Loss Of Sperm Motility Research Articles

Positive In Vitro Effect of ROCK Pathway Inhibitor Y-27632 on Qualitative Characteristics of Goat Sperm Stored at Low Temperatures.

Y-27632, as a cytoskeleton protector, is commonly used for low-temperature preservation of cells. Goat sperm are prone to damage to the cytoskeleton under low-temperature conditions, leading to a loss of sperm vitality. However, the Y-27632 small molecule has not yet been used in research on low-temperature preservation of goat semen. This study aims to address the issue of low temperature-induced loss of sperm motility in goats by using Y-27632, and explore the regulation of Y-27632 on goat sperm metabolism. At a low temperature of 4 °C, different concentrations of Y-27632 were added to the sperm diluent. The regulation of Y-27632 on the quality of low temperature-preserved goat semen was evaluated by detecting goat sperm motility, antioxidant capacity, mitochondrial activity, cholesterol levels, and metabolomics analysis. The results indicated that 20 µM Y-27632 significantly increased plasma membrane integrity (p < 0.05), and acrosome integrity (p < 0.05) and sperm motility (p < 0.05), increased levels of superoxide dismutase (SOD) and catalase (CAT) (p < 0.01), increased total antioxidant capacity (T-AOC) (p < 0.05), decreased levels of malondialdehyde (MDA) and reactive oxygen species (ROS) (p < 0.01), and significantly increased mitochondrial membrane potential (MMP). The levels of ATP, Ca2+, and TC in sperm increased (p < 0.01). Twenty metabolites with significant differences were identified, with six metabolic pathways having a significant impact, among which the D-glutamic acid and D-glutamine metabolic pathways had the most significant impact. The artificial insemination effect of goat semen treated with 20 μM Y-27632 was not significantly different from that of fresh semen. This study indicates that Y-27632 improves the quality of low-temperature preservation of sperm by protecting the sperm plasma membrane, enhancing sperm antioxidant capacity, regulating D-glutamine and D-glutamate metabolism, and promoting the application of low-temperature preservation of semen in artificial insemination technology.

Analysis of sperm separation protocols for isolating cryopreserved human spermatozoa.

Sperm cryopreservation is a valuable tool for the long-term preservation of male fertility. Thus, determining the optimal technique for isolating spermatozoa post-thaw is vital to ensure recovery of the highest quality spermatozoa with minimal iatrogenic damage. This not only enhances the chances of successful conception but also reduces the risk of genetic damage in the embryo. To address this issue, human semen samples were cryopreserved using a slow freezing protocol and Quinn's Advantage™ Sperm Freeze medium. The samples were subsequently thawed and subjected to three types of sperm isolation procedures: direct swim-up, density gradient centrifugation, and electrophoretic separation using the Felix™ device. Cryopreservation led to the anticipated loss of sperm motility and vitality in association with increases in lipid peroxidation and DNA damage. Following sperm selection, all three isolation techniques resulted in an increase in sperm motility which was particularly evident with the swim-up and Felix™ procedures. The latter also significantly improved sperm vitality. There were no differences between sperm separation techniques with respect to morphology, and mitochondrial reactive oxygen species generation remained essentially unchanged when cell vitality was taken into account. By contrast, major differences were observed in DNA integrity and lipid aldehyde formation, where Felix™ isolated cells exhibiting significantly less DNA damage than the other isolation procedures as well as lower levels of 4-hydroxynonenal formation. Electrophoretic sperm isolation, therefore, offers significant advantages over alternative separation strategies, in terms of the quality of the gametes isolated and the time taken to achieve the isolation. Long-term storage of sperm is vital to assisted reproductive technology because it permits the preservation of fertility that might be compromised as a result of factors such as chemotherapy or vasectomy. This goal can be achieved via cryopreservation - the freezing of cells to -196°C. When the sperm are subsequently required for conception, they must be carefully separated from the cryopreservation medium in a manner that maximizes the chances of successful conception and minimizes the risk of genetic defects in the offspring. In this paper, three isolation techniques were compared for their ability to separate ideal sperm from semen and media following cryopreservation. It was found that cryopreservation led to lower levels of motility and vitality and created higher levels of DNA and cell membrane damage. Of the three techniques compared, only cells separated on the basis of their size and electric charge (electrophoretic isolation) exhibited significantly lower levels of DNA fragmentation.

