Effect of quercetin or butylated hydroxytoluene on cooled or frozen-thawed ram sperm quality

Cooling and freezing processes cause physical and chemical damage to sperm by cold shock and oxidative stress. This study aimed to evaluate the effect of two antioxidants on sperm parameters of cooled and frozen-thawed ram semen diluted in an egg yolk-based extender. Semen was collected from 30 rams and processed in two consecutive experiments to test the inclusion of different concentrations of quercetin and butylated hydroxytoluene (BHT) in an egg yolk-based semen extender. Dimethyl sulfoxide (DMSO) was added as a solvent to the semen extender in a ratio of 1 mL DMSO for 90 mg of quercetin and 1 mL DMSO for 880 mg of BHT. After collection, semen was diluted at 200 × 106 motile sperm/mL (control) and split into different groups in each experiment. In experiment 1, semen was diluted with the extender containing quercetin (Q5, 5 μg/mL; Q10, 10 μg/mL; Q15, 15 μg/mL) or DMSO alone (DMSO1, 0.055 μL DMSO per mL; DMSO2, 0.165 μL DMSO per mL). In experiment 2, semen was diluted with the extender with BHT (BHT1, 0.5 μg/mL; BHT2, 1 μg/mL; BHT3, 1.5 μg/mL) or DMSO alone (DMSO3, 0.375 μL DMSO per mL; DMSO4, 1.125 μL DMSO per mL). After dilution, the semen was divided into two aliquots. Treated ram sperm samples were also subjected to different storage methods. The first set of samples was cooled at 5 °C for 24 h, whereas the second set of samples was frozen-thawed. Sperm motility parameters and plasma membrane integrity (PMI) were evaluated immediately after dilution (0h) and 24 h after cooling and in the frozen-thawed samples via computer-assisted sperm analysis and epifluorescence microscopy, respectively. The inclusion of quercetin or BHT did not affect sperm motility parameters or PMI of fresh, cooled, or frozen-thawed sperm in this study (P < 0.05). However, further studies are needed to test the effects of these antioxidants on the fertility of cryopreserved ram semen.

The antioxidant roles of L-carnitine and N-acetyl cysteine against oxidative stress on human sperm functional parameters during vitrification

Objective: Amino acids can protect sperm structure in cryopreservation due to their antioxidant properties. Therefore, the present study aimed to investigate the protective effect of L-carnitine (LC) and N-acetyl cysteine (NAC) on motility parameters, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), DNA damage, and human sperm intracellular reactive oxygen species (ROS) during vitrification. Methods: Twenty normal human sperm samples were examined. Each sample was divided into six equal groups: LC (1 and 10 mM), NAC (5 and 10 mM), and cryopreserved and fresh control groups. Results: The groups treated with LC and NAC showed favorable findings in terms of motility parameters, DNA damage, and MMP. Significantly higher levels of intracellular ROS were observed in all cryopreserved groups than in the fresh group (p≤0.05). The presence of LC and NAC at both concentrations caused an increase in PMI, MMP, and progressive motility parameters, as well as a significant reduction in intracellular ROS compared to the control group (p≤0.05). The concentrations of the amino acids did not show any significant effect.Conclusion: LAC and NAC are promising as potential additives in sperm cryopreservation.

Effect of Glutathione on Sperm Quality in Guanzhong Dairy Goat Sperm During Cryopreservation

