IN VITRO PRODUCTION OF BUFFALO (Bubalus bubalis) EMBRYOS

Abstract

Two experiments (Exp) were carried out to test the effect of different media on in vitro maturation (IVM) (Exp 1; n = 1142 ovaries with 2666 oocytes) and to study in vitro fertilization (IVF) of buffalo oocytes (Exp 2; n = 110 ovaries with 330 oocytes). In exp 1 the collected cumulus oocyte complex (COCs) was allocated to three maturational media; TCM-199 supplemented with hormones (M1), TCM-199 supplemented with epidermal growth factor (M2) and m-SOF (M3) overlaid with paraffin oil before incubating under 5 % CO2 in air at 38.5 C. A part of COCs was fixed to determine the stages of nuclear maturation after 24 hr. In exp 2 the matured oocytes were allotted with sperm suspension before incubating under 5 % CO2 in air at 38.5 C, for 22-24 hr. Presumptive embryos were placed, individually in 96 wells petri dish to determine at 12 hr intervals the kinetics of cleavage up to blastocyst stage. Average of collected COCs per ovary was 2.4 with 86 % of excellent and good graded COC’s. M1 and M2 showed higher (P<0.05) maturation rate vs. M3. After culturing oocyte diameter and perivitalline space increased (P<0.05) relative to preculture diameters, while diameter of ooplasm and thickness of zona pellucida non significantly decreased . Fertilized ova and cleaved embryos (%) were 80.2 and 77.8 %, respectively. Early embryonic cleavage up to 32 cell stage lapsed took 84 hr, while corresponding periods to reach morula, and blastocyst stage were 101 and 134.5 hr, respectively. Out of the fertilized ova 31.5 % reached excellent and good graded blastocyst stage.