132 TIMING OF THE FIRST ZYGOTIC CLEAVAGE WAS NOT RELATED TO THE SHIFT IN SEX RATIO OF BOVINE BLASTOCYST IN VITRO

Abstract

It has been shown that embryos which cleaved earlier (<30 h postinsemination, hpi) have greater chance to develop to blastocyst. It has been shown that in in vitro conditions male embryos develop faster than females. Further, spermatozoa with the Y chromosome more frequently penetrate oocytes during the first 6 hpi. The aim of this experiment was to investigate the sex-related embryo growth rate in relation to the timing of the first zygotic cleavage and GH presence during IVM. Oocytes were matured in TCM-199 supplemented with fatty acid free BSA and hormones (FSH and GH) and then inseminated (Parrish et al. 1998 Biol. Reprod. 38, 1171–1180), whereas embryos were cultured in sequential media (Lane et al. 2005 Theriogenology 60, 407–419). All embryos that cleaved by 30 hpi (early cleavers, EC) were selected and cultured separately. The remaining embryos cleaved by 48 hpi (non early cleavers, NEC) were also incubated in separate drops. Blastocysts of proper morphology were collected at 176 hpi and subjected to sex determination by PCR (AMGL gene). The significance of developmental stage, timing of the first zygotic cleavage and GH supplementation in relation to the sex ratio was evaluated by the chi-square test. All straws with frozen semen of 2 bulls used in this experiment were derived from single ejaculates. The sex ratio of sperm samples used for IVF was evaluated by FISH with locus-specific probes. The experiment was done on 266 embryos obtained from 1249 oocytes. A significant predominance of male blastocysts regardless of the experimental conditions was observed [the male to female ratio (M:F) 2:1, P < 0.001]. FISH analysis revealed that there was no deviation from the expected 1:1 ratio of X and Y spermatozoa in sperm samples used for IVF. When the timing of the first zygotic cleavage was considered, shift in M:F ratio in favor of males (P < 0.01) in blastocysts derived from EC zygotes was noticed. Due to a very low number of NEC embryos, this category was not included in analysis. Although the M:F ratio was shifted towards males, the rate of female embryos was greater in the control group (25F/38M) v. GH group (16F/39M) of EC expanded blastocysts. This phenomenon was not observed among hatched blastocysts; however, GH presence caused an increase (P < 0.01) in the number of females at this stage. Therefore GH may stimulate embryonic growth, especially embryos of reduced quality, through its positive influence on cytoplasmic oocyte maturation. In conclusion, the predominance of male blastocysts observed in this study may be attributed to the applied IVF procedure because the X:Y ratio in spermatozoa was not different from the expected 1:1 ratio. Moreover, significantly fewer females among analyzed blastocysts may suggest that the developmental potential of female embryos in applied in vitro conditions was somehow reduced when compared with males. This became evident when the transition from expanded to hatched blastocysts was observed.

This work was supported by the project No. N302 046 31/3780 of the Ministry of Science and Higher Education, Poland.