- Email: [email protected]
Effect of Selection for Fertility of Frozen-Thawed Semen in Chickens on the Fertility of Fresh and Stored Semen
BREEDING AND GENETICS Effect of Selection for Fertility of Frozen-Thawed Semen in Chickens on the Fertility of Fresh and Stored Semen Y. F. YOUSIF, 1 G. A. ANSAH, and R. B. BUCKLAND Department of Animal Science, Macdonald College of McGill University, Ste-Anne de Bellevue, Quebec, Canada H9X 1 CO (Received for publication March 21, 1983)
1984 Poultry Science 63:1475-1480 INTRODUCTION Studies o n t h e d e v e l o p m e n t of techniques to store chicken semen in vitro b y freezing to — 79 or —196 C have used diluents with differing composition and levels of cryopreservative agents (Polge et al, 1 9 4 9 ; Lake, 1 9 6 8 ; Sexton, 1976; Lake and Stewart, 1 9 7 8 ) . Mitchell et al. ( 1 9 7 7 ) froze chicken semen to - 1 9 6 C in a diluent containing 13.6% glycerol as the major cryopreservative agent and demonstrated t h e feasibility of improving t h e fertility of frozen-thawed semen by selection. T h e y reported a heritability estimate of .12 for t h e fertility of frozen-thawed semen. S e x t o n et al. (1978) e m p l o y e d t w o freezing procedures to
1 Department of Poultry Science, University of British Columbia, Vancouver, BC V6T 2A2.
freeze t h e semen of a line of chickens selected for improved fertility of frozen-thawed semen and t h a t of t h e control line (Generation 3 males of t h e lines used in the present s t u d y ) in t w o diluents containing either glycerol or d i m e t h ylsulphoxide (DMSO) as the major cryopreservative agent. T h e y reported t h a t resistance or susceptibility t o freeze damage was n o t cryopreservative or p r o c e d u r e specific. Mitchell et al. ( 1 9 7 7 ) obtained low p h e n o typic correlations between the fertility of frozen-thawed and fresh semen and suggested t h a t t h e limiting factors with respect to these traits are probably different. Scott et al. ( 1 9 8 0 ) and Ansah and Buckland ( 1 9 8 3 ) have, however, observed positive correlated responses in t h e fertility of fresh semen through selection for increased d u r a t i o n of fertility of frozen-thawed semen.
1475
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 22, 2014
ABSTRACT Studies were conducted using 68 males from the seventh generation of a line of meat-type breeder chickens selected for increased duration of fertility of frozen-thawed semen and 69 males of the randomly selected control line. These chickens were used to determine the presence or absence of differences between the selected and control lines with respect to fertility and spermatozoal motility when the semen was subjected to the following preinsemination treatments: 1) frozen in a diluent containing 13.6% glycerol; 2) frozen in a diluent containing 1.7% glycerol; 3) fresh undiluted; 4) fresh diluted; 5) diluted and stored for 24 hr; and 6) diluted and stored for 48 hr. Semen from each male was inseminated into 4 to 6 White Leghorn tester hens per experiment. The effects of genotype (line) by preinsemination semen treatment interaction on fertility and spermatozoal motility were also examined. When semen was frozen (Treatments 1 and 2), the selected line had significantly (P<.01) higher duration of fertility, percent fertility for 7 days postinsemination, and percent fertility for the duration of fertility and spermatozoal motility than the control line. For fresh semen (Treatments 3 and 4), the selected line had significantly (P<.01) higher fertility estimates and spermatozoal motility than the control line except for percent fertility for the duration of fertility (P>.05) of undiluted semen. For stored semen (Treatments 5 and 6), the selected line only had significantly (P<.05) higher duration of fertility after 24 hr storage and percent fertility for the duration of fertility after 48 hr storage than the control line. When the effects of line by preinsemination semen treatment interaction on fertility estimates and spermatozoal motility were examined in a series of comparisons involving the treatment used in previous generations of selection (Treatment 1) and each of the other treatments (2, 3,4, 5, and 6), there was no effect for the fertility of frozen-thawed semen (Treatment 2) and the fertility of fresh semen (Treatments 3 and 4) except for the percent fertility for the duration of fertility of fresh semen. However, the effect of the interaction was significant (P<.05) for the fertility and spermatozoal motility of stored semen (Treatments 5 and 6) and the spermatozoal motility of frozen-thawed semen (Treatment 2) and undiluted fresh semen (Treatment 3). (Key words: chicken, selection, fertility, frozen-thawed semen, stored semen, fresh semen)
1476
YOUSIF ET AL.
