Sperm Mitochondrial Membrane Potential Research Articles

Hyaluronic acid capacitation induces intracellular signals modulated by membrane-associated adenylate cyclase and tyrosine kinase involved in bovine in vitro fertilization

Heparin is the most commonly used in vitro capacitation inducer in the bovine. However, hyaluronic acid (HA) has been recently used for capacitation induction as well as for other reproductive biotechnologies, such as sperm selection and in vitro fertilization (IVF). Our aim was to induce sperm capacitation with heparin or HA in order to study mAC and TK intracellular signals and their relation with cleavage and blastocyst rates after IVF as well as with the oxidative status of the potential bovine embryos. 2,5-dideoxyadenosine and genistein were used as mAC and TK inhibitors, respectively. Sperm capacitation was analyzed using CTC technique, sperm plasma membrane and acrosome integrity were determined using trypan blue stain and differential interference contrast, and mitochondrial activity was evaluated using fluorochrome JC-1. Cleavage rate was analyzed 48h and blastocyst production 7–8 days after IVF, while cytosolic oxidative activity was determined using RedoxSensor Red CC-1 fluorochrome 7h after IVF. When mAC and TK inhibitors were added to sperm samples, only capacitation decreased significantly both in HA and heparin treated samples (P < 0.05), but plasma membrane and acrosome integrity percentages were not affected in any of these groups (P > 0.05). Sperm mitochondrial membrane potential only decreased in heparin treated samples in the presence of both inhibitors (P < 0.05). Oocytes activated with HA sperm treated samples with the addition of 2,5-dideoxyadenosine and genistein presented a lower cytosolic oxidative status than those activated with sperm treated with HA alone (P < 0.05). On the other hand, oocytes activated with heparin treated sperm samples presented a lower cytosolic oxidative status only in the presence of 2,5-dideoxyadenosine (P < 0.05). Therefore, mAC and TK present a differential participation in heparin and HA sperm induced capacitation and mitochondrial function as well as in IVF.

Evaluation of Sperm Mitochondrial Function: A Key Organelle for Sperm Motility.

Introduction: The role of nutraceuticals in the treatment of male infertility, especially in the “idiopathic form”, remains the subject of significant debate. Many antioxidants improve sperm motility but the exact mechanism by which they act is still unclear. Although several studies have shown a correlation between sperm motility and mitochondrial function, the effects of antioxidant therapy on mitochondrial membrane potential (MMP) are poorly studied. The first aim of this review was to evaluate the efficacy of antioxidants on mitochondrial function and, consequently, on sperm motility in male infertile patients. Material and Methods: we performed a systematic search of all randomized controlled and uncontrolled studies available in the literature that reported sperm motility and MMP at baseline and after antioxidant administration in-vivo and in-vitro in patients with idiopathic asthenozoospermia. Pubmed, MEDLINE, Cochrane, Academic One Files, Google Scholar and Scopus databases were used. Results: Unexpectedly, among 353 articles retrieved, only one study met our inclusion criteria and showed a significant effect of myoinositol on both MMP and sperm motility. We then summarized the main knowledge on anatomy and metabolism of sperm mitochondria, techniques allowing to assess sperm mitochondria function and its relationships with low sperm motility. Finally, we paid special attention to the effect of antioxidant/prokinetic molecules for the treatment of asthenozoospermia. Conclusions: This is the first systematic review that has attempted to evaluate the effects of antioxidants on MMP and sperm motility. Although results are not conclusive due to the dearth of studies, the close relationship between mitochondria and sperm motility is clear. The investigation of this correlation could provide valuable information to be exploited in clinical practice for the treatment of male infertility.

Comparison of TNC and standard extender on post-thaw quality and in vivo fertility of Thai native chicken sperm

