81 Effects of Day 5 or 6 HEPES-Buffered Transportation Culture Medium on Developmental Competence of Bovine In Vitro-Produced Embryos

Abstract

Most in vitro-produced (IVP) bovine embryos are transferred fresh. Use of a HEPES/bicarbonate embryo culture medium for transportation would offer flexibility for embryo shipment and transfer. We hypothesized that embryos cultured for 36 (Day 6 embryos) or 60 h (Day 5 embryos) in a novel SCF1T medium (SOF for Conventional Freezing 1 supplemented with HEPES) would maintain developmental competence compared with bicarbonate-buffered medium SCF1 (control). In 5 replicates, IVP embryos were produced by aspirating cumulus–oocyte complexes (COC) from 2-to 8-mm follicles of abattoir ovaries. The COC (n = 1036) were matured for 23 h, fertilized with semen from 1 of 3 bulls, and cultured in SCF1 at 38.5°C, 5% CO₂, 5% O₂, and 90% N₂ (Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130). Randomly, on Day 5 and 6 after fertilization, a subset of presumptive embryos were moved into 500-µL polystyrene vials containing 100 µL of SCF1T medium, covered with 300 µL of sterile mineral oil and cultured in a portable incubator (MicroQ iQ2, Scottsdale, AZ, USA) at 38.5°C for 60 and 36 h, respectively. On Day 7.5 post-fertilization, blastocyst rates were evaluated and embryos (n = 8) from each group were stained with 1 µg mL⁻¹ Nile Red for lipid quantification, and 300 nM Mitotracker Red CMX-Rosamine (Thermo Fisher Scientific, Waltham, MA, USA) for mitochondrial polarity. Images were obtained with confocal microscopy and fluorescent intensity (AFU) was measured by Image J software (National Institutes of Health, Bethesda, MD, USA). Data were analysed by one-way ANOVA and means separated by Tukey’s HSD. Results (Table 1) indicate similar blastocyst rates and lipid content between embryos cultured for 36 to 60 h in SCF1T and control media (P > 0.05). However, mitochondrial polarity was lower in the Day 5 group (P < 0.05) compared with Day 6 and control groups. Results suggest that culturing embryos in SCF1T medium for 36 h maintains developmental competence compared with bicarbonate-buffered media and offers an alternative for shipment and transfer of IVP embryos. Studies involving evaluation of pregnancy rates of the present study are ongoing.

Table 1.  Effects of Day 5 or 6 SCF1T embryo culture medium on development, lipid content, and mitochondrial polarity