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.

Sirtuin 1 (SIRT1), the most conserved nicotinamide adenine dinucleotide-dependent protein deacetylase, is involved in the regulation of energy metabolism, genomic stability, and development. SIRT1 knockout (SIRT1) mice exhibit decreased energy expenditure and hypersensitivity to a high-fat diet (HFD). SIRT1 deficiency in the testis has also been shown to cause male infertility in animal models. Therefore, the present study was conducted to examine the alteration of the testicular function of SIRT1 mice on HFD. Six-week-old mice were fed ad libitum as wild type (WT) and SIRT1 male mice with either a control diet or with HFD for 32 weeks and then were sacrificed. The levels of biomarkers for hepatotoxicity, sex hormones, and cytokines were analyzed in the serum and blood-testis barrier, and the sperm morphology was examined in the testis and epididymal spermatozoa. Interestingly, an enlargement of seminal vesicles was observed in the SIRT1 mice fed with HFD. A significantly higher level of hepatotoxicity was also seen in these mice. The concentration of serum testosterone increased in HFD-fed SIRT1 mice compared to the controls. The levels of interleukin-1β and TNF-α increased in both HFD-fed WT and SIRT1 mice. In RT-PCR, the m RNA expression of tight junction protein 2 and claudin 3 significantly decreased in HFD-fed SIRT1 compared to those of the controls. Degenerative spermatocytes and spermatids were detected in the HFD-fed SIRT1 mice testicular section. Sperm motility decreased in WT and SIRT1 with HFD feeding, and sperm concentration decreased significantly in WT-HFD and SIRT1 mice with or without HFD feeding. Taken together, HFD can alter energy and steroid metabolism in SIRT1-deficient mice, which can lead to imbalances in motility and production of sperm and testosterone that can result in male reproductive disorders.

Aluminum (Al) is one of the most widely applied metals in various industries. Anthropogenic activities of industrial waste result in increased accumulation of Al in natural water resources and aqueous organisms, leading to increased heavy metal pollution in the environment. This will ultimately associate with health risks to all living beings including humans. The present study addresses the possible toxic effects of Al on the motility and regeneration of planarians, using Dugesia japonica as the experimental animal model. Planarians were exposed to varying concentrations of Al (50–1,200 mg/L) for 1 hr, and subsequently evaluated for their motility, seizure-like behaviors, regeneration and alterations of the cell-organelles in their body. Results of the study exhibit that increasing Al concentrations lead to decreased motility and increased seizure-like behaviors, especially the c-type and head bob behaviors in planarians (p<0.05). The regeneration ability in the tail segments was reduced by increased Al concentrations, and the eyespot and optic nerves were more clearly observed in the control groups than planarians exposed to Al. Organelle analysis revealed morphological abnormalities in the mitochondria, golgi apparatus, endoplasmic reticulum, and cilia in the head region of planarians, as compared to the control groups. Taken together, our results indicate that exposure to Al alters the behavior of planarians and delays the regeneration of amputated body parts. Therefore, we suggest that Al exposure adversely can affect the lives of aquatic organisms, and induce toxicity such as cell abnormalities in animals.

Lead (Pb) is a major heavy metal that augments environmental pollution and is a health risk to living organisms. This study was performed to investigate the effect of lead on characteristics of planarian, Dugesia japonica. Briefly, planarians were cultivated in water containing different concentrations (0–400 mg/L) of Pb, and incubated for varying durations (1, 3, and 5 hr). After termination of the incubation time, motility and seizure-like behaviors (c-like, head-bop, snake-like, and screw-like) of the planarians were counted in fresh water. Results showed that increasing Pb concentration and time resulted in decreased motility of the planarians (p<0.05). Increasing concentrations of Pb also resulted in significant increase in the seizure-like behavioral patterns, in particular the c-like and head-bop behaviors. In order to examine eye regeneration, the head region was cut, and rest of the body was incubated in water in the absence or presence of Pb, until emergence of the eye extrusion. Formation of eye spots was initiated in amputated fragments of the control group (without Pb) on day 4 after incubation, whereas appearance of the eye spots was delayed in planarian exposed to 200 mg/L Pb. Moreover, immunohistochemistry revealed that formation of the optic nerve was delayed in planarians exposed to Pb. Thus, our studies determined that planarians exposed to high concentrations of Pb resulted in decreasing motility and induction of seizure-like behaviors, as well as delayed eye regeneration. Results of the current study therefore validate that exposure to lead has a negative effect on the lifespan of aquatic organisms and can cause disturbance of the nervous system in animals, thereby implying the possibility of threatening health.

