Effects of extender and packaging method on morphological and functional characteristics of cryopreserved Ossimi ram semen

Objective: To test the effects of extenders and packaging methods on morphological and functional characteristics of frozen thawed Ossimi ram semen.Methods: Ram semen was pooled,diluted in 3 different extenders:Tris-egg yolk(TEY),Tris-soybean lecithin(TSBL),and Tris-butylatedhydroxytoluene(TBHT),equilibrated at 5 ℃ for 4 h,and packaged in straws or pellets for freezing.Semen was evaluated for sperm progressive motility,viability,abnormality,and membrane integrity after dilution,equilibration and thawing.The percentages of viable,early apoptotic,apoptotic,and necrotic spermatozoa as well as comet assay parameters were determined in post-thawed semen.Total antioxidants capacity,malondialdehyde and lactic dehydrogenase were assayed in thawed seminal plasma.Results: After equilibration,only sperm membrane integrity was significantly higher(P<0.05)in TEY and TSBL than in TBHT.After thawing,TEY or TSBL in straws significantly improved sperm progressive motility and vitality(P<0.05).In thawed seminal plasma,TBHT or TSBL in straws and TBHT in pellets significantly reduced malondialdehyde,and TBHT in pellets significantly increased lactic dehydrogenase(P<0.05).TEY in straws increased viable sperm,while significantly decreased early apoptotic and apoptotic sperm(P<0.05).DNA damage was significantly decreased(P<0.05)in straws with TEY and TSBL,and tail moment decreased in straws with all extenders(P<0.05).Conclusions: Despite the disadvantages of dilution of cryopreserved semen with egg yolk,ram semen cryopreserved with TEY gives the best physical,morphological and functional characteristics in straws compared with pellets,followed by semen diluted with TSBL.However,semen diluted with TBHT or TSBL,regardless of packaging method,showed the highest impact on antioxidant status of cyopreserved ram semen.