Semen extender has a vital role in preservation of sperm cells properties in terms of sperm viability, motility, acrosome integrity, and mitochondrial membrane potential. The objective of the present study was to evaluate a new extender, known as Thai native chicken (TNC) extender compared to BHSV-based and modified Sasaki extenders for freezing chicken semen. Semen from Thai native roosters was collected, pooled, and randomly divided into three groups. Semen was frozen with a simple freezing method using nitrogen vapor and dimethylformamide. In the first experiment, post-thaw motion parameters, viability, acrosome integrity, mitochondrial function, and lipid peroxidation levels were analyzed using computer-assisted sperm analysis, propidium iodide, fluorescein isothiocyanate-conjugate peanut agglutinin, JC-1, and the thiobarbituric acid reaction. Results showed that the type of extender had no effect on the percentage of total motile and curvilinear velocity. The percentage of progressive motile, straight-line velocity, and average path velocity of post-thawed semen were significantly lower in TNC compared to the modified Sasaki extender. However, the percentages of post-thawed acrosome integrity and active mitochondria were significantly higher in TNC extender (P < 0.05). For the second experiment, semen was thawed by using each of extenders thereafter, was inseminated to 48-layer breeder hens to determine the fertility rate. Among the three extenders used, the highest fertility rate was found in TNC extender. In conclusion, TNC extender can be recommended as an appropriate and useful cryopreservation media for native chicken semen since it maintains the quality of rooster semen and fertility after freezing and thawing process.

Taurine effects on Bisphenol A-induced oxidative stress in the mouse testicular mitochondria and sperm motility.

Objectives:This study was performed to investigate the protective effects of taurine (2-aminoethanesulfonic acid, TAU) on oxidative stress in the isolated mouse testicular mitochondria, mitochondrial membrane potential (MMP), viability and motility of the exposed sperms to the BPA.Methods:We treated epididymal spermatozoa obtained from mice and isolated mouse testicular mitochondria with BPA (0.8 mmol/mL) and various doses of TAU (5, 10, 30 and 50 µmol/L). We used the MTT assay and Rhodamine 123 uptake to assess sperm viability and MMP. We assessed the oxidative stress through measuring ROS (reactive oxygen species), MDA (malondialdehyde), GSH (glutathione), and SOD (super-oxide dismutase) levels in the testicular mitochondrial tissue.Results:BPA significantly elevated ROS, MDA and MMP levels, and markedly reduced SOD and GSH levels in the isolated mitochondria. BPA also considerably impaired spermatozoa viability and motility. Pretreatment with 30 and 50 µmol/L of TAU could considerably suppressed mitochondrial oxidative stress, enhanced MMP, and improved sperm motility and viability.Conclusion:TAU may attenuate the BPA-induced mitochondrial toxicity and impaired sperm motility via decreasing oxidative stress.

114 Omega-3-enriched diet improves fertilization competence of cryopreserved sperm

Intensive reproductive management in dairy herds is mostly based on AI using high-merit bulls. Therefore, semen quality of bulls is of high importance. An association between semen quality and fatty acid content in feed has been suggested. Accordingly, the aim of this study was to examine the effect of omega-3 supplementation on sperm traits and fertilization competence. Fifteen Israeli Holstein bulls were assigned to three experimental groups. Bulls were fed over 13 weeks with a standard ration top-dressed with encapsulated-fat supplementation-fish oil (FO) or flaxseed oil (FLX; i.e. omega-3 sources), or saturated fatty acids (SFA, control). Ejaculates were collected before initiation of the study, during the feeding trial, and 1 month after feeding trial. Ejaculates were treated according to the routine procedure of the Israeli AI centre (Sion Ltd.), frozen, and stored in straws. Frozen-thawed samples were subjected to “swim-up” procedure, and spermatozoon viability, mitochondrial membrane potential, reactive oxygen species (ROS) level, acrosome membrane integrity, and DNA fragmentation were evaluated via flow cytometry, using sperm-specific kits (EasyCyte, IMV Technologies). Feeding with FO, FLX, or SFA did not affect the viability or mitochondrial membrane potential of sperm collected before, during, or after the feeding trial. On the other hand, a reduced proportion of sperm with ROS expression was recorded in the FLX samples compared to the SFA sample at the end of the feeding trial (42.2±1.2 vs. 47.3±4.3%, respectively; P&amp;lt;0.05) and one month later (36.3±2.2 vs. 41.6±4.6%, respectively; P&amp;lt;0.05). A low proportion of sperm with damaged acrosomal membrane was observed in both FLX and FO samples compared with SFA at the end of the feeding trial (48.8±3.4 and 41.7±2.7 vs. 59.8±3.4%, respectively; P&amp;lt;0.05). The proportion of sperm with fragmented DNA was lower in the FLX group than in the SFA group, collected one month after the end of the feeding trial (2.3±0.6 vs. 5.4±1.2%, respectively; P&amp;lt;0.05). To examine fertilization competence, oocytes were aspirated from ovaries collected from a local abattoir, then matured (n=216; 3 replicates) and fertilized invitro with a pool of samples from each group, collected one month after the end of the feeding trial (n=5 samples per group). The proportions of 2- to 4-cell-stage embryos and of blastocysts were determined 42h and 8 days after fertilization, respectively. Although the proportion of cleaved embryos did not differ between groups, a higher blastocyst formation rate was recorded in the FLX group (P&amp;lt;0.05), and a moderate increase was noted in the FO group, relative to the SFA group (28.1±4.4, 19.1±2.6, and 11.9±3.4%, respectively). Results imply that feeding bulls with omega-3 originating from FLX improves sperm quality, most likely due to improved redox status and decreased DNA fragmentation. This nutritional approach seems to be an effective tool for improving bull fertility competence. Nevertheless, invivo examination is required.

