Sperm Lipid Peroxidation Research Articles

CHANGES IN THE METABOLISM OF NATIVE AND DECONSERVED BULL SPERMATOZOA UNDER THE ACTION OF MOLECULAR HYDROGEN

Background. The process of storing sperm in a deeply frozen state causes structural and functional disorders of spermatozoa, which reduces their fertilizing ability. The main mechanism of the negative effect of cryopreservation on spermatozoa is the development of oxidative stress. Molecular hydrogen has some advantages as a potential antioxidant molecule - it is a selective effect on certain reactive oxygen species, the ability to overcome cell membranes, the absence of toxic effects. Purpose. To study the effect of molecular hydrogen on the functional status of native and deconserved sperm cells of breeding bulls. Materials and methods. The object of the study was the ejaculates of bulls before cryopreservation and after defrosting. The native sperm diluted with the "BioXcell" diluent, the native sperm diluted with the "BioXcell" diluent enriched with molecular hydrogen, the sperm after deep freezing and the sperm after deep freezing pretreated with molecular hydrogen were studied. The intensity of free radical processes and the activity of antioxidant enzymes were determined in spermatozoa. Results. After cryopreservation of sperm, the processes of lipid peroxidation in spermatozoa are significantly activated. The content of malondialdehyde, diene and triene conjugates increases. The use of molecular hydrogen to correct the quality of sperm production after cryopreservation gave positive results. A decrease in the concentration of primary and intermediate products of lipid peroxidation was noted. The activity of superoxide dismutase and catalase in spermatozoa significantly increases. Conclusion. Molecular hydrogen has the potential as a new and effective antioxidant, the widespread use of which is possible for veterinary purposes. Sponsorship information. The study was funded by the Russian Science Foundation grant No. 23-26-00205.

The Effects of Supplementation of the Freezing Extender with Silymarin on the Quality Parameters of Frozen-Thawed Arabian Stallion Sperm: A Preliminary Evaluation.

This study evaluated the effects of supplementation of the freezing extender with different concentrations of silymarin on the quality of frozen-thawed Arabian stallion spermatozoa. Semen samples from three stallions (1, 2, and 3) were suspended in the freezing extender without or with silymarin (0, 25 μg/mL, 50 μg/mL, 75 μg/mL, and 100 μg/mL) and cryopreserved in 0.5 mL straws. After 1 month of storage, the frozen semen samples in straws were thawed and evaluated in terms of viability, mitochondrial membrane potential, kinematic parameters, total and progressive motility, plasma membrane integrity, lipid peroxidation, and DNA fragmentation. The findings indicated that 25-100 μg/mL of silymarin significantly improved viability and mitochondrial membrane potential while reducing the stallion sperm lipid peroxidation, DNA fragmentation, and apoptosis compared with the control group (p < 0.05). Silymarin concentrations of 75 μg/mL and 100 μg/mL significantly increased progressive motility and plasma membrane integrity (p < 0.05). Based on our findings, it can be inferred that silymarin exhibited a dose-dependent enhancement in the frozen-thawed Arabian stallion sperm quality. The most favorable outcomes were observed when 100 μg/mL silymarin was used.

Chiral thioacetyl derivatives of proline as novel potential agents for beluga reproduction

The aim of the research was to study antioxidant activity and protective potential of new phenol-containing thioacetyl derivatives of D- and L-prolines as potential agents on beluga sperm and survival in the early stages of ontogeny of bester (Huso x Acipenser ruthenus) development. The study indicated that both enantiomers demonstrated the equal scavenging activity towards DPPH•, ABTS•+, O2•–, H2O2 and NO, and that the activity towards all of these radicals, with the exception of nitric oxide, exceeded the activity of BHT. No significant differences were registered between the enantiomers in the inhibition of long-term lipid peroxidation of beluga sperm in vitro. The enantiomers revealed the opposite effect on the motility duration time of beluga spermatozoa, the rate of their fertilization, and survival at the early stages of bester ontogeny. Application of the L-proline derivative for the beluga sperm treatment resulted in the increase of the total period of movement of beluga sperm cells, the raise of the fertility of sterlet eggs and the survival of bester offspring at the prelarval stage, which led to an increase in the number of produced larvae. The introduction of the D-proline derivative or BHT decreased the motility duration time of beluga sperm, the fertility of sterlet eggs and the survival of offspring at the prolarval stage, which led to a decrease in the number of obtained larvae.

