11 INFLUENCE OF SPERM STIMULANTS ON EQUINE EPIDIDYMAL SPERM APOPTOSIS

Alterations to a sperm cell that are similar to apoptosis reduce the sperm cell longevity in the female reproductive tract. The aim of the present study was to evaluate the effect of sperm motility factors [caffeine, heparin, and penicillamine, hypotaurine, and epinephrine (PHE)] on phosphatidylserine translocation in equine epididymal semen. Equine epididymal samples were obtained from 10 stallions using retrograde flush with 40 mL of BotuSemen® (BS) per epididymal cauda. The samples were divided into 3 groups: BS (control), Fert-TALP (Fert), and Sperm-TALP (SP), and diluted 1 : 1 with these media. Samples containing 100 × 106 sperm cells were loaded into straws, and then incubated at room temperature (25°C) for 15 min. The samples were centrifuged (1000g for 10 min), the supernatant was removed and the pellets were resuspended in BotuCrio®. Samples were loaded into 0.5-mL straws and cooled at 5°C for 20 min. Then, they were frozen in nitrogen vapor (6 cm above the level of LN) for an additional 20 min and finally plunged into nitrogen and stored. The straws were thawed at 46°C/20 min. Sperm motility was evaluated by computer-assisted semen analysis (CASA; HTM-IVOS 12). To evaluate the pancreatic secretory trypsin inhibitor (PSTI), the Annexin V-FITC Apoptosis Detection Kit (6710KK; Pharmingen, San Jose, CA, USA) was used according to the manufacturer’s recommendations. The samples were diluted in Annexin V buffer solution to a concentration of 2 × 106 spermatozoa mL–1 and then 5 mL of Annexin V-FITC, 5 mL of propidium iodide (PI, 50 mg mL–1), and 2 mL of Hoechst 33342 dye (H342, 40 mg mL–1) were added. The samples were then homogenized and incubated for 15 min. Then, 400 µL of Annexin V buffer solution was added to obtain a final concentration of 1 × 106 spermatozoa mL–1. The samples were evaluated. Flow cytometry was carried out at BD LSR II (Becton Dickinson, Mountain View, CA, USA). Mean and standard deviation were calculated, and then the normality test was evaluated by the Kolmogorov-Smirnov test. An analysis of variance and Tukey test with a P