MATERIALS AND METHODS A maximum of 68 males from Generation 7 of a line of meat-type breeder chickens selected for increased duration of fertility of frozenthawed semen and 69 males of the randomly selected control line which were about 11 months of age were used. Glycerol was the major cyropreservative agent in the diluent (Lake and Stewart, 1978) used to freeze the semen in each of the previous six generations. The two lines that originated from a male broiler breeder control population and the selection procedures have been described previously (Mitchell et al., 1977; Ansah and Buckland, 1983). Two to 4 males in the selected line and 1 to 3 males in the control line were chosen randomly as semen donors from each of the 20 and 33 sire families in the respective lines of the population. Semen was collected from individual males in groups of 12 (6 selected and 6 control) by the massage technique (Burrows and Quinn, 1937), and individual samples were subjected to the following preinsemination treatments: 1) diluted in a diluent containing 13.6% glycerol (the protocol used in the previous generations of selection) and frozen in liquid nitrogen to
— 196 C at an average rate of 6 C per min; 2) diluted in Extender II of Macpherson et al. (1969) containing 1.7% glycerol and frozen as in (Treatment 1); 3) fresh undiluted; 4) fresh diluted; 5) diluted and stored at 5 C for 24 hr; 6) diluted and stored at 5 C for 48 hr. In Treatments 4 to 6, semen was diluted 1:3 with a 7.05 pH diluent (Table 1). A separate ejaculate was collected for each treatment. Two experiments (Trials 1 and 2) were conducted for each preinsemination semen treatment except for fresh undiluted semen which involved a single experiment. White Leghorn hens of the same age as the males were used to test the fertility of the males in each treatment. All inseminations were intravaginally between 1300 and 1500 hr. To determine the fertility of frozen semen, the frozen samples were thawed in an ice bath (2 to 5 C), the glycerol removed by sequential dilution with a diluent containing no glycerol (Lake and Stewart, 1978) (Table 1) and centrifugation at 700 X g for 10 min. After the removal of supernatant following centrifugation, the samples were allowed to stand for about 1 min and the remaining supernatant removed by aspiration. The spermatozoa were resuspended in the diluent containing no glycerol to approximately the initial volume of the undiluted semen. A constant volume of .07 ml of resuspended spermatozoal containing approximately 2.3 x 10 8 spermatozoa was inseminated into each of 4 to 6 White Leghorn tester hens per male. For fresh semen, the individual ejaculates were inseminated into hens either in the undiluted or diluted state. A constant volume of .05 ml of undiluted and .1 ml of diluted semen containing approximately 4.0 x 10 8 and 3.6 X 10 8 spermatozoa, respectively, was inseminated into each of 4 to 6 tester White Leghorn hens per male. For stored semen, after storage at 5 C for either 24 hr or 48 hr, a constant volume of .1 ml of diluted semen containing approximately 4.1 X 10 8 spermatozoa was inseminated into each of 4 to 6 tester White Leghorn hens per male. The motility of spermatozoa was determined microscopically in duplicate immediately after each insemination from the remaining samples. A subjective score of 0 to 5 was assigned with 0 indicating no movement and 5 indicating very vigorous wave-line movement (Allen and Champion, 1955).
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 22, 2014
Wilcox et al. (1961) observed significant breed differences in the fertility of semen stored at 10 C but found no differences with respect to the fertility of fresh semen. Buckland (1971a) observed greater family differences for semen stored at 2 to 5 C for 48 hr than for fresh semen. Furthermore, Buckland (1971b) and Phillip et al. (1974) reported that the correlation coefficients between stored (2 to 5 C) and fresh semen were generally low and that the fertility of fresh semen was a poor indicator of fertility after in vitro storage. The present study examined the presence or absence of differences in fertility and spermatozoal motility between a line of chickens selected over seven generations for increased duration of fertility of frozen-thawed semen using a diluent containing 13.6% glycerol (Lake and Stewart, 1978) as the major cryopreservative agent and the control line when the semen was: 1) frozen in two diluents containing either 13.6% or 1.7% glycerol; b) inseminated fresh in the undiluted and diluted states, or diluted and stored for 24 and 48 hr. The effects of genotype (line) by preinsemination semen treatment interaction on fertility and spermatozoal motility were also examined.
SELECTION FOR SEMEN FERTILITY
1477
TABLE 1. Composition of diluents used Frozen semen
Compound 1 Sodium glutamate-HjO Sodium acetate (anhydrous)
pH
Osmolarity 1
Fresh and stored semen (Lake and Ravie, 1979)
Macpherson's Extender II (Macpherson etal., 1969)
1.9200
1.3805
0800
Deglycerolizing solution (Lake and Stewart, 1978)
1.5200
1.9200 5100
0800
0800
.0244 1280 5000 1280 8000
6000 .1500 11.000
6000 3 0500
3000
1.0000 3.3 ml 5 8 ml
13 6400 6 80
2581
1.7 ml 6.40 2158
7 05 411
6 75 365
Values are in grams unless otherwise stated, dissolved and made up to 100 ml with distilled water.
Eggs were collected daily, stored at 3 to 5 C for 5 to 7 days, and incubated. Fertility was determined by candling on Day 7 of incubation. Duration of fertility was estimated as the number of days from Day 2 after insemination to the day before three consecutive infertile eggs were laid. Percent fertility for 7 days postinsemination was the percentage of fertile eggs laid from Days 2 to 8 after a single insemination. Percent fertility for the duration of fertility was the percentage of fertile eggs laid. All percentage data were transformed to arc sin for the analyses of variance. The data were analyzed by the General Linear Model (GLM) procedure of the Statistical Analysis System (SAS) (Barr et al, 1976) to determine the significance (P<.05) of the main effects (line, trial, day, and time of day of insemination) for each semen treatment where applicable. In a series of comparisons involving the treatment used in previous generations of selection (Treatment 1) and each of the other preinsemination treatments (2, 3, 4, 5 and 6) individually, the significance (P<.05) of line by semen treatment interaction was tested. The model assumed was: Yijkmno = M + aj + dj + pk + t m + l n + tlmn + e ijkmno
where: Yijkmno denotes the kjkmnoth observation, M ai d
J
Pk tm In tlmn
e
ijkmno
population mean, effect of the it h trial (i = 1, 2), effect of the j t n day (j = 1, 2, . . .11), effect of the k t n time of day of insemination (k = 1, 2, 3), effect of the m t n semen treatment (m = 1, 2,), effect of the n t n line (n = 1, 2), effect of the interaction between the m t n semen treatment and n t n line, the error associated with the ijkmno t n observation assumed to be independently distributed (0, op.
