Genetic effects of aging on fitness and nonfitness traits in laying hens housed three per cage.

Three White Leghorn strains, their two-way crosses, and two commercial lines were used to study the effects of aging on several parameters related to performance of fitness and nonfitness traits during the first laying cycle of hens housed three per cage. Egg number of the survivors (EPF) and egg number including mortality and morbidity (EPM) were divided into 12 periods of 28 d each, starting at age at sexual maturity. Egg weight (EW), specific gravity (SG), and albumen height (AH) were measured at 240, 350, and 450 d of age. Mean heterosis was significant over time, except for AH, increasing in magnitude with age for EPF, EPM, EW, and AH. Reciprocal effects were more important for egg quality than for egg production traits and were influenced by age. The age-related changes in additive, Z-chromosome, and heterotic effects varied among strains, showing that strains differ in their genetic schemes in response to aging. Nonadditive, environmental, and phenotypic variances increased with age for all traits. Additive variance increased with age for EPF, EW, and AH. Z-chromosome variance increased with age for EW and AH. Heritabilities decreased with age, except for EPF and AH. On average, genetic variance increased with advancing age. Improvement in lifetime performance may be obtained by selecting birds at older ages. As the relative increase with age in additive variance was larger for egg production than for egg quality traits, selection for the latter could be performed at early stages.

Genetic effects of aging on egg quality traits in the first laying cycle of White Leghorn strains and strain crosses.

Three White Leghorn strains, their two-way crosses, and two commercial lines were used to evaluate the effects of aging on heterosis (H), reciprocal effects, and additive (A), Z-chromosome (Z), and heterotic effects and their variances on egg quality traits during the first laying cycle. Egg weight (EW), specific gravity (SG), Haugh unit (HU), and albumen height (AH) were measured at 240, 350, and 450 d of age from hens housed one per cage in a randomized block design. The mean heterosis was significant over time only for EW. For EW, heterosis increased in magnitude with age. The mean heterosis for both HU and AH was also influenced by age. Reciprocal effects were significant, on average, across periods for all traits and were influenced by age. The age-related changes in additive, Z-chromosome, and heterotic effects varied significantly among strains, indicating differences by genetic group in response to aging for egg quality traits. The heterotic, environmental, and phenotypic variances increased with age for all traits, except for AH. The additive and Z-chromosome variances did not always increase with age. Their age trend varied, depending on the trait. Heritabilities decreased with advancing age, suggesting that selection to improve lifetime performance of egg quality traits can be done early in the cycle.

Trait association of a genetic marker near the IGF-I gene in egg-laying chickens.

The insulin-like growth factor I (IGF-I) gene was screened for genetic variants associated with trait means and trait correlations. Analysis of an unselected randomly mated White Leghorn population revealed a PstI restriction fragment length polymorphism (RFLP) in the 5' region of the gene which segregated at a frequency of 0.83 for the PstI(+) allele (presence of a PstI restriction site). A comparison of the three genotypic classes revealed that the PstI(-/-) genotype was associated with a significantly lower egg weight measured in three different time periods, while the PstI(+/-) genotype was significantly associated with a higher eggshell weight estimated from the egg weight and egg specific gravity. For eggshell weight, the effect was age dependent and significant only for the last two periods of egg laying. No genotype associations were found for body weight, feed consumption, and egg laying rates. Significant dominance effects of the IGF-I genotype were observed for two of the egg weight measurements and three of the eggshell weight estimates. Partial correlation analyses in the two most frequent genotypic classes, PstI(+/+) and PstI(+/-), revealed the presence of a regulatory loop between feed consumption, body weight, egg weight, and the rate of egg laying. Several aspects of this regulatory loop were different between the two genotypic classes. In particular, for the PstI(+/+) genotype, feed consumption was positively associated with egg weight, while there was no significant association for the PstI(+/-) genotype. Further, the degree of association of body weight with egg weight decreased with age in the genotypic class PstI(+/-), while it was constant for the PstI(+/+) genotype. The results indicated that the marker in the IGF-I gene was not only associated with changes in some trait means, but also with changes in the stability of the coordination between feed intake, body weight, and egg production traits.

Genetic effects of aging on egg production traits in the first laying cycle of White Leghorn strains and strain crosses.

