annals of animal science - Instytut Zootechniki PIB

Transcription

ANNALS
OF ANIMAL SCIENCE
NATIONAL RESEARCH INSTITUTE OF ANIMAL PRODUCTION
Vol. 6
KRAKÓW 2006
No. 1
EDITORIAL BOARD
Je˛drzej Krupiński (Chairman) — Kraków-Balice,
Franciszek Brzóska — Kraków-Balice,
Clas Elwinger — Uppsala,
Tibor Gere — Gyöngyös,
Kay-Uwe Götz — Poing-Grub,
Ingemar Gustavsson — Uppsala,
Eugeniusz Herbut — Kraków-Balice,
Dymitr Kaliszewicz — Olsztyn,
Jolanta Kurył — Jastrze˛biec,
Andrzej Potkański — Poznań,
Eligiusz Rokicki — Warszawa,
Marian Różycki — Kraków-Balice,
Yasuo Shioya — Ibaraki,
Zdzisław Smora˛g — Kraków-Balice,
Vasyl Vlizlo — Lviv,
Stanisław We˛żyk — Kraków-Balice,
Jacek Wójtowski — Poznań
EDITORIAL STAFF
Editor-in-Chief — Ewa Słota
Deputy Editors-in-Chief — Marian Duniec, Mariusz Pietras
Secretary — Halina Lach
Editing — Danuta Dobrowolska, Halina Lach, Jerzy Pilawski
Cover design — Beata Barszczewska-Wojda
Address of Editorial Office — Instytut Zootechniki
ul. Sarego 2, 31-047 Kraków, Poland
The ‘‘Annals of Animal Science” are derived from the journal
‘‘Roczniki Naukowe Zootechniki” which has been published since 1974
This publication was supported by the Ministry of Education and Science
 Copyright by National Research Institute of Animal Production
PL ISSN 1642-3402
Ann. Anim. Sci., Vol. 6, No. 1 (2006)
CONTENT
Genetics and farm animal breeding
1. M. Bugno, M. Owczarek-Lipska, A. Pieńkowska-Schelling, E. Słota — Variation in the size
of the Y chromosome in four breeds of domestic horse . . . . . . . . . . . .
2. T. Za˛bek, A. Żyga, A. Radko, E. Słota — Analysis of genetic variation in Małopolski
horses using molecular and pedigree data . . . . . . . . . . . . . . . .
3. U. Czarnik, T. Zabolewicz, C.S. Pareek, R. Ziemiński, K. Walawski — Evaluation of putative
relationship between PRNP octapeptide repeat polymorphism and variability of milk production
traits in cattle . . . . . . . . . . . . . . . . . . . . . . . . . .
4. A. Felenczak, A. Fertig, E. Gardzina, M. Ormian, J. Trela — Technological traits of milk
of Simmental cows as related to κ-casein polymorphism . . . . . . . . . . .
5. J. Komisarek, K. Waśkowicz, Z. Dorynek — Analysis of the relationship between two
single nucleotide polymorphisms of the butyrophilin (BTN1A1) gene and milk production
traits in Jersey cattle . . . . . . . . . . . . . . . . . . . . . . . .
Biology, physiology and animal reproduction
6. J. Pytlewski, I. Antkowiak, Z. Dorynek — Relationship between interpregnancy interval
and lifetime productivity of cows . . . . . . . . . . . . . . . . . . .
7. A. Mazanowski, Z. Bernacki, M. Adamski, T. Kisiel — Analysis of time trends for reproductive and meat traits in randomly mated conservation flocks of northern variety geese .
8. A. Mazanowski, E. Samorek-Salamonowicz, M. Urbanowski — Level and duration of persistence of antibodies in the blood serum of native varieties of geese after vaccination against
Derzsy’s disease . . . . . . . . . . . . . . . . . . . . . . . . .
5
13
29
37
45
53
59
75
Animal nutrition and feedstuffs
9. F. Brzóska — Effect of rapeseed meal protected with calcium salts of fatty acids from linseed
oil on cows’ yield and milk and blood parameters . . . . . . . . . . . . . 87
10. E. Hanczakowska, J. Urbańczyk, I. Kühn, M. Świa˛tkiewicz — Effect of glucanase and
xylanase supplementation of feed for weaned piglets . . . . . . . . . . . . . 101
11. A. Szewczyk, F. Borowiec, E. Hanczakowska — Fatty acid and cholesterol content of meat
of broilers fed linseed oil or different linseed varieties . . . . . . . . . . . . 109
12. S. Nowaczewski, H. Kontecka, E. Pruszyńska-Oszmałek — Effect of feed supplementation
with vitamin C on haematological indices, corticosterone concentration in blood and duration
of tonic immobility in pheasants . . . . . . . . . . . . . . . . . . . 117
Environment, hygiene and animal production technology
13. S. Kornaś, B. Nowosad, M. Skalska — Dynamics of small strongyle (Cyathostominae)
infection in horses under different management systems . . . . . . . . . . . .
14. P. Paraponiak — Effect of crossbreeding on pasture rearing of lambs and chemical
and sensory properties of slaughter material . . . . . . . . . . . . . . . .
129
139
4
15. Hannu T. Korhonen, L. Jauhiainen, T. Rekilä — Effects of year-round nestbox availability
and temperament on welfare and production performance in blue foxes (Alopex lagopus)
16. I. Skomorucha, E. Herbut — Use of an earth-tube heat exchanger to optimize broiler house
climate during the summer period . . . . . . . . . . . . . . . . . . .
149
169
Ann. Anim. Sci., Vol. 6, No. 1 (2006) 5 – 12
VARIATION IN THE SIZE OF THE Y CHROMOSOME IN FOUR BREEDS
OF DOMESTIC HORSE*
M o n i k a B u g n o1, M a r t a O w c z a r e k - L i p s k a2, A l d o n a P i e ń k o w s k a - S c h e l l i n g2,
E w a S ł o t a1
1
Department of Immuno- and Cytogenetics, National Research Institute of Animal Production,
32-083 Balice n. Kraków, Poland
2
Department of Genetics and Animal Breeding, Agricultural University, Wołyńska 33,
60-637 Poznań, Poland
Abstract
The mean relative lengths of the Y chromosome were measured for each of four horse breeds (pure
Arab, noble half-bred, Hutsul and Shetland pony). The measurement included the length of the
X and Y chromosomes as well as the constitutive heterochromatin block of the Y chromosome.
Using statistical methods, highly significant within-species differences were shown in the ratios of
the constitutive heterochromatin block to Y chromosome length and the ratio of Y chromosome
length to X chromosome length.
Key words: horse, length polymorphism, Y chromosome
The Y chromosomes of different mammalian species vary widely in the
morphology, size and content of genes. The smallest Y chromosome is found in
marsupials: it is only 10 Mb in size and has no pseudoautosomal region, which
means that no pairing with the X chromosome and thus no formation of
synaptonemal complexes and no recombination with the X chromosome, take place
(Toder et al., 2000). The human Y chromosome has a completely different
structure. It is considerably larger (approximately 60 Mb) and has two pseudoautosomal regions localized in the distal part of the short and long arms
(Quintana-Murci et al., 2001). Most of the human Y chromosome sequences (as
much as 95%) are defined as non-recombining regions (NRY). They can occur in
euchromatic, centromeric and heterochromatic regions (Foote et al., 1992).
* The work was conducted as part of NRIAP statutory activity, project no. 3207.1.
6
M. Bugno et al.
In horses, the size of the Y chromosome corresponds with the smallest
acrocentric chromosomes, while the X chromosome is a large submetacentric. After
CBG staining, constitutive heterochromatin emerges in the area of almost the entire
Y chromosome and on an additional band on the q arm of the X chromosome
(Bugno et al., 2005).
Because of the different degrees of chromosome spiralization during the course
of mitosis, which prevents the absolute values from being compared, the relative
value of the Y chromosome was evaluated in all the studies with the aim of showing
the polymorphism of the Y chromosome. This value was expressed as the
centromeric index, the long arm to short arm ratio (q : p), or the percentage length
of the haploid set of autosomes plus the length of the X chromosome (Sławomirski
et al., 1979; Świtoński and Podra˛b, 1983; Kozubska-Sobocińska et al., 1995).
Animals of the same breed were compared (Świtoński and Podra˛b, 1983;
Świtoński, 1984; Kołodziejski et al., 1997; Kołodziejski and Pietrzak, 1999) and
possible between-breed differences were also explored (Sławomirski et al., 1979;
Popescu, 1990; Kozubska-Sobocińska et al., 1995). It was found that the differences
in Y chromosome size were observed both between animals of the same breed and
between different breeds.
To investigate Y chromosome polymorphism, this study compared the size of
the Y chromosome in relation to the X chromosome as well as measured the
constitutive heterochromatin block and its relation to the entire Y chromosome.
This allowed for an objective evaluation of polymorphism in the pure Arab, noble
half-bred, Hutsul and Shetland pony breeds.
Material and methods
Y chromosome polymorphism was classified based on measurements of sex
chromosome lengths in four horse breeds of different utility types: pure Arab, noble
half-bred, Hutsul and Shetland pony. Cytogenetic analysis covered pure Arab,
noble half-bred and Hutsul stallions (three per breed) and four Shetland Pony
stallions. Metaphase plates, obtained by routine lymphocyte culture, were CBG
stained (Sumner, 1972) to reveal constitutive heterochromatin and to identify sex
chromosomes.
Using the LUCIA computer image analysis system, the lengths of the X, Y and
pair 1 chromosomes were measured on ten different metaphase plates, taking into
account the mean length of two chromatids.
The relative length of the Y sex chromosome was calculated from the ratio of
the mean length of the X chromosome chromatids (Y : X). In addition, the size of
the constitutive heterochromatin block and its ratio to the entire Y chromosome
were determined. Due to considerable differences in the length of sex chromosomes
within the metaphase plates of the same animal, means from 10 measurements of
the relative length of the Y chromosome were calculated for each stallion. The
mean lengths of the Y chromosome for each breed were calculated based on
7
Variation in the size of the Y chromosome in the horse
30 measurements and compared by statistical methods using one-way analysis of
variance and the Fisher-Snedecor test to identify the Y chromosome polymorphism
between the analysed breeds. This test analyses continuous random variables (in
this case, the measurements taken).
Results
Statistical analysis did not show any significant differences in the relative
Y chromosome length or heterochromatin block lengths between the pure Arab,
noble half-bred, Hutsul and Shetland pony breeds (Tables 1 and 2).
Table 1. Analysis of variance of the relative length of the heterochromatin region of the Y chromosome
in horses between pure Arab, noble half-bred, Hutsul and Shetland pony breeds
Source
of variation
Total
Between breeds
Within breeds
Degrees
of freedom
Sum of square
deviations (SS)
Mean square
deviations (MS)
12
3
9
0.026
0.007
0.019
0.0023
0.002
F value
1.5
Table 2. Analysis of variance of the relative length of the Y chromosome in horses between pure Arab,
noble half-bred, Hutsul and Shetland pony breeds
Source
of variation
Total
Between breeds
Within breeds
Degrees
of freedom
12
3
9
Sum of square
deviations (SS)
3097
0.02
3.95
Mean square
deviations (MS)
0.01
0.44
F value
0.02
Analysis of variance for the size of the Y chromosome and constitutive
heterochromatin block of this chromosome for all the stallions revealed highly
significant differences between the animals (Tables 3 and 4). In this case, detailed
comparisons were analysed (Table 5).
Table 3. Analysis of variance of the relative length of the Y chromosome in all investigated horses
Source
of variation
Total
Between animals
Within animals
Degrees
of freedom
Sum of square
deviations (SS)
Mean square
deviations (MS)
129
12
117
0.80
0.18
0.62
0.02
0.005
F value
2.89**
8
M. Bugno et al.
Table 4. Analysis of variance of the relative length of the heterochromatin region of the
Y chromosome in all investigated horses
Source
of variation
Degrees
of freedom
Sum of square
deviations (SS)
Mean square
deviations (MS)
129
12
117
0.862
0.214
0.648
0.018
0.005
Total
Between animals
Within animals
F value
3.22 xx
xx — highly significant differences at P ≤ 0.01.
When analysing the ratio of the constitutive heterochromatin length to the entire
Y chromosome length and the ratio of the Y chromosome to X chromosome length,
it was found that animal no. 13 (Table 5), a Shetland pony, showed the greatest
highly significant and values in within-species variation in the first measurement,
compared to the other animals. Meanwhile, animal no. 5 (Table 5), a noble
half-bred horse, showed highly significant within-species variation in the second
measurement compared to the other animals.
Table 5. Results of the detailed NIR-Fisher test for the animals investigated
No.
Breed
Number and frequency of the objects compared using the NIR-Fisher test
heterochromatin/Y chromosome
Y chromosome/X chromosome
α = 0.05
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Pure Arab
Pure Arab
Pure Arab
Noble half-bred
Noble half-bred
Noble half-bred
Hutsul
Hutsul
Hutsul
Shetland pony (mini)
Shetland pony
Shetland pony
Shetland pony
3 (6.52%)
5 (10.87%)
3 (6.52%)
4 (8.70%)
5 (10.87%)
5 (10.87%)
4 (8.70%)
2 (4.35%)
1 (2.17%)
1 (2.17%)
3 (6.52%)
1 (2.17%)
9 (19.57%)
α = 0.01
1
2
1
3
3
3
2
(4.55%)
(9.09%)
(4.55%)
(13.63%)
(13.63%)
(13.63%)
(9.09%)
0
0
1 (4.55%)
0
0
6 (27.28%)
α = 0.05
α = 0.01
0
1 (3.12%)
1 (3.12%)
1 (3.12%)
11 (34.38%)
1 (3.12%)
4 (12.50%)
3 (9.38%)
1 (3.12%)
2 (6.26%)
3 (9.38%)
1 (3.12%)
3 (9.38%)
2 (8.33%)
1 (4.17%)
1 (4.17%)
1 (4.17%)
10 (41.66%)
1 (4.17%)
1 (4.17%)
0
1 (4.17%)
1 (4.17%)
2 (8.33%)
1 (4.17%)
2 (8.33%)
The examples of different length Y chromosomes are shown in Figure 1.
9
Figure 1. The examples of different length Y chromosomes between horse breeds
Discussion
The earliest Y chromosome polymorphism studies were carried out in humans.
At a Denver conference in 1960, it was stated that the Y chromosome varies the
most in terms of total length and arm length ratio. Two size variants of the
Y chromosome were observed and both the small Y chromosome (Genest and
Lejeune, 1972; Genest, 1981) and the large Y chromosome (Unnerus et al., 1967)
were shown to be heritable traits.
As animal cytogenetic studies developed, attempts were made to determine the
phenomenon of chromosomal polymorphism in different species (Vorontsov et al.,
1978; Eldridge et al., 1983; Słota et al., 1985; Kozubska-Sobocińska et al., 1995).
Y chromosome polymorphism has been described more often in cattle than in any
other species of farm animal. In bovines, highly significant between-breed differences were identified, and Y chromosome polymorphism was found to be heritable
(Cribiu and Popescu, 1974; Cribiu, 1975; Hansen and Ellebby, 1975; de Giovanni
and Cribiu, 1977; Świtoński and Podra˛b, 1983; Świtoński, 1984; Kozubska-Sobocińska et al., 1995).
Studies of Y chromosome length polymorphism have also been carried out in
sheep (Słota et al., 1985), and in pigs (Słota, 1998). Significant differences in the
relative length of chromosome Y between investigated pig breeds and lines were
observed. The polymorphic variants of the chromosome Y length were found to be
a characteristic feature of every breed.
Observations made by Pieńkowska-Schelling et al. in 2004 on Canidae animals
provide a significant source of information on differences in the Y chromosomes of
the maned wolf and fennec. It was found that the Y chromosome of the maned wolf
10
M. Bugno et al.
is characterized by a very large constitutive heterochromatin block, which affects
the size of the chromosome, unlike in the fennec, in which this block is practically
non-existent and the Y chromosome is very small (Pieńkowska-Schelling et al.,
2004).
The first observations and measurements of the Y chromosome in horses were
made in the 1980s by Stranzinger (1980) and Hansen (1984). They suggested that
Y chromosomes can be of different lengths, as confirmed by the studies of Power,
which revealed that the largest Y chromosome was twice the length of the smallest
Y chromosome (Power, 1988).
The present measurements were subjected to statistical analysis to reveal
significant differences in the size of the Y chromosome. Highly significant differences were found for the ratio of the constitutive heterochromatin block to Y chromosome length and the ratio of Y chromosome length to X chromosome length.
Preliminary detailed analysis showed that a Shetland pony horse made the
greatest contribution to within-species variation of Y chromosome length polymorphism as measured by the ratio of the constitutive heterochromatin block to the
Y chromosome, and a noble half-bred horse had the greatest within-species
variation as measured by the ratio of Y chromosome length to X chromosome
length. Until now no correlation has been observed between Y chromosome size
and same phenotypical or physiological traits.
It can be concluded from the present study that variation is an animal-specific
rather than a breed-specific trait. However, it would be beneficial to continue this
study with a greater number of animals per breed to provide extensive information
on between-breed variation in the length of the Y chromosome.
References
B u g n o M., S ł o t a E., P i e ń k o w s k a - S c h e l l i n g A., S c h e l l i n g C. (2005). Efektywna metoda
diagnozy aneuploidii chromosomu X — najcze˛stszej aberracji kariotypu koni. Rocz. Nauk. Zoot.,
32, 1: 11 – 14.
C r i b i u E.P. (1975). Variation interraciale de la taille du chromosome Y chez Bos Taurus L. Ann.
Genet. Sel. Anim., 7, 2: 139 – 144.
C r i b i u E.P., P o p e s c u C.P. (1974). Un cas de chromosome Y anormalement long chez Bos Taurus L.
Ann. Genet. Sel. Anim., 6: 387 – 390.
E l d r i d g e F.E., F a r v e r K o e n i n g J.L., H a r r i s N. (1983). Y chromosome variation in the bovine.
Abstr. 3th North Amer. Symp. Cytogenet. Cell Biol. Domest. Anim., Medison; p. 245.
F o o t e S., V o l l r a t h D., H i l t o n A., P a g e D.C. (1992). The human Y chromosome: overlapping
DNA clones spanning the euchromatic region. Science, 258: 60 – 66.
G e n e s t P. (1981). Etude complementaire sur la nature d’un petit Y multicentenaire. Ann. Genet., 24, 4:
165 – 166.
G e n e s t P., L e j e u n e J. (1972). Recherche sur l’origine d’un petit chromosome multicentenaire. Ann.
Genet., 15, 1: 51 – 53.
G i o v a n n i A. de, C r i b i u E.P. (1977). Etude des variations du chromosome Y dans quatre races
bovines italiennes. Ann. Genet. Sel. Anim., 9: p. 527.
H a n s e n K.M. (1984). Two different length of the Y chromosomes of the domestic horse (Equus
caballus). 6th Eur. Coll. Cytogenet. Domest. Anim., pp. 172 – 176.
11
H a n s e n K.M., E l l e b b y F. (1975). Chromosome investigation of Danish A.I. beef bulls. Nord.
Vet. – Med., 27: 102 – 105.
K o ł o d z i e j s k i Z., P i e t r z a k S. (1999). Określenie polimorfizmu chromosomu Y u ogierów
wybranych ras krajowych. Aktualne kierunki hodowli i użytkowania koni w Europie. Symp.
mie˛dz., Kraków, 17 – 19.09.1999, pp. 360 – 366.
K o ł o d z i e j s k i Z., J a s z c z a k K., S y s a P. (1997). Polimorfizm chromosomu Y u koni ras krajowych.
XXV Ogólnopolska Jubileuszowa Konferencja Naukowa Sekcji Fizjologii i Patologii Konia
PTNW, Pawłowice, 23 – 24.05.1997, pp. 4 – 5.
K o z u b s k a - S o b o c i ń s k a A., S ł o t a E., D a n i e l a k - C z e c h B., R e j d u c h B. (1995). Classification of the chromosome Y polymorphism in four cattle breeds based on the measurements of
sex chromosome length. Rocz. Nauk. Zoot., 22, 2: 29 – 36.
P i e ń k o w s k a - S c h e l l i n g A., Z a w a d a M., S c h e l l i n g C. (2004). Initial Cytogenetic Studies in
three wild canid species. Cytogenet. Genom Res., 106: p. 13.
P o p e s c u P.C. (1990). Chromosomes of the cow and bull. Adv. Vet. Sci. Comp. Med., 34: 41 – 71.
P o w e r M.M. (1988). Y chromosome length variation and its significance in the horse. J. Hered., 79:
311 – 313.
Q u i n t a n a - M u r c i L., K r a u s z C., M c E l r e a v e y K. (2001). The human Y chromosome: function,
evolution and disease. Forensic Sci. Int., 118: 169 – 170.
S ł a w o m i r s k i J., S y s a P.S., L i w s k a J. (1979). Charakterystyka morfologiczna metafazalnych
chromosomów płciowych u bydła domowego (Bos Taurus dom., L.). Med. Wet., 4: 236 – 237.
S ł o t a E. (1998). Polimorfizm chromosomów świni. Rocz. Nauk. Zoot., Rozp. hab., nr 7.
S ł o t a E., K o z u b s k a A., K r u p i ń s k i J. (1985). Charakterystyka chromosomów płciowych trzech ras
owiec hodowanych w Polsce. Rocz. Nauk. Zoot., 12, 1: 15 – 22.
S t r a n z i n g e r G. (1980). Zytogenetic im dienste der Nutztierzucht. Jahrbuch Schweiz. Naturforsch.
Gessell. Wiss., 3: 68 – 79.
S u m n e r A.T. (1972). A simple technique for demonstrating centromeric heterochromatin. Exp. Cell.
Res., 75: 303 – 306.
Ś w i t o ń s k i M. (1984). Cytogenetic examination of bulls. Variants of the Y chromosome. Genet. Pol.,
25, 4: 427 – 434.
Ś w i t o ń s k i M., P o d r a˛b A. (1983). Zmienność parametrów charakteryzuja˛cych morfologie˛ chromosomów płci u buhajów. VIII Zjazd PTG, streszcz.; p. 59.
T o d e r R., W a k e f i e l d M.J., G r a v e s J.A.M. (2000). The minimal mammalian Y chromosome — the
marsupial Y as a model system. Cytogenet. Cell Genet., 91: p. 285.
U n n e r u s V., F e l l m a n J., C h a p e l l e A. de la (1967). The length of the human Y chromosome.
Cytogenetics, 6: 213 – 227.
V o r o n t s o v N.N., B o r i s o v Y.M., K a r t a v e v a I.V. (1978). Variability in mammalian sex
chromosomes. XIV Int. Congr. Genet., Moscow; p. 281.
Accepted for printing 27 IV 2006
MONIKA BUGNO, MARTA OWCZAREK-LIPSKA, ALDONA PIEŃKOWSKA-SCHELLING,
EWA SŁOTA
Zróżnicowanie wielkości chromosomu Y u koni czterech ras
STRESZCZENIE
W oparciu o pomiary długości chromosomów X, Y, jak również bloku heterochromatyny
konstytutywnej chromosomu Y u koni czterech ras (czysta krew arabska, szlachetna półkrew, hucuł i kuc
szetlandzki) obliczono średnie wzgle˛dne długości chromosomu płciowego Y dla każdej rasy. Posługuja˛c
12
M. Bugno et al.
sie˛ metodami statystycznymi wykazano wysoko istotne różnice wewna˛trzgatunkowe w stosunku bloku
heterochromatyny konstytutywnej do długości chromosomu Y oraz długości chromosomu Y do długości
chromosomu X.
ANALYSIS OF GENETIC VARIATION IN MAŁOPOLSKI HORSES
USING MOLECULAR AND PEDIGREE DATA*
T o m a s z Z a˛b e k, A g a t a Ż y g a, A n n a R a d k o, E w a S ł o t a
Department of Animal Immuno- and Cytogenetics, National Research Institute of Animal Production
Abstract
Małopolski horses have different proportions of Anglo-Arabian and oriental horse breeds in their
pedigrees. Since 1999 a steady reduction in the population of this breed has been observed. Studies
on genetic variation using 18 microsatellites were conducted in 5 Małopolski subpopulations and in
Thoroughbred and Arabian horses, the breeds used in Małopolski breeding. Analysis of the genetic
structure of the investigated Małopolski population revealed that the gene pool characteristic of its
original population could be reduced by a strong influence from other horse breeds, especially
Thoroughbreds. The variation levels expressed by expected heterozygosity for Małopolski horses
were close to those reported for other crossbred populations of horses. The studied Małopolski
population is not in danger of inbreeding, and the main source of genetic variation in Małopolski
horses is the presence of a number of breeds in their pedigrees used for breeding purposes in this
breed. Phylogenetic analysis revealed that Thoroughbreds have made the greatest contribution to
the gene pool of the current Małopolski population.
Key words: Małopolski horse, microsatellites, genetic variation
The Małopolski breed derives from horses bred in the south-eastern part of
Poland in the 15th century. Initially, local horses from this region were improved
with oriental horses such as Persian, Turkish, Turkmenian and purebred Arabian
horses, leading to the formation of halfbred oriental type. Since the second half of
the 19th century, in addition to the use of halfbred stallions from oriental
Austro-Hungarian lineages (Schagya, Gidran, Dahoman, Amurath, Gazlan, Furioso,
Przedświt, Nonius etc.), English part-bred and Thoroughbred stallions and also
Arabian and Anglo-Arabian stallions have been intensively used for reproduction.
The great contribution of Thoroughbred and Arabian blood has produced horses of
the halfbred Anglo-Arabian type. Ultimately, the Małopolski breed was formed by
* This work was conducted as part of the research project no. 3P06D 001 24, financed by the State
Committee for Scientific Research.
14
T. Za˛bek et al.
horses with different percentages of Anglo-Arabian blood, which had many
characteristics of the oriental-type horses. The stud book for the Małopolski breed
was founded in 1963. Since the reduction in demand for farm work horses,
Małopolski horses have been increasingly used in leisure riding and sport. Since
1999 a steady reduction in the effective population size of this horse has been
observed. The number of registered mares and sires is becoming smaller, and it is
not known whether the size of the population will enable viable breeding in view of
the genealogical differences between breeding lines with avoidance of inbred
matings (http://www.pzhkm.pl/rasa.php). One of the first visible effects of inbreeding might be a reduction in the breeding value of Małopolski stallions in
comparison to the stallions of other Polish warmblood breeds, reported by
Dobrowolski and Geringer (2003).
The purpose of our study was to evaluate the genetic variation of the current
population of Małopolski horses with regard to the influence of Thoroughbred and
purebred Arabian horses, using molecular marker loci (DNA microsatellite sequences). Highly polymorphic microsatellites are markers of choice for population study
in a number of wild and domesticated animal breeds. In Poland, the polymorphism
of these markers has been determined in purebred Arabian (Gralak et al., 1998),
Thoroughbred (Gralak et al., 1998; Niemczewski and Żórkowski, 2000; Za˛bek et
al., 2003), Silesian (Za˛bek et al., 2003), Biłgoraj (Za˛bek et al., 2005) and Polish
Primitive horses (Gralak et al., 2001). The present study involved analysing the
genetic structure of the three horse populations on the basis of 18 microsatellite
loci, studying the genetic variability within and between Małopolski subpopulations
(studs), and determining molecular genetic relationships between them, including
the influence of Arabian and Thoroughbred horses on the Małopolski gene pool.
The investigated Małopolski population (M) (408 individuals) included mainly
Anglo-Arabian horses with different proportions of Thoroughbred (TH), Arabian
(AR), Anglo-Arabian (AA) and purebred Anglo-Arabian (*AA*) horses in pedigrees (Table 1). The five largest studs were: Walewice (WM), Prudnik (PM), Udórz
(UM), Janów Podlaski (JM) and Ochaby (OM). Some other breeds also occurred in
the pedigrees of the studied M horses, such as Polish pure half-bred (SP), Belgian
warmblood (BWP), Hanoverian (HAN) and Selle-Français (SF) horses. The forty
Thoroughbred horses included in this study were derived from 3 Polish studs
(Strzegom, Moszna and Stubno). The fifty horses of the Arabian breed came from
the Janów Podlaski and Michałów studs.
Hair root or blood samples of horses were the source of genomic DNA,
prepared using a modified version of the method of Kawasaki (1990). Genotyping
was performed on 18 microsatellite loci: AHT4, AHT5 (Binns et al., 1995), ASB2,
ASB17 (Breen et al., 1997), HMS1, HMS2, HMS3, HMS6, HMS7 (Guerin et al.,
1994), HTG4, HTG6 (Ellegren et al., 1992), HTG7, HTG10 (Marklund et al.,
Analysis of genetic variation in Małopolski horses
15
1994), VHL20 (Van Haeringen et al., 1994), ASB23 (Irvin et al., 1998),
UCDEQ425 (Eggleston-Stott et al., 1997), LEX3 (Coogle et al., 1996), and LEX33
(Coogle, 1996). The DNA microsatellites were amplified by polymerase chain
reaction (PCR) and fluorescently labelled PCR products were subjected to vertical
electrophoresis in denaturing 4% polyacrylamide gel on a Genetic Analyser (ABI
PRISM 377, Applied Biosystems, Foster City, USA). Allele sizes were determined
after processing of raw data using the software packages GENESCAN 2.0 and
GENOTYPER 2.1 (Applied Biosystems).
Pedigree records were used to determine the number of progeny of the parents
of particular breeds and to derive the mare families and male lines of the
investigated Małopolski horses. For the pedigree analysis, data from the Studbook
of Małopolski horses (volumes II to VI) were used. Part of the information also
came from annual specifications of breeding plans released by particular studs.
Estimation of allelic frequency and a test for the presence of Hardy-Weinberg
equilibrium (H-W) following the method of Guo and Thompson (1992) were
performed using the TFPGA program (Miller, 1997). The variation level in selected
groups of horses was established according to the observed (Ho) and expected (He)
heterozygosity (Nei, 1978) measured using the MSA program (Dieringer and
Schlötterer, 2002). Genetic differentiation among the studied horse populations was
measured using the fixation coefficients (Fis and global Fst) calculated according to
Weir and Cockerham (1984) using the FSTAT program (Gaudet, 1995). The Fis
estimator was used as an inbreeding coefficient for the evaluation of genetic
equilibrium in the studied populations. The P-values of the F-statistic permutation
test were adjusted according to the sequential Bonferroni method (Holm, 1979).
Pairwise Fst coefficients were used to demonstrate genetic differences between the
investigated horse groups. To illustrate the gene flow between groups of horses, the
number of effective migrants per generation (Nm) was calculated based on Fst
estimates using the formula described by Wright (1969). The distance based on
proportion of shared alleles Dps (Bowcock et al., 1994) was calculated using the
MSA program (Dieringer and Schlötterer, 2002). Using distance matrices the
UPGMA tree was generated using the Phylip 3.2 package (Felsenstein, 1989) and
phylogenetic dendrograms were constructed using TreeView 1.6 software (Page,
1996).
Results
The number of progeny of mares and sires of particular breeds, as well as the
number of determined mare families and male lines in Małopolski studs, are
presented in Tables 1, 2 and 3, respectively.
The majority of the investigated Małopolski horses are the progeny of AA dams
(390 horses). From the paternal side of pedigrees, less than half (170 horses) of the
studied Małopolski population is the progeny of sires of other breeds such as TH,
*AA*, AR and SP. The greatest breed variability is present in the parental
16
tab. 1
T. Za˛bek et al.
tab. 2
17
18
T. Za˛bek et al.
generation of M horses from the Walewice stud. The smallest number of breeds
among parents was observed in Prudnik, with dominance of AA parents. Considering the proportion of pure breeds in the parental generation of M horses, a large
number of the studied individuals are the progeny of TH sires (95 individuals), especially at the Ochaby (29), Udórz (25) and Walewice studs (22)
(Table 1).
Sixty-six mare families were determined in the studied population of Małopolski horses. Each stud is characterized by a variety of mare families with purebred
Anglo-Arabian (*AA*), part-bred Anglo-Arabian (AA), Arabian (AR), Thoroughbred (TH), English part-bred, Austro-Hungarian, and Lipizzan (L) ancestors. Twenty
families are present in Walewice, 16 in Ochaby, 13 in Prudnik, 9 in Udórz and 8 in
the Janów Podlaski stud. The determined mare families are characterized by
unrelated pedigrees until at least the 5th generation. Only two of all families are
present in different studs (Table 2).
Table 3. Male lines present in the studied Małopolski population
Stud
Male line*
Campetot
Lirnik
Kwartet
Pick Wick
Rahman
Hippies
Hetman
First Des Termes
Vice Versa
Veritas
Jalienny
Decoration
Elsing
Lais
Parysów
Dakota
Chef Supreme
Mehari
Dzielżan
Arcus
Cynik
Saroyan
Szafir
Fordon
Breed
Walewice
*AA*
*AA*
AA
AA
AA
AA
AA
AA (France)
AA (France)
AA (France)
AA (France)
AA (France)
AR
SP
TH
TH
TH
TH
TH
TH/AA
TH/AA
TH/AA
TH/AA
TH/AA
10**
Prudnik
Udórz
Janów
Podlaski
Ochaby
1
9
8
7
12
6
4
1
1
5
3
5
3
3
1
2
4
3
4
9
5
12
6
4
4
9
8
5
1
1
19
4
8
10
22
1
3
7
5
1
4
7
2
6
1
13
1
6
8
1
10
3
* The furthest common male ancestor.
** Number of genotyped horses belonging to a given male line.
6
19
Fifty-seven stallions that are sires of horses from 5 Małopolski stud belong to
23 male lineages originated from Arabian (1 lineage), purebred (2 lineages) and
halfbred (10 lineages) Anglo-Arabian, as well as Thoroughbred (10 lineages)
ancestors (Table 3). Sires belonging to 6 TH lines are Thoroughbred horses. The
other 4 lines are formed by Anglo-Arabian sires derived from TH ancestors,
designated as TH/AA in this study. A high number of identified male lines are
present in different studs. For example, most of the TH lines are present in the
Ochaby, Walewice and Udórz studs. In Walewice and Prudnik, one line of the AR
ancestor is present. Some lines are found in one stud only: 2 lines in Prudnik (Lirnik
*AA* and Hetman AA), 3 in Janów (Vice Versa AA, Veritas AA, Decoration AA)
and 3 lines in Ochaby (Dzielżan TH, Chef Supreme TH, Szafir TH/AA).
Electrophoretic separation of PCR products exhibited 116, 97 and 166 microsatellite variants in the Arabian, Thoroughbred and whole Małopolski population,
respectively (Table. 4).
Six allelic variants (HMS3-S, HMS7-P, HTG6-F, ASB17-Q, ASB23-H and -V)
were found only in AR horses, two (ASB2-W and HTG6-R) in TH horses and fifty
(AHT4-L and -N; AHT5-I and -S; ASB2-D, -J, -S, -T and -U; HMS2-O, -T and -U;
HMS3-H; HMS6-J and -N; HMS7-H, -I and -S; HTG10-H, -N and -P; HTG4-P,
HTG6-E, -L and -N; HTG7-P; VHL20-H, -J, -K and -R; ASB17-F, -H, -J, -K, -L
and -S; ASB23-R, -T and -W; HMS1-H, -K and -O; LEX3-M; LEX33-F, -R and -S;
UCDEQ425-G, -H and -P) were found only in the Małopolski population. Despite
there being two allelic variants (ASB2-J and HTG4-P) in the M and one (HTG6-R) in
the TH population these occur with very low frequency (<0.05). In the Arabian
population in particular, a number of variants with higher frequency can be
distinguished (AHT4-I; ASB2-A; HMS3-K; HMS6-N; HTG10-Q; HTG4-L; VHL20P; HMS1-N; LEX3-H and -K), which occur with extremely low frequency (<0.05) or
were not detected in Małopolski or Thoroughbred horses. Additionally, a group of
characteristic alleles can be found in both M and TH horses (HMS6-M; HMS7-N and
-O; HTG10-I and -M; VHL20-M; ASB17-G; ASB23-U; LEX3-J and -R; LEX33-L;
UCDEQ425-J), with no occurrence or very low frequency in the Arabian breed.
The exact test for the presence of the Hardy-Weinberg equilibrium showed
deviation at locus AHT4 in Arabian horses (P < 0.05), ASB23 in TH horses
(P < 0.01) and ASB2, ASB23, HTG10 and LEX3 in M horses (P < 0.01). The
number of observed homozygous genotypes exceeded the expected number
calculated from the Hardy-Weinberg proportions.
The mean estimates of Fis did not deviate from 0 in any of the investigated
horse groups (Table 5). Fis values were highly significant only when calculated for
the whole M population (Fis = 0.036, P < 0.01). The Fis obtained indicated a 3.6%
loss of heterozygotes in relation to the expected number of heterozygous genotypes
in the whole M population (Table. 5). With the exclusion of the Arabian and
Thoroughbred populations, the observed heterozygosity (Ho) was highest for the
M subpopulation at the Walewice stud (Ho = 0.723) and lowest in the subpopulation from Prudnik (Ho = 0.683). In Prudnik, the observed heterozygosity, Ho,
was much lower than the expected heterozygosity, He (Table 5).
20
tab. 4
T. Za˛bek et al.
cd. tab. 4
21
22
T. Za˛bek et al.
Table 5. Genetic variation of the studied populations at 18 marker loci
Group
AR
TH
M
WM
PM
UM
JM
OM

33
26
279
90
26
61
55
47

17
14
129
23
20
34
27
25
Total
individuals
Total allele
no.
Mean Ho
Mean He
50
40
408
113
46
95
82
72
116
97
166
125
96
131
127
105
0.677
0.671
0.709
0.723
0.683
0.716
0.689
0.719
0.69
0.687
0.736
0.732
0.719
0.74
0.71
0.698
Mean Fis
0.018
0.024
0.036 xx
0.012
0.04
0.033
0.031
– 0.030
xx — P < 0.01.
The global Fst value (overall Fst = 0.048, P < 0.05) describing the differentiation
within the population indicated that about 5% of the total genetic variation is
explained by breed differences, with the remaining 95% corresponding to differences between individuals. Considering the variation of the M population, 2.6% of the
total variation (overall Fst = 0.026, P < 0.05) can be accounted for by differences
between the 5 M subpopulations. The pairwise Fst coefficient used as a distance
measure was lowest between the M subpopulation from Ochaby and Thoroughbred
horses (Fst = 0.014) (Table 6). The Nm value was the greatest between both horse
groups (Nm = 17.063). Excluding Fst and Nm between TH and AR horses, the
highest Fst (0.117) and lowest Nm values (1.882) were obtained between the
M population in Ochaby and the group of Arabian horses. At subpopulation level,
the lowest Fst value (0.02) was between Walewice and Udórz group, which is
correlated with the highest Nm value (12.389) between both Małopolski studs. The
highest Fst and lowest Nm values were reported between the Prudnik and Ochaby
(Fst = 0.038 and Nm = 6.252) and Prudnik and Janów Podlaski studs (Fst = 0.037 and
Nm = 6.436) (Table 6). Relationships between the investigated horse groups revealed
by pairwise Fst estimator were confirmed graphically by a dendrogram based on the
distance calculated from the proportion of shared alleles (Dps) (Figure 1).
Figure 1. Dendrogram based on Dps distance
23
Table 6. Genetic relationships between TH and AR horses, and each of the M subpopulations
AR
AR
TH
0.476
PM
UM
JM
OM
0.4
0.418
0.42
0.398
0.417
0.221
0.274
0.246
0.271
0.179
0.241
0.204
0.215
0.193
0.238
0.254
0.254
0.236
0.226
WM
TH
0.141
(1.528)1
WM
0.096
(2.36)
0.026
(9.369)
PM
0.104
(2.143)
0.043
(5.546)
0.03
(8.108)
UM
0.105
(2.141)
0.03
(8.136)
0.02
(12.389)
0.025
(9.814)
JM
0.096
(2.349)
0.044
(5.396)
0.024
(9.962)
0.037
(6.436)
0.03
(8.103)
OM
0.117
(1.882)
0.014
(17.063)
0.021
(11.868)
0.038
(6.252)
0.029
(8.38)
0.224
0.035
(6.862)
Above diagonal — Dps.
Below diagonal — pairwise Fst.
Nm — in brackets.
Discussion
Population data based on 18 microsatellite markers describe the genetic state of
the current Małopolski population in the largest 5 studs in different parts of Poland.
Analysis of the genetic structure of the investigated populations revealed the
presence of numerous rare alleles characteristic for the Małopolski population.
These alleles may have occurred more frequently in the original population of
Małopolski horses, and most of them have been lost from the current gene pool of
this breed. A group of characteristic alleles found for both M and TH horses shows
that the gene pool of the current Małopolski population is strongly influenced by
Thoroughbred horses. In relation to TH horses, the microsatellite pool of Arabian
horses is more different from the pool of alleles detected in the M population. Great
similarities between the gene pool of M and TH horses were also revealed in the
study by Za˛bek et al. (2005) with the use of 12 microsatellites.
The exact test for Hardy-Weinberg proportions revealed a significant deviation
from genetic equilibrium at four marker loci in the M population, where an increase
in homozygous genotypes was observed. Also, the significant mean value of Fis
(0.036, P < 0.01) indicated an inbreeding-like effect for the whole Małopolski
population. However, the mean values of Fis calculated for subpopulations from
5 Małopolski studs were not significant and were close to 0. Therefore, the increase
in homozygosity in the whole M population results rather from population
subdivision (Wahlund, 1928), where Arabian and Thoroughbred horses are genetically distinct groups contributing to the gene pool of AA horses from the
investigated studs.
24
T. Za˛bek et al.
The study of genetic variation revealed high mean estimates of expected
heterozygosity for the overall Małopolski population, typical of horse breeds
improved with TH horses such as American Quarter horses (Bowling et al., 1997),
Czech warmblood horses (Hamanova et al., 2001) and Oldenburg and Polish
Silesian horses (Za˛bek et al., 2003). The levels of variation estimators differ to
some extent between the subpopulations from particular Małopolski studs and
depend on the proportion of particular breeds in the parental generation of the
studied horses. Higher variation levels expressed by the observed heterozygosity
are present at the Walewice, Ochaby and Udórz studs, probably because of the
greater number of progeny of TH sires and sires of other breeds at these studs
(Table 1). In contrast, horses from Prudnik are characterized by lower variability of
breeds in the parental generation with most parents being AA (Table 1). Meanwhile, the subpopulation from Janów Podlaski includes the smallest number of
mare families, which could reduce the variation level there in comparison with
other Małopolski studs (Table 2).
The differentiation described using the global Fst values calculated at inter- and
intrapopulation level in this study was substantially lower than the differentiation
between geographically isolated horse populations (Canon et al., 2000; Achmann et
al., 2004). The levels of gene exchange expressed as migration rate between some
M subpopulations were higher than the gene exchange between geographically
isolated subpopulations of Lipizzan horses (Achmann et al., 2004). Despite a small
number of individuals derived from two families being present in 3 Małopolski
studs, pedigree records did not show closer relationships between studs of known
mare families. Therefore the presence of common male lines for a number of
M subpopulations and the use of particular stallions for breeding purposes in
different studs may be more important for the degree of relationship between
the investigated Małopolski subpopulations.
It seems that the large contribution of Thoroughbreds to the pedigree of
Małopolski horses determines the genetic differences between the investigated
M subpopulations. The fact that the smallest pairwise Fst and Dps distance was
found between the Walewice and Udórz studs may result from the presence of
a high number of progeny of TH and SP sires at both studs (Table 2). The cluster of
the TH population with the horse group from Ochaby on the phylogenetic tree
expresses the strongest influence of TH horses on this M subpopulation. Distance
data show greater genetic divergence of the Prudnik and Janów group from the
cluster of other M subpopulations because of the presence of specific male lines at
these studs (Table 3) and a smaller number of progeny of TH sires, especially at the
Prudnik stud (Table 1). The greatest phylogenetic divergence of AR horses
expressed as Dps distance and pairwise Fst values shows that the direct contribution
of the AR breed to the present Małopolski population is much smaller in relation to
TH horses.
The genetic similarities described between Małopolski and Thoroughbred
horses are similar to those found in studies of these relations between TH and Czech
warmblood horses (Hamanova et al., 2001) and between TH and German
25
Oldenburg horses (Müller-Eckert et al., 1999; Za˛bek et al., 2003). These result from
a strong tendency towards using TH horses for improvement of a number of warm
blood horse breeds (Müller-Eckert et al., 1999).
Analysis of the genetic structure of the investigated Małopolski population
revealed that its primary gene pool could have been changed by a strong influence
from other horse breeds used for breeding purposes in the Małopolski breed. The
present molecular and pedigree data show that the described M population is not in
danger of inbreeding, and breed variability in pedigrees of Małopolski horses is the
main factor behind the maintenance of genetic variation in this breed. A strong
influence of Thoroughbred horses on the current Małopolski gene pool was
documented.
References
A c h m a n n R., C u r i k I., D o v c P., K a v a r T., B o d o I., H a b e F., M a r t i E., S ö l k n e r J., B r e m G.
(2004). Microsatellite diversity, population subdivision and gene flow in the Lipizzan horse. Anim.
Genet., 35: 285 – 292.
B i n n s M.M., H o l m e s N.G., H o l l i m a n A., S c o t t A.M. (1995). The identification of polymorphic
microsatellite loci in the horse and their use in Thoroughbred parentage testing. Brit. Vet. J., 151: 9 – 15.
B o w c o c k A.M., R u í z - L i n a r e s A., T o m f o h r d e J., M i n c h E., K i d d J.R., C a v a l l i - S f o r z a
L.L. (1994). High resolution human evolutionary trees with polymorphic microsatellites. Nature,
368: 455 – 457.
B o w l i n g A.T., E g g l e s t o n - S t o t t M.L., B y r n s G., C l a r k R.S., D i l e a n i s S., W i c t u m E.,
(1997). Validation of microsatellite markers for routine horse parentage testing. Anim. Genet., 28:
247 – 252.
B r e e n M., L i n d g r e n G., B i n n s M.M., N o r m a n J., I r v i n Z., B e l l K., S a n d b e r g K.,
E l l e g r e n H. (1997). Genetical and physical assignments of equine microsatellites — First
integration of anchored markers in horse genome mapping. Mamm. Gen., 8 (4): 267 – 273.
C a n o n J., C h e c a M.L., C a r l o s C., V e g a - P l a J.L., V a l l e j o M., D u n n e r S. (2000). The genetic
structure of Spanish Celtic horse breeds inferred from microsatellite data. Anim. Genet., 31: 39 – 48.
C o o g l e L. (1996 a). Equine dinucleotide repeat loci from LEX025 to LEX033. Anim. Genet., 27:
289 – 290.
C o o g l e L., B a i l e y E., R e i d R., R u s s M. (1996). Equine dinucleotide repeat polymorphisms at loci
LEX002, -003, -004, -005, -007, -008, -009, -010, -011, -013 and -014. Anim. Genet., 27: 126 – 127.
D i e r i n g e r D., S c h l ö t t e r e r C. (2002). Microsatellite analyser (MSA): a platform independent
analysis tool for large microsatellite data sets. Mol. Ecol. Notes, 3 (1): 167 – 169.
D o b r o w o l s k i M., G e r i n g e r H. (2003). Porównanie ogierów szlachetnych półkrwi, małopolskich
i wielkopolskich zdaja˛cych próby dzielności w Zakładach Treningowych w latach 1977 – 2000 na
podstawie wartości hodowlanej określonej metoda˛ BLUP Animal Model. Mat. 68 Zjazdu Nauk.
PTZ: Zasoby genetyczne zwierza˛t w Polsce, Kraków, 9 – 12.09.2003, p. 192.
E g g l e s t o n - S t o t t M.L., D e l V a l l e A., B a u t i s t a M., D i l e a n i s S., W i c t u m E., B o w l i n g A.T. (1997). Nine equine dinucleotide repeats at microsatellite loci UCDEQ136, UCDEQ405,
UCDEQ412, UCDEQ425, UCDEQ437, UCDEQ467, UCDEQ487, UCDEQ502 and UCDEQ505.
Anim. Genet., 28 (5): 370 – 371.
E l l e g r e n H., J o h a n s s o n M., S a n d b e r g K., A n d e r s s o n L. (1992). Cloning of highly
polymorphic microsatellites in the horse. Anim. Genet., 23: 132 – 133.
G r a l a k B., K u r y ł J., Ł u k a s z e w i c z M., Ż u r k o w s k i M. (1998). Applicability of nine microsatellite DNA sequences vs eleven polymorphic blood protein and enzyme systems for the parentage
control in Polish Arabian and Thoroughbred horse. Anim. Sci. Pap. Rep., 16 (4): 209 – 218.
26
T. Za˛bek et al.
G r a l a k B., N i e m c z e w s k i C., J a w o r s k i Z. (2001). Genetic polymorphism of 12 microsatellite
markers in Polish Primitive Horse. Anim. Sci. Pap. Rep., 19 (4): 277 – 283.
G u é r i n G., B e r t a u d M., A m i g u e s Y. (1994). Characterization of seven new horse microsatellites:
HMS1, HMS2, HMS3, HMS5, HMS6, HMS7 and HMS8. Anim. Genet., 25, p. 62.
G u o S.W., T h o m p s o n E.A. (1992). Performing the exact test of Hardy-Weinberg proportion for
multiple alleles. Biometrics, 48: 361 – 372.
H a e r i n g e n H. van, B o w l i n g A.T., L e n s t r a J.A., Z w a a g s t r a K.A., S t o t t M.L. (1994).
A highly polymorphic horse microsatellite locus VHL20. Anim. Genet., 25: p. 207.
H a m a n o v a K., M a j z l i k I., G l a s n a k V., S c h r ö f f e l o w a D., (2001). Characterization and
comparison of some horse breeds in Czech Republic as based on microsatellite markers
polymorphism. Anim. Sci. Pap. Rep., 19, 3: 219 – 230.
H o l m S. (1979). A simple sequentially rejective multiple test procedure. Scand. J. Statist., 6: 56 – 70.
I r v i n Z., G i f f a r d J., B r a n d o n R., B r e e n M., B e l l K. (1998). Equine dinucleotide repeat
polymorphisms at loci ASB21, 23, 25 and 37 – 43. Anim. Genet., 29: p. 67.
K a w a s a k i E.S. (1990). Sample preparation from blood, cells and other fluids. In: Innis M.A., Gelfand
D.H., Sninsky J.J., White T.J. (eds), PCR Protocols: a Guide to Methods and Applications, Acad.
Press. New York, pp. 146 – 152.
M a r k l u n d S., E l l e g r e n H., E r i k s s o n S., S a n d b e r g K., A n d e r s s o n L. (1994). Parentage
testing and linkage analysis in the horse using a set of highly polymorphic microsatellites. Anim.
Genet., 25: 19 – 23.
M ü l l e r - E c k e r t A., C h o l e w i ń s k i G., B e u i n g R., E r h a r d t G. (1999). Charakterisierung der
verwandschaftlichen Beziehungen von Schweren Warmblutpopulationen auf oldenburgischostfriesischer Grundlage und Deutschen Reitpferdepopulationen anhand von polymorphen Genorten. Züchtungskunde, 71: 359 – 370.
N e i M. (1978). Estimation of average heterozygosity and genetic distance from a small number of
individuals. Genetics, 89: 583 – 590.
N i e m c z e w s k i C., Ż u r k o w s k i M. (2000). The genetic structure of four families of Thoroughbred
Horse as determined on the basis of the polymorphism of chosen class I and II genetic markers.
Anim. Sci. Pap. Rep., 1: p. 5.
P a g e R.D.M. (1996). TREEVIEW: An application to display phylogenetic trees on personal computers.
Computer Appl. Biosci., 12: 357 – 358.
W a h l u n d S. (1928). Zusammensetzung von Populationen und Korrelationserscheinungen von Standpunkt der Verebungslehre aus betrachtet. Hereditas, 11: 65 – 106.
W e i r B.S., C o c k e r h a m C.C. (1984). Estimating F-statistics for the analysis of population structure.
Evolution, 38: 1358 – 1370.
W r i g h t S. (1969). The theory of gene frequencies: evolution and the genetics of populations. Vol. 2.
Chicago University Press, Chicago, USA.
Z a˛b e k T., D u n i e c M., B u g n o M. (2003). Genetic relationships between Silesian, Thoroughbred
and Oldenburg horses based on DNA microsatellite polymorphism. Ann. Anim. Sci., 3, 2:
213 – 224.
Z a˛b e k T., N o g a j A., R a d k o A., N o g a j J., S ł o t a E. (2005). Genetic variation of endangered Polish
Biłgoraj horses and two common horse breeds at the microsatellite loci. J. Appl. Genet., 46 (3):
299 – 305.
Accepted for printing 21 II 2006
27
TOMASZ ZA˛BEK, AGATA ŻYGA, ANNA RADKO, EWA SŁOTA
Analiza zmienności genetycznej koni małopolskich, na podstawie badań molekularnych i danych
rodowodowych
STRESZCZENIE
Konie małopolskie posiadaja˛ w rodowodach różny udział krwi anglo-arabskiej i koni w typie
orientalnym. Od 1999 roku obserwuje sie˛ systematyczny spadek wielkości populacji tych koni.