RESULTS AND DISCUSSION
The means and standard deviations of fertility and spermatozoal motility estimates of frozen-thawed, fresh, and stored semen are presented in Table 2. Frozen-Thawed Semen. The selected line was significantly higher than the control line when semen was frozen in either Lake's diluent
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 22, 2014
Mg Acetate • 4 H 2 0 Mg Chloride-6H 2 0 K Citrate-H 2 0 K Acetate (anhydrous) Tri-KCitrate-H 2 0 Fructose Glucose Lactose N, N-bis[2-Hydroxyethyl] 2-aminoethane sulfonic acid Polyvinylpyrrolidinone N,N Dimethylacetamide N-NaOH Glycerol
Lake's diluent (Lake and Stewart, 1978)
1478
YOUSIFETAL. TABLE 2. Means and standard deviations of fertility and spermatozoal motility of semen after various preinsemination treatments
Duration of fertility (days)
Line
% Fertility for 7 days
% Fertility for the duration of fertility
Spermatozoal motility
Frozen-thawed (Lake's diluent) 58 57
5.0+ 2.7** 2.7 ± 2.5
Selected Control
61 62
6.1 ± 2.9** 4.1 ± 2.9
45.4 ±25.2** 27.9 ± 24.0
Selected Control
68 61
13.3 + 1.4** 12.0+ 3.5
90.1 + 8.4* 81.7 ± 21.8
Selected Control
66 66
12.8 ± 1.9** 11.5 ± 3.2
88.3 ± 11.0* 78.5 ± 21.7
Selected Control
65 63
7.4 ± 3.2* 6.5 ± 3.3
Selected Control
66 69
3.5 ± 3.1 2.8 ± 2.8
39.1 ± 23.5** 20.6 ± 19.6
49.7 + 23.1** 31.9 ± 25.5
2.7 ± 1.2** 1.7 ± 1.2
Frozen-thawed (Macpherson's Extender II) 53.0 ±21.5** 40.5 ± 24.6
2.0 ± 1.2** 1.6 ± 1.1
80.5 + 7.3 74.0 ± 17.5
3.8+ .9* 3.3 ± 1.0
80.9 ± 8.4* 73.8+ 15.9
3.8 ± .9* 3.2 ± 1.2
51.4 ± 22.0 45.9 ± 23.2
55.1 ± 19.3 50.1 ± 20.9
3.4+ 1.3* 3.0 ± 1.4
24.1 ± 21.5 19.2 ± 19.8
37.2 ± 22.1* 30.1 ± 21.6
2.7 ± 1.3 2.3 ± 1.2
Fresh undiluted
Fresh diluted
Stored (24 hr)
1
n = Number of males tested.
*Significant line differences (P<.05). **Significant line differences (P<.01).
or Macpherson's E x t e n d e r II for each of t h e four parameters: d u r a t i o n of fertility, percent fertility for 7 days, p e r c e n t fertility for t h e d u r a t i o n of fertility, and spermatozoal motility. T h e difference b e t w e e n t h e selected and c o n t r o l lines for d u r a t i o n of fertility was similar for t h e two semen t r e a t m e n t s ( 1 , 2). Furtherm o r e , t h e correlated responses in t h e o t h e r fertility estimates were also similar. T h e r e was n o line b y semen t r e a t m e n t interaction for t h e fertility of frozen-thawed semen using Macpherson's E x t e n d e r II in comparison with t h a t of Lake's diluent. These results indicate t h a t selection has n o t been specific to a particular diluent and level of glycerol (13.6 vs. 1.7%). T h u s , resistance of chicken s p e r m a t o z o a to freeze damage m a y n o t be diluent specific for diluents containing glycerol. S e x t o n et al. ( 1 9 7 8 ) reported t h a t resistance or susceptibility of spermatozoa to freeze damage was n o t cryopreservative-specific. F o r spermatozoal m o -
tility, t h e interaction was significant ( P < . 0 1 ) , indicating t h a t the magnitude of response of the selected line varied u n d e r t h e t w o freezing t r e a t m e n t s . Fresh Semen. T h e selected line was significantly higher t h a n t h e control line for three of t h e four parameters measured for fresh undiluted semen. T h e p e r c e n t fertility for t h e d u r a t i o n of fertility was higher in t h e selected line t h a n t h e control line, b u t t h e difference was n o t significant. F o r diluted fresh semen, the selected line was significantly higher than the control line for t h e four parameters. These correlated increases indicate t h a t selection for improved fertility based on frozen-thawed semen m a y yield improved fertility of fresh semen. Scott et al. ( 1 9 8 0 ) and Ansah and Buckland ( 1 9 8 3 ) tested the males of Generations 3, and 4 through 8, respectively, of t h e two lines used in t h e s t u d y reported herein for fertility of undiluted fresh semen and observed
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 22, 2014
Selected Control
1479
SELECTION FOR SEMEN FERTILITY
Stored Semen. The selected line was above t h e control line for t h e four parameters. However, significant ( P < . 0 5 ) differences occurred only for t h e d u r a t i o n of fertility and sperm a t o z o a l motility for semen stored 2 4 hr and percent fertility for t h e d u r a t i o n of fertility for semen stored 48 hr. T h e r e was a significant ( P < . 0 5 ) line b y semen t r e a t m e n t interaction for the fertility and s p e r m a t o z o a l motility of semen stored for either 2 4 or 48 hr in comparison with frozen-thawed semen. This was attributed to t h e narrowing of t h e difference in fertility and spermatozoal m o t i l i t y b e t w e e n the selected and control lines with stored semen as opposed to frozen-thawed semen. These results suggest t h a t different factors m a y be responsible for t h e p r o t e c t i o n and s u b s e q u e n t fertility of s p e r m a t o z o a t h a t are frozen and thawed and those stored in t h e nonfrozen state. This suggestion is consistent with t h e r e p o r t s b y Ansah and Buckland ( 1 9 8 2 a , b ) o n fowl semen
