Short-term preservation of fowl sperm in buffered potassium chloride.

Previous research demonstrated that sperm motility is dependent upon mitochondrial calcium cycling. Thus, sperm are inactivated when extracellular calcium ions are chelated. Mitochondrial calcium cycling, however, is driven by extracellular sodium ions. The hypothesis that sperm inactivation is subject to 2 variables was tested in the present work. Sperm motility was evaluated with computer-assisted sperm motion analysis in the first experiment. Sperm became immotile within minutes when suspended in buffered isotonic potassium chloride containing calcium ions. This outcome set the stage for the second experiment in which sperm were inactivated by centrifugation through 12% (wt/vol) Accudenz prepared with potassium chloride and tetrasodium 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Sperm mobility was the end point in the second and subsequent experiments. Potassium chloride was a suitable medium in regard to sperm inactivation with BAPTA followed by storage for 2 h at 10°C. Consequently, sperm so inactivated were reactivated after 1, 2, 3, 4, and 5 h of storage in the third experiment. Whereas pre- and postwash sperm mobility were equivalent, sperm mobility declined with time (P < 0.001) without exogenous energy in the storage medium. Therefore, the effect of 5 mM glucose was tested in the fourth experiment. In this case, recovery of sperm mobility was independent of time when sperm were stored at 10°C for 5 h (P > 0.05). Potassium chloride was replaced with potassium glutamate in the last experiment. Whereas reactivation was once again independent of time when sperm were stored with glucose (P > 0.05), greater variability was observed among observations in comparison with the potassium chloride-based medium. In summary, sperm motility was inactivated when calcium was chelated and extracellular sodium was replaced with potassium. Sperm reactivation was most consistent when chloride was the predominant extracellular anion. Collectively, these experiments demonstrate that short-term sperm storage can be achieved with simple media that promote sperm glycolysis and minimize energy demands imposed by the active transport of calcium and sodium ions.

Validation of a spectrophotometer-based method for estimating daily sperm production and deferent duct transit.

The objectives of the present work were 3-fold. First, a new method for estimating daily sperm production was validated. This method, in turn, was used to evaluate testis output as well as deferent duct throughput. Next, this analytical approach was evaluated in 2 experiments. The first experiment compared left and right reproductive tracts within roosters. The second experiment compared reproductive tract throughput in roosters from low and high sperm mobility lines. Standard curves were constructed from which unknown concentrations of sperm cells and sperm nuclei could be predicted from observed absorbance. In each case, the independent variable was based upon hemacytometer counts, and absorbance was a linear function of concentration. Reproductive tracts were excised, semen recovered from each duct, and the extragonadal sperm reserve determined by multiplying volume by sperm cell concentration. Testicular sperm nuclei were procured by homogenization of a whole testis, overlaying a 20-mL volume of homogenate upon 15% (wt/vol) Accudenz (Accurate Chemical and Scientific Corporation, Westbury, NY), and then washing nuclei by centrifugation through the Accudenz layer. Daily sperm production was determined by dividing the predicted number of sperm nuclei within the homogenate by 4.5 d (i.e., the time sperm with elongated nuclei spend within the testis). Sperm transit through the deferent duct was estimated by dividing the extragonadal reserve by daily sperm production. Neither the efficiency of sperm production (sperm per gram of testicular parenchyma per day) nor deferent duct transit differed between left and right reproductive tracts (P > 0.05). Whereas efficiency of sperm production did not differ (P > 0.05) between low and high sperm mobility lines, deferent duct transit differed between lines (P < 0.001). On average, this process required 2.2 and 1.0 d for low and high lines, respectively. In summary, we developed and then tested a method for quantifying male reproductive tract throughput. This method makes the study of semen production amenable to systems biology.

An expressed sequence tag analysis of the chicken reproductive tract transcriptome.

Analysis of the chicken reproductive tract transcriptome is important in comparative biology for analysis of reproductive tract development and evolution. In addition, molecular analysis of the reproductive tract is important for identification of genes affecting fertility in the poultry industry. We sampled the chicken reproductive tract (ovary, oviduct, and testis) transcriptome, generating 5,328 expressed sequence tags that assembled into 4,518 contigs. We identified 475 contigs with no match in the current expressed sequence tag databases or in GenBank. The novel contigs included 31 with no match to the current assembly of the chicken genome, 119 representing spliced transcripts, and 309 that were unspliced. More detailed molecular characterization of the 428 novel contigs present in the assembly will be important to gene discovery and annotation of the chicken and other vertebrate genomes.