W prezentowanej pracy określono zmienność genetyczna˛ 5 subpopulacji koni małopolskich, koni pełnej
krwi angielskiej oraz koni czystej krwi arabskiej, z wykorzystaniem 18 markerów mikrosatelitarnych.
Analiza struktury genetycznej wykazała, że pula genów typowych dla wyjściowej populacji koni
małopolskich mogła ulec ograniczeniu w wyniku stosowania dużego dolewu krwi koni innych ras,
a szczególnie pełnej krwi angielskiej. Poziom zróżnicowania genetycznego koni małopolskich, wyrażony heterozygotycznościa˛ oczekiwana˛, jest zbliżony do zmienności innych ras powstałych w wyniku
mie˛dzyrasowych krzyżówek. Badana populacja koni małopolskich nie jest zagrożona wzrostem inbredu,
a głównym źródłem zmienności genetycznej populacji tych koni jest obecność innych ras w rodowodach
koni małopolskich, używanych do ich uszlachetniania. Analiza dystansu genetycznego wykazała
najwie˛kszy wpływ koni pełnej krwi angielskiej na pule˛ genów obecnej populacji koni małopolskich.
EVALUATION OF PUTATIVE RELATIONSHIP BETWEEN PRNP
OCTAPEPTIDE REPEAT POLYMORPHISM AND VARIABILITY
OF MILK PRODUCTION TRAITS IN CATTLE
U r s z u l a C z a r n i k 1, T a d e u s z Z a b o l e w i c z 1, C h a n d r a S . P a r e e k 1,
R y s z a r d Z i e m i ń s k i 2, K r z y s z t o f W a l a w s k i 1
1
Department of Animal Genetics, University of Warmia and Mazury, Oczapowskiego 5,
10-718 Olsztyn, Poland
2
Department of Cattle Breeding and Milk Production, Agricultural University, Chełmońskiego 38C,
51-631 Wrocław, Poland
Abstract
Previous investigations have focused on the association between prion protein (PRNP) and BSE
susceptibility. The usefulness of PRNP polymorphism as a possible quantitative trait locus
(QTL)-linked marker has generally been overlooked. The aim of this study was to evaluate PRNP
octapeptide repeat polymorphism as a putative factor of milk production trait variability. This
experiment was designed to separately investigate the segregation effects of PRNP 6 and
PRNP 5 alleles caused by the sire. The study covered 495 cows from large herds of Black-andWhite cattle, made up of 222 randomly tested cows representing an outbreeding population and
two half-sibling families of 106 cows related to the PRNP 6/5 sire and 167 cows related to the
PRNP 6/6 sire. The milk production trait database was comprised of milk, fat and protein yield
and content, evaluated over a 305-day lactation I period. Statistically significant differences were
found in fat yield and protein content (P ≤ 0.05) between animal groups representing PRNP 6/6 and
PRNP 6/5 genotypes. However, the results within animal groups revealed highly significant
(P ≤ 0.01) and significant (P ≤ 0.05) differences only in the case of animal groups representing
PRNP 6/6, where significant differences (P ≤ 0.05) were found for fat yield and protein yield and
highly significant differences (P ≤ 0.01) for fat content and protein content, in the case of progenies
originating from the PRNP 6/6 sire and randomly tested cows. Finally, significant differences
(P ≤ 0.05) in protein content were also obtained in the progenies originating from the PRNP 6/6 and
PRNP 6/5 sires.
Key words: Black-and-White cattle, PRNP polymorphism, milk production traits, QTL marker
Conformational destabilization of the prion protein (PRNP) molecule has been
recognized as a causal factor in neurodegenerative disorders. PRNP is a glycoprotein with four α-helixes, while its pathogenic variant, after refolding of the two
30
U. Czarnik et al.
α-helixes, is transformed into a β-helix structure. The physiological role of
normal PRNP has not been fully explained. However, a relationship with synaptic
function (Collinge et al., 1994) and neuronal survival (Chen et al., 2003) has been
postulated.
The genetic background of transmissible encephalopathies has been well
researched (Weissmann, 1996; Agrini et al., 2002; Mead et al., 2003; Goldfarb et
al., 2004). In cattle, a PRNP gene was initially located within the U 11 syntenic
group (Ryan and Womack, 1993) and physically mapped on BTA-13q17 (Schläpfer
et al., 1998). Three exons comprising a total of 795 bp (Yoshimoto et al., 1992),
a partial genomic nucleotide sequence of 4244 bp (Horiushi et al., 1998) and
a complete 78056 bp of genomic DNA sequences (Hills et al., 2001) were identified
in the bovine PRNP gene. It was found that in cattle, mainly only exon 3 is
translated. Initially, two mutations were detected, i.e. deletion of 23 nucleotides
within the ORF region of exon 3 as well as CT transition within the 3’ flanking
fragment (Goldmann et al., 1991). Further studies (Hills et al., 2001) found many
other point mutations and the total number of mutations is currently considered to
be about 60 (Sander et al., 2004).
Polymorphism of the octarepeat fragment of Pro-His/Gln-Gly(Gly)-Gly-TryGly-Gln amino acids is expressed by the occurrence of three allelic variants. In all
the examined cattle breeds, the six-repeat allele (PRNP 6) is the most common. The
five-repeat allele (PRNP 5) is less common (McKenzie et al., 1992; Brown et al.,
1993; Hunter et al., 1997; Premzl et al., 2000; Walawski and Czarnik, 2003), and
the seven-repeat allele (PRNP 7) has been identified exclusively in one breed of
Brown Alpine cattle (Schläpfer et al., 1999; Leone et al., 2002).
Cattle population studies to date have been dominated by the search for
a relationship between PRNP polymorphism and susceptibility to BSE (McKenzie
et al., 1992; Neibergs et al., 1994; Hunter et al., 1997; Sander et al., 2004).
Occurrence of the PRNP 5/5 homozygous genotype in the infected animal group
has not yet been found. However, the very low frequency of the PRNP 5/5 genotype
has caused real difficulties in the statistical verification of this regularity. Recently,
in studies involving BSE-infected and healthy animals with PRNP 6/6 and PRNP
6/5 genotypes (Sander et al., 2004), statistically significant differences in PRNP
allele frequency were found. The PRNP 5 allele was more frequent in infected than
in healthy animals. On the other hand, genome-wide analysis (Hernandez-Sanchez
et al., 2002) indicates putative markers associated with BSE in chromosome 5, but
not in chromosome 13.
In the studies completed to date, the possible use of PRNP polymorphism as
a QTL marker has not been mentioned. The aim of the current study was to revise
commonly observed abnormalities in the segregation of PRNP 6 and PRNP
5 alleles as well as to make a preliminary assessment of the putative relationship
between PRNP octapeptide repeat polymorphism and variability of milk production
traits.
Relationship between PRNP polymorphism and milk traits in cattle
31
This study examined a total of 495 contemporary cows at lactation I, originating
from large herds of Black-and-White cattle. The experiment was designed to enable
verification of the co-segregation effect caused by PRNP 6 and PRNP 5 alleles
transmitted by sires and cows and expressed by variability of milk production traits.
Therefore, three groups of cows, comprised of 222 randomly tested animals from
two herds and related to 28 AI sires as well as a half-sib group of 106 cows related
to a PRNP 6/5 sire and a half-sib group of 167 cows related to a PRNP 6/6 sire
originating from five herds, were qualified for experimental analysis.
The sources of the genomic DNA were the spermatozoa of the sire semen and
cows’ peripheral blood leukocytes. Polymorphism caused by deletion/insertion of
23 nucleotides in the ORF region of PRNP gene exon 3 was determined using the
PCR method according to previously described procedures (Walawski and Czarnik,
2003). The PCR products were subjected to electrophoresis in 1.5% Amplisize
Agarose (Bio-Rad) gel. Polymorphism was recorded using a Fluor STM Multimager
(Bio-Rad).
The milk trait database was comprised of milk yield and fat and protein yield, as
well as the fat and protein content of milk from the 305 days of lactation I.
Particular PRNP genotype groups, represented by an outbreeding herd of randomly
examined cows as well as half-sib cow groups originating from PRNP 6/5 and
PRNP 6/6 sires, were characterized on the basis of arithmetical mean values and
standard deviation.
The relationship between prion protein gene polymorphism (PRNP) and milk
production traits was verified independently in subsequent sire families using the
statistical program STATISTICA 6. For this, the ANOVA analysis was performed
in the non-orthogonal form with interactions. The effect of milk production traits on
animal groups as well as genotype groups was also analysed as the dependent
factor, using the following model:
yijk = µ + aj + bj + (ab)ij + eijk
where:
yijk — milk production trait values,
µ — population mean,
aj — influence of genotypes,
bj — influence of animal groups,
(ab)ij — interaction,
eijk — random error.
The test of significant differences between average arithmetical means in
animal groups was verified using Scheffe’s test.
32
U. Czarnik et al.
Results
Table 1 shows the distribution of analysed animals (n = 495) with respect
to PRNP genotype originating from outbreeding herd and half-sib sire families.
In the analysed group of 495 animals, 368 cows were identified as PRNP 6/6
genotypes and 124 cows as 6/5 genotypes. This result indicated that the specific
genetic structure of the cow group originating from the PRNP 6/6 sire, which
is manifested in progeny by a very low percentage of PRNP 6/5 heterozygous
animals, indicates the possible effect of restricted transmission of the PRNP 5 allele
by dams.
Table 1. Numbers of animals (n) investigated for the PRNP octapeptide repeat polymorphism and
variability of milk production traits in Polish Black-and-White cattle
Genotype
groups
PRNP 6/6
PRNP 6/5
PRNP 5/5
Total
Animal groups
progeny of sire genotype
PRNP 6/5
PRNP 6/6
50
53
3
106
157
10
0
167
randomly tested
cows
Total
161
61
0
222
368
124
3
495
However, very few animals were genotyped as PRNP 5/5. This genotype was
registered in 3 cows for progenies originating from the sire genotyped as PRNP 6/5.
The differences between PRNP genotypes and milk production traits, analysed
between the animal groups representing PRNP 6/6 and PRNP 6/5 genotypes and
within the animal groups representing half-sib cows originating from PRNP 6/5 and
PRNP 6/6 sires and in the randomly tested cows, are presented in Table 2.
Comparison of the results obtained for the animal groups representing PRNP
6/6 and PRNP 6/5 genotypes revealed that the mean values of milk fat yield and
protein content differ significantly (P ≤ 0.05) in the analysed animal groups for milk
production traits. The mean values recorded were higher for milk fat yield and
lower for protein content in the PRNP 6/5 genotypes.
However, based on the results within animal groups, highly significant
(P ≤ 0.01) and significant effects (P ≤ 0.05) were observed only in the case of animal
groups representing PRNP 6/6. Significant differences (P ≤ 0.05) were observed
between progenies originating from the PRNP 6/6 sire and randomly tested cows in
the case of fat yield and protein yield. Likewise, highly significant differences
(P ≤ 0.01) were observed between progenies originating from the PRNP 6/6 sire and
randomly tested cows for fat content and protein content. Additionally, significant
differences in protein content (P ≤ 0.05) were found between progenies originating
from PRNP 6/6 and PRNP 6/5 sires.
33
Table 2. Differences between PRNP genotypes and milk production traits in half-sib cows originating
from PRNP 6/5 and PRNP 6/6 sires and in randomly tested cows
Genotype
groups
Animal groups
n
Statistical Milk
measures yield
(kg)
Fat
yield
(kg)
Protein
content
(%)
yield
(kg)
content
(%)
PRNP 6/6 progeny originating
from sire genotype
PRNP 6/5
50
x̄
SD
6252 278.65
1410
63.72
4.47
0.45
207.08
44.63
3.32 a
0.19
progeny originating
from sire genotype
PRNP 6/6
157
x̄
SD
6469 294.07 a
1122
54.17
4.56 A
0.49
220.58 a
36.52
3.41 Ab
0.21
randomly tested cows 161
x̄
SD
6431 275.86 b
1228
61.74
4.32 B
0.60
209.34 b
43.59
3.29 B
0.19
Total
368
x̄
SD
6424 283.94 x
1209
59.40
4.44
0.55
213.81
41.19
3.35 x
0.21
PRNP 6/5 progeny originating
from sire genotype
PRNP 6/5
53
x̄
SD
6810 317.44
1329
70.64
4.62
0.61
225.02
48.06
3.27
0.22
progeny originating
from sire genotype
PRNP 6/6
10
x̄
SD
6323 287.10
917
61.83
4.52
0.56
212.00
31.14
3.35
0.14
randomly tested cows
61
x̄
SD
6569 288.22
1457
65.72
4.42
0.55
215.38
45.83
3.29
0.18
124
x̄
SD
6653 300.79 x
1364
68.63
4.52
0.58
219.28
45.78
3.29 x
0.20
Total
x — Differences between the PRNP genotypes were significant (P ≤ 0.05). A, B — Within the genotypic group the
differences between animal groups were highly significant (P ≤ 0.01), a, b — within the genotypic group the differences
between animal groups were significant (P ≤ 0.05).
Discussion
The results of this and previous studies (Walawski et al., 2003) suggest that the
statistically significant effect of natural or breeding selection in discriminating
animals with PRNP 6/5 genotype may also concern PRNP 6/6 homozygous
animals.
The distribution of PRNP 6 and PRNP 5 generally exhibits an aberration from
independent allele segregation. The PRNP 5/5 genotype occurs very rarely in some
populations and exclusively in female animals. In studies of Black-and-White cattle
(Walawski et al., 2003), irregular segregation of PRNP alleles was found in
the offspring of heterozygous PRNP 6/5 parents. The genotype distribution
(PRNP 6/6 — 44.8%, PRNP 6/5 — 32.8%, PRNP 5/5 — 22.4%) found in progeny
groups indicates a drastic deformation of the expected genotype frequency rate
(25%: 50%: 25%). A considerable number of PRNP 6/5 heterozygous individuals
34
U. Czarnik et al.
are supposed to be subject to natural elimination in the prenatal period or in the first
weeks after birth. This indicates possible PRNP linkage with an as yet unidentified
lethal gene locus which occurs alternatively in the cis and trans phase. Based on the
results of this and previous studies, it is supposed that the statistically significant
effect of natural selection discriminating animals with PRNP 6/5 may also concern,
in some cases, the PRNP 6/6 homozygous genotype.
The speculative character of the alleged positive and negative effects of
alternative linkage phases of cis and trans between the PRNP locus and other genes
of the BTA 13 chromosome requires further study to verify possible detection of
diversified expression of milk production traits in cows with PRNP 6/6 and PRNP
6/5 genotypes.
Hitherto, in a limited number of experimental results, in only a few cases has the
possible occurrence of QTL microsatellite markers for milk production traits in the
BTA 13 chromosome been indicated. However, statistically significant relationships have been observed between BMS1742, BMC1222, HUJ616 (Mosig et al.,
2001) and BMS1352 (Olsen et al., 2002) markers and for differentiation of protein
yield and content. Another interesting element of the studies completed to date has
been the highly significant association between the occurrence in BTA13 of QTL
markers for milk production traits, as well as for udder conformation traits
(Schrooten et al., 2004). Based on the results of the previous studies, the BTA13
chromosome could be an interesting object of studies to identify the QTL for milk
production traits.
However, the suggested relationships between PRNP polymorphism and fat
yield (our own studies) and protein yield/content and differentiation in milk somatic
cell count (other authors’ studies) require further confirmation. Additionally, the
hypothesis of cis and trans alternative variants of the conjugation of the PRNP
locus and some anonymous loci expressed by lethal and subvital effects needs to be
verified in further research.
References
A g r i n i U., C o n t e M., M o r e l l i L., D i B a r i M.A., D i G u a r d o G., L i g i o s C., A n t o n u c c i G.,
A u f i e r o G.M., P o z z a t o N., M u t i n e l l i F., N o n n o R., V a c c a r i G. (2002). Animal
transmissible spongiform encephalopathies and genetics. Vet. Res. Commun., 27: 31 – 38.
B r o w n R., Z h a n H.M., N i e s e S.K. de, A x R.J. (1993). Bovine prion gene allele frequencies
determined by AMFLP and RFLP analysis. Anim. Biotech., 4: 47 – 51.
C h e n S., M a n g e A., D o n g L., L e h m a n S., S c h a c h n e r M. (2003). Prion protein as trans-acting
partner for neurons is involved in neurite outgrowth and neuronal survival. Mol. Cell Neurosci., 22:
227 – 233.
C o l l i n g e J., W h i t t i n g t o n M.A., S i d l e K.C., S m i t h C.J., P a l m e r M.S., C l a r k e A.R.,
J e f f e r y s J.G. (1994). Prion protein is necessary for normal synaptic function. Nature, 370:
295 – 297.
G o l d f a r b L.G., C e r v e n k o v a L., G a j d u s e k D.C. (2004). Genetic studies in relation to kuru: an
overview. Curr. Ml. Med., 4: 375 – 384.
35
G o l d m a n n W., H u n t e r N., M a r t i n T., D a w s o n H., H o p e J. (1991). Different forms of the
bovine PrP gene have five or six copies of a short G-C element within the protein-coding region.
Gen. Virol., 72: 201 – 204.
H e r n a n d e z - S a n c h e z J., W a d d i n g t o n D., W i e n e r P., H a l e y C.S., W i l l i a m s J.L. (2002).
Genome-wide search for markers associated with bovine spongiform encephalopathy. Mamm.
Genome, 13: 164 – 168.
H i l l s D., C o m i n c i n i S., S c h l a e p f e r J., D o l f G., F e r r e t t i L., W i l l i a m s J.L. (2001).
Complete genomic sequence of the bovine prion gene (PRNP) and polymorphism in its promoter
region. Anim. Genet., 32: 231 – 232.
H o r i u s h i M., I s h i g u r o N., N a g a s a w a H., T o y o d a Y., S h i n g a w a H. (1998). Genomic
structure of the bovine PrP gene and complete nucleoside sequence of bovine PrP cDNA. Anim.
Genet., 29: 37 – 40.
H u n t e r N., G o l d m a n n W., S m i t h G., H o p e J. (1997). Frequencies of PrP gene variants in healthy
cattle and cattle with BSE in Scotland. J. Vet. Record., 135: 400 – 405.
L e o n e P., C a s t i g l i o n i B., S e h i T., C a s s a n i P., S t e l l a A. (2002). Prion gene octa-peptide
variability in the Italian cattle breeds. Proc. 7th World Congress on Genetics Applied in Livestock
Production, 13: p. 40.
M c K e n z i e D.I., C o w a n C.M., M a r s h R.F., A i k e n J.M. (1992). PrP gene variability in the US
cattle population. Anim. Biotech., 3: 309 – 315.
M e a d S., S t u m p f M.P., W h i t f e l d J., B e c k J.A., P o u l t e r M., C a m p b e l l T., U p h i l l J.B.,
G o l d s t e i n D., A l p e r s M., F i s h e r E.M., C o l l i n g e J. (2003). Balancing selection at the prion
gene consistent with prehistoric kuru-like epidemics. Sciece, 300: 640 – 643.
M o s i g M.O., L i p k i n E., K h u t o r e s k a y a G., T c h o u r z i n a E., S o l l e r M., F r i e d m a n n A.
(2001). A whole genome scan for quantitative trait loci affecting milk protein percentage in Israeli
Holstein cattle, by means of selective DNA pooling in a daughter design, using an adjusted false
discovery rate criterion. Genetics, 157: 1683 – 1698.
N e i b e r g s H.L., R y a n A.M., W o m A c k J.E., S p o o n e r R.L., W i l l i a m s J.L. (1994). Polymerase
analysis of the prion gene in BSE-affected and unaffected cattle. Anim. Genet., 25: 313 – 317.
O l s e n H.G., G o m e z - R a y a L., V a g e D.I., O l s a k e r I., K l u n g l a n d H., S v e n d s e n M.,
S a b r y A., K l e m e t s d a G., S c h u l m a n N., K r a m e r W., T h a l l e r G., N i n g e n K.R., L i e n S.
(2002). A genome scan for Quantitative Trait Loci affecting milk production in Norwegian Dairy
Cattle. J. Dairy Sci., 85: 3124 – 3130.
P r e m z l M., B o z i c P., G a m u l i n V. (2000). PRNP octapeptide repeat allele genotype frequencies
among the modern and rare cattle breeds in Croatia. Anim. Genet., 31: 408 – 409.
R y a n A.M., W o m a c k J.E. (1993). Somatic cell mapping of the bovine prion protein gene
and restriction fragment length polymorphism studies in cattle and sheep. Anim. Genet., 24:
23 – 26.
S a n d e r P., H a m a n n H., P f e i f e r I., W e m h e u e r W., B r e n g B., G r o s c h u p M.H., Z i e g l e r U.,
D i s t l O., L e e b T. (2004). Analysis of sequence variability of the bovine prion protein gene
(PRNP) in German cattle breeds. Neurogenetics, 5: 19 – 25.
S c h l ä p f e r J., G a l l a g e r D.S. Jr., B u r z l a f f J.D., W o m a c k J.E., S t e l l y D.M., T a y l o r J.F.,
D a v i s S.K. (1998). Comparative mapping of bovine chromosome 13 by fluorescence in situ
hybridization. Anim. Genet., 29: 265 – 272.
S c h l ä p f e r J., S a i t b e k o v a N., G a i l l a r d C., D o l f G. (1999). A new allelic variant in the bovine
prion protein gene (PRNP) coding region. Anim. Genet., 30: 386 – 389.
S c h r o o t e n M.C., B i n k A.M., B o v e n h u i s H. (2004). Whole genome scan to detect chromosomal
regions affecting multiple traits in dairy cattle. J. Dairy Sci., 87: 3550 – 3560.
W a l a w s k i K., C z a r n i k U. (2003). Prion protein octapeptide-repeat polymorphism in Polish
Black-and-White cattle. J. Appl. Genet., 44: 191 – 195.
W a l a w s k i K., C z a r n i k U., W o j c i e c h o w s k i R., P a r e e k C.S. (2003). Abnormal segregation of
prion protein octa-peptide repeat alleles in cattle. J. Appl. Genet., 44: 375 – 378.
W e i s s m a n n C. (1996). The Ninth Datta Lecture. Molecular biology of transmissible spongiform
encephalopathies. FEBS Lett., 389: 3 – 11.
36
U. Czarnik et al.
Y o s h i m o t o J., J i n u m a T., I s h i g u r o N., H o r i u s h i M., S h i n a g a w a M. (1992). Comparative
sequence analysis and expression of bovine PrP gene in mouse. Virus Genet., 6: 343 – 356.
Accepted for printing 11 XII 2005
URSZULA CZARNIK, TADEUSZ ZABOLEWICZ, CHANDRA S. PAREEK,
RYSZARD ZIEMIŃSKI, KRZYSZTOF WALAWSKI
Analiza zwia˛zku mie˛dzy polimorfizmem powtórzeń ośmiopeptydowego fragmentu genu
białka prionowego PRNP i cechami użytkowości mlecznej bydła
STRESZCZENIE
Dotychczasowe badania koncentrowały sie˛ na poszukiwaniu zależności mie˛dzy polimorfizmem
genu białka prionowego (PRNP) a podatnościa˛ bydła na BSE. Przydatność polimorfizmu PRNP jako
potencjalnego markera QTL była dotychczas pomijana. Celem prezentowanej pracy była ocena
możliwości wykorzystania polimorfizmu PRNP jako czynnika przejawiaja˛cego sie˛ zróżnicowaniem
cech użytkowości mlecznej. Materiał obejmował 495 krów rasy czarno-białej utrzymywanych w dużych
stadach, w tym 222 losowo testowane krowy reprezentuja˛ce populacje˛ masowa˛ i dwie grupy półsióstr
— 106 krów pochodza˛cych po buhaju PRNP 6/5 i 167 krów stanowia˛cych potomstwo buhaja PRNP 6/6.
Kontrolowane cechy użytkowości mlecznej obejmowały: wydajność mleka, wydajność tłuszczu i białka,
zawartość tłuszczu i białka w okresie 305-dniowej I laktacji. Stwierdzono statystycznie istotne różnice
mie˛dzy wydajnościa˛ tłuszczu a zawartościa˛ białka (P ≤ 0,05) pomie˛dzy grupami zwierza˛t o genotypach
PRNP 6/6 i PRNP 6/5. Wyniki w obre˛bie grup ujawniły wysoko istotne (P ≤ 0,01) i istotne (P ≤ 0,05)
różnice tylko w przypadku zwierza˛t o genotypie PRNP 6/6, dla których stwierdzono istotne różnice
(P ≤ 0,05) w wydajności tłuszczu i wydajności białka oraz wysoko istotne różnice (P ≤ 0,01) w zawartości tłuszczu i zawartości białka w przypadku potomstwa pochodza˛cego od buhaja PRNP 6/6 i losowo
testowanych krów. Istotne różnice (P ≤ 0,05) w zawartości białka uzyskano także dla potomstwa
pochodza˛cego od buhajów o genotypach PRNP 6/6 i PRNP 6/5.
TECHNOLOGICAL TRAITS OF MILK OF SIMMENTAL COWS
AS RELATED TO κ-CASEIN POLYMORPHISM
A n d r z e j F e l e n c z a k 1, A n n a F e r t i g 1, E w a G a r d z i n a 1, M a r i a n O r m i a n 1, J a n T r e l a 2
1
Department of Cattle Breeding, Agricultural University, al. Mickiewicza 24/28, 30-059 Kraków, Poland
2
Department of Farm Animal Genetic Resources Conservation,
National Research Institute of Animal Production, 32-083 Balice n. Kraków, Poland
Abstract
The composition and technological properties of milk and milk protein polymorphism were
studied in Simmental cows. κ-casein polymorphism was shown to have a significant differentiating
effect on milk composition and properties. The milk of cows with BB and AB CSN3 genotypes was
characterized by a more favourable composition, i.e. a higher fat, total protein and casein content,
shorter clotting time and higher curd cheese yield. The results obtained indicate that CSN3
genotypes can serve as an additional and valuable source of information in cattle selection.
Key words: cow, milk, κ-casein polymorphism, technological properties
A review of studies on the genetic polymorphism of milk proteins has revealed
that the genes of these proteins are the most studied genes in cattle. Their
chromosomal localization and nucleotide sequence are known. The genes of casein
proteins are localized on chromosome 6, and the main whey protein (β-lactoglobulin) is determined by a gene locus on chromosome 11 (Mercier and Vilotte,
1993). In cattle, a species that is most utilized for milk, genetic polymorphism has
been shown for the main six proteins of milk: αS1-casein (CSN1S1), αS2-casein
(CSN1S2), β-casein (CSN2) and κ-casein (CSN3) as well as β-lactoglobulin (LGB)
and α-lactoalbumin (LALBA) (Eigel et al., 1984).
The aim of studies on milk protein polymorphism is to explain the relationships
between the genetic variants of some proteins and the productive traits and
technological and nutritive properties of milk. The occurrence of such relationships
may be conditioned by the fact that the locus of the analysed proteins is located on
the same chromosome and in the neighbourhood of the loci of genes that determine
desirable traits. Studies to find the markers of dairy production traits in cattle have
been carried out by Czarnik and Walawski (2003), among others.
38
A. Felenczak et al.
The principal milk protein is casein, which occurs in native state in the form of
large, colloidal molecules known as micelles. A significant role in proteins is
played by one of its fractions, κ-casein, which is a major stabilizing factor of the
protein structure as it forms an external layer of casein micelles. The processes
undergone by κ-casein during milk processing are the basis for gel formation and
milk clotting. The CSN3 gene polymorphism affects the production of a specific
protein, which, in turn, influences the course of technological processes during
cheese-making. Previous studies by many authors have shown that milk containing
BB and AB CSN3 polymorphic fractions is characterized by a more favourable
composition and is more suitable for processing than milk with the AA CSN3
fraction (Aaltonen and Antila, 1987; Litwińczuk et al., 1997; Ng-Kwai-Hang, 1994;
Strzałkowska et al., 2000; Ziemiński et al., 2002).
The aim of the study was to examine the composition and technological
properties of cow’s milk and to determine the relationship between the occurrence
of polymorphic fractions of κ-casein and milk traits.
Milk from 100 Simmental cows was investigated. Cows in the herd were milk
recorded using the A4 method and were under constant veterinary supervision. Milk
samples for analysis were collected once a month during test milking throughout lactation. Prior to milk sampling, udder health was examined using the area cell-mediated
test and the somatic cell count (SCC) was determined using a Milkoscan device.
Cow feeding in the herd was divided into summer and winter periods. The
average milk yield of the cows for lactation was around 5400 kg. During the winter
feeding period, the cows’ ration contained maize silage, meadow hay and
concentrate. In the summer period, the basic feed was pasture grass supplemented
with brewer’s grain and concentrate.
Proteins in the analysed milk were separated using horizontal starch gel
electrophoresis, according to the method of Mitjutko et al. (1974), to determine
polymorphic fractions of proteins (αS1-, β- and κ-casein (CSN) and β-lactoglobulin
(LGB)) and the occurrence and frequency of alleles and genotypes.
Milk samples were taken from healthy cows between the 3rd and 8th months of
lactations to determine milk composition and properties. Each time, around 100 ml
of milk per cow was collected for analysis. The milk was analysed using
a Milkoscan device for basic chemical composition, i.e. levels of total protein, fat,
lactose and solids, as well as the casein content according to the method of Walker,
density using the aerometric method, thermostability (alcohol number), potential
acidity according to the method of Soxhlet-Henkel, clotting time using the rennet
method, and cheese yield using the enzymatic method (PN-68/A-86122).
The results were analysed statistically using a multivariate analysis of variance
and the Statistica 6.0 packet. The significance of differences between the means
was analysed using the Scheffe test.
Technological properties of cow’s milk and κ-casein polymorphism
39
Results
The separation of proteins using starch gel electrophoresis allowed simultaneous determination of the polymorphism of four milk protein fractions: betalactoglobulin and alphas1-, beta- and kappa-casein (Table 1). The largest number of
genotypes in the analysed Simmental cattle was found in the β-lactoglobulin (AA,
AB, BB, BD) and β-casein (AA, AB, BB, BC) systems. In each of the other protein
systems, three genotypes were found. In the LGB system, the highest frequency
(0.488) was characteristic of the AB genotype, while the frequency of AA and BB
homozygotes was similar (0.236 and 0.252, respectively). Analysis of Simmental
cows showed the occurrence of a rare genotype, BD LGB (frequency of 0.024).
Table 1. Frequency of genes and genotypes of milk proteins in the Simmental cows
Milk proteins
Genotypes
No. of cows
Frequency
of genotypes
Gene
frequency
β-lactoglobulin
(LGB)
AA
AB
BB
BD
77
166
82
4
0.236
0.488
0.252
0.024
A = 0.486
BB
BC
CC
239
85
5
0.733
0.247
0.020
B = 0.856
AA
AB
BB
BC
240
58
6
25
0.669
0.235
0.020
0.076
A = 0.818
AA
AB
BB
89
171
69
0.281
0.498
0.221
A = 0.530
αS1-casein
(CSN1S1)
β-casein
(CSN2)
κ-casein
(CSN3)
B = 0.502
D = 0.012
C = 0.144
B = 0.144
C = 0.038
B = 0.470
κ-casein polymorphism manifested itself in the presence of two alleles, A and B,
which condition the occurrence of three genotypes of this protein: AA, AB and BB.
The highest frequency was characteristic of the AB genotype (0.498) and the
frequency of the BB CSN3 genotype, which is commonly regarded as beneficial,
was also high. The frequency of the CSN3 A allele was 0.530 and that of the B allele
was slightly lower (0.470).
The results obtained showed that the milk of Simmental cows is characterized
by high levels of fat (4.20%), total protein (3.62%), casein (2.72%) and solids
(13.59%). The milk was characterized by high density (1.0295) and a relatively
short protein coagulation time (343 s). The average somatic cell count in the
analysed milk was 220,000/cm3.
40
A. Felenczak et al.
The present study showed that CSN3 polymorphism is a significant differentiating factor of milk composition, i.e. the total protein and casein content (Table 2).
The milk containing the polymorphic BB CSN3 fraction was characterized by the
highest total protein and casein content, and the difference in relation to the milk
with the AA CSN3 type was 0.19% in both cases. The milk with the AB CSN3 type
was characterized by an intermediate protein content, which was significantly
higher than that of the milk with the AA CSN3 type. The percentage of fat in the
milk containing the AB and BB CSN3 polymorphic fractions was similar but higher
than that in the milk of AA CSN3 homozygous cows.
Table 2. Technological milk properties of cows with different κ-casein genotypes
CSN3 genotypes
Traits
AA
x̄
N
Fat (%)
Crude protein
(%)
Casein (%)
Lactose (%)
Solids (%)
Density
(g/cm3)
Acidity (oSH)
Thermal
stability
AB
SD
x̄
161
4.13
3.50 AB
2.62 AB
4.76
13.23
BB
SD
x̄
307
SD
138
0.62
4.22
0.60
4.20
0.64
0.43
0.28
0.42
0.90
3.66 A
2.74 A
4.80
13.83
0.43
0.26
0.36
0.63
3.69 B
2.81 B
4.78
13.49
0.39
0.31
0.40
0.92
1.0292 ab
7.09
0.018
0.06
1.0296 a
7.17
0.015
0.07
1.0297 b
7.26
0.016
0.06
4.99
0.31
4.98
0.32
5.12
0.38
Values in rows with the same letter are significantly different: aa — P ≤0.05; AA — P ≤ 0.01.
Significant differences between the CSN3 genetic groups were also observed for
milk properties, i.e. protein clotting time, density and cheese yield. The shortest
clotting time (308 s) was characteristic of the milk of BB CSN3 homozygous cows.
It was 48 s shorter than in the AA CSN3 genotype group (Figure 1). Similarly, the
highest cheese yield was found for the milk of cows with the BB CSN3 genotype
and the difference in relation to AA homozygotes exceeded 4% (Figure 2). In
addition, the milk containing the polymorphic BB CSN3 fraction was characterized
by the highest density and a high alcohol number, which indicates that its thermal
stability was good. The milk containing the AB CSN3 fraction had the highest levels
of fat and solids. As regards the other milk traits, no large differences were found
between the genotype groups.
41
Figure 1. Protein clotting time from milk of cows with different κ-casein genotypes
Figure 2. Cheese yield from milk of cows with different κ-casein genotypes
Discussion
Total protein and casein are particularly important parameters of cow’s milk
composition, both in terms of the nutritive value of milk and its suitability for
processing. Milk with a higher level of these components has superior technological
properties as it is more useful for cheese-making. The results obtained indicate
a positive effect of the B CSN3 allele on the above traits. These findings are
confirmed by Ikonen (2000) and Wan and Ng-Kwai-Hang (1997). Likewise,
Mackle et al. (1998) obtained statistically significant differences in the total protein
and casein content in cows with the AA and BB CSN3 genotype. They found that the
milk of cows with the homozygous type BB CSN3 contained as much as 0.43%
more total protein than that of AA homozygotes.
Clotting time and cheese yield are very important technological parameters of
milk. In light of the results available, many authors hold the view that the selection
of dairy cows with the CSN3 genotype might help to improve these parameters
(Dikkeboom et al., 2000; Fitzgerald, 1998; Felenczak et al., 2002).
42
A. Felenczak et al.
In the present study, we found that the B CSN3 allele has a significant effect on
protein clotting time and cheese yield. This influence was additive. Likewise,
Ikonen (2000) indicated the B CSN3 allele as a factor affecting milk clotting
processes through significant shortening of the flocculation time and better
compactness of the casein clot.
According to Summer et al. (2002), milk containing the homozygous form of
the B CSN3 fraction is characterized by considerably higher level of CSN3 in solids,
and thus a higher proportion of micelles of low diameter. Such micelles, in turn,
show better reactivity with the rennet, which directly produces a greater amount of
casein clots. All these characteristics have a considerable effect on the clotting
properties of cow’s milk and on cheese yield. In the studies by Fitzgerald (1998),
milk of the BB CSN3 type had a 7 min shorter clotting time and an 8% higher
cheese yield in relation to milk of the AA CSN3 type. Buchberger and Dovč (2000)
reported that milk from cows with the BB CSN3 genotype was characterized by
a 3 – 6% higher Cheddar cheese yield.
It is concluded that the milk of the analysed cows was characterized by a high
total protein, casein and fat content. CSN3 polymorphism was a factor that
significantly differentiated milk composition and properties. The milk of cows with
the BB and AB CSN3 genotypes was characterized by a more favourable composition, shorter clotting time and higher curd cheese yield. The research results
obtained so far demonstrate that CSN3 genotypes can be an additional and valuable
source of information in the selection of dairy cattle.
References
A a l t o n e n M.L., A n t i l a V. (1997). Milk rennet properties and the genetic variants of proteins.
Milchwissenschaft, 42, 8: 490 – 492.
B u c h b e r g e r J., D o v č P. (2000). Lactoprotein genetic variants in cattle and cheese making ability.
Food Techn. Biotechn., 38 (2): 91 – 98.
C z a r n i k U., W a l a w s k i K. (2003). Cicha mutacja genu podjednostki CD18 ITGB2 jako potencjalny
marker cech użytkowości mlecznej bydła. Zesz. Nauk. PTZ, Prz. Hod., 69: 9 – 17.
D i k k e b o o m A.L., C h e n C.M., J a e g g i J.J., J o h n s o n M.E., T r i c o m i W.A., Z i m b r i c M.G.
(2000). Milk proteins and cheese composition — the influence of genetic variants. Dairy Pipeline,
12, 3: 6 – 7.
E i g e l W.N., B u t l e r J.E., E r n s t r o m C.A., F a r r e l l H.P., H a r w w a l k a r V.R., J e n n e s s R.,
W h i t n e y R.M. (1984). Nomenclature of proteins of cow’s milk: fifth revision. J. Dairy Sci., 67:
1599 – 1631.
F e l e n c z a k A., S z a r e k J., G a r d z i n a E. (2002). Polymorphism of proteins versus composition and
properties cow milk. Proc. Int. Sci. Conf.: XX Genetic Days, Brno, 2002; pp. 49-51.
F i t z g e r a l d R.J. (1998). Genetic variants of milk proteins — Relevance to milk composition and
cheese production. End of project report. Dairy Prod. Res. Centre, 19: 3 – 11.
I k o n e n T. (2000). Possibilities of genetic improvement of milk coagulation properties of dairy cows.
University of Helsinki, Department of Animal Science, http://ethesis.helsinki.fi/julkaisut/maa/kotie/
vk/ikonen/contents.html.
L i t w i ń c z u k Z., L i t w i ń c z u k A., B a r ł o w s k a J., C h a b u z W. (1997). Porównanie wydajności
i składu mleka krów rasy czarno-białej i jersey oraz ich mieszańców F1 (cb × jersey) ze
43
szczególnym uwzgle˛dnieniem polimorfizmu białek. Ann. Univ. M.C.-S., Lublin, Polonia, XV, 2:
7 – 13.
M a c k l e T.R., B r y a n t A.M., P e t c h S.F., H i l l J.P., A u l d i s t H.J. (1998). Nutritional influences on
the composition of milk from cows of different protein phenotypes in New Zealand. J. Dairy Sci.,
82: 172 – 180.
M e r c i e r J.C., V i l o t t e J.L. (1993). Structure and function of milk protein genes. J. Dairy Sci., 76, 10:
3079 – 3098.
N g - K w a i - H a n g K.F. (1994). Genetic variants of milk proteins and cheese yield. Int. Dairy Feder.,
Brussels, Special Issue, 9402: 160 – 166.
S t r z a ł k o w s k a N., K r z y ż e w s k i J., R y n i e w i c z Z. (2000). Wpływ genotypu beta-laktoglobuliny
i kappa-kazeiny na wydajność, skład chemiczny i podstawowe parametry technologiczne mleka
krów cb. Pr. Mat. Zoot., 56: 107 – 119.
S u m m e r A., M a l a c a r n e M., M a r t u z z i F., M a r i a n i P. (2002). Structural and functional
characteristics of modenese cow milk in parmigiano-reggiano cheese production. Ann. Fac. Medic.
Vet., Di Parma, 22: 163 – 174.
W a n X i a o c h u n, N g - K w a i - H a n g K.F. (1997). Effect of genetic variants of κ-casein and
β-lactoglobulin on cheese yielding capacity of Ayrshire milk. www.mcgill.ca/animal/publications/1997/ ‘‘Research reports 1997”: pp. 67 – 70.
Z i e m i ń s k i R., J u s z c z a k J., W a l a w s k i K. (2002). Association between milk protein polymorphism and lifetime production traits in herds of Black-and-White and Red-and White cattle
improved by Holstein-Friesian sires. Ann. Anim. Sci., 2 (1): 29 – 40.
Accepted for printing 23 III 2006
ANDRZEJ FELENCZAK, ANNA FERTIG, EWA GARDZINA, MARIAN ORMIAN, JAN TRELA
Cechy technologiczne mleka krów rasy Simental oraz ich zwia˛zek z polimorfizmem κ-kazeiny
STRESZCZENIE
Badano skład, właściwości technologiczne oraz polimorfizm białek mleka krów rasy Simental.
Wykazano, że polimorfizm κ-kazeiny był czynnikiem różnicuja˛cym istotnie skład i właściwości mleka.
Mleko pochodza˛ce od krów o genotypach BB i AB CSN3 charakteryzowało sie˛ korzystniejszym składem,
tj. wyższa˛ zawartościa˛ tłuszczu, białka ogólnego i kazeiny, a także krótszym czasem koagulacji i wyższa˛
wydajnościa˛ sera twarogowego. Uzyskane wyniki wskazuja˛, że genotypy CSN3 moga˛ stanowić
dodatkowe, cenne źródło informacji w selekcji bydła.
ANALYSIS OF THE RELATIONSHIP BETWEEN TWO SINGLE
NUCLEOTIDE POLYMORPHISMS OF THE BUTYROPHILIN (BTN1A1)
GENE AND MILK PRODUCTION TRAITS IN JERSEY CATTLE*
J o l a n t a K o m i s a r e k, K r y s t y n a W a ś k o w i c z, Z b i g n i e w D o r y n e k
Department of Cattle Breeding and Milk Production, Agricultural University,
Wojska Polskiego 71 A, 60-625 Poznań, Poland
Abstract
The aim of the present study was to analyse the relationship between the P35Q and K468R single
nucleotide polymorphisms of the butyrophilin (BTN1A1) gene and milk production traits in cattle.
The investigation was conducted on a population of 219 Jersey cows. The following allele
frequencies were found: P35Q — 0.84 (A) and 0.16 (C); K468R — 0.66 (A) and 0.34 (G). The
analysis did not reveal a significant association for P35Q polymorphism. For the K468R mutation,
the AA genotype was characterized by significantly higher milk, fat, and protein yields than the AG
and GG genotypes. The relationship has not, however, been confirmed by within-sire family
analysis.
Key words: cattle, butyrophilin, gene polymorphism, milk traits
Butyrophilin (BTN1A1) is a protein specifically expressed on the apical surface
of the mammary epithelial cells in the final stage of pregnancy and during lactation
(Aoki et al., 1997; Franke et al., 1981; Ogg et al., 1996). It is also the most abundant
protein in the membrane surrounding milk-fat droplets (Mather et al., 1980; Mondy
and Keenan, 1993). Although the function of BTN1A1 is not fully understood, its
expression profile suggests an important role in lactation. Recently, a generation of
BTN1A1-ablated mice supplied evidence that butyrophilin is required for proper
milk-fat secretion (Ogg et al., 2004).
Butyrophilin is a type I transmembrane glycoprotein containing an N-terminal
exoplasmic domain with two Ig-like folds, a centrally located single membrane
anchor, and a C-terminal cytoplasmic domain with a highly conserved B30.2 region
(Banghart et al., 1998). The presence of Ig-like folds within the protein structure
* This work was conducted as part of the research project no. 2 P06D 017 26, financed by the State
Committee for Scientific Research.
46
J. Komisarek et al.
makes butyrophilin a member of the Ig superfamily (Gardinier et al., 1992) and
indicates its possible immunologic function, unrelated to milk-fat secretion.
The BTN1A1-encoding gene has been described in several mammalian species,
including cattle (Jack and Mather, 1990), humans (Taylor et al., 1996 b) and mice
(Ishii et al., 1995). Both gene structure and protein amino acid sequence show
a high degree of conservation (Ogg et al., 1996; Taylor et al., 1996 b).
The bovine BTN1A1 gene has been mapped to chromosome 23 (Ashwell et al.,
1996 a; Taylor et al., 1996 a). In the same genome region, several quantitative trait
loci (QTLs) for health and production traits have been identified. Most of them
were found to influence the somatic-cell content of milk (Ashwell et al., 1996 b;
1998; Heyen et al., 1999; Holmberg and Andersson-Eklund, 2004), but the
presence of putative genes affecting milk yield and composition was also reported
(Ashwell et al., 1997; Bennewitz et al., 2003; 2004; Zhang et al., 1998).
Butyrophilin might serve as a possible candidate influencing the variation in the
QTLs detected.
The aim of the present study was to analyse the relationship between P35Q and
K468R single nucleotide polymorphisms (SNPs) and milk production traits in
Jersey cattle. P35Q is due to the C → A substitution in exon 3 of the BTN1A1 gene
(Seyfert and Lüthen, 1998) resulting in the amino acid change from proline
to glutamine at the IgI domain, whereas K468R is the A → G mutation in
exon 8 (Taylor et al., 1996 a) that leads to the lysine arginine replacement within
the B30.2 region.
The study included 219 Jersey cows born between 1996 and 2002 and kept on
the Siedlec farm belonging to the Horse Stud Farm in Iwno. The studied animals
were the progeny of 21 sires. The average number of daughters per sire was 10.4
and ranged from 1 to 51.
Genomic DNA was isolated from blood using the phenol-chloroform method,
according to the standard protocol. Genotypes were determined using the PCRRFLP technique. Primer sequences for PCR were established on the basis
of the gene sequence available in the GenBank database (entry No. Z93323),
using PRIMER3 software (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3 —
www.cgi):
P35Q-F: 5’ — TGGTAGGTCAGGAAGCCATC — 3’
P35Q-R: 5’ — GTATTCAGCCATCTCCTCGC — 3’
K468R-F: 5’ — TGGAGCTCTATGGAAATGGG — 3’
K468R-R: 5’ — ACCCTTTGGGTTTTCTGCTT — 3’
The PCR reaction volume of 10 µl contained approximately 50 ng of genomic
DNA, 0.5 units of Taq DNA polymerase (Fermentas), 1 × PCR buffer with
(NH4)2SO4 (Fermentas), 2 mM MgCl2, 5% DMSO, 1 µM of each primer, and
200 µM of each dNTP. Thermal cycling conditions included an initial denaturation
Relationship between polymorphisms of BTN1A1 gene and milk production traits in cattle
47
at 94oC for 5 min, followed by 30 cycles at 94oC for 30 s, 58oC (P35Q) or 56oC
(K468R) for 30 s, and 72oC for 40 s, followed by a final extension at 72oC for 5 min.
The PCR reactions were carried out using a TGradient thermocycler (Biometra).
The amplified P35Q and K468 fragments were digested overnight at 37oC with
5 units of BcnI and BsuRI (HaeIII) restriction endonucleases (Fermentas), respectively, then subjected to electrophoretic separation in 2.5% ethidium bromidestained agarose gel (BASICA LE GQT, Prona).
The frequencies of the alleles and genotypes were estimated, and a chi-square
analysis was performed to test whether the genotype distributions obtained
complied with the Hardy-Weinberg law. The effect of genotypes on milk traits was
tested using the GLM procedure of the SAS package (SAS, 1989). The statistical
model included the effects of BTN1A1 genotypes, the sire effect, and the effect of
lactation number and year and season of calving. The analysis was carried out on
the group of 185 cows with at least one 305-day complete lactation (185 animals
with one lactation, 123 with two lactations, and 91 with three lactations). The
additional test was performed on two families headed by those sires with the highest
number of daughters (51 and 47 cows, respectively). The model used for the
within-family analysis included the effects of BTN1A1 genotypes (K468R), the
effect of lactation number, and the effect of the year and season of calving. Data for
305-day lactations, including overall milk, fat and protein yields as well as fat and
protein contents, originated from the routine control of milk performance and were
obtained from the farm documentation.
Results
The PCR amplifications resulted in 574 bp and 780 bp long DNA products for
P35Q and K468R butyrophilin gene polymorphisms, respectively. Five BsuRI
restriction sites were found in the K468R lysine-encoding A allele, at PCR product
positions 162, 175, 185, 556 and 693. In the arginine-encoding G allele, an
additional restriction site at position 218 was present. The P35Q fragment, digested
with the BcnI restriction enzyme, was visible on the gel as two (74 and 500 bp) or
three (74, 96 and 404 bp) bands for the A (glutamine) and C (proline) alleles,
respectively.
Among the 219 cows examined, the P35Q AA genotype was identified in 154,
AC in 59, and CC in 6 animals, giving allele frequencies of 0.84 (A) and 0.16 (C).
For the K468R mutation, the following genotype and allele frequencies were
obtained: AA — 0.45, AG — 0.43, and GG — 0.12; A — 0.66, and G — 0.34,
respectively. All genotypes were distributed according to the Hardy-Weinberg
equilibrium.
The effects of BTN1A1 genotypes on milk production traits were tested in the
group of 185 Jersey cows. The results obtained are presented in Table 1. The
analysis did not reveal any significant association for P35Q polymorphism.
Animals with the AA genotype of the K468R SNP, however, were characterized by
48
J. Komisarek et al.
higher (P ≤ 0.05) milk, fat and protein yields than cows with the AG and
GG genotypes. Fat and protein levels did not seem to be affected. To prove
the significant results obtained to be true, an additional analysis was carried out
on the two heterozygous sire families with the highest number of daughters
(Table 2). The relationship between K468R and yield traits was not confirmed.
Instead, in the half-sib family headed by sire 1, a relationship with milk-fat content
was found.
Table 1. Overall means and their standard deviations for milk production traits in different P35Q
and K468R genotypes
Genotype
Trait
P35Q
AA
No. of animals
130
Milk yield
(kg)
4454 ± 724
Fat yield (kg) 248 ± 43
Fat content
(%)
5.58 ± 0.54
Protein yield
(kg)
174 ± 28
Protein
content (%)
3.91 ± 0.22
K468R
AC
CC
AA
AG
GG
50
5
78
83
24
4592 ± 834
256 ± 43
5.59 ± 0.53
4352 ± 547
236 ± 22
5.46 ± 0.58
177 ± 31
166 ± 19
3.86 ± 0.26
3.83 ± 0.25
4616 ± 753 ab 4411 ± 755 a 4311 ± 635 b
257 ± 42 ab
245 ± 43 a
243 ± 37 b
5.59 ± 0.58
178 ± 28 ab
3.89 ± 0.24
5.57 ± 0.54
5.57 ± 0.36
171 ± 29 a
168 ± 27 b
3.92 ± 0.23
3.90 ± 0.17
a, b — columns with the same superscript differ at P ≤ 0.05.
Table 2. Overall means and their standard deviations for milk production traits in cows with different
K468R genotypes being the progeny of two heterozygous sires
Trait
Genotypes of sire 1 daughters
AA
AG
GG
Genotypes of sire 2 daughters
AA
No. of
daughters
10
29
8
21
Milk yield
(kg)
4460 ± 493
4365 ± 680 4307 ± 611
4498 ± 572
Fat yield
(kg)
265 ± 41
249 ± 42
243 ± 34
248 ± 30
Fat content
(%)
5.94 ± 0.44 Aa 5.71 ± 0.45 a 5.53 ± 0.38 A 5.54 ± 0.51
Protein yield
(kg)
179 ± 22
174 ± 29
172 ± 26
169 ± 20
Protein
content (%) 4.00 ± 0.21
3.99 ± 0.22
3.99 ± 0.17
3.76 ± 0.24
AG
GG
20
10
4601 ± 676
4361 ± 626
246 ± 40
237 ± 35
5.41 ± 0.38
5.44 ± 0.27
172 ± 28
166 ± 24
3.74 ± 0.15
3.80 ± 0.16
A, a — columns with the same superscript differ at P ≤ 0.05 (small letters) or at P ≤ 0.01 (capital letters).
49
Discussion
BTN1A1 is a likely candidate gene that affects economically important traits in
dairy animals because it is specifically expressed in the lactating mammary
epithelial tissue and the gene product may play a role in the secretion of milk lipid.
However, although several polymorphic forms of BTN1A1 have been identified
(Husaini et al., 1999; Seyfert and Lüthen, 1998; Taylor et al., 1996 a; Zegeye et al.,
1999), their impact on production traits in cattle has not been studied extensively.
