The Thai Journal of Veterinary Medicine


The present study evaluated the accuracy of four methods, i.e. eosin-nigrosin, trypan blue, hypo-osmotic swelling test (HOST) and Hoechst 33342 (H342)/propidium iodide (PI) for assessing bovine sperm vitality. Frozen-thawed semen (n = 30) of Holstein bulls was layeredon 40%/80% percoll solutions to isolate viable spermatozoa. An aliquot of viable spermatozoa was kept at 37°C (live sample); the rest was submitted to cold shock (dead sample). The two aliquots were mixed in three proportions, corresponding to 0%, 50% and100% of viable cells; the sperm vitality was analyzed. The percentages of viable spermatozoa evaluated with the four methods significantly correlated with the expected vitality (rs= 0.92 –0.94, P<0.001). Comparing methods of evaluation, the sperm vitality assessed by eosin-nigrosin and trypan blue was comparable (P>0.05) when live and dead samples were used. For the live sample, eosin-nigrosin (82.37 ± 1.48%) and trypan blue (81.00 ± 1.67%) yielded significantly greater results than HOST (50.14 ± 1.68%) and H342/PI (56.69 ± 1.76%) (P<0.001). The percentage of viable spermatozoa in the dead sample (13.45 ± 0.86%) was highest when HOST was exploited (P<0.001); still, the sperm vitality acquired by this technique become the lowest when the live sample was evaluated (P<0.001 to P=0.03). In conclusion, while HOST and fluorescence stains H342/PI with the protocol used in this study are not trustworthy methods, eosin-nigrosin and trypan blue are accurate techniques for sperm vitality assessment in frozen-thawed Holstein bull semen.



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