159 Laparoscopic insemination method in sheep allows the use of an animal protein-free and inexpensive freezing medium

Animal-derived products are widely used in sperm cryopreservation for their cryoprotective properties. These components, however, must be replaced because of sanitary risks. STEMALPHA.CRYO3 (Ref. 5617, Stem Alpha), called CRYO3, is a chemically defined preservation medium currently used for freezing human tissue and adult stem cells. The aim of this study was to evaluate the effects of a CRYO3-based medium and of two cooling rates on invitro parameters and invivo fertility of ram sperm. Six rams (Blanche du Massif Central) were subjected to sperm collection four times using an artificial vagina. Sperm were split and frozen in three media: an egg yolk and milk-based medium (positive control), a CRYO3-based medium (tested medium), and a medium without additives (negative control). The two cooling rates were related to the distance between the straws and the surface of liquid nitrogen during the freezing process (5 and 20cm). Sperm membrane integrity (propidium iodide/SYBR-14), acrosome integrity (fluorescein isothiocyanate-peanut agglutinin/propidium iodide; FITC-PNA/PI), and mitochondrial membrane potential (JC-1) were assessed using flow cytometry, whereas functional membrane integrity was assessed using a hypo-osmotic swelling test and motion characteristics were evaluated using computer-assisted sperm analysis. Pregnancy rate, parturition rate, and prolificacy were evaluated after performing laparoscopic inseminations (n=75 ewes). Moreover, we characterised the freezing media thermodynamically using a differential scanning calorimeter. Statistical analyses were performed using R software. Invitro parameters were assessed using a mixed model including the time and the medium as fixed effects and the ram as a random effect. Pregnancy and parturition rates, following a binomial distribution, and prolificacy, assumed to follow a Poisson distribution, were analysed using generalised linear models, including the medium as a fixed effect and the ram as a random effect. Differences with P&amp;lt;0.05 were considered statistically significant. The cooling rates had no significant effect except on the wobble motion parameter. The positive control medium showed significantly higher results than the CRYO3-based medium and the negative control medium for all invitro parameters except for straightness motion parameter. Conversely, field trials showed no significant difference between the media for pregnancy rate (71, 64, and 74%), parturition rate (68, 61, and 74%) and prolificacy (2.0, 2.1, and 1.7), for the positive control, CRYO3-based medium, and the negative control, respectively. This study showed that the product, CRYO3, cannot replace egg yolk and milk in freezing extenders. Moreover, we showed that laparoscopic inseminations allowed a 74% parturition rate due to an easy and inexpensive medium comprising only a Tris buffer and glycerol. Although it could not be used on a large scale, this medium remains an option for international transport or long-term storage of genetic diversity.

Supplementation of l-tryptophan (an aromatic amino acid) in tris citric acid extender enhances post-thaw progressive motility, plasmalemma, mitochondrial membrane potential, acrosome, and DNA integrities, and in vivo fertility rate of buffalo (Bubalus bubalis) bull spermatozoa