Deduction of a calcium ion circuit affecting rooster sperm in vitro.

Four premises for rooster sperm preservation were outlined previously. Understanding mitochondrial Ca cycling in terms of whole-cell Ca flux was one premise. The present work tested the hypothesis that sperm mitochondria can be damaged by intracellular as well as extracellular Ca. Sperm were washed by centrifugation through 12% (wt/vol) Sperm were washed by centrifugation through 12%(at/vol) Accudenz to procure sperm at a physiological concentration within a chemically-defined suspension. Five solutions were tested. Each solution contained 30 m glucose, and had an osmolality of 320 mmol/kg and a pH of 7.4. Washed sperm were diluted to 2.0 × 10 sperm/mL. Each replicate sperm suspension was cooled to 10°C. Sperm mobility was measured after 1, 2, 4, 8, 12, and 24 h. Data were plotted as a function of time in each experiment. Function type was confirmed by lack of fit analysis. A parabola with a maximum at 3.7 h was observed when sperm were suspended in 205 m taurine buffered with 50 m-tris[hydroxyl-methyl]methyl-2-amino-ethanesulfonic acid (TES). This effect was attributed to a Ca flux from the nuclear envelope into mitochondria. An exponential decay was observed when TES-buffered taurine contained 2 m Ca. This effect was attributed to mitochondrial Ca overload induced by uptake of extracellular Ca. Exponential decay also was observed when TES-buffered taurine contained a Ca chelator. This effect was attributed to a Ca flux from the nuclear envelope through mitochondria and then into an extracellular Ca sink. This possibility was supported by the response of sperm to thapsigargin. Specifically, inhibition of sarcoendoplasmic reticulum Ca-ATPase compromised sperm mobility relative to a buffer control. Finally, a 60 m phosphate buffer containing 2 m citrate yielded a linear relationship in contrast to the TES-buffered solutions tested. Sperm mobility after 24 h of storage in the phosphate buffer was 92% of that observed for prewashed sperm. The linear response was attributed to weak chelators providing resistance within a Ca circuit and thereby preventing mitochondrial Ca overload. Fertility, however, was compromised when hens were inseminated with mobile sperm recovered after either 8 or 24 h of storage at 10°C. In conclusion, sperm cell Ca homeostasis was proven to be critical for maintaining sperm mobility in vitro, but mitochondrial Ca uptake is not the sole phenomenon that compromises sperm function during in vitro storage.

Evaluation of a semen extender from a bioenergetic perspective.

Four premises for sperm preservation were previously outlined. The present work tested 2 of these. The first premise was that sperm mobility phenotype affects procedural efficacy. Random bred roosters were phenotyped with the sperm mobility assay. A normal frequency distribution was observed with 35% (SD = 16.4) mobile sperm. Test subjects had values >51% (high) or between 19 and 35% (below average). Phenotypes were confirmed by repeated measure analysis. Ejaculates were pooled by phenotype. Sperm were washed by centrifugation through 12% (wt/vol) Accudenz. Washed sperm were suspended in Beltsville Poultry Semen Extender (BPSE) at 2 × 10 sperm/mL. Such sperm were stored at 10°C for 24 h. In the case of highly mobile sperm, an exponential decay was observed with a -intercept of 72% and an asymptote of 53%. In contrast, postwash values for below-average males decreased linearly from a -intercept of 31 to 17% after 24 h. A logistic decay was observed when sperm from high phenotype subpopulation males were extended with BPSE rather than washed before storage. Whereas -intercepts were equivalent between experiments, end points were not, that is, 53 vs. 17% mobile sperm. This difference was attributed to the extent of cytotoxic edema. The second premise tested was that the sperm mobility assay can predict the status of sperm cell mitochondria in response to sperm manipulation. Highly mobile sperm were washed and then suspended in either saline or glucose-free extender. Solution pH and osmolality were equivalent. Extender osmolality was controlled by replacing glucose with mannitol. Sperm were stressed by incubation at 2 × 10/mL at 20°C for 8 h. In each case, loss of sperm mobility approximated a logistic function. Whereas -intercepts were equivalent, the time at which loss of function was half maximal was prolonged with the extender ( < 0.01). This difference was attributed to a diminution of the process whereby energy-deprived sperm were rendered immobile by cellular edema. An a posteriori analysis was limited to pretreatment data from males categorized a priori with the high phenotype. Phenotype was independent of time ( = 0.81) during the 14-wk interval in which experiments were performed. In summary, extender efficacy was affected by sperm mobility phenotype as well as the means by which the extender was used. To date, such effects have not been addressed in attempts to preserve chicken sperm in vitro.

