101 EXTERNAL PARAMETRIC INDICATORS OF IN VITRO DEVELOPMENTALLY COMPETENT WATER BUFFALO OOCYTES

Abstract

External parametric indicators for in vitro developmentally competent water buffalo oocytes were determined. Oocytes were retrieved from ovarian follicles and classified based on the 1) density of surrounding cumulus cells (Rank A, n = 94: with >5 layers, Rank B, n = 73: with 3 to 5 layers, Rank C, n = 73: with <3 layers, Rank D, n = 63: with irregular or denuded from cumulus cells, and Rank E, n = 42: with expanded cumulus cells, and 2) granulation of ooplasm (Homogeneous, n = 164: evenly granulated, Heterogeneous, n = 180: not evenly granulated where some part is either light or dark), 3) size of the ooplasm, n = 647 (<100, n = 87; 100–119, n = 312; 120–139, n = 164; ≥140 μm, n = 84), and 4) size of the donor antral follicle, n = 688 (<2, n = 244; 2 to 3.9, n = 221; 4 to 5.9, n = 116; 6 to 7.9, n = 61; ≥8 mm, n = 46). Oocytes classified based on these parameters were matured for 22 to 24 h and the nuclear maturation was examined with cleavage rate and blastocyst development rate assessed after in vitro fertilization. To validate the hypothesis that oocytes with compact cumulus (n = 248) are at growing phase while those with loose cumulus (n = 270) are at developmental phase, they were matured and fertilized in vitro at shorter (20 to 22 h) or longer (24 to 26 h) period and embryo development was assessed. Each study was replicated 5 to 10 times. Data were statistically analysed by chi-square test, Fisher’s exact test, and correlation analysis. Results showed that oocytes surrounded by multi-layers (>5 layers) of cumulus cells had highest developmental competence. Oocytes with a diameter of <100 μm lacked developmental competence, evidenced by the failure to develop to metaphase II (MII) after in vitro maturation (IVM), whereas oocytes with diameter of ≥100 μm developed to MII and cleaved after IVF. Optimum cleavage (96.8%) and blastocyst development (27.0%) was observed in oocytes with ≥120 μm. The size of the donor follicle was linearly correlated with oocyte developmental competence with follicles ≥6 mm containing highly developmentally competent oocyte. Based on the findings, oocytes surrounded by >3 layers of compact or loose cumulus with evenly granulated and with ∼110 μm diameter ooplasm and derived from ≥4 mm follicles are developmentally competent. Oocytes with a compact cumulus required 24 to 26 h of IVM while those with loose cumulus required 20 to 22 h of IVM for optimum blastocyst development. These results suggest that the density and compactness of the surrounding cumulus, and the diameter of ooplasm and donor follicles are positive indicators for oocytes with developmental competence.