Comparison of different methods for sperm vitality assessment in frozen-thawed Holstein bull semen

Abstract

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 layered on 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% and 100% 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 eosinnigrosin and trypan blue was comparable (P>0.05) when live and dead samples were used. For the live sample, eosinnigrosin (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.