly damaged. Thus, embryos
fertilized by such sperm might be less capable of surviving.
The combined effects of dietary inclusions of M. oleifera and G. latifolium on fertility and
hatchability characteristics of hens inseminated with semen from treated toms is presented in
Table below:
70
Table 12: Combined Effect of M. oleifera and G. latifolium on Fertility and Egg
Hatchability of Turkey Tom’s semen.
Fertility indices
Treatments
B
25
Overall Egg Total
A
25
Percentage Infertility
22.83
24.70
Percentage Fertility
76.17
Dead-Shell-Embryos (%)
Percent Hatchability
Combinations
C
25
D
25
E
25
MSE
13.60
29.75
19.94
0.26NS
75.17
86.39
69.25
80.06
5.68NS
19.98 b
28.06a
16.99c
30.13 a
19.12b
5.36*
76.67c
75.17c
88.39a
66.87 d
83.33b
4.27*
Means within the same row with different superscripts are significantly different. *=P<0.05,
NS= Not significant, LOS= Level of Significances, A=Control, B=1.5kgMO+1.5kgGL, C=
3kgMO+1.5kgGL, D= 1.5kgMO+3kgGL, E =3kgMO+3kgGL
4.5.1 Combined Effects of M. oleifera and G. latifolium on Percentage Fertile and infertile Eggs
The result of this study indicate that there is no significant difference (P > 0.05) in the
combined treatments at various level of inclusions of M. oleifera and G. latifolium and the
control group on percent fertile and infertile eggs. This implies that, statistically, the
treatment combinations at level B, C, D and E had no significant difference from treatment A
in percent fertile and infertile eggs. This result concur with the report of Machebe et al.
(2013) who recorded no significant difference in percentage fertile eggs of hens fed diet
containing herbs when compared with the
control group. However, numerically, the
treatment combinations C and E had higher percent values of 86.39% and 80.6% respectively
better than 76.17% recorded for the control, which shows that higher fertility can be obtained
when toms diets are supplemented with 3kgMO+ 1.5kgGL and 3kgMO+3kgGL.
M. oleifera and G. latifolium are two known powerful antioxidant, but studies have shown
that pronlong treatment with only G. latifolium might exerted adverse effects on male
fertility. Interestingly, co-treatment with M. oleifera seems to suppress the antifertility
properties of GL thereby improving fertility of the toms. According to Pastuszewska et al.,
2006) plants with high alkaloid contents were reported to be responsible for disrupting
hormone functions, to the extent of inhibit gonadothrophin action of the testis and subsequent
impairment of male fertility. Therefore, alkaloids inherent in those levels of inclusions are to
be considered higher enough to cause significant decrease on the fertility of the treated toms.
71
Fig. 5: Hatched live poult and dead in-shell embryos
4.5.2 Combined Effects of M. oleifera and G. latifolium on Percent Dead-in -Shell Embryos
Percentage dead-in-shell embryos were significantly higher in eggs fertilized by toms from
treatment D which is statistically the same with treatment B followed by treatment A
(control) and E which are also statistically and numerically the same. A lowest value of
16.99% was recorded in treatment combination C (3kgMO+1.5kgGL). The findings of this
study showed that the percentage dead- in-shell embryos were slightly lower than the range
20±1.68 to 50±2.36% reported by Machebe et al. (2013) for hens treated with the plant
extract. However, toms fed 3kgMO+3kgGL recorded 19.12% dead- in- shell is close to 20%
late dead- in –shell embryos reported by Machebe et al. (2013) for hens treated with Okra
seed extract. In another similar study with broiler breeder Mahmoud et al. (2011) reported
that dietary supplementation of breeder male with Zinc increased fertility, hatchability of
total eggs, hatchability of fertile eggs, sperm egg penetration and a reduction in embryonic
mortality
The result of this study, suggest that the reduction in percent dead-in-shell embryos could be
associated with powerful antioxidant present in these herbs which might have extend their
effects to the developing embryos, when compared to the control. The combination with
higher levels of G. latifolium recorded higher percent dead-in-shell embryos which could be
due to the reduced semen quality associated with the herb and possible incubation mistakes
(over heating or under heating and inappropriate turning). Natural antioxidants, including
vitamin E and C, Selenium, and carotenoids play important roles in avian reproduction by
maintaining antioxidant defenses of the spermatozoa and embryonic tissues (Surai et al.,
2006). However, seminal plasma is considered to be the central source of antioxidants
72
protecting the seminal components against oxidative damage (Agarwal et al., 2004).