In this paper, the P35Q and K468R polymorphisms were examined. P35Q was
first identified in a Holstein-Friesian (HF) population (Seyfert and Lüthen, 1998),
but, until now, no association analysis has been performed. The allele distribution
obtained in the present study suggests that the most frequent allele A (0.84) might
have been favoured by selection for dairy production. However, the direct impact of
this mutation on milk production seems to be questionable. First, no relationship
between P35Q and milk-related traits in Jersey cows has been found. Second, the
polymorphism leads to an amino acid change within one of two exoplasmic Ig-like
folds of the butyrophilin peptide. The IgI domain might be responsible for the
immunologic function of BTN1A1, but is probably not involved in milk synthesis.
Thus, the accumulation of the A allele could merely be a result of indirect selection
on other, closely linked QTLs. On the other hand, the possibility that P35Q
polymorphism has an effect that was not observed in the present investigation
because of the low number of animals and low C allele frequency cannot be
excluded.
K468R is more likely the causative mutation affecting milk traits in cattle. The
lysine to arginine substitution appeared within the highly conserved region of
the butyrophilin cytoplasmic domain, known as B30.2, that probably participates in
the protein-protein interactions (Henry et al., 1997; Ishii et al., 1995). One of the
potential interactive partners is a xanthine dehydrogenase/oxidase (XDH/XO). It is
postulated that the BTN1A1-XDH/XO protein complex interacts with lipid droplets
at the apical surface of mammary epithelial cells and is essential for milk-fat
secretion (for reviews, see Mather and Keenan, 1998; Murphy and Vance, 1999).
On the other hand, both lysine and arginine are similar amino acids within the basic
R-group. The substitution should not, therefore, affect the protein structure or its
binding affinity.
The K468R allele frequencies estimated for Jersey cows in the present study
(A — 0.66 and G — 0.34) differ from those reported previously for HF cattle. In
Holstein-Friesians, the G allele frequency was established to be equal to approximately half these values, ranging from 0.12 (Komisarek and Dorynek, 2003)
to 0.15 (Husaini et al., 1999). Nevertheless, both breeds are characterized by the
preponderance of the A variant, suggesting that it might be favoured by selection
for milk performance. The association analyses performed so far have produced
uncertain results. Zegeye et al. (1999) did not reveal a relationship between K468R
(HaeIII) polymorphism and milk production in five grand-sire HF families. Also,
comparison of the breeding values of HF bulls carrying the AA and AG genotypes
50
J. Komisarek et al.
(Komisarek and Dorynek, 2003) resulted in no significant association, although for
one trait (milk-fat percentage) the difference came close to a significance level of
P ≤ 0.05. In the present study, Jersey cows with the AA genotype were characterized
by higher milk, fat and protein yields than cows with two other genotypes. The
association was not, however, confirmed by within-family analysis. Thus, K468R is
probably not the causative mutation affecting milk production in cattle. It might,
however, be linked to a locus with a direct effect, which remains undetected due to
the lack of causative gene allele segregation in two analysed families or because of
the small number of daughters per sire. One of the potential candidates could be the
prolactin (PRL)-encoding gene. It is located on chromosome 23, close to the
BTN1A1 locus (Hallerman et al., 1988). The role of prolactin in lactation
physiology is well known, and some relationships between bovine PRL polymorphisms and milk production traits have been found (Chung et al., 1996; Dybus, 2002;
Brym et al., 2005). On the other hand, BTN1A1 cannot be definitively excluded as
a source of variability of milk-related traits. Further investigations, conducted on
larger populations, are therefore needed to verify the effect of butyrophilin in cattle.
References
A o k i N., I s h i i T., O h i r a S., Y a m a g u c h i Y., N e g i M., A d a c h i T., N a k a m u r a R.,
M a t s u d a T. (1997). Stage specific expression of milk fat globule membrane glycoproteins in
mouse mammary gland: comparison of MFG-E8, butyrophilin, and CD36 with a major milk
protein, β-casein. Biochim. Biophys. Acta, 1334: 182 – 190.
A s h w e l l M.S., O g g S.L., M a t h e r I.H. (1996 a). The bovine butyrophilin gene maps to chromosome
23. Anim. Genet., 27: 171 – 173.
A s h w e l l M.S., R e x r o a d C.E. Jr., M i l l e r R.H., V a n r a d e n P.M. (1996 b). Mapping economic trait
loci for somatic cell score in Holstein cattle using microsatellite markers and selective genotyping.
Anim. Genet., 27: 235 – 242.
A s h w e l l M.S., R e x r o a d C.E. Jr., M i l l e r R.H., V a n R a d e n P.M., D a Y. (1997). Detection of loci
affecting milk production and health traits in an elite US Holstein population using microsatellite
markers. Anim. Genet., 28: 216 – 222.
A s h w e l l M.S., D a Y., V a n r a d e n P.M., R e x r o a d C.E. Jr., M i l l e r R.H. (1998). Detection of
putative loci affecting conformational type traits in an elite population of United States Holsteins
using microsatellite markers. J. Dairy Sci., 81: 1120 – 1125.
B a n g h a r t L.R., C h a m b e r l a i n C.W., V e l a r d e J., K o r o b k o I.V., O g g S.L., J a c k L.J.,
V a k h a r i a V.N., M a t h e r I.H. (1998). Butyrophilin is expressed in mammary epithelial cells from
a single-sized messenger RNA as a type I membrane glycoprotein. J. Biol. Chem., 273: 4171 – 4179.
B e n n e w i t z J., R e i n s c h N., G r o h s C., L e v e z i e l H., M a l a f o s s e A., T h o m s e n H., X u N.,
L o o f t C., K u h n C., B r o c k m a n n G.A., S c h w e r i n M., W e i m a n n C., H i e n d l e d e r S.,
E r h a r d t G., M e d j u g o r a c I., R u s s I., F o r s t e r M., B r e n i g B., R e i n h a r d t F., R e e n t s R.,
A v e r d u n k G., B l u m e l J., B o i c h a r d D., K a l m E. (2003). Combined analysis of data from
two granddaughter designs: A simple strategy for QTL confirmation and increasing experimental
power in dairy cattle. Genet. Sel. Evol., 35: 319 – 338.
B e n n e w i t z J., R e i n s c h N., G u i a r d V., F r i t z S., T h o m s e n H., L o o f t C., K ü h n C.,
S c h w e r i n M., W e i m a n n C., E r h a r d t G., R e i n h a r d t F., R e e n t s R., B o i c h a r d D.,
K a l m E. (2004). Multiple quantitative trait loci mapping with cofactors and application of
alternative variants of the false discovery rate in an enlarged granddaughter design. Genetics, 168:
1019 – 1027.
51
B r y m P., K a m i ń s k i S., W ó j c i k E. (2005). Nucleotide sequence polymorphism within exon 4 of the
bovine prolactin gene and its associations with milk performance traits. J. Appl. Genet., 45:
179 – 185.
C h u n g E.R., R h i n T.J., H a n S.K. (1996). Association between PCR RFLP markers of growth hormone
and prolactin genes and production traits in dairy cattle. Korean J. Anim. Sci., 38: 321 – 336.
D y b u s A. (2002). Associations of growth hormone GH and prolactin PRL genes polymorphism with
milk production traits in Polish Black and White cattle. Anim. Sci. Pap. Rep., 20: 203 – 212.
F r a n k e W.W., H e i d H.W., G r u n d C., W i n t e r S., F r e u d e n s t e i n C., S c h m i d E.,
J a r a s c h E.D., K e e n a n T.W. (1981). Antibodies to the major insoluble milk fat globule
membrane-associated protein: specific location in apical regions of lactating epithelial cells. J. Cell
Biol., 89: 485 – 494.
G a r d i n i e r M.V., A m i g u e t P., L i n i n g t o n C., M a t t h i e u J.M. (1992). Myelin/oligodendrocyte
glycoprotein is a unique member of the immunoglobulin superfamily. J. Neurosci. Res., 33:
177 – 187.
H a l l e r m a n E.M., T h e i l m a n n J.L., B e c k m a n n J.S., S o l l e r M., W o m a c k J.E. (1988).
Mapping of bovine prolactin and rhodopsin genes in hybrid somatic cells. Anim. Genet., 19:
123 – 131.
H e n r y J., R i b o u c h o n M.-T., O f f e r C., P o n a r o t t i P. (1997). B30.2-like domain proteins:
A growing family. Biochem. Biophys. Res. Commun., 235: 162 – 165.
H e y e n D.W., W e l l e r J.I., R o n M., B a n d M., B e e v e r J.E., F e l d m e s s e r E., D a Y.,
W i g g a n s G.R., V a n R a d e n P.M., L e w i n H.A. (1999). A genome scan for QTL influencing
milk production and health traits in dairy cattle. Physiol. Genom., 1: 165 – 175.
H o l m b e r g M., A n d e r s s o n - E k l u n d L. (2004). Quantitative trait loci affecting health traits in
Swedish dairy cattle. J. Dairy Sci., 87: 2653 – 2659.
H u s a i n i Y., W i l k i n s R.J., D a v e y H.W. (1999). Identification of five point mutations, including an
AluI RFLP, in the bovine butyrophilin gene. Anim. Genet., 30: 400 – 401.
I s h i i T., A o k i N., N o d a A., A d a c h i T., N a k a m u r a R., M a t s u d a T. (1995). Carboxy-terminal
cytoplasmic domain of mouse butyrophilin specifically associates with a 150-kDa protein of
mammary epithelial cells and milk fat globule membrane. Biochim. Biophys. Acta, 1245:
285 – 292.
J a c k L.J., M a t h e r I.H. (1990). Cloning and analysis of cDNA encoding bovine butyrophilin, an apical
glycoprotein expressed in mammary tissue and secreted in association with the milk-fat globule
membrane during lactation. J. Biol. Chem., 265: 14481 – 14486.
K o m i s a r e k J., D o r y n e k Z. (2003). Polymorphism of BTN and GHR genes and its impact on bulls’
breeding value for milk production traits. J. Anim. Feed Sci., 12: 681 – 688.
M a t h e r I.H., K e e n a n T.W. (1998). Origin and secretion of milk lipids. J. Mammary Gland Biol.
Neoplasia, 3: 259 – 273.
M a t h e r I.H., T a m p l i n C.B., I r v i n g M.G. (1980). Separation of the proteins of bovine milk-fatglobule membrane by electrofocusing with retention of enzymatic and immunological activity. Eur.
J. Biochem., 110: 327 – 336.
M o n d y B.L., K e e n a n T.W. (1993). Butyrophilin and xanthine oxidase occur in constant molar
proportions in milk lipid globule membrane but vary in amount with breed and stage of lactation.
Protoplasma, 177: 32 – 36.
M u r p h y D.J., V a n c e J. (1999). Mechanisms of lipid-body formation. Trends Biochem. Sci., 24:
109 – 115.
O g g S.L., K o m a r a g i r i M.V.S., M a t h e r I.H. (1996). Structural organization and mammary-specific
expression of the butyrophilin gene. Mamm. Genome, 7: 900 – 905.
O g g S.L., W e l d o n A.K., D o b b i e L., S m i t h A.J.H., M a t h e r I.H. (2004). Expression of
butyrophilin (Btn1a1) in lactating mammary gland is essential for the regulated secretion of
milk-lipid droplets. Proc. Natl. Acad. Sci., USA, 101: 10084 – 10089.
S e y f e r t H.-M., L ü t h e n F. (1998). The structure of the bovine butyrophilin encoding gene differs
grossly from mouse concerning promoter localization and exon organization of the S-untranslated
region. Proc. 6th World Congr. Genet. Appl. Livest. Prod., 25: 51 – 54.
52
J. Komisarek et al.
T a y l o r C., E v e r e s t M., S m i t h C. (1996 a). Restriction fragment length polymorphism in
amplification products of the bovine butyrophilin gene: assignment of bovine butyrophilin to
bovine chromosome 23. Anim. Genet., 27: 183 – 185.
T a y l o r M.R., P e t e r s o n J.A., C a r i a n i R.L., C o u t o J.R. (1996 b). Cloning and sequence analysis of
human butyrophilin reveals a potential receptor function. Biochim. Biophys. Acta, 1306: 1 – 4.
Z e g e y e A., A s h w e l l M., O g g S., R e x r o a d C., M a t h e r I.H. (1999). RFLP markers in the bovine
butyrophilin gene. Anim. Genet., 30: 385 – 386.
Z h a n g Q., B o i c h a r d D., H o e s c h e l e I., E r n s t C., E g g e n A., M u r k v e B., P f i s t e r g e n s k o w M., W i t t e L.A., G r i g n o l a F.E., U i m a r i P., T h a l l e r G., B i s h o p M.D. (1998).
Mapping quantitative trait loci for milk production and health of dairy cattle in a large outbred
pedigree. Genetics, 149: 1959 – 1973.
Accepted for printing 5 I 2006
JOLANTA KOMISAREK, KRYSTYNA WAŚKOWICZ, ZBIGNIEW DORYNEK
Analiza asocjacji mie˛dzy dwoma polimorfizmami genu butyrofiliny (BTN1A1)
a cechami produkcji mleka u bydła rasy Jersey
STRESZCZENIE
Celem pracy była analiza zależności mie˛dzy polimorfizmem P35Q i K468R butyrofiliny (BTN1A1)
a cechami produkcji mleka u bydła. Badaniem obje˛to populacje˛ 219 krów rasy Jersey. Uzyskano
naste˛puja˛ce frekwencje alleli: P35Q — 0.84 (A) i 0.16 (C); K468R — 0.66 (A) i 0.34 (G). Analiza nie
wykazała istotnych asocjacji dla polimorfizmu P35Q. W przypadku mutacji K468R, genotyp AA
charakteryzował sie˛ istotnie wyższa˛ wydajnościa˛ mleka, tłuszczu i białka w stosunku do genotypów AG
i GG. Zależność ta nie została jednak potwierdzona w analizie przeprowadzonej w grupach półrodzeństwa.
RELATIONSHIP BETWEEN INTERPREGNANCY INTERVAL
AND LIFETIME PRODUCTIVITY OF COWS
J a r o s ł a w P y t l e w s k i, I r e n e u s z A n t k o w i a k, Z b i g n i e w D o r y n e k
Department of Cattle Breeding and Milk Production, Agricultural University, Wojska Polskiego 71 A,
Abstract
The aim of the study was to investigate the relationship between the length of the interpregnancy
interval and the lifetime productivity of Black-and-White cows. No clear correlation was found
between the length of the interpregnancy interval and milk production traits. Cows in which the
mean length of the interpregnancy interval ranged from 121 to 160 days had the longest lifespan,
productive life and milking period, as well as the highest lifetime production of milk, butterfat and
protein.
Key words: cow, interpregnancy interval, lifetime productivity
A trend towards deteriorating health and fertility is observed in cows with
increasing milk yields. This pattern is confirmed by the results obtained by Pösö
and Mäntysaari (1996), who showed a negative genetic correlation between milk
yields and reproduction indices. A consequence of this relationship is that the
productive life of animals becomes shorter.
Many researchers have shown that it is possible to make higher profits from
cows with prolonged lactations resulting from increased lifetime milk production,
extended cow productive life and decreased herd replacement costs.
The aim of the study was to investigate the relationship between the length of
the interpregnancy interval and the lifetime productivity of Black-and-White cows.
The study included 407 culled Black-and-White cows with varying percentages
of Holstein-Friesian genes (47.4% on average) in their genotypes. Animals were
removed from the herd belonging to ‘‘Maja˛tek Rogalin” Ltd. in the years
1991 – 2004. Feeding of cows was based on feeds produced on the farm. Starting
54
J. Pytlewski et al.
from 1997, feed rations were formulated based on the INRA feeding standards.
Culled cows were characterized on the basis of the following parameters: days of
life, days of productive life, days of milking, mean length of the interpregnancy
interval and lifetime production, as well as yields per day of life, day of productive
life and day of milking. Milk production was expressed in kg of milk, butterfat,
protein and fat-corrected milk (FCM). Cows were divided into groups based on the
mean length of their interpregnancy interval: ≤ 95, 96 – 120, 121 – 160 and > 160
days. The study analysed relationships between the length of the interpregnancy
interval and the lifetime productivity of cows. In order to perform statistical
analyses the MEANS procedure of the SAS (ver. 9.1) software package was
applied for means and standard deviations, and the GLM procedure for the analysis
of variance. The calculations included the following effects: year of the study,
lactation number, genotype, age at first lactation, causes of cow culling and mean
interpregnancy interval. A detailed comparison of the means was performed using
the LSD (least significant differences) test.
Results
Table 1 presents the relationships between the length of the interpregnancy
interval and the lifetime productivity of cows. Cows with a mean interpregnancy
interval within the range of 121 – 160 days had the longest lifespan (2418 days), the
longest productive lives (1539 days) and the longest milking period (1098 days).
The least advantageous values for the above-mentioned parameters were found in
cows with a mean interpregnancy interval of ≤ 95 days. As a result of the statistical
analysis performed for the lifespan and length of productive life, highly significant
differences were found between cows with a mean interpregnancy interval ranging
from 121 to 160 days and the group of animals with a mean interpregnancy interval
of ≤95 days. Significant differences were found between the population of animals
with a mean interpregnancy interval ranging from 121 to 160 days and the groups of
animals with mean interpregnancy intervals from 96 to 120 days and > 160 days, as
well as cows with interpregnancy intervals of > 160 days and ≤ 95 days. In the
analysis of the number of milking days, the group of cows with an interpregnancy
interval from 121 to 160 days differed (P ≤ 0.01) from the populations of animals
with mean interpregnancy intervals amounting to ≤ 95 days and > 160 days.
In our study, the mean length of the interpregnancy interval was found to have
an effect on lifetime production of cows. The highest yields of milk, butterfat,
protein and FCM were observed in cows with a mean interpregnancy interval
ranging from 121 to 160 days. Their lifetime productivity was 15 892 kg milk,
674 kg butterfat, 471 kg protein and 16 461 kg FCM. The lifetime milk production
of cows with a mean interpregnancy interval ranging from 121 to 160 days differed
highly significantly from the lifetime milk yields of animals with a mean
interpregnancy interval of ≤ 95 days and significantly from the population of
animals with the mean interpregnancy interval of > 160 days. Analysis of the
Relationship between interpregnancy interval and lifetime productivity of cows
tab. 1
55
56
tab. 2
J. Pytlewski et al.
Relationship between interpregnancy interval and lifetime productivity of cows
57
lifetime production of 4% FCM showed that the population of cows with a mean
interpregnancy interval ranging from 121 to 160 days differed from the population
of animals with a mean interpregnancy interval of ≤ 95 days at P ≤ 0.01. Moreover,
statistically significant differences were found between the above-mentioned
groups of cows in terms of butterfat and protein yields.
Table 2 gives the productivity of cows per day of life, day of productive life and
day of milking in terms of the length of the mean interpregnancy interval. In terms
of data expressed per day of milking period only, cows with mean interpregnancy
intervals of > 160 days and ≤ 95 days differed highly significantly in the production
of FCM and significantly in milk yield. More advantageous values for the analysed
traits were found in animals with a longer interpregnancy interval. Analysis of these
milk production traits in terms of values expressed per day of life and day of
productive life did not reveal any statistically significant differences between
groups of cows with differing interpregnancy intervals.
Discussion
In the opinion of Butler and Smith (1989) and Senatore et al. (1996), the energy
deficit in cows after calving caused by high yields may disturb energy metabolism
and result in ketosis, as well as problems with reproduction extending beyond the
interpregnancy interval. Szarek (1998) reported that the present high productivity of
cows requires breeders to change the system from 12 months to an extended
production cycle of 15 or even 18 months, which improves the health of cows and
results in an extension of their lifespan and productive life. A shortening of the
lifespan of cows causes a shortening of their productive life, which in turn causes
a decrease in their lifetime yields. According to Antkowiak et al. (2001), extension
of the productive life of cows may be one of the essential factors in improving the
profitability of milk production. The most advantageous interpregnancy interval
(121 – 160 days), found in this study in terms of the lifetime productivity of cows, is
consistent with the value of this index considered appropriate by Kaczmarek
(1987). A study by Strzałkowska et al. (2004), meanwhile, showed that the
optimum length of the interpregnancy interval in cows producing approximately
9000 kg milk in one lactation is 110 days.
The following conclusions can be drawn from the investigations conducted:
— no clear correlation was found between the length of the interpregnancy interval
and milk production traits,
— cows in which the mean length of the interpregnancy interval ranged from 121
to 160 days had the longest lifespan, productive life and milking period, as well as
the highest lifetime production of milk, butterfat and protein.
58
J. Pytlewski et al.
References
A n t k o w i a k I., P y t l e w s k i J., S t a n i s ł a w s k i D. (2001). Intensywność i przyczyny brakowania
krów w gospodarstwie farmerskim „Paruszewo”. Zesz. Nauk. PTZ, Prz. Hod., 59: 66 – 77.
B u t l e r W.R., S m i t h R.D., (1989). Interrelationships between energy balance and postpartum
reproductive function in dairy cattle. J. Dairy Sci., 72: 767 – 783.
K a c z m a r e k A. (1987). Praca hodowlana nad doskonaleniem bydła o użytkowości mlecznej. Mat.
konf.: Obecny stan i przyszłościowe metody zwie˛kszenia produkcji bydle˛cej. Kalisz, 1987;
ss. 1 – 73.
P ö s ö J., M ä n t y s a a r i E.A. (1996). Genetic relationships between reproductive disorders, operational
days open and milk yield. Livest. Prod. Sci., 46: 41 – 48.
S e n a t o r e E.M., B u t l e r W.R., O l t e n a c u P.A. (1996). Relationship between EB and post-partum
ovarian activity and fertility in first lactation dairy cows. Anim. Prod., 62: 17 – 23.
S t r z a ł k o w s k a N., K r z y ż e w s k i J., R e k l e w s k i A., D y m n i c k i E. (2004). Relationship
between the strained length of calving intervals, some reproduction traits and adjusted cow’s milk
yield. Med. Wet., 60: 1312 – 1316.
S z a r e k J. (1998). Perspektywiczny cykl produkcji u krów mlecznych. Zesz. Nauk. PTZ, Prz. Hod., 38:
45 – 55.
Accepted for printing 9 V 2006
JAROSŁAW PYTLEWSKI, IRENEUSZ ANTKOWIAK, ZBIGNIEW DORYNEK
Zależność mie˛dzy długościa˛ okresu mie˛dzycia˛żowego a produkcyjnościa˛ życiowa˛ krów
STRESZCZENIE
Celem pracy było zbadanie zależności mie˛dzy długościa˛ okresu mie˛dzycia˛żowego a produkcyjnościa˛ życiowa˛ krów czarno-białych. Nie stwierdzono jednoznacznej zależności mie˛dzy długościa˛ okresu
mie˛dzyciażowego a badanymi cechami użytkowości mlecznej. Krowy, u których średnia długość okresu
mie˛dzycia˛żowego wynosiła od 121 do 160 dni, żyły najdłużej, miały najdłuższy okres użytkowania
i dojenia oraz najwyższa˛ wydajność życiowa˛ mleka, tłuszczu i białka.
ANALYSIS OF TIME TRENDS FOR REPRODUCTIVE
AND MEAT TRAITS IN RANDOMLY MATED CONSERVATION FLOCKS
OF NORTHERN VARIETY GEESE*
A d a m M a z a n o w s k i 1,2, Z e n o n B e r n a c k i 1, M a r e k A d a m s k i 1, T o m a s z K i s i e l 2
1
Department of Poultry Breeding, University of Technology and Agriculture, Mazowiecka 28,
85-084 Bydgoszcz, Poland
2
Waterfowl Genetic Resources Station Dworzyska, National Research Institute of Animal Production,
62-035 Kórnik, Poland
Abstract
The study was designed to analyse time trends for reproductive and meat traits in randomly mated
conservation flocks of northern variety geese — Kartuska (Ka), Rypińska (Ry) and Suwalska
(Su) — and to evaluate the effect of reproductive traits, which increased during the study period,
on the meat traits of their offspring. Suwalska geese were characterized by the greatest number of
eggs (31) per goose and the highest egg weight (171.3 g) compared to Kartuska and Rypińska geese.
The highest egg fertility (68.8%) and the highest hatching percentage from fertilized eggs (62.9%)
were characteristic of Rypińska geese. Kartuska and Suwalska geese of both sexes had higher body
weight (4541 and 4512 g) and longer breastbone (16.3 and 16.4 cm) than Rypińska geese (16.1 cm).
The breast muscle thickness of the northern variety geese was similar (2.2 cm). Meat percentage
was highest in Rypińska geese (32.2%) and fat percentage was highest in Kartuska geese (14.4%)
compared to the other varieties of northern geese, which were less muscled (31.8%) but at the same
time less fatty (14.0 – 14.3%). Time trends for the number of eggs per goose were positive, and time
trends for hatching percentage from fertilized eggs were positive and statistically significant. The
effect of the increasing reproductive trait values was noticeable mainly in the positive time trends
for breastbone length and meat percentage. Time trends for fat percentage were similar
throughout the study. During this time, statistically significant decreases in body weight and breast
muscle thickness were also found. Random mating of the geese increased reproductive trait values,
decreased body weight and breast muscle thickness, and increased breastbone length and carcass
meat percentage.
Key words: goose, regional varieties, reproductive traits, meat traits, time trends
Kartuska (Ka), Rypińska (Ry) and Suwalska (Su) geese, derived from greylag
geese (Anser anser L.), represent the medium heavy northern variety (Mazanowski,
* This work was conducted as part of NRIAP statutory activity, project no. 17110.2.
60
A. Mazanowski et al.
1986; Smalec, 1991). Geese of these varieties were collected in 1973 – 1975 to form
conservation flocks at the Waterfowl Breeding Farm in Dworzyska, which belongs
to the National Research Institute of Animal Production. The term conservation
flock refers to birds of one variety that are kept without any infusion of foreign
blood and subjected to random mating in which the selection of next generation
animals is not based on breeding criteria (Smalec, 1991). The goose varieties
discussed in this study are included in the World Watch List for Domestic Animal
Diversity (FAO, 2000).
The preservation of farm animal (including bird) diversity is dictated not only
by economic, breeding and scientific considerations, but also by biological,
environmental, educational, cultural, ethnographic and even emotional considerations. In 1995, Poland ratified the Convention on Biological Diversity, which
obliges each signatory state to preserve the diversity of plants and animals within its
borders, not only in protected areas, but also in utilized agricultural areas. In
addition to wild species, the convention covers livestock breeds and varieties
developed by breeders, especially those facing extinction due to limited commercial
use (FAO, 2000; Mazanowski, 1986; Smalec, 1991).
The aim of the present study was to analyse time trends of reproductive
(1982 – 2000) and meat traits (1982 – 1998) in randomly mated conservation flocks of
northern variety geese (Kartuska, Rypińska and Suwalska) and to evaluate the effect of
reproductive traits, which changed during the study, on the meat traits of the offspring.
The study was carried out in 1982 – 2000 at the Waterfowl Genetic Resources
Station Farm in Dworzyska, which belongs to the National Research Institute of
Animal Production. During the whole study period, the number of females in the first
year of egg production was 2408 for Kartuska geese, 2408 for Rypińska geese and 2291
for Suwalska geese, which were grouped with ganders at a 1 : 3 ratio (Mazanowski,
1984). The number of birds intended for conservation flocks and reared each year
ranged from 40 to 80 for ganders and from 120 to 200 for geese. Ganders and geese
were reared to 6 weeks of age in a rearing house without access to free range. Later,
they were reared outdoors until the end of reproduction, in partially roofed pens with
rye straw bedding. Geese of each variety were placed in four subgroups.
From 1 to 6 weeks of age, geese were fed ad libitum with a complete diet
containing 20.0% crude protein and 2827 kcal (11.82 MJ) metabolizable energy per
kg feed. Later, until the end of reproduction, they received a diet containing 17.3%
crude protein and 2665 kcal (11.14 MJ) metabolizable energy. During rearing and
reproduction, geese were given, in separate troughs, a mineral mixture for poultry
(MM-D), chalk, and gravel, mixed at a volumetric ratio of 1 : 1 : 4. Throughout the
study, all the geese received mixtures of similar composition.
The number of eggs laid was recorded daily in each conservation flock during
successive reproduction periods, and the egg weight was monitored individually for
Analysis of time trends for reproductive traits in geese
61
2 weeks at the peak egg production. The percentages of fertilized eggs and
hatchability of healthy goslings from fertilized eggs were systematically recorded
each year in all the conservation flocks, during the entire reproductive period. At 12
weeks of age, ganders and geese were weighed and the length of their keel and
forearm was measured. Breast muscle thickness with skin and subcutaneous fat was
measured using a needle catheter, at a distance of 4 cm from the start of the keel and
2.5 cm laterally, perpendicular to the edge of the breastbone.
Multiple regression equations (Bochno et al., 1981; Wawro et al., 1985) were
used to estimate the weight of meat (M) and fat (F) in 12-week-old geese:
M = 0.233 X1 + 18.915 X2 + 60.178 X3 — 113.944,
F = 0.279 X1 — 63.252 X2 + 623.302,
where:
X1 — body weight (g),
X2 — forearm length (cm),
X3 — thickness of breast muscles with skin and subcutaneous fat (cm).
The meat and fat percentages of goose carcasses were calculated from the
proportion of meat and fat weight to body weight.
Patterns of reproductive and meat traits in the analysed years were shown as
time trends plotted using linear regression equations (Mazanowski et al., 1999;
2000). In this equation, y = a + bx, a — value of the analysed trait in the year of
evaluation, b — directional coefficient expressing the annual increment in the value
of the trait, and x — time in years. The time trends made it possible to interpret and
compare trends in productive traits in conservation flocks of randomly mated geese.
The means of traits and standard error of the mean were calculated, and linear
trends of traits were plotted. Two-way analysis of variance was used in the
calculations. The significance of the differences was analysed using Duncan’s test.
Results
Suwalska geese were characterized by the greatest number of eggs per goose,
the highest egg weight, and the lowest egg fertility compared to Kartuska and
Rypińska geese (Table 1). Kartuska and Rypińska geese demonstrated similar egg
production but different egg weights. The highest egg fertility and the highest
hatchability of goslings from fertilized eggs were characteristic of Rypińska geese.
The time trends for the number of eggs per goose were positive, and in
Suwalska geese even statistically significant (Figure 1). The trend towards egg
weight was positive in Rypińska geese only (Figure 2) and remained at a similar
level in the other geese of the northern variety. The time trends for egg fertility were
positive in Kartuska and Suwalska geese, and negative in Rypińska geese (Figure
3). The time trends for hatchability of goslings from fertilized eggs were positive
and statistically significant (Figure 4).
Figure 2. Time trends for egg weight
Figure 1. Time trends for number of eggs per goose; *Time trends for trait differ significantly (P ≤ 0.05)
62
Figure 4. Time trends for healthy goslings from fertilized eggs; * Time trends for trait differ significantly (P ≤ 0.05)
Figure 3. Time trends for fertilized eggs
63
64
Table 1. Mean values (x̄) and standard errors of the mean (SEM) for reproductive traits in geese
(1982 – 2000)
Symbol
of geese
Number of eggs
per goose
Egg weight
(g)
Fertilized eggs
(%)
Healthy goslings
from fertilized
eggs (%)
Ka
x̄
SEM
25.0 b
0.02
169.4 a
2.18
67.8 b
3.13
57.9 b
3.11
Ry
x̄
SEM
25.0 b
0.06
166.1 b
2.21
68.8 a
2.41
62.9 a
2.63
Su
x̄
SEM
31.0 a
0.08
171.3 a
2.83
64.9 c
3.83
62.1 a
2.45
Mean values for traits in columns followed by different letters differ significantly (P ≤ 0.05).
Table 2. Mean values (x̄) and standard errors of the mean (SEM) for meat traits in 12-week-old geese
(1982 – 1998)
Traits
Symbol of geese — sex
Su
Ka
Ka
Ry
4885 a
7.94
16.8 b
0.04
2.3 a
0.01
4567 b
5.81
16.7 b
0.03
2.3 a
0.01
4827 a
5.60
17.0 a
0.03
2.2 b
0.01
4197 a
3.33
15.8 b
0.01
2.1 a
0.01
3911 b
3.11
15.6 b
0.01
2.1 a
0.01
4197 a
2.80
15.9 a
0.02
2.1 a.
0.01
31.5 a
0.01
14.6 a
0.04
31.9 a
0.01
14.2 a
0.04
31.6 b
0.01
14.4 a
0.04
32.1 b
0.01
14.2 a
0.02
32.4 a
0.01
13.9 a
0.02
32.0 b
0.01
14.1 a
0.03
males
Body weight (g)
x̄
SEM
Breast bone length x̄
(cm)
SEM
Thickness of breast x̄
muscles with skin SEM
and fat (cm)
Proportion of meat x̄
(%)
SEM
Proportion of fat x̄
(%)
SEM
Ry
Su
females
Mean values for traits in rows separately for males and females followed by different letters differ significantly
(P ≤ 0.05).
Table 3. Mean values (x̄) and standard errors of the mean (SEM) for meat traits in 12-week-old ganders
and geese (1982 – 1998)
Traits
Body weight (g)
Breast bone length (cm)
Thickness of breast muscles
with skin and fat (cm)
Proportion of meat (%)
Proportion of fat (%)
Ka
x̄
SEM
x̄
SEM
x̄
SEM
x̄
SEM
x̄
SEM
4541 a
3.45
16.3 a
0.02
2.2 a
0.01
31.8 b
0.67
14.4 a
1.88
Symbol of geese
Ry
4239 b
2.84
16.1 b
0.01
2.2 a
0.01
32.2 a
0.74
14.0 b
2.00
Mean values for traits in rows followed by different letters differ significantly (P ≤ 0.05).
Su
4512 a
2.61
16.4 a
0.02
2.2 a
0.01
31.8 b
0.57
14.3 b
2.17
* Time trends for trait differ significantly (P ≤ 0.05)
Figure 5. Time trends for body weight in 12-week-old ganders and geese;
65
Figure 6. Time trends for breast bone length in 12-week-old ganders and geese
66
Figure 7. Time trends for thickness of breast muscles in 12-week-old ganders and geese
* Time trends for trait differ significantly (P ≤ 0.05)
67
Figure 8. Time trends for meat proportion in 12-week-old ganders and geese
68
Figure 9. Time trends for fat proportion in 12-week-old ganders and geese
69
70
Kartuska and Suwalska ganders and geese had significantly higher body
weights than Rypińska ganders and geese (Table 2). The longest breastbone was
characteristic of Suwalska ganders and geese. Neither breast muscle thickness with
skin and subcutaneous fat, nor carcass fat percentage, differed significantly between
the conservation flocks of geese. The highest percentage of meat was found in
Rypińska ganders and geese, and a significantly lower percentage in Kartuska and
Suwalska ganders and geese.
Body weight and keel length were significantly greater in Kartuska and
Suwalska than in Rypińska ganders and geese (Table 3). Breast muscle thickness
was similar in all the varieties of northern geese. Meat percentage was highest in
Rypińska birds of both sexes and fat percentage was highest in Kartuska geese
compared to the other northern varieties of geese, which had slightly poorer
muscles but were less fatty.
The time trends for the body weight of 12-week-old ganders and geese, as well
as those for breast muscle thickness with skin and subcutaneous fat, were negative
and statistically significant (Figures 5 and 7). The time trends for keel length
(Figure 6), meat percentage (Figure 8) and fat percentage (Figure 9) were positive
but the values were not significant.
Discussion
Suwalska geese were characterized by the greatest number of eggs per goose
and the highest egg weight, while Rypińska geese had the highest percentages of
fertilized eggs and hatchability of goslings from fertilized eggs, with lower values
for the other reproductive traits. The lowest reproductive trait values were found in
Kartuska geese. The time trends for reproductive traits were positive, while the
percentage of gosling hatchability from fertilized eggs was positive and statistically
significant. The increase in the time trends for egg weight was the least noticeable
because during random mating, progress in reproductive traits was influenced more
by egg production, egg fertility and gosling hatchability than by egg weight. It
appears that the positive time trends for reproductive traits could be related to the
reproduction model adopted, in which the random mating of geese gave priority to
those birds producing the greatest number of offspring, which were later used in
further reproduction (Mazanowski, 1984; Mazanowski, 1986; Smalec, 1991). In
another study (Kisiel and Ksia˛żkiewicz, 2004), the weight of eggs from geese of
regional northern varieties did not differ significantly and ranged from 164.8 to
172.3 g.
In Rhine and Roman geese, characterized by reproductive traits similar to those
of regional geese of northern varieties, a negative time trend was found over
15 years of study for the number of eggs per goose and a positive but nonsignificant time trend for the percentage of fertilized eggs and gosling hatchability
from fertilized eggs. The time trends for egg weight were negative in Rhine geese
and positive in Roman geese (Mazanowski et al., 1999). The direction of the
71
changes found in these geese was similar to that found in the northern variety geese,
but not as clear for the number of eggs per goose and hatchability of goslings from
fertilized eggs.
In an earlier study (Mazanowski, 1986), the number of eggs per goose was 22
for Kartuska, 24 for Rypińska and 30 for Suwalska birds. The egg weight, 150-159
g, was lower than in the present study, while egg fertility did not exceed 51%. In
another study (Tilki and Inal, 2004), egg weight during the first year of egg
production ranged from 144.2 to 148.5 g according to goose variety. Similar results
were obtained by Smalec (1991), who analysed 8 generations of geese for the mean
values of reproductive traits during the first period of egg production. These
reproductive traits were lower than in our study. Mazanowski et al. (2005 a) found
that Suwalska geese produced 31 eggs per layer and Kartuska geese laid 23 eggs per
layer. Egg fertility was 64.8% in Kartuska and 53.8% in Suwalska geese. In the
same experiment, hatchability of goslings from fertilized eggs was 86.4% in
Kartuska geese and 84.4% in Suwalska geese.
Due to their good muscling and high reproductive trait values, ganders
of northern varieties have also been used to create utility crossbreds (Mazanowski
and Kiełczewski, 1999; Mazanowski and Kisiel, 2004). In Suwalska or Kartuska
ganders mated to White Kołuda and Cuban crossbreds, egg fertility was 60.8
and 55.8%, and hatchability from fertilized eggs was 76.5 and 75.5%, respectively.
Suwalska or Kartuska and White Kołuda crossbred ganders mated to White Kołuda
and Cuban crossbred geese gave offspring of both sexes weighing 5118 – 5124 g
at 12 weeks of age, which is higher than for Suwalska or Kartuska geese. Kartuska
and Suwalska geese were characterized by very good egg production (46 and
58 eggs per layer) and high egg weight (177 g), while Kartuska birds of both
sexes were also characterized by a significantly higher body weight than the other
birds.
Kartuska and Suwalska ganders and geese had higher body weight and longer
breastbone than Rypińska geese, but they did not differ in the thickness of breast
muscles with skin and fat. Carcass meat percentage was the highest in Rypińska
geese and carcass fat percentage in Kartuska geese. The increase in time trends for
the number of eggs per goose and the statistically significant increase in the time
trends for gosling hatchability from fertilized eggs, as well as the increased
percentage of fertilized eggs, could have a negative effect on the body weight of
ganders and geese.
With the increased time trends for reproductive traits and significantly declining
time trends for the body weight of birds, positive time trends were found for keel
length in geese of all the northern varieties. Keel length increases as a result of
improving reproductive traits and is usually connected with a decrease in fatness
and an increase in muscling (Cheng et al., 2003; Romanov, 1999; Schneider, 1987).
Breast muscle thickness did not differ between the goose varieties. The time trends
for the breast muscle thickness of ganders and geese were negative and statistically
significant. This may be evidence of the negative effect the assumed mating scheme
had on breast muscle thickness with skin and subcutaneous fat, both in ganders and
72
in geese. The small and positive time trends for carcass fat percentage were similar
and statistically non-significant throughout the study. The time trends for carcass
meat percentage were non-significant but assumed higher positive values than the
time trends for carcass fat percentage in 12-week-old ganders and geese.
The above findings suggest that the negative trends for breast muscle thickness
with skin and fat were related more to decreased thickness of skin with fat than to
the thickness of breast muscles. The reproductive trait values increased as a result
of the reproductive use of ganders and geese obtained from random mating, which
led to decreased body weight, increased breastbone length and increased carcass
meat percentage in 12-week-old ganders and geese, and carcass fatness being
maintained at a similar level throughout the study (Schneider, 1987). The latter fact
is connected with a significant decrease in the time trends for breast muscle
thickness with skin and fat.
Analysis of the time trends for reproductive and meat traits in Rhine geese
selected for body weight, increased meatiness and decreased carcass fatness showed
a favourable effect of selection on the positive time trends for breastbone length and
meat percentage and on the negative time trends for carcass fat percentage. In
12-week-old Roman ganders and geese, which, just like the geese from the
conservation flocks, were randomly mated, positive time trends were found for keel
length and meat percentage. There were also favourable negative time trends for
carcass fat percentage (Mazanowski et. al., 2000).
Kłosowicz and Kukiełka (1958) were the first to evaluate the meat traits of
northern varieties of geese. The body weight of 22-week-old geese was 4700 g for
Suwalska, 4563 g for Rypińska and 4872 g for Kartuska birds. During oat fattening
the geese of northern varieties had weight gains of 815 – 1146 g. Their carcasses
were very well muscled (43.7%), with only 22.5% fat in the carcasses. In another
study, 12-week-old ganders and geese weighed 4475 g (Suwalska), 4211 g (Rypińska) and 4450 g (Kartuska). Breastbone length ranged from 15.4 to 15.7 cm and
breast muscle thickness with skin and fat was 2.3 cm (Mazanowski, 1986). In
a study by Smalec (1991), body weights were 4780 g for Suwalska, 4472 g for
Rypińska, and 4700 g for Kartuska geese. The muscling of northern variety geese
was good and carcass fatness low. The values of meat traits were similar to those
obtained in our study. Meanwhile, the body weight of 12-week-old four-way
crossbreds with wild greylag geese averages 4354 g, and breastbone length is
16.8 cm (Mazanowski et al., 2005 b).
In geese of northern varieties, randomly mated in 1982 – 2000, positive time
trends were found for the number of eggs per goose as well as significant and
positive time trends for the percentage of gosling hatchability from fertilized eggs.
The time trends for egg weight and egg fertility were mostly positive. The effect of
the increasing reproductive trait values was reflected primarily in positive time
trends for breastbone length and meat percentage. The time trends for fat
percentage were similar throughout the study period. There was a statistically
significant decrease in the time trends for body weight and breast muscle thickness
with skin and subcutaneous fat. Random mating of the geese increased the
73
reproductive trait values, decreased body weight and breast muscle thickness, and
increased breastbone length and carcass meat percentage.
References
B o c h n o R., L e w c z u k A., W a w r o E., W a w r o K. (1981). Badania nad opracowaniem równań
regresji wielokrotnej przydatnych do szacowania zawartości mie˛sa, tłuszczu i kości w tuszkach
ge˛si. Rocz. Nauk. Zoot., 8, 2: 33 – 44.
C h e n g Y.S., R o u v i e r R., H u Y.H., T a i J.J.L., T a i C. (2003). Breeding and genetics of waterfowl.
World’s Poultry Sci. J., 59, 4: 509 – 519.
FAO (2000). World Watch List for Domestic Animal Diversity. 3rd edition. FAO, UNDP, pp. 350 – 352.
K i s i e l T., K s i a˛ż k i e w i c z J. (2004). Physical traits and hatching results of eggs from geese of polish
regional varieties. Ann. Anim. Sci., 4, 1: 33 – 42.
K ł o s o w i c z W., K u k i e ł k a E. (1958). Charakterystyka wartości użytkowych odmian ge˛si krajowej.
Rocz. Nauk Roln., 72-B-4: 615 – 643.
M a z a n o w s k i A. (1984). Metody zachowania rezerw genetycznych ptactwa wodnego. Biul. Inf. IZ,
22, 3: 14 – 23.
M a z a n o w s k i A. (1986). Rezerwa genetyczna ge˛si w Polsce. Mat. konf.: Hodowla, chów i patologia
ge˛si. IZ, Kraków, pp. 15 – 29.
M a z a n o w s k i A., K i e ł c z e w s k i K. (1999). Wyniki reprodukcji ge˛si ze stad rezerwy genetycznej
i ge˛si białych kołudzkich w dwóch okresach nieśności. Rocz. Nauk. Zoot., 26, 1: 55 – 72.
M a z a n o w s k i A., K i s i e l T. (2004). Cechy reprodukcyjne i mie˛sne ge˛si wybranych stad zachowawczych. Rocz. Nauk. Zoot., 31, 1: 21 – 38.
M a z a n o w s k i A., K o k o s z y ń s k i D., S z u k a l s k i G. (1999). Analiza trendów czasowych cech
reprodukcyjnych w stadach rezerwy genetycznej ge˛si zagranicznych. BTN Bydgoszcz, Pr. Wydz.
Nauk Przyrod., 45: 19 – 28.
M a z a n o w s k i A., K o k o s z y ń s k i D., S z u k a l s k i G. (2000). Analiza trendów czasowych cech
mie˛snych w stadach rezerwy genetycznej ge˛si zagranicznych. Zesz. Nauk. AT-R Bydgoszcz, Zoot.,
227, 32: 61 – 74.
M a z a n o w s k i A., K i s i e l T., A d a m s k i M. (2005 a). Evaluation of some regional varieties of geese
for reproductive traits, egg structure and egg chemical composition. Ann. Anim. Sci., 5, 1: 67 – 83.
M a z a n o w s k i A., B e r n a c k i Z., K i s i e l T. (2005 b). Meat traits and meat chemical composition in
hybrids of Graylag (Anser anser L.) with White Kołuda and Slovakian geese. Anim. Sci. Pap. Rep.,
23, 1: 15 – 32.
R o m a n o v M.N. (1999). Goose production efficiency as influenced by genotype, nutrition and
production systems. World’s Poultry Sci. J., 55, 3: 281 – 294.
S c h n e i d e r K.H. (1987). Erhöhung der Legeleistung bei Gänsen durch eine effektive Selektion auf
Eizahl. Tierzucht, 41, 9: 416 – 418.
S m a l e c E. (1991). Zróżnicowanie ge˛si rezerwy genetycznej pod wzgle˛dem cech użytkowych
i polimorfizmu białek surowicy krwi. Zesz. Nauk. Drob., 3: 6 – 87.
T i l k i M., I n a l S. (2004). Quality traits of geese eggs: 2. Effects of goose origin and storage time of
eggs. Arch. Geflügelk., 68, 5: 230 – 234.
W a w r o E., B o c h n o R., W a w r o K., J a n i s z e w s k a M. (1985). Zastosowanie równań regresji
wielokrotnej do oceny umie˛śnienia i otłuszczenia ge˛si Włoskich, Kubańskich i mieszańców tych
ras. Pr. Mat. Zoot., 36: 77 – 88.
74
ADAM MAZANOWSKI, ZENON BERNACKI, MAREK ADAMSKI, TOMASZ KISIEL
Analiza trendów czasowych cech reprodukcyjnych i mie˛snych w rozmnażanych losowo stadach
zachowawczych ge˛si odmian północnych
STRESZCZENIE
Celem pracy była analiza trendów czasowych cech reprodukcyjnych i mie˛snych w kojarzonych
losowo stadach zachowawczych ge˛si odmian północnych kartuskich (Ka), rypińskich (Ry) i suwalskich
(Su) oraz ocena oddziaływania zwie˛kszaja˛cych sie˛ w badanym okresie cech reprodukcyjnych na cechy
mie˛sne potomstwa. Ge˛si suwalskie cechowała najwie˛ksza liczba jaj od 1 ge˛si (31 sztuk) i najwie˛ksza
masa jaja (171,3 g) w porównaniu z ge˛siami kartuskimi i rypińskimi. Najwie˛ksze zapłodnienie jaj
(68,8%) i najwie˛kszy procent wyle˛gu piskla˛t z jaj zapłodnionych (62,9%) miały ge˛si rypińskie.
Ge˛si kartuskie i suwalskie obojga płci miały wie˛ksza˛ mase˛ ciała (4541 i 4512 g) oraz dłuższy
mostek (16,3 i 16,4 cm) niż rypińskie (16,1 cm). Grubość mie˛śni piersiowych była podobna u ge˛si ze
wszystkich odmian północnych (2,2 cm). Procentowy udział mie˛sa był najwie˛kszy u ge˛si rypińskich
(32,2%), a procentowy udział tłuszczu u ge˛si kartuskich (14,4%), w porównaniu z pozostałymi
odmianami ge˛si północnych, które były gorzej umie˛śnione (31,8%), ale równocześnie mniej otłuszczone
(14,0 do 14,3%).
Trendy czasowe liczby jaj od 1 ge˛si były dodatnie, a trendy czasowe procentów wyle˛gu piskla˛t z jaj
zapłodnionych dodatnie i statystycznie istotne. Trendy czasowe masy jaj i zapłodnienia jaj były
w wie˛kszości przypadków dodatnie. Wpływ zwie˛kszania wartości cech reprodukcyjnych zaznaczył sie˛
przede wszystkim w dodatnich trendach czasowych długości mostka oraz w procentowym udziale mie˛sa.
Trendy czasowe procentowego udziału tłuszczu były podobne w całym okresie badań. W tym czasie
stwierdzono też statystycznie istotne zmniejszenie masy ciała i grubości mie˛śni piersiowych. Kojarzenie
losowe ge˛si spowodowało zwie˛kszenie wartości cech reprodukcyjnych, a zmniejszenie masy ciała
i grubości mie˛śni piersiowych oraz zwie˛kszenie długości mostka i procentowego udziału mie˛sa w tuszce.
LEVEL AND DURATION OF PERSISTENCE OF ANTIBODIES
IN THE BLOOD SERUM OF NATIVE VARIETIES OF GEESE
AFTER VACCINATION AGAINST DERZSY’S DISEASE
A d a m M a z a n o w s k i 1,3 E l ż b i e t a S a m o r e k - S a l a m o n o w i c z 2,
M a r i u s z U r b a n o w s k i3
1
Waterfowl Genetic Resources Station Dworzyska, National Research Institute of Animal Production,
62-035 Kórnik, Poland
2
Laboratory for Diagnosis of Poultry Viral Diseases, National Veterinary Research Institute,
Partyzantów 57, 24-100 Puławy, Poland
3
Department of Poultry Breeding, University of Technology and Agriculture, Mazowiecka 28,
85-084 Bydgoszcz, Poland
Abstract
The antibody level and duration of persistence were determined after vaccination against Derzsy’s
disease in native varieties of geese compared to White Kołuda geese. Lubelska (Lu), Kielecka (Ki)
and Podkarpacka (Pd) southern varieties and Kartuska (Ka), Rypińska (Ry) and Suwalska (Su)
northern varieties of ganders and geese were investigated. White Kołuda (WK) geese were the
control group. Each group included 45 ganders and 45 geese during rearing and 12 ganders and
24 geese during reproduction. The geese were immunized using a Palmivax vaccine (Merial). The
first vaccination was performed in 4-week-old geese of both sexes and the second in adult birds
before they went into egg production. Blood was drawn five times from 8 ganders and 8 geese of
each group to determine antibody levels using the ELISA test. The level of maternal antibodies was
determined in day-old goslings (0.153), four weeks after the first vaccination (0.247), on the day
of the second vaccination (0.272), four weeks after the second vaccination (0.398) and during the
final period of reproduction (0.226). The level of antibodies against Derzsy’s disease throughout
reproduction was higher in geese (0.270) than in ganders (0.248) and was the highest in all the
groups of geese four weeks after the second vaccination. Lubelska (0.320) and Kielecka (0.284)
geese of both sexes had the highest level of post-vaccination antibodies compared to the geese
of other varieties at four weeks after the first and second vaccinations. Towards the end of
reproduction, the level of antibodies decreased in all the groups of native goose varieties to the level
of maternal antibodies on the first day of goslings’ life. In White Kołuda geese, the high level of
antibodies persisted until the end of reproduction. The immune response of geese following
vaccination against Derzsy’s disease, expressed as the level of antibodies, was found to vary
according to the origin of the birds.
Key words: goose, conservation flocks, Derzsy’s disease, seroconversion after vaccination,
antibodies
76
Derzsy’s disease, found in geese and Muscovy ducks, is one of the most
dangerous of contagious diseases. It is caused by a parvovirus known as the
Derzsy’s disease virus or goose parvovirus (Kisary, 1993). Transmission among
goslings is horizontal via the digestive tract, while vertical infections are negligible
(Derzsy, 1967; Kisary, 1993). The virus replicates in the intestines, and the target
organs are the liver and heart, as well as the kidneys, thymus and pancreas (Derzsy,
1967; Kisary, 1986). Infected birds excrete a large number of molecules of the
contagious virus, which spread in the environment. The incidence, clinical
symptoms and mortality depend on the age and immune status of goslings.