Sperm mobility determines the outcome of sperm competition in the domestic fowl.

The aim of this study was to establish whether the mobility of sperm of the domestic fowl, as measured by an in vitro assay, predicted the outcome of sperm competition. Thirteen pairs of New Hampshire roosters, comprising one male categorized as having high-mobility sperm and the other as having average-mobility sperm, were used. Each male provided 25 x 10(6) sperm, which were mixed and artificially inseminated into between four and seven New Hampshire hens, each of which produced 2-11 offspring. The experiment was conducted twice, such that the same pair of males inseminated the same females. Paternity was assigned by using microsatellite markers. There was a clear effect of sperm-mobility phenotype on the outcome of sperm competition: in all 13 pairs the high-mobility male fathered the majority of offspring (75.3% overall; p < 0.0001). The proportion of offspring fathered by the high-mobility male within pairs varied significantly between male pairs (p < 0.0005). This effect was associated with the difference in sperm-mobility scores between males within pairs; there was a significant positive relationship between the proportion of offspring fathered by the high-mobility male and the ratio of mobility scores between males (p < 0.05). In addition, compared with their success predicted from the non-competitive situation, in the competitive situation high-mobility males were disproportionately successful in fertilizing eggs compared with average-mobility males. This may occur because female sperm storage is limited in some way and a greater proportion of high-mobility sperm gain access to the female's sperm storage tubules. There was no evidence that female effects accounted for any of the variation in paternity.

Sperm-bound amidase activity as a marker for acrosomal integrity in bull spermatozoa.

An assay for sperm-bound amidase activity was validated using bovine spermatozoa and N-benzoyl-DL-arginine p-nitroanilide as substrate. The assay had intra- and interassay coefficients of variations of 5 and 12%, respectively. It is an inexpensive, simple and rapid assay since 100 samples can be evaluated in 2 hours and it requires only 4 X 10(6) spermatozoa per sample. Sperm-bound amidase activity was proportional (r = 0.95) to the percentage of spermatozoa with an intact acrosome, as determined by differential interference-contrast microscopy. A change of five percentage units in the incidence of damaged spermatozoa was detectable. Using this procedure, assessment of sperm-bound amidase activity is therefore a sensitive and efficient means of evaluating acrosomal integrity.

Onset of spermatozoal degeneration in low-fertility Delaware roosters and test for autoimmune basis.

The objectives of this study were 1) to determine the onset of a heritable reproductive disorder in the rooster that is characterized by extensive spermatozoal degeneration within the ductus deferens, and 2) to determine if autoimmunity was associated with spermatozoal degeneration. Seventy-five percent of the affected roosters did not ejaculate large percentages of degenerate spermatozoa at 20 wk of age, approximately the age of sexual maturity. Rather, seminal quality gradually declined over the next 6 wk, as both ejaculate volume and number of spermatozoa ejaculated increased. The evaluation of testicular and excurrent duct tissues via immunofluorescence failed to reveal either IgY or IgA associated with spermatozoa. While histological examination revealed greater lymphocyte numbers (P less than .05) in the proximal ductus deferens, these cells were not associated with spermatozoa nor spermatozoal clumping. While spermatozoal degeneration tends to be latent at the onset of semen production, it does not appear to be due to spermatozoal autoimmunity.

Semen donor selection by in vitro sperm mobility increases fertility and semen storage in the turkey hen.

