149 Uptake of C_18:0 from culture media during in vitro culture decreases cryosurvival rates of bovine embryos

Abstract

Cryosurvival of in vitro-produced bovine embryos is lower than that of in vivo-produced embryos, limiting their usability in the field. Previous work showed that the embryo’s lipid composition relates to its quality and cryosurvival. The present study aimed to investigate the effects of free fatty acid (FA) additions to embryo culture media during the oviduct phase of embryonic development on the improvement of cryosurvival of in vitro-produced blastocysts. Bovine cumulus–oocyte complexes (n = 1675, 3 replicates) were harvested from slaughterhouse ovaries, in vitro matured (23 h), and subsequently fertilized (18–20 h). Embryos were cultured until Day 5 post-fertilization in synthetic oviductal fluid (SOF) with (1) bovine serum albumin (BSA; control, n = 253); (2) delipidified BSA (>96% FA free, n = 460); (3), delipidified BSA complexed with 25 µM unsaturated oleic acid (C_18:1, n = 455); or (4) with saturated stearic acid (C_18:0, n = 507) with a stoichiometry of 5:1. At Day 5, SOF was refreshed and embryos were cultured without supplementation. At Days 7 and 8, blastocyst rates were determined. Blastocysts were LD540 stained for lipid droplets (LD), and the LD number and size were analysed by ANOVA. Cryosurvival %, defined by re-expansion of the blastocoel, was analysed by logistic regression. Additionally, fresh and frozen–thawed blastocysts were stained for apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labelling, TUNEL), necrosis (EthD-1), and DNA (Hoechst 33342) and analysed using negative binomial regression. Group differences were tested using a post hoc Tukey test. Statistical analysis was performed in R Studio (version 3.4.2), and P-values <0.05 were considered significant. FA-free culture delayed and decreased blastocyst rates to 19% compared with any FA supplementation: 35%, 27%, and 29% for control, C_18:1, and C_18:0, respectively (P < 0.04). Cryosurvival doubled with culture in FA-free SOF (58%) and C_18:1 (63%) compared with C_18:0 (23% P = 0.01 and P < 0.01, respectively) and control (29%; P = 0.15 and P < 0.02, respectively), approaching cryosurvival rates of donated multiple ovulation embryo transfer (MOET) embryos (CRV Company; 67%). C_18:0 exposure also resulted in elevated necrosis levels after cryopreservation (5–8% of cells), compared with all groups (2–4%; P < 0.016). The LD size increased in blastocysts cultured with C_18:1 compared with all groups (3.1 µm² vs. 2.4–2.7 µm²; P < 0.016). C_18:0 addition to SOF during embryo culture in vitro, as well as a mixture of FA in control SOF (including C_18:0), caused a reduction of ∼50% in blastocyst cryosurvival compared with MOET blastocysts. Interestingly, either C_18:1 addition or the complete omission of FA in SOF during embryo culture in vitro restored the cryosurvival of blastocysts to the level of MOET blastocysts. Currently, we are investigating whether the free FA conditions in the oviduct endorse the distinct quality between in vivo- and in vitro-produced embryos.