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

Ham's F-10 with or without gonadotrophins and supplemented with either 20 % buffalo oestrous serum (BES) or fetal calfserum (FCS). The maturation rates ofoocytes cultured in TCM-199 or Ham's F-10 medium supplemented with 20 % BES were 47\m=.\4\m=+-\17\m=.\8and 44\m=.\8\m=+-\25\m=.\6,respectively. Addition of luteinizing hormone (LH) (5\g=m\g ml\m=-\1) significantly improved the maturation rate in the Ham's F-10 medium supplemented with 20 % BES (76\m=.\8\m=+-\18\m=.\3),but follicle-stimulating hormone (FSH) (0\m=.\5 \g=m\gml\m=-\1) and oestradiol (1 \g=m\gml\m=-\1) failed to synergize with LH(71\m=.\7\m=+-\19\m=.\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\m=.\7\m=+-\1\m=.\4and 81\m=.\7\m=+-\14\m=.\5,respectively; P < 0\m=.\05).Frozen\p=n-\thawedspermatozoa prepared in Bracket and Oliphant (BO) medium and treated with 5 mmol caffeine 1\m=-\1 \m=+-\10 \g=m\gheparin showed a higher fertilization rate (29\m=.\8%)than those treated in Hepes\p=n-\Talpand treated with 10 \g=m\gheparin ml\m=-\1 (19\m=.\6%). Fertilization rate was significantly improved when fresh ejaculated spermatozoa treated with 5 mmol caffeine 1\m=-\1and 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\m=.\1and 36\m=.\8%, respectively) than with frozen\p=n-\thawedspermatozoa (27\m=.\0and 22\m=.\0%,respectively). Development rate was enhanced when fertilized ova were cultured in ligated rabbit oviduct (28\m=.\0%)than when co-cultured on oviductal cell monolayers (8\m=.\2%).The results indicate that oocytes cultured in medium supplemented with BES and gonado-trophins reveal high rates of maturation and development to the blastocyst stage after fertilization with fresh ejaculated spermatozoa