Three White Leghorn strains, their two-way crosses, and two commercial lines were used to evaluate the effects of aging on mean heterosis, reciprocal, additive, Z-chromosome, and heterotic effects and their variances in two egg production traits during the first laying cycle. Egg number of the survivors (EPF) and egg number including mortality and morbidity (EPM) were evaluated from hens housed one per cage in a randomized block design. For analyses, egg number was divided into 12 periods of 28 d each. Synchronization of the records was achieved by starting recording at age at first egg. The mean heterosis was significant over time and increased with age for both traits (P<0.05). Reciprocal effects were not significant across periods but increased in magnitude with age for EPF. The pattern of age changes in additive, Z-chromosome, and heterotic effects varied among strains, indicating genotypic differences in response to aging. These differences increased, on average, with age. The additive, heterotic, environmental, and phenotypic variances increased with age for both traits. A decline with physiological age was observed in heritabilities, which was due to a faster increase in environmental variance than to an increase in additive variance. Genetic variance of egg production increased with age, indicating that improvement of lifetime performance of layers is possible. This improvement could be achieved by selecting animals at older ages or by favoring individuals with better DNA repair or those who had more genes turned on or off during the course of aging.

Genetic effects of ageing on fertility and hatchability in the first laying cycle of three white leghorn strains and their two-way crosses.

1. Fertility and hatchability of 3 White Leghorn strains and their 2-way crosses were examined in the 1st laying cycle to evaluate the effects of ageing on mean heterosis, reciprocal effects, additive (A), Z-chromosomne (Z) and heterotic effects (H), as well as on their variances. 2. Fertility and hatchability were evaluated at 37, 47 and 70 weeks of age for hens housed 1 per cage in a randomised block design. The mean heterosis was significant over time and increased with advancing age for both traits. Reciprocal effects were not significant across periods and did not show a consistent trend with age. 3. The pattern of age changes in A, Z and H effects varied among strains, indicating genotypic differences in response to ageing. The additive, heterotic, environmental and phenotypic variances increased at the end of the cycle for both traits, except for the additive variance of hatchability, which increased until 47 weeks of age but declined thereafter. 4. Heritability increased with age for fertility but did not show a consistent pattern across the cycle for hatchability. Genetic variance for fertility and hatchability increased at the end of the cycle, stggesting that improvement in performance during the 1st production cycle of layers could be achieved by selecting animals at older ages.

A genetic marker in the growth hormone receptor gene associated with body weight in chickens.

A genomic clone spanning 16 kb of the GH receptor gene was mapped and used as a probe for identifying restriction fragment length polymorphisms (RFLPs) in chickens. Several strains of meat-type and egg laying chickens were found to segregate for an HindIII RFLP located in the intron preceding exon 4. The polymorphic HindIII site overlapped with a poly(A) signal. Association of the HindIII RFLP with traits was analyzed in a random-bred White Leghorn strain in three generations using either selective or random genotyping. Both methods revealed significant association of the HindIII+ allele (presence of the poly(A) signal) with an increased juvenile body weight (130 days of age). In two meat-type strains divergently selected for size of the abdominal fat pad, the HindIII+ allele was coselected with leanness. The results indicate the presence of a genetic variant of the GH receptor gene which affects growth and abdominal fat deposition and which is relatively frequent in egg laying as well as in meat-type chickens.

Prediction of heterosis from DNA fingerprints in chickens.

To assess the value of DNA fingerprints for the prediction of heterosis in chickens, retrospective analyses of data from three crossbreeding experiments and DNA fingerprints (DEP) of parental strains were conducted using two minisatellite and one middle-repetitive DNA probes. DEP bands were assessed on pooled DNA samples of 10-15 individuals per parental genetic group. The number of DEP bands evaluated in the experiments ranged from 81 to 139. The probes varied in their predictive value, but predictability of heterosis generally increased with multiple probes. Highly significant correlations (0.68-0.87) between band sharing ratios (SH) and heterosis were found in 25 crosses of White Leghorns in the first egg production cycle for age at sexual maturity, egg production, and mature body weight: traits with heterosis of 10% or more of the means. Regressions on SH explained 78.4% of the variation in heterosis in age at sexual maturity, 60.2% in egg production and 46.4% in mature body weight. For "broiler" traits with heterosis of < 1%, none of the correlations, based on 13 crosses, were significant. It was concluded that multilocus probe DFP of pooled DNA samples show promise as predictors of heterosis.

The effect of simultaneous selection on the genetic correlation.

