Using Resveratrol and Epigallocatechin-3-Gallate to Improve Cryopreservation of Stallion Spermatozoa With Low Quality

다국어 초록 (Multilingual Abstract)

<P><B>Abstract</B></P> <P>Reactive oxygen species levels in semen with low quality increased from 25% to about 40%. Resveratrol (Res) is a natural grape-derived phytoalexin, and epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea (<I>Camellia sinensis</I>); both molecules are known to have high levels of antioxidant activity. The aim of this study was to investigate the effects of this antioxidant's supplementation on cryopreservation of stallion semen with poor quality. Experiment 1: Experiment design to asses stallions semen quality (n = 3). Arabian stallions were managed for breeding with fresh, extended, or chilled semen during 2 years (in two breeding season) with a resulting per cycle pregnancy rate (103/41 estrus cycle/conception) of 39.80%. A total of 81 ejaculates were collected and evaluated in fresh condition (total and progressive motility, membrane integrity, acrosome defects, morphology, and abnormal DNA condensation). Evaluation of several ejaculates revealed that sperm production and semen quality mostly did not change during the breeding season, maintaining normal sperm production with poor quality. Experiment 2: 18 ejaculates, collected with Missouri model artificial vagina from Arabian stallion (n = 3). Extended semen was centrifuged at 600 × g for 10 minutes. To eliminate individual stallion variability, the resulting sperm pellets were pooled and divided in seven different treatments in a final concentration of 100 × 10<SUP>6</SUP> sperm/mL by using semiskimmed milk extender containing no supplements (control), and the following supplements with three different concentrations: Res 5, 10, and 20 μM (Res 1, Res 2, Res 3, respectively) and EGCG 25, 50, 100 μM (EGCG 1, EGCG 2, and EGCG 3, respectively). After thawing, all samples were maintained at 37°C for 10 minutes. Evaluation of motility, plasma membrane integrity, viability, mitochondria activity, DNA fragmentation, and lipid peroxidation was determined. For all semen parameters, the lower concentration of Res (5 μM) and EGCG (25 μM) did not show significant differences compared to control (<I>P</I> > .05). Relative to the control group, Res 2 had better (<I>P</I> < .05) postthaw total and progressive motility, yet the higher concentrations (Res 3) showed a negative effect on total motility. Percentage of membrane integrity, viability, and mitochondrial activity was significantly greater in Res 2 and EGCG 2 extenders in comparison with the control group, respectively (<I>P</I> < .05). Results showed that malondialdehyde concentration was lower in Res 2 and EGCG 2 groups. In conclusion, the present study showed that the antioxidant Res prevents DNA damages in our stallions. The addition of Res and EGCG before cryopreservation can ameliorate mitochondrial activity in stallions with low-quality semen. Although further studies are needed, the present work showed that Res and EGCG could be considered as cryopreservation procedures in stallions with low-quality sperm and possibly equine, to avoid or minimize DNA damages, and preserve sperm plasma membrane integrity and mitochondrial activity.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The aim of this study was to investigate the effects of resveratrol (Res) and epigallocatechin-3-gallate (EGCG) supplementation on cryopreservation of stallion (n = 3) spermatozoa with poor quality. </LI> <LI> Five micromolar of Res and 50 μM EGCG in semiskimmed milk extender before cryopreservation can ameliorate total and progressive motility, percentage of membrane integrity, and viability in stallions with low quality. </LI> <LI> In conclusion, the present study showed that the 5 μM Res could prevent DNA damages in subfertile stallions. </LI> <LI> Although further studies are needed, the present work showed that Res and EGCG could be considered as cryopreservation procedures in stallions with low-quality sperm and possibly equine, to avoid or minimize DNA damag