Use of Hoechst 33342 stain to evaluate live fresh and frozen bull sperm by computer-assisted analysis.

The objective of this research was to investigate possible procedures for evaluating living bull sperm stained with Hoechst 33342 while in a simple medium and in commonly used complex egg yolk-glycerol-Tris (EYGT) and whole milk-glycerol (WMG) extenders. The two semen extenders provide good cryoprotection, but the latter one virtually obscures the sperm. To evaluate sperm motion characteristics when static nonsperm particles are present, a new Hamilton Thorne epifluorescent optical system (UV) with a strobe light was developed for potential use with DNA-stained sperm. This system permitted examination for the first time of sperm motion characteristics in milk. In Experiment 1 (four bull semen replicates with five dye concentrations and three incubation times), 2.5 microg/ml of Hoechst 33342 stained live and dead sperm sufficiently in a modified Tyrode's solution to measure all sperm characteristics without depressing motility, which was validated by using phase-contrast to analyze stained and unstained controls. In Experiments 2a and 2b, each using semen from four bulls with a 5 x 5 factorial arrangement, it was determined that 40 to 60 microg/ml of dye in EYGT or WMG, with UV illumination for 20 minutes, was optimal. There was no detrimental effect on sperm motility. In Experiment 3, analyses of two ejaculates, from each of eight bulls, confirmed that motion characteristics of sperm in EYGT and WMG were not depressed when the sperm were stained with Hoechst 33342. These experiments demonstrate that the dye concentrations and exposure times developed for use with the new epifluorescent optics facilitate evaluating bull sperm frozen in particle-filled whole milk and should be useful for sperm evaluation of a variety of species when nonsperm particulate matter may otherwise interfere.

Computer-assisted sperm analysis for assessing initial semen quality and changes during storage at 5 degrees C.

Computer-assisted sperm analysis equipment was used to evaluate bull sperm initially in a modified Tyrode's solution, in Cornell University extender, and in egg yolk-glycerol-Tris extender (following cooling and storage in the latter two extenders). Two ejaculates of semen were collected from each of eight bulls. Semen was divided into aliquots using a factorial arrangement. The semen, diluted to approximately 20 x 10(6) sperm/ml, was loaded into two 20-micron chambers, and six microscope fields from each chamber were videotaped for each treatment of each ejaculate of semen. Eight sperm characteristics analyzed with the Hamilton Thorne integrated visual optical system (Hamilton Thorne, Beverly, MA) were reported, and some of these characteristics differed significantly among bulls. The initial values of motile sperm in modified Tyrode's solution, Cornell University extender, and egg yolk-glycerol-Tris extender were 87, 79, and 66%; little change followed cooling and storage at 5 degrees C in the latter two extenders. Also, there was a small but significant decline in sperm velocity during 3 d of storage. Hyperactive sperm increased slightly during storage. The procedures used can rapidly and accurately measure many sperm characteristics in fresh semen and in semen stored in egg yolk extenders, and differences among bulls can be detected.

Media and dilution procedures tested to minimize handling effects on human, rabbit, and bull sperm for computer-assisted sperm analysis (CASA).

Proper handling of semen prior to computer-assisted sperm analysis (CASA) is critical if the analysis is to be representative of the fresh sample. The effects of diluting medium or dilution and holding time before CASA on multiple sperm characteristics were studied. Four replicates of unselected semen samples from each of eight human donors were diluted with phosphate-buffered saline (PBS)-glucose plus bovine serum albumin (BSA), with Tyrode's albumen lactate pyruvate (TALP), and with high-potassium TALP (K-TALP) to a concentration of approximately 25 x 10(6) sperm/ml. The diluted semen was held for 0, 1, and 2 hours at approximately 30 degrees C before CASA, with little difference between the three diluents in all 12 variables measured. There was a decline of 3-6% in the proportion of motile sperm over a 2-hour period (P < 0.05). Donors were the largest source of differences (P < 0.05). Rabbit sperm (five bucks, four ejaculates per buck) were processed in a manner similar to that of the human sperm. There was a major effect of media. The average percentages of motile sperm over 2 hours in TALP, K-TALP, and PBS were 76, 42, and 29%, respectively (P < 0.05), with a decline of only 3% in TALP during the 2 hours. Hyperactivity and other characteristics were affected by treatment. Donors were a large source of variation. Bull semen (10 bulls, two ejaculates per bull) either was not diluted or diluted with TALP 2x or 4x and held for 0, 1, and 2 hours at 30 degrees C. It was then diluted to 25 x 10(6) sperm/ml with TALP. There was little change in most sperm characteristics in any treatment during the first hour, although many of the changes were statistically significant. The percentage of motile sperm in undiluted semen declined from 87% to 82% over 2 hours. Modified TALP was a suitable medium for sperm from all three species, and a simple PBS-glucose-BSA medium can be used for human sperm.

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.

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.