Growth Dynamic and Threshold Values for Spermicidal Effects of Multidrug-Resistant Bacteria in Extended Boar Semen.

The aim of this study was first to examine the prevalence of bacteria-associated loss of sperm quality in samples from insemination centers during a seven-year semen monitoring program and, second, to investigate the growth dynamic of four different multidrug-resistant bacterial species and their impact on sperm quality during semen storage. A reduced sperm quality associated with bacterial contamination was found in 0.5% of 3219 of the samples from insemination centers. In samples spiked with Serratia marcescens and Klebsiella oxytoca, bacterial growth by six log levels was seen during storage at 17 °C, causing loss of sperm motility, membrane integrity, membrane fluidity, and mitochondrial membrane potential at >107 CFU/mL (p < 0.05). Storage at 5 °C in the Androstar Premium extender efficiently inhibited their growth. Achromobacter xylosoxidans and Burkholderia cepacia showed limited growth up to two log levels at 17 °C and did not impair sperm quality. In conclusion, spermatozoa tolerate moderate loads of multidrug-resistant bacteria, and hypothermic, antibiotic-free semen storage effectively limits bacterial growth. The constant use of antibiotics in semen extenders should be reconsidered.

Quantitative proteomics of sperm tail in asthenozoospermic patients: exploring the molecular pathways affecting sperm motility.

Asthenozoospermia, characterized by low sperm motility, is one of the most common causes of male infertility. While many intrinsic and extrinsic factors are involved in the etiology of asthenozoospermia, the molecular basis of this condition remains unclear. Since sperm motility results from a complex flagellar structure, an in-depth proteomic analysis of the sperm tail can uncover mechanisms underlying asthenozoospermia. This study quantified the proteomic profile of 40 asthenozoospermic sperm tails and 40 controls using TMT-LC-MS/MS. Overall, 2140 proteins were identified and quantified where 156 proteins have not been described earlier in sperm tail. There were 409 differentially expressed proteins (250 upregulated and 159 downregulated) in asthenozoospermia which by far is the highest number reported earlier. Further, bioinformatics analysis revealed several biological processes, including mitochondrial-related energy production, oxidative phosphorylation (OXPHOS), citric acid cycle (CAC), cytoskeleton, stress response, and protein metabolism altered in asthenozoospermic sperm tail samples. Collectively, our findings reveal the importance of mitochondrial energy production and induced stress response as potential mechanisms involved in the loss of sperm motility in asthenozoospermia.

Duration effects of alcohol graded concentrations on the extent of lipid peroxidation, testis morphology and sperm quality assessment in Wistar rats

Alcohol consumption is known to cause an array of alcohol-induced biochemical changes in a biological system. This study investigated the durations effects of different alcohol concentrations (30%, 40%, and 50%) on malondialdehyde levels, testes histology, and sperm characteristics in matured male Wistar rats. The rats were divided into four groups namely thus; control, 30%, 40% and 50%. Control group was orally administered 0% alcohol while, group 30%, 40% and 50% received orally 30%, 40% and 50% of alcohol concentrations (3.20 g/ Kg body weight) respectively for maximum durations of 28 days. On the day 1, 7, 14, 21, and 28, five rats from each group (control, 30%, 40% and 50% alcohol) were sacrificed, and malondialdehyde levels, testes histology, and sperm characteristics were examined.Graded alcohol concentrations caused different detrimental effects on sperm characteristics and induced pathological lesions in the testes. Significant increases in serum, liver and testes malondialdehyde levels were durations independent but almost entirely concentrations dependent.Ultimately, administration of alcohol graded concentration led to loss of sperm motility and testicular degeneration in concentration and durations dependent manner without a concomitant increase in the malondialdehyde levels.

