Influence of the duration of gamete interaction on cleavage,growth rate and sex distribution of in vitro produced bovine embryos

Various factors including the length of gamete interaction and embryo culture conditions are known to influence the rate of development and sex ratio of mammalian embryos produced in vitro. While the duration of gamete interaction deemed optimum would vary depending upon the species involved and the preferred sex in the outcome of in vitro procedures, the mechanisms favoring the selection of embryos of one sex over the other, or the exact time of post-fertilization stage at which a sex-related difference in growth rate is manifested, are not fully understood. In order to determine the optimum length of gamete co-incubation and the impact of male gamete 'aging' on the growth rate and sex ratio of bovine embryos, a series of experiments was carried out using in vitro matured (IVM) oocytes. In experiment 1, IVM oocytes were co-incubated with sperm from two different bulls for 6, 9, 12 and 18 h and the presumptive zygotes were cultured for approximately 7.5 days (178-180 h post-insemination (hpi)) prior to assessing the cleavage rate, blastocyst yield and the sex ratio of blastocysts in each co-incubation group. In experiment 2, the blastocysts obtained from different co-incubation groups were subjected to differential staining to determine the total cell number (TCN) and the proportion of cells allocated to the inner cell mass (ICM) in male and female embryos to test for sex-related differences in cell proliferation or in differentiation of the two embryonic cell lineages in the blastocysts. In experiment 3, IVM oocytes co-incubated for 6, 9, 12 and 18 h with sperm from a single bull, were cultured for 3 days (72 hpi) and the pre-morulae, categorized according to the specific stage of early development, were sexed to determine if a sex-dependent difference is detectable before the blastocyst stage. In experiment 4, IVM oocytes exposed to prolonged co-incubation (18 and 24 h) were compared with those co-incubated with "aged" (pre-incubated) sperm to determine if "aging sperm" is a factor affecting the growth rate and sex ratio of the out come. Our experiments showed that (1) the shortest period (6 h) allowed the highest proportion of cleaved oocytes to reach the blastocyst stage regardless of the semen donor, (2) males out number females (over 2 to 1) among blastocysts when co-incubation of gametes is reduced to 6 h, (3) the male blastocysts display higher total cell count, and (4) the faster growth rate of the male embryos does not affect the early differentiation and allocation of cells to the ICM. Furthermore, our results indicate that the disruption of the expected 1:1 ratio for male and female embryos in the short term co-incubation group is evident as early as the 4-cell stage and peaks at the 8-cell stage and that prolonged gamete interaction tends to reduce the blastocyst yield to even out the sex ratio. Absence of a significant effect on the yield and sex ratio of blastocysts in the prolonged co-incubation groups irrespective of the type of sperm (aged versus non-aged) used suggest that the preponderance of male embryos in short term gamete interaction group may be dependent upon the in vitro advantage of the Y-chromosome bearing sperm. This advantage, manifested in the precocious development during the pre-morulae stage is confined to a short duration that is neutralized when gamete interaction is allowed to proceed beyond 6h.