The isolation and in vitro culture of bovine preantral and early antral follicles of different size classes

Abstract

The ovary of cattle contains thousands of oocytes which are enclosed primarily in the preantral follicles. Methods of culturing preantral follicles are now being developed. The aim of this study was to investigate the effect of the size of bovine preantral and early antral follicles and culture media on their in vitro growth. Individual follicles isolated by microdissection of the ovarian slices were sorted into the following size classes: 75 to 124, 125 to 174, 175 to 224, 225 to 274, 275 to 324 and ≥ 325 μm. The follicles were cultured individually in TCM 199 + fetal calf serum (FCS)+supplements (FSH, estradiol-17β, insulin, transferrin, sodium selenite, sodium pyruvate, 1-glutamine and hypoxanthine) or in Menezo B 2 + FCS + supplements (Experiment 1) and in TCM 199 + steer serum (SS) with or without additional supplements (Experiment 2). The total number of isolated follicles of different size classes was similar in heifers and cows. No significant difference in the growth rate of follicles of different sizes was seen in the 2 media (TC 199 and B 2). However, the culture of follicles in the TCM 199 that was supplemented only with SS and contained no other additives significantly reduced follicular survival and growth in comparison with follicles cultured in the supplemented medium. The survival time of follicles was related to their initial size at the beginning of culture. The longest period of growth was for follicles 275 to 324 μm in diameter (i.e., 10.7±5.7; 12.1±6.2 and 12.2±2.7 d, respectively, for culture in supplemented Menezo B 2, TCM 199+FCS and TCM 199+SS). Survival and growth of some follicles was maintained for 23 d.