A comparison of in vitro maturation in buffalo (Bubalus bubalis) and bovine oocytes using confocal microscopy.

Understanding the timing of oocyte maturation in the mediterranean italian buffalo (Bubalus bubalis) is of paramount importance for optimizing IVF and subsequent embryo development. The aim of this study was to compare in vitro maturation of buffalo and bovine oocytes by means of confocal microscopy. The trial was carried out using 280 buffalo and 172 bovine COCs with intact compact cumulus cell layers, recovered from slaughtered animals. COCs were washed several times in Hepes-buffered TCM 199, supplemented with 10% (v/v) FCS, and in vitro matured (IVM) into Bicarbonate-buffered TCM 199, supplemented with 10% (v/v) FCS, 0.5 mcg ml-1 FSH, 5 mcg ml-1 LH, 0.1 mcg ml-1 17-beta-estradiol. 50 or 100 mcM cysteamine was added respectively to the maturation medium for buffalo and bovine oocytes. COCs were cultured in incubator under 5% CO2 in humidified air at 38.5°C (buffalo) and 39°C (bovine). Oocytes were fixed in paraformaldehyde 3.7% in PBS for 15 minutes, treated with Triton x-100 for 12-15 h at 0, 5, 10, 15, 16, 17, 18, 19, 20, 22, 24 h post IVM start, washed in PBS 1x and stained with propidium iodide (10mcg/ml in PBS 1x). A confocal microscope (Olympus-fluoview system) was used for observation and images were acquired with an adobe photoshop program. Statistical differences on proportion data were assessed by ANOVA, using arcsin transformation, as reported by Snedecor and Cochran (1989). Buffalo oocytes remained at the germinal vesicle (gv) stage until 10 h and completed the first meiotic division within 14 h. Most of buffalo oocytes at 15 h and at 16 h had reached metaphase II (M II). Between 17-19 h and 20-24 h respectively 76-87% and 47-50% of oocytes in M II were observed. A significant increase in complete chromosome extrusion (chromosomes were detected outside the cells) was observed in oocytes at 20-24 h (31,9-36,8%) compared with oocytes at 18 h. Oocytes characterized by a complete absence of DNA were observed at 17 h, 19 h, 20 h (13.6%) and 24 h (10.5%). These unusual findings would need further investigations. Anaphase II (A II) and Telophase II (T II) was observed between 16-24 h. Statistical difference (P<0.01) was shown between 15-19 h and 20-24 h with regard to incidence of oocytes in M II. In bovine oocytes gv was observed until 15 h (35.7%) and the M I was reached within 18 h. Most of oocytes were in M II between 18 and 24 h. In contrast to buffalo species both oocytes characterized by a complete extrusion of chromosomes and total absence of DNA were not observed in bovine. Buffalo oocytes complete both meiotic divisions in an in IVM system earlier than bovine oocytes.