Folic acid protects against fluoride-induced oxidative stress and testicular damage in rats

Objective: To investigate the effects of folic acid on testicular oxidative damage in sodium fluoride-induced male Wistar rats. Methods: A total of 24 male Wistar rats were divided into 4 groups: the control, sodium fluoride (fed with 100 mg/L sodium fluoride through drinking water orally for 21 days), folic acid (36 μg/kg body weight/day, orally), and sodium fluoride plus folic acid (received similar dose orally) groups. At the end of 21 days, epididymal sperm parameters, biochemical analysis of testicular tissue, and serum hormonal levels were performed along with histopathological studies. Results: Sodium fluoride intoxication resulted in marked reduction in gonado somatic index, serum luteinizing hormone, and testosterone level along with 3 β -hydroxysteroid dehydrogenase and 17 β -hydroxysteroid dehydrogenase activities. In addition, reduction in sperm density, as well as loss of sperm motility and sperm viability, were also observed. Besides, increased levels of testicular malondialdehyde, nitrite, interleukin-6 and tumor necrosis factor- α as well as decreased levels of superoxide dismutase and catalase activities and reduced glutathione content were found to be associated with this toxicity. Folic acid co-treatment, on the other hand, could prevent all the sodium fluoride-induced testicular pathophysiology and oxidative stress related parameters. Histological examinations of testicular sections from the experimental rats supported these results. Conclusions: Combining all, this study suggests that being an antioxidant, folic acid plays a beneficial role against fluoride-induced adverse effects on the male reproductive system.

Monitoring the reactive oxygen species in spermatozoa during liquid storage of boar semen and its correlation with sperm motility, free thiol content and seasonality.

Oxidative stress is an important factor affecting the quality of spermatozoa during liquid storage of boar semen; however, monitoring of reactive oxygen species (ROS) that provides direct insight into the oxidative status is not yet attempted. This study aimed to monitor ROS in boar sperm during liquid semen storage to determine its correlation with sperm motility and free thiol (SH) content, and seasonality. Ejaculate was collected from mature Duroc boars in a commercial farm in autumn and spring, diluted in Mulberry III extender, stored at 15°C, and examined daily for sperm ROS level, SH content and motility. The ROS levels in spermatozoa prepared during autumn and spring were constantly low until days 4 and 5 of storage, respectively, which thereafter progressively increased in association with the loss of sperm motility. The increased sperm ROS level correlated with the higher SH level and lower motility, which was accentuated from day 4 of storage and was higher in September, or early autumn. This study indicates that increased sperm ROS levels during liquid storage results in oxidative damage, causing loss of sperm motility, presumably through decreased sperm viability, suggesting that sperm ROS monitoring effectively evaluates the quality of boar semen.

High-throughput droplet vitrification of stallion sperm using permeating cryoprotective agents

Stallion sperm is typically cryopreserved using low cooling rates and low concentrations of cryoprotective agents (CPAs). The inevitable water-to-ice phase transition during cryopreservation is damaging and can be prevented using vitrification. Vitrification requires high cooling rates and high CPA concentrations. In this study, the feasibility of stallion sperm vitrification was investigated. A dual-syringe pump system was used to mix sperm equilibrated in a solution with a low concentration of CPAs, with a solution containing a high CPA concentration, and to generate droplets of a defined size (i.e., ~20 μL) that were subsequently cooled by depositing on an aluminum alloy block placed in liquid nitrogen. Mathematical modeling was performed to compute the heat transfer and rate of cooling. The minimum CPA concentration needed for vitrification was determined for various CPAs (glycerol, ethylene glycol, propylene glycol, dimethyl sulfoxide) and combinations thereof, while effects of droplet size and carrier solution were also identified. Sperm vitrification was eventually done using a glycerol/propylene glycol (1/1) mixture at a final concentration of 45% in buffered saline supplemented with 3% albumin and polyvinylpyrrolidon, while warming was done in standard diluent supplemented with 100 mM sucrose. The sperm concentration was found to greatly affect sperm membrane integrity after vitrification-and-warming, i.e., was found to be 21 ± 12% for 10 × 106 sperm mL−1 and 54 ± 8% for 1 × 106 sperm mL−1. However, an almost complete loss of sperm motility was observed. In conclusion, successful sperm vitrification requires establishing the narrow balance between droplet size, sperm concentration, CPA type and concentration, and exposure time.

