Influence of cooling rates and extenders upon post-thaw viability of bovine spermatozoa packaged in .25- and .5-ml French straws.

Post-thaw survival of bovine spermatozoa was compared for semen packaged in either .25- or .5-ml French straws and frozen at three different cooling rates in moving N2 vapor. Using a split ejaculate technique, nine ejaculates were extended in heated skim milk, egg yolk-citrate and egg yolk-Tris and packaged in .25- and .5-ml French straws. Semen packaged in the .25- and .5-ml straws was frozen simultaneously at initial N2 vapor temperatures of -140, -110 or -80 C. Semen was thawed in a water bath at 35 C for 1 min. Recovery of spermatozoa was evaluated immediately post-thaw (0 h) and again after 3 h of incubation at 37 C. Motility estimates and motility counts were made using phase contrast microscopy; percentage of spermatozoa possessing intact acrosomes was quantitated using differential interference contrast microscopy. There was a packaging unit X cooling rate interaction (P less than .05) for all three viability measures. However, there was no consistent trend with regard to cooling rate or packaging unit among the three extenders examined. Post-thaw viability for each characteristic varied (P less than .01) among extenders, but not for cooling rate or packaging unit (P greater than .05). Spermatozoa extended and frozen in egg yolk-Tris had greater (P less than .05) post-thaw viability than those extended in skim milk or egg yolk-citrate regardless of cooling rate or volume of the seminal package.

Effect on fertility of freezing large numbers of straws of bovine spermatozoa in a mechanical freezer.

Fertility of semen frozen in a mechanical forced-vapor freezer was compared with that frozen in static nitrogen (N) vapor. Semen from 10 Holstein bulls was extended in milk-10% glycerol at 30 x 10(6) progressively motile spermatozoa/ml and packaged in .5-ml French straws. Straws were divided into three equal groups . bull-1 . collection day-1 and frozen in a mechanical freezer at full or half loads (7,250 +/- 250 or 3,500 +/- 250 straws in 5-straw goblets on metal canes) or in static N vapor (330 +/- 30 straws held singly on horizontal racks). For field distribution, straws frozen in static N vapor also were placed in 5-straw goblets on canes after being frozen. Each of the two goblets on a cane held a different treatment. Based on 75-d nonreturn rates for 23,137 first service inseminations, fertility was 1.6 percentage points higher (P less than .05) for semen frozen in static vapor (66.3%) than that frozen in a fully loaded mechanical freezer (64.7%). The difference of 1.3 percentage points in fertility favoring static vapor over freezing a half load in a mechanical freezer (65.0%) approached significance (P = .06); half and full loads did not vary (P greater than .05). In conclusion, the small increase in fertility favoring static over forced-vapor freezing supports use of the static vapor method unless the savings in time and labor of freezing large numbers of straws at one time in a mechanical freezer are considered to offset the small difference in fertility.

Effect of long term ejaculation at high frequency on output of sperm, sexual behavior, and fertility of Holstein bulls; relation of reproductive capacity to high nutrient allowance.

Eighteen Holstein bulls were used to determine the long term effect of high frequency ejaculation on reproductive performance. From 1 to 7 yr of age, six bulls were ejaculated weekly, and six pairmates were ejaculated six times weekly. For comparison, six bulls were ejaculated one or six times weekly from 1 to 2 yr of age, placed on sexual rest, and then ejaculated six times weekly for 6 wk and twice daily for 14 days at 3, 4, and 5 yr of age. Six time bulls yielded 3.3 times more motile sperm per week than one time bulls. Sexual activity, which was similar for one and six time bulls, was relatively constant to 5 yr of age and then gradually decreased. Sexual rest did not affect subsequent weekly output of sperm. Postthaw motility of sperm was similar for the one time, six time, and sexually rested groups. Fertility at 4, 6, or 8 yr of age did not differ between one and six time bulls. In conclusion, continuous high frequency of ejaculation from 1 to 7 yr of age greatly increased the harvest of sperm from a bull without harming his growth, reproductive capacity, or fertility. Ten other Holstein bulls fed concentrate ad libitum from 17 wk to 2 yr of age attained puberty 2 wk earlier (37 wk of age) and yielded 1.4 times more sperm per week on six times at 3 yr of age than the six time bulls fed limited energy. Raising bulls on maximum energy allowance enables sampling in a progeny testing program by 13 to 14 mo of age.

