Less is more: Reduced nutrient concentration during in vitro culture improves embryo production rates and morphophysiology of bovine embryos.

Reproducing the environment to which the embryo is naturally exposed may be an alternative to improve viability of embryos produced in vitro. In the first part of this work, we describe a novel culture media, namely Embryonic Culture Supplementation (ECS100). The composition of this media was based on the contents of carbohydrates and amino acids found in oviductal and uterine fluids. Because it was a new formulation, we investigated the performance of ECS100 in comparison with conventionally used SOFaa, and possible benefits to embryo development. Embryo production rates (cleavage, morula and blastocyst conversion, blastocyst and hatching rates) and morphophysiological parameters (total cell number, cell allocation, Mitochondrial membrane potential (MMP), Reactive Oxygen Species (ROS), NADH, FAD+ and ATP content) were similar between ECS100 and SOFaa. Next, we tested if a reduction of ECS100 concentration could positively contribute to embryo viability by resembling the more dynamic availability of nutrients that reach the embryos in vivo. Therefore, embryos were cultured in ECS100 or in its serial dilution (ECS75, 50 and 25). Despite the fact that the lowest concentration (ECS25) still supported blastocyst formation, halving the concentration of metabolites (ECS50) actually improved embryo production rates. Thus, embryos produced in ECS100 or ECS50 were submitted to further analyses on Days 4 and 7. Embryos cultured in ECS50 presented better developmental rates and morphophysiological profile than embryos cultured in ECS100. Additionally, physiological traits (MMP, ROS and NADH levels) of embryos cultured in ECS50 presented the expected pattern for embryos produced in vivo. In conclusion, we presented a novel, more personalized and effective culture media for bovine IVP embryos. And although the ECS media formulation was based on the contents of female reproductive fluids, it is worth mentioning that adaptations must be specifically directed for in vitro conditions rather than reproduced exactly from in vivo state.

Lipid characterization of in vitro-produced bovine embryos with distinct kinetics of development.

Human embryo studies have proposed the use of additional morphological evaluations related to the moment of the first cell divisions as relevant to embryo viability. Nevertheless, there are still not enough data available related to morphokinetic analysis and its relationship with lipid composition in embryos. Therefore, the aim of this study was to address the lipid profile of bovine embryos with different developmental kinetics: fast (four or more cells) and slow (two or three cells) at 40 h post-insemination (hpi), at three time points of in vitro culture (40, 112 and 186 hpi) and compare these to profiles of in vivo embryos. The lipid profiles of embryos were analyzed by matrix-assisted laser desorption ionization mass spectrometry, which mainly detected pools of membrane lipids such as phosphatidylcholine and sphingomyelin. In addition to their structural function, these lipid classes have an important role in cell signalling, particularly regarding events such as stress and pregnancy. Different patterns of lipids in the fast and slow groups were revealed in all the analyzed stages. Also, differences between in vitro embryos were more pronounced at 112 hpi, a critical moment due to embryonic genome activation. At the blastocyst stage, in vitro-produced embryos, despite the kinetics, had a closer lipid profile when compared with in vivo blastocysts. In conclusion, the kinetics of development had a greater effect on the membrane lipid profiles throughout the embryo culture, especially at the 8-16-cell stage. The in vitro environment affects lipid composition and may compromise cell signalling and function in blastocysts.