326 FOLLICULAR SIZE, BUT NOT STAGE OF REPRODUCTION OR SEASON, INFLUENCES MEIOTIC MATURATION OF DOMESTIC DOG OOCYTES

Abstract

The current in vitro maturation system (IVM) for dog oocytes is inefficient. On the average, only 15% of ovarian oocytes complete nuclear maturation in vitro. For unknown reasons, the ability of oocytes to develop to the metaphase II stage (MII) varies markedly among bitches (Songsasen et al., 2002, Mol. Reprod. Dev. 62, 407–415). The objective of this study was to identify the cause(s) underlying these significant variations in nuclear maturation. Initially, we retrospectively analyzed data obtained during the past 3 years;; 1661 oocytes were obtained from 74 bitches where stage of reproduction for the donor was known based on ovarian morphology. Oocytes were cultured in TCM 199 + 0.1% polyvinyl alcohol at 38.5°C in 5% CO₂ in humidified air under various experimental conditions. Analysis of variance (ANOVA) was performed to compare differences in meiotic competence of oocytes obtained at various reproductive stages and during different seasons. Stage of reproduction did not influence meiotic abilities of oocytes. Percentages of oocytes obtained during proestrus/estrus (n = 468 oocytes), diestrus/metestrus (n = 333), anestrus (n = 331) or prepuberty (6–8 months of age, n = 479) and developing to MII were 17.9 ± 2.9%, (mean ± SEM), 24.0 ± 6.0%, 20.8 ± 4.7%, and 17.8 ± 5.2%, respectively (P > 0.05). A similar analysis across seasons (spring, summer, fall, winter) also indicated no influence of time of year on nuclear maturation (P > 0.05). Because there is a known strong link between follicular growth and meiotic competence of goat oocytes (De Smedt et al., 1994 J. Exp. Zool. 269, 128–139), we also examined the impact of follicular size on nuclear maturation. The cortex of ovaries from 15 bitches was horizontally dissected (5 mm thickness) so follicles could be observed and divided into three classes: (1) <0.5 mm diameter (n = 60); (2) ≥0.5 to <1 mm (n = 110); and (3) 1–2 mm (n = 72). Follicles were separated according to these size classes;; oocytes were recovered and cultured in TCM 199 + 0.25 mM pyruvate, 2 mM glutamine, 25 mM β-mercaptoethanol, 10 ng/mL epidermal growth factor (Basal TCM) supplemented with 0.5 IU/mL equine chorionic gonadotropin for 1 h. Oocytes then were cultured in Basal TCM for 48 h before staining with 1% orcein to assess nuclear status. Follicular size influenced meiotic competence of the oocytes (ANOVA, P < 0.05). Mean percentages of MII oocytes were 14.2 ± 7.2, 15.6 ± 4.5, and 30.9 ± 8.2, for oocytes recovered from <0.5-mm, ≥0.5 to <1-mm and 1–2-mm diameter follicles, respectively. This study revealed that stage of reproduction and season have no impact on in vitro nuclear maturation of the dog oocyte. However, the findings demonstrate that dog oocytes acquire meiotic competency during follicular development. Because the source of most dog oocytes for IVM are small follicles, results suggest that oocytes may be incapable of completing nuclear maturation under in vitro conditions that are designed for fully-grown oocytes.