Oxidative stress on alpaca epididimal sperm frozen with different concentrations of seminal plasma

Objective. To assess the oxidative stress on alpaca epididimal sperm frozen with different concentration of seminal plasma. Materials and methods. 29 post mortem alpaca epididymis were used. Epididymal sperm were obteined thought cuts and suspended in a diluent based on skim milk, egg yolk and fructose and dimethylacetamine. Samples were separated into four aliquots which were supplied with seminal plasma (v/v) in concentrations of 0, 10, 25 and 50% respectively; then, sperm motility was assessed before freezing; then, motility was assessed before freezing with an automatic freezer machine. After thawing, mitochondrial membrane activity, sperm viability, lipid peroxidation and sperm apoptosis were assessed using flow cytometry with images. Results. Sperm exposed to 50% seminal plasma showed the lowest post-thaw motility (3.5±3.7%) and the highest percentages of apoptotic cells (71.43±20.6%). No significant differences were found in the rest of parameters. Conclusions. This study constitutes non-conclusive evidence that presence of seminal plasma does not affect viability, mitochondrial activity, or lipid peroxidation of alpaca epididymal sperm during cryopreservation processes; however, when it is absent or added up to 25% in the cryopreservation process, it may be possible to obtain better results in terms of sperm motility and sperm apoptosis.

In-Vitro Effects of Perfluorooctanoic Acid on Human Sperm Function: What Are the Clinical Consequences?

Background: Lifestyle and environmental pollution harm male fertility. Perfluoroalkyl substances (PFAS) are bio-accumulates in the environment as well as in several human tissues, and one of the most common PFAS is perfluorooctanoic acid (PFOA). Therefore, this study aimed to evaluate the in vitro effects of PFOA with hydrophobic and waterproofing properties on motility and bio-functional sperm parameters. Methods: To accomplish this, 50 healthy men with normozoospermia and not exposed to high doses of PFAS were enrolled. Their spermatozoa were incubated for 3 h with increasing concentrations of PFOA (0, 0.01, 0.1, and 1 mM) to evaluate its effects. In particular, we evaluated the effects of PFOA on total and progressive sperm motility and, by flow cytometry, on the following bio-functional sperm parameters: degree of chromatin compactness, viability, early and late apoptosis, mitochondrial membrane potential, the degree of lipoperoxidation, and concentrations of mitochondrial superoxide anion. Results: The results showed that PFOA decreased both total and progressive sperm motility, impaired chromatin compactness, and increased sperm lipid peroxidation and mitochondrial superoxide anion levels. Conclusions: This study showed that PFOA alters several sperm parameters and thus it may play a negative role in male fertility.

Impact of Seminal Plasma Antioxidants on DNA Fragmentation and Lipid Peroxidation of Frozen-Thawed Horse Sperm.

Cryopreservation is a stressful process for sperm, as it is associated with an increased production of reactive oxygen species (ROS). Elevated ROS levels, which create an imbalance with antioxidant capacity, may result in membrane lipid peroxidation (LPO), protein damage and DNA fragmentation. This study aimed to determine whether the membrane LPO and DNA fragmentation of frozen-thawed horse sperm relies upon antioxidant activity, including enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and paraoxonase type 1 (PON1)); non-enzymatic antioxidant capacity (Trolox-equivalent antioxidant capacity (TEAC), plasma ferric reducing antioxidant capacity (FRAP) and cupric reducing antioxidant capacity (CUPRAC)); and the oxidative stress index (OSI) of their seminal plasma (SP). Based on total motility and plasma membrane integrity (SYBR14+/PI-) after thawing, ejaculates were hierarchically (p < 0.001) clustered into two groups of good- (GFEs) and poor-(PFEs) freezability ejaculates. LPO and DNA fragmentation (global DNA breaks) were higher (p < 0.05) in the PFE group than in the GFE group, with LPO and DNA fragmentation (global DNA breaks) after thawing showing a positive relationship (p < 0.05) with SP OSI levels and ROS production. In addition, sperm motility and membrane integrity after thawing were negatively (p < 0.05) correlated with the activity levels of SP antioxidants (PON1 and TEAC). The present results indicate that LPO and DNA fragmentation in frozen-thawed horse sperm vary between ejaculates. These differences could result from variations in the activity of antioxidants (PON1 and TEAC) and the balance between the oxidant and antioxidant components present in the SP.

