Protocol 1. Cryopreservation of Human Semen by Manual Method Cooling/freezing phase (1) Allow sample to liquefy at room temperature for approximately 30 min (2) Determine basic semen parameters manually or using CASA (3) Slowly add equal volume of Test-Yolk Buffer containing

In general, the CPA of choice for semen/ sperm cryopreservation is glycerol. Usually a solution of about 6% v/v glycerol is used in a buffered medium containing TES (N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid), TRIS (tris (hydroxymethyl) aminomethane) and citrate buffers mixed with 20% (v/v) egg yolk and antibiotics 19,20. The concentration of glycerol is initially doubled so that after dilution with an equal volume of semen, the final desired concentration is achieved. After adding the medium to the semen over 10-15 min to allow gradual replacement of cell water, the mixture is placed in a beaker of water at ambient temperature and refrigerated (2-6ºC) for 30-60 min to complete equilibration and cool slowly (approximately –0.2ºC/min). The mixture is then aliquoted into labeled and refrigerated plastic cryovials in volumes ranging from 0.5 to 0.8 ml (although

smaller volumes may also be frozen). The vials are transferred either to a freezing tray for manual freezing or directly into a programable freezer (see Protocols 1 and 2).