Alpaca spermatozoa quality can be difficult to measure using conventional techniques such as CASA and flow cytometry due to its low concentration and oscillatory motility. The aim of this experiment was to validate a heterologous zona pellucida-binding assay for alpaca spermatozoa.

Alpaca, sheep and cattle ovaries were collected from the abattoir and their follicles aspirated using a needle and syringe. Oocytes were placed in DPBS with BSA (4 mg/mL) and hyaluronidase (1 mg/mL) to facilitate removal of cumulus cells, then washed in DPBS and stored in oocyte salt storage solution at 5˚C. Prior to use, oocytes were washed in DPBS and placed in 50 µL drops of sperm capacitating medium (CM; sperm TALP plus 10 µg/mL heparin) under embryo culture oil and equilibrated for one hour at 38˚C under 5% CO₂.

Semen was collected from one alpaca, one bull and one ram using species appropriate techniques. The ejaculates were diluted in TALP, centrifuged to remove seminal plasma and the pellets resuspended in CM containing Hoechst 33342 fluorescent stain. Two million motile spermatozoa were added to each oocyte-containing drop for co-incubation over 1 hour. Each of the three species’ spermatozoa was co-incubated with each species’ oocytes. After co-incubation, the oocytes and their attached spermatozoa were washed in TALP, fixed in formalin and stored in Quench solution. The number of attached spermatozoa was determined by visualising the oocytes under fluorescent microscopy.

The spermatozoa from all three species were able to bind to the oocytes of all three species. There was a significant difference between the number of alpaca and ram spermatozoa bound to sheep oocytes but no difference between the binding of alpaca and bull spermatozoa to cattle oocytes. These results suggest that cattle oocytes would be more suitable than sheep oocytes for use in an alpaca zona-binding assay.