Factors affecting the analysis and interpretation of sperm quality in frozen/thawed stallion semen

In the current study, we examined: 1) the agreement (bias) between fluorescence-based methods (NucleoCounter-SP100 [NC] vs. flow cytometry [FC]) for determining the viability (VIAB) of frozen/thawed stallion sperm; 2) the agreement between post-thaw sperm total motility (TMOT) and VIAB; 3) whether a difference between TMOT and VIAB [VIAB – TMOT] in frozen/thawed stallion sperm could be explained by the level of lipid peroxidation in viable sperm (VLPP); 4) the repeatability of post-thaw analysis of sperm quality; and 5) the effect of final post-thaw semen dilution (10, 30, or 50 x 106 sperm/mL) on sperm motion characteristics. Post-thaw VIAB was similar between NC and FC (P > 0.05), and the agreement between these two methods was high (bias: 1 to −3). The agreement between post-thaw TMOT and VIAB decreased as the pre-freeze percentages of morphologically normal sperm and DNA quality decreased: bias – 4 to – 25. The bias between [VIAB – TMOT] and VLPP ranged from – 5 to 7. Differences in post-thaw sperm quality (TMOT, PMOT, VIAB, and sperm concentration) were not observed when analyzing one or three straws per ejaculate (P > 0.05). There was no effect of post-thaw sperm concentration (i.e., 10 vs. 30 vs. 50 x 106 sperm/mL) on sperm motion characteristics (P > 0.05). This study reports factors other than post-thaw sperm motility that warrant further consideration when analyzing frozen/thawed stallion sperm.

Effects of 4G mobile phone radiation exposure on reproductive, hepatic, renal, and hematological parameters of male Wistar rat.

Mobile phones have become a vital part of human life. Due to drastic increase in the number of mobile phone subscribers, exposure to radiofrequency radiation (RFR) emitted from these phones has increased dramatically. Hence, the effect of RFR on humans is an area of concern. This study was performed to determine the impact of 4G mobile phone radiation on the male reproductive system, liver, kidney, and hematological parameters. Seventy-day-old Wistar rats were exposed to 4G radiation (2350MHz for 2h/day for 56days). Sperm parameters such as sperm count, viability, sperm head morphology, mitochondrial activity, total antioxidant activity, and lipid peroxidation of sperm were evaluated. Histopathology of the testis, prostate, epididymis, seminal vesicle, liver, and kidney was carried out. Complete blood count, liver and kidney function tests, and testosterone hormone analysis were done. At the end of the experiment, results showed a significant (p < 0.05) decrease in sperm viability with alterations in the histology of the liver, kidney, testis, and other reproductive organs in the exposed group of rats. A reduced level of testosterone, total antioxidant capacity, and decreased sperm mitochondrial function were also observed in the exposed rats. Moreover, the exposed rats showed an increase in sperm lipid peroxidation and sperm abnormality. Hematological parameters like hemoglobin, red blood cells (RBC), and packed cell volume (PCV) showed a significant (p < 0.05) increase in the exposed rats. The results indicate that chronic exposure to 4G radiation may affect the male reproductive system, hematological system, liver, and kidney of rats.

