319 IN VITRO MATURATION OF EQUINE OOCYTES IN A COMPLETELY DEFINED MEDIUM SUPPLEMENTED WITH PROGESTERONE

Abstract

A completely defined medium for in vitro maturation (IVM) of equine oocytes has not yet been developed, since most of the media used for IVM are supplemented with serum or BSA. Furthermore, in this species there is no report about the influence of progesterone on maturation, although it has already been used as supplement (500 ng mL⁻¹) in EMMI (Maclellan LJ et al., 2001, Theriogenolgy 55, 310 abst). The aims of this study were to develop a completely defined medium for equine oocyte maturation and to investigate the effect of progesterone on nuclear maturation. Equine oocytes were collected by follicular scraping of abattoir-derived ovaries between April and June. The basal medium for maturation was SOFaa supplemented with pFSH-LH 0.1 IU mL⁻¹ (Pluset, Laboratorios Calier, Barcelona, Spain), EGF^* 50 ng mL⁻¹, ITS (Insulin, Transferrin, Sodium selenite), L-cysteine 1.2 mM, Maturation SOF (MSOF). Compact cumulus-oocyte complexes were selected, washed three times in H-SOF and matured in one of the following media (15–20 oocytes mL⁻¹): (1) MSOF + FCS 10% (MSOF-FCS), (2) MSOF + progesterone 100 ng mL⁻¹ (MSOF-P4), (3) MSOF. After 24 h of culture in 5% CO₂ in air at 38.5°C, the oocytes were denuded by gently pipetting in a 0.25% trypsin solution, washed and stained with Hoechst 33258 (10 μg mL⁻¹ in PBS) for 30 min at room temperature. Oocytes were examined under a fluorescent microscope to assess nuclear maturation. Only oocytes with an evident polar body and metaphase II plate (MII) were considered mature. The experiment was done in 6 replicates. Chi Square test was used for statistical analysis (Statistica for Windows – Stat Soft Inc., Tusla, OK, USA). Significance was assessed for P < 0.05. The results of this study show that MSOF can be considered a suitable completely defined medium for IVM of equine oocytes. Adding progesterone significantly (P < 0.05) increases the nuclear maturation rate at 24 h of culture. It can be speculated that although cumuls cells produce this hormone, supplementation is useful to reach progesterone concentrations similar to those present in follicular fluid (early dominant 63.4 ± 19.3 ng mL⁻¹, healthy preovulatory follicle 1094.3 ± 170.9 ng mL⁻¹; Gerard N et al., 2002, Reproduction 124, 241–248). Further studies are needed to investigate the influence of progesterone on cytoplasmic maturation and to test the effect of different progesterone concentrations and time of maturation in a completely defined system.

^*All chemicals were purchased from Sigma, St. Louis, MO, USA, unless otherwise stated.

Table 1 
Maturation of equine oocytes in different media