This study was undertaken to evaluate the toxic effect of cadmium sulfate (Cds) on planarians (Dugesia japonica), and the suitability of planarians as an alternative animal model for toxicity studies. Planarians were exposed to varying concentrations of experimental solutions containing 0.1–25 mg/L Cds, and incubated for 1, 3, and 5 hrs. Motility, seizure-like behaviors (c-like, head-bop, snake-like, and screw-like) and regeneration ability of the amputated fragment were subsequently evaluated. Results showed decreased motility with increasing concentrations of Cds (p<0.05), with the lowest motility being observed at the highest concentration of 25 mg/L Cds. Results also indicate that seizure-like behavior patterns were significantly affected by increments in the Cds concentrations, especially the c-like and head-bop behaviors were notably increased. Compared to the control, the regeneration ability of the planarians was decreased in the experimental solutions containing Cds. Planarians exposed to Cds showed either delayed eye formation or no eye regeneration during incubation. Moreover, increased concentrations of Cds resulted in failure to regenerate and death of the planarians. In conclusion, this study confirm that the heavy metal Cds exerts a toxic effect on planarians. Furthermore, the performances of the planarians in the experimental period exhibit their suitability as an alternative animal model for toxicity studies.

Cyanobacteria (blue green algae) blooms formed in natural water resources due to the environmental pollution produce toxic compounds as secondary metabolites, causing health hazards to both humans and other living beings. Microcystin is a well-known toxin produced by cynobacteria. The present study was undertaken to evaluate varying concentrations and exposure times of two different forms of microcystin, viz., -LR (MCLR) and -LA (MCLA), on the motility and seizure-like behavior of planarian (Dugesia japonica). Compared to control, reduced motility was observed in both the MCLR or MCLA treated groups, but did not differ significantly with increasing concentrations of microcystin. However, the number of seizure-like behaviors were increased dose-dependently in planarian exposed to MLCR or MCLA. Exposure time to microcystine also affected the motility and seizure-like behaviors of planarians; 24 hrs incubation with MCLR, and 48 and 96 hrs exposure to MCLA, showed significantly (p<0.05) lower motility, as compared to the control. Assessing regeneration of the planarians revealed the simultaneous completion of eye formation at day 9 in planarians incubated in the absence or presence of MCLR or MCLA, thereby indicating that exposure to microcystin has no effect on the process. In conclusion, we determined that exposure to microcystins resulted in decrease in the number of motility, and induced abnormal behavior pattern in planarians. Further studies are required to identify the toxicity of microcystin that affects aquatic ecosystems.

Nicotine is a component of tobacco products and is one of the most commonly abused substances that leads to addiction. Therefore, the present study was performed to investigate the behavioral pattern and toxicity by nicotine exposure in planarians. Basically, planarians were exposed to different concentration of nicotine for 5 min. To investigate detoxification effect, planarians were exposed to nicotine for 5 min, and treated glycyrrhizin for 5 min, then motility and seizure-like behavior were observed for 5 min. As a result, the motility of nicotine-exposed planarians decreased approximately more than 50% compared to freshwater control. However, the motility of glycyrrhizin-exposed planarians recovered than nicotineexposed planarians. In the assessment of seizure-like behavioral pattern, planarians exposed to nicotine showed head-bop or c-like type rather than screw-like or snake-like patterns. However, planarians exposed to glycyrrhizin showed no seizure-like behavior. To examine the oxidative stress response, planarians were cultured in fresh water containing 1 mM nicotine for 1 day. Planarians were homogenized and extracted to assay the contents of reactive oxygen species (ROS), lipid hydroperoxides (LH), glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD). The result showed that a significantly higher level of ROS, LH indicated in planarians exposed to nicotine, on the other hand, glycyrrhizin-exposed planarians were significantly decreased ROS, LH levels. In conclusion, the motility decreased when planarians were exposed to nicotine, in a dose-dependent manner, whereas seizurelike behavior increased. Nicotine induced behavioral disturbances and cell toxicity in planarians were recovered by glycyrrhizin, suggesting a candidate substance for nicotine addiction treatment.

