REPLACEMENT OF SERUM AND HORMONE ADDITIVES WITH FOLLICULAR-FLUID IN THE IVM MEDIUM - EFFECTS ON MATURATION, FERTILIZATION AND SUBSEQUENT DEVELOPMENT OF BUFFALO OOCYTES IN-VITRO

Buffalo follicular fluid was used in the IVM medium in place of serum and hormone additives for stimulating nuclear and cytoplasmic maturati on of buffalo oocytes in vitro. Follicular fluid (buFF) was aspirated from visible surface follicles from buffalo ovaries. Cumulus oocyte co mplexes (COCs) were matured for 24 to 26 h at 38.5 degrees C, 5% CO2 i n air in the maturation medium (TCM-199). When used, the concentration of fetal bovine serum (FBS) was 10% and that of FSH-P was 5 mu g/ml. In Experiment 1 TCM-199 was supplemented with 1) FBS, 2) FBS + FSH-P, 3) 20% buFF and 4) 40% buFF. The matured oocytes were denuded and stai ned with Giemsa stain to study nuclear maturation. The proportion of o ocytes which completed nuclear maturation was similar in medium contai ning FSH (74%) and 20 or 40% buFF (67%), which was higher (P < 0.05) t han in medium with FBS but without FSH or buFF (47%). In Experiment 2, which was aimed at examining the effects of buFF on cumulus expansion and rates of fertilization and subsequent development to the blastocy st stage after IVF, the maturation medium was supplemented with 1) FBS + FSH-P, 2) 20% buFF and 3) 40% buFF. The matured in medium containin g 20 or 40% buFF had significantly higher (P < 0.01) cumulus expansion than those matured in medium with FBS+FSH-P. Of the COCs matured in m edium with FBS+FSH-P and 20 or 40% buFF, the fertilization rates indic ated by the incidence of cleavage (56, 51 and 52%, respectively) and t he proportion of cleaved COCs developing to morula (58, 54 and 57%, re spectively) and blastocyst stage (30, 31 and 35%, respectively) were n ot significantly different. In Experiment 3, supplementation of the ma turation medium with 1) FBS + FSH-P and 2)FBS + FSH-P + 20% buFF resul ted in similar rates of morulae (41 and 38%, respectively) and blastoc ysts (31 and 25%, respectively), indicating that simultaneous presence of FBS, FSH-P and buFF did not have an additive effect on embryo yiel d. The results show that the gonadotropin and serum source in the IVM medium can be replaced by buFF at the 20% level to achieve comparable morula and blastocyst yields. (C) 1997 by Elsevier Science Inc.