Sperm Protection Research Articles

Influence of antifreeze protein III on canine sperm cryopreservation.

Sperm cryopreservation is crucial in reproductive biotechnology; however, the longevity of frozen and thawed semen is limited by the deterioration of sperm cell integrity. This study aimed to examine the effects of adding antifreeze protein III (AFP III) to the diluent, using samples from eight healthy mature dogs. The ejaculates were divided into aliquots and diluted with a standard Tris-fructose-egg yolk extender containing AFP III at concentrations of 0, 0.75, 1.0, and 2.0μg/ml. After thawing, the samples were analyzed for kinematic parameters, membrane Integrity, lipid peroxidation, viability, acrosome integrity, intracellular hydrogen peroxide, mitochondrial membrane potential and apoptotic metrics. The results show that while motility and velocity were not significantly different between the treated and control groups (p>0.05), the treated groups generally performed better. Specifically, the 0.75 and 1.0μg/ml groups exhibited better movement compared to the 2.0μg/ml group. Additionally, there was a significant difference (p<0.05) in membrane integrity between the control and treated groups, though no differences were observed among the treated groups. Significant differences (p<0.05) were also observed in viability and acrosome integrity, with the 0.75 and 1.0μg/ml groups outperforming the control and 2.0μg/ml groups. There were no significant variations (p>0.05) in phosphatidylserine translocation, lipid peroxidation, mitochondrial membrane potential, or hydrogen peroxide levels. However, the 0.75 and 1.0μg/ml groups demonstrated superior effects compared to both the control and the 2.0μg/ml groups. These results suggest that the addition of antifreeze proteins, specifically AFP III, markedly improves the protection of canine sperm during cryopreservation. This enhancement is evident in various parameters, underscoring the beneficial effects of AFP III in maintaining sperm quality.

Antioxidant activity and protective effect of hydroxy derivatives of chalcones for sterlet (Acipenser ruthenus, Linnaeus, 1758) sperm.

The aim of this work is to study the effect of adding hydroxy derivatives of chalcones to the basic cryomedium on the ability of sterlet sperm to utilize superoxide and hydrogen peroxide, the intensity of lipid peroxidation of male fish germ cells, and their viability both before cryopreservation and after 3 days of freezing at liquid nitrogen temperature. The ability of phenolic derivatives of chalcones to increase the superoxide dismutase and catalase activities of sterlet sperm and to reduce the intensity of lipid peroxidation has been established. The antioxidant activity of the derivatives exceeds the effect of Trolox, which inhibits the functioning of the enzyme component of the antioxidant protection of fish sperm and promotes lipid peroxidation of fish sperm before cryopreservation. A beneficial effect of hydroxy derivatives of chalcones on the motility parameters of thawed sperm has been shown, indicating their ability to increase the cryoresistance of sperm in such a promising aquaculture species as sterlet.

Влияние пирролидиновых производных 2,6-ди-трет-бутилфенола на супероксид- и пероксид водорода-утилизирующую активность спермы сибирского осетра

The effect of (3.5-di-tert-butyl-4-hydroxyphenyl)-pyrrolidine-2-carboxylic acid derivatives on the ability of Siberian sturgeon (Acipenser baerii, Brandt, 1869) sperm to utilize superoxide anion radical (O2–•) generated in a model system of adrenaline oxidation in an alkaline medium, as well as exogenous hydrogen peroxide (H2O2) before and after freezing at liquid nitrogen temperature for 3 days was studied. The study of this activity of phenolic derivatives was carried out in comparison with the reference antioxidant Trolox, a water-soluble analogue of vitamin E. When phenolic derivatives were added at a concentration of 0.1 mM to the basic Stein cryomedium containing 12.5% egg yolk, 12.5% DMSO, 5 mM KCl, 130 mM NaCl, 20 mM NaHCO3 and 5.5 mM glucose, a multidirectional effect of the compounds on the ability of Siberian sturgeon sperm to utilize superoxide anion radical and hydrogen peroxide before and after the freezing-defrosting process was established. The studied compounds increase the H2O2-utilizing activity of reproductive cells, while reducing their superoxide-utilizing activity. Pyrrolidine derivatives of phenol were found to be more effective in stimulating the H2O2-utilizing activity of defrosted sperm of the Siberian sturgeon than Trolox. A decrease in the rate of O2– • and H2O2 utilization by fish sperm was found after cryopreservation in the control and in the presence of (3.5-di-tert-butyl-4-hydroxyphenyl)-pyrrolidine-2-carboxylic acid derivatives. A protective effect of new phenolic derivatives on the enzymatic link of antioxidant protection of Siberian sturgeon sperm during cryopreservation was shown. The obtained results indicate the prospects for further studies of the protective activity of (3.5-di-tert-butyl-4-hydroxyphenyl)-pyrrolidine-2-carboxylic acid derivatives in the process of cryopreservation of reproductive cells of rare, endangered and commercially valuable fish species.