Goose parvovirus (GPV) is resistant to physical and chemical agents (Kisary,
1993), which makes Derzsy’s disease control on goose farms a challenge that
requires concerted action aimed at eliminating the virus from the environment as
well as preventing birds from contracting the infection, using vaccinations. The
vaccine given to laying geese produces the antibodies, which are later transmitted to
the progeny through the egg (Kisary, 1977; Kisary et al., 1978; SamorekSalamonowicz et al., 1989 b). Maternal antibodies protect goslings from infection
during the time when they are most vulnerable, i.e. the first weeks of life (Kisary,
1986; Samorek-Salamonowicz, 1989 a). Despite the introduction of mass vaccinations, Derzsy’s disease continues to account for 15 – 20% of the mortality of
geese and Muscovy ducks (Gough, 1997; Samorek-Salamonowicz et al., 1998).
Analysis of disease cases has shown that the disease mostly affects goslings hatched
towards the end of reproduction, at a time when inadequate maternal antibody titre
is found (Samorek-Salamonowicz et al., 1989 b).
In addition to White Kołuda geese — produced as a result of long-term
selection, characterized by high reproductive and meat trait values and thus suitable
for intensive breeding — there are less productive native varieties of geese in
Poland that are suited to extensive breeding in small farms (Smalec, 1991). These
birds are resistant to harsh environmental conditions and poor nutrition. There are
no data on the resistance of native geese to the disease or on the induction of
immune processes. It is not entirely known whether the number of maternal
antibodies and post-vaccination immunity are the same in native geese and
high-producing geese (Samorek-Salamonowicz et al., 1997). Neither is it known
whether the immunity against Derzsy’s disease in unselected geese of native
varieties is the same as in White Kołuda geese, which have been selected over many
generations for their productive traits.
The aim of the study was to determine the level and duration of persistence of
serum antibodies after vaccination against Derzsy’s disease in six varieties of native
geese and in White Kołuda geese, during rearing and reproduction.
The study was carried out at the Waterfowl Genetic Resources Station Dworzyska, belonging to the National Research Institute of Animal Production. Lubelska (Lu),
Level of antibodies in geese vaccinated against Derzsy’s disease
77
Kielecka (Ki) and Podkarpacka (Pd) southern varieties and Kartuska (Ka), Rypińska
(Ry) and Suwalska (Su) northern varieties of ganders and geese were investigated.
White Kołuda (WK) geese were the control group. Each group included 45 ganders
and 45 geese during the 28-week rearing period and 12 ganders and 24 geese during
the reproductive period. The environmental and feeding conditions were identical for
all the geese. The birds were clinically healthy. Additional parasitological tests
conducted prior to vaccination and at the start and peak of lay were negative.
The geese were immunized against Derzsy’s disease using a Palmivax vaccine
(Merial, France), that is a lyophilized live vaccine for geese and Muscovy ducks.
The vaccine contained the Hoekstra strain of the DDV virus and was administered
intramuscularly, according to the manufacturer’s instructions, at a dose of 102.5
TCID50, 0.5 ml per bird. Geese were vaccinated twice under the programme. The
first vaccination was performed in 4-week-old geese of both sexes, following the
disappearance of maternal antibodies, and the second in adult birds before they
went into egg production. Blood was collected randomly from 8 ganders and
8 geese of each native variety to determine antibody levels against Derzsy’s disease
using the ELISA test. In order to allow comparison with the experimental geese, the
serum level of antibodies was also examined in White Kołuda geese.
Blood was taken from the geese five times to determine the level of antibodies
against Derzsy’s disease. The proportion of maternal antibodies was determined in
day-old goslings, four weeks post-vaccination, on the day of the second vaccination, four weeks after the second vaccination, and towards the end of
reproduction. Prior to blood sampling, food was withheld from geese for 12 hours.
Blood was taken from the wing vein (Vena ulnaris cutanea). The ELISA test was
performed according to the method of Samorek-Salamonowicz and Czekaj (1995).
Nunclon polystyrene dishes were coated with an antigen made from the B-38 strain.
Control and test sera were diluted at 1/100. Rabbit immunoglobulin against goose
IgG, labelled with horseradish peroxidase, was used as the conjugate, with ABTS
(Sigma) serving as a substrate. The results of colour reactions were read in
a spectrophotometer at a wavelength of 405 µm. Positive samples were those in
which the mean value of optical density (OD), which determines the level of
antibodies, exceeded 0.200. Samples with OD ranging from 0.150 to 0.200 were
considered doubtful, and those with OD below 0.150 were considered negative
(Samorek-Salamonowicz and Czekaj, 1995).
For geese of native varieties and White Kołuda geese, mean antibody levels
(OD) against Derzsy’s disease were calculated on each serum collection day and for
the whole study period. Time trends were also calculated for the level of antibodies
in native geese (southern and northern variety) of both sexes and in White Kołuda
geese, taking into account dates of serum evaluation. The time trends were plotted
using a linear regression equation y = a + bx, where a is the value of the analysed
trait on test dates, b is the directional coefficient expressing the increment in the
value of the trait on test dates, and x is the time expressed as the test dates. The time
trends made it possible to interpret and compare antibody levels (OD) on serum test
dates.
78
The results were analysed statistically (mean values of traits, standard errors of
the mean, analysis of variance, time trends of traits) using a statistical package.
Two-way analysis of variance was used in the calculations. The significance of
differences was verified using Duncan’s test.
Results
In day-old goslings, the mean antibody level, expressed in OD units, was
negative (0.153). The highest antibody level was found in Kartuska (0.184) and
Rypińska goslings (0.182). Four weeks after the first vaccination, the serum
antibody level was positive (0.247) except in Rypińska and White Kołuda goslings.
On the day of the second vaccination, the antibody level in all the goose groups was
positive and averaged 0.272 (Table 1). Four weeks after the second vaccination, the
antibody level (0.398) was the highest in the whole study period, and the difference
was statistically significant. At this time, a statistically significantly higher antibody
level was found in Lubelska and Kielecka geese. Towards the end of reproduction,
the level of antibodies declined dramatically in all the native varieties of
geese. White Kołuda geese represented an exception, as in these birds the antibody
level was significantly higher than in other birds (0.340 in ganders and 0.353
in geese).
The time trends for the antibody level in southern variety geese of both sexes
were positive (Figure 1). The highest antibody level was found four weeks after the
second vaccination, and the lowest on the first day of life and towards the end of
reproduction. The time trends for the antibody level in northern variety geese of
both sexes showed a slight increase, especially in Kartuska geese (Figure 2). Four
weeks after the second vaccination, a statistically significantly higher level of
antibodies was found in these geese. Towards the end of reproduction, the level of
antibodies decreased markedly. In White Kołuda geese, the time trend for the
antibody level showed a statistically significant increase throughout the study
(Figure 3). This increase became noticeable at the second vaccination (9 February
2000) and persisted until the end of reproduction (25 May 2000).
The mean levels of antibodies against Derzsy’s disease in the serum of ganders
and geese during the entire study period are given in Table 2. The highest level of
antibodies was found in Lubelska (0.320) and Kielecka ganders and geese (0.284),
as well as in White Kołuda geese (0.273). The mean antibody levels during the
entire study period were positive in native variety and White Kołuda geese. The
standard errors of the antibody level were low in the geese of all native varieties and
in White Kołuda geese.
tab. 1
79
Figure 2. Time trends of antibodies after vaccination against Derzsy’s disease in ganders and geese of northern varieties Kartuska (Ka),
Rypińska (Ry) and Suwalska (Su) on serum collection days.
Mean values for traits followed by different letters differ significantly (P ≤ 0.05). Date of test given in Table 1.
Figure 1. Time trends of antibodies after vaccination against Derzsy’s disease in ganders and geese of southern varieties Lubelska (Lu), Kielecka (Ki)
and Podkarpacka (Pd) on serum collection days.
Mean values for traits followed by different letters differ significantly (P ≤ =0.05). Date of test given in Table 1.
80
81
Figure 3. Time trends of antibodies after vaccination against Derzsy’s disease in ganders and geese of
White Kołuda (WK) variety on serum collection days.
Mean values for trait followed by different letters differ significantly (P ≤ 0.05).
* Time trends for traits differ significantly (P ≤ 0.05).
Date of test given in Table 1.
Table 2. Mean values (x̄) and standard errors of the mean (SEM) for level of antibodies (OD) against
Derzsy’s disease in blood serum of ganders and geese throughout the research period
Symbol of
conservation flock
Lu
Ki
Pd
Ka
Ry
Su
BK
x̄
SEM
Sex of geese

0.316
0.254
0.222
0.210
0.230
0.226
0.280
0.248
0.006

a
bc
c
d
bcd
bcd
b
0.324
0.314
0.257
0.242
0.241
0.247
0.266
0.270
0.006
a
b
c
c
c
c
bc
0.320
0.284
0.239
0.226
0.235
0.236
0.273
0.259
0.04
a
b
cd
d
c
cd
bc
Mean values for traits in columns followed by different letters differ significantly (P ≤ 0.05).
Discussion
The immune processes taking place in geese have received little study. It is
unknown whether unselected native geese are characterized by the same immunity
to disease and immune process dynamics after vaccination (Samorek-Salamonowicz et al., 1997) as White Kołuda geese that have been immunized against
Derzsy’s disease. The level of antibodies produced after vaccination was determined using the immunoenzymatic ELISA test, which proved highly useful for the
diagnosis of Derzsy’s disease, and produced results that showed high conformity
with the results obtained using other tests (Kardi and Szegletes, 1996; SamorekSalamonowicz and Czekaj, 1995).
82
In day-old goslings, the level of antibodies was low, although statistically
significant differences were found between goose varieties. The highest level of
antibodies was found in day-old Kartuska and Rypińska goslings. Goslings acquire
immune activity with age. Based on their own research, Kisary et al. (1978)
suggest that until the end of the first month of life, the immune system of goslings is
not mature, and their blood contains antibody inhibitors. Therefore, in this
experiment we vaccinated 4-week-old goslings in accordance with the current
immune prophylaxis calendar.
Vaccination against Derzsy’s disease generated antibodies, the levels of which
after the first vaccination were the highest in Lubelska and Kielecka ganders and
geese, and the lowest in Podkarpacka, Kartuska and Rypińska geese. In White
Kołuda geese, the level of antibodies (OD) was only 0.189 and in practical terms
can be regarded as negative. This level was similar in ganders (0.194). The reason
for the slower immunization of White Kołuda geese, although non-significantly
different from that of ganders and geese of some native varieties, is obscure,
because feeding and rearing conditions were the same for all the geese of native
varieties and White Kołuda geese. The geese were clinically healthy and their
rearing followed a normal course. Nevertheless, vaccination of White Kołuda geese
evoked a weaker immune response.
The first vaccination in 4-week-old geese and the second vaccination before the
start of lay provide the progeny with antibodies that protect them for two thirds of,
or even the entire, reproductive period (Kisary, 1986; Samorek-Salamonowicz et
al., 1989 a). This only concerns White Kołuda geese, in which the level of
antibodies rises slowly, but unlike the antibody level in native variety geese, is still
high (0.353) by the end of reproduction.
On the day of the second vaccination against Derzsy’s disease, there was
a marked drop in the level of antibodies in Lubelska and Kielecka geese and
a smaller decline in Kartuska and Suwalska geese, compared to the antibody level
four weeks after the first vaccination. In Podkarpacka, Rypińska and White Kołuda
geese, the level of antibodies increased. Overall, the level of antibodies on
the day of the second vaccination was significantly higher in all the geese
together compared to the antibody level four weeks after the first vaccination.
In White Kołuda geese, there was a marked increase in antibody titre on the day
of the second vaccination, compared to the previous test. It is likely that the
spread of the vaccine virus after the first vaccination against Derzsy’s disease
caused an immune reaction and had an effect on the production of antibodies for
a long time.
Four weeks after the second vaccination, in a similar way to after the first,
antibody levels were the highest in Lubelska and Kielecka ganders and geese. In the
other native varieties, the antibody level ranged from 0.242 to 0.346 in ganders and
from 0.312 to 0.381 in geese. These values, found in laying geese, guarantee that
specific antibodies will be transmitted through the egg to the progeny. These
antibodies will be sufficient to protect goslings from Derzsy’s disease during the
first weeks of life.
83
At the end of the reproductive period, there was a marked decline in the level of
antibodies in native variety geese. Antibody levels ranged from 0.174 in Kartuska
ganders to 0.273 in Lubelska ganders, and from 0.169 in Lubelska geese to 0.242
in Suwalska geese. The level of antibodies was significantly high only in White
Kołuda geese of both sexes. In these geese, the level of antibodies increased
gradually until the end of reproduction. The results obtained by other authors
(Kisary et al., 1978; Samorek-Salamonowicz et al., 1989 a, b; 1997) indicate
a decrease in the antibody titre of geese during the final period of reproduction. Therefore, the maintenance of, and even the increase in, the level of
antibodies in White Kołuda geese towards the end of reproduction requires further
study.
The level of serum antibodies against Derzsy’s disease in southern and northern
variety ganders and geese were also presented as time trends. The time trend for the
serum level of antibodies in Lubelska geese achieved a significantly higher value
four weeks after the second vaccination, then diminished to a low value during
reproduction, similar to the value found on the first day of life. The same types of
changes were also found in Kielecka geese. In Podkarpacka geese of both sexes, the
significantly higher time trends were found for the level of antibodies on the day of
the second vaccination and four weeks after the second vaccination.
In northern variety geese, the antibody level did not show such a clear positive
time trend as in southern variety geese. For this reason, the decrease in the level of
antibodies against Derzsy’s disease in northern variety geese was not as large
at the end of reproduction as in the blood serum of southern variety geese, in
which the level of antibodies increased rapidly four weeks after the second
vaccination and decreased rapidly during reproduction. In Kartuska and Rypińska
geese, no clear increase in the time trend for the level of antibodies against Derzsy’s
disease was found four weeks after the second vaccination or during the final
period of reproduction. This trend appeared only in Suwalska geese. The time
trend for the level of antibodies against Derzsy’s disease in White Kołuda geese
increased from the first collection of blood from day-old goslings to the fourth
collection, which was performed four weeks after the second vaccination. Towards
the end of reproduction, the level of antibodies in White Kołuda geese did not
fall.
The level of antibodies against Derzsy’s disease, calculated separately for
ganders and geese of native southern and northern varieties and for White Kołuda
geese during the whole study period, was the highest in Lubelska and Kielecka
geese, and the lowest in Kartuska geese. Standard errors of the mean for the
antibody level were low. Foreign references reveal no studies on the immune
response after vaccination against Derzsy’s disease in geese of different origin.
Polish studies (Samorek-Salamonowicz et al., 1997) have compared the immune
response of White Kołuda and greylag geese and their reciprocal crosses after
two-time vaccination against Derzsy’s disease. The highest antibody titres were
found in greylag geese and White Kołuda ganders crossed with greylag geese,
which proved the most immunocompetent.
84
During the entire study period it was shown that the level of antibodies against
Derzsy’s disease was slightly higher in geese than in ganders. In native geese of
southern and northern varieties and in White Kołuda geese, the antibody levels were
the highest four weeks after the second vaccination. In southern variety Lubelska
and Kielecka geese, compared to the other varieties of geese, the highest level of
post-vaccination antibodies was found four weeks after the first and second
vaccination. In the final period of reproduction, the level of antibodies decreased
markedly in all the groups of native variety geese and was similar to or lower than
the level of maternal antibodies on the first day of goslings’ life. Only in White
Kołuda geese was the level of antibodies against Derzsy’s disease similar four
weeks after the second vaccination and towards the end of reproduction. The
resistance of White Kołuda geese against Derzsy’s disease increased slowly but
lasted longer than in native variety geese. The highest antibody level in the whole
study period was found in Lubelska, Kielecka and White Kołuda ganders and geese.
In summary, the intensification of the immune response in geese after vaccination
against Derzsy’s disease, expressed as production of antibodies, varied according to
the origin of the birds.
References
D e r z s y D. (1967). A viral disease of goslings. Acta Vet. Hung., 17: 443 – 448.
G o u g h R. E. (1997). Goose parvovirus infection. Diseases of Poultry X ed. Calnek B.W., MosbyWolfe, pp. 777 – 783.
K a r d i V., S z e g l e t e s E. (1996). Use of ELISA procedures for the detection of Derzsy’s disease virus
of geese and of antibodies produced against it. Avian Pathol., 25: 25 – 34.
K i s a r y J. (1977). Immunological aspects of Derzsy’s disease in goslings. Avian Pathol., 6: 327 – 334.
K i s a r y J., D e r z s y D., M e s z a r o s J. (1978). Attenuation of the goose parvovirus strain B.
Laboratory and field trials of the attenuated mutant for vaccination against Derzsy’s disease. Avian
Pathol., 7: 397 – 406.
K i s a r y J. (1986). Acute virus infections of poultry. Martinus Nijhoff, Dordrecht, Netherlands,
pp. 239 – 242.
K i s a r y J. (1993). Virus infections of birds, Martinus Nijhoff, Dordrecht/Boston/Lancaster, pp. 157 – 162.
S a m o r e k - S a l a m o n o w i c z E., C z e k a j H., T o m c z y k G. (1989 a). Kształtowanie sie˛ odporności
matczynej u ga˛sia˛t pochodza˛cych od matek szczepionych przeciwko chorobie Derzsy’ego. Med.
Wet., 45: 36 – 38.
S a m o r e k - S a l a m o n o w i c z E., C z e k a j H., T o m c z y k G., M u s i a l i k M. (1989 b). Serokonwersja po szczepieniu przeciwko chorobie Derzsy’ego u ge˛si niosek oraz ich potomstwa. Med. Wet.,
45: 336 – 338.
S a m o r e k - S a l a m o n o w i c z E., C z e k a j H. (1995). Opracowanie testów do szybkiej diagnostyki
choroby Derzs’yego u ga˛sia˛t. Mat. Sesji Naukowej „Stan badań weterynaryjnych w świetle wyników
uzyskanych w zakończonych projektach badawczych finansowanych przez KBN”, Puławy.
S a m o r e k - S a l a m o n o w i c z E., C h e ł m o ń s k a B., K o z d r u ń W., C h r z a n o w s k a M., Czekaj
H. (1997). Serokonwersja po szczepieniu przeciwko chorobie Derzsy’ego u różnych ras ge˛si. Mat.
II Mie˛dzynarodowego Symp. Naukowego „Aktualne problemy w rozrodzie ptaków”, Wrocław, 31:
251 – 253.
S a m o r e k - S a l a m o n o w i c z E., C z e k a j H., K o z d r u ń W. (1998). Choroby zakaźne wyste˛puja˛ce
u drobiu wodnego. Med. Wet., 54: 305 – 308.
85
S m a l e c E. (1991). Zróżnicowanie ge˛si rezerwy genetycznej pod wzgle˛dem cech użytkowych
i polimorfizmu białek surowicy krwi. Zesz. Nauk. Drob., COBRD, Poznań (rozpr. hab.), 3: 5 – 87.
ADAM MAZANOWSKI, ELŻBIETA SAMOREK-SALAMONOWICZ, MARIUSZ URBANOWSKI
Poziom i czas utrzymywania sie˛ przeciwciał w surowicy krwi ge˛si odmian krajowych
po szczepieniu przeciwko chorobie Derzsy’ego
STRESZCZENIE
Określono poziom i czas utrzymywania sie˛ przeciwciał po szczepieniu przeciwko chorobie
Derzsy’ego u ge˛si odmian krajowych, w porównaniu z ge˛siami białymi kołudzkimi. Materiał doświadczalny stanowiły ge˛siory i ge˛si odmian południowych — lubelskie (Lu), kieleckie (Ki) i podkarpackie
(Pd) oraz północnych — kartuskie (Ka), rypińskie (Ry) i suwalskie (Su). Ge˛si białe kołudzkie (BK)
tworzyły grupe˛ kontrolna˛.
W czasie wychowu każda grupa liczyła 45 ge˛siorów i 45 ge˛si, a w okresie reprodukcji 12 ge˛siorów
i 24 ge˛si. W celu uodpornienia ge˛si przeciwko chorobie Derzsy’ego zastosowano szczepionke˛ Polmivax
firmy Merial. Pierwsze szczepienie przeprowadzono u czterotygodniowych ge˛si obojga płci, a drugie
u ptaków dorosłych przed nieśnościa˛. W celu określenia poziomu przeciwciał testem ELISA, pobrano
pie˛ciokrotnie krew od 8 ge˛siorów i 8 ge˛si z każdej grupy. Poziom przeciwciał matczynych określono
u jednodniowych ga˛sia˛t (0.153), naste˛pnie w cztery tygodnie po pierwszym szczepieniu (0.247), w dniu
drugiego szczepienia (0.272), w cztery tygodnie po drugim szczepieniu (0.398) i w końcowym okresie
reprodukcji (0.226).
Poziom przeciwciał przeciwko chorobie Derzsy’ego w całym okresie reprodukcji był wie˛kszy
u ge˛si (0.270) niż u ge˛siorów (0.248) i we wszystkich grupach ge˛si najwie˛kszy w cztery tygodnie po
drugim szczepieniu. Ge˛si obojga płci — lubelskie (0.320) i kieleckie (0.284), w porównaniu z ge˛siami
innych odmian, miały najwyższy poziom przeciwciał poszczepiennych w cztery tygodnie po pierwszym
i po drugim szczepieniu. W końcowym okresie reprodukcji poziom przeciwciał zmalał we wszystkich
grupach ge˛si odmian krajowych do poziomu przeciwciał matczynych w pierwszym dniu życia ga˛sia˛t.
Natomiast u ge˛si białych kołudzkich wysoki poziom przeciwciał utrzymał sie˛ do końca reprodukcji.
Stwierdzono, że nasilenie odpowiedzi immunologicznej u ge˛si po szczepieniu przeciwko chorobie
Derzsy’ego, wyrażone poziomem przeciwciał, było różne i zależało od pochodzenia ptaków.
EFFECT OF RAPESEED MEAL PROTECTED WITH CALCIUM SALTS
OF FATTY ACIDS FROM LINSEED OIL ON COWS’ YIELD
AND MILK AND BLOOD PARAMETERS
F r a n c i s z e k B r z ó s k a
Department of Animal Nutrition and Feed Science, National Research Institute of Animal Production,
Abstract
The experiment involved 32 Red-and-White cows assigned equally to 4 groups, with 4 periods of 32
days each. Cows were fed on pasture and additionally received field forage, fresh brewer’s grains
and feed mixture. The feed mixture contained 20% rapeseed meal unprotected from rumen
degradation (control group) and 20, 25 or 30% rapeseed meal protected with calcium salts of fatty
acids (CSFA) from linseed oil (experimental groups). This corresponded to a rapeseed meal intake
of 1.56, 1.61, 1.80 and 2.15 kg/day, respectively. The amount of undegradable protein in the diets
was 26, 28, 30 and 32% crude protein, respectively. Feed intake by the cows averaged 22.3 kg dry
matter/day, including feed mixture intake of 7.54 kg dry matter/day. The milk yield of cows was
25.47 in the control group and 26.76, 27.72 and 26.08 kg/day in the experimental groups,
respectively, and differed significantly between the groups. The amount fat and protein
corrected milk (FPCM) was 25.14 in the control group and 25.80, 25.97 and 26.50 kg/day,
respectively, in the experimental groups, and the difference compared to the control group was
significantly higher. Feeding protected rapeseed meal increased the cows’ yield by 1 – 2.5 kg/day
compared to feeding unprotected rapeseed meal. The fat and protein content of milk showed an
upward tendency, but the differences were not significant. Giving protected rapeseed meal
significantly increased the amount of fat and protein secreted in milk. Fat yield was 986 g/day in
the control group and 982, 1120 and 1067 g/day in the experimental groups, respectively. No
significant differences were found in the N fractions of milk or between groups of cows in terms of
milk acidity, renneting time or density. There were no differences in plasma levels of glucose, total
protein or triglycerides between cows from particular groups. Giving cows rapeseed meal
protected with CSFA significantly increased blood urea levels. No significant differences were
found between groups of cows in the fatty acid content of milk, including saturated and
unsaturated fatty acids, or in their mutual proportions. It is concluded from the study that giving
rapeseed meal protected with CSFA from linseed oil to cows with an average yield of 26 kg
milk/day significantly increases their milk yield, increases daily production of fat and protein, and
has no effect on other parameters of milk quality or on the overall metabolism of the cows, with an
increase in the concentration of urea N in milk.
Key words: protected rapeseed meal, milk yield, protein content, milk fatty acids
Abbreviation key: PRSM — protected rapeseed meal, Control — not protected rapeseed meal,
CSFA — calcium salts of fatty acids, FPCM — fat and protein corrected milk, CLA — conjugated
linoleic acid
88
F. Brzóska
In addition to soybean meal, rapeseed meal is one of the principal protein feeds
used in cow nutrition in Poland. In terms of amino acid composition, the biological
value of rapeseed protein does not differ significantly from the nutritive value of
soybean meal. Earlier studies showed that rumen degradation of rapeseed meal is
approximately 57 – 81% after 12 – 24 h of in sacco digestion (Pieszka and Brzóska,
2000). This means that over two-thirds of rapeseed meal is degraded in the rumen
with less than one-third passing to the duodenum and the small intestine. Considering
the high degradability of bulky feeds (including pasture forage, silage and hay),
which negatively affects cows’ fertility (Canfield et al., 1990), in our climatic zone
there is a shortage of feeds with reduced rumen degradability to formulate diets in
accordance with the latest ruminant feeding requirements (Tamminga et al., 1994;
INRA, 1988; NRC, 2001). There are several known methods for protecting feed protein
against rumen degradation, such as the use of high temperature (Schroeder et al., 1995),
formaldehyde (Subuh et al., 1996), lignosulphonates (Standford et al., 1995) and xylose
(Nakamura et al., 1992). In recent years, CSFA have been used to protect oil meal
protein (Rohr et al., 1993; Rossi et al., 1999). In this method, particles of high-protein
feed are covered with a layer of CSFA. The fact that calcium salts melt at a higher
temperature than the rumen temperature results in protein passing through the rumen
without degradation by proteolytic bacteria. However, the effective protection of
protein feeds requires fairly large amounts of calcium salts (200 – 300 g/kg feed), which
reduces the overall level of protein in the feed (Pieszka and Brzóska, 2000; 2001 a). At
present, CSFA are produced from vegetable oils, especially linseed oil, and enrich
protein feeds in energy, including unsaturated fatty acids (Klusmayer et al., 1989),
which may modify the fatty acid composition of milk. Preliminary studies on the
efficiency of giving soybean meal and rapeseed meal protected with CSFA to silage-fed
cows in the winter period have shown that these have a positive effect on milk yield and
increase milk fat content (Pieszka and Brzóska, 2001 b).
We hypothesized that giving pastured cows a feed mixture containing rapeseed
meal protected with CSFA would have a stimulatory effect, leading to increased
milk production and protein and fat synthesis in milk as a result of the increased
supply of amino acids to the small intestine. Another hypothesis was that CSFA
from linseed oil would increase the level of unsaturated fatty acids in milk. The aim
of the study was to test these hypotheses.
Experimental design and animals
The study was carried out in the Grodziec Śla˛ski Experimental Station at the
Lipowa farm (Silesian province), using 32 Red-and-White cows assigned equally to
4 groups (8 cows per group), with 4 periods of 32 days each. The experimental
animals were chosen from a herd of 135 cows with an average yield of
6,000 – 7,000 kg milk. The cows were aged 5 – 7 years, were between their third and
fifth lactations, and calved between late February and early April. The 138-day
Effect of protected rapeseed meal on cows’ yield and blood parameters
89
experiment began on 14 May. The cows’ ration contained grazed pasture grass as
well as field-cultivated forage, brewer’s grains and feed mixture given in the
cowhouse. The feed mixture was given at amounts of 0.28 kg/kg of milk obtained.
The pasture forage contained mostly orchard-grass, perennial ryegrass and meadow
fescue. The field forage contained a mixture of red clover and grasses. The feed
mixture contained unprotected rapeseed meal (control) and rapeseed meal protected
with CSFA (experimental), given at amounts of 20, 25 and 30% of the feed mixture
(Table 1). Protected rapeseed meal was purchased for the experiment from the
INNFOSS Company in Poznań, Poland. The grazing area was allocated every
2 days based on pasture yield, so as to ensure intake of approximately 8 – 10 kg dry
matter during 8-hour grazing. The grazing area was surrounded by an electric fence.
Cows had constant access to water. Cows received brewer’s grains and field forage
in the afternoon, when they left the pasture. Feed mixtures were made on the farm
from cereals, not protected or protected rapeseed meal, supplemented with Bovimix
mineral mixture (BASF Kutno, Poland) at amounts of 1%, and administered twice
a day during milking. In the cowhouse, cows were fed individually and intake of
bulky feeds and feed mixture was determined.
Samples and analytical methods
Feed samples (500 g) were taken at 2-week intervals, and dried and pooled for
each group and period separately. Feeds were assayed for dry matter, basic
nutrients, acid detergent fibre (ADF) and neutral detergent fibre (NDF) content. The
nutrient content was used to calculate the nutritive value of the feeds, using
INWAR 1.3 software. Cows were milked twice a day. On the last 3 days of each
experimental period, the milk yield of the cows was checked and milk samples were
taken for analyses. The amount of milk drawn was checked using a Tru-Test milk
meter (model FV). Milk samples were preserved with 2-bromo-2-nitro-1,3-propanediol (GROPOL) and stored frozen until further analyses. On the last day of each
period, milk samples from the jugular vein were taken into heparinized tubes and
centrifuged to obtain plasma. Glucose was determined in plasma samples after
collection, and the other analyses in plasma were performed after thawing. The
plasma was also assayed for total protein, urea and triglycerides.
The basic nutrient content of the feeds was determined by way of standard
procedures (AOAC, 1990) using nitrogen, fat (Büchi) and crude fibre analysers
(Foss-Tecator). Feed dry matter was determined by drying at 105oC. The NDF and
ADF content was determined according to Goering and van Soest (1970). Protein,
fat and lactose in milk were determined using a MilkoScan FT 120 (Foss Electric),
and milk pH, specific gravity and density were determined in accordance with
standard PN-68/A-86112. Nitrogen fractions of milk were determined using the
method of Kjeldahl according to the procedure described by Gordon and Kolan
(1983). Milk fatty acids were determined as methyl esters of fatty acids using gas
chromatography (GC Varian 3400 on a CP-Wax 58 column, 60 m, 0.53 mm, 1.0
micron) according to the method of Atwal et al. (1990). The acid determination
method is described in detail by Brzóska (2004).
90
F. Brzóska
The levels of glucose, total protein, urea and triglycerides in plasma were determined
enzymatically using diagnostic kits (Cormay Diagnostyka S.A., Lublin, Poland).
FPCM yield was calculated using the formula of Subnel et al. (1994), where:
FPCM = Fat and Protein Corrected Milk (kg) = [0.337 + 0.116 × fat (%) +
0.06 × protein (%)] × milk (kg)
The results were analysed statistically using analysis of variance and the new
multiple range test using Statgraphics 6.0 software.
Results
The unprotected rapeseed meal content of the feed mixture for the control group
was 20%, which corresponded to an intake of 1.56 kg/day. The protected rapeseed
meal content of the feed mixture in the experimental groups was 20, 25 and 30%,
respectively (Table 1), which corresponded to an intake of 1.61, 1.80 and
2.15 kg/day, respectively (Table 2). Assuming that CSFA used to protect protein
Table 1. Composition of diets (on DM basis)
Item
Control1
Protected RSM2
20
25
30
39.3
20.7
5.3
20.4
6.9
43.5
18.7
5.6
17.5
6.5
42.5
19.3
5.8
16.1
6.5
0.1
7.3
0.1
8.1
0.1
9.7
0.1
Chemical composition:
crude protein
crude fat
NDF
ADF
ash
17.8
3.1
46.0
20.5
6.0
17.3
3.6
46.0
20.6
6.2
17.9
3.9
47.9
21.7
6.5
18.0
3.9
47.7
21.4
6.5
Estimated RUP, NEL3:
RUP (% CP)
NEL (Mcal/kg)
(MJ/kg)
26.0
7.15
1.71
28.0
7.07
1.69
29.5
7.07
1.69
31.5
7.07
1.69
Ingredients:
meadow pasture
arable land forage
brewer’s barley grain
ground wheat
ground barley
rapeseed meal, control
rapeseed meal, protected
mineral-vitamin mixture
44.4
13.2
6.0
21.6
7.4
7.3
1
Control — non protected rapeseed meal, 20% of concentrate; 2 protected rapeseed meal, 20, 25 and 30% of
concentrate respectively; 3 rumen undigested protein computed from INRA (1988) tables as CP % values for diet
ingredients.
91
represented 25% of the meals, the actual protected rapeseed intake in the experimental groups was 1.21, 1.35 and 1.61 kg/day, respectively. The level of total
protein in the control feed mixture was 159.4 g/kg and ranged from 152.8 to 164.6 g/kg
in the experimental feed mixtures. Dry matter intake averaged 22.3 kg/day, including
approximately 7.54 kg dry matter/day from feed mixtures, and did not differ significantly between the groups (Table 2). The milk yield of the cows was 25.47 kg/day
in the control group and 26.85 kg/day in the experimental groups, and differed
significantly between the groups (P < 0.01). The amount of FPCM was 25.14 kg in the
control group and 26.76 kg in the experimental groups, and was significantly higher
than in the control group (P < 0.01). The fat and protein content of milk did not differ
significantly, although it showed an upward tendency. Significantly greater amounts
of protected fat (P < 0.01) and protected protein (P < 0.05) were found to be secreted
in the milk of cows receiving rapeseed meal. There were no significant differences in
the N fraction content of milk (Table 3). Protected meal had no effect on physical
traits of milk such as pH, renneting time, or density. The cows’ body weights varied
greatly. No significant differences were found in the plasma levels of glucose, total
protein, or triglycerides, with a significant increase in the urea content of blood in
cows from the PRSM-30 group (P < 0.01) (Table 4).
Table 2. Milk yield, milk composition, milk physical parameters and cows’ body weight
Item
Dry matter intake (kg/day):
total
meadow pasture
arable land forage
brewer’s barley grain
concentrate
NSBM in concentrate
PSBM in concentrate
Body weight (kg)
Milk yield (kg/day)
FPCM (kg/day):
fat (%)
protein (%)
lactose (%)
fat (g/day)
protein (g/day)
lactose (g/day)
urea
Acidity (oSH)
Renneting time (s)
Density (g/cm3)
BW change (g/day)
Control1
21.20
9.35
2.79
1.28
7.78
1.56
521
25.47 cC
25.14 cC
3.87 b
3.35
4.88
986 cC
853 b
1243
0.038
1.59
194
1.031
+ 186
20
Protected RSM2
25
30
23.28
9.16
4.83
1.23
8.06
22.32
9.72
4.18
1.24
7.18
22.10
9.40
4.27
1.28
7.15
1.61
1.80
2.15
534
26.76 bB
25.80 cC
3.67 b
3.35
4.92
982 cC
896 a
1317
0.036
1.59
163
1.0296
– 37
525
27.72 aA
27.97 aA
4.04 a
3.38
4.90
1120 aA
931 a
1358
0.036
1.65
148
1.0297
–8
558
26.08 bB
26.50 bB
4.09 a
3.41
4.87
1067 bB
889 a
1270
0.039
1.58
195
1.0302
+ 95
a, b, c — values in the same rows with different letters differ significantly (P < 0.05).
A, B — values in the same rows with different letters differ significantly (P < 0.01).
1–2
for abbreviations see Table 1.
SEM
0.22
0.18
0.09
0.05
0.09
1.03
0.93
0.14
0.04
0.03
0.13
39
33
0.00
0.03
17
0.00
92
F. Brzóska
Table 3. Milk nitrogen fractions (%)
Control1
Item
Total N
True protein N
NPN
Whey protein N
Casein N
N true (% total N)
N casein (% total N)
0.538
0.501
0.036
0.098
0.403
93.12
74.91
Protected RSM2
20
25
30
0.538
0.502
0.036
0.096
0.406
93.31
75.46
0.542
0.504
0.036
0.101
0.403
92.99
74.35
0.547
0.506
0.034
0.102
0.404
92.50
73.86
SEM
0.007
0.007
0.001
0.003
0.005
0.34
0.55
All values in the table were not significant.
1–2
Table 4. Physiological parameters of cows’ blood plasma (mg/100 ml)
Control1
Item
Glucose
Total protein
Urea
Triglycerides
76.32
8.41
39.96 bB
6.92
Protected RSM2
20
25
74.88
8.60
38.24 bB
7.61
30
74.34
8.63
40.34 AB
7.11
SEM
74.70
0.08
8.48
0.11
46.05 aA 1.30
7.25
0.34
a, b — values in the same rows with different letters differ significantly (P < 0.05).
1–2
Giving the cows protected rapeseed meal significantly increased the unsaturated
fatty acid (UFA) content of the milk of cows in the PRSM-25 group, including
monounsaturated fatty acids (MUFA) (P < 0.01). In this group, the protected meal
caused a significant improvement in the level of hypocholesterolemic fatty acids
(DFA) in milk and the hypo- to hypercholesterolemic (DFA/OFA) acid ratio
(P < 0.01). The conjugated linoleic acid (CLA) content remained similar across
groups, with no significant differences (Table 5).
Table 5. Fatty acid content of fat milk (g/100 g)
Item
Control1
1
2
Estimated:
C8
C 10
C 12
C 14
C 16
C 16:1
C 18
2.10
4.09
4.47
12.59
30.94
2.14
12.20
aA
aA
abAB
aA
b
20
Protected RSM2
25
30
3
4
5
2.08
4.01
4.26
12.68
30.92
1.91
12.73
aAB
aAB
abAB
bAB
ab
1.80
3.20
3.43
11.31
29.63
1.96
13.40
bB
bB
bB
abAB
ab
2.01
3.59
3.84
12.41
31.36
1.80
13.72
SEM
6
abAB
abAB
aA
bB
a
0.05
0.10
0.10
0.19
0.20
0.03
0.19
93
Table 5 — contd.
1
C 18:1
C 18:2
C 18:3 n-6
C 18:3 n-3
CLA
C 20
C 20:4
C 20:5 EPA
C 22
C 22:6 DHA
Calculated:
SFA
UFA
MUFA
PUFA
PUFA 6
PUFA 3
DFA
OFA
UFA/SFA
DFA/OFA
PUFA/SFA
MUFA/SFA
2
3
4
26.35 b
2.57
0.19
0.51
1.61
0.15 bB
0.10 aA
0.047 a
0.067 b
0.004
25.98 b
2.62
0.19
0.51
1.69
0.19 aAB
0.09 abAB
0.045 ab
0.090 a
0.007
29.47 a
2.75
0.19
0.53
1.65
0.18 abAB
0.08 bcBC
0.038 bc
0.077 ab
0.005
26.12 b
2.57
0.19
0.54
1.74
0.20 aA
0.07 cC
0.037 c
0.091 a
0.007
0.41
0.03
0.00
0.01
0.04
0.00
0.00
0.001
0.002
0.000
66.55
33.45
28.21
4.95
2.86
0.57
45.65
54.35
0.51
0.87
0.07
0.43
66.95
33.05
27.89
5.16
2.91
0.56
45.75
54.22
0.50
0.85
0.08
0.42
64.26
36.67
31.43
5.25
3.02
0.58
50.07
49.93
0.58
0.93
0.08
0.50
66.84
33.17
28.01
5.15
2.84
0.58
46.89
53.11
0.50
0.89
0.08
0.42
0.50
0.47
0.43
0.06
0.03
0.01
0.49
0.49
0.01
0.02
0.00
0.01
b
b
bB
aA
bAB
bB
bAB
b
b
bB
aA
bAB
bB
bB
5
a
a
aA
bB
aA
aA
aA
6
b
b
abAB
abAB
bAB
bAB
bB
A, B, C — values in the same rows with different letters differ significantly (P < 0.01).
1–2
Discussion
The intake of cows on pasture was 7 – 10 kg dry matter containing 18 – 24%
crude protein, of which just 20% was rumen undegradable protein (McCormick et
al., 1999). This could result in a deficiency of intestinally digested protein. Earlier
studies in which cows were fed grass silages showed that giving soybean and
rapeseed meal protected with CSFA to cows yielding an average of 26 – 30 kg
milk/day increases their productivity by 1 – 2 kg, with increases in the protein and
fat content of milk (Pieszka and Brzóska, 2001 b). A similar effect was obtained by
giving cows protected soybean meal during the pasture period (Brzóska, 2005). The
efficiency of using protected protein in cow nutrition depends on whether the
requirement for intestinally digested amino acids is fulfilled. The amino acid deficit
in cow nutrition increases with increasing milk yield. Schingoethe (1996) reported
that cows producing more than 5 kg milk/day per 100 kg body weight respond to
the intake of ruminal escape protein with a further increase in milk production. The
positive effect of undegradable protein on the yield of cows has been confirmed in
many studies (DePeters and Cant, 1992; Schor and Gagliostro, 2001), including
94
F. Brzóska
a study in which casein was infused into the abomasum (Whitelaw et al., 1986). In
other studies, protected protein had no significant effect or did not affect the cows’
milk yield (Schingoethe, 1996; McCormick et al., 2001). In cow feeding standards,
28 – 30% crude protein is considered to be the optimal proportion of undegradable
protein in the ration (NRC, 2001).
Summer feeding with pasture forage causes the cows to have a relatively high
intake of dry matter with a high level of protein, which is rapidly degraded in the
rumen into ammonia (Horngherholt and Muller, 1998). This may lead to a deficiency of undegradable protein in early lactation. Increasing the amount of undegradable protein in feed mixtures for cows is considered important when feeding lactating
cows (Santos et al., 1998). In many studies, blood meals, meat meals and feathers
have been used as undegradable protein (Palmquist and Weiss, 1994). Their use in
cow nutrition has been banned by law in the EU to help prevent the incidence of
BSE in cattle. Fish meals (Schroeder and Gagliostro, 2000) and plant products such
as maize gluten (Erdman and VanDersall, 1983), which are characterized by low
rumen degradability, have also been used in cow nutrition. The search continues for
efficient means of protecting plant protein against rumen degradation in our
climatic zone, as an alternative to the methods currently used. The Megapro
preparation, which is a rapeseed meal protected with calcium salts of palmitic acid,
is available on the Polish market. Studies performed with cows yielding 32 – 35 kg
milk/day have demonstrated a positive effect of Megapro supplementation in terms
of milk yield and increased fat content, with a tendency towards higher milk protein
content (Kraszewski et al., 2003). In other studies in cows with similar milk yield,
which were fed maize gluten diets containing protein degraded to varying degrees
in the rumen, there were no differences in cows’ milk yield or milk components
(Erdman and VanDersall, 1983). An original method for the protection of soybean
and rapeseed meal with Erafet friable fat was developed in Poland (E. Foss —
personal communication). This resulted in the creation of two preparations
characterized by 35 – 40% rumen degradability of protein, known as Soyafos and
Rapefos (Pieszka et al., 2000).
The present results indicate that giving cows rapeseed meal protected with
CSFA at rates of 20, 25 and 30% of feed mixtures increases milk production in
Red-and-White cows during the pasture period by 1 – 2 kg/day, with a significant
increase in the fat content of milk and a tendency towards higher milk protein
content. This results in increased production of FPCM as calculated according to
the method of Subnel et al. (1994). Giving the cows rapeseed meal protected with
CSFA did not adversely affect the physical traits of milk, including milk density,
pH and renneting time, indicating that the milk of these cows is suitable for
processing and cheese-making.
No significant differences were found in the metabolic indicators based on
analysis of cows’ blood serum, including carbohydrates and fats. There were no
significant differences in the levels of these metabolic indicators in blood and these
were in accordance with the reference data for lactating cows reported by Kłopocki
and Winnicka (1992). In the group of experimental cows with the highest
95
proportion of protected meal, the blood serum urea content was significantly higher
than in the other groups. The urea content of blood is closely linked to the level of
urea in the rumen and to the degree of protein degradation to ammonia (Davidson et
al., 2003). Giving cows protected protein should reduce the level of ammonia
nitrogen in the rumen and thus the level of urea in blood serum. In this study, the
reaction was reverse, which is hard to justify. A possible reason is that fat protected
both the protein and the non-protein part of the whole crude protein found in
rapeseed meal. The non-protein part contains plant nitrogen in the form of
ammonia, which may be a source of increased urea synthesis in the body. The
positive effect of giving early-lactation cows feeds with reduced rumen degradability during the pasture period has been reported by several authors (Rogers
et al., 1980; Schroeder and Gagliostro, 2000), although this effect did not occur in
other studies (Dunlap et al., 2000; McCormick et al., 2001). Schor and Gagliostro
(2001) attributed the mechanism of increased milk yield to two factors: increased
dry matter intake or increased fat mobilization in cows receiving protected protein.
In this study, giving protected rapeseed meal increased the intake of pasture forage
and field forage, which seems to support this theory. Higher fat mobilization was
not found in cows from the experimental groups, as greater body weight decreases
were found in these animals during the experiment. The latter authors also reported
that it is unclear how the protected protein absorbed can affect the mobilization of
fat. In another study, Rossi et al. (1999) showed unprotected soybean meal to have
greater intestinal digestibility than fat-protected meal. They speculated that this is
because the process of digestion by proteolytic enzymes is disturbed by the
presence of a fat matrix. Studies in bulls on the intestinal digestibility of ruminally
undegraded soybean and rapeseed meal protected with Erafet (Pieszka and Brzóska,
2000; 2001a) showed that it is 92% and 74%, respectively, with no differences in
digestibility between meals protected and not protected with fat. These findings
confirm the data reported by Żebrowska et al. (1997) for unprotected rapeseed
meal. Opinions vary as to the intestinal digestibility and availability of amino acids
from soybean and rapeseed meal. Comparative studies performed on intestinally
cannulated heifers showed that compared to soybean meal, rapeseed meal increases
the flow rate of total amino acids (including sulphur amino acids) to the small
intestine, and its ruminal degradation is significantly lower (Lardy et al., 1999).
Protection of high-protein meals is effective when fairly large amounts of CSFA
are used. A daily intake of 1 – 2 kg protected meal corresponds to an intake of
0.20 – 0.40 kg of CSFA. Salts are fairly abundant in unsaturated fatty acids, which
can modify the composition of milk fatty acids. In a study on the pasture feeding of
cows receiving soybean meal protected with Erafet, increased milk fat content and
increased milk production in 24-h cycles were found, while the increase in the level
of unsaturated fatty acids fat in the cows’ milk was small (Brzóska, 2005).
Analysis of milk nitrogen fractions showed that the proportions of true N and
casein N in milk in relation to the total N content of milk conformed with standard
values for cow’s milk and were in keeping with the results of earlier studies
performed on Red-and-White cows (Brzóska et al., 1999; Ruppert et al., 2003).
96
F. Brzóska
It was expected that giving cows protected rapeseed meal would increase the
supply of amino acids to the small intestine, which would, in turn, increase the
protein content of milk and its 24-h synthesis. A slight increase in the protein
content of the cows’ milk was found in an earlier study in which pastured cows
were given soybean meal protected with CSFA (Brzóska, 2005). In this study there
was no increase in the protein content of milk, although there was a tendency
towards higher protein levels in the milk of cows receiving protected rapeseed
meal. A significantly higher 24-h protein synthesis result was obtained. This
suggests a need to study the amino acid composition of rumen escape protein in
protected feeds, including rapeseed meal, so as to determine the flow rate and
supply of amino acids to the small intestine using cows with a daily yield of
35 – 40 kg milk.
In terms of physical traits, which together with casein content are of great
technological importance for processing milk into cheese, the milk did not differ
significantly across the groups. These parameters did not differ fundamentally from
the earlier results of studies of Red-and-White cows (Brzóska et al., 1999; Pieszka
and Brzóska, 2000; Brzóska, 2005), indicating that the level of undegradable
protein in the rumen does not affect these parameters of milk.
In summary, giving pastured cows with an average yield of approximately
26.0 kg milk/day rapeseed meal protected with CSFA from linseed oil increased
their yield in the pasture period by 1.0 – 2.0 kg/day, with a significant increase in
the fat content of milk and an increase in the daily synthesis of protein and fat. It is
expected that protein protected with CSFA would have a greater impact in cows
with a higher milk yield and a higher requirement for amino acids. Considering the
protected protein purchase price of 2.0 – 2.3 zloty/kg and the milk purchase price of
1.0 – 1.2 zloty/kg, the increase in the cows’ milk yield obtained was close to the
limit of economic efficiency for milk production, and did not compensate for the
increased feeding costs of the cows. It is expected that the results would be better
directly after calving, at a milk yield exceeding 30 kg milk/day.
References
AOAC (1990). Official Methods of Analysis of the Association of Official Analytical Chemists. Ed. by
Kenneth Helrich, 15th Edition, Arlington, Virginia, USA.
A t w a l A.S., H i d i r o g l o u M., K r a m e r J.K.G., B i n n g s M.R. (1990). Manipulation of the fatty acid
composition of milk by feeding protected canola seeds. J. Dairy Sci., 75: 1090 – 1096.
B r z ó s k a F. (2004). Effect of copper inhibitors in diet on cows’ yield, milk composition and cholesterol
level in milk and blood plasma. Ann. Anim. Sci., 4 (1): 43 – 55.
B r z ó s k a F. (2005). Effect of soybean meal protected with Ca salts of fatty acids on cows yield, protein
and fat components in milk and blood. Ann. Anim. Sci., 5 (1): 111 – 123.
B r z ó s k a F., G a˛s i o r R., S a l a K., Z y z a k W. (1999). Effect of linseed oil fatty acid calcium salts and
vitamin E on milk yield and composition. J. Anim. Feed Sci., 8: 367 – 378.
C a n f i e l d R.W., S n i f f e n C.J., B u t l e r W.R. (1990). Effects of excess degradable protein on
postpartum reproduction and energy balance in dairy cattle. J. Dairy Sci., 73: 2342 – 2349.
97
D a v i d s o n S., H o p k i n s B.A., D i a z D.E., B o l t S.M., B r o w n i e C., F e l l n e r V., W h i t l o w L.W.
(2003). Effects of amounts and degradability of dietary protein on lactation, nitrogen utilization,
and excretion in early lactation Holstein cows. J. Dairy Sci., 86: 1681 – 1689.
D e P e t e r s E.J., C a n t J.P. (1992). Nutritional factors influenced the nitrogen composition of bovine
milk: a review. J. Dairy Sci., 75: 2043 – 2070.
D u n l a p T.F., K o h n R.A., D o u g l a s s L.W., E r d m a n R.A. (2000). Diets deficient in rumen
undegraded protein did not depress milk production. J. Dairy Sci., 83: 1806 – 1812.
E r d m a n R.A., V a n D e r s a l l J.H. (1983). Effect of rumen degradability on milk yield of dairy cows in
early lactation. J. Dairy Sci., 66: 1873 – 1880.
G o r d o n W.G., K o l a n E.B. (1983). Protein in milk. In: Fundamentals of Dairy Chemistry. 2nd
Edition. B.W. Webb, A.H. Johnson, J.A. Alford (eds). AVI Publ. Co., Inc., West Port, CT, 78 pp.
H o r n g h e r h o l t D.D., M u l l e r L.D. (1998). Supplementation of rumen-undegradable protein to the
diets of early lactation Holstein cows on grass pasture. J. Dairy Sci., 81: 2204 – 2214.
INRA (1988). Ruminant Nutrition: Recommended Allowances and Feed Tables. R. Jarrige (ed.), INRA,
John Libbey Eurotext, Paris.
K l u s m a y e r T.H., L y n c h G.L., C l a r k J. H. (1989). Effects of source of protein and calcium salts of
long chain nutrients to the small intestine of lactating dairy cows. J. Dairy Sci., 67, Suppl., 1: p. 482.
K r a s z e w s k i J., W a w r z y ń c z a k S., M a n d e c k a B. (2003). Ocena efektywności preparatu
MEGAPRO w żywieniu wysoko produkcyjnych krów. Rocz. Nauk. Zoot., 30 (1): 69 – 77.
L a r d y G.P., A d a m s D.C., K l o p f e n s t e i n T.J., C l a r k R.T. (1999). First limiting nutrient for
summer calving cows grazing autumn-winter range. J. Range Management, 52 (4): 317 – 326.
M c C o r m i c k M.E., F r e n c h D.D., B r o w n T.F., C u o m o G.J., C h a p a A.M., F e r n a d e z J.M.,
B e a t t y J.F., B l o u i n D.C. (1999). Crude protein and rumen undegradable protein effects on
reproduction and lactation performance of Holstein cows. J. Dairy Sci., 82: 2697 – 2708.
M c C o r m i c k M.E., W a r d J.D., R e d f e a r n D.D., F r e n c h D.D., B l o u i n D.C., C h a p a A.M.,
F e r n a n d e z J.M. (2001). Supplemental dietary protein for grazing dairy cows: effect on pasture
intake and lactation performance. J. Dairy Sci., 84: 896 – 907.
N a k a m u r a T., K l o p f e n s t e i n T.J., O w e n F.G., B r i t t o n R.A., G r a n t R.J., W i n o w s k i T.S.
(1992). Non-enzymatically browned soybean meal for lactating dairy cows. J. Dairy Sci., 75:
3519 – 3523.