Commercial turkey production relies on the artificial insemination (AI) of pooled semen. However, semen quality ultimately depends on the quality of individual ejaculates. The purpose of this study was to evaluate semen from individual toms by means of an objective sperm-mobility assay. Semen was then pooled by mobility phenotype and inseminated into hens, and the percentages of fertile and hatched eggs were determined after egg incubation. To indirectly evaluate hens' sperm storage, we determined the number of sperm holes in the perivitelline layer (PL) of freshly laid eggs. Semen from individual ejaculates (two trials, total of 169 toms) was evaluated by use of the sperm-mobility test (SMT). Semen was diluted to 1 x 10(9) sperm/ml, in prewarmed N-tris-[hydroxymethyl] methyl-2-amino-ethanesulfonic acid (TES)-buffered saline, and was placed over 3 ml of a 2% (w/v) Accudenz solution at 41degrees C. After a 5-minute incubation period, the cuvette was placed in a densimeter, and percentage transmission was recorded after 1 minute. Semen samples from toms ranked, according to sperm mobility, in the highest 10% and the lowest 10%, after three evaluations, were pooled by group and were used to inseminate hens weekly (trial 1: n = 20 hens/group, for 10 weeks, AI dose 150 x 10(6) spermatozoa inseminated fresh and after 24-hour in vitro storage at 5 degrees C; trial 2: n = 60 hens/group, for 16 weeks, AI dose = 75 x 10(6) spermatozoa inseminated fresh). Each week, eggs from day 6 post-AI were evaluated for holes in the PL, vestiges of acrosomal induced hydrolysis. Spermatozoa from toms of different mobility phenotypes were also evaluated individually, for sperm chromatin structure and motility variables, by use of the Hobson Sperm Tracker. Toms characterized by high and low in vitro sperm-mobility phenotype were categorized as "high mobility" and "low mobility," respectively. The percentage of fertile eggs from hens inseminated with semen from the high-mobility toms was higher than the percentage of fertile eggs from the low-mobility group, in each trial (95.8+/-1.3% vs. 90.4+/-2.2%, and 88.7+/-4.0% vs. 82.4+/-0.4%, trials 1 and 2, respectively; P < 0.05). More sperm holes were observed in the PL of eggs fertilized by the high-mobility toms than in the PL of eggs fertilized by the low-mobility toms (P < 0.05). No differences in susceptibility of sperm nuclear DNA to denature in situ, as measured by the flow-cytometric sperm chromatin-structure assay, were detected between toms of differing mobility phenotypes. Sperm-motility variables, straight-line velocity, and average-path velocity were significantly greater for high-mobility toms compared to low-mobility toms (P < 0.05). Sperm-mobility differences between toms (detected by means of the SMT) influenced sperm storage, as indicated by the number of sperm in the PL and by the percentage of fertile eggs produced.

Identification of ferrous sulfate toxicity in a commercial broiler flock.

A 2.5% mortality rate was observed in a flock of 19,000 commercial one-day-old broiler chicks that had been placed 24 hours previously on litter treated with ferrous sulfate heptahydrate. Ulcerative ventriculitis and severe hepatopathy were the primary lesions observed grossly and microscopically. Pooled digesta contained 6854 ppm iron. Lesions identical to those found in the field case were reproduced experimentally.

Inhibition of motility of bovine, canine and equine spermatozoa by artificial vagina lubricants.

The effects of four vaginal lubricants on progressive spermatozoal motility were evaluated. Neat semen was exposed to 0, 5, or 10% (w/v) of H-R, sterile K-Y, nonsterile K-Y or Maxilube lubricating jellies for 10 min at 37 degrees C and then extended to 10x10(6) spermatozoa/ml. Spermatozoal motility was evaluated after 0, 1, 2, 4 and 6 or 8 h of incubation at 37 degrees C. For bovine spermatozoa, sterile K-Y jelly at 10% suppressed motility (P<0.05), but nonsterile K-Y, H-R and Maxilube jellies had no effect. Maxilube was toxic (P<0.01) to canine spermatozoa and is not recommended for use during collection or insemination of canine semen. Exposure of equine semen to 10% H-R jelly had no effect on spermatozoal motility, whereas 10% sterile K-Y, nonsterile K-Y or Maxilube jellies suppressed (P<0.05) motility. For all three species, the new, nonsterile K-Y jelly was no more deleterious to spermatozoal motility than the old, sterile K-Y jelly, and H-R jelly also was satisfactory. Fertility tests are required to determine the effect of these products on fertility.

Effects of centrifugation, glycerol level, cooling to 5 degrees C, freezing rate and thawing rate on the post-thaw motility of equine sperm.