Rubus coreanus is known to have diverse biological properties, such as free radical scavenging activity and antibacterial activity. In the present study, Rubus coreanus leaf and stem extract (RLSE) was used in boar semen preservation whether it has a beneficial effect on assisted reproductive technology (ART) in mammals. Boar spermatozoa were preserved in Beltsville thawing solution (BTS) in the presence of varying concentrations of RLSE (0-10 μg/mL). Sperm motility, sperm viability, and intracellular reactive oxygen species (ROS) levels were examined after 2 days of preservation. The percentage of total motile spermatozoa and progressive motile spermatozoa improved in the spermatozoa preserved with 0.5 μg/mL RLSE. Higher proportions of viable spermatozoa were seen in the presence of 0.5 and 1 μg/mL RLSE than in the control. Intracellular ROS levels decreased when the spermatozoa were preserved in BTS with 0.1–1 μg/mL RLSE. In order to examine the bacterial growth, E. coli was added to liquid semen diluted with antibiotics-free BTS in the presence or absence of RLSE. No anti-bacterial activity of RLSE against E. coli was observed during liquid semen preservation. Although there was no inhibition of E. coli growth, the addition of RLSE might help improve sperm motility and viability during boar semen preservation, suggesting it as a potential reagent for ART in mammals.

This study was carried out to investigate the effects of cryoprotectants, warming solution and removal of lipid on open pulled straw vitrification (OPS) method of porcine embryos produced by nuclear transfer (NT) of fetal fibroblasts. All solutions used during vitrification were prepared with holding medium consisting of 25 mM Hepes buffered TCM199 medium containing 20% fetal bovine serum (FBS) at 38.5℃. The blastocysts derived from NT with or without lipid were vitrified in each medium of different concentrations of dimethyl sulfoxide (DMSO) and ethylene glycol (EG). Also, blastocysts after cryopreservation were warmed into different concentrations of sucrose in warming solution. The optimal concentrations of cryoprotectants in vitrification solution were 10% DMSO + 10% EG in vitrification solution 1 (VS1) and 20% DMSO + 20% EG in vitrification solution 2 (VS2). The optimal concentrations of sucrose were 0.3 M sucrose in warming solution 1 (WS1) and 0.15 M sucrose in warming solution 2 (WS2). Lipid removal from oocytes before NT enhanced the viability of NT embryos after vitrification. Our results show that use of the OPS method in conjunction with lipid removal provides effective cryopreservation of porcine nuclear transfer embryos.

This study was designed to evaluate effects of BSA, PVA, gonadotropins and follicle shell during IVM of porcine oocytes and subsequent development to the blastocyst stage after IVF. Cumulus oocyte complexes (COCs) were cultured in TCM-199 media containing 4 mg/ml BSA and 1 mg/ml PVA during IVM for 44 hr. To compare the effect of gonadotropins on oocyte maturation, COCs were cultured with FSH+LH, FSH, LH and FSH-LH-free media during IVM, respectively. Also, different number of follicle shells (0, 2, 4 and 6) was used to examine whether the presence of follicle shell in culture medium affects oocyte maturation. The percentages of fertilization and blastocyst formation, respectively, were higher in the medium containing the PVA (49.0 and 17.9%) than those containing the BSA (40.0 and 12.2%). Significantly higher rates of MII oocytes were in the presence of FSH+LH and FSH (88.6 and 85.1%) compared to other treatments (64.0 and 53.4% at LH and FSH-LH-free media). Co-culture with inverted follicle shells in 2 ml maturation medium enhanced the developmental competence of porcine oocytes. In conclusion, PVA could be used as a macromolecules instead of BSA, and FSH and follicle shell played important roles in maturation of porcine oocytes.