In the process of cryopreservation of dairy goat semen, it will face many threats such as oxidative damage, which will affect the motility and plasma membrane function of sperm. As an endogenous antioxidant in animals, glutathione (GSH) can significantly improve the quality of thawed sperm when added to the frozen diluent of semen of pigs and cattle. In this study, different concentration gradients of GSH [0 mmol/L (control), 1, 2, 3, 4 mmol/L] were added to the frozen diluent of Guanzhong dairy goat semen. By detecting the sperm motility parameters, acrosome intact rate and plasma membrane intact rate after thawing, the effect of GSH on the cryopreservation of dairy goat semen was explored. Sperm motility parameters were measured with the computer-aided sperm analysis (CASA) system (total power, TM; forward power, PM; linearity, LIN; average path speed, VAP; straight line speed, VSL; curve speed, VCL; beat cross frequency, BCF). The sperm acrosome integrity rate after thawing was detected by a specific fluorescent probe (isothiocyanate-labeled peanut agglutinin, FITC-PNA), and the sperm plasma membrane integrity rate after thawing was detected by the hypotonic sperm swelling (HOST) method. Reactive oxygen species (ROS) kit, malondialdehyde (MDA) kit, superoxide dismutase (SOD) kit, glutathione peroxidase (GSH-PX) kit were used to detect various antioxidant indicators of thawed sperm. in vitro fertilization experiment was used to verify the effect of adding glutathione on sperm fertilization and embryo development. The results showed that when the concentration of glutathione was 2 mmol/l, the sperm viability, plasma membrane intact rate, and acrosome intact rate were the highest after thawing, reaching 62.14, 37.62, and 70.87% respectively, and they were all significantly higher. In terms of antioxidant indexes; the values of SOD and GSH-PX were 212.60 U/ml and 125.04 U/L, respectively, which were significantly higher than those of the control group; The values of ROS and MDA were 363.05 U/ml and 7.02 nmol/L, respectively, which were significantly lower than the control group. The addition of 2 mmol/L glutathione significantly improves the fertilization ability of sperm. In short, adding 2 mmol/l glutathione to the semen diluent can improve the quality of frozen Guanzhong dairy goat sperm.

In vitro effect of ferrous sulphate on bovine spermatozoa motility parameters, viability and Annexin V-labeled membrane changes

The aim of this study was to assess the dose- and time-dependent in vitro effects of ferrous sulphate (FeSO4.7H2O) on the motility parameters, viability, structural and functional activity of bovine spermatozoa. Spermatozoa motility parameters were determined after exposure to concentrations (3.90, 7.80, 15.60, 31.20, 62.50, 125, 250, 500 and 1000 μM) of FeSO4.7H2O using the SpermVisionTM CASA (Computer Assisted Semen Analyzer) system in different time periods. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, and the Annexin V-Fluos was applied to detect the membrane integrity of spermatozoa. The initial spermatozoa motility showed increased average values at all experimental concentrations compared to the control group (culture medium without FeSO4.7H2O). After 2 h, FeSO4.7H2O stimulated the overall percentage of spermatozoa motility at the concentrations of ≤ 125 μM. However, experimental administration of 250 μM of FeSO4.7H2O significantly (P < 0.001) decreased the spermatozoa motility but had no negative effect on the cell viability (P < 0.05) (Time 2 h). The lowest viability was noted after the addition of ≥ 500 μM of FeSO4.7H2O (P < 0.001). The concentrations of ≤ 62.50 μM of FeSO4.7H2O markedly stimulated (P < 0.001) spermatozoa activity after 24 h of exposure, while at high concentrations of ≥ 500 μM of FeSO4.7H2O the overall percentage of spermatozoa motility was significantly inhibited (P < 0.001) and it elicited cytotoxic action. Fluorescence analysis confirmed that spermatozoa incubated with higher concentrations (≥ 500 μM) of FeSO4.7H2O displayed apoptotic changes, as detected in head membrane (acrosomal part) and mitochondrial portion of spermatozoa. Moreover, the highest concentration and the longest time of exposure (1000 μM of FeSO4.7H2O; Time 6 h) induced even necrotic alterations to spermatozoa. These results suggest that high concentrations of FeSO4.7H2O are able to induce toxic effects on the structure and function of spermatozoa, while low concentrations may have the positive effect on the fertilization potential of spermatozoa.