Evaluation of the effects of hydroxytyrosol on human sperm parameters during cryopreservation

Human sperm cryopreservation is a routine procedure in assisted reproductive technology, but it has detrimental effects on different sperm parameters due to oxidative stress. Our objective was to assess the impacts of hydroxytyrosol (HT), as an antioxidant, on human sperm parameters following cryopreservation. In the first phase, 20 normal human semen samples were cryopreserved using the rapid freezing method with different concentrations of HT including 0, 50, 100, 150, and 200 μg/mL. In the second phase, 20 normal semen samples were collected and cryopreserved with 50 and 100 μg/mL HT. The beneficial effects of HT were determined by evaluation of motility (computer-assisted sperm analysis; CASA), viability (Eosin-nigrosine stain), DNA integrity (sperm chromatic dispersion test, SCD), reactive oxygen species (DCF and DHE staining by flowcytometry) lipid peroxidation (malondialdehyde, MDA test) and mitochondrial membrane potential (JC1 staining by flowcytometry) of sperm after cryopreservation. After thawing, sperm motility had an increasing trend in 50 and 100 μg/mL HT groups in comparison with other groups, althought the difference was not significant. However, sperm viability was significantly increased at 50 and 100 μg/mL HT. Our data also showed that sperm DNA fragmentation was significantly decreased after thawing at 100 μg/mL in comparison with 0 and 50 μg/mL HT. However, the level of intracellular reactive oxygen species, lipid peroxidation and mitochondrial membrane potential were not significantly different between groups. Our results showed that HT may have protective effects on the viability and DNA integrity of human sperm during the freezing-thawing process.

Correlation between in vitro sperm kinetic, oxidative stress assessments and field fertility of cryopreserved bull semen

This study assessed kinetic parameters and oxidative stress in bull sperm after post-thaw (PT) or after sperm selection by Percoll™ gradient, and thermo resistance test (SS + TRT) to identify useful indicators of field fertility. For the experiment, commercial doses of frozen semen were obtained from six Aberdeen Angus bulls. Three of the bulls were classified as high fertility and three as low fertility according to the IFert™ index provided by the international breeding company CRV Lagoa. Pooled semen samples were distributed between two treatment groups for analysis: post-thaw (PT) or sperm selection (SS) (Percoll™) and thermal resistance test (SS + TRT). The samples were evaluated using sperm kinetics (CASA) (motility %, progressive motility %, VCL µm/s, VSL µm/s, VAP µm/s, LIN %, STR % and WOB%), production of reactive oxygen species (ROS), lipid peroxidation, superoxide dismutase (SOD) enzyme activity and total antioxidant capacity. Data were analyzed using Two-Way ANOVA, considering the fertility index, the treatment used in the samples as effects, and the interaction between these factors. When a significant effect was observed, the values were compared using the Bonferroni test. A Pearson Correlation analysis was performed between the fertility indices and the sperm parameters analyzed in vitro, to evaluate the relationship between sperm quality and the fecundity rate obtained by the bulls. Sperm kinetic parameters, including total motility, progressive motile, and beat cross-frequency, were higher in low fertility compared to high fertility bulls (P &lt; 0.05). However, curvilinear velocity was greater in high fertility bulls followed by SS + TRT. Straight-line velocity, average path velocity, linearity, and beat cross-frequency beat were higher in high fertility bulls after SS + TRT. Reactive oxygen species was correlated with fertility after SS. In addition, there was a decrease in lipid peroxidation was observed only in high fertility bulls. However, lipid peroxidation and high fertility were correlated after PT and SS + TRT. The combination of in vitro sperm kinetic parameters predicted in vivo fertility more accurately than individual kinetic parameters. The lipid peroxidation of sperm is an important indicator of fertility in bulls. High fertility bulls appeared to be more susceptible to lipid peroxidation, which was only reduced in high fertility bulls, suggesting that their sperm can repair the damage induced by oxidative stress.