NRC (2001). Nutrient requirements of dairy cattle. 7th ed., National Academy Press, Washington, DC.
P a l m q u i s t D.L., W e i s s W.P. (1994). Blood and hydrolyzed feather meals as sources of undegradable protein in high fat diets for cows in early lactation. J. Dairy Sci., 77: 1630 – 1643.
P i e s z k a M., B r z ó s k a F. (2000). Rumen degradability and intestinal digestibility of rapeseed meal
protein and dry matter protected by calcium salts of fatty acid. Ann. Anim. Sci. — Rocz. Nauk.
Zoot., 27 (4): 279 – 292.
P i e s z k a M., B r z ó s k a F. (2001 a). Wpływ osłony białka paszowego przed rozkładem w żwaczu przy
użyciu soli wapniowych kwasów tłuszczowych na procesy trawienne w żwaczu i jelicie cienkim.
Rocz. Nauk. Zoot., 28 (2): 237 – 249.
P i e s z k a M., B r z ó s k a F. (2001 b). Effect of protected rapeseed or soybean meal supplementation on
milk yield and physico-chemical composition in cows fed grass silage. Ann. Anim. Sci., 1 (2):
75 – 87.
P i e s z k a M., B r z ó s k a F., S a l a K. (2000). Wpływ osłaniania solami wapniowymi kwasów
tłuszczowych białka pasz wysokobiałkowych na jego strawność w żwaczu i jelicie cienkim. Rocz.
Nauk. Zoot., Suppl., 6: 420 – 424.
R o g e r s G.I., P o s t e r R.H.D., C l a r k T., S t e w a r t J.A. (1980). Effect of protected casein
supplementation on pasture intake, milk yield and composition of cows in early lactation. Austr.
J. Agric. Res., 31: 1147 – 1152.
R o h r K., L e b z i e n P., D a e n i c k e R., E n g l i n g F.P. (1993). Milk yield and milk composition of
high-producing cows as influenced by calcium salts of plants oil fatty acids in combination with
protected protein or grain maize. J. Anim. Physiol. Anim. Nutr., 69 (5): 251 – 259.
R o s s i F., F i o r e n t i n i L., M a s o e r o F., P i v a G. (1999). Effect of fat coating on rumen degradation
and intestinal digestibility of soybean meal. Anim. Feed Sci. Techn., 81 (3/4): 309 – 318.
98
F. Brzóska
R u p p e r t L.D., D r a c k l e y J.K., B r e m m e r D.R., C l a r k J.H. (2003). Effect of tallow in diets based
on corn silage or alfalfa silage on digestion and nutrient use by lactating dairy cows. J. Dairy Sci.,
86: 593 – 609.
S a n t o s F.A.P., S a n t o s J.E.P., T h e u r e r C.B., H u b e r J.T. (1998). Effects or rumen-undegradable
protein on dairy cow performance: A 12-year literature review. J. Dairy Sci., 81: 3182 – 3213.
S c h i n g o e t h e D.J. (1996). Dietary influence on protein level in milk and milk yield in dairy cows.
Anim. Feed Sci. Techn., 60: 181 – 190.
S c h o r A., G a g l i o s t r o G.A. (2001). Undegradable protein supplementation to early lactation dairy
cows in grazing conditions. J. Dairy Sci., 84: 1507 – 1606.
S c h r o e d e r G.F., G a g l i o s t r o G.A. (2000). Fishmeal supplementation to grazing dairy cows in early
lactation. J. Dairy Sci., 83: 2899 – 2906.
S c h r o e d e r G.F., E r a s m u s L.J., L e e u w K.J., M e i s s n e r H.H. (1995). Effects of roasting on
ruminal degradation intestinal digestibility and absorbable amino acid profile of cottonseed and
soybean oil cake meals. South. Afr. J. Anim. Sci., 25: 109 – 117.
S t a n d f o r d K., M c A l l i s t e r T.A., X u Z., P i c k a r d M., C h e n g K.J. (1995). Comparison of
lignosulfonate-treated canola meal and soybean meal as rumen undegradable protein supplements
for lambs. Can. J. Anim. Sci., 75: 371 – 377.
S u b n e l A.P.J., M e i j e r R.G.M., S t r a a l e n W.M. Van, T a m m i n g a S. (1994). Efficiency of milk
protein production in the DVE protein evaluation system. Liv. Prod. Sci., 40: 215 – 224.
S u b u h A.M.H., R o w a n T.G., L a w r e n c e T.L.J. (1996). Effect of heat or formaldehyde treatment on
the rumen degradability and intestinal tract apparent digestibility of protein in soybean meal and in
rapeseed meal of different glucosinolate content. Anim. Feed Sci. Techn., 57: 257 – 265.
T a m m i n g a S., S t r a a l e n W.M. Van, S u b n e l A.P.J., M e i j e r R.G.M., S t e g A., W e v e r C.J.G.,
B l o k M.C. (1994). The Dutch protein evaluation system: The DVE/OEB system. Livest. Prod.
Sci., 40: 139 – 155.
W h i t e l a w F.G., M i l n e J.S., Ø r s k o v E.R., S m i t h J.S. (1986). The nitrogen and energy metabolism
of lactating cows given abomasal infusions of casein. Br. J. Nutr., 55: 537 – 556.
Ż e b r o w s k a T., D ł u g o ł e˛ c k a Z., P a j a˛k J.J., K o r c z y ń s k i M. (1997). Rumen degradability of
concentrate protein, amino acids and starch, and their digestibility in the small intestine of cows.
J. Anim. Sci., 6: 451 – 470.
Accepted for printing 28 III 2006
FRANCISZEK BRZÓSKA
Wpływ śruty rzepakowej chronionej solami Ca-KT oleju lnianego na wydajność krów
i parametry mleka oraz krwi
STRESZCZENIE
Doświadczenie wykonano na 32 krowach rasy czb, podzielonych na 4 grupy w układzie
analogowym, z czterema okresami, każdy po 32 dni. Krowy żywiono na pastwisku, a ponadto
otrzymywały one zielonke˛ z upraw polowych, młóto browarniane świeże i mieszanke˛ paszowa˛.
Mieszanka paszowa zawierała śrute˛ poekstrakcyjna˛ rzepakowa˛ nie chroniona˛ przed rozkładem żwaczowym w ilości 20% (grupa kontrolna) i śrute˛ poekstrakcyjna˛ rzepakowa˛ chroniona˛ solami Ca-KT z oleju
lnianego w ilości 20, 25 i 30% mieszanki paszowej (grupy doświadczalne). Odpowiadało to pobraniu
śruty rzepakowej w ilości 1,56 i odpowiednio 1,61; 1,80; 2,15 kg/dobe˛. Ilość białka nierozkładalnego
w dietach wynosiła odpowiednio 26, 28, 30 i 32% białka ogólnego a pobranie paszy przez krowy —
średnio 22,3 kg s.m./dzień, w tym mieszanek paszowych 7,54 kg s.m./dzień. Wydajność mleczna krów
99
wynosiła w grupie kontrolnej 25,47, a w grupach doświadczalnych odpowiednio 26,76, 27,72
i 26,08 kg/dzień i różniła sie˛ pomie˛dzy grupami istotnie. Ilość mleka skorygowanego na zawartość
białka i tłuszczu to 25,14 w grupie kontrolnej, a grupach doświadczalnych odpowiednio 25,80, 25,97
i 26,50 kg/dzień i była ona istotnie wyższa. Podawanie śruty rzepakowej chronionej w porównaniu z nie
chroniona˛ zwie˛kszyło wydajność krów o 1 – 2,5 kg/dzień. Zawartość tłuszczu i białka w mleku wykazała
tendencje˛ rosna˛ca˛, lecz różnice były statystycznie nieistotne. Podawanie śruty rzepakowej chronionej
istotnie zwie˛kszyło ilość tłuszczu i białka wydzielana˛ w mlekiem. Wydajność tłuszczu wynosiła
986 g/dzień w grupie kontrolnej i odpowiednio 982, 1120 i 1067 g/dzień w grupach doświadczalnych.
Nie stwierdzono istotnych różnic w zawartości frakcji N mleka i pomie˛dzy grupami krów w kwasowości, czasie krzepnie˛cia i ge˛stości mleka. Nie zauważono różnic w zawartości glukozy, białka całkowitego
i trójglicerydów w osoczu krwi krów z poszczególnych grup. Podawanie krowom śruty rzepakowej
chronionej solami Ca-KT istotnie zwie˛kszyło w krwi zawartość mocznika. Nie stwierdzono istotnych
różnic pomie˛dzy grupami krów w zawartości kwasów tłuszczowych w mleku, w tym nasyconych
i nienasyconych oraz ich wzajemnych proporcjach.
Na podstawie wykonanych badań można stwierdzić, że podawanie krowom, o przecie˛tnej
wydajności 26 kg mleka/dzień, śruty poekstrakcyjnej rzepakowej chronionej solami Ca-KT z oleju
lnianego zwie˛ksza wydajność mleczna˛ istotnie oraz zwie˛ksza dobowa˛ produkcje˛ tłuszczu i białka,
natomiast nie wpływa na pozostałe wskaźniki jakości mleka, a także ogólny metabolizm krów, przy
wzroście ste˛żenia N mocznikowego w mleku.
EFFECT OF GLUCANASE AND XYLANASE SUPPLEMENTATION
OF FEED FOR WEANED PIGLETS*
E w a H a n c z a k o w s k a 1, J e r z y U r b a ń c z y k 1, I m k e K ü h n 2, M a ł g o r z a t a Ś w i a˛t k i e w i c z 1
1
Department of Animal Nutrition and Feed Science,
2
AB Enzymes GmbH, D-64293 Darmstadt Felabergstrasse 78, Germany
Abstract
Two experiments were conducted to estimate the effect of the preparation Econase containing
beta-glucanase or xylanase on the performance of early-weaned piglets. All piglets were fed with
barley, wheat and soybean diets. In experiment I, piglets (n = 146) were weaned at 28 days of age
and received a basal diet (control) or a diet supplemented with beta-glucanase: group II —
26000 BU and group III — 39000 BU per kg of feed. Group IV received xylanase — 24000 BXU.
The design of experiment II was the same as experiment I but piglets were weaned at 42 days of
age. Experiment I was concluded at 99 days and experiment II at 84 days of age. It was found that
beta-glucanase was more efficient in younger piglets. Between 28 and 56 days of age, the weight
gains of experimental animals were up to 36% higher than those of control animals. Between
56 and 70 days this difference was smaller (up to 26%). In the whole of experiment I, a lower
supplement of glucanase improved weight gains by 7.2% and a higher supplement by 17%, while
xylanase improved weight gains by 14%. In older piglets (experiment II), only the difference
between the control and xylanase groups was statistically significant, although all the experimental
animals grew faster than the control animals.
Key words: piglets, beta-glucanase, xylanase
The time when piglets are weaned from a sow is always a very stressful
moment. Such a change in the environment and feed requires a change in the
animal’s ability to secrete endogenous enzymes. In order for piglets to be able to
fully utilize feed postweaning it might be appropriate to supplement their diet with
exogenous enzymes. An additional problem is caused by the removal of feed
antibiotic, which negatively affects the health status and nutrient digestibility in
piglets.
102
E. Hanczakowska et al.
Non-starch polysaccharides (NSP) present in the cell walls of cereal grains are
unfavourable, especially for young monogastric animals. Many experiments with
NSP hydrolyzing enzyme preparations have been performed on poultry. Glucanase
and/or xylanase produced good results in the nutrition of laying hens (Lazaro et al.,
2003), broiler chickens (Meng et al., 2005) and turkeys (Mathlouthi et al., 2003).
Fewer studies have been carried out using these preparations in pig feeding. Li
et al. (1996) supplemented hulled barley- or wheat-soybean meal diets with
beta-glucanase. They found higher gross energy and crude protein ileal digestibility
in the case of the barley diet, but no such improvement was found when the enzyme
was added to the wheat-soybean meal diet. Higher apparent ileal digestibility of
amino acids in pigs fed wheat-based diets after supplementation with xylanase was
also reported by Barrera et al. (2004). Omogbenigun et al. (2004) reported increased
total-tract digestibility of nutrients using multienzyme preparations containing the
enzymes glucanase and xylanase, among others.
The objective of this experiment was to determine the effect of different doses
of beta-glucanase or xylanase supplements in a barley-wheat-soybean diet on piglet
performance.
Animals and diets
Experiment I. The experiment was carried out on 146 piglets originating from 16
sows (Polish Landrace × Polish Large White) mated to a Duroc × Pietrain boar.
Before the start of the experiment, piglets were fed with a standard PP prestarter
mixture with no enzyme supplement. Piglets were assigned to four groups, with
4 litters in each. Animals were fed with the same basal barley-wheat-soybean diet,
differing in additives (Table 1).
Group I (control) — basal diet with no enzyme supplement.
Group II — basal diet supplemented with beta-glucanase at 26000 BU*/kg of feed.
Group III — basal diet supplemented with beta-glucanase at 39000 BU*/kg of feed.
Group IV — basal diet supplemented with xylanase at 24000 BXU*/kg of feed.
Each litter was kept in a separate pen, fed ad libitum until weaning and then
restricted. The experiment lasted until day 99 of age (about 32 kg of body weight)
and piglets were weaned at 28 days of age. The body weight of piglets was recorded
at 28, 56, 70, 84 and 99 days of age.
Experiment II. The design of the experiment, feed composition and enzyme
supplementation were the same as in experiment I. A total of 110 piglets of the
same origin as in the former experiment were weaned at 42 days of age and kept
in the experiment until 84 days of age (about 25 kg of body weight). Each feeding
group comprised 3 litters. Piglets were weighed at 42, 56, 70 and 84 days of age.
* BU and BXU — units of enzyme activity.
Effect of glucanase and xylanase supplementation of feed for piglets
103
Table 1. Composition and nutritional value of the experimental feed mixture
Components
Content (%)
Barley
Wheat
Rapeseed meal
Soybean meal
Dried whey
Rapeseed oil
Limestone
NaCl
Lys
Premix 0.5% Standard starter1
Dicalcium phosphate
49.90
14.00
6.00
20.00
5.00
2.00
1.20
0.25
0.15
0.50
1.00
Metabolizable energy (MJ)
Dry matter (%)
Crude protein (%)
Ether extract (%)
Crude fibre (%)
ADF (%)
NDF (%)
ADL (%)
Crude ash (%)
N-free extractives (%)
Lys (g)
Met + Cys (g)
Thr (g)
Trp (g)
Ca (g)
P (g)
13.37
89.51
19.09
3.52
4.20
5.21
20.23
0.17
4.93
57.77
11.12
6.58
7.14
2.32
9.80
6.99
1
Premix composition: Vitamin: A — 2700000 IU; D3 — 400000 IU; E — 8.0 g; K3 — 0.5 g; B1 — 0.5 g; B2 —
0.8 g; B6 — 0.8 g; B12 — 0.008 g; pantothenic acid — 2.8 g; choline cloride — 70 g; folic acid — 0.2 g; nicotinic
acid — 5.0 g; magnesium — 10 g; manganese — 12 g; iodine — 0.1 g; zinc — 30 g; iron — 20g; copper — 32 g;
cobalt — 0.06 g; selenium — 0.04 g; complete limestone to 1000 g.
The feed intake of each litter was measured and feed utilization of each litter
was calculated.
Chemical analysis
The gross composition of feed and its components was analysed using standard
methods (AOAC, 1990). The amino acid (lysine, sulphur amino acids, tryptophan
and threonine) content of the feed was analysed using a Beckmann automatic
analyser. Fractions of feed fibre (ADF, NDF and ADL) were estimated according to
Goering and Van Soest (1970).
Statistics
The results obtained were analysed using the one-way ANOVA procedure and
the significance of differences between mean values was estimated using Duncan’s
test (Statistica, 1995).
104
Results
Experiment I
Piglets weaned at 28 days of age reacted positively to beta-glucanase and
xylanase supplements (Table 2). Body weight gains between 28 and 56 days of age
were significantly higher in the groups receiving beta-glucanase (by 20 – 36%)
and in the group receiving xylanase (by 19%) in comparison to the control group. In
the case of glucanase, these differences were smaller (13 – 26%) but still statistically significant in the older animals (between 56 – 70 days of age). During the whole
Table 2. Piglets’ rearing indexes (Exp. I)
Supplement
betabetaglucanase
glucanase
26000 BU/kg 39000 BU/kg
xylanase
24000
BXU/kg
Item
Control
No. of piglets in group
Average no. of piglets in litter
No. of dead piglets
No. of piglets with diarrhoea
Average body weight of 1 piglet
at days of age (kg):
28
56
70
84
99
Average daily weight gains
of 1 piglet at days of age (g):
28 – 56
56 – 70
70 – 84
84 – 99
28 – 99
Daily feed intake per piglet
28 – 56
56 – 70
70 – 84
84 – 99
28 – 99
Average feed utilization per kg
body weight gain at days
of age (kg):
28 – 56
56 – 70
70 – 84
84 – 99
28 – 99
38
9.50
4
16
37
9.25
3
7
35
8.75
3
9
36
9.00
2
8
6.20
9.39
13.66
20.07
29.82
5.73
9.58
14.43
21.54
31.12
5.97
10.31
15.67
22.49
33.67
6.38
10.20
15.47
22.57
33.31
114
305
458
650
333
a
a
a
a
137
346
508
639
357
ab
ab
a
ab
155
383
487
745
390
b
b
b
b
136
376
507
716
379
ab
ab
ab
ab
SEM
0.109
0.222
0.355
0.512
0.689
5.69
12.52
13.45
14.73
8.84
0.260 a
0.766
1.074
1.425
0.767
0.311 ab
0.843
1.104
1.432
0.816
0.332 b
0.865
1.042
1.523
0.851
0.294 ab
0.867
1.078
1.548
0.830
0.012
0.027
0.029
0.031
0.018
2.28
2.51
2.35
2.19
2.30
2.26
2.43
2.17
2.23
2.27
2.14
2.26
2.14
2.05
2.18
2.15
2.30
2.12
2.16
2.19
0.083
0.096
0.073
0.050
0.047
a, b — mean values in the same row with different letters differ significantly at P ≤ 0.05.
105
experimental period, glucanase supplements improved weight gains by 7.2% in
group II and by 17% in group III, while in the group fed xylanase mean weight
gains were improved by about 14%. Feed intake in the control group was lower than
that in the experimental groups during the whole experiment, but the difference
between groups I and III was statistically significant only after weaning (28 – 56
days of age).
Diarrhoea was observed in 16 piglets in the control group and in only
7 – 9 piglets in each experimental group.
Experiment II
Enzyme supplements improved the body weight gains of the piglets weaned at
42 days of age (Table 3). At the end of the experiment (70 – 84 days) piglets
receiving xylanase had significantly higher weight gains (by 16.7%) than the
Table 3. Piglets’ rearing indexes (Exp. II)
Supplement
betabetaglucanase
glucanase
26000 BU/kg 39000 BU/kg
xylanase
24000
BXU/kg
Item
Control
No. of piglets in group
Average no. of piglets in litter
No. of piglets with diarrhoea
Average body weight of 1 piglet
42
56
70
84
Average daily weight gains
of 1 piglet at days of age (g):
42 – 56
56 – 70
70 – 84
42 – 84
Daily feed intake per piglet
42 – 56
56 – 70
70 – 84
42 – 84
Average feed utilization per
kg body weight gain of litter
at days of age (kg)
42 – 56
56 – 70
70 – 84
42 – 84
27
10.5
18
26
10.75
8
28
9.5
6
29
8.75
3
9.67 ab
11.19
16.81
24.62
8.78 a
10.90
16.41
24.40
10.21 b
12.03
18.02
26.86
8.65 a
10.92
16.80
25.90
108
401
557 a
356 a
151
394
571 a
372 ab
130
428
631 ab
396 ab
162
420
650 b
411 b
SEM
0.20
0.24
0.35
0.47
9.63
9.85
13.38
8.66
0.260
0.868
1.350
0.826
0.347
0.801
1.318
0.822
0.310
0.865
1.357
0.856
0.313
0.780
1.274
0.789
0.05
0.04
0.07
0.04
2.39
2.16
2.42 b
2.32 b
2.29
2.03
2.31 ab
2.21 ab
2.38
2.02
2.15 ab
2.16 ab
1.93
1.86
1.96 a
1.92 a
0.04
0.03
0.05
0.06
a, b — mean values in the same row with different letters differ significantly at P ≤ 0.05.
106
control piglets and this group was the only one that significantly differed from the
control group (I). Average feed conversion during the same period was 19% better
in piglets fed xylanase compared with the control piglets, and this difference was
also significant (P ≤ 0.05).
During the whole experimental period, no piglets died or were culled (Table 3).
However, some cases of diarrhoea were observed, especially in group I (18 animals)
and in group II (8 animals). In groups III and IV only 6 and 3 animals were ill,
respectively.
Discussion
The results of both experiments suggest that the enzyme supplement produced
the best results early after weaning. In the first experiment, weight gains of
xylanase-supplemented piglets were 19% better than in the control group in the
period 28 – 56 days after weaning, and only 10% better in the period 84 – 99 days
postweaning. In the second experiment, the differences in mean weight gains
between these two groups in the first period were even greater, especially in the
case of piglets receiving dietary xylanase. This could be due to the increasing
endogenous enzyme activity in pigs, as was suggested by Graham et al. (1988). In
such a situation the addition of exogenous enzymes is less effective. It also could be
due to higher feed consumption by piglets receiving enzymes. However, such early
weaning was not beneficial because on day 84 of age piglets weaned on day 28 were
characterized by lower individual body weight than those weaned on day 42.
A positive effect of enzyme preparations in young (25-day-old) piglet feeding was
found by Omogbenigun et al. (2004), but in their experiment all piglets were weaned
earlier and the effect of enzyme supplementation in animals weaned later was not
stated. The authors concluded that the use of enzyme additives may allow for more
cost-effective and environmentally friendly formulation of diets for young pigs.
A similar improvement in the weight gains of pigs after supplementing feed
with beta-glucanase and xylanase was found in an earlier experiment by Bedford et
al. (1992). According to Mathlouthi et al. (2002) beta-glucanase and xylanase
reduce the viscosity of feedstuffs. Lower viscosity may result in better feed
digestibility and higher weight gains by piglets. It is possible that enzyme
supplementation also had this effect in the present experiment. A reduction in the
intestinal viscosity of cereal-based feeds, especially those based on barley, and an
increase in apparent nutrient digestibility after supplementing feed with betaglucanase and xylanase, were found by Lazaro et al. (2003) in an experiment with
laying hens. The higher weight gains of piglets receiving beta-glucanase or
xylanase could also be a result of improved protein digestibility (Barrera et al.,
2004) and/or other nutrient digestibility (Diebold et al., 2004).
The fact that the control group contained the highest number of piglets suffering
from diarrhoea could result from the antibiotic-free diet and lower availability of
nutrients for animals with no enzyme supplementation.
107
In the present experiment the preparation was more effective when a higher
dose of glucanase was added. This is in accordance with the findings of Lazaro et
al. (2003) who found in the above-mentioned experiment that an excess of enzymes
had no deleterious effect.
In summary, it is concluded that preparations containing beta-glucanase or
xylanase added to feed containing wheat and barley improve the weight gains and
feed conversion of weaned piglets, especially early after weaning.
References
AOAC. (1990). Official Methods of Analysis. Association of Official Analytical Chemists, 15th Ed.,
Arlington, Virginia, USA.
B a r r e r a M., C e r v a n t e s M., S a u e r W.C., A r a i z a A.B., T o r r e n t e r a R., C e r v a n t e s M.
(2004). Ileal amino acid digestibility and performance of growing pigs fed wheat-based diets
supplemented with xylanase. J. Anim. Sci., 82: 1997 – 2003.
B e d f o r d M., P a t i e n c e J.F., C l a s s e n H.L., I n b o r r J. (1992). The effect of dietary supplementation of rye- and barley-based diets on digestion and subsequent performance in weaning pigs.
Can. J. Anim. Sci., 72: 97 – 105.
D i e b o l d G., M o s e n t h i n R., P i e p h o H.P., S a u e r W.C. (2004). Effect of supplementation of
xylanase and phospholipase to a wheat-based diets for weanling pigs on nutrient digestibility and
concentration of microbial metabolites in ileal digesta and feces. J. Anim. Sci., 82: 2647 – 2656.
G r a h a m H., L o w g r e n W., P e t t e r s o n D., A m a n P. (1988). Effect of enzyme supplementation on
digestion of a barley/pollard-based pig diet. Nutr. Rep. Int., 38: 1073 – 1079.
L a z a r o R., G a r c i a M., A r a n i b a r M.J., M a t e o s G.G. (2003). Effect of enzyme addition to wheat-,
barley- and rye-based diets on nutrient digestibility and performance of laying hens. Brit. Poultry
Sci., 44: 256 – 265.
L i S., S a u e r W.C., H u a n g S.X., G a b e r t V.M. (1996). Effect of ß-glucanase supplementation to
hulless barley- or wheat-soybean meal diets on the digestibilities of energy, protein, ß-glucans, and
amino acids in young pigs. J. Anim. Sci., 74: 1649 – 1656.
M a t h l o u t h i N., J u i n H., L a r b i e r M. (2003). Effect of xylanase and beta-glucanase supplementation of wheat- or wheat- and barley-based diets on the performance of male turkeys. Brit. Poultry
Sci., 44: 291 – 298.
M a t h l o u t h i N., S a u l n i e r L., Q u e m e n e r B., L a r b i e r M. (2002). Xylanase, beta-glucanase, and
other side enzymatic activities have greater effects on the viscosity of several feedstuffs than
xylanase and beta-glucanase used alone or in combination. J. Agric. Food Chem., 50: 5121 – 5127.
M e n g X., S l o m i n s k i B.A., N y a c h o t i C.M., C a m p b e l l L.D., G u e n t e r W. (2005). Degradation
of cell wall polysaccharides by combinations of carbohydrase enzymes and their effect on nutrient
utilization and broiler chicken performance. Poultry Sci., 84: 37 – 47.
O m o g b e n i g u n F.O., N y a c h o t i C.M., S l o m i n s k i B.A. (2004). Dietary supplementation with
multienzyme preparations improves nutrient utilization and growth performance in weaned pigs.
J. Anim. Sci., 82: 1056 – 1061.
108
EWA HANCZAKOWSKA, JERZY URBAŃCZYK, IMKE KÜHN,
MAŁGORZATA ŚWIA˛TKIEWICZ
Wpływ dodatku glukanazy i ksylanazy do paszy odsadzonych prosia˛t
STRESZCZENIE
W dwóch doświadczeniach oceniano wpływ preparatu Econase, zawieraja˛cego beta-glukanaze˛
lub ksylanaze˛ na użytkowość wcześnie odsadzanych prosia˛t. Wszystkie prosie˛ta żywiono je˛czmieniem,
pszenica˛ i soja˛. W doświadczeniu I, prosie˛ta (n = 146) odsadzane w 28. dniu życia otrzymywały diete˛
podstawowa˛ (kontrolna˛) lub diete˛ z dodatkiem beta-glukanazy: grupa II — 26000 BU, grupa III —
39000 BU na kg paszy. Grupa IV otrzymywała ksylanaze˛ — 24000 BXU. Układ doświadczenia II był
taki sam jak w przypadku doświadczenia I, jednak prosie˛ta odsadzano w wieku 42 dni. Doświadczenie
I zakończono w 99. dniu, natomiast doświadczenie II w 84. dniu ich życia.
Stwierdzono wie˛ksza˛ skuteczność beta-glukanazy u młodszych zwierza˛t. Mie˛dzy 28. a 56. dniem
życia przyrost masy ciała zwierza˛t doświadczalnych był do 36% wyższy niż w przypadku zwierza˛t
kontrolnych. Mie˛dzy 56. a 70. dniem życia różnica ta była mniejsza (do 26%). W całym doświadczeniu I
niższy dodatek glukanazy poprawił przyrosty masy ciała o 7,2%, natomiast wyższy dodatek o 17%.
Przyrost masy ciała zwierza˛t pod wpływem ksylanazy poprawił sie˛ o 14%. U starszych prosia˛t
(doświadczenie II), tylko różnica mie˛dzy grupami kontrolna˛ i otrzymuja˛ca˛ dodatek ksylanazy była
statystycznie istotna, choć wszystkie zwierze˛ta doświadczalne przyrastały szybciej niż zwierze˛ta
kontrolne.
FATTY ACID AND CHOLESTEROL CONTENT OF MEAT OF BROILERS
FED LINSEED OIL OR DIFFERENT LINSEED VARIETIES*
A g n i e s z k a S z e w c z y k 1, F r a n c i s z e k B o r o w i e c 2, E w a H a n c z a k o w s k a 1
1
Department of Animal Nutrition, National Research Institute of Animal Production,
2
Department of Animal Nutrition, Agricultural University, al. Mickiewicza 24/28,
30-059 Kraków, Poland
Abstract
A growth experiment was performed on broilers to estimate the effect of different varieties of
linseed on the fatty acid composition and cholesterol content of breast and leg meat. A total of 100
broilers were divided into five groups. Birds were housed individually and fed ad libitum one of five
diets: control (I), supplemented with 4% linseed oil (II), or supplemented with 10% Omega seeds
(III) or Opal seeds (IV) or Linola seeds (V). The fatty acid content was estimated in seeds, feed and
leg and breast meat. The cholesterol content of meat and blood serum was also analysed. No
significant differences in broilers’ weight gains were found. The meat of experimental broilers had
higher levels of polyunsaturated fatty acids (PUFA) than the meat of control broilers but lower
levels of saturated (SFA) and monounsaturated (MUFA) acids. Linseed supplementation lowered
the cholesterol content of meat. The Opal variety had the highest hypocholesterolemic activity.
Key words: chicken, feeding, linseed, fatty acid, cholesterol
Normalization of the plasma lipid profile is the goal of nutritional intervention
to prevent or reduce the development of atherosclerosis. To reach this goal, expert
groups, including the 2001 National Cholesterol Education Program Adult Treatment Panel III, the American Heart Association (AHA), and the Canadian Working
Group on Hypercholesterolemia and Other Dyslipidemia, recommended replacing
saturated fats with unsaturated fats rather than with carbohydrates. According to the
AHA diet, the proportion of saturated lipids should be reduced to ≤ 10% of total
energy and cholesterol consumption limited to < 300 mg/d (Beauchesne-Rondeau et
al., 2003).
110
A. Szewczyk et al.
Alpha-linolenic acid (C 18:3 n-3) is the fatty acid precursor for the synthesis of
eicosapentaenoic acid (EPA; C 20:5 n-3) and docosahexaenoic acid (DHA;
C 22:6 n-3), which play a major role in the control of cardiovascular diseases (KrisEtherton et al., 2004).
As a result of these benefits, it is now widely recommended that the
consumption of long-chain n-3 polyunsaturated fatty acids (PUFA) be increased in
the human diet. This is the reason for increasing the supply of long-chain n-3 PUFA
by changing the diet of farm animals such as pigs, cows and poultry.
The fatty acid profile in the body lipids of broiler chickens depends to a large
extent on the composition of the fat contained in feed mixtures (Gonzalez-Esquerra
and Leeson, 2000).
The addition of linseed increases the energy concentration in feeds and makes it
possible to fulfil requirements for essential fatty acids (Borowiec et al., 2001;
Lopez-Ferrer et al., 2001; Bean and Leeson, 2003). Linseeds are rich in alphalinolenic acid, which is why the addition of traditional linseed varieties to broiler
feeds leads to a reduction in saturated (SFA) and monounsaturated fatty acids
(MUFA) and next to a possible reduction in the level of total cholesterol in broilers’
tissues and blood plasma.
The aim of this study was to investigate the effect of feeding broiler chicks with
diets supplemented with the Linum usitatissimum brown-seeded variety, Opal; the
yellow-seeded varieties, Linola or Omega; or linseed oil on the fatty acid content of
meat fat and the level of cholesterol in meat and blood plasma.
The experiment was conducted on 100 Starbro broilers divided into five groups
of 20 birds each, from 14 to 42 days of age. The broilers were housed individually
under standard conditions in metabolic cages fitted with individual feeders and
automatic drinkers. The birds were fed ad libitum with complete feeds according to
the Nutrient Requirements of Poultry (Table 1).
The control feed (I) was not supplemented with fat, while diet II was
supplemented with 4% low-linolenic linseed oil, diet III with 10% Omega seeds,
diet IV with 10% Opal seeds, and diet V with 10% Linola seeds. The energy content
of the diets for the experimental groups (II – V) was similar and ranged from 4130.2
to 4463.6 kcal/kg. The diets were prepared weekly and fed as mash. At 7 weeks of
age, 7 chickens from each group were decapitated and left breast muscle, abdominal
fat and livers were sampled. The samples were placed in plastic bags and stored at
–18oC until analysis. The tissues from individual birds were homogenized and
lipids were extracted using the method of Folch et al. (1957). The fatty acid profile
was determined in feed fats, diets, and tissue lipids using a Varian 3400CX gas
chromatograph equipped with an FID detector, with argon as the carrier gas (DB-23
column, column temperature 100 – 205oC, sample injector 200oC, detector 240oC).
Blood was taken from 7 birds and analysed. Total cholesterol (TC) in serum was
111
Fatty acid and cholesterol content of meat of broilers fed linseed
analysed using the POCH biochemical test. The HDL fraction was analysed using
the POCH test, and the LDL fraction of cholesterol was calculated as the difference
between total and HDL cholesterol.
Table 1. Composition and nutrient content of experimental diets (g/kg)
Item
Ground wheat
Ground maize
Soybean meal
Bone-blood meal
Linseed oil
Omega seeds
Opal seeds
Linola seeds
Mineral and vitamin premix
In 1 kg of diet:
crude protein
ether extract
crude fibre
NDF
ADF
ADL
gross energy (kcal/kg)
Control
Linseed
oil
Yellowseeded
variety
Omega
Brownseeded
variety
Opal
Yellowseeded
variety
Linola
I
II
III
IV
V
335
350
203
80
315
320
213
80
40
305
310
173
80
315
320
153
80
305
310
173
80
100
100
32
32
32
32
100
32
18.44
3.60
2.10
11.91
5.50
20.90
4130.21
18.13
7.61
2.16
12.22
7.00
2.98
4463.60
18.69
7.70
2.45
13.90
6.24
2.29
4439.51
18.00
7.78
2.69
14.34
7.09
2.78
4407.71
18.25
7.81
2.71
13.07
6.67
2.31
4424.42
The results were subjected to statistical analysis using one-way analysis of
variance. Duncan’s test was used to determine the significance of differences
between means for groups.
Results
The lipid fraction of Omega and Opal seeds contained the highest amount of
linolenic acid (Table 2), while diets supplemented with linseed oil and Linola seeds
were rich in linoleic acid (51.7 – 56.2%). The experimental diets contained more fat
than the control diet therefore the weight gain was lowest in the control group
(1662 g), while the average weight gain in the experimental groups ranged from
1790 to 1812 g.
The leg meat of broilers receiving linseed oil or Linola seeds contained the
highest amount of linoleic acid (Table 3) and this difference was statistically
significant (P<0.01). The leg meat of all the experimental chicks contained more
linolenic acid than that of the controls. The highest level of this acid was found in
112
A. Szewczyk et al.
the leg meat of chicks fed Omega and Opal linseed (5.06 and 5.01%, respectively).
The accumulation of PUFA in broilers’ leg meat in group II increased at the
expense of MUFA.
Table 2. Fatty acid content of feed mixtures and linseed (%)
Mixture
Fatty
acid
C
C
C
C
C
C
C
C
C
14
16
16:1
18
18:1
18:2
18:3
20
20:1
Seed
control
linseed
oil
yellowseeded
variety
Omega
brownseeded
variety
Opal
yellowseeded
variety
Linola
I
II
III
IV
V
0.89
13.54
1.43
5.17
28.94
38.85
2.07
0.28
1.36
0.75
12.84
1.00
4.67
24.55
51.73
2.90
0.17
0.78
0.53
11.41
0.62
5.11
28.66
26.35
25.18
0.28
0.84
0.58
12.21
0.72
6.45
28.73
26.89
23.87
0.10
0.31
0.48
11.97
0.61
4.77
22.95
56.24
2.47
0.20
yellowseeded
variety
Omega
brownseeded
variety
Opal
Yellow
seeded
variety
Linola
5.82
5.31
6.81
3.89
29.96
16.43
43.93
4.66
23.15
19.13
47.75
3.40
14.79
71.36
3.63
Table 3. Fatty acid composition in leg meat of broiler chickens (%)
Item
C 14
C 16
C 16:1
C 18
C 18:1
C 18:2
C 18:3
C 20:1
C 20:2
C 20:3
C 20:4
MUFA
PUFA
SFA
UFA
Control
Linseed
oil
Yellowseeded
variety
Omega
Brownseeded
variety
Opal
Yellowseeded
variety
Linola
I
II
III
IV
V
0.83
23.97
4.37
11.28
39.23
14.40
0.88
0.81
0.37
0.56
2.29
44.42
18.52
36.10
62.94
Total fat in meat (%)
2.24
*Cholesterol in meat
(mg/100 g)
137.6
B
b
Bbc
A
A
B
Bc
Aa
b
A
0.75
22.19
3.37
10.19
35.68
22.00
1.58
0.76
0.35
0.47
2.10
39.82
26.52
33.14
66.34
2.31
127.6
A
a
Aa
C
A
B
Aa
Cc
a
B
0.87
22.05
3.20
11.23
39.94
14.31
5.06
0.86
0.22
0.42
1.20
44.01
21.23
34.16
65.24
2.07
128.6
A
a
Bc
A
B
A
Bb
ABb
ab
AB
0.80
22.66
3.85
10.85
39.19
13.92
5.01
0.82
0.31
0.56
1.26
43.87
21.08
34.32
64.96
0.90
22.62
3.34
10.46
37.91
19.08
1.88
0.76
0.34
0.49
1.34
42.02
23.16
34,00
65.19
AA
ab
Bbc
A
B
A
Bb
ABb
ab
AB
1.86
112.8
2.19
131.4
A
a
Bb
B
A
A
ABb
Bb
ab
AB
SEM
0.02
0.18
0.14
0.25
0.36
0.63
0.39
0.03
0.02
0.03
0.11
0.42
0.56
0.36
0.37
0.05
1.65
113
The ratios of particular fatty acids in the breast meat of broilers (Table 4) were
similar to those found in their leg meat.
Table 4. Fatty acid composition in breast meat of broiler chickens (%)
Item
C 14
C 16
C 16:1
C 18
C18:1
C 18:2
C 18:3
C 20:1
C 20:2
C 20:3
C 20:4
MUFA
PUFA
SFA
UFA
Control
Linseed
oil
Yellowseeded
variety
Omega
Brownseeded
variety
Opal
Yellowseeded
variety
Linola
I
II
III
IV
V
0.92
24.62
3.17
12.18
39.07
13.61
1.23
0.77
0.50
0.64
2.36
43.02
18.37
37.74
61.39
Total fat in meat (%)
Cholesterol in meat
(mg/100 g)
Bc
B
Bb
Aa
Aa
ab
b
b
AB
b
Aa
Bb
ABa
1.25
96.9
0.66
22.40
3.06
10.33
34.85
21.28
1.69
0.62
0.49
0.64
3.54
38.54
27.65
33.40
66.20
Aa
A
Aa
Cb
ABa
ab
b
b
B
a
Bc
Aa
Cb
1.62
97.1
0.98
24.97
3.16
11.53
36.36
15.32
3.15
0.55
0.24
0.45
2.06
40.08
21.23
37.49
61.32
Bc
AB
ABab
ABa
Bb
a
a
a
AB
ab
Aab
Bb
ABa
1.40
94.1
0.94
24.39
2.80
13.04
38.37
12.90
2.86
0.88
0.32
0.51
1.34
42.06
17.93
38.35
60.00
Bbc
B
ABb
Aa
Bb
b
ab
b
A
b
Aa
Bc
Aa
1.10
89.4
SEM
0.83
23.22
2.51
11.33
37.19
19.23
1.27
0.87
0.43
0.62
2.03
40.57
23.59
35.37
64.16
0.04
ABab 0.25
0.13
AB
0.25
ABab 0.44
BCb 0.71
Aa
0.21
b
0.05
ab
0.03
b
0.03
AB
0.20
ab
0.53
ABbc 0.87
ABab 0.52
BCb 0.45
1.30
0.10
87.9
3.39
Table 5. Lipid profile of serum in broiler chickens
Item
Total cholesterol —
TC (mg/dl)
HDL-C fraction
(mg/dl)
LDL-C fraction
(mg/dl)
HDL-C/TC ratio
Triacylglycerols
(mg/dl)
Total lipids (mg/dl)
Control
Linseed
oil
Yellowseeded
variety
Omega
Brownseeded
variety
Opal
Yellowseeded
variety
Linola
I
II
III
IV
V
SEM
154.35
128.61
141.32
152.2
146.96
3.91
115.16
109.25
98.38
104.73
110.43
3.25
39.18 Aa
0.74 Aa
29.40
357
19.36 Bb
0.85 Bb
28.38
333
42.93 Aa
0.69 Aa
24.48
330
47.49 Aa
0.68 Aa
30.81
303
a, b — values in the same rows with different letters differ significantly (P < 0.05).
36.52 Aa
0.75 Aa
31.15
338
2.82
0.02
1.72
10.75
114
A. Szewczyk et al.
The cholesterol content of meat was lowered by linseed supplementation. The
highest hypocholesterolemic activity was characteristic of the brown-seeded variety, Opal, which lowered the cholesterol content of breast meat from 96.9 to 89.4 and
that of leg meat from 137.6 to 112.8 mg/100 g.
Supplements of linseed and linseed oil lowered the TC and HDL-C content of
blood (Table 5) but these differences were not statistically significant. Linseed oil
significantly lowered the LDL-C fraction in broilers’ blood when compared to the
control and other experimental groups. The highest (best) HDL-C to TC ratio was
also found in this group.
Discussion
According to Schuman et al. (2000), feeding laying hens flaxseed reduced body
weight and hepatic fat as compared to the control hens receiving animal or other
vegetable oil. In this experiment, a lower level of total fat in meat was found only in
the experimental group fed the Opal linseed variety.
Banno et al. (1997) showed a higher cholesterol ester content in the liver
and blood of chickens fed saturated fat (palm oil) than in those receiving linseed oil.
Liver triacylglycerol and free cholesterol levels significantly decreased in chicks
fed a diet containing n-3 fatty acids (linseed oil or fish oil). These authors also
reported increased levels of linoleic and linolenic acids in chicks’ tissue. The results
of our experiment are consistent with the results of Lopez-Ferrer et al. (2001), who
found lower amounts of SFA and MUFA but higher amounts of PUFA in the leg
meat of chicks fed linseed oil. In an experiment by Gonzales-Esquerra and Leeson
(2000), linolenic acid was preferentially deposited in dark meat and other
long-chain omega-3 fatty acids in white meat. In this experiment, also, alphalinolenic acid was deposited mainly in dark meat.
Cholesterol levels were lowered by linseed, especially in the case of dark meat
(leg) compared to breast meat, although these differences were not statistically
significant. There were also some differences in the level of blood cholesterol,
found to be lower in the experimental groups than in the control group, which
concurs with the results of An et al. (1997) and Eder et al. (2005). Wiesenfeld et al.
(2003) showed that flaxseed meal significantly reduces serum cholesterol and
increases levels of alpha-linoleic and eicosapentaenoic acids in the serum and
tissues of rats. Other authors found that feeding animals extracts of linseed
and rapeseed increases the level of alpha-linolenic acid by 20- to 40-fold in
eggs, 10-fold in chickens, 6-fold in pork, and less than 2-fold in beef (Bourre,
2005).
It is concluded from this experiment that different varieties of linseed have
different fatty acid contents and can change the fatty acid profile of chicks’ meat
fat. They also have different hypocholesterolemic activity.
115
References
A n B.K., B a n n o C., X i a Z.S., T a n a n k a K., O h t a n i S. (1997). Effects of dietary fat sources on lipid
metabolism in growing chicks (Gallus domesticus). Comp. Biochem. Physiol. Biochem. Mol. Biol.,
116, 1: 119 – 125.
B a n n o C., X i a Z.S., T a n a k a K, O h t a n i S. (1997). Effects of dietary fat sources on lipid metabolism
in growing chicks (Gallus domesticus). Comp. Biochem. Physiol. Biochem. Mol. Biol., 116(1):
119 – 225.
B e a n LD., L e e s o n S. (2003). Long-term effects of feeding flaxseed on performance and egg fatty acid
composition of brown and white hens. Poultry Sci., 82(3): 388 – 394.
B e a u c h e s n e - R o n d e a u E., G a s c o n A., B e r g e r o n J., J a c q u e s H. (2003). Plasma lipids and
lipoproteins in hypercholesterolemic men fed a lipid-lowering diet containing lean beef, lean fish,
or poultry. Am. J. Clin. Nutr., 77: 587 – 593.
B o r o w i e c F., Z a j a˛c T., K o w a l s k i Z.M., M i c e k P., M a r c i ń s k i M. (2001). Comparison of
nutritive value of some new commercial linseed oily cultivars for ruminants. J. Anim. Feed. Sci.,
10: 301 – 308.
B o u r r e J.M. (2005). Effect of increasing the omega-3 fatty acid in the diets of animals on the animal
products consumed by humans. Med. Sci. (Paris), 21(8 – 9): 773.
E d e r K., G r u n t h a l G., K l u g e H., H i r c h e F., S p i k e J., B r a n d s c h C. (2005). Concentration of
cholesterol oxidation products in raw, heat-processed and frozen-stored meat of broiler chickens fed
diets differing in the type of fat and vitamin E concentrations. Brit. J. Nutr., 93(5): 633 – 643.
F o l c h J., L e e s M., S t a n l e y G.H.S. (1957). A simple method for the isolation and purification of total
lipids from animal tissues. J. Biol. Chem., 226: 497 – 509.
G o n z a l e z - E s q u e r r a R., L e e s o n S. (2000). Effects of menhaden oil and flaxseed in broiler diets
on sensory quality and lipid composition of poultry meat. Brit. Poultry Sci., 41(4): 481 – 488.
K r i s - E t h e r t o n P.M., H e c k e r K.D., B i n k o s k i A.E. (2004). Polyunsaturated fatty acids and
cardiovascular health. Nutr. Rev., 64(11): 414 – 426.
L o p e z - F e r r e r S., B a u c e l l s M.D., B a r r o e t a A.C., G a l o b a r t J., G r a s h o r n M.A. (2001). n-3
enrichment of chicken meat. 2. Use of precursor of long-chain polyunsaturated fatty acids: linseed
oil. Poultry Sci., 80(6): 753 – 761.
S c h u m a n B.E., S q u i r e s E.S., L e e s o n S. (2000). Effect of dietary flaxseed, flax oil and n-3 fatty
acid supplement on hepatic and plasma characteristics relevant to fatty liver haemorrhagic
syndrome in laying hens. Brit. Poultry Sci., 41(4): 465 – 472.
W i e s e n f e l d P.W., B a b u U.S., C o l l i n s T.F., S p r a n d o R., O ’ D o n n e l l M.W., F l y n n T.J.,
B l a c k T., O l e j n i k N. (2003). Flaxseed increased α-linolenic and eicosapentaenoic acid and
decreased arachidonic acid in serum and tissues of rat dams and offspring. Food Chem. Toxicol.,
41, 6: 841 – 855.
AGNIESZKA SZEWCZYK, FRANCISZEK BOROWIEC, EWA HANCZAKOWSKA
Zawartość kwasów tłuszczowych i cholesterolu w mie˛sie brojlerów żywionych olejem lnianym
lub różnymi odmianami lnu
STRESZCZENIE
W doświadczeniu wzrostowym na brojlerach oceniano wpływ różnych odmian lnu na skład
kwasów tłuszczowych i zawartość cholesterolu w mie˛sie piersi i nóg. Sto brojlerów podzielono na
116
A. Szewczyk et al.
5 grup. Ptaki utrzymywano pojedynczo i żywiono do woli jedna˛ z pie˛ciu diet: kontrolna˛ (I), z dodatkiem
4% oleju lnianepo (II), z dodatkiem 10% nasion odmiany Omega (III), Opal (IV) lub Linola (V).
Zawartość kwasów tłuszczowych oceniano w nasionach, paszy oraz mie˛sie piersi i nóg. Analizowano
także zawartość cholesterolu w mie˛sie i surowicy krwi.
Nie stwierdzono istotnych różnic w przyrostach masy ciała brojlerów. Mie˛so brojlerów doświadczalnych miało wyższa˛ zawartość wielonienasyconych kwasów tłuszczowych (PUFA) niż mie˛so
brojlerów kontrolnych ale niższa˛ zawartość kwasów nasyconych (SFA) i jednonienasyconych (MUFA).
Dodatek lnu obniżył zawartość cholesterolu w mie˛sie. Nasiona odmiany Opal wykazywały najwie˛ksze
działanie hipocholesterolemiczne.
EFFECT OF FEED SUPPLEMENTATION WITH VITAMIN C
ON HAEMATOLOGICAL INDICES, CORTICOSTERONE
CONCENTRATION IN BLOOD AND DURATION OF TONIC
IMMOBILITY IN PHEASANTS
S e b a s t i a n N o w a c z e w s k i1, H e l e n a K o n t e c k a1, E w a P r u s z y ń s k a - O s z m a ł e k2
1
Department of Poultry Science, Agricultural University of Poznań, Witosa 45,
2
Department of Animal Physiology and Biochemistry, Wołyńska 35, 60-637 Poznań, Poland
Abstract
The aim of the study was to investigate whether the inclusion of vitamin C in feed affects
haematological indices, corticosterone in blood and the duration of tonic immobility in pheasants.
In experiment I, the material comprised 72 one-year-old birds (8 cocks and 64 hens) kept in
aviaries. Investigations were conducted for 11 weeks during the reproductive season (April – June).
The birds were divided into four groups, group I — control and groups II, III and IV —
experimental in which birds were fed diets supplemented with 100, 200 and 300 mg/kg vitamin C,
respectively. At week 8 of the experiment, blood was collected once from all females. In
experiment II, the experimental material comprised 300 growing pheasants, 1 to 28 days old, kept
indoors. The birds were divided into three groups of 100 animals each, group I — control,
group II — experimental, in which the feed was supplemented with 1200 mg/kg vitamin C 24 hours
before fasting and investigating tonic immobility and group III — experimental in which pheasants
were fed diets supplemented with 1200 mg/kg vitamin C every day. Blood was sampled from
4-week-old pheasants (15 birds from each group). Tonic immobility was studied on day 21 of age
in 30 pheasants from each group. In comparison with the control and the second group, in
experiment I we observed a higher number of erythrocytes in the blood of the breeding pheasants
fed diets supplemented with vitamin C in amounts of 200 and 300 mg/kg of feed. The heterophil
to lymphocyte ratio in the pheasant hens fed diets supplemented with 100 mg/kg vitamin C
was significantly lower (by 0.19) in comparison with the control group. In experiment II, pheasants
fed daily up to 4 weeks of age, diets containing 1200 mg/kg vitamin C were characterized, in
comparison with the control group, by a higher number of erythrocytes, higher haemoglobin
levels and a higher hematocrit value. The corticosterone concentration in the blood serum of these
birds, however, was significantly lower (by 26.7 ng/ml) in comparison with the control group.
No differences in the duration of tonic immobility between the examined groups were
observed.
Key words: pheasant, vitamin C, haematological indices, corticosterone, tonic immobility
118
S. Nowaczewski et al.
The environmental conditions under which indoor-reared pheasants are kept
differ considerably from natural conditions. This is associated with the exposure of birds
to a number of stress factors (e.g. restriction of the living space, contact with man,
zootechnical and veterinary treatments), which can have a negative impact on their
health and production results. Many investigations carried out on poultry have
shown that stress is often accompanied by changes in the values of haematological
indices (Zhou et al., 1999; Yahav, 1999). It was found that some blood traits, i.e. the
heterophil to lymphocyte (H:L) ratio and corticosterone concentration, can be good
indicators of stress in poultry (Maxwell, 1993; Puvadolpirod and Thaxton, 2000).
Meanwhile, according to Gallup (1979), the duration of tonic immobility can be a good
indicator of fear in birds. Under stress conditions, the values of these indices increase.
However, many investigations (Jaffar and Bláha, 1996; Keshavarz, 1996;
Andreasen and Frank, 1999; Whitehead and Keller, 2003) have revealed that
vitamin C administered in feed can alleviate the effect of different stress factors on
birds’ organisms and thus improve their health and production results. In addition, it
was demonstrated that this vitamin can influence the values of haematological
indices, the corticosterone concentration in blood and tonic immobility in chickens
(Kutlu and Forbes, 1994; Kontecka et al., 1997; Zulkifli et al., 2000).
The objective of this study was to determine whether feed supplementation with
vitamin C can influence haematological indices in breeding pheasants and whether
one-time or daily administration of vitamin C to growing pheasants affects the
corticosterone concentration in blood or the duration of tonic immobility.