Cryobank of Mediterranean Brown Trout Semen: Evaluation of the Use of Frozen Semen up to Six Hours Post-Collection

The aim of this study was to evaluate the effects of different cold-storage time intervals between collection and semen-freezing on both fresh and cryopreserved semen motility parameters and the post-thaw fertilizing ability of Mediterranean brown trout semen. The ejaculates were split into six aliquots and stored on ice from 1 to 6 h, until freezing. Fresh and post-thaw sperm motility was evaluated by a Computer-Assisted Sperm Analysis system, whilst the fertilizing ability was assessed by in vivo trials. In fresh semen, at 3 h of storage, a significant decrease of total motility, linear movement (STR, LIN) and beat cross frequency (BCF) was recorded, whilst the amplitude of lateral displacement of the spermatozoon head (ALH) underwent a significant increase. In frozen semen, no significant difference was observed for all the motility parameters evaluated, except for the total motility between 1 and 6 h of storage and the duration of sperm movement between 1 and 5 h. Cold-storage time did not significantly affect the percentage of live embryos following the use of frozen semen. In conclusion, our results showed that, if necessary, the Mediterranean brown trout semen can be frozen even until 6 h post-collection without losing its fertilizing ability.

O-152 Investigation of the relationship of sperm motility and Kisspeptin in subfertile men

Study question Does kisspeptin administration affect the motility parameters in sperm samples of subfertile cases? Summary answer Kisspeptin administration significantly increased gene expression levels related with sperm motility as well as intracellular calcium concentrations. What is known already Sperm motility problems are among the most important causes of male infertility. In recent years, a peptide named kisspeptin has been discovered that may have effects on sperm motility. Kisspeptin is known to trigger calcium release in hypothalamic neurons. In addition, kisspeptin administration increased sperm progressive motility in studies conducted on normozoospermic individuals. Furthermore, it is suggested that kisspeptin protein in seminal plasma is positively associated with semen quality. However, there is no evidence that how kisspeptin can affect sperm in men with infertility problems. Study design, size, duration This basic research study was an in vitro experimental approach involving the use of semen samples from an infertil cases between September to December in 2020. 40 men were included in both control and experimental groups. Participants/materials, setting, methods All analyses were performed on semen samples from 10 normozoospermic (NZ), 10 asthenozoospermic (AZ), 10 oligoasthenozoospermic (OAZ) and 10 oligoastenoteratozoospermic (OATZ) men, aging between (21-40) years. Basal serum and seminal kisspeptin levels were analyzed by ELISA. Sperm were divided into two groups. Kisspeptin-13 administered in vitro. KISS1, KISS1R, CATSPER1, AKAP4 gene expressions analyzed by qRT-PCR using 2−ΔΔCt algorithm. Intracellular calcium concentration was determined with floresence spectroflurometer and laser scanning confocal microscope. Main results and the role of chance The serum kisspeptin level of NZ was significantly higher than other groups (p &lt; 0.05). The semen kisspeptin level was significantly higher than OAZ and OATZ (p &lt; 0.05), but not in NZ (p &gt; 0.05). Also, KISS1 gene expression was higher in AZ compared to other groups (p &lt; 0.05). Biochemical and gene expression analysis of kisspeptin were consistent with each other. There was a significant increase in the expression of CATSPER1 gene in AZ compared to other groups (p &lt; 0.05). Also, AKAP4 gene expression was significantly higher in OATZ compared to other groups (p &lt; 0.05). No significant difference was documented for the expression of KISS1R (p &gt; 0.05). Intracellular calcium was significantly increased in AZ and NZ after kisspeptin administration. The intracellular calcium increase is consistent with increased CATSPER1 gene expression levels in AZ. Kisspeptin administration may have a significant effect on sperm motility parameters. Limitations, reasons for caution The biochemical and gene expression levels of KISS1 were consistent. However, gene expression was explored at the mRNA level for CATSPER1 and AKAP4. The protein expression analyses of these genes may confirm the results. Also, using kisspeptin antagonists may strength the results of intracellular calcium analysis. Wider implications of the findings Kisspeptin treatment for individuals diagnosed with asthenozoospermia may have therapeutic results. KISS1 quantitation may be a determining factor for the subfertility in routine semen analysis. Trial registration number OMU KAEK 2019/462

P–036 Investigation of the 2100 MHz electromagnetic field effects on sperm CatSper calcium channel

