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.

The objective of this study was to determine the in vitro maturation rate of cattle oocytes selected with brilliant cresyl blue (BCB) stain, in tissue culture medium 199 (TCM 199) supplemented with various concentrations of hormones. Oocytes were retrieved from abattoir-derived ovaries by aspiration. Oocytes were then exposed to 26 μM BCB stain, and classified according to the colour of their cytoplasm: BCB+ (oocytes with blue cytoplasm) and BCB- (unstained oocytes). The BCB selected and the non-selected immature oocytes were randomly allocated into TCM 199 + 10% foetal bovine serum (FBS) maturation media supplemented with three concentrations of hormones as treatments (T). The T1 group was matured in the presence of 0.5 μg follicle stimulating hormone (FSH)/mL, 5 mg luteinising hormone (LH)/mL and 2 μg estradiol (E2)/mL. The T2 group was matured in 1 μg FSH, 6 mg LH and 2.5 μg E2/mL. The T3 group was matured in 1.5 μg FSH, 7 mg LH and 4.5 μg E2/mL. The maturation rate of oocytes was determined by the protrusion of the first polar bodies 24 h after maturation. Data were analysed by ANOVA using SAS. Treatment 2 yielded higher maturation rates for with BCB+ (30.5%) and without BCB (35%) oocytes, with T1 giving a lower maturation rate for BCB+ (10.7%) and without BCB (9.7%) oocytes. However, BCB- oocytes had lower polar body extrusion (0.7%, 1% and 2.7%) for T1, T2 and T3, respectively. In conclusion, immature oocytes that were exposed to BCB+ and cultured in TCM 199 supplemented with 10% FBS, 1 μg FSH, 6 mg LH and 2.5 μg E2/mL had a higher number of matured oocytes (extrusion of first polar body), similar to those that were not exposed to BCB (no BCB). Oocyte selection with BCB staining was a useful test for classifying good-quality cattle oocytes.

In vitro maturation of indigenous African cattle oocytes is a major challenge even though different maturation protocols work successfully in other breeds. The objective of this study was to determine the maturation rate of indigenous South African cattle oocytes following in vitro maturation in media supplemented with different concentrations of hormones and selected using brilliant cresyl blue (BCB) staining. Indigenous cattle ovaries were collected from the slaughterhouse and then oocytes were retrieved by aspiration method. A total of 966 oocytes were exposed to 26 µM BCB stain and 700 oocytes were not exposed to the BCB stain. Thereafter, oocytes exposed to the BCB stain were grouped according to the colour of their cytoplasm BCB+ (oocytes with blue cytoplasm, low G6PDH) and BCB– (unstained oocytes, increased G6PDH). The BCB exposed (BCB+ and BCB–) and the oocytes not exposed to BCB were then randomly allocated into tissue culture medium (TCM199) + 10% (vol/vol) fetal bovine serum (FBS) supplemented with 3 different concentrations of hormones as treatments (T). The T1 group was matured in the presence of 0.5 µg mL–1 of FSH, 5 mg mL–1 of LH, and 2 µg mL–1 of E2; the T2 group was matured in the presence of 1 µg mL–1 of FSH, 6 mg mL–1 of LH, and 2.5 µg mL–1 of E2; and the T3 group was matured in the presence of 1.5 µg mL–1 of FSH, 7 mg mL–1 of LH, and 4.5 µg mL–1 of E2. For IVM, 20 to 25 COC were placed in 50-µL droplets of IVM medium containing the 3 different levels of hormones. Maturation rate of oocytes was determined by the extrusion of the first polar body after 24 h of incubation in maturation medium. Data was analysed by ANOVA using SAS with 4 replicates per treatment. Treatment 2 yielded higher maturation rate for both BCB+ (65.6%) and not exposed to BCB (60.3%) oocytes compared to T1 (22, 3.03, and 16% for BCB+, BCB–, and not exposed to BCB, respectively) and T3 (48, 2.2, and 48% for BCB+, BCB–, and not exposed to BCB respectively). However, BCB– oocytes had lower polar body extrusion for T1, T2, and T3 (3.03, 8.1, and 2.2%, respectively) compared to BCB+ oocytes (22, 65.6, and 48% for T1, T2, and T3, respectively). In conclusion, immature oocytes that were cultured into TCM199 supplemented with 10% FBS, 1 µg mL–1 of FSH, 6 mg mL–1 of LH, and 2.5 µg mL–1 of E2 showed maturation rate for BCB+ oocytes and those not exposed to BCB. Oocytes selection using BCB staining was a useful test to classify good quality cattle oocytes. Therefore, it is suggested that treatment 2 is a suitable in vitro-maturation medium to mature indigenous South African cattle oocytes.