Sex differences in single IVF-derived bovine embryo cultured in chemically defined medium

Abstract

Single embryo culture is essential for culturing embryos derived from few oocytes obtained from elite cows through ultrasonography guidance. Bovine in vitro fertilization (IVF) and individual embryo culture is a challenge as it generally leads to impaired embryo development. In this study, we explored the embryonic development and the sex ratio of IVF-derived bovine embryo cultured individually in chemically defined two-step culture medium. Total 63 cumulus-oocyte complexes were collected, in vitro matured, in vitro fertilized and the resultant fertilized oocytes were randomly cultured individually (4 trials, 15-16 oocytes each) in microdrops of 5 µL of a chemically defined two-step culture medium. Blastocysts were counted in every trial (n = 32, 50.79%) and all of them were used for both genomic DNA and total RNA extraction, cDNA synthesis and PCR using specific primers for GAPDH, GDP6, XIST and SRY genes. Results showed significant difference in expression of XIST (positive expression in 11 blastocysts) and SRY (positive expression in 21 blastocysts) mRNAs, P < .05. This result supports the hypothesis of sexual dimorphism among the pre-implantation in vitro produced embryos and provides an efficient medium for single bovine embryos in vitro production.

Keywords: Blastocyst; Bovine; In vitro fertilization; PCR; Single embryo culture.

Fig. 1

Representative image of gel electrophoresis showing validation of amplification of Y-specific segment by PCR. A. Validation of Primer 1 and 2 (Table 1) in genomic DNA isolated from different somatic cell lines; Tet_M (transgenic male cow); B-M (brain of male cow); and Fem (female cow). B. Positive amplification of Y-specific segment by primer 2 in embryos #1, and #19 with faint expression of #17. C. Positive amplification of Y-specific segment by primer 2 in embryos #24 #27 and #30. Neg means PCR reactions without cDNA template.

Fig. 2

Representative image of gel electrophoresis showing the expression of SRY and XIST transcripts in randomly selected 9 embryos (#1, 14, 17, 19, 21, 24, 27, 28, and 30) with their expression of GAPDH and G6DP transcripts.