Non-programmable cryopreservation of sperm from industrial-farmed Murray cod (Maccullochella p peelii)

The high price of larvae is a major constraint for the expansion of Murray cod (Maccullochella p peelii) aquaculture. Sperm cryopreservation creates a ready supply of sperm for the production of larvae, reduces the number of males required by hatcheries and contributes to preserving desirable genetic traits for breeding programmes. For hatchery application, there is a requirement for easy-to-implement cryopreservation protocols. Important factors for the development of a practical non-programmable sperm cryopreservation method for industrial-farmed Murray cod were evaluated. The results showed that Murray cod sperm can be successfully cryopreserved using dimethyl sulfoxide (Me2SO), propylene glycol (PG) and methanol (MeOH) as cryoprotectant additives (CPAs). In 0.5 mL straws, 10% PG proved to be the most effective CPA (post-thaw motility 75 ± 7%), followed by 10% MeOH (62 ± 7%). At the same time, in 2 mL cryovials, 10% PG was significantly more effective than 10% MeOH at three chosen heights (P<0.05). In 0.5 mL straws, cooling rates of 11–22 °C/min from 20 °C to −5 °C provided better results than cooling rates of 4–6 °C/min in 2 mL cryovials. Post-thaw motility was greater, regardless of CPA, without a 5.0 min equilibration period (Me2SO, from 8 to 34%; PG, from 25 to 75%; MeOH, from 19 to 62%). Toxicity tests indicated a marked loss of sperm motility following dilution with cryoprotectants; MeOH was less toxic than Me2SO and PG and the toxicity of either Me2SO or PG increased according to exposure duration. Therefore, the results suggest that on-site non-programmable sperm cryopreservation with 10% PG is effective and efficient for industrial-farmed Murray cod, yielding an acceptable percentage of post-thaw motile sperm. However, minimising equilibration time before cooling and maintaining cooling rates of ~11 °C/min between 20 °C and − 5 °C may be crucial for reducing CPA-associated toxicity. Further research is required in order to confirm that the recovered sperm motilities which are shown in this study translate into fertility during the process of in vitro fertilisation.

Fertilization potential of cold-stored Fowler&#x2019;s toad (Anaxyrus fowleri) spermatozoa: temporal changes in sperm motility based on temperature and osmolality.

Asynchrony of gamete release is problematic in amphibian captive breeding programs but can be overcome by short-term storage of spermatozoa. Hormonally induced sperm from the model species Anaxyrus fowleri were used to determine storage conditions for optimal fertilisation capacity. Sperm motility was measured over time, as a function of storage temperature (4°C or 22°C) and solution osmolality (7-40mOsm/kg). Sperm at 40mOsm/kg (spermic urine) stored at 4°C exhibited higher motility compared to 22°C. Also, sperm stored at 40mOsm/kg retained higher motility compared to sperm stored below 15mOsm/kg at both temperatures. Under optimal storage conditions (40mOsm and 4°C) a 30% decrease in sperm motility occurred within 24h, however, subsequent loss of sperm motility was lower (<10%/day) for days2-8 thereafter. Sperm samples stored for 1-8days under optimal conditions were tested for fertilising capacity by conducting in vitro fertilisation trials. Sperm stored for 8days yielded 48% neurula development, similar to sperm stored for 1day, which produced 60% neurula development. Overall, sperm stored for up to 8days at 4°C as spermic urine retained fertilising capacity and thus can be used to circumvent asynchronous gamete release in assisted breeding efforts for amphibians.

Effect of Sodium Nitrate (NaNO3) on Sperm Motility and Abnormality: An In vitro Approach

Nitrates (NO3 ) are the naturally occurring inorganic ions with beneficial effects in both plants and animals. However, NO3 in excess has adverse effect on male and female reproductive systems. The study was undertaken to investigate the effect of 10mg/ ml and 100mg/ml of sodium nitrate (NaNO3 ) on sperm motility and abnormality at different time intervals (5,10,15,20,25 and 30 min). Treatment of cauda epididymal sperm suspension with 10mg/ml of NaNO3 significantly reduced the sperm motility only at 25 and 30 min time intervals compared to controls. However, 100mg/ml of NaNO3 resulted in significant reduction of sperm motility from 5 min to 30 min time interval in time dependent manner and complete loss of sperm motility was observed at 15 min of incubation. In addition, the number of abnormal spermatozoa was significantly high in 100mg/ml of NaNO3 treated groups compared to controls and 10 mg/ml of NaNO3 . These results clearly reflected the toxic effect of NO3 on spermatozoa and its capability in fertilization.

