Addition of Pentoxifylline to Skim Milk–Based Extender on Frozen-Thawed Equine Sperm

• The addition of pentoxifylline to the extender did not increase progressive motility.
• T The pentoxifylline exert positive effect on sperm velocities, ALH, BCF and STR.
• Skim milk–based extender ensures preservation and maintaining sperm motility for 2 hours.
• Toxic effects of cryoprotectant on acrosome and mitochondria reduce after dilution.

Cryopreservation impairs sperm fertilizing ability by decreasing sperm motility and motion characteristics; therefore, pentoxifylline (PTX) has been used to stimulate sperm motility. We investigated the effects induced by adding PTX to seminal extender on sperm motion characteristics, membranes, and chromatin integrity and sperm morphology of thawed equine sperm. Eight batches from a unique stallion were cryopreserved (200 × 106 spermatozoa per mL), thawed, evaluated, and divided in two aliquots: one of them was diluted (1:2) in skim milk extender, and the other was diluted (1:2) in the same skim milk extender + PTX (7.18 mM). Analyses were done at 5 (T0), 30 (T30), 60 (T60), and 120 (T120) minutes after dilution and incubation. Sperm movement was evaluated using the computer-assisted sperm analysis, integrity of plasma and acrosomal membranes, and mitochondrial membrane potential, using fluorescent probes and chromatin denaturation by toluidine blue. Sperm dilution, without or with PTX, improves progressive motility, acrosome integrity, and mitochondrial function; however, the addition of PTX does not change these. The addition of PTX to skim milk extender increases sperm velocity characteristics, lateral head displacement, and beat-cross frequency but does not affect the motility, cellular membranes, chromatin integrity, and abnormal sperm morphology when used to dilute the frozen-thawed equine sperm.