319 USE OF PENTOXIFYLLINE AND HYALURONIC ACID FOR STALLION SPERM SEPARATION

Abstract

In this study, we reduced the volume of separating media and the time of centrifugation to increase the yield of viable sperm for ICSI when few sperm are available. Hyaluronic acid (HA) was used successfully in combination with swim-up for separating bull spermatozoa (Shamsuddin and Rodriguez-Martinez 1994 Anim. Reprod. Sci. 36, 61–75). The purpose of this study was to compare the effectiveness of mini-Percoll and swim-up for low numbers of sperm treated or non-treated with HA or pentoxifylline (PX), a molecule that stimulates sperm motility (Gradil and Ball 2000 Theriogenology 54, 1041–1047). Poor-to-medium quality frozen semen (0.5-mL straws, 200 million cells/mL) from 3 stallions was used 3 times each. Two straws were thawed and mixed. Aliquots of 100 μL semen were allocated to 7 treatments. Three aliquots were incubated at 38°C for 20 min before Percoll-centrifugation (P-NT: non-treated; P-PX: 3.5 mM PX; or P-HA: 1 mg/mL HA, each in 0.25 mL chemically defined handling medium (HCDM)); one aliquot was centrifuged through Percoll without incubation (P-CON: control). Our mini-Percoll was a two-layer gradient (0.4 mL 90% and 0.5 mL 45% Percoll) in a 1.5-mL microcentrifuge tube that was centrifuged at 600g for 5 min. Then a 30-μL pellet was washed in HCDM at 300g for 5 min. At the same time, 3 aliquots of 100 μL sperm were placed in 1 mL HCDM for swim-up (S-NT: non-treated; S-PX: 3.5 mM PX; or S-HA: 1 mg/mL HA) for 30 min. A 0.65-mL aliquot of supernatant was centrifuged in HCDM at 300g for 5 min. In every treatment, the final 30-μL pellet aspirated from the bottom was evaluated. Recovery rate was determined using a hemacytometer. Sperm head, tail and acrosome membrane integrity were evaluated with Kovacs-Foote staining (Kovacs and Foote 1992 Biotech. Histochem. 67, 119–124) counting 300 sperm/sample. Cells were classified into 5 categories: intact head, tail and acrosome membrane (Intact); intact head, tail, damaged acrosome (IHITDA); intact head, damaged tail; damaged head, intact tail; damaged head, tail, acrosome. P-CON and P-PX resulted in more intact sperm compared to all swim-ups. IHITDA was nonsignificantly (P > 0.05) higher in P-PX than in P-CON (17 vs. 11%). Better recovery rates were found in P-CON, P-PX, and S-HA than in P-NT, P-HA, S-NT and S-PX. Hyaluronic acid increased recovery rate during swim-up, but not viability in any of the treatments. Pentoxifylline is beneficial if Percoll separation is delayed, but clarification of its effect on acrosome exocytosis is needed. Table 1. Intact and recovery rates (LS means ± SE)