Use of Glass-Forming Water–Salt Systems for Preparing Human Sperm to Hypothermic Semen Preservation

A protectant for hypothermic preservation of human sperm based on glass-forming water–salt systems of vital metals (magnesium, zinc, and calcium) was developed for the first time. The method of sperm preservation without preliminary washing of seminal plasma is proposed, which retains the sperm viability and sperm motility parameters comparable to the values of native sperm. The proposed protectant and method of preparing human sperm for hypothermic storage at the first stage help to slow down the metabolism as evidenced by the complete loss of sperm motility and maintenance of the nearly 100% viability of the studied sperm. Once the protectant was removed, sperm motility is restored up to 90–100% compared to the indices of native sperm.

Knockout of serine-rich single-pass membrane protein 1 (Ssmem1) causes globozoospermia and sterility in male mice†.

Globozoospermia (sperm with an abnormally round head shape) and asthenozoospermia (defective sperm motility) are known causes of male infertility in human patients. Despite many studies, the molecular details of the globozoospermia etiology are still poorly understood. Serine-rich single-pass membrane protein 1 (Ssmem1) is a conserved testis-specific gene in mammals. In this study, we generated Ssmem1 knockout (KO) mice using the CRISPR/Cas9 system, demonstrated that Ssmem1 is essential for male fertility in mice, and found that SSMEM1 protein is expressed during spermatogenesis but not in mature sperm. The sterility of the Ssmem1 KO (null) mice is associated with globozoospermia and loss of sperm motility. To decipher the mechanism causing the phenotype, we analyzed testes with transmission electron microscopy and discovered that Ssmem1-disrupted spermatids have abnormal localization of Golgi at steps eight and nine of spermatid development. Immunofluorescence analysis with anti-Golgin-97 to label the trans-Golgi network, also showed delayed movement of the Golgi to the spermatid posterior region, which causes failure of sperm head shaping, disorganization of the cell organelles, and entrapped tails in the cytoplasmic droplet. In summary, SSMEM1 is crucial for intracellular Golgi movement to ensure proper spatiotemporal formation of the sperm head that is required for fertilization. These studies and the pathway in which SSMEM1 functions have implications for human male infertility and identifying potential targets for nonhormonal contraception.

Mass Spectrometry Reveals New Insights into the Production of Superoxide Anions and 4-Hydroxynonenal Adducted Proteins in Human Sperm.

The free-radical theory of male infertility suggests that reactive oxygen species produced by the spermatozoa themselves are a leading cause of sperm dysfunction, including loss of sperm motility. However, the field is overshadowed on several fronts, primarily because: i) the probes used to measure reactive oxygen species (ROS) are imprecise; and ii) many reports suggesting that oxygen radicals are detrimental to sperm function add an exogenous source of ROS. Herein, a more reliable approach to measure superoxide anion production by human spermatozoa based on MS analysis is used. Furthermore, the formation of the lipid-peroxidation product 4-hydroxynonenal (4-HNE) during in vitro incubation using proteomics is also investigated. The data demonstrate that neither superoxide anion nor other free radicals that cause 4-HNE production are related to the loss of sperm motility during incubation. Interestingly, it appears that many of the 4-HNE adducted proteins, found within spermatozoa, originate from the prostate. A quantitative SWATH analysis demonstrate that these proteins transiently bind to sperm and are then shed during in vitro incubation. These proteomics-based findings propose a revised understanding of oxidative stress within the male reproductive tract.

Seminal plasma has limited counteracting effects following induction of oxidative stress in donkey spermatozoa.

The aim of this study was to evaluate the response of donkey spermatozoa to oxidative stress induced by hydrogen peroxide, and to determine whether the presence of seminal plasma modulates the sperm response to that stress. Nine ejaculates were collected, extended in skim milk extender and split into two aliquots. Seminal plasma was removed from the first but not second aliquot. Samples were subsequently split into four aliquots supplemented with different concentrations of commercial hydrogen peroxide (0, 100 and 250µM and 50mM). Aliquots were incubated at 37°C under aerobic conditions and several sperm parameters, namely motility, viability, intracellular levels of peroxides and superoxides and mitochondrial membrane potential, were evaluated at 0, 1 and 3h. Exposure to hydrogen peroxide markedly decreased sperm motility but had much less of an effect on sperm viability, mitochondrial membrane potential and intracellular reactive oxygen species levels. A protective effect of seminal plasma against the loss of sperm motility was not apparent, but some kinetic parameters and relative levels of superoxides were better maintained when seminal plasma was present together with high concentration of hydrogen peroxide. In conclusion, oxidative stress induced by hydrogen peroxide reduces donkey sperm motility and has a less apparent effect on other sperm parameters. Finally, seminal plasma is only able to partially ameliorate the detrimental effect of this induced stress.

