Survival of bull sperm frozen at different rates in media varying in osmolarity.

The effects of freezing procedures, osmolarity, trehalose, and sucrose on survival of bull sperm in whole milk (WM) and egg yolk-Tris (EYT), semen extenders used worldwide, were studied. Sperm were added to extenders at 25 degreesC, cooled slowly to 5 degreesC, glycerolated, packaged in 0.5-ml straws, and frozen. Different freezing rates were accomplished in two steps. Straws were transferred from +5 degreesC to nitrogen vapor at temperatures ranging from -10 to -100 degreesC in the first step and to liquid nitrogen in the second step. Straws were thawed in water at 35 degreesC. A substantial decrease in sperm motility occurred between -10 and -20 degreesC, as abrupt nucleation occurred following supercooling to -13 degreesC. To study the interactions between osmolarity x cooling rate, WM and EYT extenders were prepared to yield media measuring 220 to 420 mOsm/L. The optimal first-step range of cooling in the two-step procedure was -30 to -70 degreesC, and the highest proportions of motile sperm after freezing and thawing were 61 to 62 in 260 to 300 mOsm/L WM and 63 to 64% in 300 to 340 mOsm/L EYT, equivalent to the results with the control procedure used commercially. As the cooling rate increased (first step to -100 degreesC) sperm motility was much higher in hypertonic than in hypotonic extenders (P < 0.05), indicating the importance of partial dehydration before rapid cooling. Replacing part of EYT and WM with equivalent solutions (same mOsm/L) of sucrose or trehalose had no appreciable effect. These results provide a basis for further investigating simple freezing systems that might be more effective in preserving bull sperm than those currently available.

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.

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.

Semen quality and behavior of Holstein bulls exposed to estradiol-treated bulls for mounts.

The objectives were to test the effects of estradiol treatment of teaser bull mounts on sexual behavior and on quality and quantity of sperm obtained from sires managed as in large commercial AI breeding organizations. In a change-over design, the same teaser bulls were either untreated or treated with estradiol. Five semen-producing bulls were ejaculated twice per day on Tuesdays and Fridays after epididymal reserves were partially depleted. A 15-min period of continuous sexual preparation with three false mounts allowed was standard before each semen collection. All bulls were attracted to and licked the preputial area of the teaser bulls followed by the Flehmen response during the period of sexual preparation. Bulls usually completed the false mounts in < or = 15 min, and all thrusted vigorously with both hind feet moving forward synchronously at this time on 100% of the 80 semen collections. Major differences among bulls and between first and second ejaculates occurred in semen volume, semen concentration, and total sperm collected. An increase of 10% in total sperm output when bulls were exposed to treated teaser bulls could be of commercial benefit. The correlation between total time to first mount for the two ejaculates per bull each day and total sperm collected per bull per day was -.44. Thus, the shorter time to first mount may be useful as a low level predictor of higher sperm out-put per bull.

Effect of sucrose, trehalose, hypotaurine, taurine, and blood serum on survival of frozen bull sperm.

Factorially arranged experiments were conducted to study the effects of adding sucrose and trehalose, known to have cryoprotective properties, blood serum, and the antioxidants taurine and hypotaurine on sperm motility after freezing and thawing at different rates. At sugar concentrations of 0.1 M, osmolality of the whole milk (WM) or egg yolk-Tris freezing medium (without glycerol) was about 370 mOsmol. Survival following freezing and thawing was reduced unless osmolality was corrected by adding pure water to reduce osmolality to about 280 mOsmol. Then the post-thaw percentages of motile sperm for control WM, and WM with 0.05 or 0.10 M sucrose, or 0.05 or 0.10 M trehalose, respectively, were 62, 55, 61, 57, and 62%. Thawing semen at 5 degrees C, versus 25 degrees C resulted in 64 versus 70% motile sperm (P < 0.05). Post-thaw survival of sperm stored at 25 degrees C for 24 h in trehalose-treated WM was superior to that in WM (P < 0.05). Hypotaurine and taurine had little effect on sperm survival. Up to 10% (v/v) of heated blood serum was generally beneficial, but gave more variable responses with different bulls. Sperm survival after cooling at -25 degrees C/min was slightly superior to that after cooling at -15 degrees C/min to -100 degrees C. The effects of the compounds studied on motility of frozen-thawed sperm were small, but if they protect sperm cell membranes, as reported for other types of membranes, they may assist sperm in surviving in the reproductive tract of the cow prior to fertilization.

Fertility of bull spermatozoa frozen in whole milk extender with trehalose, taurine, or blood serum.

Fertility of bull semen processed in heated whole milk-glycerol control semen extender with various additives was compared in six field trials. The additive in field trials 1 and 2 was 25.6 g of trehalose/L of the glycerol fraction of whole milk. Whole milk was heated to 95 degrees C for 10 min, cooled, and filtered 1 d before use (trial 1) or 3 d before use (trial 2). In field trial 3, 3.0 g/L of taurine were added to the glycerol fraction of whole milk. In field trial 4, specially prepared bovine serum (15% vol/vol) was included in the glycerol fraction of whole milk. Field trials 5 and 6 were larger fertility studies with trehalose in extenders prepared the day before use (trial 5) and 1 and 3 d before use (trial 6). Control and treated semen were coded and distributed randomly over a large group of professional inseminators. The 59-d nonreturn rates for control and treated semen, respectively, were as follows: trial 1, 74.1 and 73.7%; trial 2, 71.3 and 73.1%; trial 3, 74.9 and 70.9%; and trial 4, 75.1 and 71.6%. No significant differences resulted in trials 1 to 3, but bovine serum decreased the non-return rate in trial 4. Trials 5 and 6 resulted in nonsignificant improvement in fertility with added trehalose. Thus, these additives, useful as cryopreservatives or membrane protectors in other systems, did not enhance the fertility of sperm frozen in whole milk.