cholesterol levels and t h e u p t a k e of glycerol b y spermatozoa, t h a t indicated selection for d u r a t i o n of fertility of frozen-thawed semen using glycerol as cryoprotective agent m a y be, in part, selection for increased s p e r m a t o zoa permeability t o glycerol and t h a t in t h e t w o lines used in this s t u d y s p e r m a t o z o a w i t h s t a n d freeze-damage b e t t e r as t h e y take u p m o r e glycerol. REFERENCES Allen, C. J., and L. R. Champion, 1955. Competitive fertilization in the fowl. Poultry Sci. 34:1332— 1342. Ansah, G. A., and R. B. Buckland, 1983. Eight generations of selection for duration of fertility of frozen-thawed semen in the chicken. Poultry Sci. 62:1529-1538. Ansah, G. A., and R. B. Buckland, 1982a. Genetic variation in fowl semen cholesterol and phospholipid levels and the relationships of these lipids with fertility of frozen-thawed and fresh semen. Poultry Sci. 61:623-637. Ansah, G. A., and R. B. Buckland, 1982b. The uptake of glycerol, a-aminoisobutyric acid and 2-deoxyD-glucose by spermatozoa of a line of chickens selected for fertility of frozen-thawed semen and a control line. Theriogenology 17:401—408. Barr, A. J., J. H. Goodnight, J. P. Sail, and J. T. Helwig, 1976. A User's Guide to SAS-76. SAS Inst. Inc., Raleigh, NC. Buckland, R. B., 1971a. The activity of six enzymes of chicken seminal plasma and sperm. 1. Effect of in vitro storage and full-sib families on enzyme activity and fertility. Poultry Sci. 50:1724— 1734. Buckland, R. B., 1971b. The activity of six enzymes of chicken seminal plasma and sperm. 2. The relationship between enzyme activity and fertility of fresh and stored semen. Poultry Sci. 50:1734-1742. Burrows, W. H., and J. P. Quinn, 1937. Collection of spermatozoa from domestic fowl and turkey. Poultry Sci. 16:19-24. Lake, P. E., 1968. Observations on freezing fowl spermatozoa in liquid nitrogen. VI Congr. Int. Reprod. Anim. Artif. Insem., Paris, 2:1633 — 1635. Lake, P. E., and O. Ravie, 1979. Effect on fertility of storing fowl semen for 24 hr at 5°C in fluids of different pH. J. Reprod. Fertil. 57:149-155. Lake, P. E., and J. M. Stewart, 1978. Preservation of fowl semen in liquid nitrogen — An improved method. Br. Poult. Sci. 19:187-194. Macpherson, J. W., S. Chatterjee, and G. W. Friars, 1969. Frozen turkey semen. Can. J. Comp. Med. 33:37-38. Mitchell, R. L., R. B. Buckland, and B. W. Kennedy, 1977. Heritability of frozen and fresh chicken semen and the relationship between the fertility of frozen and fresh semen. Poultry Sci. 56: 1168-1177. Phillip, L. E., R. B. Buckland, and D. E. Bernon, 1974. A note of the relationship between the fertility
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 22, 2014
positive correlated responses in t h e fertility of fresh semen. Mitchell et al. ( 1 9 7 7 ) , however, have previously suggested t h a t fertility of fresh chickens semen is a p o o r indicator of t h e fertilizing ability of frozen-thawed semen. A l t h o u g h this suggestion m a y be correct for their s t u d y considering t h e low fertility levels with frozent h a w e d semen t h e y o b t a i n e d , with improved cryogenic technology, as several investigators have accomplished for b o t h avian and mammalian spermatozoa, a positive correlation m a y exist as indicated b y t h e results of previous studies and t h e present one. There was an absence of a line b y semen t r e a t m e n t interaction for d u r a t i o n of fertility and percent fertility for 7 days postinsemination in comparison with frozen-thawed semen t h a t further demonstrates that improved fertility of frozen-thawed semen through selection m a y also hold for fresh semen. A significant ( P < . 0 5 ) interaction effect was obtained for percent fertility for t h e d u r a t i o n of fertility. This was perhaps due to chance as t h e p h e n o t y p i c correlations a m o n g d u r a t i o n of fertility, percent fertility for 7 days postinsemination, and p e r c e n t fertility for the d u r a t i o n of fertility of fresh semen o b t a i n e d in this s t u d y , which ranged from .3 7 t o . 9 3 , were positive and significant ( P < . 0 1 ) . Similar positive and high p h e n o t y p i c correlations were obtained by Mitchell et al. ( 1 9 7 7 ) , Scott et al. ( 1 9 8 0 ) , and Ansah and Buckland ( 1 9 8 3 ) . T h e interaction was also significant for t h e spermatozoal motility of undiluted semen.
