Blastocoel fluid aspiration improves vitrification outcomes and produces similar sexing results of in vitro-produced cattle embryos compared to microblade biopsy

Abstract

The potential applications of in vitro-produced (IVP) cattle embryos are significantly enhanced when combined with genotype selection and cryopreservation techniques. While trophectoderm (TE) biopsies are frequently used for genotyping, cell-free DNA (cfDNA) found in blastocoele fluid (BF) arises as a less-invasive method. Moreover, the blastocoel collapse produced by BF aspiration could be beneficial for embryo cryotolerance. This study was conducted to test the BF as a source of cell free-DNA (cfDNA) and to compare the BF to the TE biopsy in terms of sexing efficiency/accuracy, embryo survival and gene expression after vitrification/warming. IVP day 7 expanded blastocysts were artificially collapsed by aspiration of BF (VIT-Collapsed) or biopsied (VIT-Biopsied). After sample collection, embryos were vitrified/warmed by the Cryotop method and individually cultured in vitro. Intact fresh non-vitrified and vitrified/warmed blastocysts served as Fresh Control and VIT-Control, respectively. After sex identification of BF or TE biopsies and the corresponding surviving embryos, amplification efficiency and sexing accuracy were assessed. There were no differences between the BF and TE biopsy samples in terms of sexing accuracy or efficiency. Although all vitrified groups showed lower post-warming re-expansion rates (p < 0.05), the blastocyst re-expansion rates in the VIT-Collapsed group were comparable to those in the Fresh Control group whereas biopsied blastocysts showed the lowest (p < 0.05) re-expansion rates. VIT-Collapsed blastocysts had hatching rates that were comparable to those of Fresh Control blastocysts but significantly higher than those of the other vitrification treatments. Proapoptotic gene BAX was overexpressed in VIT-Biopsied embryos, whereas BCL2 transcripts were more abundant in the VIT-Collapsed group. On the other hand, VIT-Biopsied embryos showed altered ATP1B1- and AQP3-mRNA levels. The analysis of the cfDNA present in the BF is an efficient, minimally invasive approach to sex IVP cattle embryos. Besides, the artificial collapse of blastocoel prior to vitrification resulted in higher re-expansion and hatching ability than when embryos were vitrified after being biopsied.

Keywords: BRY4a; Blastocoel collapse; Blastocoel fluid; Cell-free DNA; ICSI pipette; Microblade; PCR; RT-qPCR; SAT1; Trophectoderm biopsy.