The aim of this particular study was to test in vitro sperm capacitation protocols, using heparin (100mg/ml) and calcium ionophore (5mM). Propidium iodide and carboxifluorescein diacetate (IP/CFDA) in a fluorescence microscope as well as triple stain (congo red, neutral red and Giemsa) in Phase contrast microscope were used as staining. The spermatozoa were classified according to its viability (alive or dead) and acrossome integrity (damaged or intact). They were considered as follows: capacitated (alive and damaged); non capacitated (alive and not damaged) and dead (damaged or intact). The heparin group showed a ratio of 64.54% and 39.16% of capacitated spermatozoa in IP/CFDA and triple stain, respectively. In the calcium group, 36.41% and 18.11% of spermatozoa were capacitated in IP/CFDA and triple stain, respectively. Bulls were divided into 3 groups according to their fertility rates as follows: Group A < 63%, Group B from 63 to 68% and Group C > 68%. For all three groups there was significant differences (p<0.01), regarding capacitated spermatozoa with heparin and calcium ionophore in both stains IP/CFDA and triple stain. On the other hand, there was no significant differences (p>0.01), when observed capacitated, non capacitated and dead spermatozoa among groups A and B; A and C; B and C, using heparin and calcium ionophore in both stains. No correlation was seen between capacitation and fertility rates. Therefore heparin treatment showed better sperm capacitation rates than calcium ionophore. The IP/CFDA technique showed itself as being a better method to evaluate sperm capacitation than the triple stain (p<0.01).
Sperm capacitation; Heparin; Calcium ionophore; Staining methods
