Invitro production (IVP) of embryos is designed to reproduce an environment that resembles the female reproductive tract. However, the system does not perform optimally in terms of quality and embryo production. A major setback lies in the loss of dynamics observed in a static invitro system, which might affect the availability of substrates that reach the embryo. A reduction in the amount of nutrients in media has been used as an approach to improve IVP (Ermisch et al. 2020 Sci. Rep. 10, 9263; https://doi.org/10.1038/s41598-020-66019-4). The present study aimed at describing a defined sequential medium (embryonic culture supplementation, ECS) and to investigate the effect of reducing nutrient availability on embryo production, quality, and metabolism. ECS was developed in our laboratory and is a serum-free, salt-based culture medium supplemented with the amount of energy substrates and amino acids found in bovine oviduct (Ov) and uterus (Ut) fluids as previously described (Hugentobler et al. 2007Mol. Reprod. Dev. 74, 445–454; https://doi.org/10.1002/mrd.20607; Hugentobler et al. 2008Mol. Reprod. Dev. 75, 496–503; https://doi.org/10.1002/mrd.20760). Embryos were cultured according to the following ECS supplementation: ECS100 (supplemented with 8mg mL−1 bovine serum albumin and 100% of the energy substrates and amino acids concentrations of the Ov and Ut fluids) and ECS50 (half dilution of ECS100). Bovine oocytes from abattoir ovaries were submitted to IVP using standard protocols. On Day 0 of invitro culture, presumptive zygotes were randomly divided into groups ECS100-Ov or ECS50-Ov. On Day 4, embryos were respectively transferred to ECS100-Ut and ECS50-Ut. Expanded blastocysts were collected on Day 7 to assess embryo production, morphology (total cell number by Hoescht 33342 staining; inner cell mass and trophectoderm cells by CDX2 immunostaining), and metabolic status (mitochondrial activity and reactive oxygen species content by MitotrackerTM RedCMXRos and CellROXTM Green staining, ThermoFisher Scientific; NADH and FAD+ by autofluorescence). Data were analysed by Student’s t-test (a=4%). Although cleavage rates were similar between ECS50 and ECS100 (78.13±3.73 vs. 79.70±4.18; P=0.788), blastocyst rates were positively influenced by the reduction in concentration (28.88±1.74 vs. 16.73±2.41; P=0.004). This difference likely comes from a blockage at the morula stage in group ECS100, because the conversion from morula to blastocyst was 20% lower in this group (57.73±3.81 vs. 38.15±3.45; P=0.008). In terms of morphology, blastocysts produced in ECS50 had a higher number of cells (152.4±9.61 vs. 118.3±7.22; P=0.036), which is explained by the higher number of trophectoderm cells. Finally, metabolic status was affected by nutrient reduction: embryos from ECS50 had higher mitochondrial activity, reactive oxygen species content (P<0.0001), and lower NADH (P=0.01), suggesting higher oxidative phosphorylation to produce energy, as expected at this stage. In conclusion, ECS is a functional medium, and a reduced nutrient concentration (ECS50) improves embryo production, morphological quality, and metabolic status of blastocysts, suggesting that culture conditions must be adapted to the invitro system rather than resembling invivo conditions.
This research was funded by FAPESP (2016/00350-0, 2017/18384-0).
Physiological and molecular responses of a newly evolved auxotroph of Chlamydomonas to B12 deprivation