Sreelatha and Padma (2009) also indicated that the antioxidants present in both mature and
green leaves offer superior protection against free radicals, and the antioxidants in the leaves
behave in a comparable manner as pharmaceutical grade antioxidant preparations.
4.5.3 Combined Effects of M. oleifera and G. latifolium on Percent Egg Hatchability
The result in Table 12 shows percent hatched eggs fertilized by toms semen fed diets
containing combined levels of Moringa oleifera and Gongronema latifolium were
significantly different (P< 0.05). The significant improvement in the percent embryos
survival was recorded (88.39% and 83.33%) in combined treatment at 3kgMO+1.5kgGL and
3kgMO+3kgGL respectively. On the contrary, a significant reduction in embryonic survival
was shown with decrease in percentage hatchability from 76.67% in the control group to
66.87+5.56% in tom supplemented with 1.5kgMO+3kgGL diet. This combined treatment C
and E might likely have improved survival of embryos, and might have suppressed the
adverse effects of GL on the fertility and hatchability of the eggs. This result concur with
Durape (2007) who recorded an increase in hatchability from 57.2% to 59.1% in broiler
breeder hens inseminated with semen from males fed polyherbal supplementation. Similarly,
Machebe et al. (2013) reported an increased in hatchability from 22±1.31% in the control to
51±2.52% in hens treated with okra seed extract. Vitamin C present in large quantity may
benefit fertility in its ability to promote collagen synthesis; its role in hormone production and
its ability to protect cells from free radicals (Martin et al., 1995). A consistent theme in the
reproductive literature is that oxidant stress on the egg and sperm cause damage and impair
fertility and hatchability. Because there is such a poor success rate from the incredibly
expensive fertility workups one would think more attention would be paid to the antioxidants
particularly vitamin C, selenium and glutathione in enhancing fertility.
Table 13: Chemical Composition of Semen 0f Turkey supplemented with M. oleifera or G.
latifolium
Parameters
0kg MO+0kg GL
1.5kg MO
3kg MO
1.5kg GL
3kg GL
a
ab
b
b
Fructose (mg/100ml)
3.49+1.56
3.55+1.56
3.78+1.56
3.89+1.56
5.86+1.56c
Sodium(mg/100ml)
0. 30+0.05a
0. 35+0.05a
0.39+0.05c
0.36+0.05b
0.37+0.05b
Potassium(mg/100ml)
0. 25+0.09a
0.29+0.09b
0.35+0.09c
0.27+0.09b
0.33+0.09c
Means within the same row with different superscripts are significantly different; *=P<0.05,
Keys: NS= Not significant, MO= Moringa oleifera and GL= Gongronema latifolium
73
4.6.1
Effect of M. oleifera and G. latifolium on Fructose Concentration in Toms Semen
As shown in Table 13, dietary supplementation of M. oleifera or G. latifolium affected the
fructose concentration in semen of the treated toms. Fructose concentration is statistically
(P < 0.05) higher in toms fed 3kgGL diet, but at 1.5kgGL, 1.5kgMO and 3kgMO there were
no significant difference in the fructose concentration. The result indicates that M. oleifera
had no much significant increase in fructose concentration when compared with higher dose
of G. latifolium. Toms fed varying levels of M. oleifera had mean value of 3.55+0.05
mg/100ml and 3.78+0.05mg/100ml which was slightly higher compared with 3.49+0.21
mg/100ml for the control. This observation shows that dietary supplementation of herbs fed
to turkey toms’ especially G. latifolium may significantly increase the concentration of
fructose in the semen of toms. Hence, addition of M. oleifera did not produce significant
increase in fructose concentration, when compared to the control. The result of this study is