Two experiments were carried out in 2001 at a pheasant farm belonging to the
Agricultural University in Poznań. In experiment I, the experimental material was
a group of 72 one-year-old birds kept in aviaries in flocks made up of one cock and
8 hens, at a stocking density of 1.2 birds per m2. Investigations were conducted for
a period of 11 weeks during the pheasants’ reproductive season (April – June). The
birds were fed complete diets ad libitum (Table 1). Four groups of two flocks each
(2 cockerels and 16 hens) were formed, group I — control and groups II, III and
IV — experimental, in which the birds were fed complete diets supplemented with
100, 200 and 300 mg/kg vitamin C, respectively. At 8 weeks, blood was collected
once from all hens (64 birds).
In experiment II, the experimental material included 300 birds (both
males and females) aged 1 to 28 days and maintained indoors. It was not possible to
identify the birds’ sex at that age. The birds were fed complete diets ad libitum
(Table 2). Three groups of 100 birds were established, group I — control and group
II — experimental in which the birds were fed 1200 mg/kg vitamin C 24 hours
before fasting and investigating tonic immobility, and group III — experimental in
which pheasants were fed 1200 mg/kg feed of vitamin C every day. Blood was
sampled from 4-week-old pheasants (15 birds from each group).
Effect of vitamin C supplementation of pheasant diets
119
Table 1. Diet composition of reproductive pheasants
Ingredients and analysis
Ground wheat
Soybean meal
Rapeseed meal ‘‘00”
Meat meal
Plant oil
Calcium III phosphate
Calcium carbonate
DL — methionine
Sodium chloride
Premix DJR forte
In 1 kg:
Crude protein (%)
Crude fibre (%)
Lys (%)
Met (%)
Met + Cys (%)
Thr (%)
Try (%)
Total Ca (%)
Available P (%)
Total Na (%)
NaCl (%)
Vitamin A (I.U.)
Vitamin D3 (I.U.)
Vitamin E (mg)
Vitamin K (mg)
Vitamin B1 (mg)
Vitamin B2 (mg)
Vitamin B6 (mg)
Vitamin B12 (mg)
Biotin (mg)
Folic acid (mg)
Nicotinic acid (mg)
Calcium pantothenate (mg)
Choline chloride (mg)
Manganese (mg)
Iodine (mg)
Zinc (mg)
Copper (mg)
Iron (mg)
Selenium (mg)
Calcium (g)
(%)
61.3
13.5
10.0
5.0
3.5
1.7
4.0
0.2
0.3
0.5
Quantity
11.7
19.1
3.64
1.03
0.43
0.78
0.72
0.26
2.62
0.58
0.19
0.43
12500
2500
30.00
2.50
2.50
10.00
3.00
0.02
0.05
0.40
20.00
10.00
400.00
45.00
0.35
40.00
2.50
25.00
0.15
1.14
In both experiments, blood was sampled from the subcutaneous elbow vein
(vena ulnaris cutanea) following 12-hour fasting. Heparin was used as an
anticoagulant. The following parameters were determined in blood: haemoglobin
120
content, hematocrit value, number of erythrocytes and leukocytes. Leukocytes were
classified into granulocytes (heterophils, eosinophils and basophils) and agranulocytes (lymphocytes and monocytes). The level of haemoglobin was determined
using the cyanomethemoglobin method and the hematocrit value — using the
micro-hematocrit method. The number of erythrocytes and leukocytes was counted
under a microscope in the Bürker chamber. Blood smears were dyed using the
Pappenheim method and then, using a microscope, 100 successive leukocytes were
counted and divided into granulocytes and agranulocytes. The corticosterone
concentration in the pheasants’ blood serum was determined using the standard test
of the Corticosterone 125J RIA Kit (ICN Biomedicals, Inc., Costa Mesa CA 92626 ).
Table 2. Diet composition of pheasants in the rearing period (%)
Ingredients and analysis
1
Wheat
Triticale
Soybean meal
Rapeseed meal
Yeasts
Meat meal
Calcium II phosphate
Sodium chloride
Calcium carbonate
Dry lucerne
PH premix
In 1 kg:
Crude protein (%)
Crude fibre (%)
Lys (%)
Met (%)
Met + Cys (%)
Thr (%)
Try (%)
Total Ca (%)
Available P (%)
Total Na (%)
NaCl (%)
Vitamin A (I.U.)
Vitamin D3 (I.U.)
Vitamin E (mg)
Vitamin K (mg)
Vitamin B1 (mg)
Vitamin B2 (mg)
Vitamin B3 (mg)
Vitamin B6 (mg)
Vitamin B12 (mg)
Age of pheasants (weeks)
0–3
4–6
2
3
47.0
41.9
10.0
24.0
12.0
0.5
5.0
0.8
0.1
0.7
4.0
1.0
39.0
0.9
10.0
2.0
0.1
1.0
Quantity
11.50
28.13
3.95
1.60
0.57
1.31
0.93
0.37
1.17
0.71
0.17
0.31
12500
3000
30.00
2.50
2.00
6.00
0.00
4.00
0.02
11.02
24.03
5.28
1.29
0.34
1.16
0.84
0.33
1.05
0.41
0.14
0.29
12500
3000
30.00
2.50
2.00
6.00
0.00
4.00
0.02
121
Table 2 — contd.
1
Nicotinic acid (mg)
Calcium pantothenate (mg)
Folic acid (mg)
Biotin (mg)
Choline chloride (mg)
Iron (mg)
Copper (mg)
Cobalt (mg)
Zinc (mg)
Manganese (mg)
Iodine (mg)
Selenium (mg)
2
3
20.00
12.00
0.60
0.10
600.00
35.00
2.50
0.40
55.00
65.00
0.35
0.15
20.00
12.00
0.60
0.10
600.00
35.00
2.50
0.40
55.00
65.00
0.35
0.15
Tonic immobility (TI) was checked on day 21 of age (30 birds from each group)
using the method described by Jones (1986). Each bird was placed on its back in
a wooden trough and held in the trough for 15 seconds, i.e. long enough to arouse
TI. Next the researcher left the bird and moved away to a distance of about 2 m and
registered the duration of TI until the bird raised. The minimum and maximum
durations of TI were assumed to be 10 and 480 seconds, respectively.
In both experiments, the vitamin C was administered in crystalline form
(acidum ascorbicum — Polfa, Kraków). The appropriate dose of this vitamin was
introduced into the mash mixture shortly before feeding to birds. In order to ensure
appropriate feed homogeneity, three pre-mixtures were made before administering
the feed.
Statistical calculations for haematological indices and blood hematocrit content
were performed using one-way analysis of variance. In the case of TI duration,
a trait that does not show normal distribution, the analysis of variance was preceded
by data transformation: x = log 10y, where y = trait value. The significance of
differences between means for individual groups was verified using Fisher’s test.
For statistical analysis the SAS (v. 9.1) package was used.
Results
In experiment I (Table 3), pheasants from the control group and group II were
characterized by a significantly lower (by 0.23 and 0.16 mln/µl, respectively)
number of erythrocytes in blood in comparison with the other birds. On the other
hand, none of the groups differed statistically significantly with respect to the blood
haemoglobin content or hematocrit value, or the number of leukocytes in blood.
A statistically significant difference was found between groups II and IV in the
percentage of eosinophils. The value of the H:L ratio in the blood of females from
122
group II was significantly lower in comparison with the control group. Pheasants
whose diets were supplemented with vitamin C had good health throughout the
experiment. Deaths were recorded only in the control group (approximately 16.7%
of the initial number of birds in this group).
Table 3. Effect of feed supplementation with vitamin C on haematological indices of reproductive
pheasants (exp. I)
Groups
Trait
Erythrocytes (mln/µl)
x̄
SEM
Haemoglobin (mmol/l)
x̄
SEM
Hematocrit (%)
x̄
SEM
Leucocytes (thous./µl)
x̄
SEM
Basophils (%)
x̄
SEM
Eosinophils (%)
x̄
SEM
Heterophils (%)
x̄
SEM
Lymphocytes (%)
x̄
SEM
Monocytes (%)
x̄
SEM
Heterophil to lymphocyte ratio (1:1)
(H:L)
x̄
SEM
I — control
(n = 16)
II
III
IV
100 mg vit. C 200 mg vit. C 300 mg vit. C
(n = 16)
(n = 16)
(n = 16)
2.90 a
0.04
2.97 a
0.07
3.13 b
0.04
3.13 b
0.04
6.03 a
0.29
5.85 a
0.21
6.09 a
0.22
5.72 a
0.26
34.0 a
0.85
34.0 a
0.77
33.0 a
0.79
33.0 a
1.41
16.1 a
2.06
16.8 a
0.95
14.5 a
1.23
15.1 a
0.99
12.1 a
1.37
12.2 a
1.53
10.8 a
1.23
11.1 a
1.64
2.2 ab
0.53
4.4 a
1.46
2.3 ab
0.88
1.8 b
0.39
26.3 a
2.62
18.8 a
2.74
21.1 a
2.82
25.3 a
2.02
56.1 a
2.80
62.1 a
2.78
63.2 a
2.71
59.1 a
1.77
3.4 a
0.81
2.5 a
0.52
2.7 a
0.71
2.9 a
0.89
0.51 a
0.07
0.32 b
0.05
0.37 ab
0.07
0.44 ab
0.05
Mean values in rows with different letters differ significantly (P ≤ 0.05).
123
Table 4. Effect of feed supplementation with vitamin C on haematological indices of 4-week-old
pheasants (exp. II)
Trait
Erythrocytes (mln/µl)
x̄
SEM
Haemoglobin (mmol/l)
x̄
SEM
Hematocrit (%)
x̄
SEM
Leucocytes (thous./µl)
x̄
SEM
Basophils (%)
x̄
SEM
Eosinophils (%)
x̄
SEM
Heterophils (%)
x̄
SEM
Lymphocytes (%)
x̄
SEM
Monocytes (%)
x̄
SEM
Heterophil to lymphocyte ratio (1:1) (H:L)
x̄
SEM
Corticosterone (ng/ml)
x̄
SEM
Tonic immobility (s) (TI)
x̄
SEM
Groups
I — control
(n = 15)
II
(n = 15)
III
(n = 15)
2.38 a
0.12
2.43 ab
0.12
2.48 b
0.12
7.35 a
0.36
8.37 ab
0.25
8.40 b
0.49
31.0 a
0.98
35.0 b
0.62
17.9 a
1.09
19.4 a
1.36
20.5 a
1.26
3.8 a
1.95
4.1 a
1.17
2.1 a
0.71
1.7 a
0.55
1.6 a
0.47
1.6 a
0.48
22.6 a
2.80
24.0 a
2.43
27.3 a
2.37
66.6 a
3.16
65.6 a
2.46
63.8 a
2.04
5.3 a
1.30
4.7 a
1.10
5.2 a
0.63
0.37 a
0.05
0.39 a
0.05
0.45 a
0.05
40.1 a
5.28
296 a
0.07
29.8 ab
7.98
328 a
0.05
34.0 b
0.60
13.4 b
1.76
274 a
0.08
Mean values in rows with different letters differ significantly (P ≤ 0.05).
In experiment II (Table 4), birds from group III had a significantly higher (by
0.1 mln/µl) mean number of erythrocytes, about 1.1 mmol/l higher haemoglobin in
blood and 3 percentage points higher hematocrit in blood in comparison with the
control group. On the other hand, birds from group II, in comparison with group I,
had a significantly higher (by 4 percentage points) hematocrit value. No significant
124
differences were found between the examined groups of birds in the number of
leukocytes or percentage of agranulocytes or granulocytes, or in the H:L ratio. The
corticosterone concentration in the blood of the pheasants from group I was
significantly higher (by 26.7 ng/ml) in comparison with group III. In addition, birds
from this group were characterized by a 22 second shorter TI duration than those
from the control group. However, this difference was not confirmed statistically.
The mortality of birds was similar in the examined groups and amounted to 6% on
average.
Discussion
The number of erythrocytes, the hematocrit value and the haemoglobin content
determined in the blood of breeding pheasants were similar to the values reported
by Jamroz et al. (1983). Pheasant hens fed diets supplemented with 200 or
300 mg/kg vitamin C were characterized by a higher number of erythrocytes than
the birds from the control group and group II (100 mg/kg vitamin C). A significantly higher number of erythrocytes (by about 0.56 mln/µl) in comparison with the
control group was also reported by Kontecka et al. (1997) in experiments on laying
hens fed diets supplemented with 200 mg/kg vitamin C. However, this study, as
well as experiments carried out by Torgowski and Kontecka (1998) on pheasants
fed diets supplemented with vitamin C and iron, failed to show significant
differences between the control and experimental groups with regard to the number
of leukocytes or proportions of individual types of granulocytes and agranulocytes.
However, in our study, the basophil content of the blood of pheasants from
individual groups was similar to that reported by Lucas and Jamroz after Campbell
(1995) and Torgowski and Kontecka (1998). An increased number of monocytes
accompanied by a decreased number of eosinophils may be indicative of the
organism’s response to stress factors (Maxwell et al., 1990). In the experiments
performed, no differences were found between the control group and the experimental groups with regard to the percentage of the above-mentioned granulocytes. Nevertheless, both in the control group and in the group fed 300 mg/kg
vitamin C, a trend was observed towards a reduced number of eosinophils and an
increased number of monocytes in blood.
Gross and Siegel, after Maxwell and Robertson (1998), distinguished three
stress levels (low, medium and high) in relation to the value of the H:L ratio
determined in the blood of birds (0.2, 0.5 and 0.8, respectively). The elevated value
of the H:L ratio (x̄ = 0.41) determined in experiment I indicates that the birds were
exposed to a medium stress level. This could have been caused by the weather
conditions (air temperature ranged from 25 to 30oC) that prevailed on the farm for
a few days before blood sampling. Kontecka et al. (1999) also reported a higher H:L
ratio in ducks two days after exposure of the birds to various stress factors.
The value of the H:L ratio in pheasants fed diets supplemented with vitamin C
(100 mg/kg) was significantly lower than in the control group. Moreover, the
125
200 mg dose of vitamin C did not affect this trait. Similar results were reported by
Kontecka et al. (1997) who failed to find a lower H:L ratio in laying hens fed diets
supplemented with 200 mg/kg vitamin C in comparison with the control group.
The application of a higher dose of 1200 mg vitamin C in experiment II was
motivated by the lower biosynthesis of this vitamin by young organisms and its
significantly higher metabolism (Seeman, 1991). Moreover, many experiments
have indicated that growing birds require vitamin C in quantities over 1000 mg per
kg feed or per l of water when they are exposed to stress factors such as catching,
transport or zootechnical treatments (Pardue et al., 1984; Satterlee et al., 1989;
Zulkifli et al., 2000).
Growing pheasants fed daily diets containing 1200 mg/kg vitamin C, in
comparison with the control group, were characterized by a significantly higher
number of erythrocytes, a higher hematocrit value and higher haemoglobin levels.
On the other hand, no significant differences were observed in the H:L ratio
between the birds from the experimental groups and the control group. Januszewski
(1988) added vitamin C (200 mg/l) and rutin (50 mg/l) to water for chickens and
found that, in comparison with the control group, they were characterized by
a higher number of erythrocytes, a higher hematocrit value and higher blood
haemoglobin. It should be added here that neither the above-mentioned author nor
our study found significant differences between groups in the percentage of
individual types of leukocytes.
Catching birds and keeping them in order to collect blood constitute strong
stress factors that cause increased secretion of corticosterone (Littin and Cockrem,
2001). In addition, it was demonstrated that with an increase in the amount of
vitamin C in blood, the level of this hormone decreases (Kutlu and Forbes, 1994).
The concentration of corticosterone in the blood of pheasants fed 1200 mg/kg
vitamin C every day was significantly lower (by about 67%) than in birds from the
control group. Satterlee et al. (1989) also found a decline in blood corticosterone in
chickens supplemented with vitamin C at a rate of 1200 mg/l water 24 hours before
blood sampling. However, this difference was smaller and amounted to about 10%.
Mahmoud et al. (1999) obtained similar results using a lower dose of vitamin C
(500 mg/kg) and an additional stress factor, i.e. cyclical changes of temperature in
the room (21 – 30 – 21oC).
The results of the above investigations indicate that vitamin C supplemented
daily to feed for growing pheasants could alleviate the effects of stress caused by
bird catching and collection of blood (lower corticosterone concentration in blood).
The observed lack of differences between groups in the value of the H:L ratio can
probably be attributed to the fact that the proportion of these leukocytes in blood
undergoes changes after a longer period of time from the moment the stress
stimulus occurs. This was shown by Gross after Maxwell (1993) who reported
a higher H:L ratio in broiler chickens only 18 hours after the application of the
stress factor (noise), while Kontecka et al. (1999) reported significant differences in
H:L values in ducks only 2 days after application of the stress factor (no access to
water).
126
Pheasants fed daily diets containing 1200 mg/kg vitamin C were characterized
by a shorter, but non-significant, TI duration in comparison with birds from the
other groups. Also, Satterlee et al. (1994) and Jones et al. (1996) reported, in
comparison with the control group, a shorter TI duration in broiler chickens and
Japanese quails which were administered water with 1000 mg/l vitamin C 24 hours
before sampling.
On the basis of the investigations performed, it can be stated that reproductive
pheasants fed diets supplemented with 200 or 300 mg/kg vitamin C are characterized by a higher number of erythrocytes in blood. Pheasants which were fed
100 mg/kg vitamin C in their diets were characterized by a lower H:L ratio value in
comparison with the control group.
In comparison with the control group, the blood of growing pheasants fed daily
diets containing 1200 mg/kg vitamin C was characterized by a higher number of
erythrocytes, higher haemoglobin levels and a higher hematocrit value. Moreover,
the lower corticosterone concentration obtained in the blood of pheasants from this
group leads us to assume that vitamin C probably influenced the increased
resistance of birds to the stress caused by catching and blood sampling. Our
findings also indicate that supplementing feeds given to breeding and growing
pheasants with vitamin C is useful, as it may alleviate the effects of stress under
farm rearing conditions.
References
A n d r e a s e n C.B., F r a n k D.E. (1999). The effects of ascorbic acid on in vitro heterophil function.
Avian Dis., 43: 656 – 663.
C a m p b e l l T.W. (1995). Avian hematology and cytology. Iowa State University Press. Ames Iowa,
104 pp.
G a l l u p G.G. Jr. (1979). Tonic immobility as a measure of fear in domestic fowl. Anim. Beh., 27:
316 – 317.
J a f f a r G.H., B l á h a J. (1996). Effect of ascorbic acid supplementation in drinking water on growth
rate, feed consumption and feed efficiency of broiler chickens maintained under acute heat stress
conditions. Živoč. Vyr., 41: 485 – 490.
J a m r o z D., B a r t c z a k R., G i e b e l O., M a z u r k i e w i c z M., M r ó z A. (1983). Efekty produkcyjne
i stan zdrowotny bażantów żywionych niskobiałkowymi mieszankami treściwymi, sporza˛dzonymi
z surowców krajowych. Med. Wet., 38: 357 – 360.
J a n u s z e w s k i J. (1988). Wpływ rutiny i rutinoscorbinu na stan zdrowotny i efekty tuczu kurcza˛t
brojlerów. Zesz. Nauk. AR Wroc., Wet., 44: 87 – 107.
J o n e s R.B. (1986). The tonic immobility reaction of the domestic fowl: a review. World’s Poultry Sci.,
J. 42: 82 – 96.
J o n e s R.B., S a t t e r l e e D.G., M o r e a u J., W a d d i n g t o n D. (1996). Vitamin C supplementation and
fear-reduction in Japanese quail: short-term cumulative effects. Brit. Poultry Sci., 37: 33 – 42.
K e s h a v a r z K. (1996). The effect of different levels of vitamin C and cholecalciferol with adequate or
marginal levels of dietary calcium on performance and eggshell quality of laying hens. Poultry Sci.,
75: 1227 – 1235.
K o n t e c k a H., W i t k i e w i c z K., S o b c z a k J. (1997). Wpływ dodatku witaminy C do paszy na
wskaźniki produkcyjne i hematologiczne kur nieśnych. Zesz. Nauk. PTZ, Prz. Hod., 32: 139 – 145.
K o n t e c k a H., K s i a˛ż k i e w i c z J.M., N o g o w s k i L. (1999). Effects of different stressors on laying
rate and selected blood indices in reproductive ducks. J. Anim. Feed. Sci., 8: 63 – 72.
127
K u t l u H.R., F o r b e s J.M. (1994). Response of broiler chicks to dietary ascorbic acid and corticosterone. Brit. Poultry Sci., 35: 184 – 186.
L i t t i n K.E., C o c k r e m J.F. (2001). Individual variation in corticosterone secretion in laying hens.
Brit. Poultry Sci., 42: 536 – 546.
M a h m o u d K.Z., E d e n s F.W., E i s e n E. (1999). Vitamin C reduces HSP70 and plasma corticosterone responses in broilers subjected to cyclic heat stress. Poultry Sci., Suppl. (Ann. Meet. Abstr.)
1: 106.
M a x w e l l M.H. (1993). Avian blood leucocyte responses to stress. World Poultry Sci. J., 49: 34 – 43.
M a x w e l l M.H., R o b e r t s o n G.W. (1998). The avian heterophil leucocyte: a review. World Poultry
Sci. J., 54: 155 – 178.
M a x w e l l M.H., R o b e r t s o n G.W., S p e n c e S., M c C o r q u o d a l e C. (1990). Comparison of
haematological values in restricted- and ad libitum-fed domestic fowls: white blood cells and
thrombocytes. Brit. Poultry Sci., 31: 399 – 405.
P a r d u e S.L., T h a x t o n J.P., B r a k e J. (1984). Plasma ascorbic acid concentration following ascorbic
acid loading in chicks. Poultry Sci., 63: 2492 – 2496.
P u v a d o l p i r o d S., T h a x t o n J.P. (2000). Model of physiological stress in chickens. 1. Response
parameters. Poultry Sci., 79: 363 – 369.
S a t t e r l e e D.G., A q u i l e r a - Q u i n t a n a I.A., M u n n B.J., K r a u t m a n n B.A. (1989). Vitamin
C amelioration of the adrenal stress response in broiler chickens being prepared for slaughter.
Comp. Biochem. Physiol., 94A: 569 – 574.
S a t t e r l e e D.G., J o n e s R.B., R y d e r F.H. (1994). Effects of ascorbyl-2-polyphosphate on adrenocortical activation and fear-related behavior in broiler chickens. Poultry Sci., 73: 194 – 201.
S e e m a n M. (1991). Is vitamin C essential in poultry nutrition? World’s Poultry Misset, 7: 17 – 19.
T o r g o w s k i J., K o n t e c k a H. (1998). Wpływ dodatku witaminy C i żelaza do paszy na wskaźniki
produkcyjne i hematologiczne bażantów łownych (Phasianus colchicus). Rocz. AR Pozn. 302.,
Zoot., 50: 235 – 242.
W h i t e h e a d C.C., K e l l e r T. (2003). An update on ascorbic acid in poultry. World Poultry Sci. J., 59:
161 – 184.
Y a h a v S. (1999). The effect of constant and diurnal cyclic temperatures on performance and blood
system of young turkeys. J. Therm. Biol., 24: 71 – 78.
Z h o u W.T., F u j i t a M., Y a m a m o t o S. (1999). Effects of ambient temperatures on blood viscosity
and plasma protein concentration of broiler chickens (Gallus domesticus). J. Therm. Biol., 24:
105 – 112.
Z u l k i f l i I., C h e N o r m a M.T., C h o n g C.H., L o h T.C. (2000). The effects of crating and road
transportation on stress and fear responses of broiler chickens treated with ascorbic acid. Arch.
Geflügelkunde, 65: 33 – 37.
SEBASTIAN NOWACZEWSKI, HELENA KONTECKA, EWA PRUSZYŃSKA-OSZMAŁEK
Wpływ dodatku witaminy C do paszy na wskaźniki hematologiczne, zawartość kortykosteronu
we krwi i czas trwania bezruchu tonicznego bażantów
STRESZCZENIE
Celem pracy było sprawdzenie czy podawana do paszy witamina C ma wpływ na wskaźniki
hematologiczne, zawartość kortykosteronu we krwi oraz czas trwania bezruchu tonicznego u bażantów.
W doświadczeniu I materiał badawczy stanowiły 72 jednoroczne ptaki (8 kogutów i 64 kury)
128
utrzymywane w wolierach. Badania prowadzono przez 11 tygodni w sezonie reprodukcyjnym (kwiecień – czerwiec). Utworzono 4 grupy: I — kontrolna oraz II, III i IV — doświadczalne, w których ptaki
otrzymywały w mieszance dodatek odpowiednio 100, 200 i 300 mg/kg witaminy C. W 8. tygodniu
pobrano jednorazowo krew od wszystkich samic. W doświadczeniu II materiał badawczy stanowiło 300
rosna˛cych bażantów, w wieku od 1. do 28. dnia życia, utrzymywanych w pomieszczeniu. Utworzono
3 grupy po 100 bażantów w każdej: I — kontrolna, II — doświadczalna, której na 24 godziny przed
głodzeniem i badaniem bezruchu tonicznego ptaków dodawano do paszy 1200 mg/kg witaminy C oraz
III — doświadczalna, w której bażanty żywiono codziennie pasza˛ z dodatkiem 1200 mg/kg witaminy C.
Krew pobrano od 4-tygodniowych ptaków (15 szt. z każdej grupy). Bezruch toniczny badano w 21. dniu
życia bażantów (30 ptaków z każdej grupy).
W doświadczeniu I wykazano wie˛ksza˛ niż w grupie kontrolnej liczbe˛ erytrocytów we krwi
bażantów reprodukcyjnych żywionych pasza˛ z dodatkiem 200 i 300 mg/kg witaminy C. Wartość
stosunku heterofili do limfocytów u kur bażancich otrzymuja˛cych do paszy dodatek 100 mg/kg
witaminy C była istotnie mniejsza (o 0,19) niż w grupie kontrolnej. W doświadczeniu II, bażanty
żywione codziennie pasza˛ z dodatkiem 1200 mg/kg witaminy C charakteryzowała wie˛ksza niż ptaków
z grupy kontrolnej liczba erytrocytów, zawartość hemoglobiny i wartość hematokrytu, a zawartość
kortykosteronu w surowicy krwi tych ptaków była istotnie mniejsza — o 26,7 ng/ml. Nie stwierdzono
różnic mie˛dzy badanymi grupami w czasie trwania bezruchu tonicznego.
DYNAMICS OF SMALL STRONGYLE (CYATHOSTOMINAE) INFECTION
IN HORSES UNDER DIFFERENT MANAGEMENT SYSTEMS
S ł a w o m i r K o r n a ś, B o g u s ł a w N o w o s a d, M a r t a S k a l s k a
Department of Zoology and Ecology, Agricultural University, Al. Mickiewicza 24/28,
30-059 Kraków, Poland
Abstract
On the basis of coproscopical and larvoscopical examinations carried out in 2000 – 2002, the level
of small strongyle (Cyathostominae) infection in horses was determined in four production
facilities. In two cycles of one year each, faecal samples from 291 horses kept under stable or stable
and pasture management systems were examined. The prevalence of infection and the number of
eggs per gram of faeces (EPG) were calculated for horses of different ages. The prevalence of horse
infection in the stable management system (ND) ranged from 16.7 to 79.3% in the first cycle and
from 34.2 to 80% in the second cycle. EPG was 78 – 675 and 88 – 340, respectively. The prevalence
of infection in horses under the stable and pasture management system (NS) was higher
(71.4 – 97.4% and 10.5 – 96.4%), with EPG counts of 138 – 969 and 90 – 649, respectively. At the
first riding club, pastured horses were more infected than horses kept in paddocks. In yearling and
2-year-old horses kept under the stable and pasture management system (NS), the classic dynamics
of infection in the annual cycle were ascertained, with two peaks of strongyle egg excretion — the
first in spring and the second at the end of the pasture season. An increase in egg output in spring
(April to May) was found in all the analysed facilities and in the summer-autumn months in horses
kept on pasture.
Key words: horses, infection, Cyathostominae, management system
Small strongyles (Cyathostominae) constitute a major problem in horse breeding because of their high prevalence, survival strategy in the host and environment, and ability to acquire resistance to antiparasitic drugs. The dynamics of small
strongyle egg output are closely related to their development. After being eaten
with grass, the infective larvae (L3) of the nematodes penetrate the mucous or
submucous membranes of the horse colon or caecum. After moulting to L4 they
return to the lumen of the intestine and mature to adults or arrest their development
(hypobiosis), especially during winter (Ogbourne, 1978). The development from L3
to adult stages from spring to early autumn lasts 1.5 to 3 months. Hypobiosis occurs
in late autumn (Mirck, 1982). The number of hypobiotic larvae can be very high
and can even reach 25 – 100 per cm2 of the mucous membrane of the intestines
(Mariotti et al., 1996).
130
S. Kornaś et al.
Clinical cyathostomosis occurs more commonly in young horses between late
winter and early spring. Clinical signs depend on many factors, but mainly on the
level of infection. The major symptoms are weight loss, diarrhoea, and sometimes
death in horses within 2 – 3 weeks (Love et al., 1999).
The objective of this study was to compare the dynamics of small strongyle
infection in horses of different age kept under different management systems.
The study was carried out at the national stud (NS), national depots (ND) and
two horse riding clubs (HC-1 and HC-2) in southwest Poland (Table 1). The
individual faecal samples from horses of different breeds, mainly Silesian, were
examined monthly from April 2000 to March 2002 (two annual cycles in adult
horses at NS and ND, one cycle at HC-1 and HC-2, and one cycle in yearlings and
2-year-old horses). Horses were treated with Antiverm (pyrantel embonate) except
yearlings and 2-year-old horses, which were treated with Eqvalan (ivermectin). The
McMaster method was used to estimate EPG and prevalence of infection monthly
in horses under all the management systems analysed.
Table 1. Design of the study
Type
of facility
National stud
(NS)
Management
system
stable-pasture
Number
of examined
horses
Treatment
(month)
43
Apr; Oct
48
Apr; Oct
Apr 2000 –
Mar 2001
22
Apr; Oct
stallions
Apr 2000 –
(4 – 20 years old) Mar 2001
Apr 2001 –
Mar 2002
65
Apr; Oct
74
Apr; Oct
Age
of horses
mares
Apr 2000 –
(4 – 18 years old) Mar 2001
Apr 2001 –
Mar 2002
yearling and
two years old
National depot
(ND)
stable
Annual
cycles
Horse riding
club 1 (HC-1)
stable-pasture
2 to 28 years old Apr 2000 –
Mar 2001
13
May; Oct
Horse riding
club 2 (HC-2)
stable
3 to 20 years old Apr 2000 –
with paddocks
Mar 2001
26
May, Oct
The faecal cultures were performed according to Henriksen and Korsholme
(1983) and infective larvae (100 larvae where possible) were identified according to
Soulsby (1965) to define small (Cyathostominae) or large (Strongylinae) strongyles.
Dynamics of small strongyle infection in horses
131
Results
Based on the larvae differentiation, a very low level of large strongyle
infections was found in the examined horses. Infective larvae of large strongyles
were identified only at the National Stud, at a very low percentage (0.05%). The
remaining 99.95% of larvae were small strongyles.
The dynamics of small strongyle infections were different in all the analysed
management systems. The mean prevalence of small strongyle infections in stabled
stallions (ND) ranged from 16.7% (April) to 79.3% (July) in the first cycle, and
from 34.2% (April) to 80% (November) in the second cycle (Figure 1). The
dynamics of small strongyle egg output showed a larger peak in April (675 EPG)
and a smaller peak (386 EPG) in August in the first cycle. In the second cycle, the
respective values were 340 EPG in April and 285 EPG in September (Figure 2).
The mean prevalence of mare infections under the stable and pasture system
(NS) was higher than in stabled stallions (ND), from 71.4% (May) to 97.4%
(December) in the first cycle, and from 10.5% to 96.4% in the second cycle
(Figure 1). For the dynamics of small strongyle egg output, a higher peak was
observed in August (969 EPG) than in March (484 EPG) in the first cycle, and in
September (649 EPG) than in March (360 EPG) in the second cycle (Figure 2).
On average, small strongyle infections were the most prevalent in horses under
stable and pasture management (the first riding club, HC-1) in the period from July
to October (75 – 95%) and the intensity of egg output peaked in May (792 EPG),
whereas in July-October it ranged from 672 to 910 EPG. Prevalence and EPG
decreased in June and November as a result of treatments applied in May and
October (Figures 1 and 2).
The prevalence of small strongyle infection in horses under the stable management system with paddocks from the second riding club (HC-2) was lower
(especially EPG) than in those under the other management systems analysed. The
highest prevalence of infections was in March and April (around 80%) and the
highest egg output in May (495 EPG), despite the treatment of horses in April
(Figures 1 and 2).
The dynamics of small strongyle infections in yearling and 2-year-old horses
were typical of the stable and pasture system (NS). The mean prevalence of
infection was very high (85.7 – 100%) except in the month after treatment
(Figure 3). The Cyathostominae egg output increased from April to September (633
EPG), then decreased after treatment in October (33 EPG) and slowly increased
once more until the end of the study in March (369 EPG) (Figure 4).
Figure 1. Prevalence of small strongyle infection in adult horses
132
S. Kornaś et al.
Figure 2. Dynamics of small strongyle egg output in adult horses
133
134
S. Kornaś et al.
Figure 3. Prevalence of small strongyle infection in yearling and 2-year-old horses
Figure 4. Dynamics of small strongyle egg output in yearling and 2-year-old horses
135
Discussion
The increase in small strongyle egg output was observed in spring (from April
to May) in all types of facilities and also during the summer and autumn months in
horses under the stable and pasture management system (NS, HC-1).
The peak of small strongyle egg output observed in early spring may have
resulted from the large quantities of hypobiotic larvae being released from the
mucous membrane of the horse intestines, which reached maturity and produced
many eggs excreted with horse faeces. The development of hypobiotic larvae may be
triggered not only by a temperature increase (Reinemeyer, 1986) but also by drug
treatment (after removal of adult stages of this parasite) (Gibson, 1953). The greatest
emergence of hypobiotic larvae was observed in late winter and early spring
(Reinemeyer et al., 1986). As a result of this, large numbers of sexually mature, female
small strongyles appeared in horse intestines from spring to autumn (Reinemeyer et al.,
1986), from April to June (Ogbourne, 1976) and in June (Mirck, 1982).
In the present study, the small strongyle egg output also increased from June to
October in horses under the stable and pasture system (NS and HC-1). In stabled
stallions (ND), the dynamics were similar but the number of eggs found in faeces
was considerably lower.
The lower intensity of infection during the autumn and winter months (from
November to January) observed in the present study may be possibly due to the
decrease in the adult population of small strongyles. The smallest number of adult
females was found from October to December, and a large number of females with
limited reproductive ability were found from the end of autumn and during the
winter (Ogbourne, 1976; Reinemeyer et al., 1986).
The dynamics of small strongyle egg output with two peaks in early spring and
late summer, observed in the present study in horses under the stable and pasture
system in particular, were similar to the results reported by other authors (Mage,
1996; Betlejewska, 2000). Rapid growth of infection intensity from early spring to
late summer and early autumn, followed by a decrease in the winter, has been
observed by other authors such as Slocombe et al. (1987) and Baudena et al. (2000).
Depending on local weather conditions, the grazing season in Poland lasts from
April/May to October/November. During this time horses excrete many strongyle
eggs while becoming infected with larvae, which develop from these eggs. The
development of larvae on the pasture to the infective stage depends on the latitude,
local weather conditions and type of pasture. In a study in Denmark, low pasture
infections occurred from April to June, then increased in July with a peak at the end
of pasture season (Henriksen, 1985). In a study in the Czech Republic, larvae were
present on the pasture from May to October, and the number increased from July to
October (Langrova, 1999). In a German study, pastures were more infected in June,
August and October (Hasslinger and Bittner, 1984) and in a French study, at the
turn of summer and autumn (Mage, 1996). Weather conditions in these countries
are similar to those in Poland, so the dynamics of strongyle larvae occurrence on
pasture could be similar to ours.
136
S. Kornaś et al.
The prepatent period of small strongyles lasts from 1.5 to 3 months. The
common genus Cylicocyclus spp. (subfamily Cyathostominae) develops for approximately 2 months. The pasture season in Poland is about 6 months long, which
allows for the occurrence of 2 or 3 generations of these parasites in horse intestines.
This was confirmed by an experiment conducted under German weather conditions,
which shows three peaks of infective larvae on pasture at 9, 16 and 24 weeks of the
6-month pasture season and three generations of small strongyles in horse intestines
(Bittner, 1983). In the present study, there was only one peak of small strongyle egg
output from August to October, because anthelminthics were given mostly in April
and October, which changed the seasonal dynamics of this nematode.
The dynamics of small strongyle egg output in stabled horses with paddocks
from the second riding club (HC-2) was different. At this facility, like at others, an
increased level of egg output was observed in spring (April – May). This was
probably due to the emergence of hypobiotic larvae from horse mucus or infections
with larvae surviving in small paddocks where horses stay from early spring
(Lindberg, 1976). The output of small strongyle eggs in horses housed only in
paddocks (HC-2) was constant from July to October and was lower than in spring.
The high moisture and rise in temperature in paddocks in the spring stimulated the
activity of larvae, which developed to the infective stage. No peaks in small
strongyle egg output were observed during the summer, due to the absence of
pasture which is a very important source of small strongyle infection in horses.
It is very difficult to determine the interaction between the species of
Cyathostominae subfamily using coprological tests. Knowledge of the reproductive
biology, dynamics and developmental processes of this nematode species is still
insufficient, as is knowledge of the influence of and competition between different
species or genera of small strongyles, in spite of the high prevalence and numerous
species of these parasites described in horses (Kornaś et al., 2004). Gawor (1995)
described 23 species of small strongyles in Poland. The number of small strongyle
species found in one host varies, with reports of 2 – 16 (Gawor, 1995), 4 – 16
(Ougbourne, 1976) and 2 – 11 (Reinemeyer et al., 1984).
The present study suggests that the dynamics of small strongyle infections
depend not only on the biology of this nematode, but also on environmental factors
and management systems. Knowledge of the dynamics of infection could help to
create treatment programmes adapted to local management conditions.
References
B a u d e n a M.A., C h a p m a n M.R., F r e n c h D.D., K l e i T. R. (2000). Seasonal development and
survival of equine cyathostome larvae on pasture in South Louisiana. Vet. Parasitol., 1 – 2: 51 – 60.
B e t l e j e w s k a K. (2000). The dynamics of small strongyles (Cyathostominae) invasion in horses
during an annual cycle. Med. Wet., 1: 36 – 38.
B i t t n e r G. (1983). Ein Beitrag zur Epizootologie der Strongylideninfektion des Pferdes in einem
bayerischen Gestutsareal. Tierärztliche Fakultät, Ludwig-Maximilians-Universitat München, 68.
137
G a w o r J. (1995). The prevalence and abundance of internal parasites in working horses autopsied in
Poland. Vet. Parasitol., 1 – 2: 99 – 108.
G i b s o n T.E. (1953). The effect of repeated anthelmintic treatment with phenotiazine on fecal egg
counts of housed horses, with some observations on the life cycle of Trichonema spp. in the horse.
J. Helminthol., 1/2: 29 – 40.
H a s s l i n g e r M.A., B i t t n e r G. (1984). Zur Saisondynamik der larven von Pferdestrongyliden und
deren Beziehung zum Infektionsrisiko auf der Weide. Zentralblatt für Veterinärmedizin, 1: 25 – 31.
H e n r i k s e n S.A., K o r s h o l m e M. (1983). A method for culture and recovery of gastrointestinal
strongyle larvae. Nord Vet., 35: 429 – 430.
H e n r i k s e n S.A. (1985). Seasonal variation of pasture contamination with infective larvae of equine
strongyles — preliminary studies. Dansk Vet., 7: 364 – 367.
K o r n a ś S., N o w o s a d B., S k a l s k a M. (2004). Zarażenie pasożytami przewodu pokarmowego koni
w zależności od warunków utrzymania. Med. Wet., 60: 853 – 857.
L a n g r o v a I. (1999). The importance of contaminated pastures and litter in stables for the infection
with nematodes of family Strongylidae in horses on studfarm Xaverov. Helminthologia, 4:
241 – 249.
L i n d b e r g R. (1976). Survival of infective larvae of horse strongyles on pasture grass. An introductory
field study. Svensk Vet., 11: 509 – 514.
L o v e S., M u r p h y D., M e l l o r D. (1999). Pathogenicity of cyathostome infection. Vet Parasitol., 85:
113 – 122.
M a g e C. (1996). Epidemiologie parasitaire chez les juments de trait au paturage. Rev. Med. Vet., 3:
211 – 214.
M a r i o t t i F., M a n d a r a M.T., M u g h e t t i L., V i t e l l o z z i G. (1996). La ciatostomiasi larvale del
cavallo. Aspetti anatomo-istopatologici. Ob. Doc. Vet., 5: 71 – 80.
M i r c k M.H. (1982). The prevalence, relative abundance and site distribution of Strongylidae in
Shetland ponies in the Netherlands. Parasitology, 84: 1.
O g b o u r n e C.P. (1976). The prevalence, relative abundance and site distribution of nematodes of the
subfamily Cyathostominae in horses killed in Britain. J. Helminthol., 3: 203 – 214.
O g b o u r n e C.P. (1978). Pathogenesis of cyathostome (Trichonema) infections of the horses: a review.
Commonwealth Institute of Helminthology. Commonwealth Agricultural Bureaux of the United
Kingdom, Misc. Publ., 5: 25.
R e i n e m e y e r C.R., S m i t h S.A., G a b e l A.A., H e r d R.P. (1984). The prevalence and intensity of
internal parasites of horses in the U.S.A. Vet. Parasitol., 1: 75 – 83.
R e i n e m e y e r C.R. (1986). Small strongyles. Recent advances. Vet. Clin. N. Am. Equine Prac., 2:
281 – 312.
R e i n e m e y e r C.R., S m i t h S.A., G a b e l A.A., H e r d R.P. (1986). Observations on the population
dynamics of five cyathostome nematode species of horses in northern USA. Equine Vet. J., 2:
121 – 124.
S l o c o m b e J.O.D., V a l e n z u e l a J., L a k e M.C. (1987). Epidemiology of strongyles in ponies in
Ontario. Can. J. Vet. Res., 4: 470 – 474.
S o u l s b y E.J.L. (1965). Textbook of veterinary clinical parasitology. Vol. I. Helminths. Blackwell
Scient. Public., Oxford.
Accepted for printing 23 XI 2005
138
S. Kornaś et al.
SŁAWOMIR KORNAŚ, BOGUSŁAW NOWOSAD, MARTA SKALSKA
Dynamika zarażenia słupkowcami małymi (Cyathostominae) koni z różnego systemu chowu
STRESZCZENIE
W oparciu o badania koproskopowe i larwoskopowe w latach 2000-2002 określono poziom
zarażenia koni słupkowcami małymi (Cyathostominae) w czterech typach obiektów. W dwóch cyklach
rocznych próby kału pobrano od 291 koni utrzymywanych systemem alkierzowym i alkierzowopastwiskowym. Na podstawie uzyskanych wyników obliczono ekstensywność zarażenia i średnia˛ liczbe˛
jaj słupkowców w 1 g kału (EPG) koni w różnym wieku.
Ekstensywność zarażenia koni utrzymywanych alkierzowo (Stado Ogierów) wynosiła 16,7 – 79,3%
w pierwszym cyklu rocznym i 34,2 – 80% w — drugim, a EPG odpowiednio: 78 – 675 i 88 – 340.
Zarażenie koni utrzymywanych w systemie alkierzowo-pastwiskowym (Stadnina) było wie˛ksze i wynosiło:
71,4 – 97,4% i 10,5 – 96,4%, a EPG 138 – 969 i 90 – 649 odpowiednio. W pierwszym klubie jeździeckim,
konie wypasane na pastwiskach były bardziej zarażone niż konie z klubu jeździeckiego korzystaja˛ce
z padoków. U koni jednorocznych i dwuletnich utrzymywanych systemem alkierzowo-pastwiskowym
(Stadnina) wykazano klasyczna˛ dynamike˛ zarażenia w cyklu rocznym, z dwoma szczytami wydalania jaj
słupkowców, tj. wiosna˛ i pod koniec sezonu pastwiskowego. W kale koni z wszystkich badanych
obiektów liczba wydalanych jaj słupkowców wzrastała wiosna˛ (od kwietnia do maja), a w kale koni
utrzymywanych systemem alkierzowo-pastwiskowym także w czasie miesie˛cy letnio-jesiennych.
EFFECT OF CROSSBREEDING ON PASTURE REARING OF LAMBS
AND CHEMICAL AND SENSORY PROPERTIES OF SLAUGHTER
MATERIAL*
Paweł Paraponiak
Department of Technology, Ecology and Economics of Animal Production, National Resarch Institute
of Animal Production, 32-083 Balice n. Kraków, Poland
Abstract
The aim of the study was to determine the effect of crossing Polish Mountain Sheep (PMS) ewes
with Bergschaf (BF) and Weisse Alpenschaf (WAS) rams on the fattening and slaughter traits of
the crossbreds and some chemical and sensory properties of their meat. A total of 140 ram lambs
were investigated in 7 groups: PMS, BF, WAS, BF × PMS, WAS × PMS, BF × (BF × PMS) and
WAS × (WAS × PMS), with 20 rams per group (10 animals in experiment and 10 animals in
replication). From around 4 months of age, lambs were grazed in the rotational system and
additionally fed with ground barley and ground wheat. The mean daily gains of the rams were
calculated between 2 and 200 days of age. The cold dressing percentage was determined and the
weight and proportion of valuable cuts in the right half-carcass were estimated. Detailed leg
dissection was performed. Selected chemical and sensory properties of lamb meat were studied.
WAS rams were characterized by the highest weight gains (211 g/day) and the highest empty body
weight and cold carcass weight. The mean weight gains of the crossbred rams were 12 – 37%
higher compared to those of PMS lambs. The dressing percentage of all the crossbreds was higher
than the value established for PMS rams (P ≤ 0.01). The meat obtained from all the experimental
rams was characterized by a desirable water to protein ratio, fat to protein ratio and pH value. The
results of sensory analysis showed that the meat of F1 and R1 crossbred lambs has high taste value.
In this respect, the meat of purebred WAS rams achieved lower scores.
Key words: sheep, crossbreeding, growth, meat quality, carcass quality
Since the mid-1980s, there has been a dramatic decline in the Polish sheep
population, in principle due to the marginalization of wool as a commercial product.
Several programmes oriented towards meat performance and the optimum commercial utilization of slaughter material were developed to halt this negative trend.
Considering this, and the fact that lamb meat is the only highly valued kind of meat
140
P. Paraponiak
whose production fails to meet the domestic market demand within the European
Union, a decision was made to increase the quota of imported Polish lamb by 10%
a year (Okularczyk, 2000). This limit is not used due to the weak lamb meat market
and poor reproductive and meat parameters of the native sheep breeds. The
primitive Polish Mountain breed, which is the only native breed to be optimally
suited to mountain conditions, serves as a good example. It is safe to assume that
the carcasses of crossbreds derived from PMS ewes and rams with superior
meatiness traits can be valuable export material and thus help to improve the
profitability of sheep production in Poland.
The aim of the present study was to determine the effect of crossing PMS ewes
with BF and WAS rams on the fattening and slaughter traits of the crossbreds and
the chemical and sensory properties of their meat.
The study was conducted at the Sheep Breeding Research and Implementation
Centre in Piorunka near Krynica Zdrój, located in the region of the Beskid Sa˛decki
Mountains (550 m above sea-level). There were 140 ram lambs representing
7 groups: Polish Mountain Sheep (PMS), Bergschaf (BF), Weisse Alpenschaf
(WAS), BF × PMS, WAS × PMS, BF × (BF × PMS) and WAS × (WAS × PMS),
with 20 rams per group (10 animals in experiment and 10 animals in replication).
To weaning at approximately 70 days of age, in addition to mother’s milk the lambs
received meadow hay and CJ concentrate mixture at 0.25 kg/day/animal (containing 5.33 MJ net energy and 190 g crude protein per kg). After weaning, rams
were fed according to feeding standards with farm-produced fodders (hay, ensiled
hay, ground maize) and commercial feeds (wheat and barley bran, sugar beet pulp
with added whey). From the beginning of the grazing season (middle of May,
around 4 months of age) rams were pastured in a rotational system and additionally
fed with ground barley and ground wheat at approximately 0.25 kg/day/animal.
The daily gains of the rams were calculated for the whole experiment. The body
weight of the rams was determined prior to slaughter (after 24-h fasting). Slaughter at
the age of 200 days and carcass processing were performed in accordance with the
methods used at the National Research Institute of Animal Production. Cold carcass
weight (after 24-h cooling at +4oC) and dressing percentage were determined.
The proportion of valuable cuts (leg, saddle, best end of neck and shoulder) was
calculated for the right half-carcass. Detailed leg dissection was performed to
determine the weight and percentage of muscular, adipose and bone tissue in the
leg.
The chemical parameters of the meat, i.e. the water to protein ratio (W/P), the
fat to protein ratio (F/P) and pH24 value (Radelkis device), were analysed on the
longissimus dorsi muscle. The sensory analysis of the meat (musculus semimembranosus, 5-point scale) covered determination of the taste intensity and quality of
meat samples.
Effect of crossbreeding on pasture rearing of lambs
141
The results were analysed statistically using multivariate analysis of variance,
with experimental group, year and replication being the differentiating factors. No
significant differences were found between years and replications in any of the traits
studied. The statistical differences found between the groups are given in the tables.
Post-hoc analysis was performed using Tukey’s honestly significant difference
test. The calculations were performed using the Anova/Manova procedure of
Statistica for Windows.
Results
PMS rams were characterized by the lowest dynamics of growth expressed as
daily gains, and the differences in relation to the other experimental groups were
highly significant (P ≤ 0.01). Of all the crossbreds, the highest rate of growth in the
experimental period was found in the WAS × (WAS × PMS) group (mean gains of
199 g/day), and the growth rate was higher than that found in purebred BF lambs
(185 g/day). The difference proved highly significant (P ≤ 0.01) (Table 1).
The body weight of rams after 24-hour fasting ranged from 30.9 to 44.9 kg
(Table 1). Both the PMS group (lowest body weight) and the WAS group (highest
body weight) differed highly significantly from each other and from the other
experimental groups. R1 crossbred rams were heavier than F1 crossbred rams, with
no significant differences found between them. The high variation in pre-slaughter
body weight resulted in considerable differences in cold carcass weight, with the
heaviest carcasses found in the WAS group (20.2 kg) and the lightest in the PMS
group (12.0 kg) (Table 1).
The highest dressing percentage was characteristic of the meat of WAS rams
(45.0%), and the difference in relation to the other groups, except R1 WAS × (WAS
× PMS) crossbreds (44.0%), was highly significant (Table 1).
The lowest weight of valuable cuts was found in PMS half-carcasses (3.35 kg)
and this value was highly significantly different from the analogous results obtained
in the other experimental groups, except the BF × PMS crossbreds group (Table 2).
Meanwhile, the highest proportion of valuable cuts was noted in WAS
half-carcasses (58.49%) followed by 58.28% in WAS × (WAS × PMS) crosses.
These experimental groups were significantly different in terms of this trait from
the analogous values found in the half-carcasses of PMS, BF and BF × PMS rams
(55.14, 57.31 and 57.53%, respectively).
The leg dissection results showed that the highest percentage of muscles was to
be found in WAS rams (73.64%), and the differences in relation to all the other
groups were statistically significant. The lowest meat content was found in PMS
legs (63.03%), with a slightly higher proportion in BF × PMS (64.44%) animals.
The differences in relation to WAS, WAS × (WAS × PMS) and BF animals proved
significant (Table 2). The highest proportion of adipose tissue (12.01%) occurred in
the legs of F2 rams with 75% WAS blood. The proportion of leg bone tissue ranged
from 16.55 (WAS) to 25.58% (PMS) (P ≤ 0.05).
142
Tab. 1, 2
P. Paraponiak
tab. 3
143
144
P. Paraponiak
Relative parameters of meat quality (longissimus dorsi muscle), i.e. the W/P
ratio and the F/P ratio, were determined (Table 3). The highest W/P value (3.90)
was found in the WAS × PMS experimental group and the lowest (3.76) in the
groups of F1 and F2 rams with 50 and 75% BF blood (P > 0.05). In the other
experimental groups, this trait ranged from 3.77 in PMS to 3.86 in WAS.
The lowest F/P ratio (0.09) was found in three groups (PMS, BF and BF ×
PMS), although the differences in relation to all the other groups were not
significant (Table 3).