1480
YOUSIF ET AL.
of fresh semen and that stored varying lengths of time and the effect of storage on duration and percent fertility. Poultry Sci. 53:2216-2218. Polge, C , A. U. Smith, and A. S. Parkes, 1949. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164:666. Scott, T. A., R. B. Buckland, and B. W. Kennedy, 1980. The effect of selection for fertility of frozen-thawed semen on spermatozoa oxygen uptake, motility and concentration and ejaculate volume in the chicken. Theriogenology 14:
281-298. Sexton, T. J., 1976. Studies on the fertility of frozen fowl semen. Pages 1079-1081 in Proc. Vlllth Int. Congr. Anim. Reprod. Artif. Insem., Krakow. Sexton, T. J., R. B. Buckland, and R. Lopez, 1978. Comparison of two procedures for freezing semen from cocks of high and low fertility with frozen semen. Poultry Sci. 57:550—552. Wilcox, F. H., C. S. Shaffner, and H. R. Wilson, 1961. Breed differences in storing chicken semen. J. Hered. 52:119-121.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 22, 2014
1984 Poultry Science 63:1475-1480 INTRODUCTION Studies o n t h e d e v e l o p m e n t of techniques to store chicken semen in vitro b y freezing to — 79 or —196 C have used diluents with differing composition and levels of cryopreservative agents (Polge et al, 1 9 4 9 ; Lake, 1 9 6 8 ; Sexton, 1976; Lake and Stewart, 1 9 7 8 ) . Mitchell et al. ( 1 9 7 7 ) froze chicken semen to - 1 9 6 C in a diluent containing 13.6% glycerol as the major cryopreservative agent and demonstrated t h e feasibility of improving t h e fertility of frozen-thawed semen by selection. T h e y reported a heritability estimate of .12 for t h e fertility of frozen-thawed semen. S e x t o n et al. (1978) e m p l o y e d t w o freezing procedures to
1 Department of Poultry Science, University of British Columbia, Vancouver, BC V6T 2A2.
freeze t h e semen of a line of chickens selected for improved fertility of frozen-thawed semen and t h a t of t h e control line (Generation 3 males of t h e lines used in the present s t u d y ) in t w o diluents containing either glycerol or d i m e t h ylsulphoxide (DMSO) as the major cryopreservative agent. T h e y reported t h a t resistance or susceptibility t o freeze damage was n o t cryopreservative or p r o c e d u r e specific. Mitchell et al. ( 1 9 7 7 ) obtained low p h e n o typic correlations between the fertility of frozen-thawed and fresh semen and suggested t h a t t h e limiting factors with respect to these traits are probably different. Scott et al. ( 1 9 8 0 ) and Ansah and Buckland ( 1 9 8 3 ) have, however, observed positive correlated responses in t h e fertility of fresh semen through selection for increased d u r a t i o n of fertility of frozen-thawed semen.
1475
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 22, 2014
ABSTRACT Studies were conducted using 68 males from the seventh generation of a line of meat-type breeder chickens selected for increased duration of fertility of frozen-thawed semen and 69 males of the randomly selected control line. These chickens were used to determine the presence or absence of differences between the selected and control lines with respect to fertility and spermatozoal motility when the semen was subjected to the following preinsemination treatments: 1) frozen in a diluent containing 13.6% glycerol; 2) frozen in a diluent containing 1.7% glycerol; 3) fresh undiluted; 4) fresh diluted; 5) diluted and stored for 24 hr; and 6) diluted and stored for 48 hr. Semen from each male was inseminated into 4 to 6 White Leghorn tester hens per experiment. The effects of genotype (line) by preinsemination semen treatment interaction on fertility and spermatozoal motility were also examined. When semen was frozen (Treatments 1 and 2), the selected line had significantly (P<.01) higher duration of fertility, percent fertility for 7 days postinsemination, and percent fertility for the duration of fertility and spermatozoal motility than the control line. For fresh semen (Treatments 3 and 4), the selected line had significantly (P<.01) higher fertility estimates and spermatozoal motility than the control line except for percent fertility for the duration of fertility (P>.05) of undiluted semen. For stored semen (Treatments 5 and 6), the selected line only had significantly (P<.05) higher duration of fertility after 24 hr storage and percent fertility for the duration of fertility after 48 hr storage than the control line. When the effects of line by preinsemination semen treatment interaction on fertility estimates and spermatozoal motility were examined in a series of comparisons involving the treatment used in previous generations of selection (Treatment 1) and each of the other treatments (2, 3,4, 5, and 6), there was no effect for the fertility of frozen-thawed semen (Treatment 2) and the fertility of fresh semen (Treatments 3 and 4) except for the percent fertility for the duration of fertility of fresh semen. However, the effect of the interaction was significant (P<.05) for the fertility and spermatozoal motility of stored semen (Treatments 5 and 6) and the spermatozoal motility of frozen-thawed semen (Treatment 2) and undiluted fresh semen (Treatment 3). (Key words: chicken, selection, fertility, frozen-thawed semen, stored semen, fresh semen)
1476
YOUSIF ET AL.
