145 THE ROLE OF CELL APOPTOSIS ON IN VITRO-PRODUCED BEEF CATTLE EMBRYOS

Abstract

Successful in vitro development of embryos is dependent upon maintenance of cellular function in the embryonic microenvironment. Exposure of pre-implantation embryos to a variety of cellular stresses can induce apoptosis in all or a fraction of blastomeres. Among the conditions that can induce apoptosis in cattle embryo production in vitro are heat shock, media composition, oxygen levels, and tumour necrosis factor (TNF)-α. The aim of the study was therefore to evaluate the in vitro culture environment of embryos in terms of cell apoptosis through caspase and DNA fragmentation evaluation. A total of 1200 immature oocytes were collected at slaughter from indigenous South African cow ovaries. The cumulus-oocyte complexes were randomly allocated into 2 maturation temperatures, 39°C and 41°C (200/temperature /replicated 6 times), and cultured in M199 + FSH-LH-oestradiol medium under oil at 100% humidity and 5% CO₂ for 24 h. Postmaturation, oocytes were subjected to normal subsequent embryo conditions (Rynkowska et al. 2011 Biotechnol. Comput. Biol. Bionanotechnol. 92, 45–53). All matured oocytes were fertilized for 6 h with frozen-thawed Nguni bull semen from 10 bulls replicated 5 times at a concentration of 265 × 10⁶ sperm cells/mL in Bracket and Oliphant medium under oil. The presumptive zygotes from each treatment were cultured into SOF-BSA medium under oil and incubated at 39°C for assessment of cleavage rate post IVF. Produced embryos were divided into 3 groups: 2–4 cell, ≥8 cell embryos, and blastocyst. Two- to four-cell embryos were removed at Day 2, ≥8 cell at Day 5 (mainly 8-cell, morula, and cavitated blastocyst), and expanded blastocyst at Day 7 of embryo culture to determine caspase activity and DNA fragmentation for evidence of apoptosis. Caspase was performed using colourimetric assay on a 96-well microplate reader and monitored at 450 nm reference filter. The DNA fragmentation was examined using the TUNEL assay method and imaging was done using the Alexa Fluor® 488 emission at 519 nm. Data were analysed using SAS 9.2 software (SAS Institute Inc., Cary, NC, USA), and Shapiro-Wilks test was used on the standardized residuals to test for deviations from normality, means of significant effects were compared using Student’s t-l.s.d. at the 95% confidence interval. There was no significant difference on the cleavage rates [39°C (72.0 ± 22.70) and 41°C (67.2 ± 18.9)] and blastocysts [39°C (11.4 ± 2.6) and 41°C (11.2 ± 6.3)] from both maturation temperatures. Caspase activity showed no significant difference on 2–4 cell [39°C (0.015 ± 0.001) and 41°C (0.016 ± 0.002)] and ≥8 cell embryos at both temperatures [39°C (0.022 ± 0.007^a) and 41°C (0.032 ± 0.013)]. However, blastocyst differed significantly at both temperatures [39°C (0.037 ± 0.012) and 41°C (0.053 ± 0.005)]. A higher (P < 0.05) DNA fragmentation was observed at 2–4 cell (6.5 ± 2.9), ≥8 cell (13.3 ± 3.1), and blastocyst (48.0 ± 8.2) for embryos produced from 41°C matured oocytes compared to the 39°C maturation group [2–4 cell (2.2 ± 1.2), ≥8 cell (4.5 ± 1.9), and blastocyst (9.7 ± 6.7)]. It is therefore concluded that embryos produced from 41°C matured oocytes can have the same developmental capacity as the 39°C maturation group. Moreover, both temperatures can show signs of apoptosis, however, with more apoptosis evidence in the 41°C than the 39°C maturation group.