Seminal proteoforms from bulls with contrasting semen freezability: a story deciphered by top-down mass spectrometry.

Top-down proteomics was employed to construct proteoform atlas of sperm and seminal plasma (SP) from bulls with low (LF) and high (HF) semen freezability. Sperm and seminal proteins were fractionated by tandem size exclusion chromatography (< 30 kDa) and analyzed by reversed-phase liquid chromatography-tandem mass spectrometry. This approach enabled the identification of 299 SP (from 46 families) and 267 sperm proteoforms (from 139 families). Seventy proteoforms belonging to beta-defensin 10, c-type natriuretic peptide (NPPC), caltrin, seminal ribonuclease, osteopontin, and binder of sperm protein (BSP) 3 families were unique to HF bulls' SP. LF seminal proteins had unique 77 proteoforms, including caltrin, NPPC, osteopontin, BSP3, serpin family A member 5, and β-NGF families. Proteoform families of SP in HF and LF bulls were related to Ca2+ uptake, capacitation, acrosome reaction, sperm protection, fertilization and proteolytic processes. Thirty-three proteoforms of NPPC, caltrin, and cylicin-2 families were upregulated in HF sperm. Twenty-two proteoforms of caltrin, cylicin-2, ATP synthases, and malate dehydrogenase families were among those upregulated in LF sperm. Truncated and acetylated histone H2A and non-truncated and acetylated c-Myc binding protein were prevalent in LF sperm. Cylicin-2 proteoforms were observed in HF and LF sperm, and truncated glyceraldehyde-3-phosphate dehydrogenases, only in HF sperm. In silico analyses indicated the enrichment of mitochondrial metabolic pathways in HF sperm, including fatty acid metabolism and TCA cycle. Our study brings an unprecedented description of the bovine SP and sperm proteoforms. Post-translational processing appears to define the bio-properties of semen proteins and their associations with sperm cryoresistance.

Impact of Vitamin C and Hydroxyurea on the Reproductive System Health in Male Rats

Hydroxyurea (HU) is a medication used to treat various diseases and is also being studied for its effects on spermatogenesis. Vitamin C (VC), an antioxidant, has been shown to protect sperm cells from oxidative stress, potentially improving fertility and sperm quality. In a study involving twenty-four adult male albino rats divided into four groups, Group 1 served as the control, Group 2 received 5 mg of vitamin C, Group 3 received 100 mg of hydroxyurea per body weight, and Group 4 received a combination of vitamin C and hydroxyurea to assess essential functions of the reproductive system, including hormone levels, antioxidant markers, oxidative stress indicators, histopathology, and identification of DNA damage. The study found that hydroxyurea significantly reduced testicular weight, SOD, CAT, and GSH while increasing FSH and MDA levels and causing abnormalities in sperm morphology. Hydroxyurea also caused apparent alterations in the histological structure of the testes and comet parameters. Rats treated with vitamin C showed a significant increase in absolute and relative epididymis weight compared to the control group. Moreover, vitamin C intervention reversed the adverse effects observed in rats fed hydroxyurea, indicating that low doses of vitamin C can protect against testicular damage.

The effect of myo-inositol antioxidant activity on human sperm parameters and DNA damage in ultra-rapid and conventional freezing methods

