In vitro meiosis resumption of feline oocytes isolated from vitrified ovarian tissue

Introduction and aim. In the preovulatory follicle, canine oocytes are exposed to high concentrations of progesterone (P4) and estradiol (E2), and different concentrations of gonadotrophins are present in the pre- and post-ovulatory environment, i.e. oviduct, where full nuclear maturation occurs (1,2,3). Thus, two microenvironments characterized by the presence of different hormonal concentrations contribute to the achievement of Metaphase II (MII) stage. In vitro, maturation conditions should be based on these different pre- and post-ovulatory environments and on the fact that cultural requirements for oocyte maturation may vary along the time. Hence, the aim of this work was to study the influence of different bi-phasic systems with gonadotrophins and steroids on in vitro maturation rates of canine oocytes. Materials and Methods. Ovaries were collected from 14 anestrous bitches (various breeds, age 1- 7 y) by routine ovariohysterectomy and sliced in PBS-PVA to release the cumulus-oocyte complexes (COCs). A total of 363 COCs grade I (two or more dense layers of cumulus cells and darkly pigmented cytoplasm) were selected for culture. Oocytes were randomly allocated in three different cultural systems with the base medium (BM) consisting of TCM-199 (Sigma Chemical Co., USA), with antibiotics, 10% FBS, 2.2 mg/ml sodium bicarbonate and 20 μl/ml pyruvic acid. The systems were as follows: in system A (control) oocytes (n. 91) were matured for 72h in BM with 10 i.u./ml hCG (Sigma), 1 μg/ml P4 (Sigma), 1 μg/ml E2 (Sigma); in bi-phasic system B oocytes (n. 120) were matured for 48h in BM with hCG and for 24h in BM with P4; in bi-phasic system C oocytes (n. 124) were matured for 48h in BM with hCG, P4 and E2, and for 24h in BM with P4. In systems B and C hormones were supplemented at the same doses as in system A. Cumulus-oocyte complexes were incubated at 38°C, 5% CO2 in air and, at the end of maturation (72h), were transferred to PBS containing 0.2% hyaluronidase (Sigma) for removal of cumulus cells by repeated pipetting. Oocytes were stained with Hoechst 33342 (Sigma) for evaluation of meiotic configuration. Data were analyzed by Chi-square test. Results. Anaphase/Metaphase I rates were higher (P<0.02) in oocytes cultured in bi-phasic system B (27.5%; 33/120) and C (29%; 36/124) when compared to control (13.2%; 12/91). Only three oocytes from control group reached MII stage (3.3%), while higher rates (P<0.02) of oocytes cultured in system B (11.7%; 14/120) and C (13.7%; 17/124) were able to complete meiosis. Degeneration rate was remarkably lower (P<0.05) in C system (4%; 5/124) compared to B (12.5%; 15/120) and control (19.8%; 18/91). Conclusions. These results suggest that the use of bi-phasic systems is beneficial for oocyte meiosis in vitro, when compared to the continuous exposition (72h) to the same hormonal association (control). This is the first time that a bi-phasic system with gonadotrophins and steroids had been studied. As no differences were observed between the two associations of hormones (system B and C), further investigation on the effect of other hormonal combinations and concentrations are needed. References. 1) De los Reyes et al., Theriogenology 2005;64:1-11. 2) Willingham-Rocky et al., Reproduction 2003;126:501-8. 3) Luvoni et al., Theriogenology 2005;63:41-59.