Effect of heparin, caffeine and calcium ionophore A23187 on in vitro induction of the acrosome reaction of fresh ram spermatozoa

ObjectiveTo determine the effect of different concentrations of heparin, caffeine or calcium ionophore and incubation time on motility, hyperactivity (HA) and acrosome reaction (AR) of ram sperm in vitro.MethodsSemen samples were collected by artificial vagina from three mature ram. Split fractions (0.1 mL) of the pooled semen were layered under 1 mL of S-TALP medium supplemented with either heparin (10, 25, 50, 75 and 100 μg/mL), caffeine (0.5, 0.75, 1, 2 mg/mL), or calcium ionophore A23187 (0.5, 0.75, 1, 1.55 mM/mL). Individual motility, hyperactivity percentage and acrosomal status were recorded at 0, 1, 2, 3 and 4 h post-incubation for all treatments and control. Moreover, they were examined for ability to fertilize sheep oocyte in vitro.ResultsHeparin, caffeine and calcium ionophore A23187 at a concentration of 75 μg/mL, 1 mg/mL, and 1.55 mM/mL respectively can be used as a protocol to provide the best results for in vitro cap citation and acrosome reaction in ram. The penetration rates of rate of oocytes inseminated with spermatozoa treated with calcium ionophore A23187, and heparin were higher as compared with caffeine. Moreover, heparin achieved higher fertilization rates but without significant difference with others.ConclusionThe best concentration of heparin, caffeine and ionophore A23187 are 75 μg/mL, 1 mg/mL, 1.55 mM/mL for 3, 1, 4 h incubation respectively and can be used for in vitro fertilization in sheep.