111 Serum replacement as an alternative for fetal calf serum in equine embryo culture

Equine in vitro embryo production has drastically improved during the past decade, and there is a growing interest in a serum-free defined medium that would allow greater repeatability and reproducibility in in vitro experiments. Likewise, the development of individual cultures would permit individual embryo follow-up. Therefore, the objective of this study was to determine the effect of serum replacement in individual and group culture on embryo development after intracytoplasmic sperm injection. Cumulus-oocyte complexes obtained from slaughterhouse-derived ovaries were in vitro matured for around 28 h. After maturation, oocytes were denuded, and those with a polar body (242/436; 55.5%) were injected with semen from a fertile stallion. Presumed zygotes were cultured individually in 3.3 μL droplets under parafilm oil or in group culture in 20 μL droplets at a ratio of 3 to 4 μL per zygote under oil. Embryo culture was performed in DMEM/F-12 (Gibco 21331-020, ThermoFisher) supplemented either with 10% KnockOut™ Serum Replacement (SR) (Gibco 10828-010, ThermoFisher) or 10% fetal calf serum (FCS) (Gibco 10082-147, ThermoFisher). Four groups resulted from those different conditions: (A) oocytes cultured individually in a medium supplemented with serum replacement (IndSR; n = 18); (B) oocytes cultured individually in a medium supplemented with fetal calf serum (IndFCS; n = 48); (C) oocytes cultured in group in a medium supplemented with serum replacement (GSR; n = 35); and (D) oocytes cultured in group in a medium supplemented with fetal calf serum as control (GFCS; n = 69). The culture environment was 5% CO₂, 5% O₂, and 90% N₂ at 38.2°C. For statistical analysis, the zygote/embryo was considered the unit of interest. Generalised mixed effects models were used to test the effect of serum replacement supplementation in individual and group culture on cleavage and blastocyst rate. For all the models, the replicate was set as a random effect, and results are expressed as least squares means ± standard error. Regarding cleavage rate, IndSR (58.7 ± 13.5%) showed a tendency (P < 0.1) to be lower than IndFCS and GSR, with 83.0 ± 6.3% and 85.1 ± 6.7%, respectively, and significantly inferior to GFCS (91.4 ± 3.7%; P < 0.05). No differences were present between GSR, IndFCS, and control. Regarding blastocyst rate, IndFCS (14.6 ± 5.1%) exhibited lower blastocyst formation than control (31.9 ± 5.6%), and similar results to GSR (25.7 ± 7.4%). No differences were present in group culture when FBS or SR was used, and no blastocysts were obtained from IndSR. Our results coincide with previous studies in individual embryo culture of other species, in which blastocyst formation is affected. The serum replacement group was especially affected, and the small droplet size might have had a negative influence. However, we demonstrated that serum replacement might substitute FCS in equine embryo culture in group in order to obtain a serum-free defined medium.