In Vitro Maturation of Bovine Oocytes with Fractions of Bovine Follicular Fluid Separated by Heparin Affinity Chromatography.

Abstract

To elucidate the beneficial effects of follicular fluid (FF) added to maturation medium on nuclear maturation and postfertilization development of bovine oocytes in vitro, we attempted to separate largely bovine FF (bFF) collected from small follicles into heparin-binding and -nonbinding fractions (HBF and HNF, respectively) by heparin affinity chromatography. Each fraction was dialyzed (molecular weight cut off ？？1, 000 Da) and lyophilized, then dissolved in a half original bFF volume of modified synthetic oviduct fluid (mSOF). Experiment 1: Cumulus-oocyte-complexes (COCs) were matured in mSOF in the presence or absence (control) of whole bFF (wFF), HBF or HNF at a concentration of 10% (v/v). After in vitro fertilization, oocytes were freed from COCs and then cultured in mSOF with fetal calf serum until Day 8. Nuclear maturation rates were not different among wFF, HBF, HNF and control (69.8, 75.6, 63.6 and 72.9%, respectively). However, HBF favored postfertilization development, especially to the blastocyst stage (27.2%) compared with the control (19.0%), whereas HNF showed inhibitory effects, although wFF had no effect. Experiment 2: To determine whether the effects of HBF and HNF would become more evident by high-dose supplementation, HBF or HNF was added to in vitro maturation (IVM) medium at concentrations of 10, 20 or 40% (v/v). HBF at all concentrations examined resulted in significantly higher (p<0.05) cleavage rates (80.5-86.2%) and blastocyst yield (31.1-44.2%) than the control(cleavage: 70.1%, blastocyst: 22.4%), whereas high dosages of HNF (？？20%) markedly inhibited embryonic development. These results indicated that heparin-binding fraction of bFF when added to the IVM medium was highly effective for enhancement of developmental competence of bovine oocytes in vitro.