In vitro maturation, fertilization and development of follicular oocytes from buffalo (Bubalus bubalis)

Abstract

Cumulus–oocyte complexes, 5596, were cultured for 24 h in either TCM-199 or Ham's F-10 with or without gonadotrophins and supplemented with either 20% buffalo oestrous serum (BES) or fetal calf serum (FCS). The maturation rates of oocytes cultured in TCM-199 or Ham's F-10 medium supplemented with 20% BES were 47.4±17.8 and 44.8±25.6, respectively. Addition of luteinizing hormone (LH) (5 μ g ml⁻¹) significantly improved the maturation rate in the Ham's F-10 medium supplemented with 20% BES (76.8±18.3), but follicle-stimulating hormone (FSH) (0.5 μ g ml⁻¹) and oestradiol (1 μ g ml⁻¹) failed to synergize with LH (71.7±19.5). In the TCM-199 system, LH failed to enhance the maturation rate but the addition of FSH and oestradiol significantly enhanced the proportion of mature oocytes (42.7±1.4 and 81.7±14.5, respectively; P < 0.05). Frozen–thawed spermatozoa prepared in Bracket and Oliphant (BO) medium and treated with 5 mmol caffeine l⁻¹±10 μg heparin showed a higher fertilization rate (29.8%) than those treated in Hepes–Talp and treated with 10 μg heparin ml⁻¹ (19.6%). Fertilization rate was significantly improved when fresh ejaculated spermatozoa treated with 5 mmol caffeine l⁻¹ and 10 pg heparin in BO medium (50%) was used. Rate of cleavage and development were also higher when in vitro fertilization was carried out with fresh ejaculated spermatozoa treated with caffeine and heparin (34.1 and 36.8%, respectively) than with frozen–thawed spermatozoa (27.0 and 22.0%, respectively). Development rate was enhanced when fertilized ova were cultured in ligated rabbit oviduct (28.0%) than when co-cultured on oviductal cell monolayers (8.2%). The results indicate that oocytes cultured in medium supplemented with BES and gonadotrophins reveal high rates of maturation and development to the blastocyst stage after fertilization with fresh ejaculated spermatozoa.