L-cysteine improves boar semen motility at 5 ºC but does not affect the oxidative status

Hypothermic storage has been proposed as a method to reduce bacterial loads and promoting prudent use of antibiotics. Reducing temperature, however, can lead to cold shock damage and oxidative stress in boar semen. This study verified the effect of L-cysteine on the quality of semen stored at 5 °C for 120 h. Twenty-one normospermic ejaculates were diluted in Beltsville Thawing Solution into five treatments: Positive control (Pos_Cont, storage at 17 °C without L-cysteine) and groups with 0, 0.5, 1, and 2 mmol/L of L-cysteine supplementation stored at 5 °C. Variables were analyzed as repeated measures, considering treatment, storage time, and interaction as main factors. The effects of different L-cysteine concentrations were also evaluated using polynomial orthogonal contrasts. Sperm motility and pH were higher in the Pos_Cont compared to the groups stored at 5 °C (P < 0.05). In polynomial orthogonal contrast models, total motility was affected by the interaction between L-cysteine and storage time (P = 0.04), with a linear increase in motility when increasing the amount of L-cysteine at 72 and 120 h. Progressive motility increased quadratically as the L-cysteine reached 1 mmol/L (P < 0.01). In the thermoresistance test at 120 h, sperm motility increased quadratically up to an L-cysteine dose of 1 mmol/L (P < 0.05). Sulfhydryl content linearly increased with L-cysteine supplementation (P = 0.01), with no effect on intracellular ROS and sperm lipid peroxidation (P ≥ 0.06) in 5ºC-stored doses. In conclusion, L-cysteine supplementation has a positive effect on sperm motility up to 120 h of storage at 5 °C.

Оптимизация состава криопротектора для сохранения репродуктивных клеток африканского сома

The article presents a study on the selection of the optimal composition of the protective medium for cryopres-ervation of the reproductive cells of male African catfish. Glycerol, dimethyl sulfoxide (DMSO) at concentrations of 3, 5 and 10% were studied as the main cryoprotective additives. The influence of these compounds on the quality indicators of catfish sperm (the percentage and time of sperm movement after activation), the level of sperm lipid peroxidation, and the activity of the antioxidant enzyme catalase before and after the cryopreservation process was assessed. A decrease in fish breeding indicators of the quality of African catfish sperm (the proportion of living cells, motility time) was established when DMSO was added to the basic cryomedium. The greatest decrease in the percentage of sperm motility before freezing is observed when DMSO is added at a concentration of 10%. A dose-dependent effect of a decrease in sperm activity after freezing was noted with an increase in the addition of DMSO to the cryogenic medium from 3 to 10%. It has been shown that 3% glycerol in a cryogenic medium does not contribute to the preservation of cells during freezing, cryodamage caused by the formation of ice crystals leads to a significant decrease in the motility of catfish sperm. The best indicators of sperm quality were obtained when using glycerol, the most effective concentration is 5% (40% of mobile reproductive cells within 53 s). The addition of 5% glycerol to the cryomedium contributes to the greatest decrease in the level of peroxidation of sperm lipids and an increase in catalase activity, which is fully consistent with the results on sperm motility. The calculated correlation coefficients confirm the dependence of the reproductive qualities of gametes on the antioxidant status of cells. The results indicate the importance of using antioxidants as cryoprotective additives to improve the quality of African catfish sperm breeding and indicate the need for more research to evaluate the effect of adding new potentially effective and safe antioxidants as cryoprotective additives.

Aberrant Expression of TET2 Accounts for DNA Hypomethylation in Varicocele.

Epigenetic modifications such as DNA methylation play a key role in male infertility etiology. This study aimed to explore the global DNA methylation status in testicular spermatogenic cells of varicocele-induced rats and consider their semen quality, with a focus on key epigenetic marks, namely 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC), as well as the mRNA and proteins of ten-eleven translocation (TET) methylcytosine dioxygenases 1-3. In this experimental study, 24 mature male Wistar rats (8 in each group) were assigned amongst the control, sham, and varicocele groups. Sperm quality was assessed, and DNA methylation patterns of testicular spermatogenic cells were investigated using reverse transcription-polymerase chain reaction (RT-PCR), western blot, and immunofluorescence techniques. Sperm parameters, chromatin and DNA integrity were significantly lower, and sperm lipid peroxidation significantly increased in varicocele-induced rats in comparison with control rats. During spermatogenesis in rat testis, 5-mC and 5-hmC epigenetic marks, and TET1-3 mRNA and proteins were expressed. In contrast to the 5-mC fluorescent signal which was presented in all testicular cells, the 5-hmC fluorescent signal was presented exclusively in spermatogonia and a few spermatids. In varicocele-induced rats, the 5-mC signal decreased in all cells within the tubules, whereas a strong signal of 5-hmC was detected in seminiferous tubules compared to the control group. As well, the levels of TET2 mRNA and protein expression were significantly upregulated in varicocele-induced rats in comparison with the control group. Also, our results showed that the varicocele-induced animals exhibited strong fluorescent signals of TET1-3 in testicular cells, whereas weak fluorescent signals were identified in the seminiferous tubules of the control animals. Consequently, we showed TET2 upregulation and the 5-hmC gain at testicular levels are associated with varicocele and sperm quality decline, and therefore they can be exploited as potential biomarkers of spermatogenesis.

