Capacitation refers to the physiological changes (cellular and molecular) spermatozoa must undergo in order to achieve the ability to penetrate and fertilise the oocyte. Capacitation involves remodelling of the sperm plasma membrane (loss of cholesterol and exocytosis of specific phospholipids from the inner membrane; Gadella [1] discuss plasma membrane reorganisation in detail), a change in sperm pH, tyrosine phosphorylation of sperm proteins and hyperactivated motility; ultimately leading to the acrosome reaction. In vitro capacitation of stallion spermatozoa can be achieved using a modified sperm medium containing methyl-β-cyclodextrin developed within our laboratory [2]. Spermatozoa are incubated in this medium at 37 °C for four hours, under 5% CO₂ in air. The achievement of capacitation is assessed via the acrosome reaction (using the acrosome integrity assay) and tyrosine phosphorylation assays.

Tyrosine phosphorylation is the addition of a phosphate group to the amino acid tyrosine on a protein. This process is vital for signal transduction and enzymatic activity. The extent of tyrosine phosphorylation is assessed via immunocytochemistry. Cells are fixed in paraformaldehyde and mounted on a microscope coverslip, after which they are permeabilised to allow the antibodies entry into the cell. During antibody incubation, the primary antibody binds to the antigen―phosphotyrosine―and the secondary antibody, with conjugated fluorophore, binds to the primary antibody. Any unbound antibodies are removed by washing. Coverslips are visualised using fluorescent microscopy. Spermatozoa that have capacitated will fluoresce green.