Characterization of calf follicular fluid and its ability to support cytoplasmic maturation of cow and calf oocytes

Abstract

The aims of the present study were to characterize the follicular fluid from prepubertal calf follicles of known size and quality and to study the ability of follicular fluid to support cytoplasmic maturation of calf and cow oocytes. Follicular fluid was obtained from 67 calf follicles classified according to size (S: small < 6 mm, M: medium 6-8 mm and L: large > 8 mm in diameter) and quality (HY: healthy, EA: early atretic and A: atretic). Quality was first determined by mitosis:pycnosis ratios in granulosa cell smears and confirmed by insulin-like growth factor binding protein (IGFBP) patterns. There was approximately 90% agreement between the two methods of follicle classification and on this basis the calf follicular fluid was pooled into nine groups. The accuracy of this pooling was confirmed by evaluation of oestradiol concentrations in the nine pools of follicular fluid using radioimmunoassay. Increases in follicle size were characterized by a decreased intensity of bands for IGFBP-2, IGFBP-5 and IGFBP-4 an increase in the proportion of healthy follicles and a decrease in the proportion of follicles in the early stages of atresia. This finding is in agreement with previously published results in cows. All classes of calf follicular fluid contained lower concentrations of oestradiol than previously reported for corresponding classes of cow follicular fluid. Cow oocytes were matured in M199 alone, or supplemented with 10% fetal calf serum (FCS), or 10% calf follicular fluid from one of three pools (LHY, LEA, LA), fertilized, and cultured for 8 days in synthetic oviduct fluid. Addition of FCS or calf follicular fluid to cow oocytes during in vitro maturation increased the yield of blastocysts on day 8 over the control (23%, 21/91), FCS (39%, 37/96, P < 0.05), LA (41% 21/52, P < 0.05), LEA (32%, 28/88), LHY (36%, 32/88), although not significantly in all cases. The rate of hatching of blastocysts was also improved: control (38%, 8/21), FCS (54%, 20/37), LA (62%, 13/21), LEA (75%, 21/28, P < 0.02), LHY (59% 19/32). In contrast, the addition of either FCS, calf follicular fluid or cow follicular fluid did not improve development of calf oocytes compared with the unsupplemented control. In conclusion, it is probable that serum and follicular fluid contain factors that stimulate the acquisition by oocytes, during maturation, of developmental competence and to which prepubertal oocytes are unable to respond. Specific receptors for these factors may develop only around puberty.