Viability of fresh and frozen bull sperm compared by two staining techniques

Abstract

Semen cryopreservation results in sublethal damage to sperm due to membrane deterioration and an increased number of spermatozoa that undergo acrosome reaction, these damages lead to fertility reduction. The objective of this study was to compare two methods of staining in order to evaluate bull sperm viability after cryopreservation. A single batch of fresh and frozen semen from eight Holstein bulls was obtained for assessment of sperm viability and acrosomal status. Fresh and frozen-thawed semen samples were stained with eosin-nigrosin (EoNig) and the triple staining technique (TriSt) and evaluated under a light microscope. The results showed a significant decrease (P < 0.05) in the number of viable cells after cryopreservation with both techniques. A significant difference (P < 0.05) in viability on both fresh and frozen thawed sperm was observed when the two stains were compared. Differences in viability among bulls using both staining techniques were found on fresh samples, whereas in frozen-thawed sperm no differences were observed with the TriSt (P < 0.05). A marked decline (41.4 ± 11 S.D., P < 0.001) in the mean number of acrosome intact live sperm (AILS) was observed after cryopreservation using TriSt. In contrast, the EoNig technique overestimated the mean number of living sperm that could maintain their fertilizing ability on the frozen-thawed semen samples. In frozen-thawed semen TriSt showed a mean number of 33.4 ± 6.2 S.D. Concluding that EoNig as well as the TriSt, are useful tools for evaluating fresh semen samples, but when evaluating cryopreserved bovine semen, the TriSt offers more reliable results.