Male fertility preservation is still challenged by cell damage induced during sperm cryopreservation and impaired sperm structure and function. Sperm ultra-rapid freezing, despite a higher protective effect compared to conventional freezing method, is still associated with suboptimal sperm cryosurvival and needs to be modified to increase its efficiency in sperm protection. Sperm freezing media supplemented with antioxidants can improve sperm parameters following freezing-warming process. In this study, we aimed to investigate the effect of employing ultra-rapid freezing and myo-inositol on sperm cryosurvival. Thirty semen samples with normal sperm parameters were collected and each one was divided into four portions to cryopreserve by conventional freezing, ultra-rapid freezing, conventional freezing + myo-inositol 2 mg/ml, and ultra-rapid freezing + myo-inositol 2 mg/ml. Sperm samples warmed after at least 24 h of freezing and sperm cryosurvival were analyzed by evaluation of sperm motility, viability, morphology and DNA fragmentation index (DFI). Freezing method had a significant influence on post-thaw sperm DFI and morphology (p < 0.05) and the interaction between freezing method and antioxidant supplementation significantly affected sperm morphology (p < 0.05). The highest percentage of sperm normal morphology and minimal DFI was achieved using ultra-rapid freezing supplemented by myo-inositol antioxidant compared to other groups (P < 0.05). The highest sperm DNA damage after freezing-warming was observed following the conventional freezing method. In conclusion, sperm freezing method was identified as factor strongly influencing sperm DFI and morphology after thawing/warming. Sperm samples can be rapidly frozen using the modified freezing media supplemented by myo-inositol without impacting sperm DNA and morphology.

Effect of seminal plasma cholesterol and triacylglycerides concentrations and sperm morphology on semen freezability in domestic cats (Felis silvestris catus)

There is scarce information about the effect of sperm morphology and seminal plasma composition on cat semen freezability. Thus, this study aims to assess the effect of cat sperm morphology and seminal plasma cholesterol (CHOL) and triacylglyceride (TAG) concentrations on sperm post-thaw survival. Ejaculates (n = 49) were evaluated, and seminal plasma was separated and frozen until CHOL and TAG concentrations were measured. The sperm pellet was diluted in a tris-based egg yolk extender, frozen (n = 38), or processed for sperm ultrastructure study (n = 11). Abnormalities recorded were abnormal head shape and size, detached heads, knobbed or ruffled acrosomes, eccentric mid-piece insertion, proximal and distal cytoplasmic droplets, folded and coiled tails, and Dag defect. Ultramicroscopic evaluation detected several sperm abnormalities in fresh semen and some sperm damage in frozen semen. Seminal plasma lipids components were positively correlated with post-thaw motility and acrosome integrity. Higher freezability indices for motility and acrosome integrity were observed in frozen-thawed semen with high seminal plasma CHOL and TAG concentrations. No freezability differences were observed between teratozoospermic and normozoospermic ejaculates. Our results showed that even when seminal plasma was removed before cryopreservation, sperm survival after thawing was significantly higher in samples with high seminal plasma CHOL and TAG concentrations, indicating a rapid adherence to these compounds to the sperm plasma membrane, protecting sperm cells from temperature changes. Nevertheless, there were no differences in sperm freezability by sperm morphology.

A spider mating plug functions to protect sperm.

Mating plugs in animals are ubiquitous and are commonly interpreted to be products of mating strategies. In spiders, however, mating plugs may also take on functions beyond female remating prevention. Due to the vagaries of female genital (spermathecal) anatomy, most spiders face the problem of having to secure additional, non-anatomical, protection for transferred sperm. Here, we test the hypothesis that mating plugs, rather than (or in addition to) being adaptations for mating strategies, may serve as sperm protection mechanism. Based on a comparative study on 411 epigyna sampled from 36 families, 187 genera, 330 species of entelegyne spiders, our results confirm the necessity of a sperm protection mechanism. We divided the entelegyne spermathecae into four types: SEG, SED, SCG and SCD. We also studied detailed morphology of epigynal tracts in the spider Diphya wulingensis having the SEG type spermathecae, using 3D-reconstruction based on semi thin histological series section. In this species, we hypothesize that two distinct types of mating plug, the sperm plug and the secretion plug, serve different functions. Morphological details support this: sperm plugs are formed on a modified spermathecal wall by the spilled sperm, and function as a temporary protection mechanism to prevent sperm from leaking and desiccating, while secretion plugs function in postcopulation both as a permanent protection mechanism, and to prevent additional mating. Furthermore, with the modified spermathecal wall of S2 stalk, the problem of shunt of sperm input and output, and the possibility of female multiple mating have been resolved. Variation in spermathecal morphology also suggests that the problem of sperm protection might be resolved in different ways in spiders. Considering mating plugs of varying shapes and origins in the vast morphospace of spiders, we conclude that mating plugs might serve different purposes that relate both to mating strategies, as well as to sperm protection.

Effect of Conditioned Medium from Human Adipose-Derived Mesenchymal Stem Cells on Human Sperm Quality During Cryopreservation.

