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This study aimed to investigate the effect of different cryoprotectants, glycerol (GLY) or ethylene glycol (EG) or dimethyl sulfoxide (DMSO) on sperm characteristics, and in vivo fertility of frozen-thawed buffalo-bull semen. A total of 85 ejaculates collected by artificial vagina from buffalo-bulls of proven fertility were used in this study. The collected ejaculates were examined for volume, motility, viability, morphology, and sperm cell concentration. The qualifying ejaculates (≥ 3 mass motion, > 70% progressive motility and viability, 1 x 10 9 sperm cells/mL) were pooled and diluted with Tris-based diluent containing either 7% GLY or 5% EG or 5% DMSO. After 4 h equilibration time, the diluted semen was loaded in 0.5 mL straws, labeled, sealed and frozen stored until analysis. Frozen straws were thawed and evaluated for progressive motility, viability, hypo-osmotic swelling test (HOST), acrosomal membrane integrity, and acrosome reaction (AR) in response to calcium ionophore A23187. Moreover, in vivo fertility was calculated after artificial insemination (AI) of 75 buffalo-cows (25 female/cryoprotectant) treated with double doses of prostaglandin F 2α (PGF 2α ) 11 days interval. The proportions of progressive motility, viability and intact-acrosome were higher ( p < 0.05) in extender containing 7% GLY compared to 5% EG and 5% DMSO. The proportion of intact-plasma membrane was comparable ( p ≥ 0.05) between GLY and EG but higher than that of DMSO. A time-dependent increase in the % AR and % relative AR was recorded in the three cryoprotectants with clear significant ( p < 0.01) difference among them at 30 and 60 min incubation, respectively. Moreover, GLY yielded higher pregnancy rate (52%) than EG (32%) and DMSO (16%). In conclusion, GLY is recommended for preservation of buffalo-bull semen in order to maintain sperm plasma membrane integrity and improve in vivo fertility of frozen-thawed buffalo-bull semen. Key words : buffalo-bull; frozen-thawed semen; cryoprotectant; estrus synchronization; In vivo fertility

EFFECT OF DIFFERENT CRYOPROTECTANTS ON THE POST-THAW SPERM CHARACTERISTICS AND IN VIVO FERTILITY OF BUFFALO (Bubalus bubalus) BULL SEMEN