Background: Cryopreservation of Arabian stallion semen is important in order to improve the function and fertility of frozen/thawed semen in this breed.
Objective: To investigate the effects of centrifugation, type of semen extenders, and type of cryoprotectants on the quality of frozen/thawed Arabian stallion spermatozoa.
Materials and methods: Semen samples collected from four adult Arabian stallions (one ejaculate per week for 10 consecutive weeks) were either processed directly without centrifugation (no centrifugation; NC) or subjected to centrifugation on the gel-free fraction. Centrifugation protocols were divided into six categories; 600 x g for 10 min (C1), 600 x g for 15 min (C2), 900 x g for 10 min (C3), 900 x g for 15 min (C4), 1200 x g for 10 min (C5), or 1200 x g for 15 min (C6) (Experiment 1). Two semen extenders, INRA-82 and modified Kenney's were compared (Experiment 2). Three different cryoprotectants, [namely 5% glycerol, 5% dimethylformamide (DMF) and 2.5% glycerol] plus 2.5% DMF were used (Experiment 3). Following freezing and thawing, motility, viability, plasma membrane integrity, acrosome status and viability index were evaluated.
Results: Centrifugation at 600 x g for 15 min before cryopreservation increased (P< 0.05) sperm motility, viability, membrane integrity and percentage of spermatozoa with intact acrosome compared to other centrifugation protocols. Dilution of Arabian stallion semen with INRA-82 before cryopreservation improved (P< 0.05) sperm quality after freezing and thawing compared to modified Kenney's extender. Supplementation of semen diluent INRA-82 with 5% DMF improved (P< 0.05) the quality of frozen/thawed Arabian stallion spermatozoa compared to 5% glycerol.
Conclusion: These findings suggest that optimized conditions such as centrifugation, types of semen extenders and cryoprotectants play an important role in processing Arabian stallion spermatozoa for cryopreservation.