The theoretical effect of simultaneous selection on the genetic correlations between two traits over 20 generations was examined using simulation. For each generation, a population of 50 male and 50 female diploid gen otypes with 15 loci, each with two alleles, was synthesized. None of the loci exhibited dominance. Five loci affected only trait 1, 5 loci only trait 2 and 5 were pleiotropic (affected both traits). Initial allelic frequencies were equal at each locus. Phenotypes were created by adding a random normal deviation for each trait to the genotype. The size of this deviation for each trait determined its heritability (h(2)). Index selection with h(2) combinations of (0.15, 0.15), (0.15,0.45) and (0.45,0.45) and relative economic weights of (1, 1) and (1, 3) for each h(2) combination was employed. In each generation, the highest ranking 25 genotypes of each sex were used to generate the next generation with single-pair matings, each producing two male and two female offspring. One hundred replicates were run for both negative and positive correlations. With a positive initial value, the genetic correlation tended to decline (toward zero). The rates of change were moderately affected by index weights and h(2). With a negative initial value, the genetic correlation tended to decrease (towards -1). However, unequal heritabilities and unequal relative economic weights slowed the rate of change with the greatest imbalances tending to hold the correlation constant or move it toward zero. These simulations illustrate that changes in parameters over time can affect the selection practiced. Under some of the conditions simulated, the use of initial genetic parameter values without change could have potentially negative effects on overall genetic gain.

Embryonic adenosine triphosphate:phosphodiesters ratios obtained with in vivo nuclear magnetic resonance spectroscopy (phosphorus-31): a new technique for selecting leaner broiler chickens.

Phosphorus-containing compounds of 18-d-old embryos from two broiler chicken lines, differing in body fat content, were measured using in vivo 31P nuclear magnetic resonance (NMR) spectroscopy. Subsequently, the same birds were slaughtered at 8 wk of age and the whole body was analyzed for body fat content. The birds of the fat line had lower (P = .002) embryonic adenosine triphosphate (ATP):phosphodiester (PDE) ratios, higher (P = .002) body fat content when adjusted to common BW, and higher (P = .047) dry matter content than the lean line. No differences (P > .05) were detected for BW between the two lines. Females of the two lines had lower (P = .002) ATP:PDE ratios, lower (P = .001) BW, higher (P = .003) adjusted fat contents, and higher (P = .003) dry matter content than the males. No interaction (P > .05) between sexes and lines was detected for any variables. Regression equations indicating a linear negative relationship (Ybody fat content = 369.05 - 407.27 XATP:PDE + .1295 XBW, R2 = .62 to .78; or Ybody fat % = 30.57 - 19.4 XATP:PDE, R2 = .49 to .71) between embryonic ATP:PDE ratios and body fat content were developed.

In vivo nuclear magnetic resonance spectroscopy of chicken embryos from two broiler strains of varying fat content.

In vivo nuclear magnetic resonance (NMR) imaging and spectroscopy techniques were used to monitor changes in P- and H-containing molecules in embryos of two broiler strains (30 and 31) differing genetically in fat content and ranging in age from 0 to 20 days of incubation. Chemical analysis showed that Strain 30 has more carcass fat than Strain 31 at market age (7 wk). Proton (1H) and 31P spectra were obtained on four eggs per strain at Days 0, 2, 4, 6, 8, 11, 12, 14, 16, 17, 19, and 20 of incubation. Fat:water, phosphomonoester (PME):phosphodiester (PDE), and adenosine triphosphate (ATP):PDE ratios were calculated. Chicks were hatched, grown to market weight (2,000 g for females and 2,300 g for males at 7 wk), and the whole intact carcasses were analyzed for crude fat. Hydrogen-1 NMR spectroscopy studies of incubated eggs indicated no significant difference (P > .05) in the fat:water ratio between the two strains. The difference in the PME:PDE ratios between the two strains as obtained by 31P-NMR spectroscopy over all days of incubation analyzed was not significant (P > .05); however, there was a significant difference in this ratio between the two strains at Days 4, 6, and 11. Up to Day 16, Strain 30 had a slightly, but not significantly (P > .05), higher ATP:PDE ratio as shown on 31P-NMR spectra, whereas after Day 17 the ATP:PDE ratio was significantly higher (P < .01) for Strain 31. Strain 31 birds had a significantly lower (P < .05) crude fat content. There was a significant difference (P < .05) in 7-wk carcass fat content between sexes, males having significantly (P < .01) less fat than females, which was correlated with a significantly higher (P < .01) ATP:PDE ratio in male embryos. It might be possible to use ATP:PDE ratios obtained during embryonic development by 31P-NMR to select strains of birds for low fat content at market weight and to distinguish between sexes during late embryonic development.

