Effect of cholesterol inclusion on kinetics, mitochondrial potential and membrane stability of equine spermatozoa after thawing

Cholesterol loss occurs in equine semen during cryopreservation and induces early capacitation and reduced viability in the female reproductive tract (Blanch et al., Reproduction in Domestic Animals. 2017;47(6):959-64). In order to increase cell resistance and longevity, the addition of cholesterol has shown satisfactory results. However, attention to the amount of cholesterol added is important as high concentrations may inhibit sperm capacitation (Contreras et al, Theriogenology. 2022;189:1-10). The object of the present study was to test the effect of cholesterol inclusion on equine semen characteristics, thus, addition of cholesterol-loaded cyclodextrin (CLC) was performed as described by Purdy and Graham (Cryobiology. 2004;48:36-45). Twelve healthy Mangalarga Marchador stallions, aged 4 to 14 years were assigned into four groups for the same ejaculate in a cross-over experimental design: without cholesterol (G1; control) or inclusion of CLC at concentrations of 1mg/120 × 106 sperm per mL (G2), 1.5mg/120 × 106 sperm per mL (G3) and 2mg/120 × 106 sperm per mL (G4).. After centrifugation, the pellet was resuspended in a freezing egg yolk-based extender (Botu-CrioTM, Botupharma, Brazil) at a final concentration of 100 × 106 sperm/mL. Semen samples were packed into 0.5 mL straws. Sperm kinetics were performed using CASA (Sperm Class Analyzer, SCA® v.4.0 Microptic®) and longevity wasevaluated by thermoresistance test at 37°C. For statistical analysis, the ANOVA F-test was performed. Evaluations were carried out at four times: T0 (immediately after thawing); T40 (after 40 minutes); T80 (after 80 minutes); and T120 (after 120 minutes). At T0, mitochondrial potential (probe JC-1, Sigma-Aldrich®) and plasma membrane destabilization (probe M540, Sigma-Aldrich®) were evaluated by flow cytometry (BD FACS Verse TM®) and Tukey test was performed for statistical analysis. At T0, higher total motility (TM) and progressive motility (PM) parameters were observed in G3 than in G1 (p<0.05). Progressive linear velocity (VSL) was higher in G3 when compared to G2 and in G4 (p<0.05). However, no difference was observed between G1 and G3. Mitochondrial potential and plasma membrane stability were similar in all experimental groups at T0 (p>0.05). After incubation at 37°C, at T40, T80 and T120, MT, MP and RAP parameters did not differ among groups. However, parameters related to cellular velocity (VCL, VSL and VAP) were higher in G3 than in G2 at T40 (VCL), T80 (VCL, VSL and VAP) and T120 (VCL and VAP). G4 showed superior VCL and VSL values at T80 than G2 (p<0.05). Therefore, 1.5mg and 2.0mg of CLC inclusion resulted in higher sperm kinetics, in addition it did not interfere with mitochondrial potential and the stability of plasma membrane and may therefore be a good option for cryopreservation of equine semen.