354 EFFECT OF OOCYTE-SECRETED FACTORS (OSF) ON EMBRYO DEVELOPMENT OF OOCYTES FROM SMALL FOLLICLES OF PREPUBERTAL GOATS

Abstract

Prepubertal goat ovaries contain a great number of small follicles, between 2.5 and 3 mm in diameter (Martino A et al. 1994 Theriogenology 41, 969-980). Oocyte-secreted factors (OSFs) act on granulosa cells to perform multiple functions required for an appropriate development of the oocyte. In bovines, co-culture of COCs with denuded oocytes (DOs) during IVM improved COCs embryo development dueto OSFs secreted byDOs (Hussein T et al. 2006 Dev Biol. 296, 514-521). In each step of follicle growth, the OSFs secreted are different (Eppig JJ et al. 2001 Reproduction. 122, 829-838). Our hypothesis is that oocytes from small follicles could improve their embryo development by co-culturing with DOs which secrete additional OSFs. Oocytes from prepubertal goats were classified according to their follicular origin: large follicles (>3 mm), small follicles (<3 mm), and control (oocytes recovered by slicing technique and selected by their morphology). The COCs from small follicles were divided in 3 groups and each one transferred into 150-μL droplets of IVM (TCM-199 with serum, hormones and cysteamine): 1) 30 COCs alone (small group); 2) 30 COCs co-cultured with 75 denuded oocytes from small follicles (SDO group); 3) 30 COCs co-cultured with 75 denuded oocytes from large follicles (LDO group). After 27 h of IVM, COCs were fertilized in vitro and the presumptive zygotes were cultured for 8 days in SOF with 10% FCS. Two samples of oocytes were used as control groups: oocytes fertilized (IVF group) and oocytes activated (activated group).At 48 h and 8 days post-insemination (pi), respectively, cleavage and blastocyst rates were recorded. Results are shown in Table 1. Differences between treatment groups were assessed using Fisher’s exact test (Graph-Pad software, San Diego, CA, USA). Values with P < 0.05 were considered statistically significant. Oocytes from small follicle had a significantly lower cleavage and blastocyst rates than IVF and activation oocyte groups. The co-cultured of COCs with SDOs and LDOs increased significantly the blastocyst rate compared to COCs from small follicles. In conclusion, additional OSFs secreted by DOs, improved blastocyst yield of oocytes coming from small follicles. However, not differences were found between OSFs secreted by small or large follicles of prepubertal goat oocytes.

Table 1.