High concentrations of resveratrol in boar insemination doses is harmful to total motility, plasma and acrosomal membranes integrity and mitochondrial membrane potential

The cellular metabolism generates reactive oxygen species (ROSs) which are responsible for causing oxidative stress; which, in turn is a deleterious processes resulting from an imbalance between the excessive formation of ROS and limited antioxidant defenses. Oxidative stress impairs sperm motility and integrity of plasma and acrosomal membrane. In this context, Resveratrol (RVT) polyphenol compost with an intense capacity to inhibit ROS formation has been used to improve sperm motility of different animals species. The current study was performed to evaluate whether the addition of RVT would improve sperm motility, simultaneous integrity of plasma and acrosomal membranes and mitochondrial membrane potential of boar sperm cooled at 17˚C for 72 h. Thus, six ejaculates from six boars (n=36) were collected. After raw semen analysis, samples were extended in BTS medium and RVT was added, giving rise to the following treatments: T0 (BTS + RVT 0 mM) without RVT, T1 (BTS + RVT 0.01 mM), T2 (BTS + RVT 0.1 mM) and T3 (BTS + RVT 1 mM). Treatments were cooled at 17oC and were evaluated at 0, 24, 48 and 72 h to total motility (SCA- Microptic, Microptic S.L., Barcelona, Spain), membrane mitochondrial membrane potential and simultaneous integrity of plasma and acrosomal membranes by flow cytometry. There was interaction between time and treatment (p < 0.05) to all variables analyzed. Addition of 1 mM of RVT in boar insemination doses was deleterious for the total motility in all times of evaluation (78.17 ± 1.50a ; 77.50 ± 1.57a ; 75.90 ± 1.50a ; 5.19 ± 0.82; percent of motile sperm for T0, T1, T2 and T3, respectively), and to mitochondrial membrane potential, the highest concentration of RVT (1mM) decrease JC-1 fluorescence intensity only after 24 and 48 hours of incubations at 17oC (0 hours: 29.50 ± 1.70; 30.65 ± 1.37; 31.44 ±1.66; 33.30 ± 1.41 / 24 hours: 29.61 ± 1.39; 32.85 ± 1.53; 29.49 ± 1.42; 22.46 ± 1.11 / 48 hours: 28.57 ± 1.12; 30.31 ± 0.99; 27.15 ± 0.77; 21.76 ± 1.17 / 72 hours: 31.09 ± 1.43; 32.66 ± 1.38; 28.43 ± 1.68; 27.10 ± 1.84; mean fluorescence intensity x103 [arbitrary units] for T0, T1, T2 and T3, respectively for all times assessed). The highest concentration of RVT (1mM) was not deleterious (p < 0.05) to plasma and acrosomal membranes integrity compared to absence of RVT at 0 hours of incubation, but it was harmful to this characteristics compared to lower concentrations (85.98 ± 1.78ab; 88.26 ± 1.64a ; 88.35 ± 1.66; 80.86 ± 1.88b ; percentage of membrane integrity for T0, T1, T2 and T3, respectively). Insemination doses with 1 mM of RVT showed lower percentages of simultaneous plasma and acrosomal membrane integrity in the others three times of incubation, compared with others treatments (24 hours: 86.69 ± 1.58a ; 88.54 ± 1.42a ; 88.65 ± 1.44a ; 70.13 ± 2.58b ; T0, T1, T2 and T3, respectively / 48 hours: 87.25 ± 1.61a ; 88.24 ± 1.37a ; 87.79 ± 1.63a ; 56.08 ± 2.64b ; T0, T1, T2 and T3, respectively / 72 hours: 85.17 ± 1.71a ; 85.87 ± 1.73a ; 86.75 ± 1.53a ; 59.73 ± 3.56b ; T0, T1, T2 and T3, respectively). In conclusion, concentrations higher than 1mM of RVT in boar insemination doses was deleterious to mitochondrial membrane potential resulting in decrease of total motility, and also reduce integrity of plasma and acrosomal membrane. However, additional studies with RVT in boar insemination doses are necessary to understand the effect of lower concentrations of RVT.