The cryopreservation procedure decreases sperm quality, causing certain changes at structural and molecular levels affecting fertilizing ability. We aimed to investigate the impacts of human adipose-derived mesenchymal stem cells (HAd-MSCs) conditioned medium (CM) on the protection of human sperm from cryoinjury. Thirty normal semen specimens were evaluated in this study. Each specimen was separated into six groups and enhanced with varying concentrations of human Ad-MSCs-CM (0, 10, 30, 50, 70, and 100%). Sperm motility, viability, morphology, apoptosis, mitochondrial potential, and lipid peroxidation, and DNA fragmentation were evaluated before freezing and after thawing. The results showed that the total motility was preserved in 10% human Ad-MSCs-CM group. Also, DNA fragmentation was significantly lower in 10% compared to 0% human Ad-MSCs-CM (63.62 ± 17.72% vs.76.46 ± 4.87%, respectively, P < 0.004). Human Ad-MSCs-CM in groups of 10, 30, 50, and 70% reduced lipid peroxidation. The normal sperm morphology rate, mitochondrial membrane potential, and apoptosis showed no significant differences across various groups. It seems that human Ad-MSCs-CM can protect the sperm parameters during the cryopreservation by decreasing cryoinjury.

The proteomic landscape of sperm surface deciphers its maturational and functional aspects in buffalo.

Buffalo is a dominant dairy animal in many agriculture-based economies. However, the poor reproductive efficiency (low conception rate) of the buffalo bulls constrains the realization of its full production potential. This in turn leads to economic and welfare issues, especially for the marginal farmers in such economies. The mammalian sperm surface proteins have been implicated in the regulation of survival and function of the spermatozoa in the female reproductive tract (FRT). Nonetheless, the lack of specific studies on buffalo sperm surface makes it difficult for researchers to explore and investigate the role of these proteins in the regulation of mechanisms associated with sperm protection, survival, and function. This study aimed to generate a buffalo sperm surface-specific proteomic fingerprint (LC-MS/MS) and to predict the functional roles of the identified proteins. The three treatments used to remove sperm surface protein viz. Elevated salt, phosphoinositide phospholipase C (PI-PLC) and in vitro capacitation led to the identification of N = 1,695 proteins (≥1 high-quality peptide-spectrum matches (PSMs), p < 0.05, and FDR<0.01). Almost half of these proteins (N = 873) were found to be involved in crucial processes relevant in the context of male fertility, e.g., spermatogenesis, sperm maturation and protection in the FRT, and gamete interaction or fertilization, amongst others. The extensive sperm-surface proteomic repertoire discovered in this study is unparalleled vis-à-vis the depth of identification of reproduction-specific cell-surface proteins and can provide a potential framework for further studies on the functional aspects of buffalo spermatozoa.

The function of mating plugs in the spider Neriene emphana: mating strategy or sperm protection?

IntroductionIt is generally thought that mating plugs, where present, impede or reduce the possibilities of female subsequent mating. Behavioral studies on numerous spiders, where mating plugs are common, have generally supported this function. However, mating plugs in spiders could plausibly serve other functions as well. Namely, the structure of entelegyne spermathecae—the morphology of most spiders—could require a mechanism that would prevent sperm from leakage, desiccation, and backflow. Although the form and function of mating plugs in several spider species imply their potential adaptation for sperm protection, this function has never been empirically tested.MethodsHere, we test whether mating plugs in the sheet-web spider Neriene emphana serve as a sperm protective device by investigating its genital morphology, its copulation process, and the precise formation of its amorphous mating plugs. ResultsThis species constructs secretion plugs through male-female cooperation. Additionally, we found sperm plugs to be formed as a side product of sperm transfer, as well as an intermediate type of secretion plugs. These plug materials are transferred in different mating stages as documented by variations in the rhythm of male palpal application during copulation. We showed that complete copulations always resulted in formation of secretion plugs at spermathecal entrances via laborious deposition of male materials. DiscussionWhile our findings do not reject that secretion plugs in N. emphana prevent females from subsequent mating, we suggest that they must have evolved to provide sperm protection.

Understanding seminal plasma in male infertility: emerging markers and their implications.

