97 ONE-DAY PROTEIN-FREE CULTURE SELECTS FOR BOVINE BLASTOCYSTS WITH IMPROVED LONG-TERM VIABILITY AFTER VITRIFICATION

Abstract

Single embryo culture in SOF with BSA allows efficient noninvasive analysis of culture medium (CM). Defined protein-free CM avoids contaminants present in commercial BSA. In this work we studied the performance of a single culture step with and without protein. In vitro-matured and -fertilized oocytes were cultured in SOF+6 mg mL^–1 BSA. From Day-6 onwards, n = 1351 morulae and early blastocysts were singly cultured in 12 µL SOF+6 mg mL^–1 BSA or 0.5 mg mL^–1 polyvinyl-alcohol (PVA; 20 replicates). Development was recorded as % Day-6 cultured embryos. Day-7 and Day-8 expanded blastocysts were vitrified and cultured upon warming in SOF+10% FCS for 48 h. Expression of genes of stress, metabolism, and imprinting was measured by RT-qPCR in Day-7 fresh expanded and in vitrified/warmed hatched blastocysts. Day-7 vitrified blastocysts were allowed to re-expand for 2 h before embryo transfer (recipients used once or twice). Day 40 pregnancy, miscarriage and birth rates, and morphometry and weight of calves, were recorded. Data were analysed by GLM and REGWQ test. Protein removal reduced Day 7 blastocyst rate (71.3 ± 2.7 v. 51.6 ± 2.7; P < 0.0001) and tended to reduce expansion rate (46.6 ± 3.1 v. 38.4 ± 3.0; P = 0.054). Survival rates of all vitrified embryos cultured with PVA tended to increase over BSA at 2 h and 24 h after warming (97.2 ± 1.7 v. 93.6 ± 1.6; P = 0.09, and 96.4 ± 1.9 v. 91.8 ± 1.8; P = 0.07, respectively). Interestingly, almost all vitrified Day-7 embryos cultured without protein reexpanded 2 h after warming (138/139 v. 137/144 with BSA). This allows suppressing the culture period before embryo transfer. Fresh embryos cultured without protein showed higher expression of IGF2R and the ER stress genes ATF4 and DDIT3 (P < 0.03), and DNMT3A tended to increase (P = 0.062). After warming, only G6PD was overexpressed in protein-free embryos (P < 0.01). Day 40 pregnancy rates did not differ, but birth rates were higher in PVA [58% (11/19)] than in BSA [31% (14/45); P = 0.039]. Pregnancy losses from Day 40 increased with [30% (6/20)] v. without protein [8% (1/12); P = 0.034]. Calf weight, size, thorax circumference, and gestation length did not vary (P > 0.41; not shown). Protein starvation limits blastocyst development but selects for improved viability. Such a profile agrees with the quiet embryo hypothesis (Leese et al. 2008 Mol. Hum. Reprod. 14, 667–672), by which the most viable embryos use endogenous nutrients efficiently and cope with stress successfully. Differences in gene expression seen before vitrification are abolished after warming, with G6PDH perhaps reflecting superior ability of protein-free cultured embryos to counteract stress induced by cryopreservation. Without protein, more embryos reach birth as morphologically normal calves.

Research was supported by MICINN (AGL2012–37772) and FEDER; AM: SENESCYT-Ecuador-II Fase 2013. The authors are members of the COST Action FA1201 (Epiconcept).