P.B. Farrell

QUANTIFICATION OF BULL SPERM CHARACTERISTICS MEASURED BY COMPUTER-ASSISTED SPERM ANALYSIS (CASA) AND THE RELATIONSHIP TO FERTILITY

Two experiments were conducted to evaluate semen quality of bulls housed under controlled conditions at a large AI facility and relate results to fertility. In Experiment 1 semen was collected from six 6-yr-old bulls twice daily at 3- to 4-d intervals for 3 d. In Experiment 2 eleven 6- to 11-yr-old bulls were used. Extensive breeding information was available and semen was collected as in Experiment 1 but replicated 4 times. Standard semen analysis and computer-assisted sperm analysis (CASA) with the Hamilton Thorne IVOS, model 10 unit, were performed on 36 first and second ejaculates in Experiment 1 and on 44 first ejaculates in Experiment 2. Sixteen fields (2 chambers with 8 fields per chamber) were examined per sample. In Experiment 1 the correlation between estimated sperm concentration by spectrophotometry and CASA was 0.91 (P < 0.01). Among bulls the range in the percentage of motile spermatozoa was 52 to 82 for CASA versus 62 to 69 for subjective measurements made by highly experienced technicians. Thus, CASA, with high repeatability, provided a more discriminating estimate of the percentage of motile sperm cells than did the subjective procedure. Bull effect was much greater than any other variable in the experiments. Chamber differences were small and so the results for the 2 chambers with 8 fields each were combined. One to five CASA values were correlated with bull fertility, defined as 59-day nonreturn rates corrected for cow and herd effects. The percentage of motile spermatozoa accounted for a small fraction of the total variation in fertility (r2 = 0.34). However higher r2 values (0.68 to 0.98) were obtained for 2 to 5 variables used in the multiple regression equations. The results are promising, and further testing will determine more precisely which of these CASA variables are most useful in estimating bull fertility potential.

Repeatability of measurements on human, rabbit, and bull sperm by computer-assisted sperm analysis when comparing individual fields and means of 12 fields

OBJECTIVE To compare repeatability of measurements of human, rabbit, and bull sperm on two Hamilton Thorne Integrated Visual Optical System (IVOS) units, software version 10. DESIGN Semen samples from seven normal human subjects, six rabbits, and eight bulls were obtained at regular intervals. The samples were diluted, two chambers were filled, and 12 fields were recorded, using high resolution recorders. Computer-assisted sperm analysis (CASA) was performed nearly simultaneously with two Hamilton Thorne IVOS units. SETTING Reproduction Research Laboratories, Cornell University and Hamilton-Thorne Research, Beverly, Massachusetts. RESULTS Optimal settings were established for evaluating by CASA sperm from three species. Fifteen variables were analyzed. The correlation coefficients for most variables characterizing sperm motion and concentration, when means of 12 fields were calculated, were 0.95 to 1.00. There were too few hyperactive sperm to obtain a reliable correlation for human sperm (r = 0.63) and repeatability of elongation was lower only for human sperm (r = 0.75). CONCLUSIONS Two units of Hamilton Thorne IVOS, software version 10, were capable of providing nearly identical estimates of many CASA variables of human, rabbit, and bull sperm. Correlations for the paired estimates of many motion characteristics and sperm concentration usually exceeded 0.97.

Comparison of six-day bovine embryo development in uterine tube (oviduct) epithelial cell co-culture versus in vivo development in the cow.

A study was designed to evaluate and compare the appearance of embryos recovered from donor cows on Day 6 to embryos from in vivo fertilized cow zygotes developed to Day 6 on uterine tube (oviduct) epithelial cell co-culture using serum-free CZB medium. Embryo stage of development and quality score were assessed. Hoechst 33342 DNA stain was then used to determine the total number of blastomeres, the number of poor nuclei and the number of nuclei in mitosis. Mean cell counts did not differ for the 70 embryos evaluated in each group (65 cells in vivo, 61 cells in vitro). The percentage of transferable emryos (excellent, good or fair quality), in each group also did not differ (57% in vivo, 56% in vitro). There were no significant differences in any of the measured parameters. Our findings suggest that co-culture of in vivo produced cow zygotes can result in embryos comparable in developmental stage and quality to embryos developed in vivo in the cow for 6 d.

