174 DEVELOPMENT OF IN VITRO-FERTILIZED BOVINE EMBRYOS AFTER EXPOSURE TO TRYPLE^™EXPRESS (RECOMBINANT TRYPSIN)

Abstract

TrypLE^™Express (1X; GIBCO, Grand Island, NY, USA) is a recombinant fungal trypsin-like protease that may provide an alternative to animal-origin trypsin for inactivation of embryo-associated virus. This experiment was designed as an embryo safety study to determine if different exposure times of TrypLE^™Express on 7-day bovine in vitro-fertilized (IVF) embryos would inhibit normal embryonic development in vitro. Good quality oocytes were harvested from ovaries of slaughtered animals and matured in vitro (Looney et al. 1994 Theriogenology 41, 67). Oocytes were fertilized (Day 0) with frozen-thawed, Percoll-separated Brangus spermatozoa in TALP-fertilization medium for 18 h. Presumptive zygotes were cultured in 0.5 mL of G1.3/G2.3 medium (Lane et al. 2003 Theriogenology 60, 407) supplemented with 8 mg/mL of Pentex BSA (Serologicals Corporation, Norcorss, GA, USA) under oil for 3 days in a 5% CO₂, 5% O₂, 90% N₂ humidified modular incubator (Billups-Rothenberg, Inc., Del Mar, CA, USA) at 38.5°C. On Day 4, embryos were washed and moved to new culture wells containing G2.3 medium. Cleavage rates of 74% (750/1014) from three replicates produced 322 viable embryos on Day 7 which were then evenly distributed by grade and stage into three treatment groups (1-, 5-, and 10-minute exposure times to TrypLE^™Express). Embryos were washed in accordance with International Embryo Transfer Society standards (Stringfellow, IETS Manual, 1998, 79–84) substituting TrypLE^™Express with phenol red for the porcine-origin trypsin. Washed embryos were cultured in G2.3 medium with hatched rates assessed at 24, 48, and 72 h. Hatched embryos were fixed and stained using a modified Hoechst method (Damiani et al. 1996 Mol. Reprod. Dev. 45, 521–534) to count cells. The results are presented in Table 1. There were no significant differences (P < 0.05, chi-square analysis) between treatment groups for hatched rates or average cell counts. The results indicate that exposure to TrypLE^™Express did not inhibit normal embryonic development. The efficacy of TrypLE^™Express to remove or inactivate certain embryo-associated viruses is being investigated. Establishing an appropriate protocol for treatment of bovine embryos with TrypLE^™Express will depend on the safety and efficacy of exposing embryos to this recombinant protease.

Table 1. Comparison of hatched IVF bovine embryo rates and average cell counts after exposure to TrypLE^™Express

This work was funded by Invitrogen Corporation.