The aromatic amino acid l-tryptophan is an essential and versatile molecule, acts by transferring an electron to free radicals and protects the plasma membrane from injuries. The aim of the present study was to investigate the effects of l-tryptophan in extender on semen quality parameters, in vitro longevity and in vivo fertility rate of buffalo spermatozoa during cryopreservation. Two ejaculates were collected from each bull (n = 2 ejaculates and n = 4 bulls) with artificial vagina at 42 °C followed by initial evaluation for volume, motility, concentrations and were diluted in five extenders (C = lacking l-tryptophan, D1 = 25 μ M l-tryptophan, D2 = 50 μ M l-tryptophan, D3 = 75 μ M l-tryptophan, and D4 = 100 μ M l-tryptophan) respectively, and cryopreserved. The experiment was repeated four times (n = 4 replicates). At post-dilution, sperm plasma membrane integrity (PMI, %), supravital plasma membrane integrity (SVPMI, %), hypo-resistivity (HR, %) and acrosome integrity (ACR-I, %) were significantly higher (P < 0.05) in extender supplemented with D4 than control. At post-thawing, progressive motility (PM, %), PMI, SVPMI, HR, ACR-I, and DNA-I of buffalo bull spermatozoa were significantly higher in D4 than control. Sperm in vitro longevity (%) assessed in terms of PM, SVPMI, and ACR-1 were significantly higher in D4 than control. Sperm mitochondrial membrane potential (%) was higher in treated groups than the control. The in vivo fertility rate was significantly higher in D4 than control (60.17% vs. 44.17%, P < 0.05). It is concluded that the supplementation of l-tryptophan in tris citric acid extender improves semen quality parameters, in vitro longevity and in vivo fertility rate of buffalo spermatozoa during freezing and thawing process.

The Effect of N-N-Dimethylformamide on the Membrane Characteristics of Canine Spermatozoa After Cryopreservation, and its Relationship With Post-Thaw Motility

Some studies have demonstrated that glycerol is superior to amides in preserving sperm motion characteristics of canine sperm; however, little is known about the effect of these cryoprotectants on the membrane characteristics of canine spermatozoa after freezing/thawing. In this study, the effects of using either N-N-dimethylformamide (DMF) or glycerol (GLY) on the integrity and function of the canine sperm, after cryopreservation were determined. We hypothesized that the use of a multiparametric approach for assessing the effect of DMF on the membranes of canine sperm would explain the lower values reported for post-thaw motility. Ejaculates from 12 dogs were collected, split into 2 groups, and frozen using a tris-fructose-citrate-egg yolk-based extender containing either 7% (v/v) GLY or 7% (v/v) DMF. Frozen straws (n = 120) were thawed and analyzed for subjectively-assessed sperm progressive motility, normal morphology, plasma membrane integrity, plasma membrane function (HOST+), acrosome membrane integrity, high mitochondrial membrane potential, and simultaneous assessment of sperm membrane integrity and function by a triple-staining fluorescent procedure. Overall, sperm motility and membrane intactness/function were higher when GLY was used as a cryoprotectant, as compared to DMF (P < .05). A model to explain the variation in progressive motility using the values obtained from the sperm integrity and function parameters was designed. The percent HOST+ sperm and high mitochondrial membrane potential sperm were mostly associated with the changes observed in the progressive motility (r2 = 0.84; P = .043) when either GLY or DMF were used as cryoprotectants. These results may explain the overall reduced sperm quality observed after cryopreservation, as a reflection of sublethal damage sustained by the sperm membranes.

Freezability biomarkers in bull epididymal spermatozoa

Sperm cryopreservation is an important tool for storing genetic traits and assisted reproduction techniques. Several studies have developed semen cryopreservation protocols. However, the sperm proteome is different between ejaculated and epididymal spermatozoa and little is known about cryopreservation effects on epididymal spermatozoa. Therefore, our study aimed to (i) investigate the differences of sperm parameters based on the freezing tolerance of spermatozoa and (ii) identify potential markers to predict the freezability of bull epididymal spermatozoa. Our preliminary study demonstrated that spermatozoa from individual bulls differ in cryopreservation freezability. We categorized spermatozoa into high freezing-tolerant spermatozoa and low freezing-tolerant spermatozoa group based on sperm motility after freezing/thawing. We evaluated several sperm functional parameters, including sperm motility/motion kinematics, sperm speed parameters, viability, mitochondrial activity, and capacitation status. Our results demonstrated that motility, sperm speed parameters, viability, and mitochondrial membrane potential had significant differences between the two groups but motion kinematics and capacitation status did not. In addition, the concentration of three proteins - glutathione s-transferase mu 5, voltage-dependent anion-selective channel protein 2, and ATP synthase subunit beta, differed between both groups. Thus, our research highlighted differences in bull epididymal spermatozoa freezability upon cryopreservation and these proteins might be useful markers to select high freezing-tolerant epididymal spermatozoa.