MATERIALS AND METHODS A maximum of 68 males from Generation 7 of a line of meat-type breeder chickens selected for increased duration of fertility of frozenthawed semen and 69 males of the randomly selected control line which were about 11 months of age were used. Glycerol was the major cyropreservative agent in the diluent (Lake and Stewart, 1978) used to freeze the semen in each of the previous six generations. The two lines that originated from a male broiler breeder control population and the selection procedures have been described previously (Mitchell et al., 1977; Ansah and Buckland, 1983). Two to 4 males in the selected line and 1 to 3 males in the control line were chosen randomly as semen donors from each of the 20 and 33 sire families in the respective lines of the population. Semen was collected from individual males in groups of 12 (6 selected and 6 control) by the massage technique (Burrows and Quinn, 1937), and individual samples were subjected to the following preinsemination treatments: 1) diluted in a diluent containing 13.6% glycerol (the protocol used in the previous generations of selection) and frozen in liquid nitrogen to
— 196 C at an average rate of 6 C per min; 2) diluted in Extender II of Macpherson et al. (1969) containing 1.7% glycerol and frozen as in (Treatment 1); 3) fresh undiluted; 4) fresh diluted; 5) diluted and stored at 5 C for 24 hr; 6) diluted and stored at 5 C for 48 hr. In Treatments 4 to 6, semen was diluted 1:3 with a 7.05 pH diluent (Table 1). A separate ejaculate was collected for each treatment. Two experiments (Trials 1 and 2) were conducted for each preinsemination semen treatment except for fresh undiluted semen which involved a single experiment. White Leghorn hens of the same age as the males were used to test the fertility of the males in each treatment. All inseminations were intravaginally between 1300 and 1500 hr. To determine the fertility of frozen semen, the frozen samples were thawed in an ice bath (2 to 5 C), the glycerol removed by sequential dilution with a diluent containing no glycerol (Lake and Stewart, 1978) (Table 1) and centrifugation at 700 X g for 10 min. After the removal of supernatant following centrifugation, the samples were allowed to stand for about 1 min and the remaining supernatant removed by aspiration. The spermatozoa were resuspended in the diluent containing no glycerol to approximately the initial volume of the undiluted semen. A constant volume of .07 ml of resuspended spermatozoal containing approximately 2.3 x 10 8 spermatozoa was inseminated into each of 4 to 6 White Leghorn tester hens per male. For fresh semen, the individual ejaculates were inseminated into hens either in the undiluted or diluted state. A constant volume of .05 ml of undiluted and .1 ml of diluted semen containing approximately 4.0 x 10 8 and 3.6 X 10 8 spermatozoa, respectively, was inseminated into each of 4 to 6 tester White Leghorn hens per male. For stored semen, after storage at 5 C for either 24 hr or 48 hr, a constant volume of .1 ml of diluted semen containing approximately 4.1 X 10 8 spermatozoa was inseminated into each of 4 to 6 tester White Leghorn hens per male. The motility of spermatozoa was determined microscopically in duplicate immediately after each insemination from the remaining samples. A subjective score of 0 to 5 was assigned with 0 indicating no movement and 5 indicating very vigorous wave-line movement (Allen and Champion, 1955).
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 22, 2014
Wilcox et al. (1961) observed significant breed differences in the fertility of semen stored at 10 C but found no differences with respect to the fertility of fresh semen. Buckland (1971a) observed greater family differences for semen stored at 2 to 5 C for 48 hr than for fresh semen. Furthermore, Buckland (1971b) and Phillip et al. (1974) reported that the correlation coefficients between stored (2 to 5 C) and fresh semen were generally low and that the fertility of fresh semen was a poor indicator of fertility after in vitro storage. The present study examined the presence or absence of differences in fertility and spermatozoal motility between a line of chickens selected over seven generations for increased duration of fertility of frozen-thawed semen using a diluent containing 13.6% glycerol (Lake and Stewart, 1978) as the major cryopreservative agent and the control line when the semen was: 1) frozen in two diluents containing either 13.6% or 1.7% glycerol; b) inseminated fresh in the undiluted and diluted states, or diluted and stored for 24 and 48 hr. The effects of genotype (line) by preinsemination semen treatment interaction on fertility and spermatozoal motility were also examined.
SELECTION FOR SEMEN FERTILITY
1477
TABLE 1. Composition of diluents used Frozen semen
Compound 1 Sodium glutamate-HjO Sodium acetate (anhydrous)
pH
Osmolarity 1
Fresh and stored semen (Lake and Ravie, 1979)
Macpherson's Extender II (Macpherson etal., 1969)
1.9200
1.3805
0800
Deglycerolizing solution (Lake and Stewart, 1978)
1.5200
1.9200 5100
0800
0800
.0244 1280 5000 1280 8000
6000 .1500 11.000
6000 3 0500
3000
1.0000 3.3 ml 5 8 ml
13 6400 6 80
2581
1.7 ml 6.40 2158
7 05 411
6 75 365
Values are in grams unless otherwise stated, dissolved and made up to 100 ml with distilled water.
Eggs were collected daily, stored at 3 to 5 C for 5 to 7 days, and incubated. Fertility was determined by candling on Day 7 of incubation. Duration of fertility was estimated as the number of days from Day 2 after insemination to the day before three consecutive infertile eggs were laid. Percent fertility for 7 days postinsemination was the percentage of fertile eggs laid from Days 2 to 8 after a single insemination. Percent fertility for the duration of fertility was the percentage of fertile eggs laid. All percentage data were transformed to arc sin for the analyses of variance. The data were analyzed by the General Linear Model (GLM) procedure of the Statistical Analysis System (SAS) (Barr et al, 1976) to determine the significance (P<.05) of the main effects (line, trial, day, and time of day of insemination) for each semen treatment where applicable. In a series of comparisons involving the treatment used in previous generations of selection (Treatment 1) and each of the other preinsemination treatments (2, 3, 4, 5 and 6) individually, the significance (P<.05) of line by semen treatment interaction was tested. The model assumed was: Yijkmno = M + aj + dj + pk + t m + l n + tlmn + e ijkmno
where: Yijkmno denotes the kjkmnoth observation, M ai d
J
Pk tm In tlmn
e
ijkmno
population mean, effect of the it h trial (i = 1, 2), effect of the j t n day (j = 1, 2, . . .11), effect of the k t n time of day of insemination (k = 1, 2, 3), effect of the m t n semen treatment (m = 1, 2,), effect of the n t n line (n = 1, 2), effect of the interaction between the m t n semen treatment and n t n line, the error associated with the ijkmno t n observation assumed to be independently distributed (0, op.