supported by the report of Shirley et al. (1963) and Moule et al. (1966), that nutritional
factors also have been shown to influence fructose level. Although it appear not to be any
recent report to compare avian seminal plasma fructose concentration, Mann (1954) reported
that total concentration of fructose in cock semen is about 4mg/100ml. Similarly, in 1964
Baker et al. studied fructose concentration in the bull, and reported that Atropine (a
poisonous alkaloid of plant source) had the reverse effect on semen characteristics but
significantly increased the concentration of fructose in the bull semen from 4.27to
6.28mg/ml, but reduced the total content of fructose in the ejaculate to one-half that of the
control semen. On the other hand, Pilocarpine had no significant influence on the
concentration of fructose, but increased the total content of fructose to twice that of the
control (4.27).
Plants with high alkaloid contents were observed to be responsible for
increase serum concentration of estradiol and prolactin. This has the capacity to inhibit
gonadothrophin action of the testis and subsequent impairment of male fertility (McGarvey et
al., 2001; Weber et al., 2001; Pastuszewska et al., 2006).
4.6.2 Cations Concentration in Tom Semen fed varying levels of M. oleifera and G.latifolium
There were significant effects of M. oleifera and G.lafolium on Cations Concentration in
turkey tom semen fed diet supplemented with the two herbs (Table 13). The toms fed various
levels of M. oleifera and G.latifolium diets were characterized by higher concentrations of Na
and K than in the control group. The 3kgMO fed toms had the higher concentrations of the
biochemical elements (Na and K). It is interesting to note that, M. oleifera (1.5kg and 3kg)
treated toms had the highest means values of 3.5+0.05mg/100ml and 0.39+0.05mg/100ml for
74
Na, 0.29+0.25mg/100ml and 0.35+0.33mg/100ml for K concentration, when compared with
the values recorded for G. lafolium supplemented toms 0.36+0.05mg/100ml and
0.37+0.05mg/100ml for Na, 0.27+0.09 mg/100ml and 0.33+0.09 mg/100ml for K. This result
suggest that M. oleifera significantly increase the concentration of Na, K and antioxidant
activities in semen, this is confirmed by the report of Tvrdá, et al. (2013) who observed that
Na, Cu, Fe, Mg, and Zn exhibited positive correlations with motility and antioxidant
parameters. Positive effects on the sperm cell motility, morphology, and concentration were
reported particularly for Zn, Mg, Se, and Ca (Eghba et al., 2008; Atig et al., 2012; Sørensen
et al., 1999). Here, we can infer that higher concentration of Na and K in the semen of M.
oleifera treated toms had a significant improvement on semen characteristics, better than the
G. latifolium and the control groups.
Mass´anyi et al. (2008) detected high positive correlation between sodium and potassium (r =
0.899) in turkey semen. In comparison, the values obtained in this study are slightly close to
3.96μg/ml and 3.14μg/ml (sodium) and 2.88μg/ml and 3.42μg/ml (potassium) for roster and
turkey toms respectively as reported by Mass´anyi et al. (2008). Chemical elements plays a
role in natural antioxidant structures, as some minerals are required for cellular defense
systems against free radicals (Marzec-Wr´oblewska et al., 2012). It has been demonstrated
that disturbances in their concentrations may lead to a reduction of antioxidant activities and
subsequently increase the risk of oxidative stress development (Marzec-Wr´oblewska et al.,
2012, Mass´anyi et al., 2008). Thus, seminal plasma is considered to be the central source of
antioxidants protecting the seminal components against oxidative damage (Agarwal et al.,
2004).
