Developmental Competence of Buffalo Oocytes from Follicles of Different Diameters Selected by Brilliant Cresyl Blue Staining

The objective of this study was to increase efficiency of blastocysts production from buffalo by selection of more competent oocytes for in vitro embiyo production. Oocytes were selected on the basis of brilliant cresyl blue (BCB) staining correlated with follicular origin. Buffalo Cumulus oocyte complexes (COCs) were recovered from ovaries by follicles isolation and classified into 3 categories according to their diameter (small, <3mm; medium 3-5 mm and large >5-8mm). In experiment I. oocytes were placed immediately into culture without exposure to BCB stain (control) or stained with BCB for 90 min before culture. One part of the control and treated oocytes were fixed and stained to determine chromatin configuration at the time of recovery. Other part was cultured in in-vitro maturation medium for 24 h and fixed to evaluate meiotic competence of oocyte. In experiment 2: oocytes were culture for 24 h fertilized in vitro for determined cleavage rate after 2 days post IVF and blastocyst obtained after 8 days from IVF. The results showed that there was no difference in meiotic configuration at time of recoveiy. In contrast, the maturation rate to metaphase II was significantly (P <0.05) higher for BCB+ and control than BCB-oocytes in all follicles diameters. Furthermore the cleavage rate after IVF was significantly (p<0.05) low for BCB-oocyte originated from all follicles size. Moreover, the BCB+ oocyte yielded a significantly (p<0.05) higher proportion of blastocyst rate from medium and large follicles. In contrast, the lowest proportion of blastocyst was obtained for BCB oocytes from small follicles. Both BCB+ and control had significantly (p<0.05) higher blastocysts than BCB-oocytes in all follicle diameters. In conclusion, the staining of buffalo COCs from different follicle diameters before in vitro maturation may be used to select more developmentally competent oocytes for IVF (BCB+ oocytes originated from 3-8 mm follicles).