Infertility affects a significant proportion of the reproductive-aged population, with male-associated factors contributing to over half of the cases. However, current diagnostic tools have limitations, leading to an underestimation of the true prevalence of male infertility. While traditional semen parameters provide some insights, they fail to determine the true fertility potential in a substantial number of instances. Therefore, it is crucial to investigate additional molecular targets responsible for male infertility to improve understanding and identification of such cases. Seminal plasma, the main carrier of molecules derived from male reproductive glands, plays a crucial role in reproduction. Amongst its multifarious functions, it regulates processes such as sperm capacitation, sperm protection and maturation, and even interaction with the egg's zona pellucida. Seminal plasma offers a non-invasive sample for urogenital diagnostics and has shown promise in identifying biomarkers associated with male reproductive disorders. This review aims to provide an updated and comprehensive overview of seminal plasma in the diagnosis of male infertility, exploring its composition, function, methods used for analysis, and the application of emerging markers. Apart from the application, the potential challenges of seminal plasma analysis such as standardisation, marker interpretation and confounding factors have also been addressed. Moreover, we have also explored future avenues for enhancing its utility and its role in improving diagnostic strategies. Through comprehensive exploration of seminal plasma's diagnostic potential, the present analysis seeks to advance the understanding of male infertility and its effective management.

Characteristics and Kinematics of Bali Bull Sperms after Thawing Using Tris Soy Lecithin

Soy functions as an extracellular cryoprotectant, which can maintain the integrity of spermatozoa cell membranes with its main content is lecithin. Lecithin from soybeans protects sperm cells from cold stress and reduces the effects of oxidative stress during cryopreservation. This study aimed to analyse the effect of various levels of lecithin diluent on the quality of Bali bull semen during cryopreservation. Semen collection of Bali bull was carried out once a week during four times consecutively using an artificial vagina. The semen was then diluted using the essential ingredient Tris Aminomethan with the addition of powdered soy lecithin; P1 (1 %), P2 (3 %) and P3 (5 %), respectively. Andromed® (K1) as a positive control and Tris without soy lecithin (K2) as a negative control. The parameters observed were motility, progressive motility, kinematics, viability, membrane integrity, and acrosome integrity. The results of this study showed that the dilution of semen with soy lecithin before and after thawing the semen quality was not significantly different (P&lt; 0.05) in motility, viability, plasma membrane integrity, and acrosome integrity. Meanwhile, the kinematics of VAP, VCL, VSL, DAP, DCL, and DSL showed that the average quality increased at P3 compared to K1, K2, P1, and P2, which decreased after thawing or were significantly different (P&lt; 0.05). It can be concluded that Bali bull semen diluted with 3% and 5% of tris soy lecithin produces good characteristics and kinematics, can protect spermatozoa from cold shock.

Minimizing sperm oxidative stress using nanotechnology for breeding programs in rams

BackgroundArtificial insemination (AI) is a routine breeding technology in animal reproduction. Nevertheless, the temperature-sensitive nature and short fertile lifespan of ram sperm samples hamper its use in AI. In this sense, nanotechnology is an interesting tool to improve sperm protection due to the development of nanomaterials for AI, which could be used as delivery vehicles. In this work, we explored the feasibility of vitamin E nanoemulsion (NE) for improving sperm quality during transport.ResultsWith the aim of evaluating this proposal, ejaculates of 7 mature rams of Manchega breed were collected by artificial vagina and extended to 60 × 106 spz/mL in Andromed®. Samples containing control and NE (12 mmol/L) with and without exogenous oxidative stress (100 µmol/L Fe2+/ascorbate) were stored at 22 and 15 ºC and motility (CASA), viability (YO-PRO/PI), acrosomal integrity (PNA-FITC/PI), mitochondrial membrane potential (Mitotracker Deep Red 633), lipoperoxidation (C11 BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (SCSA®) monitored during 96 h. Our results show that NE could be used to maintain ram spermatozoa during transport at 15 and 22 ºC for up to 96 h, with no appreciable loss of kinematic and physiological characteristics of freshly collected samples.ConclusionsThe storage of ram spermatozoa in liquid form for 2–5 d with vitamin E nanoemulsions may lead more flexibility to breeders in AI programs. In view of the potential and high versatility of these nanodevices, further studies are being carried out to assess the proposed sperm preservation medium on fertility after artificial insemination.Graphical

The Impact of Oxidative Stress on Male Reproductive Function: Exploring the Role of Antioxidant Supplementation.