Synergism between the endogenous viral loci ev6 and ev9 in inducing immunological tolerance to avian leukosis virus.

1. The course of infection by exogenous avian leukosis virus was followed in a commercial strain of White Leghorn domestic fowls by measuring viral antigen in feather pulp and egg albumin. Ten days after hatching, 2 out of 360 birds tested positive and at 286 days of age about 60% of the birds had been antigen positive at least once. 2. Among the antigen positive birds, two groups could be distinguished: those which permanently and those which transiently expressed viral antigen. Permanent antigen expression was associated with low antibody titres, while transient antigen expression was associated with high antibody titres. 3. The strain segregated for the two endogenous viral genes ev6 and ev9, both of which express endogenous viral envelope protein, and have been implicated in affecting immune-responsiveness. The antibody titre in individuals positive for both ev6 and ev9, was significantly lower than in those which had none or only one of the two ev-genes. In addition, individuals positive for both ev-genes occurred more frequently in the group permanently positive for viral antigen than in the group transiently antigen positive. 4. The results indicate that there was a strong synergism between ev6 and ev9 in reducing the antibody response to exogenous avian leukosis virus infection, perhaps by inducing immune tolerance or interfering with antibody formation.

The effect of warming velocity on motility and acrosomal integrity of boar sperm as influenced by the rate of freezing and glycerol level.

The effect of thawing velocities ranging from 10 degrees C/min to 1,800 degrees C/min on the motility and acrosomal integrity of boar spermatozoa frozen at 1 degree C/min (suboptimal), 5 degrees C/min, and 30 degrees C/min (optimal) rate was studied with the sperm suspended for freezing in diluent containing 2, 4, or 6% of glycerol (v/v). The influence of thawing on sperm survival depends on the rate at which the sperm had been frozen. In semen frozen at a suboptimal rate of 1 degree C/min, the percentage of motile sperm (FMP) initially fell to 3.5-4.0% when the thawing rose to 200 degrees C/min, but, with further increases in thawing rate, increased and reached peak values (10.3-11.0% FMP) after thawing at 1,800 degrees C/min. The percentage of sperm with normal apical ridge (NAR) also increased moderately with thawing rate, but the degree of improvement decreased as the glycerol level was increased. In semen frozen at 1 degree C/min, acrosomal integrity (NAR) was best maintained in 2% glycerol, reaching 22.9% NAR after thawing at 1,800 degrees C/min. In semen frozen at the optimal rate of 30 degrees C/min, the increases in thawing rates above 200 degrees C/min substantially improved motility. Motility was generally higher in semen protected by 4 or 6% glycerol, with the peak values of 44 or 46% FMP, respectively, after thawing at 1,200 degrees C/min. The proportion of sperm with NAR also increased with thawing rate, but as in the case of suboptimally frozen sperm it was influenced negatively by the glycerol concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of markers associated with quantitative trait loci in chickens by DNA fingerprinting.

Three approaches for identifying VNTR alleles associated with quantitative traits in chickens are described. One approach is based on the comparison of well-defined selected and non-selected control strains. The second approach is based on analyzing chickens within a breeding population ranked according to specific traits and the third approach involves segregation analysis. In this latter approach a large number of offspring of a single male segregating for a quantitative trait are produced and tested for trait association of the male DNA fingerprinting bands. In all cases pooled DNA samples of birds, rather than individual samples, are analyzed and band intensity is assumed to reflect the relative frequency of an allele. Examples from the literature and from our laboratory indicate that these methods permit the identification of DNA fingerprinting bands associated with quantitative traits. After developing locus-specific probes for these bands it should ultimately be possible to detect and map quantitative trait loci.

Assessment of inbreeding by DNA fingerprinting: development of a calibration curve using defined strains of chickens.

By analyzing DNA fingerprints of chickens from seven well-defined genetic groups, a calibration curve was established relating the degree of inbreeding with the average band frequency, allelic frequency and band sharing. The probe used was bacteriophage M13 DNA and digestion of the genomic DNA was carried out with the MspI restriction enzyme. The analysis also provided an estimate of the average allelic frequency at a hypervariable locus and the average mutation frequency per locus and generation. The values of 0.24 and 1.7 X 10(-3), respectively, are similar to the estimates for humans using other probes and hybridization protocols. It is suggested that the calibration curve established can be used for determining inbreeding not only in chickens, but also in other species.

