Short- and long-term outcomes of the absence of protein during bovine blastocyst formation in vitro.

In cattle, individual in vitro embryo culture after Day 6 benefits development, allowing non-invasive analysis of culture medium. However, undefined supplements in culture reduce analytical reliability. In this study we assayed the short- and long-term performance of embryos after bovine serum albumin removal over a 24-h period in individual culture. The absence of protein decreased embryo development and cell counts in the inner cell mass without affecting blastocyst sex ratio. However, the absence of protein produced embryos with an improved tendency to survive vitrification after 24h in culture (P=0.07). After transfer to recipients, birth rates of embryos that had been cultured with protein tended to decrease (P<0.06) mostly as a result of a higher number of miscarriages (P<0.013), reflecting lower viability. Birthweight, gestation length, height and thorax circumference did not differ between embryos cultured with or without protein. In fresh blastocysts cultured without protein, gene expression analysis showed higher abundance (P<0.05) of insulin-like growth factor 2 receptor (IGF2R; imprinting) and activating transcription factor 4 (ATF4) and DNA-damage-inducible transcript 3 (DDIT3; endoplasmic reticulum stress) transcripts, with DNA methyltransferase 3A (DNMT3A; imprinting) tending to increase (P=0.062). However, in hatched blastocysts that survived cryopreservation, glucose-6-phosphate dehydrogenase (G6PD) was overexpressed in embryos cultured without protein (P<0.01). The absence of protein results in fewer blastocysts but improved long-term viability after cryopreservation.