Male reproductive function is highly susceptible to oxidative stress, which arises from an imbalance between reactive oxygen species (ROS) production and antioxidant defense mechanisms. Oxidative stress can significantly impair sperm quality, including count, motility, morphology, and DNA integrity, leading to male infertility. Antioxidants play a crucial role in maintaining reproductive health by neutralizing ROS and protecting sperm cells from oxidative damage. This review article explores the impact of oxidative stress on male reproductive function and investigates the potential benefits of antioxidant supplementation in mitigating its detrimental effects. A comprehensive literature search was conducted to gather relevant studies examining the effects of oxidative stress on male fertility and the outcomes of antioxidant supplementation. The findings reveal that antioxidant supplementation can improve sperm quality, DNA integrity, and fertility outcomes in some individuals. However, conflicting research findings and limitations in study design highlight the need for further investigation. Factors such as individual variations, underlying causes of infertility, dosage, and duration of supplementation should be carefully considered. Lifestyle modifications, including a healthy diet and exercise, are crucial in reducing oxidative stress and optimizing male reproductive health. This review article provides valuable insights into the complex relationship between oxidative stress and male reproductive function, emphasizing the potential role of antioxidant supplementation as a supportive strategy. Further research is warranted to establish optimal protocols, identify specific subgroups that may benefit the most, and explore advancements in antioxidant therapies to improve male fertility outcomes.

Immunolocalization and expression of Siglec5 protein in the male reproductive tract of dromedary camel during rutting season.

Lectins are carbohydrate-binding proteins that are highly selective for sugar groups on other molecules. Siglec5 is a cell-surface lectin that belongs to the sialic acid-binding Ig-like lectins (Siglecs) and acts as a suppressor of immune responses. In this study, immunohistochemistry, western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of Siglec5 in the male reproductive tract of dromedary camels during the rutting season. Siglec5 displayed strong immunostaining in the cranial and caudal testicular regions and moderate immunostaining in the rete testis. Different parts of the epididymis showed varying immunoreactions to Siglec5. The spermatozoa in the testes and epididymis also showed positive immunostaining for Siglec5, whereas, the vas deferens showed negative immunostaining for the protein. The results obtained by western blotting confirmed the immunohistochemical detection of the protein in the testicular and epididymal tissues. The results of qRT-PCR showed that Siglec mRNA was expressed differently in each part of the testis and epididymis; the highest levels of expression were observed in the caudal part of the testis and in the head of the epididymis. In conclusion, the present study revealed that Siglec5 is mainly located in the testis and epididymis, where sperm production and maturation occur. Therefore, this protein may play an essential role in the development, maturation and protection of camel sperm.

Three-dimensional imaging of vascular development in the mouse epididymis.

Long considered an accessory tubule of the male reproductive system, the epididymis is proving to be a key determinant of male fertility. In addition to its secretory role in ensuring functional maturation and survival of spermatozoa, the epididymis has a complex immune function. Indeed, it must manage both peripheral tolerance to sperm antigens foreign to the immune system and the protection of spermatozoa as well as the organ itself against pathogens ascending the epididymal tubule. Although our knowledge of the immunobiology of this organ is beginning to accumulate at the molecular and cellular levels, the organization of blood and lymphatic networks of this tissue, important players in the immune response, remains largely unknown. In the present report, we have taken advantage of a VEGFR3:YFP transgenic mouse model. Using high-resolution three-dimensional (3D) imaging and organ clearing coupled with multiplex immunodetections of lymphatic (LYVE1, PDPN, PROX1) and/or blood (PLVAP/Meca32) markers, we provide a simultaneous deep 3D view of the lymphatic and blood epididymal vasculature in the mature adult mouse as well as during postnatal development.

Beneficial effects of trehalose and gentiobiose on human sperm cryopreservation.

The protection of human sperm during cryopreservation is of great importance to infertility. Recent studies have shown that this area is still a long way from its ultimate aim of maintaining the maximum viability of sperm in cryopreservation. The present study used trehalose and gentiobiose to prepare the human sperm freezing medium during the freezing-thawing. The freezing medium of sperm was prepared with these sugars, and the sperm were then cryopreserved. The viable cells, sperm motility parameters, sperm morphology, membrane integrity, apoptosis, acrosome integrity, DNA fragmentation, mitochondrial membrane potential, reactive oxygen radicals, and malondialdehyde concentration was evaluated using standard protocols. A higher percentage of the total and progressive motility, rate of viable sperm, cell membrane integrity, DNA and acrosome integrity, and mitochondrial membrane potential were observed in the two frozen treatment groups compared to the frozen control. The cells had less abnormal morphology due to treatment with the new freezing medium than the frozen control. The higher malondialdehyde and DNA fragmentation were significantly observed in the two frozen treatment groups than in the frozen control. According to the results of this study, the use of trehalose and gentiobiose in the sperm freezing medium is a suitable strategy for sperm freezing to improve its motion and cellular parameters.

