86 IMPROVED BOVINE EMBRYO PRODUCTION USING NOVEL IN VITRO CULTURE SYSTEMS

Abstract

Development and testing of new embryo production components is important to improve the outcome following in vitro production of bovine embryos. The objective of this study was to compare media used in two bovine embryo production systems (control and EmbryoTrans Biotech: ETB). In Exp. 1, abattoir-derived cumulus-oocyte complexes were randomly assigned and in vitro matured (IVM) in either control [Medium 199 with Earles salts (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 1% penicillin/streptomycin (Invitrogen), 0.2 mM sodium pyruvate, 2 mM L-glutamine (Sigma Chemical Co., St. Louis, MO, USA), and 5.0 µg mL^–1 of Folltropin^®-V (Vetoquinol, Pullman, WA, USA)] or ETB BO-IVM medium for 21 to 24 h. IVF was conducted in 500 µL of pre-equilibrated modified Tyrode-lactate medium for control (Pryor et al. 2011 Theriogenology 75, 24–33) or ETB BO-IVF in Nunclon^® 4-well multi-dishes (VWR Scientific, Pittsburgh, PA, USA). Seventeen hours post-insemination, presumptive zygotes were cleaned of cumulus cells and cultured in either Bovine Evolve (Zenith Biotech, Guilford, CT, USA) supplemented with 4 mg mL^–1 of Probumin BSA (EMD Millipore, Norcross, GA, USA), under oil (Irvine Scientific, Santa Ana, CA, USA) or ETB BO-IVC medium under BO-oil for 7 days (8 days post-IVF). All cultures were performed at 38.5°C in a humidified atmosphere of 5% CO₂, 5% O₂, and 90% N₂ using BT37 incubators (Planer Plc, Sunbury, UK). For Exp. 2, all conditions were maintained except a modified ETB BO-IVCA medium was used. On Day 8 of IVC, grade 1 and 2 blastocysts (BL) through hatching blastocysts (HBL) were counted and used to calculate total viable rates. In Exp. 2, these 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 cells (n = 49 and 107 for control and ETB, respectively). Each experiment was replicated 3 times with a total of 425 oocytes in Exp. 1 and 430 in Exp. 2, divided equally between treatments. Percentage data were transformed using arcsine square root function before analysis and means compared using a paired Student’s t-test. For Exp. 1, there were no differences in rates of cleavage or viable embryos between control and ETB systems (81.3% and 42.9% v. 80.5% and 48.4%, respectively). In Exp. 2, ETB was superior to control for percent viable, HBL, and combined HBL/expanded BL (51.9, 23.9, 45.8% v. 29.2, 5.8, 20.5, respectively; P < 0.05). Differences between mean cell counts for viable embryos were significant (control = 127.0 ± 6.7 s.e.m. and ETB = 162.7 ± 5.7; P < 0.0001). Embryo viability decreased in control media between Exp. 1 and 2 (42.9 v. 29.2%; P < 0.05). Seasonal differences may have contributed via heat stress with temperatures ranging from 23.8°C for Exp. 1 to 33.8°C for Exp. 2. Interestingly, embryo development in the ETB media did not decrease under the same conditions. In conclusion, ETB media produced more high-quality embryos than control under varying conditions experienced by commercial IVF companies.