Effect of FertTalp addition to stallion semen on kinetic parameters and production of intracellular hydrogen peroxide after thawing

Abstract

Motility stimulants increase sperm energy production and their addition after semen thawing will increase motility (Stephens et al., J. Equine Vet. Sci. 2013;33:615-621), and stimulate metabolic pathways responsible for sperm capacitation (Guasti et al., Anim. Reprod. Sci. 2017;179:27-34). Semen of 12 Mangalarga Marchador stallions was cryopreserved with addition of different concentrations of cholesterol-loaded cyclodextrin (CLC): G1 (control – without cholesterol), G2 (1mg of cholesterol), G3 (1.5mg of cholesterol) and G4 (2mg of cholesterol). To run the experiment, two straws from each stallion per experimental group were thawed and divided into two aliquots of 400µL. This was supplemented with 100µL of Fert Talp, a Tyrode's albumin lactate pyruvate medium with (FT) or without (No-FT) heparin, a motility stimulant. This subdivided the four groups into eight. Sperm kinetics were performed analyzed by CASA (Sperm Class Analyzer, SCA® v.4.0 Microptic®). Sperm longevity was analyzed by using a thermoresistance test at 37°C (TTR). Statistical analysis was done by ANOVA F-test. Complementary to sperm kinetics, the production of intracellular hydrogen peroxide was estimated by flow cytometry using Dichlorodihydrofluorescein diacetate (DCFDA®). Results were classified as: Q1 [Plasma membrane (PM) injured without intracellular hydrogen peroxide]; Q2 (PM injured with intracellularhydrogen peroxide); Q3 (intact MP with intracellular hydrogen peroxide); Q4 (intact PM without intracellular hydrogen peroxide) and Tukey test was performed for statistical analysis. Immediately after thawing, there was no difference between FT and No-FT (p>0.05). After incubation at 37°C, the addition of FT showed higher values for RAP (percentage of rapid sperm, %), VAP (average path velocity, μm/s) and LIN (linearity of the curvilinear trajectory, %) after 80 minutes of incubation and also superior rates of RAP, VSL (straight line velocity, μm/s), VAP and LIN after 120 minutes of incubation (p<0.05), demonstrating satisfactory results both with regard to sperm kinetics and longevity. It can be concluded that the use of FT was beneficial for sperm velocity. These findings suggest that the addition of this motility stimulant after thawing may improve fertility because sperm velocity – VCL (curvilinear velocity, μm/s), VSL and VAP are the most reliable parameters to assess sperm quality (Verstegen et al. Theriogenology, 2002;57:149-179). Significant differences were also observed for Q4 (intact PM, without hydrogen peroxide), where the G2 FT group demonstrated superiority when compared to all groups not supplemented with FT (p>0.05), providing satisfactory plasmatic membrane results against oxidative stress.