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Bovine Blastocyst Development In Vitro: Timing, Sex, and Viability Following Vitrification¹

Center for Regenerative Biology,³ University of Connecticut, Storrs, Connecticut 06269-4243 Research Group for Applied Animal Genetics and Biotechnology,⁴ Hungarian Academy of Sciences and Szent Istvan University, Godollo 2103, Hungary Department of Animal Biology,⁵ Agricultural Biotechnology Center, Godollo 2100, Hungary

Selection of blastocysts based on their morphological characteristics and rate of development in vitro can skew the sex ratios. The aim of this study was to determine whether an embryo's developmental rate affects its survival after vitrification, and whether male and female embryos survive vitrification differently. In vitro fertilized bovine oocytes were cultured in potassium simplex optimized medium (KSOM) + 0.1% BSA for 96 h, and then into KSOM + 1% BSA (KSOM) or in sequential KSOM + 0.1% BSA for 96 h, and then into synthetic oviduct fluid (SOF) + 5% FBS (KSOM-SOF). In part 1 of this study, embryos cultured in each medium that had developed into blastocysts at approximately 144, 156, or 180 h were recovered from culture, graded, and then vitrified. After warming, blastocyst survival rates were immediately evaluated by reexpansion of the blastocoels. In the second part of the study, all blastocysts (n = 191) were sexed by polymerase chain reaction 48 h after warming. When cultured in KSOM medium, more 144-h blastocysts survived vitrification (68%) than blastocysts vitrified at 180 h (49%). Blastocysts derived at 156 h in KSOM-SOF survived vitrification better (87%) than blastocysts vitrified at either 144 h or 180 h, and subsequently hatched at a greater rate than those vitrified at 180 h. The overall blastocyst survival rates did not differ significantly whether embryos were cultured in KSOM or sequential KSOM-SOF. Blastocysts derived at 144 and 156 h in KSOM or KSOM-SOF were predominately male, and significantly more of them survived vitrification 48 h after warming. However, blastocysts cultured in KSOM-SOF, and then vitrified at 180 h were predominately female. Overall, blastocysts that survived vitrification, and subsequently hatched 48 h after warming, were male. In summary, embryos that reached the blastocyst stage earlier were predominantly males; these males had better morphology, endured vitrification, and subsequently hatched at a greater rate than did female blastocysts.

¹ This research was supported by a joint grant (FY2001) from the Internship Program for Early Career South African Scientists (U.S. Department of Agriculture, South African National Department of Agriculture, Agricultural Research Council, and Colorado State University).

² Correspondence: X. Cindy Tian, Connecticut Center for Regenerative Biology/Department of Animal Science, University of Connecticut, 1392 Storrs Road, U-4243, Storrs, CT 06269-4243. FAX: 860 486 8809; xtian{at}canr.uconn.edu

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