Combined effect of glycerol concentration and cooling velocity on motility and acrosomal integrity of boar spermatozoa frozen in 0.5 ml straws.

The interaction of glycerol concentrations of 0-10% and cooling rates from 1 to 1,500 degrees C/min with boar spermatozoa motility and acrosomal integrity (proportion of spermatozoa with normal apical ridge) was studied after thawing 0.5 ml straws at a constant rate. While increasing the glycerol concentration from 0 to 4% progressively improved motility, the percentage of spermatozoa with a normal apical ridge gradually decreased. The magnitudes of the respective changes depended on cooling rate. A peak value of 48.1% and rating 3.8 were obtained in semen protected with 4% glycerol, frozen at 30 degrees C/min. Increasing the glycerol levels above 6% resulted in a gradual decrease in motility. The proportion of spermatozoa with normal apical ridge was highest in semen protected with 0-1% glycerol after cooling at 30 degrees C/min (64.4% and 66.1%, respectively), but at these glycerol concentrations the percentage of motile spermatozoa was low. At the 30 degrees C/min cooling rate, the decline in the proportion of cells with normal apical ridge due to increasing the glycerol levels to 3 and 4% was relatively slow (57.3% and 49.4%, respectively). Cooling at 1 degrees C/min was detrimental to acrosomal integrity, which decreased with increasing glycerol concentration, in contrast to increasing motility, which even at its maximum, remained low. The direct plunging of straws into liquid nitrogen (1,500 degrees C/min) resulted in damaged acrosomes in all spermatozoa with the total loss of motility. Balancing motility and acrosomal integrity, freezing boar semen protected with 3% glycerol by cooling at 30 degrees C/min resulted in optimal survival for boar semen frozen in 0.5 ml French straws.

Influence of selection for egg production and Marek's disease resistance on the incidence of endogenous viral genes in White Leghorns.

The influence of selection on the frequencies of endogenous viral (ev) genes related to the avian leukosis virus was studied in two genetically distinct sets of White Leghorn strains. Each set consisted of four strains: an unselected control strain, two strains selected for egg production traits, and a strain selected for Marek's disease (MD) resistance as well as egg production traits. Eight different ev genes were observed in Set I and seven in Set II, four being common to both sets. Selection for egg production traits resulted in significant changes of the frequency of four ev genes in both sets. In Set I, increased frequencies were observed for ev-4, ev-7, and ev-8; a decreased frequency for ev-9 was observed. The ev-9 gene expresses the viral envelope protein, whereas the others are transcriptionally silent, with the possible exception of ev-7. In Set II, increased frequencies were observed for the transcriptionally silent ev-8 and for ev-15, a gene which consists of a solitary long terminal repeat. Decreased frequencies were observed for ev-18, which codes for infectious endogenous virus, and for a second ev gene of unknown phenotype. In the resistance-selected strains the frequencies of the ev genes were intermediate between those of the control strains and the strains selected for egg production traits with the exception of ev-6, which expresses the viral envelope protein, and ev-3, which expresses internal viral proteins as well as the envelope protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of glycerol-related osmotic changes on post-thaw motility and acrosomal integrity of ram spermatozoa.

Ram semen, collected by artificial vagina, was diluted and processed for long-term storage as described by P. S. Fiser, L. Ainsworth, and R. W. Fairfull (Canad. J. Anim. Sci. 62, 425-428, 1982). The concentration of the cryoprotectant, glycerol, was adjusted to 4% in the diluted semen prior to freezing by a one-step addition at 30 degrees C (Method 1), by cooling the semen to 5 degrees C and addition of the glycerol gradually over 30 min (Method 2), by one-step addition of glycerol prior to equilibration for 2 hr (Method 3), or by cooling to 5 degrees C, followed by a holding period of 2 hr at 5 degrees C, and the one-step addition of glycerol just prior to freezing (Method 4). After thawing, the glycerol concentration of the semen was reduced by stepwise dilution from 4 to 0.4% over 15 or 30 min or by a one-step ten-fold dilution. The average post-thaw percentage of motile spermatozoa was significantly lower after addition of glycerol by Method 1 (39.9%) than when the glycerol was added by the other three methods (range, 44.0-46.4% averaged over the glycerol dilution). The average post-thaw percentage of intact acrosomes (61.2%), highest in semen in which the glycerol was added by Method 2, was not significantly different from those in which glycerol was added to semen by Methods 3 and 4, but it was significantly higher than that found in semen in which the glycerol was added by Method 1 (54.4%). However, when averaged over the method of glycerolation, the post-thaw percentage of motile spermatozoa (range, 43.7-44.2%) and the percentage of intact acrosomes (range, 56.8-59.5%) did not differ significantly in semen subjected to gradual decrease in glycerol concentration and diluent osmolality (over 15 and 30 min) or by a one-step, 10-fold dilution. These data indicate that post-thaw survival of spermatozoa can be influenced by the way in which glycerol is added prior to freezing. However, post-thaw spermatozoa motility and acrosomal integrity can be maintained even after a rapid decrease in glycerol concentration such as that which accompanies insemination or dilution of semen for assessment of motility.

