210 COMPARISON OF A RECOMBINANT TRYPSIN v. THE PIG PANCREATIC EXTRACT ON IN VITRO-PRODUCED BOVINE EMBRYOS

Abstract

According to the Manual of the International Embryo Transfer Society, trypsin can be used to remove certain pathogenic agents from in vivo-derived embryos. Research is currently in progress to determine whether trypsin can also remove pathogenic agents from semen. The original research on embryos involved the use of trypsin from pig pancreatic extracts. Because of stricter guidelines from international regulatory agencies on the use of animal products, several recombinant serine protease products are now becoming available. TrypZean (Sigma, St. Louis, MO, USA) is a recombinant developed from corn and is the first bovine sequence recombinant trypsin to contain no animal by-products. As part of our ongoing research on the effects of trypsin on sperm, the goal of this investigation was to examine the development of bovine embryos produced from sperm treated with the recombinant TrypZean compared with pig pancreas trypsin (Sigma) and a control (no trypsin). Oocyte aspiration, maturation, fertilization, and embryo culture were performed using standard methods in 9 replications. Semen was collected and pooled from Bos taurus and frozen in an egg-yolk cryodiluent (Biladyl, Minitube, Verona, WI). The semen was processed using density gradient centrifugation composed of 1 mL of 30% Percoll (Sigma), layered over 2 mL of 45% Percoll containing either 0.25% TrypZean (n = 972 oocytes), 0.25% trypsin (n = 1040 oocytes), or no trypsin for the control group (n = 1024 oocytes). The bottom layer for the 2 treatments and control was 2 mL of 90% Percoll containing 10 µg mL^–1 of soy-based protease inhibitor (Sigma). The density gradients were centrifuged at 700g for 30 min, after which time the pellets were washed in 5 mL of prewarmed TL HEPES Solution (Cambrex) and centrifuged at 300g for 10 min. The resulting sperm pellets were then resuspended in a volume calculated to provide 1 × 10⁶ sperm mL^–1 for in vitro insemination. The results were compared using one-way ANOVA. There were no statistically significant differences (P > 0.05) between any of the measures of embryonic development for the control and either of the treatment groups. Cleavage rates were measured for TrypZean (n = 689, 70.9%), trypsin (n = 729, 70.1%), and the control (n = 757, 73.9%) groups. More embryos reached the morula to blastocyst stages with the TrypZean (n = 367, 53.3%) and trypsin (n = 389, 53.4%) groups than the control (n = 369, 48.7%) group; however, these differences also were not statistically significant (P = 0.91) because of the large variation within the groups. In conclusion, the TrypZean and pig pancreas trypsin treatments of sperm prior to insemination showed no detrimental effects on IVF-derived bovine embryo development.