The acrosome is a cap-like structure over the anterior half of the sperm head. Following successful capacitation, the outer acrosomal membrane partially fuses with the plasma membrane of the sperm head, thereby exposing the contents of the acrosome. This process results in the exocytosis of acrosome digestive enzymes, such as hyaluronidase and acrosin, that facilitate the breakdown of the zona pellucida, subsequently enabling the sperm plasma membrane to fuse with the oolemma, eventually leading to fertilisation. The ability or failure to acrosome react is directly associated with male fertility [1, 2]. Raudsepp [49] investigated the genomic factors governing the failure to acrosome react in stallion spermatozoa.

Following capacitation, spermatozoa are incubated with a calcium (Ca2+) ionophore (A23187), which transports extracellular Ca2+ into cells or releases Ca2+ from intracellular stores [3], and eventually stimulates the acrosome reaction [4]. Acrosome reaction is assessed, as previously discussed, using the acrosome integrity assay. Spermatozoa that have acrosome reacted will not display any fluorescence, while spermatozoa that failed to acrosome react will fluoresce green. The vitality counterstain (red) enables us to distinguish between ‘true’ acrosome reaction and pathological acrosome loss which has occurred following a loss of vitality (dead cells with acrosome loss).