Cryopreservation of alpaca spermatozoa has been hindered by the high viscosity of their seminal plasma. Recently, the proteolytic enzyme papain has been shown to reduce viscosity without damaging spermatozoa, facilitating renewed attempts to develop cryodiluents for this species. This study aimed to determine the optimal egg yolk and glycerol concentrations for use in an alpaca cryodiluent.

Semen was collected from four males, diluted with a lactose-based diluent and treated with papain (0.1mg/mL final conc.) to reduce viscosity. In experiment 1: semen was further extended with lactose diluent supplemented with 5, 10 or 20% egg yolk and 5% glycerol, chilled to 5˚C over 2h and frozen in 0.25mL straws. The same procedure was followed in experiment 2, except the samples were chilled in the diluent with 5% egg yolk and 2, 5 or 10% glycerol. After thawing, samples were incubated for 2h and assessed for motility, plasma membrane and acrosome integrity pre-freeze and 0, 30, 60 and 120 minutes post-thaw.

In experiment 1, spermatozoa were found to have higher post-thaw motility, acrosome integrity and plasma membrane integrity when frozen with 5% egg yolk compared to 10 or 20% (P<0.001). The mean decline in motility from pre-freeze to 0h post-thaw was only 18.0±2.8% for the 5% egg yolk treatment. In experiment 2, glycerol concentration did not affect the motility (P=0.12) or acrosome integrity (P=0.21) of spermatozoa post-thaw. However, 10% glycerol had lower plasma membrane integrity than the 2 or 5% treatments (P=0.002). These results suggest that papain-reduced alpaca spermatozoa should be frozen in a diluent containing 5% egg yolk and 2 or 5% glycerol.