Effects of various concentrations of gonadotropins and 17β estradiol on the in vitro maturation of cattle oocytes selected using brilliant cresyl blue staining

Abstract

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.

An Update on South African Indigenous Sheep Breeds’ Extinction Status and Difficulties during Conservation Attempts: A Review

“…Follicular fluid retrieval methods: Oocytes can be retrieved either by slicing or aspiration [73]. According to Shiraz et al [74], slicing yields better oocytes (grade A) considerable for in vitro maturation than the aspiration method.…”