121 Effect of pentoxifylline on motility of good- and poor-quality frozen-thawed equine sperm

Abstract

Equine semen used for intracytoplasmic sperm injection (ICSI) is typically frozen-thawed and may be of poor quality. To prepare sperm for ICSI, semen is typically centrifuged to remove freezing extender. However, centrifugation can cause damage to sperm, which is especially meaningful if sperm quality is already poor. We evaluated a method for selection of sperm without centrifugation, using a “swim-over” technique, and assessed the effect of pentoxifylline, a phosphodiesterase inhibitor that increases sperm motility in other species. To mimic poor-quality semen, we thawed frozen semen (1×) and re-froze it three additional times (4×). Aliquots (0.25 µL; 50,000 sperm) of 1× or 4× semen were placed at the bottom of the right leg of an “H,” made using 15 µL of medium by tracing a template placed below a Petri dish. The medium used (Hanks’ balanced salt solution with 40 mg mL BSA and added lactate and pyruvate) contained different concentrations of pentoxifylline (0, 0.5, 1, 2 or 4 mg mL⁻¹). One µL of medium was removed from the tip of the left arm of the H after 15 and 30 min incubation, and the number of sperm were counted. In a second study, we evaluated the effect of pentoxifylline on sperm motility parameters using computer-assisted sperm motility analysis. After thawing, 1× and 4× semen was washed to remove freezing extender and resuspended in the same medium but with 7 mg mL⁻¹ bovine serum albumin (BSA), containing the different pentoxifylline concentrations. In Study 1, the number of collected sperm did not differ significantly for 1× sperm exposed to 0 to 4 mg mL⁻¹ pentoxifylline (means of 15 to 23 sperm at 15 min, and 18 to 25 sperm at 30 min). Similarly, in 4× frozen semen, there was no significant difference in number of collected sperm between 0 mg mL⁻¹ and 2 or 4 mg mL⁻¹ pentoxifylline concentrations (<1 to 6 at 15 min; 5 to 6 at 30 min). In Study 2, at 0 min, % total motility was significantly higher in 1 and 2 mg mL⁻¹ pentoxifylline than in 0 mg mL⁻¹ for 1× sperm (47.8 ± 1.7 and 49.3 ± 1.9, vs. 32.1 ± 3.9, respectively; P = 0.018) and significantly higher for 1, 2, and 4 mg mL⁻¹ pentoxifylline than for 0 mg mL⁻¹ for 4× sperm (3.9 ± 0.9, 5.7 ± 0.4, and 8.2 ± 0.5, vs. 1.2 ± 0.4; P = 0.0001). Similar results were found at 15 and 30 min for 1×, and at 15 min for 4×. Pentoxifylline at 1 to 4 mg mL⁻¹ significantly increased the percentage of progressive motility in 1× sperm at 30 min (17.8 ± 1.3, 21.8 ± 2.7, and 20.3 ± 1.2, vs. 10.0 ± 0.4; P = 0.002) and, at 4 mg mL⁻¹, increased the percentage of progressive motility in 4× sperm at 0 min (1.43 ± 0.1 vs. 0.2 ± 0.1; P = 0.005) and 15 min (1.4 ± 0.2 vs. 0.1 ± 0.0; P = 0.0001). Exposure of poor-quality semen to pentoxifylline at 4 mg mL⁻¹ improved total and progressive motility but did not increase the recovery of motile sperm in a swim-over collection preparation.