344 FOLLICULAR FLUIDS COLLECTED FROM LARGE FOLLICLES IMPROVED THE PROGRESSION OF IN VITRO NUCLEAR MATURATION OF PIG OOCYTES

Abstract

The present study examined the effects of supplementation of in vitro maturation medium with porcine follicular fluids (pFFs) on the progression of nuclear maturation, subsequent fertilization, and development of pig oocytes. The pFFs were collected from follicles of different diameters: small follicles (SFs, 3–4 mm in diameter) and large follicles (LFs, 5-6 mm). In experiment 1, oocytes were collected from follicles 3–6 mm in diameter. The oocytes were cultured in maturation medium (NCSU-37) supplemented (10% v/v) with pFF from SFs (SpFF) or LFs (LpFF); the kinetics of nuclear maturation, fertilization rate, and developmental competence of oocytes were evaluated. In experiment 2, oocytes were collected from SFs or LFs, and the effects of supplementation of SpFF and LpFF on the nuclear maturation of these oocytes were examined. In experiment 3, each pFF was denaturated by heat treatment (60°C for 30 min) or trypsin treatment (200 µg mL^-1), and the effects of these pFFs on the nuclear maturation of pig oocytes were examined. In experiment 4, pFFs were ultrafiltered, and separated into fractions greater and less than 30 kDa; the effects of the separated pFFs on nuclear maturation were examined. We used Student's t-test to examine the differences among the results; a P value less than 0.05 was considered to be significant. In experiment 1, the progression of nuclear maturation was significantly faster when the maturation medium was supplemented with LpFF than with SpFF (MI rates at 36 h after culture: LpFF, 20.0%, and SpFF, 35.0%; MII rates at 36 h after culture: LpFF, 33.3%, and SpFF, 20.0%). The rates of polyspermic fertilization were significantly lower in the maturation medium with LpFF than in that with SpFF (LpFF, 31.7%; SpFF, 66.7%). Moreover, the rates of monospermic fertilization (LpFF, 45.0%; SpFF, 21.7%) and blastocyst formation (LpFF, 15.2%; SpFF, 3.0%) were significantly greater in the medium supplemented with LpFF than with SpFF. In experiment 2, we observed that LpFF accelerated the progression of nuclear maturation of pig oocytes collected from LFs (MII rates at 36 h after culture: LpFF, 61.7%; SpFF, 35.5%), but not those collected from SFs (MII rates at 36 h after culture: LFs, 15.0%; SFs, 11.7%). In experiment 3, the accelerating effect of pFF on nuclear maturation was eliminated by heat (MII rates at 36 h after culture: LpFF, 30.0%; SpFF, 25.0%) or trypsin treatment (MII rates at 36 h after culture: LpFF, 20.0%; SpFF, 21.7%). In experiment 4, only LpFF with a molecular weight greater than 30 kDa had positive effects on the progression of nuclear maturation (MII rates at 36 h after culture with LpFF: >30 kDa, 30.0%; <30 kDa, 11.7%). The present study suggests that LpFF contains some proteins that improve the progression of nuclear maturation and the developmental competence of oocytes.