Study question Does electromagnetic field (EMF) effect sperm motility through CatSper calcium channels in rat? Summary answer 2100 MHz EMF may reduce sperm motility by acting on CatSper calcium channels. What is known already EMF exposure has become a serious concern in infertility patients. The effects of EMF through by using mobile phone and laptop have been explored previously, mostly focusing on sperm motility and DNA fragmentation. EMF activates the voltage gated calcium channels and increases calcium concentration. As a result of the EMA exposure, the sperm motility may increase. However, if this happens while sperms are in non-progressive motile phase in the epididymis, it may result with the depletion of limited energy stores. Sperms may become immotile and they can’t move forward in the progressive motile phase in the female reproductive system. Study design, size, duration This basic research study was an in vivo experimental approach involving the use of 50 male rats. Wistar-Albino rats (n = 10) weighing ∼320 –350g were included in each groups. The duration of EMF exposure was 1 hour per day for 28 days. Amlodipine (1 mg/kg, 28 days) was used as a calcium channel blocker. The experiment was held between July to December in 2020. 20 female rats were recruited for mating test. Participants/materials, setting, methods 50 rats were divided into five groups. Group 1; Pure control. Group 2; Sham. Group 3; EMF exposure, Group 4: EMF+Amlodipine, Group 5: Amlodipine positive control. After four weeks of exposure, rats were sacrificed and sperm were collected from cauda epididymis. Sperm motility parameters were analyzed. Intracellular calcium levels were determined with two different method, fluorescence spectrophotometer and laser scanning confocal microscope. Before sacrifice, rats were mated with female rats to evaluate mating ratios. Main results and the role of chance The mating score and live birth rates did not vary significantly among the groups (p &gt; 0.05). The sperm motility (A+B, 47.62±16.92 versus 34.19±14.62) and intracellular calcium levels (2.46±0.20 versus 1.85±0.18) were significantly decreased in the EMF group (p &lt; 0.05). The results of fluorescence spectrophotometer and laser scanning confocal microscopy with fluorescent attachment were consistent with each other. There were no significant differences found among the other groups in terms of investigated parameters. Statistical analysis was performed with Kruskal-Wallis test followed by the Dunn-Bonferroni’s test. Limitations, reasons for caution The Catsper 1, 2, 3, 4 gene expression levels are still under analyses. These gene expression levels will be helpful to understand possible changes of the sperm motility. The determination of other motility related gene expressions may strength the results. Wider implications of the findings: EMF exposure may have a significant effect on sperm motility parameters. Mobile phones carried very close to the reproductive organs may adversely affect the motility of sperm cells due to its emitted radiation levels Trial registration number Not applicable

Could the Storage Time Post-collection Affect the Post-thaw Motility and Fertilizing Ability of Mediterranean Brown Trout Semen?

The aim of this study was to evaluate the effect of different cool storage time intervals between collection and semen freezing on both fresh and cryopreserved semen motility parameters and the post-thaw fertilizing ability of Mediterranean brown trout semen. The ejaculates were split into six aliquots and stored on ice for 1 to 6 hours, until freezing. Fresh and post-thawing sperm motility were evaluated by Computer-Assisted Sperm Analysis system, whilst the fertilizing ability was assessed by in vivo trials. In fresh semen, at 3 h of storage, a significant decrease of total motility, linear movement (STR, LIN) and beat cross frequency was recorded, whilst the amplitude of lateral displacement of the spermatozoon head underwent a significant increase. Velocity parameters (VCL, VAP and VSL) were not affected by the cold storage time, whilst the duration of sperm movement was significantly higher at 1h compared to the other times tested. Freezing procedure overall decreased almost all post-thaw sperm motility parameters, however no significant differences was observed over time, both in term of fast and linear movement. Cool storage time did not significantly affect the percentage of post-thaw eyed embryos. Our results showed that Mediterranean brown trout semen can be stored on ice even up to 6 hours before freezing, without decreasing its post-thawing quality and fertilizing ability.