Influence of cryopreservation on structure and function of mammalian spermatozoa: an overview.

Cryopreservation is a useful approach to preserve male fertility for assisted reproduction technique and other evaluations. In spite of extensive development in the cryopreservation field, there are biological and biochemical points including DNA fragmentation and antioxidant which should be illuminated to preserve fertility in safe form. Several molecular markers such as coding and noncoding RNAs are transferred from spermatozoa into oocyte via fertilization. These biomarkers affect fertility potential during the cryopreservation. Despite its impact, sperm cryopreservation has some destructive effect including loss of sperm motility and viability, acrosomal damage, mitochondrial membrane depolarization, plasma membrane permeability alteration even nuclear, and DNA damage. There is a growing concern about the impact of sperm cryopreservation on biological factors which can interrupt successful fertility procedures such as in vitro fertilization. Additionally, cryo-damage can decrease embryonic development extent. Promoting cryopreservation method via investigating the wide range of non-invasive factors may be increasingly important to have access to safe approach of freezing. Therefore, the aim of this study is the assessment of biological factors which can penetrate the fertility potential during the freezing procedure, explicate innovative methods in fertility preservation.

Perfluoro-octanoic acid impairs sperm motility through the alteration of plasma membrane.

Perfluoroalkyl-substances (PFAS) are chemical additives considered harmful for humans. We recently showed that accumulation of perfluoro-octanoic acid (PFOA) in human semen of exposed subjects was associated with altered motility parameters of sperm cells, suggesting direct toxicity. To determine whether direct exposure of human spermatozoa to PFOA was associated to impairment of cell function. Spermatozoa isolated from semen samples of ten normozoospermic healthy donors were exposed up to 2h to PFOA, at concentrations from 0.1 to 10ng/mL. Viability and motility parameters were evaluated by Sperm Class Analyser. Cell respiratory function was assessed by both mitochondrial probe JC-1 and respiratory control ratio (RCR) determination. Sperm accumulation of PFOA was quantified by liquid chromatography-mass spectrometry. Expression of organic ion-transporters OATP1 and SLCO1B2 was assessed by immunofluorescence and respective role in PFOA accumulation was evaluated by either blockade with probenecid or membrane scavenging through β-cyclodextrin (β-CD). Plasma membrane fluidity and electrochemical potential (ΔΨp) were evaluated, respectively, with Merocyanine-540 and Di-3-ANEPPDHQ fluorescent probes. Compared to untreated controls, a threefold increase of the percentage of non-motile sperms was observed after 2h of exposure to PFOA regardless of the concentration of PFOA, whilst RCR was significantly reduced. Only scavenging with β-CD was effective in reducing PFOA accumulation, suggesting membrane involvement. Altered membrane fluidity, reduced ΔΨp and sperm motility loss associated with exposure to PFOA were reverted by β-CD treatment. PFOA alters human sperm motility through plasma-membrane disruption, an effect recovered by incubation with β-CD.

Development of an efficient and standardized method for the cryopreservation of Arctic charr milt and its use in the fertilization of brook trout eggs to produce ‘sparctic’ hybrids

The general objective of this study was to establish a standardized procedure for the cryopreservation of Arctic charr milt and its use in the production of ‘sparctic’ hybrids. The specific objectives of this study were to determine: (i) the optimal final sperm concentration as well as (ii) the final glucose concentration, (iii) the effect of post-thaw storage time on sperm motility characteristics, and (iv) the fertilization ability of cryopreserved Arctic charr milt at 0 and 60 min after thawing at sperm:egg ratios of 500,000:1 and 1,000,000:1. We observed high post-thaw sperm motility (49–57%) at final sperm concentrations ranging from 0.5 to 2.0 × 109 spermatozoa ml−1 in straw. We determined that a final glucose concentration of 0.15–0.19 M in 7.5% methanol was optimal (post-thaw sperm motility 48–60%). Milt cryopreserved at optimal conditions (final concentrations: 0.17 M of glucose, 7.5% methanol and 1.5 × 109 spermatozoa ml−1) could be stored for up to 15 min after thawing without the loss of sperm motility. The ability of cryopreserved milt to fertilize brook trout eggs was high (68–72%) and did not differ between the tested sperm:egg ratios (500,000:1 and 1,000,000:1) when milt was used immediately after thawing or after 60 min of post-thaw storage. The milt used in this study was obtained from two hatcheries and was characterized by significantly different values of sperm concentrations. In conclusion, a standardized and efficient cryopreservation method for Arctic charr milt has been developed and used for the production of ‘sparctic’ hybrids. Species-specific requirements for final sperm and glucose concentrations and the post-thaw storage time were determined. The hatchery of origin of Arctic charr should be taken into consideration, as high variability in sperm characteristics may be expected. The development of this method and the positive results of the fertilization trials indicate promising potential for the implementation of cryopreserved milt in hatchery practice.