Protective influence of rosiglitazone against time-dependent deterioration of boar spermatozoa preserved at 17°C.

Spermatozoa are highly specialized cells, and energy metabolism plays an important role in modulating sperm viability and function. Rosiglitazone is an antidiabetic drug in the thiazolidinedione class that regulates metabolic flexibility and glucose uptake in various cell types, but its effects on boar sperm metabolism are unknown. In this study, we investigated the potential effect of rosiglitazone against time-dependent deterioration of boar spermatozoa during liquid preservation at 17°C. Freshly ejaculated semen was diluted with Beltsville Thawing Solution (BTS) containing different concentrations of rosiglitazone, and the motility, membrane and acrosome integrity of sperm were detected. Besides, we measured glucose uptake capacity, l-lactate production level, mitochondrial membrane potential, adenosine triphosphate (ATP) content and mitochondrial reactive oxygen species (mROS) production of sperm after boar semen had been incubated with or without rosiglitazone, iodoacetate (glycolysis inhibitor) and rotenone (electron transport chain inhibitor) for 5days. The addition of rosiglitazone significantly enhanced sperm quality and had a strong protective effect on the sperm membrane and acrosome integrity during storage. BTS containing 50μM rosiglitazone maintained the total motility of liquid-preserved sperm above 60% for 7days. Rosiglitazone improved sperm quality by regulating energy metabolism manner of preserved sperm, protected the sperm mitochondrial membrane potential, enhanced sperm ATP production and in the meanwhile reduced mROS through enhancing glycolysis but not oxidative phosphorylation. The data suggested the practical feasibility of using rosiglitazone for improving boar spermatozoa quality during semen preservation.

Potent humanin analogue (HNG) protects human sperm from freeze-thaw-induced damage

This study was aimed to investigate the protective effect of potent humanin analogue (HNG) supplementation to freezing media on freezing-thawing induced human sperm damage. We collected semen samples with normal sperm parameters from 15 healthy men. After the swim-up processing, the motile spermatozoa from each of the men were allocated to four equal groups: In the control group, the spermatozoa were frozen in media without HNG supplementation. In the other three groups, the spermatozoa were frozen in media supplemented with different concentrations of HNG (2 μM, 10 μM and 20 μM, respectively). We analyzed the sperm motility, viability, sperm mitochondrial membrane potential, apoptosis, sperm DNA fragmentation index (DFI), reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and caspase-3 activity for the sperm in each group. As a result, supplementation of HNG with 2 μM, 10 μM and 20 μM to the freezing media all significantly improved sperm motility and viability (all p < 0.05) when compared with the control group. Similarly, we found that supplementation of HNG reduced the damage to the mitochondrial membrane and DNA integrity, and inhibited the reaction of oxidative stress and the activity of caspase-3 in sperm. Although these protective effects increased with the elevated concentration of HNG in the freezing media, a final HNG concentration of 20 μM failed to exert significant improvements when compared with the concentration of 10 μM (all p > 0.05). In conclusion, our results suggested that HNG supplementation to the freezing media could protect sperm cells from freezing-thawing induced sperm damage.

Characterization of the Sperm Proteome and Reproductive Outcomes with in Vitro, Fertilization after a Reduction in Male Ejaculatory Abstinence Period

Semen samples from men after a short ejaculatory abstinence show improved sperm quality and result in increased pregnancy rates, but the underlying mechanisms remain unclear. Herein, we report that ejaculates from short (1-3 h) compared with long (3-7 days) periods of abstinence showed increases in motile sperm count, sperm vitality, normal sperm morphology, acrosome reaction capacity, total antioxidant capacity, sperm mitochondrial membrane potential, high DNA stainability, and a decrease in the sperm DNA fragmentation index (p, < 0.05). Sperm proteomic analysis showed 322 differentially expressed proteins (minimal fold change of ±1.5 or greater and p, < 0.05), with 224 upregulated and 98 downregulated. These differentially expressed proteins are profoundly involved in specific cellular processes, such as motility and capacitation, oxidative stress, and metabolism. Interestingly, protein trimethyllysine modification was increased, and butyryllysine, propionyllysine, and malonyllysine modifications were decreased in ejaculates from a short versus, long abstinence (p, < 0.05). Finally, the rates of implantation, clinical pregnancy, and live births from in vitro, fertilization treatments were significantly increased in semen samples after a short abstinence. Our study provides preliminary mechanistic insights into improved sperm quality and pregnancy outcomes associated with spermatozoa retrieved after a short ejaculatory abstinence.