Omega 6/Omega 3 Ratio Is High in Individuals with Increased Sperm DNA fragmentation.

An imbalance between omega-6 and omega-3 fatty acids in sperm has been linked with lipid peroxidation and DNA damage in sperm, indicating a possible correlation to fertility potential. This cross-sectional study involved 56 infertile men (aged 25-45), and assessed the relationship between the omega-6 to omega-3 fatty acid ratio in sperm and seminal plasma with sperm DNA fragmentation. Individuals were categorized based on high or low levels of sperm DNA fragmentation according to two tests (TUNEL and SCSA assay less or greater than 10 and 30%, respectively), and their fatty acid composition, as well as sperm functional tests, were analyzed. Results showed that men with high DNA fragmentation exhibited higher percentages of total saturated, monounsaturated, and omega-6 to omega-3 fatty acid ratios in both sperm (P < 0.001) and seminal plasma (P < 0.001) compared to men with low DNA fragmentation. The percentage of sperm lipid peroxidation, and residual histone (P < 0.05) were higher, while the percentage of sperm motility (P < 0.001) was lower in the former compared to the latter group. Moreover, Pearson's correlation revealed positive associations between the omega-6 to omega-3 fatty acid ratio with sperm lipid peroxidation, DNA fragmentation, and residual histones in both sperm and seminal plasma. Overall, these observations suggest that consumption of omega-3 fatty acids may be related to male fertility potential, as it appears that individuals with a high percentage of omega-3 fatty acids have better sperm quality compared to men with a lower omega-3 fatty acid.

Evaluation of sperm and hormonal assessments in Wagyu, Nellore, and Angus bulls.

Wagyu bulls are known to have a highly exacerbated libido, as shown by the intense sexual interest of young calves. Therefore we believe that Wagyu male animals have specialized Sertoli and Leydig cells that are directly involved with the sexual precocity in this breed as mature bulls have a small scrotal circumference. This study aimed to evaluate whether there were differences in the hormone and sperm characteristics of Wagyu bulls compared with the same characteristics of subspecies Bos indicus and Bos taurus sires. Frozen-thawed semen from Wagyu, Nellore, and Angus sires were analyzed for sperm kinetics (computer-assisted sperm analysis), plasma membrane integrity, chromatin integrity, acrosome status, mitochondrial activity, lipid peroxidation and hormone [luteinizing hormone (LH) and testosterone] serum concentration. The results showed that Wagyu had lower total motility and an increased number of sperm with no motility when compared with Nellore and Angus bulls. Wagyu breed did not differ from those breeds when considering plasma and acrosome membranes integrity, mitochondrial potential, chromatin resistance, sperm lipid peroxidation or hormone (LH and testosterone) concentrations. We concluded that Wagyu sires had lower total motility when compared with Nellore and Angus bulls. Wagyu breed did not differ from these breeds when considering plasma and acrosome membranes integrity, mitochondrial potential, chromatin resistance, sperm lipid peroxidation, or hormone (LH and testosterone) concentrations.