The lowest pH, measured 24 h postmortem, was characteristic of the meat of
WAS rams (5.54), followed by BF × PMS (5.55) and PMS (5.57) rams. These
measurements in the above three groups were significantly lower than those
obtained in all the other rams. The highest pH (5.69) was measured in the meat of
BF × (BF × PMS) F2 crosses (Table 3).
The lowest score for taste (4.09 points) was awarded to the meat of WAS rams.
This value was highly significantly lower than the values obtained in all the other
experimental groups (P=0.01). The best score for taste was awarded to the meat of
WAS × (WAS × PMS) (4.58 pts), followed by BF × (BF × PMS) (4.54 pts),
WAS × PMS (4.53 pts), BF × PMS (4.49 pts) and BF (4.44 pts) crossbreds. The
differences between these groups were not significant. The taste intensity of PMS
meat (4.31 pts) was significantly lower than that found in the meat of WAS × PMS,
WAS × (WAS × PMS) and BF × (BF × PMS) crosses (Table 3).
The results of meat taste evaluation followed a similar pattern to the scores
given for taste intensity. The meat of WAS rams was given a significantly lower
score for taste (4.08 pts) compared to the score obtained in all the other
experimental groups (P ≤ 0.01). The most favourable level of this trait was obtained
by WAS × (WAS × PMS) (4.59 pts), BF × (BF × PMS) (4.54 pts) and WAS × PMS
rams (4.53 pts). The differences in this trait between the above three experimental
groups and WAS and PMS animals were statistically significant (Table 3).
Discussion
PMS rams were characterized by the lowest weight gains obtained throughout
the experiment. In a study by Roborzyński and Petkowski (1989), PMS lambs had
an inadequate growth rate, which averaged 87 – 137 g/day. BF rams had more
dynamic weight gains than F1 and R1 crosses with 50 and 75% BF blood, and their
weight gains during the period 2 – 200 days of age were lower than those
of intensively fattened lambs. This applies to both purebred BF lambs —
230 – 305 g/day (Ringdorfer, 1990; Niżnikowski and Ringdorfer, 1994; Ringdorfer,
1997) and F1 crosses of BF ewes with WAS rams — 322 – 405 g/day (Ringdorfer,
1998). WAS rams were characterized by the highest weight gains, which averaged
211 g/day, and were higher than the 199 g/day reported by Marguerat et al. (1995)
and Schneeberger (1997). In accordance with the findings of Ciuruś et al. (1980)
and Roborzyński (1984), a beneficial effect of crossing PMS with meat breeds on
145
daily weight gains was found. The mean weight gains of crossbred rams were 12%
(F1 — BF × PMS) to 37% [WAS × (WAS × PMS)] higher compared to the weight
gains of PMS rams. The weight gains for the whole rearing period were higher in F1
crosses than in PMS lambs by 5-28% (Roborzyński, 1984; Roborzyński and
Petkowski, 1989; Kieć, 1997).
The lowest empty body weight (30.9 kg) was found in PMS rams. The results
obtained by other authors concerning the body weight of PMS rams, reared to
180-210 days of age and fed in a similar way as in the present experiment, were
lower and did not exceed 27 kg (Ciuruś and Drożdż, 1988). Roborzyński and
Petkowski (1989) reported a higher pre-slaughter weight of PMS animals (34.0 kg).
Compared to PMS rams, the empty body weight of crossbred rams was 9.1 and
15.2% higher in F1 and R1 rams with BF breeding and 26.2 and 33.0% higher in F1
and R1 crosses sired by WAS fathers. Drożdż and Ciuruś (1996) showed that
compared to the maternal breed, pasture-reared F1 crosses (PMS ewes × Friesian
rams) experienced a 20-25% increase in body weight. Crossbred rams of the R1
generation — WAS × (WAS × PMS) — achieved the highest body weight of all the
crossbred groups. The results of an experiment by Roborzyński et al. (2000)
confirm the special predisposition of crossbreds sired by RAS rams to pasture
fattening.
The cold carcass weight of PMS rams was lower than that reported by
Roborzyński and Petkowski (1989), who reared lambs on pasture to a higher final
body weight of 34 – 35 kg. The weight of the heaviest carcasses from WAS animals
was 68.3% higher than the analogous values obtained in PMS carcasses.
The dressing percentage (38.8-45.0%) was close to the analogous results for
pasture fattening reported by Ciuruś et al. (1995) and Roborzyński et al. (2000). The
lowest dressing percentage was characteristic of PMS rams (38.8%). The value
reported by Ciuruś and Drożdż (1988) was slightly lower (37.8%).
Previous efforts to improve meatiness traits, in particular to increase the
dressing percentage in PMS animals through the intensification of feeding, have not
been successful. The dressing percentage of intensively fattened PMS rams reported
by Kieć (1997), 38.2%, is similar to our results obtained for pasture fattening.
A highly significantly higher dressing percentage, compared to that of PMS
animals, was obtained in BF rams (42.7%), and this value was similar to the results
obtained by Roborzyński et al. (2000). According to foreign language sources
(Ringdorfer, 1988; Niżnikowski and Ringdorfer, 1994; Ringdorfer, 1995; Ringdorfer, 1997), the dressing percentage of BF rams raised in Austria ranges from 45.1 to
49.5% and is considerably higher than the Polish results. These differences can be
attributed to the use of different management systems for BF sheep. In Austria, BF
lambs are fattened intensively to 22 – 42 kg final body weight using a feed rich in
protein and energy (Ringdorfer, 1997).
As in the study by Roborzyński et al. (2000), WAS meat rams were characterized by the highest dressing percentage (45.0%) of all the experimental groups.
Our results were 1.4 percentage units lower than the analogous results obtained
during pasture fattening in Switzerland (Marguerat et al., 1995), but higher than the
146
P. Paraponiak
results obtained in Poland by Roborzyński et al. (2000). In all the groups of
crossbred rams, the dressing percentage was significantly higher than in PMS rams.
In terms of this trait, R1 crossbred rams were superior to F1 crossbreds derived from
the same paternal breeds. This relationship may be linked to the increasing and
beneficial effect of paternal breeds in the second generation. Contrary to the
findings of Roborzyński et al. (2000), no significant differences were found in the
above-mentioned trait between F1 rams originating from fathers of different breeds.
One of the most important parameters of carcass meatiness is the weight and
percentage of valuable cuts with the highest commercial and eating value. We
found that the crossbreeding scheme used had an effect on the valuable cut content
of carcass, with results similar to the findings of Roborzyński (1984).
The poor meatiness of PMS animals in comparison with the other experimental
breeds and crossbreds is confirmed by the fact that these animals had the lowest
proportion of valuable cuts (55.14%). The percentage of valuable cuts in the
half-carcasses of F1 and F2 crossbreds with the BF paternal component (57.53 and
57.95%, respectively) was significantly higher than the result obtained for the
half-carcasses of PMS animals and did not show significant differences in relation
to purebred BF rams (57.31%). The results we obtained for BF rams are consistent
with those reported by Roborzyński et al. (2000) — 57.68%.
The half-carcasses of WAS meat rams were characterized by the highest
proportion of valuable cuts (58.49%). This result is similar to that reported by
Roborzyński et al. (2000) — 58.52%.
A very important indicator of carcass quality is the tissue composition of
carcass, in particular that of the leg. The more muscle tissue it contains in relation to
adipose and bone tissue, the more valuable a carcass cut is.
The fat content of leg in all the experimental groups is considered desirable. The
percentage of muscle tissue in WAS rams was inversely proportional to the fat
content, and these animals achieved the highest value (73.64%), with a statistically
significant difference compared to all of the other experimental groups. These
results are consistent with the results of a study by Roborzyński et al. (2000), in
which WAS rams had a significantly higher meat content (71.31%) and a low fat
content (10.24%) compared to the other animals.
The W/P ratio in meat ranged from 3.76 to 3.90 and was close to the results
obtained by Ke˛dzior (1995). The Feder number in adult slaughter animals ranges
from 3.3 to 3.9. The level of this trait in the present study leads us to conclude that
the analysed meat was derived from animals of normal somatic maturity.
The F/P ratio ranged from 0.09 to 0.11 and the value of this ratio was reflected
in the stable fat and protein content of the muscles of experimental rams. Covington
et al. (1970) showed that a lower degree of the physiological maturity of muscles is
associated with a high water content and low amount of intramuscular fat in the
muscles.
The mean pH values of the analysed meat ranged from 5.54 in the group of
purebred WAS rams to 5.69 in the group of BF × (BF × PMS) crossbreds, and
despite statistically significant differences these values were typical of meat with
147
normal properties (5.3 – 5.8) (Hofmann, 1987). These results are similar to those
obtained by other authors (Freudenreich et al., 1985; Ke˛dzior, 1995).
High scores for taste intensity and quality were characteristic of the meat of F1
rams — WAS × PMS and F2 rams — WAS × (WAS × PMS) and BF × (BF × PMS).
The taste of WAS ram meat was given lower scores compared to the analogous
results determined for the meat samples of rams from the other groups. The results
of Roborzyński et al. (2000) have confirmed the poorer sensory value of the meat of
WAS lambs.
It is concluded that crossbred rams are characterized by more favourable
fattening and slaughter traits compared to purebred PMS rams. The desirable level
achieved for the selected chemical and sensory parameters of the meat obtained
from the rams of all the experimental groups, in particular the crossbreds, is
evidence of the high eating quality of the meat.
References
C i u r u ś J., D r o ż d ż A., K r u p i ń s k i J. (1980). Przydatność do tuczu i wartość rzeźna mieszańców F1
z krzyżowania towarowego polskiej owcy górskiej z trykami ras mie˛snych. Rocz. Nauk. Zoot., 7, 2:
125 – 135.
C i u r u ś J., D r o ż d ż A. (1988). Porównanie wartości rzeźnej jagnia˛t polskiej owcy górskiej i jej
mieszańców trójrasowych. Rocz. Nauk. Zoot., 15, 1: 69 – 78.
C i u r u ś J., D r o ż d ż A., K o w a l s k i Z.M. (1995). Próby zwie˛kszenia przyrostów masy ciała jagnia˛t
w odchowie i tuczu pastwiskowym do 6. miesia˛ca życia. Rocz. Nauk. Zoot., 22, 1: 247 – 258.
C o v i n g t o n R.C., T u m a H.J., G r a n t D.L., D a y t o n A.D. (1970). Various chemical and histological
characteristices of beef muscle as related to tenderness. J. Anim. Sci., 30: 191.
D r o ż d ż A., C i u r u ś J. (1996). Wartość rzeźna jagnia˛t mlecznych owiec górskich i ich mieszańców.
Rocz. Nauk. Zoot., 23, 2: 43 – 55.
F r e u d e n r e i c h P., W o l l n y C., W a s s m u t h R. (1985). Untersuchungen an Lämmern verschiedener
Rassen und Kreuzungen. II. Chemische, physikalische und sensorische Ergebnisse. Mitteilungsblatt
der Bundesanstalt für Fleischforschung. Kulmbach, 90: 6694 – 6699.
H o f m a n n K. (1987). Der pH — Wert. Ein Qualitätskriterium für Fleisch. Fleischwirtschaft, 67, 5:
557 – 562.
K e˛ d z i o r W. (1995). Towaroznawcza charakterystyka jakości mie˛sa jagnia˛t. Zesz. Nauk. AE Krak.,
Monogr., 123.
K i e ć W. (1997). Badania nad wykorzystaniem owiec górskich do produkcji jagnia˛t rzeźnych. Mat.
mie˛dz. konf. nauk.: Rola owczarstwa górskiego w realizacji krajowych programów hodowlanych
dla owiec. IZ, Balice, 14.11.1997, ss. 33 – 40.
M a r g u e r a t C., L u c h i n g e r R., L e u e n b e r g e r H., K u n z i N. (1995). Lammfleischproduktion auf
der Weide. Die Grune, 2: 22 – 23.
N i ż n i k o w s k i R., R i n g d o r f e r F. (1994). Lammfleischproduktion im Alpenraum mit Bergschafen
und deren Kreuzungen mit Merino und Schwarzköpfigem Fleischschaf. Züchtungskunde, 66, 1:
73 – 81.
O k u l a r c z y k S. (2000). Ekonomiczne i rynkowe prognozy produkcji owczarskiej i koziej w Polsce.
Zesz. Nauk. AR Wroc., 399: 49 – 56.
R i n g d o r f e r F. (1988). Ausschlachtung und Verwertung von Schafen. Der fortschrittliche Landwirt:
Zeitgemasse Schafhaltung, 12: 5.
R i n g d o r f e r F. (1990). Gebrautskreuzungen mit Fleischrassen beim Schaf. Bericht über die 17.
Tierzuchttagung: Aktuelle Fragen der Milchvieh- und Schafhaltung, BAL Gumpenstein,
10.05.1990, ss. 1 – 9.
148
P. Paraponiak
R i n g d o r f e r F. (1995). Der Einfluss des Absetztermines auf die Zwischenlammzeit sowie auf die
Qualität der Mastlämmer beim Bergschaf. Bundesanstalt für alpenländische Landwirtschaft,
Referat für Schafe, Ziegen und Kleintiere, A-8952 Irdning.
R i n g d o r f e r F. (1997). Das Österreichische Bergschaf und seine wirtschaftliche Bedeutung. Mat.
konf. mie˛dz:. Rola owczarstwa górskiego w realizacji krajowych programów hodowlanych dla
owiec. IZ, Balice, 14.11.1997, ss. 53 – 60.
R i n g d o r f e r F. (1998). Qualitätsverbesserung mit WAS — Widder. Schafe aktuell, 3: 10 – 11.
R o b o r z y ń s k i M. (1984). Użytkowość mie˛sna jagnia˛t mieszańców F1, pochodza˛cych z krzyżowania
maciorek polskiej owcy górskiej z trykami różnych ras. Acta Agr. Silv., Ser. Zoot., 23: 53 – 65.
R o b o r z y ń s k i M., P e t k o w s k i J. (1989). Przydatność polskich owiec nizinnych, długowełnistych
i górskich utrzymywanych w warunkach gór i pogórza do produkcji jagnie˛ciny. Biul. Inf. IZ, 27,
5 – 6: 65 – 77.
R o b o r z y ń s k i M., K i e ć W., K e˛ d z i o r W., K n a p i k J., K r u p i ń s k i J. (2000). Wyniki odchowu
pastwiskowego, wartość rzeźna oraz jakość mie˛sa jagnia˛t mieszańców polskiej owcy górskiej
z trykami ras alpejskich. Rocz. Nauk. Zoot., Supl., 8: 98 – 103.
S c h n e e b e r g e r M. (1997). Das Weisse Alpenschaf — Hauptrasse für die Lammfleischerzeugung in
der Schweiz. Mat. mie˛dz. konf. nauk.: Rola owczarstwa górskiego w realizacji krajowych
programów hodowlanych dla owiec. IZ, Balice, 14.11.1997, ss. 61 – 76.
PAWEŁ PARAPONIAK
Wpływ krzyżowania mie˛dzyrasowego na wyniki odchowu pastwiskowego jagnia˛t
oraz właściwości chemiczne i sensoryczne surowca rzeźnego
STRESZCZENIE
Celem podje˛tych badań było określenie wpływu krzyżowania maciorek polskiej owcy górskiej
z trykami ras Bergschaf i Weisse Alpenschaf na cechy tuczne i rzeźne mieszańców oraz na wybrane
właściwości chemiczne i sensoryczne pozyskanego od nich mie˛sa.
Materiał doświadczalny stanowiło 140 tryczków podzielonych na siedem grup: polska owca górska
(pog), Bergschaf (BF), Weisse Alpenschaf (WAS), BF × pog, WAS × pog, BF × (BF × pog)
i WAS × (WAS × pog) po 20 szt. tryczków w każdej grupie (10 szt. — doświadczenie,
10 szt. — powtórzenie). Od około 4. miesia˛ca życia jagnie˛ta pasiono systemem kwaterowym,
dokarmiaja˛c śruta˛ je˛czmienno-pszenna˛.
Obliczono średnie przyrosty dobowe tryczków od 2. do 200. dnia życia. Określono wydajność
rzeźna˛ zimna˛ i oszacowano mase˛ oraz udział wyre˛bów wartościowych w prawej półtuszy. Przeprowadzono szczegółowa˛ dysekcje˛ udźca. Zbadano wybrane właściwości chemiczne i sensoryczne
jagnie˛ciny.
Tryczki WAS charakteryzowały sie˛ najwyższymi przyrostami masy ciała (211 g/dobe˛) oraz
najwyższa˛ masa˛ ciała po głodzeniu i masa˛ tuszy schłodzonej. Średnie przyrosty masy ciała tryczków
mieszańców były o 12 – 37% wyższe niż przyrosty uzyskane przez jagnie˛ta pog. Wydajność rzeźna
wszystkich mieszańców była wyższa od ustalonej dla tryczków pog (P ≤ 0,01). Mie˛so pozyskane od
wszystkich tryczków doświadczalnych odznaczało sie˛ poża˛danym stosunkiem wody do białka, tłuszczu
do białka i wartościa˛ pH. Na podstawie wyników oceny sensorycznej wykazano wysokie walory
smakowe mie˛sa jagnia˛t mieszańców pokolenia F1 i R1. Niżej pod tym wzgle˛dem sklasyfikowano mie˛so
czysto rasowych tryczków WAS.
EFFECTS OF YEAR-ROUND NESTBOX AVAILABILITY
AND TEMPERAMENT ON WELFARE AND PRODUCTION
PERFORMANCE IN BLUE FOXES (ALOPEX LAGOPUS)
H a n n u T . K o r h o n e n 1, L a u r i J a u h i a i n e n 2, T e p p o R e k i l ä 1
1
MTT Agrifood Research Finland
Animal Production Research, Fur Animals FIN-69100 Kannus, Finland
2
Data and Information Services, FIN-31600 Jokioinen, Finland
Abstract
This study was designed to evaluate the long-term effects of nestbox and temperament traits on
welfare and performance in juvenile and adult blue foxes (Alopex lagopus). Fearful and confident
animals were raised with and without nestboxes during the growing and breeding seasons.
Experimental groups formed at weaning were: 1) confident, no nestbox available; 2) confident,
with a year-round nestbox; 3) fearful, no nestbox available; and 4) fearful, with a year-round
nestbox. Each group was comprised of 60 females housed singly. The results showed that foxes
without nestboxes grew significantly (P = 0.01) better than those with nestboxes. Temperament had
no effect on final body weights. Confident foxes were more explorative (P < 0.01) than fearful ones,
and foxes without nestboxes were more explorative than those with nestboxes (P = 0.06). Capture
tests revealed that access to a permanent nestbox significantly (P < 0.001) increased fearfulness in
the long run. Foxes with nestboxes more frequently showed an escape reaction to human presence
than foxes without nestboxes. No significant differences were detected between temperament or
nestbox availability groups in the frequency of stereotypical behaviours, the cortisol:creatinine
ratio or reproductive success. The fur coat of foxes with nestboxes was dirty, which significantly
(P < 0.001) reduced the quality of fur. It can be concluded that year-round access to nestboxes
increases fearfulness in farmed blue foxes. Nestboxes may also otherwise compromise animal
welfare.
Key words: blue fox, year-round shelter, welfare
Central tools in the enhancement of animal welfare are the proper construction
and enrichments of housing conditions where farmed animals are kept (Chamove,
1989; Newberry, 1995). These modifications should be done so that they allow the
performance of species-specific behaviour and provide essential needs as well as
possible. Previously, farmed blue foxes (Alopex lagopus) were kept in a barren
wire-mesh environment, but now their cages are furnished with a netting platform
and an activity object made of wood (European Convention, 1999; Korhonen and
150
H.T. Korhonen et al.
Niemelä, 2000; Korhonen et al., 2002 a). Although these enrichments provide foxes
with an opportunity to perform behaviours such as observing, resting, playing and
chewing, they do not necessarily satisfy all of their behavioural needs. Recent
European welfare stipulations (European Convention, 1999; Hovland and Bakken,
2000) have put pressures on farmers to also enrich housing cages with year-round
nestboxes. It is assumed that foxes need a constant place to hide or seek shelter
from humans and their surroundings. Until now, boxes have only been provided for
breeding vixens as a nest or a place to deliver and nurse the cubs from April
onwards. Breeding boxes have been taken away before weaning in July.
The elimination of fear in farmed foxes is one of the main objectives in
European animal welfare legislation (European Convention, 1999). This is because
fear can be a distinct state of suffering and a significant damaging stressor (Jones,
1997; Korhonen et al., 2002 b, c). However, the provision of year-round nestboxes
may run counter to European legislative goals as they might have detrimental
effects on fox temperament (Hovland and Bakken, 2000). It seems that constant
access to cover from man allows animals the opportunity to develop a behavioural
strategy of hiding. Thus, instead of domestication it would lead to a process of
feralization, where a domesticated temperament evolves back into a wild one,
producing individuals with a fearful reaction towards humans (Korhonen et al.,
2000; Ahola, 2002). If this prediction holds true, it may have further consequences
due to the close link between fear and welfare. For example, decreased fear in blue
foxes is associated with a decreased stress response (Rekilä et al., 1997), and
positive behavioural and physiological changes have been found in silver foxes
selected for tameness or against fear (Koleshnikova et al., 1985; Osadchuk, 1992).
Moreover, fear has a negative influence on reproductive physiology. Examples
include the finding that confident silver and blue foxes tend to have a better
reproductive performance than fearful ones (Braastad, 1988; Kristensen, 1989,
Bakken 1994) or non-selected ones (Hansen, 1998; Kenttämies et al., 1997;
Jeppesen and Pedersen, 1998). Temperament appears to affect reproduction in
several ways. For example, fearful vixens may be difficult to mate, or sensitive to
abortion, or they may lose their cubs because of pronounced behavioural disorders
or poor maternal ability (Ilukha et al., 1997; Korhonen and Niemelä, 1996).
Year-round nestboxes could also pose other problems as foxes may use them as
a place to defecate. Dirty boxes may increase hygiene problems and cause
a deterioration in fur quality (Hovland and Bakken, 2000; Korhonen et al.,
2003 a, b). A dirty fur coat may also be problematic because it has a lower
insulation capacity, exposing the animal to excessive cold during the autumn and
winter (Korhonen, 1987; Korhonen et al., 2001, 2003 a). Detrimental effects of
dirty nestboxes would be more pronounced in fearful than confident animals
because the former are expected to frequently hide and seek cover in the nestbox.
Thus, whether there is an actual causal relationship between permanent access
to a nestbox and welfare, or between nestbox availability, temperament and
productive performance, is a matter of real concern and requires further investigations.
Effects of nestbox availability and temperament on blue fox welfare
151
The aim of the present study was to examine how year-round access to
nestboxes and temperament affect the welfare and production performance of
farmed blue foxes (Alopex lagopus). Animals of fearful and confident origin were
therefore housed with and without nestboxes during the growing and breeding
seasons.
Subjects and general managements
This study was carried out at the Fur Farming Research Station of MTT
Agrifood Research Finland (MTT), in western Finland (63.54oN, 23.54oE). In
mid-October 2002, 50 confident and 50 fearful primiparous vixens (Alopex
lagopus) were selected from the research farm’s breeding stock. Selection was
based on the feeding test (Rekilä et al., 1997) which was performed as follows: the
experimenter gave the tested fox a feed portion and then withdrew 50 cm from the
cage door. If the animal did not start eating within 1 min, it was considered fearful.
Foxes that dared to eat were classified as confident. Feed was withheld from the
animals for 24 h before the test.
In March 2003, vixens were artificially inseminated according to conventional
farming procedures. Cubs were born during May 2003. Until weaning, they were
housed with their mothers and littermates in conventional shed cages measuring
120 cm long × 105 cm wide × 70 cm high. At weaning on 30 July, cubs were
divided into four experimental groups: 1) confident, no nestbox available;
2) confident, with a year-round nestbox; 3) fearful, no nestbox available; and
4) fearful, with a year-round nestbox. Each group was comprised of 60 individuallyhoused females. The cage size was 120 cm long × 105 cm wide × 70 cm high for
both groups. Each cage contained a wire-mesh platform (105 cm long × 25 cm
wide) positioned 23 cm from the ceiling. A birchwood block (7 cm long × diameter
5 cm) was available as an activity and chewing objects. Nestbox groups (1 and 3) had
a permanently available wooden nestbox (40 cm wide × 70 cm long × 40 cm high).
The foxes were placed in the shed with one animal from groups 1, 2, 3 and
4 being placed after each other. Thus, every second cage contained a nestbox.
Freshly mixed fox feed was supplied twice a day by a commercial feeding
machine. The daily feed ration was the same for each group, ranging from
a minimum of 500 g (July) to a maximum of 1000 g (October) per animal daily.
Feed was made by the local feed kitchen (Kannus Minkinrehu Ltd), with the main
ingredients consisting of slaughterhouse offal, fish, fish offal and cereals, in
accordance with the standard Finnish recommendations. Fresh water was available
ad libitum from automatic watering devices. The health of the experimental animals
was visually checked daily.
Behavioural tests
Capture test: foxes were caught by using neck tongs. The reaction to capture
was classified as follows: 1=confident, accepting capture; 2=fearful, trying to
152
escape; and 3=non-reactive, passive (Harri et al., 1995). Evaluations were made
each time by the same person. After capture, body weights were measured on
a Vaakakoskinen AD-4326A balance (accuracy 20 g).
Table 1. Behaviour categories and short description of behavioural elements
Category
In locomotion
Jump onto platform
On platform
Jump off platform
Escape
Passive
Inside nestbox
On nestbox roof
Description
walking, moving on the cage floor
jump from the cage floor to the platform
sleeping, lying or sitting on the platform
jump from platform to the cage floor
escape to the nestbox, nestbox roof, platform or backwards
on cage floor
non-reactive on the cage floor, motionless
staying inside the box; animal cannot be seen
sleeping, lying or sitting on the nestbox roof
Walking test: the animals’ position in the cage and reaction towards man were
evaluated here by scanning observations. These took place from Monday to Friday,
i.e. during 5 days between August-November, and January-March. The total
number of scannings was 40 and 30 per fox during the growing and breeding
seasons, respectively. They were performed twice each day at 09.30 and 14.30. The
experimenter walked quietly and slowly along the feed alley and recorded the
animal’s location and behaviour of each animal in the cage when the experimenter
was at a distance of 1 m from the cage (Harri et al., 1998; Korhonen et al., 2001).
During each scanning observation, half of all cages were approached from the right
side and the other half from the left side. The behavioural categories recorded are
presented in Table 1. Stereotypical behaviour was evaluated on the basis of the
categorization of Korhonen et al. (2001). However, different forms of stereotypical
behaviour were not recorded separately.
Ball test: this test was used to identify foxes showing exploratory behaviour
towards novel objects. A baseball (diameter 7 cm) was placed in the cage, close to
the door opening, after which the cage door was closed. The number of animals that
made contact with the ball during 1 min were recorded. The ball was cleaned with
paper after each test (Rouvinen et al., 1999).
Feeding test: the reaction (confident vs fearful) of foxes in the presence of man
was evaluated here. The test began with the experimenter giving the fox a feed
portion and then withdrawing 50 cm from the cage door. If the fox did not begin
eating it within 1 min, it was considered fearful (Rekilä et al., 1997). Feed was
withheld from the animals for 24 h before the test. The feeding test was carried out
on the same days as the ball test.
Urine and blood analyses
Wooden trays were placed under the cages to collect samples of urine over 24 h.
The trays were covered with gauze to separate faeces from urine. Urine was
153
collected in August and October 2003, and in February and July 2004. After each
24-h collection period, the urine samples were weighed and bottled, and stored at
–20oC. The collection trays were carefully cleaned before each sampling day.
Samples were delivered frozen for analysis at the University of Kuopio (cortisol)
and Kuopio University Hospital (creatinine). The urine cortisol concentration
(nmol/l) was determined using a competitive immunoassay technique (Coat-ACount Cortisol Assay by Diagnostic Products Corporation, Los Angeles, CA). The
urine creatinine concentration was determined by kinetic Jaffe’s reaction (Lasley
and Kirkpatrick, 1991). Urinary cortisol was finally expressed as the cortisol:creatinine ratio to correct for variation in the dilution of urine.
For the blood analysis, a fox was caught with neck tongs, and a blood sample
was drawn from the cephalic vein into 5-ml sampling tubes. All samples were taken
within 2.5 min of the start of capture. Blood stabilized with K-EDTA was used for
the determination of haemoglobin (Hb), the erythrocyte count (RBC), whole blood
cell count (WBC), and haematocrit (HCT) by a Cell-Dyn 400 counter (SequoiaTurner Corp., USA). Blood samples were immediately sent to the Equine Research
Station at Ypäjä (MTT), where analysis was carried out during the next day.
Conditions of nestboxes and furs
The dirtiness of nestboxes was visually evaluated in August and October 2003,
and March 2004 on a scale of 1 – 4 where: 1=clean, 2=slightly dirty, 3=moderately
dirty and 4=very dirty (Korhonen et al., 2003 b). Evaluations were made by the
same person. The dirtiness of furs was evaluated in December 2003 on a scale of
1 – 4 where: 1=clean, 2=slightly dirty (few dirty spots), 3=moderately dirty (several
dirty spots) and 4=very dirty (most of fur covered by dirty spots).
Matings and whelpings
Before matings (20 February 2004), the occurrence of urine inflammation in
vixens was evaluated. After visual inspection, the pH, and levels of protein and
glucose in the urine were measured using Bayer urine test sticks (Bayer Diagnostics
Ltd, Bridgend, UK). The test is based on a colour reaction, providing a rough
estimate of the occurrence of inflammation in the urinary tract. Ten vixens from
each nestbox group were tested.
Breedings started on 25 March and lasted until 1 May, 2004. The animals were
artificially inseminated on the second day after the peak in vaginal electrical
resistance. Re-mating took place on the following day. Three weeks before
whelping, all vixens were provided new breeding nestboxes (40 cm wide × 70 cm
long × 40 cm high) made of wood. The number of cubs was counted on the first day
after parturition, and at 3 weeks and 7 weeks postpartum.
Statistical methods
The experimental design was the completely randomized design and it was
taken into account in each statistical analysis. Body weight, the logarithm of the
cortisol:creatinine ratio, and different variables measured from blood samples (e.g.
Hb, HCT, RBC, and WBC) were normally distributed. Body weight and the
cortisol:creatinine ratio were measured several times during the study from the
same animal. However, both variables were analysed in two periods: the growing
154
season and the breeding season, with more than one measurement time in each
periods. Repeated measurements from the same animal were correlated and the
correlation was taken into account in a standard repeated measurement analysis of
variance model (Littell et al., 1996). An unstructured covariance structure for
repeated measurements was selected in each analysis based on Akaike’s Information Criteria (Wolfinger, 1996). Simple one-way ANOVA was used to analyse
the blood sample data. The assumptions of both models were checked by graphical
methods: a box-plot for normality of errors; and plots of residuals for
constancy of error variance. Analyses were performed using the SAS system
for Windows, release 8.2 and the MIXED procedure (SAS, 1999).
All categories in the walking test, the number of cubs per vixens, the feeding
test, the ball test, capture reaction, fur quality, and dirtiness of fur and the nestbox
were measured on at least an ordinal scale. These variables were therefore analysed
using Kruskall-Wallis ANOVA or the chi-square test. Statistical comparisons for
the two main factors, temperament and nestbox, were performed with the SAS/IML
program developed by Berry (1995) using Kruskall-Wallis ANOVA. The chisquare test was performed using the SAS/FREQ-procedure and the two main factors
were tested separately.
Results
Growing season
Initial body weights were similar in each group (Table 2). Furthermore, no
significant differences were found in final body weights between animals of
confident and fearful temperament (P = 0.12). However, the nestbox-by-time
interaction was significant (P = 0.02). At the final weighing (Nov 17), foxes without
nestboxes were significantly heavier than those having nestboxes (10.7 vs 10.4 kg;
P = 0.01).
Table 2. Comparison of measured physiological, behavioural and fur variables during the growing
season 2003. P1: between the four groups, P2: effect of temperament, P3: effect of nestbox
Variable
Confident
Confident
box
Fearful
Fearful
box
P1
P2
P3
1
2
3
4
5
6
7
8
2.8
8.4
10.6
2.8
8.2
10.2
2.7
8.4
10.8
2.7
8.3
10.5
ns
ns
0.02
ns
ns
ns
ns
ns
0.01
22
53
10
40
13
35
13
25
ns
0.01
ns
< 0.01
ns
0.06
70
43
90
43
85
42
70
22
< 0.01
0.04
ns
0.06
ns
ns
Body weight (kg):
July 22
Sept 30
Nov 17
Contact with ball (%)a
July 24
Nov 18
Come to eat (%)b
July 25
Nov 18
155
Table 2 — contd.
1
Capture on July 22 (%)
confident
fearful
non-reactive
Capture on Nov 17 (%)
confident
fearful
non-reactive
Cortisol:creatinine
Aug 19
Oct 23
Red blood cells (109 cells 1– 1)
Dec 3
White blood cells (109 cells l–1)
Dec 3
Haematocrit (%)
Dec 3
Haemoglobin (g/l)
Dec 3
Clean fur coat (%)
Dec 11
Excellent fur quality (%)
Dec 11
a
b
2
3
4
5
6
7
8
12
8
80
13
8
78
0
30
70
0
32
68
< 0.001 < 0.001
68
7
25
57
22
22
49
10
41
52
27
22
< 0.01
ns
< 0.005
ns
9.6
9.5
7.6
8.3
7.6
9.2
8.2
8.3
ns
ns
ns
ns
ns
ns
8.8
8.9
8.9
8.9
ns
ns
ns
7.4
9.2
8.0
8.4
ns
ns
0.06
52
53
53
52
ns
ns
ns
164
163
165
163
ns
ns
ns
100
37
100
58
< 0.001
0.08
< 0.001
32
8
27
ns
ns
< 0.001
10
ball test.
feeding test.
Initially, the ball test (July 24) did not reveal any substantial differences in
explorative behaviour between the groups (Table 2). On November 18, however,
confident animals were more explorative than fearful ones (47 vs 30% of animals
touched the ball, P < 0.01). Furthermore, foxes without nestboxes were more explorative than those with nestboxes (44 vs 33% of animals touched the ball, P = 0.06).
Capture tests revealed significant differences between the groups (P<0.001;
Table 2). On July 22, foxes grouped initially as confident showed a more confident
temperament at capture than those initially grouped as fearful (13 vs 0% of animals,
P < 0.001). The same tendency was found on November 17 (63 vs 50% of animals).
Correspondingly, foxes initially grouped as fearful had more fearful temperament
on July 22 than those grouped as confident (31 vs 8% of animals, P<0.001). On
November 17, however, this difference was no longer significant (31 vs 23% of
animals). In the capture test in July, 74% of all animals were non-reactive
(passive), while in November 59% and 11% of these animals were changed to
behaving confidently or fearfully, respectively, and the remaining animals (30%)
were non-reactive in both tests. The change was not similar in all treatment groups
(P < 0.001). However, no statistically significant difference was found between the
two temperament groups (P = 0.21). The difference between animals with and
156
without nestbox was clear (P < 0.005). Only 3% of animals without a nestbox
changed from the non-reactive class to the fearful class, while the change for
animals with a nestbox was 18%. The corresponding percentages were 5% and 24%
(P < 0.01) when animals that were non-reactive in both tests were excluded from the
statistical analysis.
Table 3. Behavioural reactions during the walking tests (% of observations). Recorded during the
growing season 2003. P1: between the four groups, P2: effect of temperament, P3: effect of nestbox
Variable
In locomotion
July 28 – Aug 1
Sept 1 – 9
Oct 6 – 10
Nov 10 – 14
Jump onto platform
July 28 – Aug 1
Sept 1 – 9
Oct 6 – 10
Nov 10 – 14
On platform
July 28 – Aug 1
Sept 1 – 9
Oct 6 – 10
Nov 10 – 14
Jump off platform
July 28 – Aug 1
Sept 1 – 9
Oct 6 – 10
Nov 10 – 14
Escape
July 28 – Aug 1
Sept 1 – 9
Oct 6 – 10
Nov 10 – 14
Passive
July 28 – Aug 1
Sept 1 – 9
Oct 6 – 10
Nov 10 – 14
Inside nestbox
July 28 – Aug 1
Sept 1 – 9
Oct 6 – 10
Nov 10 – 14
On nestbox roof
July 28 – Aug 1
Sept 1 – 9
Oct 6 – 10
Nov 10 – 14
Confident
Confident
box
Fearful
Fearful
box
P1
P2
P3
10.5
15.5
21.5
35.6
5.0
11.2
18.2
29.8
11.6
17.5
15.9
29.3
5.4
12.2
15.6
31.8
< 0.001
ns
ns
0.07
ns
ns
ns
ns
< 0.001
0.03
ns
ns
0.3
4.5
5.2
8.8
0.2
1.2
1.5
2.9
0.2
1.8
2.5
5.7
0
0.9
3.7
2.3
ns
0.03
ns
0.09
ns
ns
ns
ns
ns
0.02
ns
ns
42.2
52.7
50.3
37.1
30.8
35.0
25.2
10.6
42.3
54.1
62.1
46.7
35.6
42.2
33.1
17.2
< 0.01
< 0.001
< 0.001
< 0.001
ns
ns
0.05
ns
< 0.005
< 0.001
< 0.001
< 0.001
0.2
3.8
4.0
3.8
1.2
5.2
2.3
3.3
0.2
2.1
1.6
2.9
1.2
3.2
1.9
2.8
0.09
0.05
ns
ns
ns
0.04
ns
ns
0.02
0.07
ns
ns
0.3
4.5
5.2
8.8
2.2
6.2
5.5
8.1
0.2
3.1
2.8
6.4
1.9
4.1
6.3
8.5
0.03
0.08
0.02
ns
ns
ns
ns
ns
< 0.005
0.04
< 0.005
ns
46.2
22.2
18.3
13.3
30.2
21.5
26.5
19.0
44.3
22.1
16.4
12.5
34.1
19.3
21.4
14.0
< 0.01
ns
0.02
ns
ns
ns
0.06
ns
< 0.001
ns
0.02
ns
10.8
7.5
7.7
17.9
8.8
5.2
8.1
15.9
ns
ns
ns
ns
ns
ns
ns
ns
15.7
6.2
8.2
2.9
11.2
5.8
5.4
1.9
ns
ns
ns
ns
ns
ns
ns
ns
157
No significant differences were found in cortisol:creatinine ratio between the
groups (Table 2). White blood cell counts were significantly higher (P = 0.06) in
foxes with nestboxes compared to those without nestboxes. No other differences
were found in blood picture parameters. All furs in animals without nestboxes were
clean whereas the furs of foxes having nestboxes were mostly very dirty.
Furthermore, fur quality was significantly poorer (P < 0.001) in animals with
nestboxes (Table 2).
Walking tests revealed several significant differences between the groups
(Table 3). Temperament had some effects on locomotion, i.e. foxes having
nestboxes were less frequently observed moving on the cage floor both in
July – August (11.1 vs 5.2% of animals in locomotion; P < 0.001) and September
(16.5 vs 11.7% of animals, P = 0.03). Foxes without nestboxes used platforms
significantly more frequently than foxes having nestboxes during each walking test
period (Table 3). Temperament affected platform use only during September when
fearful foxes were more frequently observed on the platform than confident ones
(47.8 vs 37.8% of animals, P = 0.05). Foxes with nestboxes showed more frequent
escape reaction compared to those without nestboxes. In confident foxes in August,
September, October and November the escape reaction of moving into the nestbox
comprised 1.7, 4.0, 2.3 and 2.3%, respectively, of all escapes. The corresponding
percentages in fearful foxes were 1.7, 2.5, 2.0 and 3.2. No significant differences
were recorded in the relative frequency of escaping to the nestbox between fearful
and confident foxes. Temperament did not affect the use of nestbox or nestbox roof.
Initially, foxes without nestboxes were more passive in the walking test than foxes
with nestboxes. In September, however, no differences were found, while in
October foxes with nestboxes were more passive but differences did not exist in
November (Table 3). No stereotypical behaviours were observed in August,
September and October at all. In November, the percentages of observations in
which stereotypical behaviours were recorded were 1.3, 0.8, 0.2 and 0 in Groups
1 – 4, respectively.
Breeding season
Significant differences were recorded in body weights between the groups
during the pre-mating period January-March (Table 4). Confident foxes were
significantly lighter than fearful ones (on January 7: 9.9 vs 10.2 kg; on February
5: 9.8 vs 9.6 kg; on March 16: 8.9 vs 8.6 kg). Furthermore, foxes without nestboxes
were significantly heavier than those with nestboxes (on January 7: 10.2 vs 9.9 kg;
on February 5: 9.9 vs 9.6 kg; on March 16: 8.9 vs 8.5 kg).
The ball test on January 22 (Table 4) showed that confident animals were more
explorative than fearful ones (66 vs 47% of animals). This difference was no longer
significant in February or March. Foxes with nestboxes were more explorative (63
vs 50% of animals) than those without nestboxes on February 18. Feeding tests did
not indicate any differences between the groups (Table 4). The capture test
demonstrated that foxes without nestboxes were more confident (77 vs 60% of
animals) than those with nestboxes. None of foxes were classified as non-reactive
on capture (Table 4). Originally (July 22), however, 74% of all animals were
158
non-reactive in the capture test. In March, 71% and 29% of these animals were
changed to behaving confidently or fearfully, respectively. The change was not
similar in all treatment groups (P < 0.005). However, no statistically significant
difference was found between the two temperament groups (P = 0.22). The
difference between animals with and without a nestbox was clear (P < 0.001). Only
16% of animals without a nestbox were changed from the non-reactive class to the
fearful class, while the percentage was 42% for animals with a nestbox.
Table 4. Comparison of measured physiological, behavioural and fur variables during the 2004 breeding
season. P1: between the four groups, P2: effect of temperament, P3: effect of nestbox
Variable
Body weight (kg):
Jan 7
Feb 5
Mar 16
Contact with ball (%)a
Jan 22
Feb 18
Mar 10
Come to eat (%)b
Jan 21
Feb 19
Mar 11
Capture on March 16 (%)
confident
fearful
non-reactive
Cortisol:creatinine
Feb 20
July 30
Clean fur coat (%)
Mar 16
Confident
10.1
9.8
8.8
Confident
box
9.7
9.4
8.4
Fearful
Fearful
box
P1
P2
P3
10.3
10.0
9.1
10.0
9.7
8.7
< 0.01
< 0.01
< 0.005
0.07
0.06
0.03
< 0.005
< 0.005
< 0.001
68
67
57
63
48
60
44
59
54
50
51
43
0.03
ns
ns
< 0.005
ns
ns
ns
0.04
ns
27
32
22
40
43
28
34
32
32
32
31
28
ns
ns
ns
ns
ns
ns
ns
ns
ns
80
20
0
55
45
0
74
26
0
66
34
0
0.02
ns
< 0.01
ns
ns
ns
ns
ns
ns
< 0.001
ns
< 0.001
6.2
2.5
77
7.4
2.5
15
5.9
2.3
83
6.4
2.6
27
Table 5. Condition (%) of nestboxes in confident and fearful nestbox groups
Scale 1 – 4, where 1 = clean, 2 = slightly dirty, 3 = dirty, 4 = very dirty
Variable
Aug 26:
Oct 2:
March 16:
confident
fearful
confident
fearful
confident
fearful
Clean
6.7
8.3
6.7
10.0
0
0
Slightly dirty
Dirty
65.0
70.0
31.7
45.0
16.9
24.1
28.3
21.7
41.7
36.7
18.6
20.7
Very dirty
0
0
20.0
8.3
64.4
55.2
159
The cortisol:creatinine ratio was the same order of magnitude in each group in
both February and July (Table 4). Evaluation of the dirtiness of the fur coat (on
March 16) showed that the percentage of foxes with a clean fur coat was
significantly higher among foxes without nestboxes than those having a nestbox
(80 vs 21% of animals) (Table 4). The temperament of the foxes did not affect the
dirtiness of nestboxes (Table 5).
Table 6. Behavioural reactions during the walking tests (% of observations) recorded during the 2004
breeding season. P1: between the four groups, P2: effect of temperament, P3: effect of nestbox
Variable
In locomotion
Jan 12 – 16
Feb 9 – 13
Mar 1 – 5
Jump onto platform
Jan 12 – 16
Feb 9 – 13
Mar 1 – 5
On platform
Jan 12 – 16
Feb 9 – 13
Mar 1 – 6
Jump off platform
Jan 12 – 16
Feb 9 – 13
Mar 1 – 6
Escape
Jan 12 – 16
Feb 9 – 13
Mar 1 – 5
Passive
Jan 12 – 16
Feb 9 – 13
Mar 1 – 5
Stereotypy
Jan 12 – 16
Feb 9 – 13
Mar 1 – 5
Inside nestbox
Jan 12 – 16
Feb 9 – 13
Mar 1 – 5
On nestbox roof
Jan 12 – 16
Feb 8 – 13
Mar 1 – 5
Confident
Confident
box
Fearful
Fearful
box
P1
P2
P3
26.8
13.0
13.7
24.7
24.2
22.5
24.9
11.8
11.3
24.6
21.2
20.7
ns
0.01
< 0.01
ns
ns
ns
ns
< 0.005
< 0.005
9.0
20.7
21.0
1.8
3.5
3.5
5.3
13.9
15.6
2.5
4.9
6.1
0.08
< 0.001
< 0.01
ns
ns
ns
0.02
< 0.001
< 0.005
24.2
24.8
28.8
12.2
8.5
9.3
38.4
37.9
40.5
21.5
21.4
18.5
< 0.005
< 0.001
< 0.001
0.05
< 0.01
ns
< 0.001
< 0.001
< 0.001
3.2
4.8
4.8
1.2
5.0
4.3
0.8
3.3
2.1
1.0
3.9
5.8
0.06
ns
ns
0.04
ns
ns
ns
ns
ns
9.0
20.8
21.0
4.7
8.5
7.8
5.7
14.6
16.1
4.8
9.7
10.5
ns
ns
ns
ns
ns
ns
ns
ns
ns
36.5
32.8
28.5
37.7
33.3
32.5
25.4
23.4
21.0
32.4
19.0
20.0
ns
0.05
0.02
0.05
< 0.001
< 0.01
ns
ns
ns
0
1.8
1.7
0.2
0.2
0.3
0.5
2.8
1.6
0
0
0.5
ns
ns
ns
ns
ns
ns
ns
ns
ns
4.7
3.9
4.9
4.0
4.1
4.5
ns
ns
ns
ns
ns
ns
3.3
3.5
2.8
3.6
2.0
2.4
ns
ns
ns
ns
ns
ns
160
According to walking tests (Table 6), foxes with nestboxes were significantly
more often in motion both in February (22.7 vs 12.4% of animals) and March
(21.6 vs 12.5% of animals). Furthermore, foxes with nestboxes spent significantly
less time on platform in January (16.8 vs 31.3% of animals), February (14.9 vs
31.4% of animals) and March (13.9 vs 34.7% of animals). Temperament also
affected platform use, i.e. fearful foxes stayed significantly more frequently on the
platform in January (30.1 vs 18.2% of animals), February (29.8 vs 16.7% of
animals) and March (29.7 vs 19.1% of animals). However, more confident than
fearful animals were seen to jump onto the platform (Table 6). The number of foxes
showing an escape reaction, passive behaviour or stereotypy during the walking
tests did not differ between the groups. Among confident foxes the reaction of
escaping to the nestbox comprised 0.5, 0 and 0.7% of all escapes in January,
February and March. The corresponding percentages in fearful foxes were 1.5, 2.4
and 1.9. Temperament did not affect the number of foxes staying inside nestbox or
on nestbox roof.
Urine analyses revealed no immediate signs of urinary inflammation before the
mating season (on February 20). In each group, the urine pH was 6.1. Furthermore,
no glucose was detected in the urine at all. A slight tendency for an increased
protein level in the urine (< 0.5 g/l) was found from 10 and 7 animals in confident
and fearful nestbox groups, respectively.
Table 7. Reproductive performance in experimental groups. Litter size and cub losses are given as
number of kits per vixen. P1: between the four groups, P2: effect of temperament, P3: effect of nestbox
Variable
Total N of vixens
Mated, N
Whelped, N
Breeding vixens
litter size at 1 wk
3 wks
7 wks
Mated vixens
litter size at 1 wk
3 wks
7 wks
Whelped vixens
litter size at 1 wk
3 wks
7 wks
cub losses (1 – 7 wks)
Confident
Confident
box
Fearful
Fearful
box
60
30
29
60
40
25
60
35
29
60
35
26
P1
P2
P3
ns
ns
ns
ns
ns
ns
3.9
3.1
2.9
4.0
2.8
2.7
4.4
3.4
3.1
3.6
3.1
2.9
ns
ns
ns
ns
ns
ns
ns
ns
ns
6.0
4.7
4.4
7.3
5.1
4.8
7.6
5.3
5.0
6.3
5.3
4.9
ns
ns
ns
ns
ns
ns
ns
ns
ns
9.0
7.2
6.7
2.3
10.0
7.1
6.7
3.4
9.1
7.0
6.4
2.7
9.0
7.7
7.2
1.8
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
Matings started in confident and fearful animals with nestboxes on April 1, and
in those without nestboxes on April 2. Mating was first completed in fearful animals
with nestboxes on May 5, then in fearful foxes without nestboxes on May 8, and
161
finally in confident animals with and without nestboxes on May 8. Mating
percentages were: confident, no nestbox 66.7%; confident with nestbox 50.0%;
fearful, no nestbox 59.3%; and fearful, with nestbox 58.3%. Reproductive performance is summarized in Table 7. No significant differences were found between the
groups in whelping results as calculated per breeding or per mated or whelped
vixens. Furthermore, litter size did not differ between the groups as calculated at
1 week, 3 weeks or 7 weeks.
Discussion
One of main predictions in this study was that constant access to a nestbox
intensifies fearfulness in farm-raised foxes. This prediction was directly evaluated
here by two methods, namely, using the feeding and capture tests. The former test is
assumed to elicit the conflicting motivations of hunger and fear whereas the latter
measures animal’s immediate reaction to capture. Consequently, the two tests differ
in strength (Korhonen and Niemelä, 1996; 2000), i.e. the capture test is considered
more powerful because the cage door is opened and the animal is reached for, and
finally caught. Our results showed that the number of foxes being fearful in feeding
tests was of the same order of magnitude, whether a nestbox was present or not. The
result was the same during both the growing and breeding seasons. Our capture
tests, however, demonstrated significant differences between foxes with and
without nestboxes. During the growing season the percentage of fearful animals
increased significantly more among foxes with than those without a nestbox. The
result was the same when comparing the development of a fearful temperament in
the long term, i.e. the change from July near weaning to March near matings. These
findings clearly support our prediction that permanent access to a nestbox increases
fearfulness in blue foxes.
Figure 1. Experimental set-up for animals with nestboxes, a) wooden nestbox; b) wire-mesh platform;
and c) wooden block. Animals without nestboxes had an otherwise similar set-up, except the nestbox was
lacking
162
Fearfulness can also be indirectly assessed from our walking test results. Data
from the growing season showed that an escape reaction occurred more frequently
in foxes having nestboxes compared to those without nestboxes. This is a clear
indication of greater fearfulness in foxes with nestboxes. During the breeding
season, however, the difference between foxes with and without nestboxes
disappeared. At the approach of the breeding season, hormonal changes in the foxes
may influence their temperament so that they become less trustful to humans.
Our results unambiguously proved that constant nestbox availability leads to
a dirty fur coat. This can be problematic, as a dirty coat typically increases hygiene
and disease problems (Hovland and Bakken, 2000; Korhonen et al., 2003 a, b).
Furthermore, a dirty fur coat also lowers the insulation of the coat, therefore
creating a welfare problem due to excessive heat loss particularly at cold
(Korhonen, 1987; Korhonen et al., 2000). Temperament was also found to affect the
dirtiness of the fur, as the fur coat became dirtier in fearful than in confident
animals. However, this result was found only during the growing season. During the
breeding season no differences between groups existed. A dirtier fur coat in fearful
animals was not due to a greater amount of time spent inside the nestbox, as
walking tests results indicated no substantial differences between temperament
classes. An alternative explanation could be that the nestboxes of fearful foxes were
dirtier than those of confident ones. However, no differences in the degree of
dirtiness were found between temperament classes. Thus, it remains open what
actually explains higher dirtiness of fur coat in fearful animals. Perhaps fearful
animals were more rolling and rubbing themselves to dirt.
According to Mononen et al. (1999), the litter size per mated vixen at weaning
is a practical index of reproductive performance in foxes. This figure is a net result
of various components, ranging from the proportion of barren vixens via the
number of ova shed, the litter size at birth and number of stillborn cubs, to postnatal
cub losses. More detailed information can be obtained if reproductive success is
also calculated per breeding and per whelped vixen, as we did in this study. We also
counted the number of cubs lost as well as the mating and whelping percentages.