Mitochondrial functionality modifies human sperm acrosin activity, acrosome reaction capability and chromatin integrity.

In addition to sperm motility, which major biological characteristics of sperm fertility potential are associated with mitochondrial functionality? Sperm fertilization capacities, including acrosin activity, acrosome reaction (AR) capability and chromatin integrity, are related to the mitochondria functionality as evaluated by the mitochondrial membrane potential (MMP). Correlative studies suggest a potential role of sperm MMP in predicting sperm fertilization ability and ensuring sperm motility. However, researches characterizing other determinants of sperm fertility potential according to MMP are lacking. The sperm MMP was examined in 627 young college students in the Male Reproductive Health in Chongqing College Students (MARHCS) cohort study in 2014. Among these participants, acrosin activity and chromatin integrity were measured in 378 and 604 subjects, respectively. These two determinants of sperm fertility potential were first compared among high-, moderate- and low-MMP groups in the college population. The effects of MMP collapse caused by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) on acrosin activity, AR, DNA fragmentation, reactive oxygen species (ROS) production, and ATP content in human spermatozoa were evaluated in vitro. The sperm MMP was evaluated by using JC-1 staining, acrosin activity was measured using a N-α-benzoyl-dl-arginine-para-nitroanilide HCl (BAPNA) substrate method, the integrity of chromatin represented by DNA fragmentation index (DFI) was measured by sperm chromatin structure assay (SCSA), AR was evaluated with chlortetracycline staining, and intracellular ROS production was evaluated with dihydroethidium. ATP concentration was determined with luciferase. Measurements were performed by spectrophotometry or flow cytometry. Nonparametric analysis revealed significantly higher acrosin activity and a lower DFI in subjects with moderate or high MMP compared to those with low MMP. After adjustment for potential confounders, increases of 7.9 and 44.4% in sperm acrosin activity and deceases of 12.0 and 25.2% in the sperm DFI were found in the moderate- and high-MMP groups, respectively. The MMP dissipation induced by CCCP caused significant declines in acrosin activity and AR capacity and increased DFI in human spermatozoa. Moreover, sperm MMP dissipation induced ROS overproduction and decreased ATP content. We cannot exclude a contribution of leukocytes to ROS production and no size gating was used to exclude these cells from the FACS measurements. No simultaneous live-dead staining was done and a contribution of dead sperm to the MMP and acrosome assays cannot be excluded. Mitochondrial functionality might be necessary to maintain sperm acrosin activity, AR and chromatin integrity. Tests of mitochondrial functionality should be developed and used independently of or in addition to conventional semen parameters in infertility diagnosis or risk-assessment processes. This study was supported by the Key Program of the National Natural Science Foundation of China (No. 81630087) and the National Natural Science Foundation of China (No. 81703254). None of the authors have any competing interests to declare.

Effects of oligomeric proanthocyanidins on quality of boar semen during liquid preservation at 17 °C

The use of antioxidants is an important aspect in the preservation of boar semen. Oligomeric proanthocyanidins (OPC) are an effective natural antioxidants that scavenges free radicals and reactive oxygen species (ROS). This study was designed to investigate the antioxidative effect of OPC on boar semen quality during liquid preservation at 17 °C. The effects of different concentrations of OPC on sperm quality variables, antioxidant effects and fertility were analyzed in this experiment. Semen collected from six Guanzhong-Black boars and was diluted with Beltsville thawing solution (BTS). During the process of liquid preservation at 17 °C, the variables assessed were measured and analyzed every 24 h. The addition of OPC improved boar sperm motility, acrosome integrity, membrane integrity, and mitochondrial membrane potential as compared with that of the control group (P < 0.05). Meanwhile, malondialdehyde content (MDA) and ROS content was less after adding OPC, thereby improving the total antioxidant capacity (T-AOC) (P < 0.05). Different concentrations of OPC have different degrees of protective effects on boar semen quality. The results indicate that 50 μg/mL of OPC was the optimum concentration, and that the conception rate, litter size, and survival rate increased at this concentration as compared with that of the control group (P < 0.05). In summary, the addition of OPC to BTS diluents can improve the quality of boar semen at 17 °C during liquid preservation. Further research is needed to understand the mechanism by which OPC provides protection to boar semen during preservation in vitro.