Effect of mitochondria-targeted antioxidant Mito-TEMPO addition to the sperm herbal extender on ram chilled sperm quality

The aim of this study was to evaluate the sperm quality preservation in sheep using mitochondria-targeted antioxidant Mito-TEMPO during chilling process. In this experiment, semen samples were collected from five Zandi rams and added to the extender containing 0, 0.5, 1, 5, 50 and 500 µM Mito-TEMPO and stored at 4 ˚C until 48 hours. Sperm quality was evaluated at 0, 24 and 48 hours after chilling and total motility, progressive motility, viability, membrane integrity, mitochondrial activity and lipid peroxidation were assessed. No difference was observed between treatments at time 0 of cooling. During 24 and 48 hours cooling periods, using 5 and 50 µM Mito-TEMPO increased total motility, progressive motility, viability, membrane integrity, mitochondrial activity and decreased lipid peroxidation in ram chilled sperm. In conclusion, using 5 and 50 µM Mito-TEMPO in chilling herbal extender could be a suitable method to conserve ram sperm quality during chilling process.

Beneficial Effect of Proline Supplementation on Goat Spermatozoa Quality during Cryopreservation.

Simple SummarySperm suffers damage from reactive oxygen species stress (ROS) during cryopreservation. Supplementation of antioxidants is suggested to reduce sperm cryodamage induced by ROS. Proline has been regarded as a powerful antioxidant. However, there is no information on the role proline plays during goat sperm cryopreservation. Thus, this study aims to investigate the effects of proline during goat sperm cryopreservation procedures. In the present study, supplementation of 2 mM proline significantly enhanced post-thaw goat sperm quality by regulating sperm redox homeostasis. Proline supplementation protects goat sperm against ROS stress through PRODH-mediated metabolism.Sperm cryopreservation contributes to the extensive utilization of artificial insemination (AI) in the daily livestock industry. However, due to the presence of few sperm with good biological function in post-thaw goat sperm, its use has been limited for AI purposes. Hence, its improvement has been the focus of many research studies. This study aimed to investigate the effects of proline supplementation of the freezing medium on goat sperm. The goat semen was cryopreserved with freezing medium supplementation of different concentrations of proline (0, 0.5, 1, 2 and 4 mM). The post-thaw sperm motility patterns, membrane integrity, acrosome integrity, lipid peroxidation (LPO) levels, malondialdehyde (MDA) levels, total antioxidant capacity (T-AOC), proline dehydrogenase (PRODH) activity, superoxide dis-mutase (SOD) activity, glutathione (GSH) levels and GSH/GSSG were evaluated. Likewise, the expression and immunofluorescent localization of PRODH in post-thaw goat sperm was also detected. It was observed that addition of 2 mM proline to the freezing medium significantly enhanced post-thaw goat sperm total motility, progressive motility, straight-linear velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), straightness (STR), linearity (LIN), membrane integrity and acrosome integrity. Interestingly, PRODH was expressed in post-thaw goat sperm, especially in the post-acrosome and sperm tail. Addition of 2 mM proline also significantly increased the post-thaw sperm PRODH activity compared to the control. Moreover, post-thaw goat sperm LPO levels and MDA levels were reduced by supplementation of 2 mM proline. Furthermore, compared to the control, the values of post-thaw goat sperm T-AOC, SOD activity, GSH level and GSH/GSSG were also significantly increased in 2 mM proline treatment. Reduction of post-thaw goat sperm apoptosis in 2 mM proline treatment was also observed as the levels of Caspase3 and Caspase9 were decreased by the supplementation with 2 mM proline. These observations suggest that the addition of 2 mM proline to the freezing medium increased post-thaw goat sperm quality by reducing oxidative stress during cryopreservation. These findings also provide novel insights into the use of proline as an efficient additive to enhance post-thaw goat sperm quality during cryopreservation.

Genetic Ablation of Na,K-ATPase α4 Results in Sperm Energetic Defects.