Effect of bulk freezing straws of bovine spermatozoa in a programmed freezer on post - thaw survival.

Twelve ejaculates were extended both in skimmilk and Tris-yolk, packaged in .5-ml French straws and frozen vertically in bulk (10,000 straws/freeze) in a programmed mechanical freezer (Linde CRFC-3). Cooling rates and post-thaw spermatozoal survival in the upper and lower thirds of straws at the following six chamber positions were compared: corner and middle within-goblet positions at center, intermediate and outer chamber locations. Cooling rates generally were faster in the upper third of straws than in the lower third and at the corner rather than the middle within-goblet positions. For combined postthaw incubation periods of 0, 3 and 6 hr at 37 C, motility (photographic and visual) and acrosomal retention (fixed and unfixed samples) were both higher (P greater than .01) for sperm in each extender in the upper third of straws than in the lower third. Spermatozoal motility and acrosomal retention generally were higher (P greater than .05) in each extender at the center rather than at the outer chamber location. Among within-goblet positions, post-thaw survival of sperm was higher (P greater than .05) at the corner than at the middle positions for Tris-yolk but not skimmilk. For comparison semen was frozen in static N vapor in straws held singly (258-straw load) on horizontal racks. For combined incubation periods, post-thaw survival of sperm did not differ (P less than .05) between bulk and static vapor- systems. There also was no difference when each of the six chamber positions was compared individually with static vapor. In conclusion, 33% more straws of semen can be frozen in a mechanical freezer than previously reported, with post-thaw spermatozoal survival comparable to that of sperm in straws frozen conventionally in static vapor.

Combinations of glycerol percent, glycerol equilibration time, and thawing rate upon freezability of bull spermatozoa in plastic straws.

Twelve ejaculates were used in a central composite experiment to test 15 combinations of glycerol (7, 9, 11, 13, or 15%), glycerol equilibration times (1, 2, 4, 8, or 16 h) and thawing rates (water at 35 C for 15 s, 50 C for 13 s, 65 C for 11 s, 80 C for 9 s, or 95 C for 7 s). Semen was diluted in heated skim milk-glycerol, packaged in .3-ml. Continental U.S. straws and frozen in liquid nitrogen vapor. Based on post-thaw progressive sperm motility after storage at -196 C for 9 to 11 days, estimated optima from multiple regression were 10.7% for glycerol, 2.0 h for glycerol equilibration time, and 76 C for thawing bath temperature. Only the linear effect for each variable was significant. Much faster thawing rates and shorter glycerol equilibration times than those for freezing bull spermatozoa in glass ampules should be used for maximum post-thaw sperm motility in straws.

Effect of sperm numbers on fertility of frozen bull spermatozoa in skim milk diluent.

Insemination doses of 10, 15, and 20 million progressively motile spermatozoa prior to freezing were compared in a split-ejaculate fertility experiment involving 4,603 first service inseminations to 34 semen samples from six Angus bulls. Based on 60- to 90-day nonreturn rates, fertility was significantly lower for inseminating doses of 10 and 15 times 10-6 than 20 times 10-6 motile sperm (61, 62, and 70%). The 1-ml doses of skim milk diluted semen were frozen in glass ampules. The post-thaw number of motile sperm per ampule after freezing and storage for 3 wk in liquid nitrogen averaged 5.5, 8.4, and 12.4 times 10-6 for corresponding pre-freeze numbers of 10, 15 and 20 times 10-6 motile sperm/ml and ranged from 4.0 to 15.7 times 10-6/ml. Frozen semen packaged in ampules should contain more than 8.4 times 10-6 motile cells post-thaw per ampule.