Semen traits and fertility of White Leghorn males shown to be positive or negative for lymphoid leukosis virus in semen and feather pulp.

1. Males from strains selected for high egg production (and other economic traits) and from unselected control strains were used to determine the frequency of shedding of lymphoid leukosis virus (LLV) into semen. The effect of the male's LLV status on semen production, fertility and hatchability was also examined in males of the unselected control strains. 2. The frequency of detection of exogenous LLV in semen by the phenotypic mixing test, and high concentrations of the viral group specific antigen in feather pulp by the complement fixation test, were both higher in control strains than in strains selected for high egg production. 3. Semen production was not reduced in LLV-shedding males. 4. Significant associations of LLV shedding with higher incidence of abnormal spermatozoa and reduced fertility were found in some populations but not in others. No significant effect of LLV shedding on hatchability was detected. 5. Tests for group specific antigen in feather pulp proved useful in identifying males that shed LLV in semen.

Evaluation of a new diluent and different processing procedures for cryopreservation of ram semen.

Ram semen was processed for freezing after initial dilution with a modified Tris-fructose diluent. Two aliquots were processed by cooling gradually to 5 degrees C, further dilution, equilibration and freezing in 0.5 ml straws either in pressurized liquid nitrogen (LN(2)) vapor (Method A) or on a block of dry ice (Method B). A third aliquot was cooled rapidly to 16 degrees C and then slowly to 5 degrees C, diluted further, equilibrated and frozen in straws in pressurized LN(2) vapor (Method C). The second dilution was carried out using a new diluent based on dextran-lactose. The diluted semen was equilibrated for 2 h before freezing. Semen was evaluated by artificial insemination (AI). The fertility of ewes bred by a double insemination with frozen-thawed semen processed by Methods A, B and C was 73% (n = 33), 67% (n = 30) and 80% (n = 30), respectively. In comparison, the fertility of ewes inseminated with fresh semen was 93% (n = 31). These preliminary data indicate an acceptable fertility can be achieved by AI with frozen-thawed semen processed using improved procedures.

The effects of rapid cooling (cold shock) of ram semen, photoperiod, and egg yolk in diluents on the survival of spermatozoa before and after freezing.

The effects of rapid cooling of semen (cold shock) from 30 degrees C to various temperatures above 0 degrees C on survival of ram spermatozoa suspended in diluents with or without egg yolk were assessed before and after freezing. Rapid cooling of extended semen from 30 to 15 degrees C had little or no effect on spermatozoa survival before or after freezing. Rapid cooling of extended semen from 30 degrees C to 10, 5, or 0 degrees C was accompanied by a progressive decrease in percentage of motile spermatozoa and percentage of intact acrosomes before freezing and a decrease in percentage of motile spermatozoa and after freezing. The ability of spermatozoa motile after cold shock to survive freezing and thawing, evaluated as cryosurvival, was not significantly (P greater than 0.05) affected by the temperature to which semen was cooled. The addition of egg yolk to the initial extender had a beneficial effect on percentage of motile spermatozoa particularly after rapid cooling of semen to 10 and 5 degrees C. Although egg yolk had little effect before freezing on semen rapidly cooled to temperatures above 15 degrees C and therefore not actually cold shocked, it substantially improved the subsequent survival of spermatozoa after freezing and thawing. Percentage of motile spermatozoa after cooling and after freezing was generally higher when the semen was collected during a decreasing photoperiod than during an increasing photoperiod.