Sperm quality characteristics and fertilization capacity after cryopreservation of rooster semen in extender exposed to a magnetic field

There has not been as much success in the effectiveness of avian semen cryopreservation as that of mammalian semen cryopreservation. This has been attributed to the unique characteristics of poultry sperm. In the area of mammalian semen, the use of magnetized extenders has enhanced the cryo-resistance of sperm in some species. The applicability of this strategy to avian semen has not yet been evaluated. The purpose of this study was to evaluate the effects on sperm quality and fertility after magnetization of extenders and use of these extenders for cryopreservation of rooster semen. Extenders were passed between two neodymium magnets that produced different magnetic induction values (0, 2000, 4000 and 6000 G). Physicochemical properties of the extenders were assessed and then semen samples were cryopreserved in the magnetized extenders. The smallest cluster sizes and zeta potentials in the extenders were observed in the extender magnetized at 2000 gauss (G). There were greater percentages of sperm total motility, viability and mitochondrial membrane potential in semen treated with the extender magnetized at 2000 G A greater percentage of sperm with enhanced membrane functionality was observed when sperm were stored in extenders magnetized with 2000 G and 4000 G. In a field trial, there were greater percentages of fertility and hatching rates of eggs using semen treated with extenders magnetized at 2000 G and 4000 G. Cryopreservation of rooster semen with magnetized extenders at 2000–4000 G resulted in enhanced quality indices of thawed sperm as well as the fertility capacity of the cryopreserved semen.

Supplementation of freezing/thawing media with GSK3 inhibitor alsterpaullone does not bypass the harmful effect of cryopreservation on boar spermatozoa

Frozen-thawed boar sperm have less motility and fertility capacity in comparison to fresh sperm. Glycogen Synthase Kinase 3 (GSK3) contributes to sperm motility in fresh semen. In addition, GSK3 inhibition in boar spermatozoa in fresh semen improves motility variables. The role of GSK3 on boar cryopreserved sperm, however, is still unknown. The hypothesis in the present study was that GSK3 pathway inhibition by alsterpaullone (AST) could result in enhancement of the quality of sperm afer cryopreservation. Two different strategies were evaluated: i) AST supplementation to the freezing medium (AST + Cryo); ii) AST supplementation after sperm thawing (AST + Thaw). Sperm motility was evaluated using the CASA system and different sperm quality variables were evaluated using flow cytometry, as well as amount of GSK3 phosphorylation of thawed spermatozoa after 30 and 90 min incubation at 38.5 °C. Results indicate that AST supplementation had detrimental effects on sperm viability (live spermatozoa) and mitochondrial membrane potential when it was added after thawing (P < 0.05) The AST supplementation after thawing, however, had a protective effect on plasma membrane lipid disorganization (P < 0.05). The percentage of motile spermatozoa was not modified by AST supplementation. Nonetheless, after 30 min post-thawing, STR and LIN variables (related to straightness of the movement) as well as the percentage of rapid lineal spermatozoa were increased with both AST supplementation protocols. The GSK3α phosphorylation was not modified through the incubation time in boar thawed sperm. In summary, results do not support the idea of adding AST to the cryopreservation/thawing medium to improve boar sperm quality after cryopreservation.

Testicular human spermatozoa cryopreservation correlation between sperm head vacuoles, DNA fragmentation and mitochondrial membrane potential

Abstract Background There are difficulties associated with testicular sperm freezing. Different methods of sperm cryopreservation developed. Not enough detailed studies about the real efficacy of these techniques exist. For sperm morphologic assessment, motile sperm organelle morphology examination (MSOME) is able to identify not only conventional morphological sperm alterations but also sperm head vacuoles. Objective To assess the effect of cryopreservation on testicular spermatozoa vacuoles by MSOME and its correlation to DNA fragmentation and mitochondrial membrane potential. Materials and methods After preparation, testicular sperm extraction samples of 15 azoospermic men, aged 20–40 years old were divided into three groups. Group 1 was assessed freshly. Group 2 was cryopreserved with vitrification method and Group 3 with cooling in liquid nitrogen vapor using droplet. Pre and post warming assessment in terms of spermatozoa head vacuoles by MSOME, DNA fragmentation, and mitochondrial membrane potential were performed. Results The number of spermatozoa with no vacuoles significantly decreased after two cryopreservation techniques (P 0.05). DNA fragmentation and mitochondrial membrane potential increased after cryopreservation (P Conclusion Cryopreservation affects spermatozoa vacuolization, DNA structure, and mitochondrial membrane potential. Using MSOME in the selection of post-thaw morphologically normal testicular spermatozoa for ICSI procedure will be of particular value.

