331 INSEMINATION OF OOCYTES WITH AGED SEMEN ALTERS SEX RATIO OF BOVINE EMBRYOS PRODUCED IN VITRO

Abstract

Preincubation of semen before insemination may alter the sex ratio of resulting embryos (Lechniak, D. et al., 2003 Reprod. Dom. Anim. 38, 224–227). The overall objective of this study was to evaluate effects of aging sperm before insemination for altering the sex ratio of bovine embryos. In the first experiment oocytes presumed mature were inseminated with frozen semen within minutes post-thaw and percoll-preparation (control) or after aging for 14 h post-thaw in a 34.4°C water bath, or after aging for 23 h post-thaw at 4°C. Sperm from 4 different bulls, representing 3 different breeds, were utilized (1 bull per experimental replicate). Sperm motility was assessed after aging and percoll preparation. Zona pellucidae were removed from cleaved embryos (43–68 h post-insemination; hpi) using 0.5% pronase. Blastomeres were dissociated and counted before transfer to PCR tubes. Sex of cleavage-stage embryos was determined using Ampli-Y^™(Bredbacka, P. 1998 Reprod. Nutr. Dev. 38, 605–613). Data were analyzed using a randomized block design with mixed models of SAS (2000) after testing for normality. Aging semen for 14 h post-thaw reduced the proportion of motile sperm and compromised the ability of presumptive zygotes to cleave after insemination (Table 1). However, ability of cleaved embryos to develop to the 8–16-cell stage was not affected. Number of blastomeres comprising cleavage-stage embryos that resulted from insemination of oocytes with aged semen was similar to that for controls. Insemination of oocytes with semen aged 14 h in a water bath increased the proportion of female embryos (Table 1).To determine if effects of aging semen on altering the sex ratio of bovine embryos was time dependent, oocytes were inseminated with frozen semen within minutes post-thaw (control) or after aging for 8 or 14 h post-thaw in a 34.4°C water bath. Sperm from 3 different bulls, representing 2 different breeds were utilized (one bull per experimental replicate). The experiment was replicated 3 times with 195–233 oocytes inseminated within each treatment. Sperm motility averaged 72.7, 65.7 and 45.0% for control and semen aged for 8 and 14 h, respectively (SEM = 10). Cleavage of inseminated oocytes (67 hpi) was similar regardless of sperm treatment (68.5, 70.2 and 70.1%; SEM 14.6, for control semen or after aging for 8 or 14 h post-thaw, respectively). Insemination of oocytes with semen aged for 14 h tended to increase the proportion of female embryos (51.6 v. 38.3 and 39.1%; sperm aged for 14 h v. 8 h or control, respectively;; P = 0.08; SEM = 9.7). Within the seven replicates across both experiments, the differences in percent female for control v. aged were 5.2, 6.3, 14.9, 18.5, 20.0, 29.7 and 60%, with the highest three being significantly different (P < 0.05) by Fisher’s Exact test. Bull or replicate variation was noted but the direction of aging for increasing proportion of females was consistent. Preliminary observations suggest that biological differences between X- and Y-bearing sperm may exist such that alternative strategies for altering sex ratio in livestock species may be possible.