Abstract #145

Section: IVF/IVP
Session: IVF/IVP
Format: Poster
Location: Rio Exhibit Hall B
# 145
M. Nkadimeng*1,2, E. van Marle-Koster2, T. R. Netshirovha1, T. L. Nedambale3, K. C. Lehloenya2, 1Agricultural Research Council, Animal Production Institute, Germplasm Conservation and Reproductive Biotechnologies, Irene, South Africa;, 2University of Pretoria, Department of Animal and Wildlife Sciences, Hatfield, South Africa;, 3Tshwane University of Technology, Faculty of Science, Department of Animal Sciences, Pretoria, Republic of South Africa.

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 ± 0.007a) 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.