Correlations of Sperm Mitochondrial Membrane Potential with Semen Parameters and Male Obesity

Background: To investigate the correlations of sperm mitochondrial membrane potential (MMP) with semen parameters and body mass index (BMI) in males with obesity. Methods: Semen samples were obtained by masturbation after 3–7 days of sexual abstinence from males who visited semen collect room of Shanghai Ninth People’s Hospital. Conventional semen analyses were performed by computer-aided sperm analysis (CASA), and sperm morphology was analyzed by modified Papanicolaou staining. Spermatozoa were stained by JC-1 to evaluate MMP through flow cytometry. Results: Sperm MMP of asthenozoospermia group (41.24% ± 9.71%) was significantly lower than that in control group (56.68% ± 11.13%). MMP was negatively correlated with BMI (r = −0.25, P < 0.01), but positively correlated with total sperm motility (r = 0.63, P < 0.01), motility of progressive sperm (r = 0.64, P < 0.01), and normal sperm morphology rate (r = 0.37, P < 0.01). In addition, MMP showed no significant correlations with age, volume of semen, sperm concentration, sperm count, and other indexes. Conclusions: Sperm MMP is an important index in the evaluation of sperm function, and detection of MMP may provide references for the diagnosis and treatment of male infertility. Key words: Body Mass Index; Conventional Semen Analyses; JC-1; Mitochondrial Membrane Potential

A mechanism by which Astragalus polysaccharide protects against ROS toxicity through inhibiting the protein dephosphorylation of boar sperm preserved at 4 °C.

Numerous studies have shown that Astragalus polysaccharide (APS) has strong antioxidant effects and high practical value for preserving semen at low temperatures in vitro. However, to date, little attention has been paid to the precise mechanism of APS in sperm preservation at 4 °C. Thus, to gain further insight into the protective effects of APS, the present study was performed to assess the changes in sperm quality parameters, antioxidant capacity, ATP content, and protein phosphorylation levels. Here, we demonstrated that supplementation with APS could effectively preserve boar sperm quality parameters such as sperm motility, acrosome integrity, and mitochondrial membrane potential. Moreover, we found that the positive effects of APS on boar sperm quality were mainly due to the elimination of excessive mitochondrial ROS, the improvement of antioxidant capacities and the enhancement of ATP levels. Interestingly, by conducting a series of studies on protein phosphorylation, we also discovered that APS could protect boar sperm from oxidative stress and energy deficiency through inhibiting the protein dephosphorylation caused by ROS via the cAMP-PKA signaling pathway. To our knowledge, this is the first exploration of the molecular mechanism underlying the protective roles of APS toward ROS toxicity from the perspective of energy metabolism and protein modification. This study comprehensively provides novel insights into the action mechanism of the protective effects of antioxidants on sperm stored at 4 °C and reveals the practical feasibility of using APS as a boar semen extender supplement for assisted reproductive technology.

Mitochondria-targeted antioxidant MitoTEMPO improves the post-thaw sperm quality

Human spermatozoa cryopreservation is an important means of assisted reproductive technology and male fertility preservation. Although this technique is particularly useful, sperm cryopreservation significantly reduces the quality of spermatozoa after freezing and thawing. The objective of the study is to examine the efficacy of mitochondria-targeted antioxidant MitoTEMPO in improving sperm quality during semen cryopreservation processes. Semen samples were collected and cryopreserved in extenders containing different concentrations (0.0, 0.5, 5, 50, and 500 μM) of MitoTEMPO. Sperm motility, viability, membrane integrity, mitochondrial membrane potential and antioxidant activities were measured and analyzed. The results showed that the addition of MitoTEMPO (5–50 μM) significantly improved post-thaw sperm motility, viability, membrane integrity and mitochondrial membrane potential (P < .05). Meanwhile, antioxidant enzymes activities were enhanced and MDA content were decreased in the group supplemented with MitoTEMPO. In conclusion, mitochondria-targeted antioxidant MitoTEMPO improves the post-thaw sperm quality and antioxidant enzymes profile.