Table 14: Chemical Composition Turkey Semen Fed Combined level of M. oleifera and G.
latifolium
Parameters
Fructose (mg/100ml)
A
2.54+1.95a
B
3.15+2.62b
C
3.67+2.84bc
sodium(mg/100ml)
0. 29+0.04a
Potassium(mg/100ml) 0. 22+0.42a
0. 32+0.19a
0.28+0.08b
0.42+0.27b
0.29+0.23c
D
4.03+2.16c
0.36+0.04ab
0.27+0.07b
E
4.26+2.86c
0.40+0.28ab
0.29+0.16bc
Means within the same row with different superscripts are significantly different; *=P<0.05
Keys: MO= Moringa oleifera and GL= Gongronema latifolium
A= Control, B = 1.5kgMO+1.5kgGL, C= 3kgMO+1.5kgGL, D= 1.5kgMO+3kgGL, E =3kgMO+
3kgGL
75
4.7.1 Effects of M. oleifera and G. latifolium on Fructose Composition of Toms Semen
The result of some biochemical parameters of turkey toms’ semen fed diets containing a
combined formula of M. oleifera and G.latifolium is presented in Table 14. The result
indicates that fructose, Na and K concentrations were all significantly different (P < 0.05)
from the control group. Fructose concentration was higher in treatment combinations D and E
which were statistically similar to treatment C, but different from treatment A and B. This result
indicates that G. latifolium had positive influence on the concentration of fructose in the
semen when compared with 3kgMO+1.5kgGL inclusion and the control group. On the other
hand, at higher inclusion of M. oleifera (3kgMO+1.5kgGL) fructose was moderately higher
than the control but lower than the combination with a higher level of G. latifolium. In the
same manner, the concentrations of Na and K were significantly increased on feeding M.
oleifera and G. latifolium to breeder toms. Here, toms fed 3kgMO+1.5kgGL diet had higher
mean value of 0.42+0.27 Na, but at lower inclusion, there was no significant increase.
Concentration of K was similar across the combined treatment except for the control which
had the lowest value (0. 22+0.42). The result of this study could not be compared, as there is
no literature available at the moment to compare and discuss our detected statistical
differences, but we assumed that treatment with either of the herbs has significant effects on
the biochemical parameters of turkey semen and the concentration of these minerals has
significant effect on the fertilizing ability of the sperm. Also, this study assumed a relation
between the increases in concentration of the biochemical parameters with improvement in
semen qualities, and antioxidant properties except for fructose which is inversely related to
sperm concentration. These assumptions are supported by the findings of G¨ur and Demirci
(2000), Tvrdá et al. (2013), who reported a positive impact of Na on all spermatozoa vitality
characteristics, assuming that Na is crucial for proper physicochemical properties of semen
and further concluded that the seminal Na is indispensable for a suitable antioxidant milieu
and activity of sperm. Tvrdá et al. (2013) also revealed that while Na exhibited generally
favorable effects on the seminal quality and antioxidant balance, K behaved inversely. G¨ur
and Demirci (2000) proposed that oxygen uptake, glycolysis, and fructolysis could be
inhibited by K and indicating that this element may adversely affect spermatozoa activity.
76
Correlation coefficients between the semen quality traits in turkey toms are presented in the
Table below.
Table 15:
Measures of Association Between Body Weight and Semen Characteristics of
Tom
Vol.(ml)
Motility (%)
Conc. (%)
Live (%)
Dead (%)
Normal (%)
Abnormal (%)
Fructose
(mg/100ml)
BW (kg)
Vol.
Motility
Conc.