Five experiments evaluated the effects of processing, freezing and thawing techniques on post-thaw motility of equine sperm. Post-thaw motility was similar for sperm frozen using two cooling rates. Inclusion of 4% glycerol extender was superior to 2 or 6%. Thawing in 75 degrees C water for 7 sec was superior to thawing in 37 degrees C water for 30 sec. The best procedure for concentrating sperm, based on sperm motility, was diluting semen to 50 x 10(6) sperm/ml with a citrate-based centrifugation medium at 20 degrees C and centrifuging at 400 x g for 15 min. There was no difference in sperm motility between semen cooled slowly in extender with or without glycerol to 5 degrees C prior to freezing to -120 degrees C and semen cooled continuously from 20 degrees C to -120 degrees C. From these experiments, a new procedure for processing, freezing and thawing semen evolved. The new procedure involved dilution of semen to 50 x 10(6) sperm/ml in centrifugation medium and centrifugation at 400 x g for 15 min, resuspension of sperm in lactose-EDTA-egg yolk extender containing 4% glycerol, packaging in 0.5-ml polyvinyl chloride straws, freezing at 10 degrees C/min from 20 degrees C to -15 degrees C and 25 degrees C/min from -15 degrees C to -120 degrees C, storage at -196 degrees C, and thawing at 75 degrees C for 7 sec. Post-thaw motility of sperm averaged 34% for the new method as compared to 22% for the old method (P<0.01).

Chicken acrosin: extraction and purification.

The objective of the present research was to identify a procedure whereby chicken acrosin could be purified. Acrosin, as evidenced by amidase activity, was extracted with urea most efficiently at a concentration of 6 M. Extraction efficiency was enhanced by spermatozoal lysis prior to admixture with 6 M urea. Lysis was induced by passage of spermatozoal suspensions through a French pressure cell. Acrosin was purified by using gel filtration, chromatofocusing, and affinity chromatography. Based on amidase activity, a 19-fold purification was obtained with a 28% recovery. Native electrophoresis resolved two major protein bands with proteolytic activity. The methods described afford the procurement of milligram amounts of chicken acrosin.

Analysis of deferent duct morphology in turkeys characterized as low or high semen volume producers.

The objective of the present research was to clarify the basis for the difference in performance of turkey toms characterized as low or high semen volume producers. Variables measured included testicular weight, deferent duct semen volume, and deferent duct morphology based upon plastic casts. Three organizational patterns emerged after viewing plastic casts of deferent ducts: helical, serpentine, and randomly folded coil. THe randomly folded coil was the principal pattern in deferent ducts from low and high semen volume producers alike. No difference in patterns was observed between low and high semen volume producers. Differential semen yield was attributed to a greater testicular mass along with a greater storage capacity of the deferent duct in high semen volume producers. However, deferent duct storage capacity appeared to be the predominant factor affecting semen yield.

Analysis of poultry fertility data. 2. Comparison of long- and short-term fertility trials.

The purpose of the present study was to determine the effect of egg collection interval on interpretation of fertility trial results. Low-fertility (LF) and randombred (RB) Delaware hens were inseminated with spermatozoa from LF or RB Delaware roosters. Three replicate trials were performed for each cross. Eggs were collected for 21 days following insemination. Fertility was analyzed with a log odds model following logit transformation. Separate analyses were performed on data collected throughout the first 7 days and the entire egg collection interval. Duration of fertility over the 21-day interval was analyzed by iterative least squares. Whereas the 21-day egg collection interval afforded detection of differences between males and between females, no differences were observed following analysis of data from the 7-day interval. Differences in reproductive efficiency also were detected when data were analyzed by iterative least squares. Thus, the value of a fertility trial is, in part, determined by the length of the egg collection interval.

Effect of extender viscosity on the insemination dose for chickens.

When semen was diluted 1:2 with Beltsville poultry semen extender (BPSE) containing 4% (w/v) carboxymethyl cellulose (CMC), seminal viscosity was 14 times greater than that of semen similarly extended with BPSE and 2.2 times more viscous than neat semen. Under these conditions, CMC was not spermicidal (P greater than .05). Increased seminal viscosity did not enhance fertility (P greater than .05) over a 20-day interval following a single insemination of 33 or 100 X 10(6) spermatozoa/hen. In the case of semen extended with BPSE as well as BPSE containing CMC, the insemination dose of 100 x 10(6) spermatozoa gave superior fertility (P less than .05) compared with results of insemination with 33 X 10(6) spermatozoa. However, when fertility data from the 1st wk of egg collection were compiled, there was no difference in fertility (P greater than .05) due to insemination dose. These results demonstrate that although a conventional insemination dose could be reduced in order to use semen more efficiently, increasing seminal viscosity may not be warranted for artificial insemination programs.

