288 KINETICS OF FERTILIZATION, DEVELOPMENT, AND SEX RATIO OF IN VITRO-PRODUCED BOVINE EMBRYOS OBTAINED WITH FOUR DIFFERENT BULLS

Abstract

The between-bulls variation in in vitro fertility and the shift of sex ratio toward male embryos are two problems affecting the in vitro production (IVP) of bovine embryos. Our objective was to evaluate the possible correlation between the kinetics of fertilization, embryo development, and the sex ratio of the resulting embryos. In a first experiment, and using frozen-thawed semen of 4 different AI bulls, the kinetics of pronucleus (PN) formation was evaluated at 8, 12, and 18 h post-in vitro insemination (hpi) after fixation and staining with Hoechst 33342. Fertilized oocytes were classified in 3 PN stages: PN1: showing the first signs of sperm head decondensation; PN2: with two pronuclei of different sizes, the two being far from each other; and PN3: showing two symmetric pronuclei of equal size, close to each other. Differences between bulls were observed at each time point, but were greater at 12 hpi than at 8 or 18 hpi. At 8 hpi and 12 hpi, bull C showed a significantly faster PN formation by comparison with the 3 other bulls (chi-square test: P < 0.05), whereas at 18 hpi, the proportion at each of the PN stages was similar to that of bulls A and D, with bull B showing delayed PN development. In a second experiment, a standard IVP procedure was conducted with the 4 bulls to determine cleavage and blastocyst rates. The timing of first cleavage was measured using time-lapse cinematography. Compared with those of bull B, the embryos generated with bull C led to significantly higher Day 7 blastocyst yields (31.3 ± 9.5% vs. 21.9 ± 6.7%; ANOVA: P < 0.05). Moreover, the embryos from bull C reaching the blastocyst stage cleaved faster (first cleavage at 23.1 ± 2.1 hpi vs. 25.4 ± 2.7 hpi for bull B; ANOVA: P < 0.05). In a third experiment, 65 to 76 Day 8 blastocysts were sexed per bull. Embryo sexing was performed by PCR using the co-amplification of a Y-specific bovine SRY sequence and an autosomal btRep-137 sequence. Only blastocysts obtained with bull C showed a shift in sex ratio toward male embryos (76.0% male embryos vs. 53.8% for bull B; chi-square test: P < 0.05), whatever the size of the blastocyst. The shift in sex ratio was already present at the 2-cell stage (64.2% male embryos; n = 53; chi-square test: P < 0.05). In conclusion, for 2 out of 4 bulls, a correlation was observed between the kinetics of PN formation, the timing of first cleavage, and the sex ratio of the resulting embryos.