Evaluation of in vitro capacitation of stallion spermatozoa

The primary aim of this study was to establish a flow cytometric technique for determining the capacitation status of stallion spermatozoa. To this en d, a flow cytometric technique that demonstrates changes in plasma membrane fluidity; namely, merocyanine 540 staining, was compared with the more con ventional Ca2+-dependent fluorescence microscopic technique, chlortetracycl ine (CTC) staining, for assessing capacitation status. In addition, the eff ect of bicarbonate/CO2 on the progress of capacitation and the acrosome rea ction (AR) and on temporal changes in sperm motility, with particular regar d to hyperactivation, was analyzed. For the study, fresh semen was washed a nd then incubated for 5 h in bicarbonate-containing or bicarbonate-free med ium, with or without Ca2+ ionophore to induce the AR, and at intervals duri ng incubation aliquots were taken and analyzed for capacitation and acrosom e status. The AR was assessed using both the CTC and fluorescein isothiocya nate-peanut agglutinin (FITC-PNA) staining techniques with similar results. In brief, it was found that merocyanine 540 detects capacitation-related c hanges much earlier than CTC does (0.5 h versus similar to3 h), and that fl ow cytometry for evaluation of capacitation and AR was a quicker (10 sec pe r sample) and more accurate (110 000 cells counted) technique than fluoresc ence microscopy. Furthermore, it was observed that Ca2+ ionophore could not induce the AR in the absence of bicarbonate, but that the ionophore synerg ized the bicarbonate-mediated induction of the AR as detected by CTC (altho ugh it was not significant when evaluated using FITC-PNA). The percentage o f hyperactive sperm in each sample was not affected by time of incubation u nder the experimental conditions studied. In conclusion, merocyanine 540 st aining is a better method than CTC staining for evaluating the early events of capacitation for stallion spermatozoa incubated in vitro. Furthermore, bicarbonate sperm activation clearly plays a vital role in the induction of the AR in stallion spermatozoa.