Evaluation of acrosome reaction in bovine sperm by a triple-staining technique

Abstract

In order to investigate the efficiency and usefulness of a simple triple-staining technique (TST) in detecting the acrosome reaction (AR) of bovine sperm, motile ejaculated bovine spern obtained by swim-up procedure were exposed to 10 µg/ml heparin and/or 100,200 and 400 µg/ml lysophosphatidylcholine (LC), or cumulus cell masses with or without bovine oocytes. The viability and AR of the sperm were assessed by TST and AR was confirmed by TEM. After exposure of sperm to heparin, heparin + 100 µg/ml LC, or 100 µg/ml LC, percentage of live/acrosome reacted sperm increased significantly (p<0.01, P<0.001, or P<0.001, respectively); exposure of sperm to 100 µg/ml LC or 200 µg/ml LC or 400 µg/ml LC with or without heparin preincubation, on the other hand, increased the percentage of dead/acrosomereacted sperm significantly (P<0.001) whereas live/acrosomereacted sperm decreased significatly (P<0.05 for 200 µg/ml LC, or P<0.01 for 400 µg/ml). Incubation with cumulus cell masses with or without oocytes induced AR in live sperm significantly (P<0.001), but showed no significant changes of AR in dead sperm. These results indicate that 1) heparin could induce AR in swim-up separated bovine sperm and the extent of the AR was increased followed by LC treatment. However the high dose of LC has detrimental effect which would cause the sperm death; 2) cumulusoocyte complexes also caused AR in bovine sperm as well as maintained their viability; and 3) TST could be used to evaluate the AR as efficient and TEM, but it would be more simple, more rapid and more economic