158 Impact of Matured Cattle Oocytes at Higher Incubation Temperature on in Vitro Embryo Production

Abstract

Heat stress during IVF is associated with reduced fertility in cattle oocytes. It may, however, enhance thermo-tolerance or cause detrimental effects on a variety of cell types or organisms, depending on the duration and intensity of the thermal challenge. The aim of this study was to evaluate the developmental potential of cumulus-oocyte complexes (COC) matured for 18 or 24 h and incubated at 39°C or 41°C. A total of 1000 immature oocytes were collected at slaughter from indigenous South African cow ovaries. The COC were randomly allocated (100/treatment) into 2 maturation times (18 or 24 h) and cultured in M199 + FSH-LH-estradiol medium under oil at 100% humidity and 5% CO2 at 39°C or 41°C. Post maturation, oocytes were subjected to normal subsequent embryo conditions. The Bracket and Oliphant medium was used for IVF. All matured oocytes were fertilised for 6 h with frozen-thawed Nguni bull semen at a concentration of 265 × 106. The presumptive zygotes from each treatment were cultured into SOF-BSA medium under oil and incubated at 39°C for assessment of cleavage rate 48 h post IVF. After Day 7 of culture, blastocyst were stained (Hoechst 33323) for nuclei cell count. Statistical analyses was performed using Genstat® software of SAS (SAS Institute, Cary, NC, USA; P < 0.05). Oocytes that were matured for 18 h in 41°C resulted in more 8-cell embryos (41%) compared with those incubated at 39°C (21.6%). However, no difference was observed for cleavage rate at both maturation times and incubation temperatures (41 or 39°C). There was more morula formation from oocytes matured for 18 h (19.6%) and 24 h (19.0%) at 41°C compared to 39°C (8.4%) group. The results further showed more blastocyst formation during 18 h at 41°C (15.2%) than at 39°C (7.4%) and during 24 h at 41°C (11.2%), 39°C (11.4%). However there was no difference in the nuclei cell number during 18 h at 41°C (45.2), 24 h (45.8), and 18 h at 39°C (43.4) of maturation. Thus, there was a significant difference in the nuclei cell numbers at 24 h on 39°C (n = 133.2) and 41°C (n = 45.8). In conclusion, oocytes that were matured for 18 and 24 h at 41°C or for 18 h at 39°C developed further to blastocyst stage on in vitro embryo production, however, with low nuclei cell numbers due to accelerated maturation temperature or shortened maturation period.

Comparison of in vitro Maturation Media on Cattle Oocytes after in vitro Embryo Production

“…This present study recorded a 2-4 cell rate ranging from 10.0-13.3%, and a total cleavage rate ranging from 10.0-23.3% using SOF BSA medium. Contrarily to these results specifically where SOF BSA medium is concerned, Nkadimeng et al (2016) reported a 2-4 cell percentage development of 65.0, 39.2% for 8 cells, and a total cleavage rate of 72.0% using SOF BSA medium at an incubation temperature of 39°C. The current results in embryo development may also be attributed to the fact that denuded presumptive zygotes using a vortexing mechanism after fertilization were used, of which the denuding of cumulus cells using this mechanism damages the cytoplasm.…”

“…Oocyte maturational (cytoplasmic and nuclear) and developmental (fertilization, pronuclear formation, and cleavage) competencies are influenced by the number of follicular fluids and the amount of the follicle from which the Cumulus-Oocyte Complex (COCs) is extracted (Vatzias and Hagen, 1999). Embryos are at risk from in vitro gamete handling since they are in a laboratory setup and can easily be harmed (Nkadimeng et al, 2016). Hence, IVM is one of the fundamental steps within the IVP process along with IVF.…”