Evaluation of the acrosomal status in Lama glama sperm incubated with acrosome reaction inducers

Abstract

The objectives of this study were to evaluate the effect of different acrosome reaction (AR) inducers on viability and acrosomal status in llama spermatozoa, by using the FITC-PNA/PI technique and evaluate if there is a positive correlation between the FITC-PNA/PI and the Coomassie blue (CB) staining techniques. After incubating twenty ejaculates in 0.1% collagenase the centrifuged pellets were resuspended in TALP–BSA medium. An aliquot was sonicated to remove the acrosomal content (positive control). The rest of the sample was incubated for 3h at 38°C with 5% CO2 and 100% humidity. TreatmentsThree aliquots were further incubated 1h with one of the following AR inducers: calcium ionophore, ionomycin or progesterone. ControlsOne without inducers and the other, incubated with dimethyl sulfoxide (vehicle of the inducing agents). Acrosomes were evaluated at time 0 and after 4h incubation. Calcium ionophore was the most potent agent for inducing the AR (67.2±14.4% live+dead AR sperm) (P<0.05). These samples showed no motility and viability was very low (0–30%). Both ionomycin and progesterone presented significantly higher (P<0.05) percentages of total AR sperm than the controls, but had similar percentages of dead reacted sperm to the controls. A positive correlation was observed between the intact acrosome FITC-PNA/PI pattern (live+dead sperm) and the acrosome-present CB pattern (r=0.64; P=0.000) in all the evaluated samples. Conclusionsthe FITC-PNA/PI technique simultaneously evaluates viability and acrosomal status in llama spermatozoa and calcium ionophore could be used as a control of AR.