Levels of DNA, Protein, Lipid Oxidation and Apoptosis Biomarkers in Semen of Men with Hyperviscous Semen: A Cross-Sectional Study.

Semen hyperviscosity is a threatening cause of abnormal spermatozoa and infertility in men. We aimed to evaluate oxidative stress, antioxidants depletion and sperm apoptosis as main reasons for poor quality of spermatozoa in men with hyperviscous semen. In this cross-sectional study, ejaculate specimens were collected from fertile (n=102) and infertile men with hyperviscous semen (n=123) and without semen hyperviscosity (n=143). Total antioxidant capacity (TAC), glutathione (GSH), malondialdehyde (MDA), protein carbonyl (PC), 8-hydroxydeoxyguanosine (8-OHdG), and were measured in semen samples to estimate oxidative stress status. Gene expression pattern of BAX, CASPASE-9, CASPASE-3, and BCL2 was assessed to estimate sperm apoptosis. The average of sperm count, normal morphology, normal motility, and sperm vitality in men with hyperviscous semen was significantly lower than infertile subjects without hyperviscous semen (P<0.01). Men with hyperviscous semen exhibited higher levels of PC (8.34 ± 1.03 nmol/mg vs. 6.01 ± 0.93 nmol/mg, P=0.008), MDA (1.14 ± 0.27 nmol/ ml vs. 0.89 ± 0.22 nmol/ml, P=0.031), 8-OHdG (259.71 ± 24.59 ng/ml vs. 197.13 ± 18.47 ng/ml, P=0.009), but lower TAC contents (1250.44 ± 66.23 μM/L vs. 1784.31 ± 89.87 μM/L, P=0.018) and GSH (3.82 ± 1.05 μM vs. 5.89 ± 0.87 μM, P=0.021) than men with non-viscous semen. The expression of BAX, CASPASE-3 and CASPASE-9 genes in men with hyperviscous semen was significantly increased by 1.39-fold (P=0.041), 1.47-fold (P=0.046), 1.29-fold (P=0.048), respectively, as compared with the infertile subjects without hyperviscous semen. However, BCL2 expression in infertile men without hyperviscous semen was higher compared to those with hyperviscous semen (1.36-fold, P=0.044). Hyperviscous semen is markedly associated with depletion of seminal plasma antioxidants, sperm membrane lipid peroxidation, DNA and protein oxidation, and sperm apoptosis. Antioxidant therapy might be considered as a valuable strategy to protect sperm cells against oxidative damage in cases with seminal fluid hyperviscosity.

The Effects of Glycerophospholipid Nanomicelles on the Cryotolerance of Frozen-Thawed Rooster Sperm.

Semen banking is an efficient method of artificial insemination for commercial breeders. However, the cryopreservation process induces severe damages to plasma membranes, which leads to reduced fertility potential of thawed sperm. The replacement of membrane lipids with oxidized membrane lipids repairs the cell membrane and improves its stability. The aim of this study was to investigate the effects of glycerophospholipid (GPL) nanomicelles on the cryosurvival of thawed rooster semen. Semen samples were collected from six 29-week Ross broiler breeder roosters, then mixed and divided into five equal parts. The samples were diluted with the Beltsville extender containing different concentrations of GPL according to the following groups: 0 (GPL-0), 0.1% (GPL-0.1), 0.5% (GPL-0.5), 1% (GPL-1), and 1.5% (GPL-1.5), then diluted semen was gradually cooled to 4°C during 3 hours and stored in liquid nitrogen. The optimum concentration of GPL was determined based on the quality parameters of thawed sperm. Our results showed sperm exposed to GPL-1 had significantly increased motion parameters and mitochondrial activity. The percentages of viability and membrane integrity were significantly higher in the GPL-1, and GPL-1.5 groups compared with the other groups (p < 0.05). Moreover, the lowest rate of apoptosis and lipid peroxidation were observed in the GPL-1 and GPL-1.5 groups in comparison with the frozen control group. Our findings indicated that membrane lipid replacement with GPL nanomicelles (1% and 1.5%) could substitute for damaged lipids in membranes and protect sperm cells against cryoinjury.