Live
Dead
Normal
Abnormal
Fructose
0.17
0.24
0.43
0.45
0.22
0.22
0.87
0.48**
0.33**
-0.37**
0.53**
-0.54**
-0.51*
0.36**
-0.38**
0.48**
-0.48**
-0.67**
-0.88**
0.34**
-0.34
0.03
-0.33**
0.33**
-0.02
-0.99**
-0.43
0.44
-
1.00**
0.17
0.24*
0.43**
-0.45**
0.22*
-0.25*
0.99**
( ∗P < 0.05; ∗∗P < 0.01). The interpretation of the results was based on the value of the
correlation coefficient: ±0.111 - ±0.333, low correlation; ±0.334 – ±0.666: moderate
correlation; ±0.667– ±0.999: high correlation.
4.8.1 Associations Between Semen Quality Parameters And Body Weight Of Treated Tom.
The result of this study shows the relationship between semen quality characteristics (semen
volume, sperm motility, sperm concentration, sperm viability, sperm morphology, body
weight and fructose) of turkey toms fed diets containing M. oleifera and G. latifolium as
significant (R < 0.05, R < 0.01) moderate or strongly and negative or positive correlation.
Table 15 shows that volume (ml) was not significantly correlate with all the measured semen
quality parameters except with body weight which was strongly (r = 1.00) and positively
correlated. Sperm motility is positively and moderately correlate with sperm concentration
(r=0.48**), live (r=0.33**) and normal (r=0.53**) sperm, but negatively correlate with dead
sperm (r= -0.37**), abnormal sperm (r= -0.54**) and fructose concentration(r=0.51*) in
semen. Sperm concentration displayed positive and moderate correlations, with live and
normal sperm and slightly with body weight, but an inverse relationship was recorded with
dead and abnormal sperm. Noteworthy, fructose concentration in semen shows a strong
negative relationship (r= -0.67**) with sperm concentration, but establish strong positive (r= 0.99**) relation with body weight. The result also indicates that body weight is moderately
correlate (r = 0.43**) with live sperm, faintly (0.22*) with normal sperm. However, live and
BW
-
77
normal sperm were highly and negatively correlated (r = -0.88, -0.99) ** with dead and
abnormal sperm respectively.
The correlation matrix in this study revealed that percentage motility is slightly related to
sperm concentration and normal sperm. Therefore, it can be suggested that motility is only
moderate indicator of sperm fertilizing ability, because Sperm count is one of the most
sensitive tests for spermatogenesis since it gives the cumulative results of all stages in sperm
production and it is highly correlated with fertility. Ekaluo et al. (2011) reported an inverse
relationship between mean sperm count and percentage of sperm head abnormality. In
addition, correlation between sperm concentration with percentage live and normal sperm are
also moderate, showing that decreased concentrations is related to decreased sperm
morphology, viability, and fertilizing potential of semen. The findings of this study contradict
the findings of Ngu et al. (2014) who recorded a non significant correlations between semen
volume and all other semen quality parameters except for live and dead, abnormal and all
other parameter which were all negatively correlated. Ngu et al. (2014), Oke and Ihemeson
(2010) reported negative correlation exist between semen concentration and semen volume in
different chicken and turkey. Wilson et al. (1979) indicated that correlations between
individual semen quality characteristics and fertility were not significant in natural mating,
but with AI, spermatozoa cell concentration, percent dead spermatozoa, and motility were
significantly correlated with fertility. Kotłowska et al. (2005) reported a non significant
correlation between volume and sperm concentration in turkey semen. It was also reported by
Mann and Parsons (1950) that there is an inverse relation between fructose and sperm
concentration in semen level, reflecting a manner and the degree of testicular hormone
activity in the male animal. Yakubu et al. (2012) reported a positively correlation between
Semen volume and seminal fructose levels and negatively associated with sperm
concentration (P<0.01). Body weight is highly related with semen volume and fructose
content of semen, this implies that as one trait increases, the other traits also increase. Stossier
(1960) found a notable lower percentage of fructose with increasing numbers of spermatozoa.