Ethylene glycol monomethyl ether effects on health and reproduction in male rabbits

Male Dutch rabbits were weighed and randomly assigned within each weight group to five groups of six animals each (plus one more in the highest dose group). They received 0, 12.5, 25.0, 37.5, or 50.0 mg of ethylene glycol monomethyl ether (EGME) per kg of body weight in the drinking water 5 d/week for 12 weeks. Feed and water consumption were monitored daily and body weight weekly. All animals consumed the water and feed, maintained body weight, and were in good health throughout the experiment. Semen was collected twice weekly for 12 weeks, and 96% of the ejaculates were obtained. By weeks 6 and 9, most males in groups receiving 50.0 or 37.5 mg of EGME per kg were oligospermic. Only minor changes in other characteristics of sperm obtained from treated animals were found, as measured by computer-assisted sperm analysis. Fertility of the males still producing sufficient sperm during week 12 to use for insemination was tested with 96 does producing 2839 oocytes, and fertility of treated males (41%) was not lower (P > 0.05) than 47% in controls. At necropsy, all vital organs were grossly normal, with no notable histopathology. However, the groups of animals receiving 37.5 and 50 mg of EGME per kg of body weight produced fewer sperm and had smaller testes than controls (P < 0.05). Although all rabbits appeared grossly normal, there was a marked disruption of spermatogenesis as ingestion of EGME increased above 25 mg/kg of body weight. Rabbit testes appear to be more sensitive to EGME than testes of rats or mice.

Pregnancy rates after the use of a gonadotropin releasing hormone agonist in bovine embryo transfer recipients

Abstract The use of the gonadotropin releasing hormone analog, Buserelin, was evaluated in a commercial embryo transfer program. Virgin Holstein heifer recipients (n=764) at two embryo transfer facilities were randomly allocated to three treatment groups: 1) control animals, 2) heifers injected with 8 μg Buserelin at time of transfer and 3) heifers receiving 8 μg of Buserelin 4 to 7 days after transfer. Fresh or frozen/thawed embryos were evaluated, equalized across treatments and transferred to recipients on Day 7 or 8 after estrus. Recipient progesterone levels were evaluated on the day of transfer. Pregnancy evaluations were done by palpation per rectum between Days 35 to 60 of gestation. There was no significant difference in pregnancy rates for the three treatment groups, with 68% of the animals pregnant in Group 1, 72% in Group 2 and 66% in Group 3. Progesterone levels at the time of transfer were similar for animals which became pregnant (2.60 ± 0.05 ng/ml) and animals which did not become pregnant (2.74 ± 0.09 ng/ml). There was no significant interaction observed between treatment and embryo quality or progesterone level, suggesting that the luteotrophic action of Buserelin at this early stage did not help support additional pregnancies over those seen in the control group.

Motility and other characteristics of human sperm can be measured by computer-assisted sperm analysis of samples stained with Hoechst 33342**Supported in part by a subcontract SBIR 2R44 ES0562 to Cornell University, Ithaca, New York, from The Small Business Innovative Research Program, National Institutes of Health, Bethesda, Maryland.††Hoechst 33342, Sigma Chemical Company, St. Louis, Missouri.

OBJECTIVE To develop methods for using a DNA-specific dye to discriminate between motile and nonmotile sperm and static particulate matter in fresh and diluted semen, using computer-assisted sperm analysis (CASA) with the Hamilton Thorne IVOS, TOX version (Hamilton-Thorne Research, Beverly, MA). DESIGN Donor semen was divided for treatment as fixed stained sperm (Hoechst 33342 stain; Sigma Chemical Company, St. Louis, MO), fresh motile and nonmotile stained sperm, and unstained control sperm. SETTING Normal human volunteers in an academic research and medical environment. PATIENTS Selected healthy student volunteers. INTERVENTIONS Delivered semen to the laboratory within 1 hour of collection. MAIN OUTCOME MEASURE Semen quality measured by CASA. RESULTS Fixed or fresh human sperm stained with Hoechst 33342 dye should be diluted to < or = 50 x 10(6) sperm/mL to count sperm accurately. Motile and nonmotile sperm were stained suitably with 5 to 10 micrograms/mL of dye when diluted with a simple diluent, but the dye concentration should be increased to 40 micrograms/mL when egg yolk is in the diluent. CONCLUSIONS The DNA-specific dye, Hoechst 33342, can be used to discriminate between motile and nonmotile sperm and other particulate matter when evaluated by CASA with instrumentation equipped with suitable optics.