RSBP15 interacts with and stabilizes dRSPH3 during sperm axoneme assembly in Drosophila

Flagellum in sperm is composed of over 200 different proteins and is essential for sperm motility. In particular, defects in the assembly of the radial spoke in the flagellum result in male infertility due to loss of sperm motility. However, mechanisms regulating radial spoke assembly remain unclear in metazoans. Here, we identified a novel Drosophila protein radial spoke binding protein 15 (RSBP15) which plays an important role in regulating radial spoke assembly. Loss of RSBP15 results in complete lack of mature sperms in seminal vesicles (SVs), asynchronous individualization complex (IC) and defective “9 + 2” structure in flagella. RSBP15 is colocalized with dRSPH3 in sperm flagella, and interacts with dRSPH3 through its DD_R_PKA superfamily domain which is important for the stabilization of dRSPH3. Moreover, loss of dRSPH3, as well as dRSPH1, dRSPH4a and dRSPH9, showed similar phenotypes to rsbp15KO mutant. Together, our results suggest that RSBP15 acts in stabilizing the radial spoke protein complex to anchor and strengthen the radial spoke structures in sperm flagella.

Electrophoretic profile of seminal proteins and their correlation with in vitro sperm characters in Black Bengal buck semen.

Aim:This study aimed to study the electrophoretic properties of seminal plasma and sperm proteins of Black Bengal buck semen and their correlation with in vitro sperm characters and freezability.Materials and Methods:Semen ejaculates from nine Black Bengal bucks were collected by artificial vagina (n=20/buck). Ejaculates were evaluated for in vitro sperm characters and electrophoretic profile of seminal protein. In vitro sperm characters were evaluated immediately after collection, after completion of equilibration period, and after freeze-thawing. For seminal protein studies, seminal plasma proteins were precipitated by ice-cold ethanol method, and sperm proteins were extracted by Triton X detergent extraction method. Discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to assess the molecular weight of seminal proteins. Correlation between in vitro sperm characters and protein bands was determined by Pearson’s correlation coefficient, and two-way ANOVA was applied to find the individual buck differences.Results:Significant difference (p<0.01) among the bucks was noticed in the in vitro sperm characters evaluated at all the three stages of semen evaluation such as immediately after collection, after completion of equilibration period, and post-freeze thawing. Progressive loss of sperm motility, membrane integrity, and other in vitro sperm characters were noticed during cryopreservation. A total of ten protein bands in the molecular weight ranging from 17 to 180 kDa were found in the SDS-PAGE of seminal plasma proteins, while nine bands of 17-134 kDa were observed in sperm proteins. Seminal plasma proteins of molecular weight 75, 62-49, 20, and 17 kDa and sperm proteins of 75, 20, and 17 kDa were present in all the nine bucks (100%) screened, and variation among the bucks was noticed for the presence of other proteins. Seminal plasma protein of 180-134 kDa showed a negative correlation with individual motility (−0.716) and functional membrane integrity of sperm cells (−0.724) in post-freeze–thaw analysis and 48 kDa protein had a positive correlation with individual motility (0.649) and functional membrane integrity of sperm cells (0.664) in post-thaw analysis. Sperm proteins of 63 kDa had a negative correlation (−0.616) with sperm concentration in neat semen.Conclusion:Variation among the bucks was noticed in the in vitro sperm characters and semen freezability. Correlation between seminal proteins and in vitro sperm characters and semen freezability had been found which might be useful as a tool to select breeding bucks.