127 VERO CELLS CONDITIONED MEDIUM IMPROVES EMBRYONIC DEVELOPMENT DURING IN VITRO CULTURE OF BOVINE EMBRYOS

Abstract

Beneficial effects on embryonic development of co-culture with Vero cells during in vitro culture have been previously reported and related to Vero cells growth factors/cytokines secretion into the medium (1998 Human Reprod. 1600–1605). However, co-culture increases the probability of microorganism contamination during culture. In this study, we aimed to overcome this problem by culturing bovine embryos in conditioned medium derived from Vero cells culture (CM). Vero cells (GenBiotech, Antibes, France) were cultured for 24 h, and then the medium was removed, filtered, and frozen until its use. A total of 701 slaughterhouse bovine cumulus oocytes complexes were matured and fertilized in vitro under standard conditions. After fertilization, zygotes were allocated to 4 different treatments [0 (control), 5, 10, and 20% of CM v/v] for in vitro culture in BBH7 medium (BoviPro^®, MOFA Global, Verona, WI, USA) at 38.5°C in 5% O₂, 5% CO₂, 90% N₂ atmosphere for 7 days. A total of 8 replicates were done. Cleavage rates were assessed at 2.5 days, blastocyst rates and embryonic stage at 7 days, and blastocyst total cell number at 7.5 days after fertilization. Data were analysed by ANOVA using GLM; the percentages were transformed using arcsin square root. If the ANOVA was significant (P < 0.05), means were separated by Tukey's procedure using Statistix 10 software (Tallahassee, FL, USA). No differences in cleavage rates were found between treatments (P > 0.1; Table 1). However, the addition of conditioned medium at all levels increased the blastocyst production at Day 7 of culture (between 11 to 19 percentage points) compared with the control group (P < 0.01; Table 1); likewise, CM addition was superior in total embryo production (Day 7 morulae plus all blastocysts; P < 0.01; Table 1), between 12.5 to 15 percentage points higher than the control group. No differences were found between treatments for blastocyst total cell number at 7.5 days of culture (P > 0.1; Table 1) and overall embryonic developmental stage (P > 0.1; Table 1). In conclusion, we found a superior embryonic development when Vero cells conditioned medium was added at Day 0 of in vitro culture; the positive effect on total embryo production was observed even at 5% of conditioned medium.

Table 1.  Means (± s.e.m.) of embryonic development of bovine embryos