Stimulation of in vitro ovine oocyte maturation with a novel peptide isolated from follicular fluid of the buffalo ( Bubalus bubalis)

Abstract

In vitro oocyte maturation is an important step in the laboratory production of embryos. The technique of in vitro maturation (IVM) has generally been standardized in sheep, but now the efforts are aimed at reducing the cost of the technology by replacing expensive inputs of the IVM medium with less expensive and chemically defined inputs. In this study, a novel peptide (Mr 26.6 kDa) isolated and partially purified from buffalo ovarian follicular fluid was incorporated at various dose levels to study its effect on the IVM of ovine oocytes. Oocytes were retrieved from sheep ovaries via a slicing technique. The basic culture medium (control) was comprised of the tissue culture medium (TCM-199) + BSA (3 mg ml −1) with gentamicin (50 ng ml −1). The isolated peptide was added to the basic culture medium at dose levels of 10, 15, 20, 25, 50, 100, 1000, 2000, 5000 ng ml −1 of culture medium. An additional treatment group was incorporated comprised of 2000 ng peptide per milliliter plus epidermal growth factor (EGF: 20 ng ml −1). A positive control group comprising of basic culture medium along with steer serum (20%, v/v) and EGF (20 ng ml −1) was also tested. The oocytes in the culture medium were placed in Petri dishes with overlaying mineral oil and incubated for 24 h at 38.5 °C, 95% relative humidity and 5% CO 2/air. The biological effect of the isolated peptide was evaluated by a cumulus expansion score and the in vitro maturation rates, which were calculated based on the cumulus expansion and extrusion of first polar body. There was a dose-dependent increment in the cumulus expansion as well as in vitro maturation rates of oocytes when the basic medium was supplemented with the isolated peptide. The in vitro maturation rates obtained with different doses of the peptide, i.e. 0, 10, 15, 20, 25, 50, 100, 1000, 2000, 5000, 2000 ng + EGF and positive control group were 18.9, 37.5, 50.7, 63.0, 64.5, 67.0, 76.5, 78.2, 81.5, 83.1, 90.4 and 88.1%, respectively. Even at a low dose level of 20 ng ml −1, the isolated peptide could stimulate the IVM rate and at 100 ng ml −1 dose, it yielded a high maturation rate (>75%). In view of the observations made in this study, the novel peptide can be incorporated at 100 ng dose in IVM medium of ovine oocytes for augmenting the efficiency of in vitro embryo production technology (IVEP) in sheep.