Effects of incubation at 4 C on calcium uptake and acrosin activity in turkey spermatozoa.

Turkey semen was diluted 1:1 with a phosphate diluent (Clemson Turkey Semen Diluent) containing radioactively labeled calcium-45 (45Ca) and stored in in vitro at 25 or 4 C to determine the effects of cold-storage on spermatozoa 45Ca permeability. In addition, acrosin activity of semen stored 24 hr at 4 C was compared with that of fresh diluted semen at 25 C. Cold-stored spermatozoa immediately sequestered 45Ca at an exponential rate until the 3 hr disintegrations per min/10(9) spermatozoa equalled that of spermatozoa stored 24 h at 4 C. Washing the spermatozoa after 3 hr incubation reduced the 45Ca of spermatozoa stored at 25 C but not at 4 C. Acrosin activity of spermatozoa diluted 1:1 with CTSD and stored at 4 C for 24 hr was 1.74 times greater than for fresh sperm held 1 hr at 25 C. These results suggest that cooling turkey spermatozoa causes an intracellular influx of calcium ions that may be associated with acrosin formation.

Analysis of poultry fertility data.

Single Comb White Leghorn (SCWL) hens were inseminated intravaginally with spermatozoa from either SCWL or subfertile Delaware roosters in three replicate fertility trials. Overall fertility was analyzed with a log odds model following logit transformation. Duration of fertility was analyzed by iterative least squares. Hens inseminated with spermatozoa from SCWL males laid a higher proportion of fertilized eggs over a longer interval than those inseminated with spermatozoa from affected Delawares. Log-odds and logistic models may have advantages over the more traditional methods of evaluation. This is particularly true in regard to the distribution and normalization of error variances via the logit transformation and removal of nonadditivity via logistic regression.

Research note: evaluation of humoral and delayed hypersensitivity responses in cockerels reared under constant light or a twelve hour light:twelve hour dark photoperiod.

Immature cockerels were reared under constant light [24 h light (L):0 h dark (D)] or a 12L:12D photoperiod in order to evaluate the effect of constant light on immunoresponsiveness. At 10 wk of age males were immunized with SRBC, with reimmunization occurring either 3 or 8 wk later. Animals reared under constant light had lower (P less than .05) anti-SRBC antibody titers than the 12L:12D males. Additionally, the pattern of the antibody response following secondary immunizations were different, with 24L:0D males exhibiting no increase in antibody titers between Days 3 and 9 postinjection. Delayed hypersensitivity responses of 24L:0D males were lower (P less than .05) than those of 12- or 16-wk-old 12L:12D males. Taken together, these results suggest that circadian light:dark cycles may play a role in modulating the immune system.

Objective measurement of sperm motility based upon sperm penetration of Accudenz.

When a suspension of rooster sperm was overlaid upon 6% (wt/vol) Accudenz, immotile sperm did not enter but motile sperm entered rapidly. The absorbance of the Accudenz layer increased as a result. These phenomena were used to measure sperm motility objectively at body temperature. The intra-assay coefficient of variation (CV) was 2.6% (n = 3). When roosters (n = 36) were ejaculated repeatedly and sperm motility data analyzed by two-way ANOVA, a male effect was observed (P < or = 0.001). When roosters were ranked by mean motility scores (n = 3 evaluations per male) and representative males selected as semen donors, a difference in fertility (P < or = 0.001) was observed between males characterized by minimal and maximal sperm motility. Frequency analysis with data from a second flock of roosters (n = 100) revealed a normal distribution. Roosters categorized by average sperm motility (n = 18) or sperm motility greater than one standard deviation above the mean (n = 17) were selected for further analysis by repeated measurements. A split-plot ANOVA revealed a difference between categories (P < or = 0.0001) and variation among males within a category (P < or = 0.0001). In contrast, sperm motility was independent of time and there was no interaction between category and time. Thereafter, five roosters from each group were ejaculated weekly and interassay CV estimated with semen pooled by category (n = 3 observations per category). During this interval, sperm motility of average roosters was 55 +/- 5.9% of that of roosters within the high motility category. Interassay CV were 18.1 and 9.2% for roosters originally categorized by average and high sperm motility, respectively. The assay described has potential for: 1) selecting males based on sperm motility, and 2) standardizing the measurement of poultry sperm motility.