RESULTS AND DISCUSSION
The means and standard deviations of fertility and spermatozoal motility estimates of frozen-thawed, fresh, and stored semen are presented in Table 2. Frozen-Thawed Semen. The selected line was significantly higher than the control line when semen was frozen in either Lake's diluent
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 22, 2014
Mg Acetate • 4 H 2 0 Mg Chloride-6H 2 0 K Citrate-H 2 0 K Acetate (anhydrous) Tri-KCitrate-H 2 0 Fructose Glucose Lactose N, N-bis[2-Hydroxyethyl] 2-aminoethane sulfonic acid Polyvinylpyrrolidinone N,N Dimethylacetamide N-NaOH Glycerol
Lake's diluent (Lake and Stewart, 1978)
1478
YOUSIFETAL. TABLE 2. Means and standard deviations of fertility and spermatozoal motility of semen after various preinsemination treatments
Duration of fertility (days)
Line
% Fertility for 7 days
% Fertility for the duration of fertility
Spermatozoal motility
Frozen-thawed (Lake's diluent) 58 57
5.0+ 2.7** 2.7 ± 2.5
Selected Control
61 62
6.1 ± 2.9** 4.1 ± 2.9
45.4 ±25.2** 27.9 ± 24.0
Selected Control
68 61
13.3 + 1.4** 12.0+ 3.5
90.1 + 8.4* 81.7 ± 21.8
Selected Control
66 66
12.8 ± 1.9** 11.5 ± 3.2
88.3 ± 11.0* 78.5 ± 21.7
Selected Control
65 63
7.4 ± 3.2* 6.5 ± 3.3
Selected Control
66 69
3.5 ± 3.1 2.8 ± 2.8
39.1 ± 23.5** 20.6 ± 19.6
49.7 + 23.1** 31.9 ± 25.5
2.7 ± 1.2** 1.7 ± 1.2
Frozen-thawed (Macpherson's Extender II) 53.0 ±21.5** 40.5 ± 24.6
2.0 ± 1.2** 1.6 ± 1.1
80.5 + 7.3 74.0 ± 17.5
3.8+ .9* 3.3 ± 1.0
80.9 ± 8.4* 73.8+ 15.9
3.8 ± .9* 3.2 ± 1.2
51.4 ± 22.0 45.9 ± 23.2
55.1 ± 19.3 50.1 ± 20.9
3.4+ 1.3* 3.0 ± 1.4
24.1 ± 21.5 19.2 ± 19.8
37.2 ± 22.1* 30.1 ± 21.6
2.7 ± 1.3 2.3 ± 1.2
Fresh undiluted
Fresh diluted
Stored (24 hr)
1
n = Number of males tested.
*Significant line differences (P<.05). **Significant line differences (P<.01).
or Macpherson's E x t e n d e r II for each of t h e four parameters: d u r a t i o n of fertility, percent fertility for 7 days, p e r c e n t fertility for t h e d u r a t i o n of fertility, and spermatozoal motility. T h e difference b e t w e e n t h e selected and c o n t r o l lines for d u r a t i o n of fertility was similar for t h e two semen t r e a t m e n t s ( 1 , 2). Furtherm o r e , t h e correlated responses in t h e o t h e r fertility estimates were also similar. T h e r e was n o line b y semen t r e a t m e n t interaction for t h e fertility of frozen-thawed semen using Macpherson's E x t e n d e r II in comparison with t h a t of Lake's diluent. These results indicate t h a t selection has n o t been specific to a particular diluent and level of glycerol (13.6 vs. 1.7%). T h u s , resistance of chicken s p e r m a t o z o a to freeze damage m a y n o t be diluent specific for diluents containing glycerol. S e x t o n et al. ( 1 9 7 8 ) reported t h a t resistance or susceptibility of spermatozoa to freeze damage was n o t cryopreservative-specific. F o r spermatozoal m o -
tility, t h e interaction was significant ( P < . 0 1 ) , indicating t h a t the magnitude of response of the selected line varied u n d e r t h e t w o freezing t r e a t m e n t s . Fresh Semen. T h e selected line was significantly higher t h a n t h e control line for three of t h e four parameters measured for fresh undiluted semen. T h e p e r c e n t fertility for t h e d u r a t i o n of fertility was higher in t h e selected line t h a n t h e control line, b u t t h e difference was n o t significant. F o r diluted fresh semen, the selected line was significantly higher than the control line for t h e four parameters. These correlated increases indicate t h a t selection for improved fertility based on frozen-thawed semen m a y yield improved fertility of fresh semen. Scott et al. ( 1 9 8 0 ) and Ansah and Buckland ( 1 9 8 3 ) tested the males of Generations 3, and 4 through 8, respectively, of t h e two lines used in t h e s t u d y reported herein for fertility of undiluted fresh semen and observed
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 22, 2014
Selected Control
1479
SELECTION FOR SEMEN FERTILITY
Stored Semen. The selected line was above t h e control line for t h e four parameters. However, significant ( P < . 0 5 ) differences occurred only for t h e d u r a t i o n of fertility and sperm a t o z o a l motility for semen stored 2 4 hr and percent fertility for t h e d u r a t i o n of fertility for semen stored 48 hr. T h e r e was a significant ( P < . 0 5 ) line b y semen t r e a t m e n t interaction for the fertility and s p e r m a t o z o a l motility of semen stored for either 2 4 or 48 hr in comparison with frozen-thawed semen. This was attributed to t h e narrowing of t h e difference in fertility and spermatozoal m o t i l i t y b e t w e e n the selected and control lines with stored semen as opposed to frozen-thawed semen. These results suggest t h a t different factors m a y be responsible for t h e p r o t e c t i o n and s u b s e q u e n t fertility of s p e r m a t o z o a t h a t are frozen and thawed and those stored in t h e nonfrozen state. This suggestion is consistent with t h e r e p o r t s b y Ansah and Buckland ( 1 9 8 2 a , b ) o n fowl semen
