Effect of storage and single layer centrifugation before cryopreservation on stallion sperm cryosurvival

Abstract

The objectives of this study were to evaluate the effect of a short, cooled storage before cryopreservation onsperm progressive motility (PM) and compare the effect of different centrifugation methods on post-thaw PM ofstored samples. Semen was diluted in chilling extender and aliquoted in 6 protocols: i) Standard centrifugation(SC) followed by freezing; ii) Single Layer Centrifugation (SLC) followed by freezing; iii) Storage for 8 h/5 ◦Cbefore SC; iv) Storage for 8 h/5 ◦C before SLC; v) Storage for 8 h/15 ◦C before SC; and vi) Storage for 8 h/15 ◦Cbefore SLC. PM was assessed before centrifugation, after centrifugation, and post-thawing. Stallions were classifiedas “good freezers” (GF) or “bad freezers” (BF). The PM in samples immediately frozen was greater than inthe stored ones (71.98 ± 14.2, 52.91 ± 17.8, 53.93 ± 18.9 for no storage, 5 ºC storage and 15 ºC storage,respectively) (P˂ 0.0001). There was an effect of storage condition (p ˂ 0.0001), centrifugation method (p ˂0.0001), and freezability (P=0.0016), with an interaction between them (P= 0.0004), on PM after centrifugation.Post-thaw PM was greater in samples treated by SLC than in samples processed by SC, for all storageconditions (p ˂ 0.05). All BF stallions ‘showed post-thaw PM ˂ 30 % when samples were previously stored.Storage at 5 ºC or 15º C for 8 h maintains an appropriate quality in GF stallions. Applying a sperm selectiontechnique as SLC is suggested to improve post-thaw motility, allowing GF straws to be frozen after storage,although BF semen should be prepared by SLC immediately after collection.