The Na,K-ATPase alpha 4 isoform (NKAα4) is expressed specifically in the male germ cells of the testes and is particularly abundant in mature spermatozoa. Genetic deletion of NKAα4 in mice (NKAα4 KO mice) results in complete infertility of male, but not female mice. The reduced fecundity of NKAα4 KO male mice is due to a series of defects, including a severe impairment in total and hyperactive sperm motility. In this work, we show that deletion of NKAα4 also leads to major defects in sperm metabolism and energetics. Thus, compared to wild-type sperm, sperm from NKAα4 KO mice display a significant reduction in the extracellular acidification rate (ECAR), indicative of impaired glycolytic flux. In addition, mitochondrial function is disrupted in sperm lacking NKAα4, as indicated by a reduction in the mitochondrial membrane potential and lower oxygen consumption rate (OCR). Moreover, the ratio between the oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD/NADH) is increased in NKAα4 KO sperm, indicating a shift in the cellular redox state. These metabolic changes are associated with augmented reactive oxygen species (ROS) production and increased lipid peroxidation in NKAα4 KO sperm. Altogether, these findings reveal a novel link between NKAα4 activity and sperm energetics, highlighting the essential role of this ion transporter in sperm physiology.

Synergistic effects of myo-inositol and melatonin on cryopreservation of goat spermatozoa.

Overproduction of reactive oxygen species (ROS) during sperm cryopreservation has a detrimental effect on sperm parameters. Therefore, the use of antioxidants in the sperm freezing extender can reduce ROS destructive effects. In this study, we investigated whether co-supplementation of melatonin and myo-inositol into the semen extender can improve the post-cryopreservation quality of goat spermatozoa. After the freeze-thawing process, sperm motility, viability, plasma membrane and acrosome intact morphology were improved in the combined myo-inositol and melatonin group compared to both individual and the control groups (p<.05). In addition, the mean of sperm ROS, DNA damage and lipid peroxidation were reduced in co-supplementation of myo-inositol and melatonin compared to their individual counterparts (p<.05). Therefore, the synergistic effects of myo-inositol and melatonin on the cryopreserved spermatozoa are highly likely mediated through the reduction in important factors involved in the sperm lipid peroxidation. Finally, we used the cryopreserved spermatozoa for in vitro production of embryos. Results showed that combined group of myo-inositol and melatonin improved the cleavage rate compared to both individual and control groups, although blastocyst rate was improved using both individual and combined groups. In conclusion, co-supplementation of melatonin and myo-inositol is a promising approach for the improvement of goat sperm cryopreservation.

Supplementation of melatonin to cooling and freezing extenders improves canine spermatozoa quality measures

BackgroundSperm freezing and cold storage are the two most common assisted reproductive technologies in the canine breeding industry. The freeze-thawing process causes significant detrimental changes in both sperm cell structure and function. Previous research has confirmed that excessive accumulation of un-scavenged free radicals (oxidative stress) plays an important role in the cryopreservation-induced damage to sperm cells. Also, the gradual accumulation of the free radicals during cold storage leads to a decline in the sperm quality markers. Melatonin is an endogenous neurohormone synthesized from tryptophan amino acid by pineal glands. Besides its several well-known physiologic roles, melatonin has a significant antioxidant potential through direct free radical scavenging properties. Therefore, the current study was designed to evaluate the potential in vitro protective properties of melatonin (0.5, 1, and 2 mM) on canine sperm cells after freezing or during long-term cold storage (9 days, 5 °C) on most important sperm in vitro fertility markers.ResultsMelatonin at 0.5, 1- or 2-mM concentrations could preserve significantly higher sperm total motility after 4 days of cold storage. However, only the 1- and 2 mM melatonin concentrations could result in better TM and PM values after 7 days of cold storage. Furthermore, melatonin supplementation could preserve higher sperm viability and acrosome integrity after 7 days of storage. Also, it could have significant protective effects on the cooled sperm DNA integrity. In the freezing section of the current research, melatonin at either 1- or 2-mM concentrations could not improve the sperm post-thaw TM and PM, whereas they improved sperm DNA integrity. Also, the post-thaw plasma membrane functional integrity and sperm velocity parameters were not affected by the treatment. Although DMSO (Dimethyl Sulfoxide) as the melatonin solvent could reduce the level of sperm lipid peroxidation and even improve the post-thaw sperm DNA integrity compared to the negative control, it reduced the post-thaw sperm progressive motility. However, the negative effects were reversed by concurrent melatonin supplementation at 1- and 2-mM concentrations.ConclusionThe addition of 1- or 2-mM melatonin to the canine sperm freezing and cooling media could improve sperm motility, viability, acrosome, and DNA integrity.