Gropper & Nikolowski, (1954); McCullagh & Schaffenburg, (1951) assumed that correlation
exist, without direct evidence, between fructose concentration in seminal plasma and certain
sperm abnormalities and indirectly with volume of ejaculate.
78
CHAPTER FIVE
SUMMARY AND RECOMMENDATIONS
5.1 Summary
This study was aimed at determining the effects of Moringa oleifera, Gongronema latifolium
and their combined formulas on semen quality, fertility, hatchability and some biochemical
parameters of local turkey toms. Toms were fed 0kg, 1.5kg or 3kg/100kg diets M. oleifera, or
G.
latifolium
and
their
combination
(1.5kgMO+1.5kgGL,
3kgMO+1.5kgGL,
1.5kgMO+3kgGL, 3kgMO+3kgGL). The results showed that toms fed higher dose (3kg) of
M. oleifera yielded the best result in most of the parameter measured, when compared with
the control group.
While, G. latifolium adversely affected semen qualities of the
supplemented tom diets especially at 3kg level of inclusion, G. latifolium treated toms had the
lowest semen qualities which was attributed to their anti-fertility properties at the same level.
Fertility and hatchability results indicate significant (P<0.05) effects of M. oleifera and G.
latifolium on percentage fertile eggs, percentage hatched eggs and percentage dead in shell
embryos and egg hatchability. The result showed that M. oleifera especially at 3kg
significantly improved most of the measured parameters. However, percent fertile and
infertile eggs were statistically the same in all the combined treatment, while other
parameters showed significant improvement in hens inseminated with semen from local toms
fed M. oleifera diet at 3kg. The mean values for dead-in-shell embryos and percentage dead
in shell embryos were higher in hens inseminated with semen of toms treated with higher
dose of G. latifolium.
The results of this study reveals that artificial insemination is the best option for improvement
in turkey production, and that male breeder’s semen can be improve by supplementing tom’s
feed with 3kg M. oleifera and if necessary combined 3kgMO+1.5kgGL in the diet of turkey
toms. Consequently improve fertility, reduce shell embryos mortality, and thus survivability
of newly hatched poult. Interestingly, G. latifolium diet significantly enhances fructose
concentration in semen better than M. oleifera, but M. oleifera significantly increase Na and
K concentration in the semen of the treated toms.
Lastly, semen quality parameters, body weight and fructose concentration was equally
correlation. Volume of the ejaculate had no significant difference, except for body weight
which was highly correlated with body weight. Sperm progressive motility was moderately
related with sperm concentration, percentage live sperm, and percentage normal. But an
79
inverse relationship was recorded between dead, abnormal and fructose with all other
parameters. Fructose was insignificantly correlated with all the parameters except body
weight which was highly positively correlated.
It can be deducted from the study that 3kg M. oleifera and, or when combine with low level
of G. latifolium (3kg M. oleifera and 1.5kg G. latifolium) have a vital role in improving
semen quality, fertility and egg hatchability of inseminated hens. However, based on the
findings of this study, it can be inferred that a strategic AI in turkey production can play a
major role in developing and propagating economically viable turkey flocks, making turkey
production more profitable and popular just like other breeds of poultry, there by sustaining
and improve the local breed. Therefore, AI in local turkey can be used as a major tool for the
reproductive improvement of turkey toms as it is considered superior to natural mating in
many aspects.
5.2 Recommendations
Based on the result of this study, it is therefore recommended that:•
Artificial insemination should be made an integral part in breeding turkey in Nigeria,
even with the local turkey. Semen should be analyzed prior to insemination.
•
Farmers should include M. oleifera 3kg/100kg diet because it will greatly improve the
quality of tom semen which could result to high fertility and hatchability.
•
Where M. oleifera and G. latiofolium are to be used because of the improved
antioxidants of the combination, higher level of M. oleifera (3kg) and lower G.
latifolium (1.5kg) should be adopted to avoid depression of some quality traits.
80
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