PARENT SESSION
Preimplantation Embryo/Fetal Development

IN VITRO CULTURED MALE BOVINE EMBRYOS MAKE THE TRANSITION FROM MORULA TO BLASTOCYST MORE SUCCESSFULLY THAN FEMALES.

Kimura, Koji¹, Larson, Melissa¹, Roberts, R.¹, ¹

ABSTRACT- Several mammalian species are capable of adjusting the sex ratio of offspring born, most likely as the result of selection prior to implantation. There have also been numerous reports that in vitro cultured male embryos advance to the blastocyst stage more quickly than females, although it is often unclear whether males develop more quickly than females during early cleavage, or whether there is a block to development, possibly dependent upon culture conditions, that affects females more than males. The goal here has been to determine whether the bias towards males that we have observed at the expanded blastocyst stage among in vitro produced bovine embryos is the result of differential growth rate or a developmental block. Oocytes were matured and fertilized by standard in vitro procedures. Zygotes were cultured in batches of approximately 25 for 48 h on glucose-free CZB medium (50ml) and then transferred to M199 containing glucose and supplemented with FBS. Under these conditions approximately 25% of the oocytes advanced to blastocysts, which were sexed retrospectively with Y-specific primers. The mean time for male and female embryos to reach blastocyst was 8.8 ± 0.1 and 8.6 ± 0.1 days, respectively, for one study (p = .07) and 8.4 ± 0.1 for both sexes in the second. However, the ratio of males to females in study 1 was 0.66:0.34 (n = 235) and 0.64:0.36 (n = 269) in study 2. To determine where the block to development of females occurred, embryos were removed from group cultures on the mornings of d3, d6, and d8. At d8, the blastocysts that had formed were classified as either early (first signs of a blastocoel), blastocyst (obvious blastocoel, but no increase in diameter of the embryo), or expanded blastocyst. An approximate 1:1 sex ratio was maintained until the embryos reached the early blastocyst stage, and males did not progress faster in development than females until d6. Morulae at d8 were predominantly female, while the majority of d8 blastocysts, other than those in the very earliest stages of development, were male. Thus, female embryos have difficulty negotiating the morula to blastocyst transition, possibly as a result of culture conditions. Supported by NIH Grant HD 21896.

KEY WORDS: blastocyst, embryo culture, IVM/IVF, sex ratio