cholesterol levels and t h e u p t a k e of glycerol b y spermatozoa, t h a t indicated selection for d u r a t i o n of fertility of frozen-thawed semen using glycerol as cryoprotective agent m a y be, in part, selection for increased s p e r m a t o zoa permeability t o glycerol and t h a t in t h e t w o lines used in this s t u d y s p e r m a t o z o a w i t h s t a n d freeze-damage b e t t e r as t h e y take u p m o r e glycerol. REFERENCES Allen, C. J., and L. R. Champion, 1955. Competitive fertilization in the fowl. Poultry Sci. 34:1332— 1342. Ansah, G. A., and R. B. Buckland, 1983. Eight generations of selection for duration of fertility of frozen-thawed semen in the chicken. Poultry Sci. 62:1529-1538. Ansah, G. A., and R. B. Buckland, 1982a. Genetic variation in fowl semen cholesterol and phospholipid levels and the relationships of these lipids with fertility of frozen-thawed and fresh semen. Poultry Sci. 61:623-637. Ansah, G. A., and R. B. Buckland, 1982b. The uptake of glycerol, a-aminoisobutyric acid and 2-deoxyD-glucose by spermatozoa of a line of chickens selected for fertility of frozen-thawed semen and a control line. Theriogenology 17:401—408. Barr, A. J., J. H. Goodnight, J. P. Sail, and J. T. Helwig, 1976. A User's Guide to SAS-76. SAS Inst. Inc., Raleigh, NC. Buckland, R. B., 1971a. The activity of six enzymes of chicken seminal plasma and sperm. 1. Effect of in vitro storage and full-sib families on enzyme activity and fertility. Poultry Sci. 50:1724— 1734. Buckland, R. B., 1971b. The activity of six enzymes of chicken seminal plasma and sperm. 2. The relationship between enzyme activity and fertility of fresh and stored semen. Poultry Sci. 50:1734-1742. Burrows, W. H., and J. P. Quinn, 1937. Collection of spermatozoa from domestic fowl and turkey. Poultry Sci. 16:19-24. Lake, P. E., 1968. Observations on freezing fowl spermatozoa in liquid nitrogen. VI Congr. Int. Reprod. Anim. Artif. Insem., Paris, 2:1633 — 1635. Lake, P. E., and O. Ravie, 1979. Effect on fertility of storing fowl semen for 24 hr at 5°C in fluids of different pH. J. Reprod. Fertil. 57:149-155. Lake, P. E., and J. M. Stewart, 1978. Preservation of fowl semen in liquid nitrogen — An improved method. Br. Poult. Sci. 19:187-194. Macpherson, J. W., S. Chatterjee, and G. W. Friars, 1969. Frozen turkey semen. Can. J. Comp. Med. 33:37-38. Mitchell, R. L., R. B. Buckland, and B. W. Kennedy, 1977. Heritability of frozen and fresh chicken semen and the relationship between the fertility of frozen and fresh semen. Poultry Sci. 56: 1168-1177. Phillip, L. E., R. B. Buckland, and D. E. Bernon, 1974. A note of the relationship between the fertility
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 22, 2014
positive correlated responses in t h e fertility of fresh semen. Mitchell et al. ( 1 9 7 7 ) , however, have previously suggested t h a t fertility of fresh chickens semen is a p o o r indicator of t h e fertilizing ability of frozen-thawed semen. A l t h o u g h this suggestion m a y be correct for their s t u d y considering t h e low fertility levels with frozent h a w e d semen t h e y o b t a i n e d , with improved cryogenic technology, as several investigators have accomplished for b o t h avian and mammalian spermatozoa, a positive correlation m a y exist as indicated b y t h e results of previous studies and t h e present one. There was an absence of a line b y semen t r e a t m e n t interaction for d u r a t i o n of fertility and percent fertility for 7 days postinsemination in comparison with frozen-thawed semen t h a t further demonstrates that improved fertility of frozen-thawed semen through selection m a y also hold for fresh semen. A significant ( P < . 0 5 ) interaction effect was obtained for percent fertility for t h e d u r a t i o n of fertility. This was perhaps due to chance as t h e p h e n o t y p i c correlations a m o n g d u r a t i o n of fertility, percent fertility for 7 days postinsemination, and p e r c e n t fertility for the d u r a t i o n of fertility of fresh semen o b t a i n e d in this s t u d y , which ranged from .3 7 t o . 9 3 , were positive and significant ( P < . 0 1 ) . Similar positive and high p h e n o t y p i c correlations were obtained by Mitchell et al. ( 1 9 7 7 ) , Scott et al. ( 1 9 8 0 ) , and Ansah and Buckland ( 1 9 8 3 ) . T h e interaction was also significant for t h e spermatozoal motility of undiluted semen.
1480
YOUSIF ET AL.
of fresh semen and that stored varying lengths of time and the effect of storage on duration and percent fertility. Poultry Sci. 53:2216-2218. Polge, C , A. U. Smith, and A. S. Parkes, 1949. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164:666. Scott, T. A., R. B. Buckland, and B. W. Kennedy, 1980. The effect of selection for fertility of frozen-thawed semen on spermatozoa oxygen uptake, motility and concentration and ejaculate volume in the chicken. Theriogenology 14:
281-298. Sexton, T. J., 1976. Studies on the fertility of frozen fowl semen. Pages 1079-1081 in Proc. Vlllth Int. Congr. Anim. Reprod. Artif. Insem., Krakow. Sexton, T. J., R. B. Buckland, and R. Lopez, 1978. Comparison of two procedures for freezing semen from cocks of high and low fertility with frozen semen. Poultry Sci. 57:550—552. Wilcox, F. H., C. S. Shaffner, and H. R. Wilson, 1961. Breed differences in storing chicken semen. J. Hered. 52:119-121.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 22, 2014