78 DIFFERENT EXTENDERS TO HARVEST EQUINE EPIDIDYMAL SPERM AND THEIR INFLUENCE ON FREEZABILITY

Abstract

Harvesting and freezing epididymal sperm is a technology that enables the preservation of the gene pool from animals that had died either unexpectedly or because of colic conditions. This technique may also be employed in animals that have to be euthanized because of traumatic injuries. Therefore, the objective of the present study was to improve the process of freezing epididymal sperm using a freezing extender without the prior centrifugation of the samples. Twelve stallions aging between 3 and 6 years and of different breeds were used (Quarter Horse, Mangalarga, and Brazilian Jumping Horse). Stallions were castrated, and the cauda epididymides were isolated from the testis. The connective tissue was carefully dissected, and the cauda epididymides were straightened. A 200-µL pipette tip was attached to a 20-mL syringe, and the cauda epididymides were flushed using 40 mL of either (A) BotuSemen^® (Nidacon, Mölndal, Sweden) or (B) BotuCrio^® (Nidacon). They were then immediately processed at room temperature (25°C). Samples flushed with B were randomly subjected to either of the 2 procedures: B1) directly loaded into 0.5-mL straws or B2) centrifuged at 600g for 10 min, the supernatant was discarded, and the pellet was resuspended with B and loaded into 0.5-mL straws. The straws were kept at 5°C for 20 minutes followed by another 20 min at 6 cm above liquid nitrogen before immersion. After thawing at 46°C for 20 s the samples were analyzed by computer-assisted semen analysis (HTM – IVOS 12) and plasma membrane integrity was assessed using fluorescent probes (carboxyfluorescein diacetate and propidium iodide). Data were analyzed by ANOVA followed by the Tukey test (P < 0.05). No differences were observed for the values of total motility (A: 57.1 ± 12.35, B1: 46.3 ± 10.0, B2: 47.2 ± 12.84), progressive motility (A: 25.5 ± 9.05, B1: 21.7 ± 9.02, B2: 20.7 ± 7.20), percentage of rapids (A: 40.6 ± 15.92, B1: 30.7 ± 10.51, B2: 32.6 ± 12.39), and plasma membrane integrity (A: 47.8 ± 9.56, B1: 45.0 ± 13.81, B2: 41.3 ± 8.74). It was found that the fluid derived from epididymal secretions, which composes seminal plasma, had no influence on sperm parameters, because there was no difference among freezing protocols. Therefore, flushing equine epididymal cauda with B and freezing the samples without centrifuging can be successfully used. Both extenders (A and B) were efficient in protecting epididymal sperm throughout the freezing process.