329 PREGNANCY RATE IN EWES INSEMINATED WITH SEXED SEMEN IN ARGENTINA

Abstract

Offspring of many species have been born from flow cytometrically sex-sorted sperm since its introduction in 1989 (LA Jonhson et al.; 1999, Theriogenology). Births of lambs after insemination of ewes with low doses of sexed semen were first reported by Hollinshead et al. (Rep. Fert. Dev. 2002). A field trial was carried out in Patagonia Argentina comparing the use of low doses of sexed and non-sexed frozen semen. This study is aimed at comparing pregnancy rates in ewes inseminated with sorted and nonsorted frozen-thawed sperm. Ejaculates from two Merino rams were diluted with modified tirode solution (Shenk et al., 1999, Theriogenology), stained with Hoescht 33342, and finally incubated at 35°C for 40 minutes. The addition of 10% ram seminal plasma to the sample was done to avoid sperm agglutination caused by dilution effect (Mann, 1964). Sorting was performed with a high speed flow cytometer (MOFLO^®). Up to 8 million of sexed spermatozoa were collected in 15-mL conical tubes containing modified tirode solution plus 10% ram seminal plasma, centrifuged at 600g 12 min and resuspended with test yolk media plus 11% skimmed milk and 5% glycerol. After being refrigerated at 4°C for at least 1.5 h, sexed and nonsexed semen were packaged in 0.25-mL straws at a concentration of 4 to 5 total million sperm and then frozen in an automatic freezing machine (IMV^®). One hundred and eighty-three ewes and hoggets were laparoscopically inseminated by the same technician with eight to ten million sorted and nonsorted frozen-thawed semen 14 h after heat detection. Pregnancy diagnosis was performed by ultrasound at day 30 post-insemination. Data were analized by Chi-square. There was significant difference in the overall pregnancy rate between sorted and nonsorted sperm, although, for one of the rams that difference was not significant. There was, also, no significant difference in pregnancy rate between ram 1 and 2 in ewes inseminated with either sexed or nonsexed semen (Table 1). These results indicate that the different performance of sorted compared to non sorted sperm is mainly explained by the lower results achieved with semen from ram 2. According to this conclusion, further studies should be done in order to evaluate the degree to which the sorting process affects the sperm fertilizing capability in different rams. This research was supported by Fundación Margarita Perez Companc.

Table 1