Heparin‐induced and caffeine or ouabain supplemented capacitation of frozen‐thawed yak (Bos grunniens) spermatozoa

Our goal was to investigate heparin-induced capacitation of frozen-thawed yak sperm and to assess the effects of caffeine or ouabain supplementation with heparin on sperm capacitation. Sperm were incubated with varying heparin concentrations, namely 0, 12.5, 25, 50 and 100 μg/ml, for 0, 15, 30 and 60 min. In every treatment, sperm capacitation was assessed using microscopic examination of the sperm acrosomal status and western blot analysis of the levels of tyrosine phosphorylation (Tyr-P). Based on our results, the optimal condition for frozen-thawed yak sperm capacitation was a 30-min exposure to 50-μg/ml heparin. Next, we incubated frozen-thawed yak sperm with 50-μg/ml heparin, along with varying concentrations of caffeine supplementation, namely 0, 2.5, 5 and 10 mM for 30 min. Interestingly, caffeine significantly increased yak sperm acrosome reaction (AR) and Tyr-P (p < .05). The optimal caffeine concentration was 5 mM, followed by 2.5 and 10 mM, with the lowest AR and Tyr-P found in sperm cells that did not receive any caffeine. To examine the effects of ouabain on sperm capacitation, we next incubated frozen-thawed yak sperm with 50-μg/ml heparin, along with varying concentrations of ouabain, namely 0, 25, 50 and 100 µM for 30 min. We demonstrated that ouabain supplementation did not alter yak sperm AR or Tyr-P in sperm cells, relative to the control (p > .05). In summary, our findings suggested that caffeine acts synergistically with heparin to increase yak sperm capacitation, but ouabain does not synergize with heparin to promote yak sperm capacitation.