200 Maturation method affects lipid accumulation in bovine oocytes

Abstract

In vitro maturation is a key step in in vitro embryo production, since its success will depend on the availability of good quality oocytes. Previous studies have shown that it is during this stage that the greatest accumulation of lipid droplets occurs, which is reflected in the amount of lipid present in embryos produced in vitro. However, this is not observed when maturation is performed in vivo. Therefore, we hypothesised that lipid accumulation would be avoided if oocyte maturation were carried out in ovarian follicles following intrafollicular transfer of immature oocytes (IFIOT). We compared lipid accumulation in oocytes matured in vitro, in vivo, and by IFIOT. A total of 90 Nellore heifers were distributed in 3 experimental groups: donors of immature oocytes (D-IMA), ovulators of IFIOT oocytes (D-OV), and superstimulated donors of in vivo-matured oocytes (D-FSH). All animals rotated through all groups during the experiment. To obtain immature oocytes, the D-IMA were submitted to ovum pickup (OPU), in which aspiration medium was supplemented with 500 μM 3-isobutyl-1-methylxanthine (IBMX), and, after selection, part of the oocytes were cultured in vitro for 22 h (MatF) and part were used for IFIOT (MatT). To perform MatT, the D-OV had their ovulation synchronized by a progesterone and benzoate oestradiol protocol, in which 30 h after the implant removal, the IFIOT was performed on the dominant follicle. The D-FSH oocytes were stimulated with 80 mg of FSH (Folltropin; Vetoquinol) over 4 days, every 12 h, in decreasing doses. At the same time that the immature oocytes were placed in MatF and IFIOT, ovulation was induced with the gonadotrophin releasing hormone (GnRH) analogue (50 µg of lecirelin) in D-OV and D-FSH groups. After 22 h, matured oocytes were either removed from culture (MatF) or recovered from follicles by OPU (MatT and MatS). From the recovered oocytes of all groups, only those with a polar body were used for lipid droplet evaluation. To quantify lipid accumulation, denuded oocytes were fixed and stained with boron-dipyrromethene (Bodipy) 493/503 (20 µg mL⁻¹) and evaluated by confocal microscopy. Captured images were evaluated in the ImageJ program (National Institutes of Health), and lipid content was determined by calculating the ratio of the area of the lipid droplets to total oocyte area. Data were analysed by ANOVA with statistical significance set at P < 0.05. A total of 95 oocytes were evaluated: 25 immature (CT), 24 in vitro (MatF), 30 in vivo (MatS), and 16 in vivo (MatT). The mean area containing lipid droplets in immature oocytes (14% ± 0.9) was similar (P > 0.05) to that observed in both in vivo maturation systems (MatS = 17.26% ± 0.8 and MatT = 14.11% ± 0.9). However, in the MatF oocytes, lipid content (24.34% ± 1) increased during maturation and was higher than in the other groups (P < 0.05). We showed for the first time that oocytes matured by IFIOT are similar to those in vivo matured with regard to lipid content, which may imply their superior quality over those matured in vitro. This new maturation method opens new possibilities for biotechnologies that need to use mature oocytes, such in vitro embryo production, oocyte and embryo cryopreservation, cloning, and transgenesis.

This study was supported by FAP-DF and Capes.