Fertilizing capacity of vitrified stallion sperm assessed utilizing heterologous IVF after different semen warming procedures

•Warming at 60 °C/5 s showed greater sperm characteristics than others water bath methods•The highest number of sperm bound to bovine oocytes was found after vitrification•Vitrification obtained 22.8 % pronuclei formation and 39.7 % embryo cleavage•Vitrification and freezing showed a similar ability to fertilize bovine oocytes The aim of this study was to evaluate the fertilizing capacity of frozen or vitrified stallion sperm after assessing different warming procedures. In Experiment 1, different warming procedures were compared after sperm vitrification: immersion in extender at 43 °C (C), or in a water bath at 37 °C/30 s (W37), 43 °C/10 s (W43) or 60 °C/5 s (W60). With the W60 treatment, there were greater values (P < 0.05) for VCL (83.93 ± 3.6 μm/s) and ALH (3.00 ± 0.2 μm) than freezing and with the C group, and greater values (P < 0.001) for PM (35.33 ± 2.5 %) than with the W43 treatment. In Experiment 2, the fertilizing capacity of vitrified and frozen sperm was assessed utilizing heterologous IVF procedures, using cattle oocytes. Vitrification resulted in greater values (P < 0.05) than freezing for the number of bound sperm (1.36 ± 0.3 and 0.69 ± 0.2, respectively). There were no differences between frozen or vitrified sperm in pronuclear formation (26 hours post-insemination - hpi; 14.08 ± 4.2 % and 22.78 ± 4.8 %, respectively) or cleavage rate (32.77 ± 4.3 % and 39.66 ± 4.6 %, respectively). In conclusion, vitrified stallion sperm warmed in a water bath at 60 ºC had the capacity to penetrate cattle oocytes, leading to pronuclear formation and hybrid embryo cleavage after heterologous IVF.