The functional roles of plant extracts have been investigated for the treatment of various diseases including subfertility. Recent studies have highlighted the benefits of ashwagandha extract (AE) in enhancing sperm production, boosting testosterone levels, and lowering reactive oxygen species (ROS) levels in mammals. The current study is to examine the effects of the addition of AE to liquid boar semen on sperm quality during storage and its potential application in assisted reproductive technology. A hot water extract of ashwagandha was prepared from the dried powder of ashwagandha roots. Boar spermatozoa were stored in Beltsville thawing solution (BTS) at 17℃ for 5 days, with various concentrations of AE (1–50 mg/mL). During storage, motility, viability, acrosomal integrity and ROS of boar spermatozoa were examined. The results have shown that sperm stored in BTS with varying quantities of AE ranging from 1–20 mg/mL exhibited higher motility compared to those without AE (control) or with 50 mg/mL AE for 5 days. Similarly, sperm viability was better maintained in sperm treated with 1–20 mg/mL AE. Moreover, sperm stored in BTS with AE led to significantly higher acrosomal integrity and chromatin stability rates than sperm stored without AE. Notably, intracellular ROS levels significantly decreased in sperm stored in BTS with AE. Particularly, spermatozoa stored at 10 mg/mL AE exhibited an effective reduction in ROS during storage. These findings suggest the potential role of AE as an additive during sperm storage maintains sperm quality and can be used during subfertility treatment in both animals and humans.

Toll-like receptor 4 (TLR4) is known to contribute to the modulation of insulin resistance and systemic inflammation seen in obesity and the metabolic syndrome. The present study was performed to investigate the fertility competence of TLR4 knock out male mice (TLR4 mice) on a high-fat diet (HFD), compared to a normal-chow diet (NCD). The controls included wildtype (WT) mice fed on a HFD or NCD. Six-week-old male mice were fed with either a NCD or HFD for 20 weeks. Body and organ weights, serum levels of glucose, triglycerides and hepatoxicity, sperm quality and spermatogenesis were observed after the sacrifice. Also, randomly selected male mice were mated with virgin female mice after feeding of 19 weeks. The weight of the body and organs increased in WT and TLR4 mice on a HFD compared to those of mice on a NCD. The weights of the reproductive organs did not vary among the treatment groups. The motility and concentration of the epididymal spermatozoa decreased in both WT and TLR4 mice fed a HFD. The pregnancy rate and litter size declined in the HFD-fed WT mice compared to the HFD-fed TLR4 mice. In conclusion, the HFD alters energy and steroid metabolism in mice, which may lead to male reproductive disorders. However, fertility competence was somewhat restored in HFD-fed TLR4 male mice, suggesting that the TLR4 is involved in testis dysfunction due to metabolic imbalance.