Cryoprotective capacity on ram spermatozoa of vitamin e and spermine combined with trehalose in hyperosmotic conditions

LÓPEZ ARMENGOL, M.F.; VENTURINO, A.; AMBROSI, C.P.; AISEN, E.G.

Tours

Congreso; Association of Applied Animal Andrology (AAAA). 10TH Biennial Meeting; 2016

Association for Applied Animal Andrology

Cryopreservation produces physical and chemicalstress on the sperm membrane, associated to oxidative processes and ROSgenerated by dead spermatozoa and molecular oxygen (Bucak et al, Small RuminantRes, 75:128, 2008). Trehalose (Tre) stabilizes the plasma membrane, increasingits fluidity after thawing. Spermine (Spm) is a polyamine with antioxidant andstabilizing properties, preventing premature capacitation. Vitamin E (VitE) ispresent in the sperm cells, being a chain breaking antioxidant in membranes (Lenziet al, Front Biosci 5:1, 2000).The aim of this study was to evaluatethe cryoprotective capacity on ram spermatozoa of vitE and Spm combined withtrehalose.Ejaculates from five Australian Merino ramswere pooled. Semen samples were diluted with six extenders: Tris-citricacid-glucose-glycerol-egg yolk extender (B), B + Spm 0,1 mM + VitE 0,1 mM(BSV), B + Tre 100 mM (T), T + Spm 0,1 mM (TS), T + VitE 0,1 mM (TV) and T +Spm 0,1 mM + ViTE 0,1 mM (TSV). After freezing-thawing, sperm cell motility(MOT), acrosome integrity (ACR), viability (Eo-Ni), hypoosmotic swelling test (HOST),4 hours post-thawing resistance (TR-MOT) and GSH content were determined.The parameters were analyzed with ANOVA (maineffects) and Fisher ?LSD post hoctests (StatSoft, Inc., 2007).Spermine improved (p<0.039) MOT (50.45 ±1.90% vs. 43.03 ± 1.92%, with and without Spm, respectively), showing highestvalues when VitE was present (52.27 ± 3.53%). Viability showed the highest valuewhen Tre was included (60.11 ± 0.95% vs. 54.86 ± 1.41%, with and without Tre).The parameters ACR and TR-MOT were higher when Tre was present, independentlyof the antioxidants added. HOST did not show significant differences. The GSHcontent was higher (p<0.003) in the diluents containing Tre (36.91 ± 6.02nmol/109 sperm cells vs. 10.95 ± 3.14 nmol/109 spermcells, with and without Tre), with a tendency to be higher when Spm and VitE werepresent (49.94 ± 11.32 nmol/109 sperm cells).In conclusion, Tre plus Spm or VitE improvemorpho-phisyological parameters of ram spermatozoa, indicating an important effecton plasma membrane stabilization, protecting the sperm cells from oxidativestress.