94 EFFECTS OF 6- OR 12-HOUR CULTURE IN A Micro Q STRAW INCUBATOR ON DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS

Abstract

Transporting in vitro-produced (IVP) embryos can be challenging when an embryo transfer destination is more than 6 h away or electricity is not available on site to unload embryos for transfer. The objective of this study was to determine if development rates would be compromised for Day 6.5 IVP embryos when placed in warmed Vigro holding medium (Vetoquinol, Pullman, WA, USA) loaded and plugged into 1/4 cc straws (Professional Embryo Transfer Services, Canton, TX, USA) for a period of either 6 or 12 h in a 38.5°C Micro Q straw block incubator (Micro Q iQ1T 64). Bovine oocytes were shipped and matured in transit from a commercial abattoir (DeSoto Biosciences, Seymour, TN, USA), fertilized (IVF = Day 0) with frozen-thawed semen, and cultured in Bovine Evolve (Zenith Biotech, Guilford, CT, USA) supplemented with 4 mg mL^–1 BSA (Probumin, EMD Millipore, Norcross, GA, USA) under oil in a 5% CO₂, 5%O₂, 90% N₂ humidified incubator (Pryor et al. 2011 Theriogenology 75, 24–33). Cleavage rates of 87.7% (664/757) from three replicates produced 273 (36.0%) viable embryos on Day 6.5 post-IVF, which were evenly distributed by IETS stage (4–7) and grade (1 and 2) into three treatment groups (0 = control, 6 or 12 h straw incubation) before in vitro culture for an additional 24 h. For each replicate, the average embryo stage was calculated by multiplying the number of embryos in each treatment by their IETS stage and dividing by total embryos per group. The change in stage for each treatment was calculated by subtracting the initial average stage from the final average stage on Day 8. Grade 1 and 2 embryos at stage 6–8 were counted and used to calculate total viable rates. Day 8 (post-IVF) embryos were fixed in cold methanol, washed in PBS/0.1%Tween 20, mounted in 10 μg mL^–1 Hoechst 33342/glycerol and viewed under UV light to count nuclei. Percentage data were transformed using arcsine square root function before analysis, and means were compared using a one-way ANOVA and Tukey’s HSD. Although viability decreased with increasing time in straw incubation, there were no statistical differences between control, 6 and 12 h treatments for total viable rates (90.8, 80.3, and 70.8%, respectively). Average embryo stage on Day 8 for control, 6 and 12 h (7.0 ± 0.66, 6.6 ± 0.24, and 6.2 ± 0.30 s.e.m., respectively) was not different, but tended to be higher in control (P = 0.08). The change in stage, however, was different between control and 12 h (1.46 ± 0.33 and 0.66 ± 0.24, respectively; P < 0.05). Likewise, cell numbers were greater in control and 6 h embryos compared with 12 h straw incubation (149.8 ± 9.14, 138.7 ± 7.94, and 101.8 ± 5.29; n = 47, 50, and 46, respectively P < 0.01). In conclusion, 6.5 day IVP embryos held in warm Vigro holding medium for 12 h in 1/4 cc straws fail to develop at the same rate and incurred lower cell counts than either control or 6 h treatments. Further research to evaluate pregnancy rates following transfer and utilising different incubation or media and/or temperature is warranted to further evaluate the utility of in straw incubation for extended periods of time.