Chromosome analysis of Murrah buffalo (Bubalus bubalis ) spermatozoa using zona-free hamster oocytes

An interspectific in vitro fertilization system was adopted to analyse sperm chromosomes of Murrah buffalo (Bubalus bubalis ). Superovulation was induced in mature female golden hamsters (Mesocricetus auratus ) to obtain a large number of oocytes. The zona pellucidae were digested by trypsin treatment. Zona free hamster oocytes were penetrated by buffalo spermatozoa capacitated with calcium ionophore A23187. Fertilized ova were cultured in TC 199 medium supplemented with 10% fetal calf serum (FCS). Podophyllotoxin and vinblastine were used to interrupt karyogamy and tubulin polymerization, respectively. Oocytes were fixed by modified gradual fixation air drying method. Slides were stained with 2% Giemsa for 45 min. Analyzable metaphase chromosome spreads were obtained from 22.4+/-3.8% of the penetrated oocytes. Of the 70 sexed spermatozoa, 38 were X-bearing and 32 were Y-bearing spematozoa.