280 EFFECTS OF HOT SEASON ON BOVINE OOCYTE QUALITY: HOW TO BYPASS THE POOR OOCYTE QUALITY DURING THIS SEASON?

Abstract

Many authors attribute the decline of reproductive activity in summer to the heat stress, a multifactorial problem in which hyperthermia affects cellular function in various tissues of the female reproductive tract (Hansen et al. 2001; De Rensis et al. 2003). In particular, the combination of high temperatures and high humidity for a long period causes a reduced blood flow to uterus, oviducts, and ovaries, leading to a rise in the concentration of the degradation products of cellular activity. Therefore, the aim of this work was to elucidate the negative effect of the hot season on bovine oocyte quality and evaluate the influence of different factors on the acquisition of meiotic competence. In particular, meiotic competence of bovine oocytes recovered from animals housed at 44°28′00″ N, 11°26′00″ E during spring (March, 4–13°C) and summer (June, 16–27°C) was evaluated. Likewise, in summer the effect of an antioxidant, myo-inositol, the use of serum replacement (SR), and the use of oocytes recovered from cycling heifers (16–18 months) as compared to cows (>24 months) were tested. A total of 1346 abattoir-derived oocytes, equally divided for different experimental groups (over 6 replicates), were in vitro matured in TCM 199 supplemented with EGF (25 ng mL^–1), IGF1 (100 ng mL^–1), ITS supplement, pFSH-LH (0.1 IU each), and 10% FBS. Myoinositol was added at a concentration of 0, 15, 30, and 50 mM, while 10% SR was used alternatively to FBS. At the end of maturation period (20–22 h), oocytes were denuded and stained with 10 μg mL^–1 of Hoechst 33342 at room temperature in the dark. After 15 min they were mounted on glass slides for evaluation of nuclear status using a Nikon Eclipse E400 microscope equipped with fluorescence filters. Nuclear configurations were classified as (a) germinal vesicle (GV), (b) germinal vesicle breakdown (GVBD), (c) metaphase I (M-I), (d) metaphase II (M-II), and (e) degenerated (DEG). Data are expressed as mean ± s.e.m. and were analysed by ANOVA (IBM SPSS Statistics) considering significance at P < 0.05. Oocyte quality of summer oocytes was significantly lower than spring counterparts as result of a higher rate of DEG (8.2 ± 0.6 v. 0.7 ± 0.6) and GV (5.4 ± 0.3 v. 0.4 ± 0.4, respectively; P < 0.05). Myo-inositol supplementation in IVM medium did not significantly affect either oocyte quality or meiotic competence in the hot season, such as the use of SR. When the oocytes were collected from cycling heifers ovaries during summer, the recovery rate of COC/ovary was significantly higher as compared to cows (4.5 v. 2.0), and a lower rate of DEG (1.8 ± 0.2; 8.2 ± 0.6) and GVBD (0.9 ± 0.6; 6.1 ± 0.3) was found (P < 0.05), even if the rate of GV (22.4 ± 0.1 v. 5.4 ± 0.3) was higher (P < 0.05) compared with cow. In conclusion, the hot season negatively affects oocyte quality, myo-inositol does not affect nuclear maturation, and SR can be used alternatively to FBS. The lower age of oocyte donor positively influenced the number of recoverable oocyte and degeneration rate.