253 COMPARISON OF THREE DIFFERENT IVF PROTOCOLS FOR BOVINE OOCYTES

Abstract

Recently, the development of commercially available defined media and sperm centrifugation gradients has offered new possibilities for increasing the efficiency of commercial in vitro fertilization (IVF) systems. The objective of this study was to compare three different IVF protocols using two different separation gradients, two fertilization media, and two embryo culture media, as follows: Group 1. sperm separation (SS): Percoll (Sigma, St. Louis, MO, USA), fertilization medium (FM): TALP-Fert (TFM), embryo culture media (ECM): G1/G2 (version 3, Vitrolife, Englewood, CO, USA). Group 2. SS: Percoll, FM: Bovine vitro Fert (Cook, Brisbane, Australia), ECM: Bovine vitro Blast/Bovine vitro Cleave (Cook); and Group 3. SS: EquiPure (Nidacon, Spectrum Technologies, Healdsburg, CA, USA), FM: TFM, ECM: G1/G2. Oocytes were obtained from slaughterhouse ovaries and matured in vitro (Looney et al. 1994 Theriogenology 41, 67). IVF was conducted using frozen/thawed semen from one bull. Semen was separated by centrifugation at 700g for 30 min in the given density gradients; Percoll was used in a 45% to 90% gradient. Sperm viability after separation was assessed by fast-green/eosin stain (Sigma). IVF was carried out in 0.5 mL of the given fertilization medium supplemented with PHE1 and heparin (10 μg/mL), in humidified 5% CO₂ in air atmosphere at 38.7°C. Final sperm concentration in the IVF wells was 1 × 10⁶/mL. In Experiment 1, a total of 368 oocytes (2 replicates) were fixed and stained (Hoechst 33342, Sigma) 24 h post-IVF to assess sperm penetration (Group 1, n = 128, Group 2, n = 108, Group 3, n = 132). In Experiment 2, a total of 400 embryos (2 replicates) were cultured in 0.5 mL of the given culture medium under mineral oil in a 5% O₂, 5% CO₂, 90% N₂ atmosphere at 38.7°C with high humidity for 112 h before fixation and staining. Embryos in Groups 1 (n = 129) and 3 (n = 139) and Group 2 (n = 132) were changed to G2 and Cleave media, respectively, at 84 h. Sperm separation with Percoll yielded lower numbers of sperm (average sperm concentration after separation of 218 vs. 383 × 10⁶ for EquiPure; P < 0.05), but resulted in higher total motility (60% vs. 41%, respectively; P < 0.05) and higher viability (93% vs. 70%, respectively; P < 0.05) of separated sperm. In Experiment 1, rates of normal fertilization were significantly lower for Group 3 (58%) than for Groups 1 and 2 (74% and 77%, respectively, P < 0.05). In Experiment 2, rates of development to <8, 9 to 16, and >16 cells at 112 h were not significantly different among groups (43, 48, and 46% for Group 1; 22, 18, and 31% for Group 2; and 35, 34, and 23% for Group 3, respectively; P > 0.1). These results indicate that the commercial separation medium, EquiPure, may be associated with lowered sperm motility, viability, and fertilization rates when compared to a standard medium (Percoll) for bovine sperm separation. Commercial fertilization and embryo culture media (Bovine vitro Fert, Cleave, and Blast) provided equivalent embryo development to that currently in use by our laboratory (TFM, G1/G2).