However, none of these figures revealed marked differences between the study
groups. Because of the multifactorial nature of reproductive performance, attempts
to relate a single environmental factor, such as a nest box type or presence of
a platform, to reproductive success have generally failed to give a significant result
(Korhonen and Niemelä, 1995; 1996; Ilukha et al., 1997). Taking this into account,
it is not actually surprising that we find no marked group differences. However, we
also found no differences between fearful and confident animals. Several previous
studies have shown that confident silver foxes in particular (Braastad, 1988;
Kristensen, 1989; Bakken, 1994; Jeppesen and Pedersen, 1998), but also blue foxes
(Hansen, 1998; Kenttämies et al., 1997), tend to have a better reproductive
performance than fearful or non-selected ones. A potential explanation for the
non-significance in our study could be that mating percentages in each group were
rather low (Ilukha et al., 1997), i.e. only slightly more than half of vixens could be
mated. Therefore, the number of vixens from which final reproductive success was
163
calculated was low. Often the variation in litter size is large in blue foxes, and it thus
requires fairly high number of vixens to reveal statistically significant group
differences. The main reason for the low mating percentage here was most likely the
presence of nestboxes in the shed. They seemed to prevent light from penetrating the
shed, thus negatively affecting the development of oestrus (Korhonen et al., 2004).
One issue worth further consideration is that although increased fearfulness due
to nestboxes did not have a significant effect on reproduction in this study, it may
have long term effects. If nestboxes are provided to foxes year after year, this could
easily multiply the number of fearful animals and lead to more pronounced effects
on the temperament.
The welfare of an animal cannot be directly measured but it must be assessed.
Unfortunately, there is no simple method, even in the assessment of welfare
(Simonsen, 1996; Veasey et al., 1996). This is because no single factor can precisely
reveal the objective welfare status of the animal in question. As a consequence,
a multi-perspective approach with different physiological and behavioural variables is
recommended to use (Harri et al., 1995). In the present study, we also measured
several parameters that are indicative of wellbeing in foxes, namely, the cortisol:creatinine ratio, blood status, the occurrence of stereotypical behaviours, explorativity, temperament, growth and reproduction. Of these, the frequency of
stereotypical behaviours and the cortisol:creatinine ratio are often considered the two
most striking indicators of welfare status (Korhonen et al., 2000, 2001). These
parameters did not reveal any substantial differences between the experimental
groups. However, the blood status, a typical indicator of the health status, revealed
one crucial difference, namely, the white blood cell count was elevated in foxes with
nestboxes in November. Nestboxes were dirty and also made animals and their fur
coat dirty. Increased numbers of white blood cells are here likely to be indicative of
some latent inflammation or disease outbreak. Thus, it seems that presence of
a permanent nestbox to a certain extent does compromise animal welfare.
Some differences found in explorativity, temperament development and growth
between foxes with and without nestboxes tempt us to conclude that nestboxes may
have some additional welfare consequences. Housing systems promoting exploration can be expected to improve animal welfare or, conversely, conditions
which do not encourage animals to behave exploratively often tend to be
unfavourable also in terms of animal welfare (Mononen, 1998). Foxes with
nestboxes were less explorative in ball tests than foxes without nestboxes. Thus, the
presence of nestboxes does not encourage animals to explore, but keeps them less
open to novel objects or situations. Furthermore, our results showed that confident
foxes were more explorative than fearful ones. This also confirms the expectation
that explorativity is positively related to welfare.
The lower weight gain in foxes with nestboxes was also a negative result
arguing against the permanent use of shelters. In theory, the main reasons for lower
growth may be that animals 1) were more active, 2) ate less or 3) the conditions in
which they were kept were otherherwise unfavourable for maximum growth
performance (Korhonen et al., 2003 b). Our walking test results affirmed that the
164
lower weights recorded were not a result of higher acitivity, on the contrary, foxes
with nestboxes tended to be less in locomotion than those without nestboxes.
Furthermore, animals in each group were fed with the same feed portion, which
eliminates explanation number 2. Thus, it appears that housing conditions otherwise
explain lower weights. For example, it is known that parasitic infections due to poor
hygienic conditions are one important reason for cessation of growth (Korhonen et
al., 2003 b). In the present study, we did not determine the occurrence of parasites
but dirty fur coats as well as elevated level of white blood cells are good indicators
of poorer hygiene in foxes with nestboxes.
It is known that foxes prefer higher places because they provide a good
opportunity to watch the surroundings (Korhonen et al., 1996). In the present study,
foxes with nestboxes used platforms less frequently than foxes without nestboxes.
This can be explained by the additional use of the nestbox roof as the walking test
results clearly indicated. The nestbox roof is thus also an elevated level that enables
observation of the environment (Mononen, 1996; Mononen et al., 1998). However,
there is no need to simultaneously provide both a nestbox roof and a platform for
the same behavioural purpose. Platforms serve this function well.
According to Hovland and Bakken (2000), the presence of nestboxes may serve
the function as an alternative option to withdrawal in challenging situations, thereby
assisting the coping attempts of the individual. In the present study, foxes with
nestboxes indeed showed more frequent escape reactions compared to foxes
without nestboxes. However, there were no differences between confident and
fearful foxes. Furthermore, our physiological and behavioural results do not support
the concept that a more frequent escape reaction in foxes with nestboxes is
beneficial to them in terms of animal welfare.
Conclusions
The breeding goal in fox farming is to produce a fur of high value with the
lowest possible production costs without compromising animal welfare. The
present results tempt us to conclude that this can be achieved by keeping blue foxes
in wire mesh cages with a platform and activity object but without permanent
access to nestboxes. To a certain extent, year-round nestboxes seem to compromise
both animal welfare and productivity.
Acknowledgements
This study was funded by the Ministry of Agriculture and Fishery, the Finnish
Fur Breeders’ Associaton and MTT Agrifood Research Finland. The staff of the Fur
Farming Research Station of Kannus (MTT) are gratefully acknowledged for their
valuable help in carrying out these experiments. Many thanks also to Seppo
Kukkonen, Juhani Sepponen and Hannele Johansson for additional help.
References
A h o l a L. (2002). Effects of social and physical housing environment on the welfare in silver foxes
(Vulpes vulpes). Ph.D. Thesis. Kuopio University Publications C. Natural and environmental
sciences 145, 69 p.
165
B a k k e n M. (1994). Infanticidal behaviour and reproductive performance in relation to competition
capacity among farmed silver fox vixens, Vulpes vulpes. Ph.D. Thesis, University of Trondheim,
Norway 1994, 35 p.
B e r r y J.J. (1995). A simulation-based approach to some nonparametric statistics problems. Observations, 1: 19 – 26.
B r a a s t a d B. (1988). Sammenhangen mellem atferd og reproduksjonsresultater hos rev. Aktuelt fra
Statens fagtjeneste for Landbruket, 1: 422 – 427.
C h a m o v e A.S. (1989). Environmental enrichment: a review. Anim. Tech., 40: 155 – 178.
H a n s e n S.W. (1998). Selection for trusting blue foxes — reproduction results and stability of
temperament. NJF seminar no 295, 7 – 9 September 1998, Bergen, Norway, 10 pp.
H a r r i M., R e k i l ä T., M o n o n e n J. (1995). Factor analysis of behavioural tests in farmed silver and
blue foxes. Appl. Anim. Behav. Sci., 42: 217 – 230.
H a r r i M., M o n o n e n J., R e k i l ä T., K o r h o n e n H., N i e m e l ä P. (1998). Effects of top nest box on
growth, fur quality and behaviour of blue foxes (Alopex lagopus) during their growing season. Acta
Agricult. Scand., 48: 184 – 191.
H o v l a n d A-H., B a k k e n M. (2000). The welfare situation of farmed foxes in relation to domestication status and compared to other farmed species. Agric. Univ. of Norway (NLH), Dept. of
Anim. Sci., 2000, 88 pp.
I l u k h a V.A., H a r r i M., R e k i l ä T. (1997). Reproductive success of farmed blue foxes. J. Anim.
Breed. Gen., 114: 465 – 474.
J e p p e s e n L.L., P e d e r s e n V. (1998). Selektionforsog for tillidsfulla solvrev i Danmark. NJF seminar
no 295, 7 – 9 September 1998, Bergen, Norway, 8 pp.
J o n e s B.R. (1997). Fear and distress. In: Appleby M.C., Hughes B.O. (eds.), Animal Welfare, CAB
International, Wallingford, Oxford, UK, Ch. 6, pp. 75 – 87.
K e n t t ä m i e s H., S m e d s K., R e k i l ä T. (1997). Preliminary results from a selection experiment
for more confident foxes. NJF seminar no 280, 6 – 8 October 1997, Helsingfors, Finland,
pp. 227 – 233.
K o l e s n i k o v a L., L u c e n k o N., T r u t L., J u r i s o v a M., B e l y a e v D. (1985). Selection of silver
foxes for domestic type of behaviour: morphological and functional characteristics of organ
systems, pp. 663 – 670. In: J. Milkovsky & V.J.A. Novak, eds. Evolution and morphogenesis.
Academia, Prague, Czech Republic.
K o r h o n e n H. (1987). Significance of sleeping plate as a thermal protection for farmed raccoon dogs
(Nyctereutes procyonoides). Comp. Biochem. Physiol., 87A: 631 – 633.
K o r h o n e n H., N i e m e l ä P. (1995). Comparison of production results between blue foxes housed
with and without platforms. Agricult. Sci. Fin., 4: 351 – 361.
K o r h o n e n H., N i e m e l ä P. (1996). Temperament and reproductive success in farmbred silver foxes
housed with and without platforms. J. Anim. Breed. Gen., 113: 209 – 218.
K o r h o n e n H., N i e m e l ä P., T u u r i H. (1996). Seasonal changes in platform use by farmed blue
foxes (Alopex lagopus). Appl. Anim. Beh. Sci., 48: 99 – 114.
K o r h o n e n H., N i e m e l ä P. (2000). Enrichment value of wooden blocks for farmed blue foxes
(Alopex lagopus). Anim. Welf., 9: 177 – 191.
K o r h o n e n H., N i e m e l ä P., J a u h i a i n e n L., T u p a s e l a T. (2000). Effects of space allowance and
earthen floor on welfare-related physiological and behavioural responses in male blue foxes.
Physiol. Behav., 69: 571 – 580.
K o r h o n e n H., J a u h i a i n e n L., N i e m e l ä P., H a r r i M., S a u n a - a h o R. (2001). Physiological and
behavioural responses in blue foxes (Alopex lagopus): comparisons between space quantity and
floor material. Anim. Sci., 72: 375 – 387.
K o r h o n e n H., J a u h i a i n e n L., N i e m e l ä P., S a u n a - a h o R. (2002 a). Wooden block and straw as
environmental enrichments for juvenile blue foxes (Alopex lagopus). Acta Ethol., 5: 29 – 37.
K o r h o n e n H., H a n s e n S.W., M a l m k v i s t J., H o u b a k B. (2002 b). Effect of capture, immobilization and handling on rectal temperatures of confident and fearful male mink. J. Anim.
Breed. Gen., 117: 337 – 345.
K o r h o n e n H., J a u h i a i n e n L., R e k i l ä T. (2002 c). Effect of temperament and behavioural
166
reactions to the presence of a human during pre-mating period on reproductive performance in
farmed mink (Mustela vison). Canad. J. Anim. Sci., 82: 275 – 282.
K o r h o n e n H., J a u h i a i n e n L., N i e m e l ä P., R e k i l ä T. (2003 a). Access to ground contact and its
welfare implications in farmed foxes. Proc. of the NJF’s 22nd Congress, Nordic Agriculture in
Global Perspective, July 1 – 4, 2003, Turku, Finland, pp. 261 – 264.
K o r h o n e n H.T., J a u h i a i n e n L., R e k i l ä T. (2003 b). In-cage sandbox as a ground substitute for
farmed blue foxes (Alopex lagopus): effects on digging activity and welfare. Canad. J. Anim. Sci.,
83: 703 – 712.
K o r h o n e n H., R e k i l ä T., K i v i n e n T., J a u h i a i n e n L. (2004). Comparison of hall and shed as
housing environments for blue foxes. Scientifur, 28 (3): 7 – 10.
K r i s t e n s e n M.P. (1989). An evaluation of exploratory and fear-motivated behaviour as a predictor of
reproductive success in silver fox vixens. Scientifur, 12: 199 – 205.
L a s l e y B.L., K i r k p a t r i c k J.F. (1991). Monitoring ovarian function in captive and free-ranging
wildlife by means of urinary and fecal steroids. J. Zoo. Wild. Med., 22: 23 – 31.
L i t t e l l R.C., M i l l i k e n G.A., S t r o u p W.W., W o l f i n g e r R.D. (1996). SAS System for Mixed
Models, Cary, NC: SAS Institute Inc, 633 pp.
M o n o n e n J. (1996). Resting platform and nest boxes for farmed blue foxes (Alopex lagopus) and silver
foxes (Vulpes vulpes): the extent of use, reasons for use and welfare effects. Kuopio University
Publications C. Natural and Environmental Sciences 52, 62 pp.
M o n o n e n J. (1998). Evaluation of the open field test. In: Proceedings of the Nordic ISAE Winter
Meeting, Tune, Denmark, 1998. Royal Veterinary and Agricultural University of Denmark,
Copenhagen, p. 6.
M o n o n e n J., K o r h o n e n H., H a r r i M., K a s a n e n S. (1998). A comparison of the use of resting
platforms and nest boxes in growing farmed silver foxes (Vulpes vulpes). Appl. Anim. Behav. Sci.,
58: 383 – 396.
M o n o n e n J., H a r r i M., S e p p o n e n J., K o r h o n e n H., R e k i l ä T., A h o l a L. (1999). A top and
a floor box as breeding nest boxes in farmed blue foxes (Alopex lagopus): reproductive
performance, use of boxes and cub carrying. Acta Agricult. Scand., Sect. A, Anim. Sci., 49:
206 – 210.
N e w b e r r y R. (1995). Environmental enrichment: increasing the biological relevance of captive
environments. Appl. Anim. Behav. Sci., 44: 229 – 243.
O s a d c h u k L.V. (1992). Endocrine gonadal function in silver fox under domestication. Scientifur, 16:
116 – 121.
R e k i l ä T., H a r r i M., A h o l a L. (1997). Validation of the feeding test as an index of fear in farmed
blue (Alopex lagopus) and silver foxes (Vulpes vulpes). Physiol. Behav. 62: 805 – 810.
R o u v i n e n K., A r c h b o l d S., L a f f i n S., H a r r i M. (1999). Long-term effects of tryptophan on
behavioural response and growing-furring performance in silver foxes. Appl. Anim. Behav. Sci.,
63: 65 – 77.
S i m o n s e n H.B. (1996). Assessment of animal welfare by a holistic approach: behaviour, health and
measured opinion. Acta Agricult. Scand., 27: 91 – 96.
V e a s e y J.S., W a r a n N.K., Y o u n g R.J. (1996). On comparing the behaviour of zoo housed animals
with wild conspecifics as a welfare indicator. Anim. Welf., 5: 13 – 24.
W o l f i n g e r R. (1996). Heterogeneous Variance-Covariance Structures for Repeated Measures. J.
Agricult., Biol. Environ. Statist., 1 (2): 205 – 230.
167
HANNU T. KORHONEN, LAURI JAUHIAINEN, TEPPO REKILÄ
Wpływ całorocznej doste˛pności skrzynek wykotowych i temperamentu
na dobrostan i produkcyjność lisów niebieskich (Alopex lagopus)
STRESZCZENIE
Celem doświadczenia była ocena długotrwałego wpływu skrzynek wykotowych i temperamentu na
dobrostan i użytkowość młodych i dorosłych lisów niebieskich (Alopex lagopus). Zwierze˛ta bojaźliwe
i pewne siebie odchowywano w okresie wzrostu i reprodukcji z użyciem skrzynek wykotowych lub bez.
Przy odsadzeniu utworzono naste˛puja˛ce grupy doświadczalne: 1) zwierze˛ta pewne siebie — brak
skrzynek wykotowych; 2) zwierze˛ta pewne siebie — skrzynka wykotowa doste˛pna przez cały rok; 3)
zwierze˛ta bojaźliwe — brak skrzynek wykotowych; 4) zwierze˛ta bojaźliwe — skrzynka wykotowa
doste˛pna przez cały rok. W każdej grupie było 60 samic, które utrzymywano pojedynczo.
Stwierdzono, że lisy pozbawione skrzynek wykotowych rosły istotnie (P = 0,01) lepiej niż lisy
maja˛ce doste˛p do skrzynek. Temperament nie miał wpływu na końcowa˛ mase˛ ciała. Zachowanie lisów
pewnych siebie było bardziej eksploracyjne (P < 0,01) niż zachowanie lisów bojaźliwych, natomiast
zachowanie lisów pozbawionych skrzynek wykotowych było bardziej eksploracyjne niż zachowanie
zwierza˛t maja˛cych doste˛p do skrzynek (P = 0,06). Testy wyłapywania ujawniły, że stały doste˛p do
skrzynki wykotowej istotnie (P < 0,001) zwie˛kszał bojaźliwość w dłuższym okresie. Lisy maja˛ce doste˛p
do skrzynek wykotowych były bardziej skłonne do ucieczki na widok człowieka aniżeli zwierze˛ta
pozbawione skrzynek. Nie stwierdzono istotnych różnic pomie˛dzy grupami zróżnicowanymi pod
wzgle˛dem temperamentu i w zależności od doste˛pu do skrzynek wykotowych w cze˛stości zachowań
steterotypowych, stosunku kortyzolu do kreatyniny i reprodukcyjności. Okrywa włosowa lisów
maja˛cych doste˛p do skrzynek wykotowych była zabrudzona, co istotnie (P < 0.001) obniżało jakość
futra. Stwierdzono, że całoroczny doste˛p do skrzynek wykotowych zwie˛ksza bojaźliwość fermowych
lisów niebieskich. Obecność skrzynek wykotowych może również w inny sposób negatywnie wpływać
na dobrostan zwierza˛t.
USE OF AN EARTH-TUBE HEAT EXCHANGER TO OPTIMIZE
BROILER HOUSE CLIMATE DURING THE SUMMER PERIOD*
I w o n a S k o m o r u c h a, E u g e n i u s z H e r b u t
Department of Technology, Ecology and Economics of Animal Production,
Abstract
This study was designed to determine the effect of an earth-tube heat exchanger (ETHE) on broiler
house climate and on the productivity of broiler chickens during the summer period. Group I was
the control broiler house in which broilers were reared under standard conditions. Group II was
the experimental broiler house equipped with an ETHE, intended to act as an air conditioner and
thus optimize thermal conditions for chicken rearing. The results obtained showed that the air
temperature in the ETHE broiler house was 2 – 3oC lower than in the control broiler house. The
lower and thus optimum temperature inside the experimental building highly significantly
increased body weights, improved feed conversion and reduced the mortality of birds compared to
the birds from the control house. The better productivity of birds from the experimental group was
reflected in a higher European Production Index (EPI) in this group.
Key words: broiler chickens, earth-tube heat exchanger, productivity
Genetic progress in poultry is making birds increasingly sensitive to environmental conditions, of which house climate plays a significant role (Dobrzański et
al., 2003). Having optimum climatic conditions inside livestock buildings guarantees that the life processes of birds will be normal, while any deviation from animal
hygiene standards may cause stress resulting in disturbed homeostasis inside the
body.
Recent practice has shown that the recommended indoor climate standards are
often exceeded during the summer heat-wave in Poland. This results in an
excessively high indoor temperature, which, in addition to excessively low or high
air humidity, causes hyperthermia in birds. Hyperthermia, or heat stress, causes
losses related to lower productivity, poorer health, and mass mortality (Sokołowicz
and Herbut, 2004).
170
I. Skomorucha and E. Herbut
Efforts are therefore being made to introduce solutions that do not increase
production costs but allow the optimum temperature to be maintained without
causing any unfavourable fluctuations in temperature or excessive cooling of birds
(Białas and Dobrzański, 2000). This is intended to increase the comfort and health
of birds, thus making production more effective. Analysis of the research findings
on the subject shows that new possibilities are offered by earth-tube heat
exchangers (ETHE), which can be used to heat or cool the inlet air stream as
required (Viedt, 1991; Shingari et al., 1995; Bieda and Herbut, 1999; Alchalabi,
2001; Bieda et al., 2001).
The aim of the present study was to determine the effect of an ETHE on house
climate and on the productivity of broiler chickens during the summer production cycle.
The experiment was performed at the Experimental Station of the National
Research Institute of Animal Production in Rossocha using 30,400 Starbro broiler
chickens, during the summer production cycle, from 10 June to 22 July 2002.
The study was carried out in two modern broiler houses (standard control and
experimental with an ETHE), each with a production area of 800 m2, fully
automated feed and water administration, and air heating with propane. The
experimental broiler house was fitted with a mechanical negative pressure ventilation system containing an ETHE, which covers 10% of the summer requirement
for ventilation air. The principle of ETHE operation consists in an exchange of heat
between the air stream flowing through the exchanger pipes and the surrounding
ground. During the summer, warm air leaving the exchanger pipes is cooled and
radiates heat towards the ground, which has a lower temperature than atmospheric air.
Used air is eliminated by way of mechanical ventilation of the broiler house only.
Broiler chickens were reared on straw bedding until 42 days of age. They were
fed standard diets prepared on the basis of starter (1 – 3 weeks of rearing), grower
(4 – 5 weeks of rearing) and finisher concentrates (6 weeks of rearing). Throughout
the study, birds had free access to water drinkers. The light regime was 16 hours
light : 8 hours dark with a light intensity of 3 – 5 lux.
Broilers were assigned to two groups at a stocking rate of 19 birds/m2, and their
response to indoor thermal conditions was examined:
Group I — control broiler house in which chickens were reared under standard
conditions,
Group II — experimental broiler house equipped with an ETHE, intended to act
as an air conditioner and thus optimize thermal conditions for chicken rearing.
Throughout the 42 days of rearing, the following basic climate parameters were
monitored every week inside and outside the buildings:
— temperature — measured three times a day at 08.00, 14.00 and 18.00,
— relative humidity, water vapour pressure, air movement and dry cooling —
measured once a day at 14.00.
Earth-tube heat exchanger and broiler house climate
171
The indoor climate was measured with wall ventilators turned on, at 3 diagonal
points in the building at a height of 30 cm above the floor, i.e. in the living area of
the broilers. Outside, the measurements were made 2 m away from each wall of the
building, at a height of around 1 m.
Temperature and air humidity measurements were taken using an Assmann
aspiration psychrometer according to the animal hygiene testing method described
by T. M. Janowski (1979). Water vapour pressure was calculated using a formula
based on the temperature and air humidity values obtained. Dry cooling was
measured with a dry-bulb katathermometer, and air movement was read from tables
based on the dry cooling values.
Body weight in 100 randomly selected birds and the number of dead birds were
checked every week, as were feed intake from 1 to 21 and from 1 to 42 days of
rearing.
Based on the production results obtained, the efficiency of broiler rearing was
expressed as the European Production Index, which was calculated using the
formula:
EPI =
m×p
t×a
× 100
where:
EPI — European Production Index,
m — mean body weight of broiler (kg),
p — survival, number of chickens reared (%),
t
— number of days of rearing,
a — feed conversion (kg feed/kg gain).
The results were analysed statistically using analysis of variance and significant
differences were estimated using Duncan’s test.
Results
The results of the climate tests are given in Tables 1 – 3.
Air temperature measured in the morning in weeks of rearing was similar in
both buildings and no statistically significant differences were found (Table 1).
A highly significantly lower temperature was found in the experimental broiler
house at 14.00 on days 21, 35 and 42 of rearing. The difference in temperature
between the buildings was 2.7, 3.6 and 3.1oC, respectively.
Analysis of the air temperature at 18.00 showed that throughout rearing it was
lowest in the ETHE broiler house. Highly significant differences were noted on
days 21 and 35 of rearing only.
In the experimental building, lower diel fluctuations in the temperature of the
inlet air stream were found. The difference in the minimum and maximum
temperature between the control and experimental buildings was 4.1 and 2.5oC on
172
day 21, 5.6 and 3.7oC on day 35, and 2.2 and 0.4oC on day 42 of the experiment,
respectively (Table 1).
Table 1. Mean air temperature (oC) inside and outside the broiler house
7
14
21
28
35
42
Inside
14.00
08.00
Day of
rearing
Outside
(14.00)
x̄
18.00
group
I
II
I
28.0 ± 0.1
25.0 ± 0.1
24.2 ± 0.1
24.8 ±0.1
23.6 ± 0.2
18.9 ± 0.1
28.2 ± 0.1
25.2 ± 0.2
23.1 ± 0.1
22.2 ±0.1
21.9 ± 0.2
17.6 ± 0.1
II
I
27.7 ± 0.3
27.2 ± 0.3
25.2 ± 0.1
25.00 ± 0.1
28.3 ± 0.4 A 25.6 ± 0.1 B
28.4 ± 0.5
26.2 ± 0.4
29.2 ± 0.4 A 25.6 ± 0.1 B
21.1 ± 0.4 A 18.0 ± 0.50 B
II
27.7 ± 0.2
24.9 ± 0.1
25.7 ± 0.2 A
27.3 ± 0.3
27.5 ± 0.2 A
18.9 ± 0.1
27.0 ± 0.2
27.0
24.8 ± 0.1
21.1
23.6 ± 0.1 B 28.9
25.8 ± 0.2
28.5
25.0 ± 0.1 B 28.2
17.7 ± 0.1
20.2
A, B — values marked with different letters differ highly significantly (P ≤ 0.01).
The measurements of relative air humidity are given in Table 2. In both
group I and group II, the lowest relative humidity was found at 21 days of rearing
(57.7 and 60.3%) and the highest at 35 days of rearing (73.3 and 77.0%,
respectively).
The water vapour pressure results were similar in both buildings to 35 days of
rearing (Table 2). On day 42 of the experiment, the water vapour pressure in
building II was 19.1 hPa and was significantly higher than in broiler house I
(17.3 hPa).
Table 2. Mean relative air humidity and water vapour pressure inside and outside the broiler house
Relative air humidity (%)
inside
group
Day
of rearing
7
14
21
28
35
42
Water vapour pressure (hPa)
inside
group
outside
I
II
x̄
71.7 ± 1.20
63.0 ± 0.58
57.7 ± 0.07
65.0 ± 1.53
73.3 ± 1.76
69.7 ± 1.45
72.7 ± 1.45
64.0 ± 0.58
60.3 ± 1.20
64.0 ± 0.58
77.0 ± 1.15
71.3 ± 1.76
65
59
54
63
70
71
I
26.5 ± 0.31
20.1 ± 0.21
22.2 ± 0.23
25.0 ± 0.24
29.8 ± 0.40
17.3 ± 0.22 a
outside
II
27.1 ± 0.71
20.3 ± 0.24
21.6 ± 0.36
26.1 ± 0.63
29.5 ± 1.05
19.1 ± 0.23 b
x̄
23.92
14.65
21.71
25.10
26.62
16.91
a, b — values marked with different letters differ significantly (P ≤ 0.05).
No statistically significant differences were found in air movement between
groups I and II (Table 3). This climate parameter ranged from 0.259 to 1.167 m/s in
building I and from 0.259 to 0.933 m/s in building II.
173
Table 3. Mean air velocity and dry cooling inside and outside the broiler house
Dry cooling (mW/cm2)
Air movement (m/s)
inside
group
Day
of rearing
7
14
21
28
35
42
inside
group
outside
outside
I
II
x̄
I
II
x̄
0.259 ± 0.01
0.377 ± 0.07
0.279 ± 0.04
0.709 ± 0.19
1.167 ± 0.18
1.072 ± 0.28
0.259 ± 0.06
0.272 ± 0.05
0.597 ± 0.11
0.933 ± 0.15
0.537 ± 0.21
0.716 ± 0.28
0.420
1.160
0.519
1.949
1.308
1.250
14.97 ± 0.55
21.30 ± 1.10
14.11 ± 1.06
18.05 ± 2.12
19.43 ± 1.76
39.61 ± 4.92
14.53 ± 1.12
19.53 ± 0.73
20.57 ± 1.27
17.68 ± 0.38
16.65 ± 2.96
31.55 ± 3.76
15.84
38.75
13.65
23.80
22.03
42.81
Building I was characterized by greater dry cooling during almost the whole
study period (Table 3), with 6.46 mW/cm2 lower cooling than in group II found on
day 21 only. The highest cooling was found in both broiler houses on day 42 (39.61
and 31.55 mW/cm2, respectively).
The production results for the broiler chickens are given in Table 4. On day 42,
the body weight of broiler chickens averaged 2017 g in group I and 2120 g in the
experimental group, and the difference was highly significant.
Analysis of the results of feed conversion per kg weight gain showed 90 g lower
feed intake by chickens from the experimental group in the first period of rearing.
For the whole experimental period, this parameter was 1950 and 1840 g,
respectively.
A markedly lower proportion of dead chickens was found in the experimental
broiler house. From 1 to 21 days of rearing, bird mortality was 1.93% in group I and
1.47% in group II. In the second period of the experiment, the difference in the
proportion of dead chickens was 0.95%. Mortality during the whole rearing period
was 4.33% in group I and 2.92% in group II.
Table 4. Reproductive performance of broiler chickens
Item
Body weight
Feed intake (g) per kg weight gain
Mortality (%)
EPI (pts)
Day of rearing
42
1 – 21
1 – 42
1 – 21
22 – 42
1 – 42
Group
I
II
2017 ± 10.14 A
1530
1950
1.93
2.4
4.33
236
2120 ± 6.73 B
1440
1840
1.47
1.45
2.92
269
A, B — values marked with different letters differ highly significantly (P ≤ 0.01).
Birds from the experimental building were characterized by a higher EPI
compared to broiler chickens reared in the control building (269 vs. 236 points)
(Table 4).
174
Discussion
The optimum temperature for broiler chickens should be gradually decreased
with age by 2 – 3oC a week to reach 18 – 20oC during the final weeks of rearing
(Journal of Laws, 03.167.1629).
Analysis of the measurements shows that air temperature standards were
exceeded in both the control and experimental broiler houses, especially during
the final weeks of rearing. However, comparison of this climate parameter in both
houses showed that in the ETHE building, air temperature was around 2 – 3.5oC
lower, especially in the afternoon hours when the building was most vulnerable to
being affected by sunrays and the external temperature. Considering that the ETHE
studied accounted for only 10% of the air exchange necessary for the summer
period, its efficiency proved relatively high. In preliminary studies on the use of
ETHE for cooling a stream of ventilation air during the summer production cycle of
broilers, Bieda et al. (2001) noted that the temperature of the internal air in the
experimental building with an ETHE was approximately 2.5 K lower compared to
the control building in which air was not cooled with an ETHE. Shingari et al.
(1995) showed that with the use of an ETHE, the air temperature in the building
studied decreased by approximately 6 – 7 K compared to the control building.
A fairly high decrease in temperature and small diel fluctuations inside the building
were reported by Alchalabi (2001). The use of double cooling of inlet air (in the
ground and by way of evaporation) resulted in a considerable decrease in indoor
temperature, and the difference between the minimum and maximum temperature
was 3.8oC, compared to a difference of 19.5oC outside the building.
It is equally important to maintain the required level of air humidity, which is
strictly connected with temperature. The Regulation of the Ministry of Agriculture
and Rural Development of 2 September 2003 (Journal of Laws, 03.167.1629)
concerning the minimum housing conditions for different animal species indicates
that the optimum relative air humidity in a broiler house should be 70, 65 and 60%
in the first, second and subsequent weeks of rearing, respectively.
In our study, relative air humidity in both groups was similar, as evidenced by
the lack of statistically significant differences, and ranged from 57.7 to 77%. It
should be noted, however, that in the second period of rearing, relative air humidity
standards were exceeded in both buildings and, as reported by Herbut and Walczak
(2001), this may increase the negative effect of heat stress, because it inhibits the
radiation of heat by the release of excess water vapour.
Niedziółka and Tombarkiewicz (1989) reported that water vapour pressure
should range from 6 to 8 mmHg (8 to 10.7 hPa), but at higher temperatures it can
reach 11 mmHg (14.7 hPa). During the summer period, when it is quite difficult to
keep water vapour pressure up to the standard level, it is assumed that the difference
in pressure inside and outside the building should be as low as possible and range
from 2 to 4 mmHg (Niedziółka and Tombarkiewicz, 1989). Our own findings
indicate that this difference, calculated from the mean values for the whole rearing
period, did not exceed the upper limit of 4 mmHg (5.3 hPa) either in the control or
175
experimental house, where it was 1.5 and 1.8 mmHg (2 and 2.4 hPa), respectively.
Cooling, which is directly related to air temperature, humidity and velocity,
is an important factor in poultry facilities. Dobrzański (1983) reported that
cooling in poultry houses should range from 16.72 to 35.44 mW/cm2, and the
permissible air velocity value should not exceed 1.0 m/s regardless of the season of
the year. In our study, dry cooling in the experimental broiler house fell below the
recommended level only on day 7 of rearing, while air velocity did not exceed
1.0 m/s.
It is well known that air temperature has a significant effect on bird bodies. In
the present study, we found that thermal and humidity conditions in broiler houses
had an effect on the productivity of broiler chickens: at 42 days of rearing, the
difference in the body weight of chickens was 103 g (P ≤ 0.01). Likewise, Yahav
and Hurwitz (1996) found significantly lower body weight in chickens exposed to
high temperature compared to the control chickens. Yalçin et al. (2001) reported
that the body weight of broiler chickens in a building with an air temperature higher
than that recommended decreased by 23%, while Özkan et al. (2003) reported
a 34% decrease in the weight gains of birds reared during the summer months in
countries with a subtropical climate. A decreased growth rate in birds exposed to
high temperature has also been reported by Cooper and Washburn (1998), Deeb and
Cahaner (2001), and Sokołowicz and Herbut (2001). During short-term heat stress
(for 2 hours, temperature elevated by 10oC in relation to the recommended
temperature), Sosnówka-Czajka and Herbut (2003) did not find lower weight gains
or final body weights, nor did they note poorer feed conversion per kg weight gain
in birds exposed to high air temperature. Different results were obtained by
Sokołowicz and Herbut (2001), who noted a 30 g higher feed intake per kg weight
gain by broiler chickens during the last week of rearing when birds were exposed to
an air temperature 12oC higher than the optimum temperature. This is confirmed by
our own findings, in which broiler chickens from group II, subject to additional
cooling through ETHE, were characterized by roughly 6% lower feed intake in both
the first and second periods of rearing. Increased feed intake by birds reared under
elevated temperature conditions has also been reported by Yahav and Hurwitz
(1996) and Cooper and Washburn (1998).
During the summer period, when poultry house temperatures exceed the
recommended values, bird mortality due to hyperthermia is very frequent. Sokołowicz and Herbut (2004) observed 100% mortality among 40-day-old broiler
chickens exposed to 3-day heat stress during the summer production cycle.
Researchers studying the issue of elevated rearing temperature for broiler
chickens often report increased bird mortality (Mennicken et al., 1994; Sokołowicz
and Herbut, 2001; Özkan et al., 2003).
In our study, we observed 1.41% higher mortality in chickens in the control
broiler house. May and Lott (2000) reported that bird mortality is the best indicator
of whether rearing temperature is correct. It can therefore be stated that thermal and
humidity conditions in the experimental broiler house were more favourable, as
reflected in the lower proportion of dead birds during the whole rearing period. In
176
contrast, the results of Cooper and Washburn (1998) fail to confirm the relationship
between elevated rearing temperature (32oC) and mortality.
Environmental conditions have a considerable effect on EPI (Krawczyk and
Sokołowicz, 2001). In the present study, chickens reared in the experimental broiler
house achieved a 33-point higher EPI compared to the control chickens. Similar
results were obtained by Skomorucha and Herbut (2005), who noted higher EPI in
the group of broilers reared in a building equipped with an ETHE.
It is concluded from the present study that the use of an ETHE to cool the
stream of ventilation air has a beneficial effect on house climate mainly as a result
of the lower temperature inside the building (by 2 – 3oC) in relation to the control
broiler house.
The lower air temperature in the ETHE broiler house improved the ‘‘thermal
comfort” of broiler chickens reared in this building. This was reflected in better
productivity and lower mortality compared to birds from the control group and, as
a consequence, in higher EPI values.
References
A l c h a l a b i D. (2001). Two-stage air cooling for very hot environments. Poultry Internat., 40, 11:
28 – 32.
B i a ł a s W., D o b r z a ń s k i Z. (2000). Ochładzanie strefy nadściólkowej w budynku brojlerni
z płaszczyznowym ogrzewaniem podłogowym. Zesz. Nauk. Politechniki Opolskiej, Seria: Budownictwo, 44: 25 – 31.
B i e d a W., H e r b u t E. (1999). Koncepcja rurowo, gruntowo-powietrznego wymiennika ciepła
działaja˛cego w systemie wentylacyjnym brojlerni. Zesz. Nauk. AR Krak., 355, Inż. Środ., 19:
153 – 163.
B i e d a W., H e r b u t E., K o ź b i a ł M. (2001). Cooling of air blown into broiler house in an air-earth
tube heat exchanger. Proc. of the International Symposium of the C.I.G.R. 2nd Technical Section.
Animal welfare consideration in livestock housing systems. Szklarska Pore˛ba, October 23 – 25:
421 – 426.
C o o p e r M.A., W a s h b u r n K.W. (1998). The relationships of body temperature to weight gain, feed
consumption, and feed utilization in broilers under heat stress. Poultry Sci., 77: 237 – 242.
D e e b N., C a h a n e r A. (2001). Genotype by environment interaction with broiler genotypes differing
in growth rate: 2. The effects of high ambient temperature on dwarf versus normal broilers. Poultry
Sci., 80: 541 – 548.
D o b r z a ń s k i Z. (1983). Zastosowanie bonitacyjno-statystycznej metody do określania zależności
mie˛dzy warunkami środowiskowymi w brojlerniach a efektywnościa˛ odchowu kurcza˛t. Zesz. Nauk.
AR Wroc., 37, Rozprawy.
D o b r z a ń s k i Z., R u d n i c k a A., S k o w r o ń s k i W. (2003). Physical factors of microclimate in
laying hens battery system as a criterion of welfare. Ann. Anim. Sci., Suppl., 1: 51 – 53.
H e r b u t E., W a l c z a k J. (2001). Aktualne wymagania cieplno-wilgotnościowe zwierza˛t gospodarskich. II Mie˛dz. Konf. Nauk. „Aktualne problemy fizyki budowli w budownictwie wiejskim”.
Kraków, 9 – 10 listopada, ss. 92 – 100.
K r a w c z y k J., S o k o ł o w i c z Z. (2001). Wpływ czynników genetyczno-żywieniowych na opłacalność produkcji kurcza˛t brojlerów. Zesz. Nauk. PTZ XII Mie˛dz. Symp. Młodych Drobiarzy P. O.
WPSA, 11 – 12.09.2001 r. Kraków, ss. 287 – 298.
M a y J.D., L o t t B.D. (2000). The effect of environmental temperature on growth and feed conversion of
broilers to 21 days of age. Poultry Sci., 79: 669 – 671.
177
M e n n i c k e n L., V a l l e - Z a r a t e A., H o r s t P. (1994). Effects of air velocity and drinking water
temperature on the performance of broiler breeder hens under tropical conditions. Proc. of the 9th
European Poultry Conference Glasgow UK, August 7 – 12th 1994, pp. 127 – 128.
N i e d z i ó ł k a J., T o m b a r k i e w i c z B. (1989). Zoohigieniczna ocena efektów ła˛cznego elektronicznego sterowania ogrzewaniem i wentylacja˛ w kurnikach. Acta Agr. Sillv, Ser. Zoot., 28: 117 – 122.
Ö z k a n S., A k b a s Y., A l t a n Ö., A l t a n A., A y h a n A., Ö z k a n K. (2003). The effects of short-term
fasting on performance traits and rectal temperature of broilers during the summer season. Brit.
Poultry Sci., 44: 88 – 95.
S h i n g a r i B.K., S i n g h A., S a p r a K.L. (1995). Earth tube exchangers. Poultry Internat., 12: 92 – 96.
S k o m o r u c h a I., H e r b u t E. (2005). Effect of an earth tube heat exchanger on broiler house climate in
the summer period. Proc. of XIIth International Congress ISAH ‘‘Animals and Environment”,
Warsaw, Poland, vol. 2: 174 – 177.
S o k o ł o w i c z Z., H e r b u t E. (2001). Effect of thermal acclimation of broiler chickens on their
performance, metabolic rate, and thyroid activity. Ann. Anim. Sci., 1, 2: 199 – 205.
S o k o ł o w i c z Z., H e r b u t E. (2004). Economic effects of disturbing broiler chicken welfare by
overheating. Ann. Anim. Sci., Suppl., 1: 197 – 199.
S o s n ó w k a - C z a j k a E., H e r b u t E. (2003). Physiological and production indicators of broiler
chickens exposed to short-term thermal stress. Ann. Anim. Sci., 3, 2: 365 – 375.
V i e d t W. (1991). Erdwärmetauscher in Mastschweine- und Abferkelställen. Landtechnik, 7/8:
377 – 379.
Y a h a v S., H u r w i t z S. (1996). Induction of thermotolerance in male broiler chickens by temperature
conditioning at an early age. Poultry Sci., 75: 402 – 406.
Y a l ç i n S., Ö z k a n S., T û r k m u t L., S i e g e l P.B. (2001). Responses to heat stress in commercial and
local broiler stocks. 1. Performance traits. Brit. Poultry Sci., 42: 149 – 152.
IWONA SKOMORUCHA, EUGENIUSZ HERBUT
Zastosowanie gruntowego wymiennika ciepła do optymalizacji mikroklimatu brojlerni
w okresie letnim
STRESZCZENIE
Celem przeprowadzonych badań było określenie wpływu gruntowego wymiennika ciepła (GWC)
na mikroklimat i wyniki produkcyjne kurcza˛t brojlerów podczas letniego cyklu produkcyjnego.
Grupe˛ I stanowiły kurcze˛ta brojlery odchowywane w warunkach standardowych, grupe˛ II doświadczalna˛ — kurcze˛ta odchowywane w brojlerni z zamontowanym gruntowym wymiennikiem ciepła
(GWC), który powinien spełniać role˛ klimatyzatora, a zatem zoptymalizować warunki termiczne
odchowu kurcza˛t.
Stwierdzono, że w brojlerni z zamontowanym GWC temperatura powietrza była o 2 – 3oC niższa
niż w brojlerni kontrolnej. Niższa, a zatem optymalna temperatura w budynku doświadczalnym
wpłyne˛ła na statystycznie wysoko istotnie wyższa˛ mase˛ ciała, lepsze wykorzystanie paszy oraz mniejsza˛
śmiertelność ptaków. Lepsze wyniki produkcyjne ptaków z grupy doświadczalnej miały swoje
odzwierciedlenie w wyższym EWW.
INSTRUCTIONS TO AUTHORS OF RESEARCH PAPERS
PUBLISHED IN THE ‘‘ANNALS OF ANIMAL SCIENCE”
I. General Rules
1. The ‘‘Annals of Animal Science” include original research papers which have not been published
either in part or as a whole in any other scientific journal, except for proceedings of symposia and
scientific conferences. The submitted papers should be written and documented so as to form an
integrated whole.
2. The ‘‘Annals of Animal Science” cover the following range of topics: genetics and farm animal
breeding; the biology, physiology and reproduction of animals; animal nutrition and feedstuffs;
environment, hygiene and animal production technology; economics and the organization of animal
production.
The assignment of a paper to a given section should be proposed by the author(s), but the final
decision rests with the Editors.
3. Papers are printed in English with a Polish summary.
4. Papers to be published should not exceed 16 manuscript pages (size A4) including tables, figures,
photographs, etc. and a summary.
5. Papers are reviewed by two reviewers who are research workers specializing in the relevant field. One
unfavourable review means that the paper will not be published. The costs of printing are covered by
the authors or by the institutions from which the papers were sent, according to current rates of paper
preparation and printing. Authors will receive 25 offprints of their paper free of charge.
II. Submission of Manuscripts
1. Manuscripts for publication are submitted to the Editor-in-Chief by research workers or the heads of
research institutions where the studies were carried out, who take responsibility for their content,
scientific value and the preparation of the text.
2. Manuscripts should be submitted in triplicate to: The Editors of ‘‘Annals of Animal Science”,
National Research Institute of Animal Production, Sarego 2, 31-047 Kraków, Poland, tel. (+48)
12 422-73-33, fax (+48) 12 422-80-65, e-mail: [email protected] on diskettes, and meet the
following requirements:
Diskettes: 3 1/2′′ format
Printout: 3 copies
Word processor: Microsoft Word
Paper size: A4 (210 × 297 mm)
Font: CG Times 11 pt or Times New Roman 12 pt
Margins: 25 mm (left, right, top, bottom)
Line spacing: double
Justification: full
Formulae: equation editor
180
Tables: table function
The ENTER key should only be used to start a new paragraph.
3. Attached to the manuscript should be the Manuscript Submission Form as appended at the end of this
Instruction.
III. Layout of the Text
1. Title page (unnumbered), not included in the paper volume, should contain: the title of the paper, the
full name(s) of author(s) with superscript numbers indicating the full postal address (postcode, street,
no.) of the department and affiliated institution where the study was carried out, e.g.:
Jan Kowalski1, Maria Anna Rokicka2, Adam Nowacki3
Department of Zoology, Jagiellonian University, św. Anny 12, 30-017 Kraków, Poland
2
Department of Immuno- and Cytogenetics, National Research Institute of Animal Production,
3
Experimental Station of the National Research Institute of Animal Production,
39-331 Chorzelów, Poland
1
abbreviated title (5-6 words as in the paper title or synonyms)
source of research financing, e.g. work financed from:
statutory activity, project no.
author’s project of the Ministry of Science and Information Society Technologies, project no.
funds of the Ministry of Agriculture and Rural Development, project no.
2. Manuscripts should be organized in the following order:
a) Abstract. Not more than 15-20 lines in length, containing the aim, principal methods and most
important results of the experiment.
b) Key words. Maximum five items that best describe the paper’s content, beginning with words
of wider meaning, e.g. ruminants, dairy cows, somatotropin, prolactin, milk.
c) Abbreviations. Abbreviations, if any, should be defined immediately below Key words.
d) Introduction. This should justify the research based on references and conclude with a clearly
formulated aim of the study or research hypothesis.
e) Material and methods. This section should contain all information needed to replicate
the experiment, e.g. experimental factors, experimental design, species, breed, sex and number
of animals, duration of experiment, feed rations and their composition, laboratory techniques
and statistical methods used. In the descriptions of methods (biological, chemical, statistical)
it is enough to refer to source material if applied accordingly. Modifications made to the methods
should be described in detail.
f) Results can be presented in tabular or graphic form (figures, diagrams, photographs) and given
a brief description. The text of the description should not repeat tabular data.
g) Discussion. This should interpret the results in terms of the influence of experimental factors
according to the aim of the experiment or to the hypothesis made in the Introduction.
The experimental results should be interpreted using the current state of knowledge to help
the reader accept or reject the hypothesis tested. This section should conclude with a summing-up
and generalization of the results obtained. The direction of further studies in the relevant field may
be also hinted at here.
h) Acknowledgments (if any).
i) References. Publications cited in the text must be organized in strict alphabetical order according
to name of author. Each citation should include the author’s name and initials, year of publication,
full title of paper, abbreviated name of journal, number of volume and issue, and initial and final
page numbers. When more than one paper published by the same author(s) in the same year
is cited, and the authors appear in the same order, the different papers should be assigned
successive letters of the alphabet (e.g. 1983 a, 1983 b) and arranged chronologically. Below are
provided examples of proper citations of references from scientific journals, congress proceedings
and books (manuals).
181
3.
4.
5.
6.
7.
Papers published in periodicals: Jenkins K.J., Hidiroglou M. (1991). Tolerance of the preruminant
calf for excess manganese or zinc in milk replacer. J. Dairy Sci., 74: 1047-1053.
Papers published in multi-author monographs, occasional publications, symposium or congress proceedings: Miller E.L. (1982). Forage protein in ruminant animal nutrition. The nitrogen
needs of ruminants. In: D.J. Thomas (Editor), Proceedings of an International Symposium on Protein
Requirements for Cattle. Kansas State University, Kansas City, KN, pp. 254 – 269.
Manuals and multi-author books: Bock H.D., Eggum B.O., Low A.G., Simon O., Żebrowska T.
(1989). Editors. Protein metabolism in farm animals: evaluation, digestion, absorption and
metabolism. Oxford, UK, Oxford University Press (1989), 452 pp.
Manuals and books: Cuhna T.J. (1991). Horse feeding and nutrition. San Diego, USA, Academic
Press, Inc. (1991). Second edition, 445 pp.
j) Summary in Polish — the same as the Abstract (Polish summary is not included in the text
volume) with full names of the author(s) and title of the paper.
Literature citations in the text. Research findings (or their authors) should be cited if strictly
connected with the study topic or research methods used. The number of citations should not exceed
the 20 most important items quoted in the text. When a citation has more than two authors, the name
of the first author should be followed with ‘‘et al.”, e.g. Nowacki et al. (1992). Unpublished papers
should be listed in the text, e.g. Błoński (personal communication) or (Błoński, unpublished data).
Tables should present the most important data. The column on the left should list the parameters studied,
while the columns in the middle and on the right should contain the results for individual experimental
factors. Tables should contain numerical data, which are the mean values for a set of observations or
measurements, replications and their statistical interpretation (e.g standard error, coefficient of variation).
Tables numbered consecutively in Arabic numerals should be submitted on separate sheets. The titles of
the tables should be brief. Each column should have a heading. Columns and lines should be spaced. No
vertical lines are allowed. Horizontal lines can only be used for strictly justified purposes.
Tabular data should not be repeated in graphic form (figures, diagrams, etc.). If there are no data for
a given parameter, leave a blank. If an explanation is necessary, use an abbreviation and explain it as
a footnote at the bottom of the table (e.g. ND — not determined or not detected). To designate the
significance of differences between two means or interaction between factors, an additional column is
recommended with the heading ‘‘significance level”, using the signs x, xx, xxx for P ≤ 0.05, 0.01 and
0.001 respectively. When the number of means is greater than two, the significance of differences
should be designated with letters which follow tabular data. Their meaning should be explained below
the bottom line of the table, e.g.:
a, b, c, d — values in rows (or columns) with different letters differ significantly (P ≤ 0.05).
A, B, C, D — as above for P ≤ 0.01.
Statistical interpretation of the results should fit the design of the experiment and the hypotheses
tested.
Figures and photographs. Research results, presented in the form of figures, charts and diagrams,
should be made on tracing paper or in other forms ready for reproduction. A single figure or diagram
must fit half of the text page. Black-and-white or colour photographs of postcard size should have
good contrast. Each figure or photograph should be provided with a brief description of its content
and, if necessary, a legend in English. References to figures or photographs in the text of the paper
should be provided with a reference mark or information.
Figures and photographs should be submitted in two sets.
Abbreviations should be explained on their first appearance. SI units of measure should be used. This
also concerns the energy value of feeds, which should be given in Joules.
Supplementary information. After receiving a review of the paper the authors should follow the
reviewers’ guidelines for changes and corrections, and return all the materials received together with
a corrected version of the manuscript within 10 days at the most. A failure to return it in due time will
delay publication by half a year. The correction of a galley-proof, confirming the final version of the
paper, should be made within 5 days of it being sent to the author(s). If this deadline is not met, the
Editors bear no responsibility for changes made.
place
date
‘‘Annals of Animal Science”
Manuscript Submission Form
AAS issue
To be filled in by Editors
I am asking you to review and print the enclosed paper in ‘‘Annals of Animal Science”
1. Author(s), title of the paper
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Notifying person:
Full name . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Address (postcode, town, street, no.) . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Telephone, fax, e-mail . . . . . . . . . . . . . . . . . . . . . . . .
Institution’s NIP number (for Polish authors only) . . . . . . . . . . . . . . .
3. Suggested section in ‘‘Annals of Animal Science”:
Genetics and farm animal breeding
Biology, physiology and animal reproduction
Animal nutrition and feedstuffs
Environment, hygiene and animal production technology
Economics and organization of animal production
4. I hereby warrant that the manuscript submitted for publication has neither in part nor as a whole been
published or submitted for publication in any other scientific journal.
5. I warrant that the co-authors of this manuscript are familiar with its content and have given consent to
its publication in the presented form.
6. I commit myself to covering the costs of the paper’s publication after it is published, in accordance
with the prices valid at the moment of printing.
...........................
Signature of the notifying person
This journal is included in the
POLISH SCIENTIFIC JOURNALS CONTENTS
— AGRIC.&BIOL. SCI.
database which can be accessed on the World Wide Web at the following
URL address:
http://psjc.icm.edu.pl
Distribution
Subscriptions and single copies of the journal can be ordered from:
Instytut Zootechniki, Zespół Wydawnictw i Poligrafii, 32-083 Balice k. Krakowa, Poland
Annual subscription price is PLN 40.00 within Poland and US$ 35.00 outside Poland.
Printing
Department of Publications and Printing, National Research Institute of Animal Production,