Antimicrobial peptides and proteins as alternative antibiotics for porcine semen preservation.

BACKGROUND: Antimicrobial resistance (AMR) is nowadays a major emerging challenge for public health worldwide. The over- and misuse of antibiotics, including those for cell culture, are promoting AMR while also encouraging the research and employment of alternative drugs. The addition of antibiotics to the cell media is strongly recommended in sperm preservation, being gentamicin the most used for boar semen. Because of its continued use, several bacterial strains present in boar semen have developed resistance to this antibiotic. Antimicrobial peptides and proteins (AMPPs) are promising candidates as alternative antibiotics because their mechanism of action is less likely to promote AMR. In the present study, we tested two AMPPs (lysozyme and nisin; 50 and 500 µg/mL) as possible substitutes of gentamicin for boar semen preservation up to 48 h of storage. RESULTS: We found that both AMPPs improved sperm plasma membrane and acrosome integrity during semen storage. The highest concentration tested for lysozyme also kept the remaining sperm parameters unaltered, at 48 h of semen storage, and reduced the bacterial load at comparable levels of the samples supplemented with gentamicin (p > 0.05). On the other hand, while nisin (500 µg/mL) reduced the total Enterobacteriaceae counts, it also decreased the rapid and progressive sperm population and the seminal oxidation-reduction potential (p < 0.05). CONCLUSIONS: The protective effect of lysozyme on sperm function together with its antimicrobial activity and inborn presence in body fluids, including semen and cervical mucus, makes this enzyme a promising antimicrobial agent for boar semen preservation.

Impact of papain on the treatment of raw diluted dromedary semen.

A variety of parameters, including liquefaction and semen viscosity, affect the sperm's ability to travel and reach the egg for fertilization and conception. Given that the details behind the viscosity of the semen in male camels have not yet been fully clarified, the purpose of this study was to ascertain how the addition of papain affected the viscosity of fresh diluted camel semen. The study examined semen samples derived from camels that had distinct viscosities. Sperm motility, viability, abnormal sperm percentage, concentration, viscosity, morphometry, acrosome integrity and liquefaction were among the evaluations following 0, 5, 10, 20 or 30 min of incubation at 37°C with papain (0.004 mg/mL, 0.04 mg/mL or 0.4 mg/mL; a semen sample without papain was used as a control). A statistically significant interaction between the effects of papain concentrations and incubation time was found (F = 41.68, p = .0001). Papain concentrations (p = .0001) and incubation times (p = .0001) both had a statistically significant impact on viscosity, according to a simple main effects analysis. A lower viscosity was found (p < .05) at 0.04 mg/mL (0.1 ± 0.0) after 10 min of incubation. A simple main effects analysis showed that papain concentrations and incubation time have a statistically significant effect on sperm motility (p = .0001). At 0.04 mg/mL papain, the sperm motility % was higher (p < .05) after 10 min (64.4 ± 4.8), 20 min (68.4 ± 6.2), and 30 min incubation (72.2 ± 6.6) compared to 0, 5 min (38.3 ± 4.1 and 51.6 ± 5.0, respectively). In conclusion, the fresh diluted camel semen had the lowest viscosity properties after 10 min of incubation with 0.04 mg/mL papain, without compromising sperm motility, viability, acrosome integrity and sperm morphology.

Associations between seminal plasma metals/metalloids and semen quality: A cross-sectional study.

Exposure to metals/metalloids is reported to potentially influence semen quality. While most studies have focused on single metal impacts, the link between exposure to multiple metals and semen quality has remained less explored. The study aimed to investigate the effects of both individual and mixed metal/metalloid exposure on semen quality. A total of 330 men were recruited from three reproductive centers in eastern China. Seminal plasma levels of 25 metals/metalloids and sperm parameters were determined. We used the Generalized Linear Model (GLM) and Restricted Cubic Spline (RCS) to assess the relationships between single metals/metalloids and semen quality. The weighted quantile sum (WQS) models were then applied to evaluate the combined effect of all these metals/metalloids. We observed positive associations of exposure to lithium (Li), zinc (Zn), and magnesium (Mg) with an increased risk of below reference values for progressive motility and total motility using a logistic regression model (P < 0.05). Additionally, our results also revealed a significant inverse relationship between aluminum (Al) and both sperm concentration and count, while cobalt (Co) demonstrated a positive association with sperm concentration (P < 0.05). Notably, the WQS model indicated a significant positive association between exposure to metal/metalloid mixtures and the risk of abnormal progressive motility (OR: 1.57; 95%CI: 1.10, 2.24) and abnormal total motility (OR: 1.53; 95%CI: 1.06, 2.19), with this association primarily driven by Li, Mg, and Zn. In summary, our findings indicate that exposure to metal/metalloid mixtures might have an adverse effect on semen quality.

Evaluation of equine semen frozen in extenders free of egg yolk using two different freezing curves.

A chemically defined cryopreservation extender that maintains seminal parameters is relevant. Fifteen ejaculates from 5 stallions (n= 5; r=3) were diluted in 5 extenders: 1) EDTA-glucose based extender with egg-yolk and dimethylformamide (EY); 2) commercial equine extender (CE); 3) CE with dimethylformamide (CE-3); 4) bovine commercial extender with liposomes (OP); 5) bovine commercial extender with soybean lecithin (BIO), and frozen using a slow and a rapid temperature descent curve. Post-thaw evaluations were: sperm kinematic parameters, viability and acrosome status, membrane lipoperoxidation and DNA fragmentation. Sperm data were analysed using an ANOVA or Friedman test (results mean ± SD). Paired comparison between the two freezing curves was analysed using the Wilcoxon test. Total and progressive motility were significantly higher (P<0.05) in the EY and CE-3 samples using the slow curve, whereas for the fast curve, total and progressive motility were significantly higher (P<0.05) in the EY samples compared to all the extenders and the samples frozen in CE-3 were significantly higher than the remaining extenders (P<0.05). The percentages of live acrosome intact sperm and of live non-peroxidized sperm were significantly higher (P<0.05) in the EY extender when using either of the freezing curves and in turn, were significantly higher (P<0.05) in samples frozen in CE-3 compared to the remaining extenders. Intact DNA was significantly lower (P<0.05) in the BIO extender, using the rapid curve. To conclude, the commercial equine extender with 3% dimethylformamide, without egg-yolk, could be a suitable alternative for extenders with egg-yolk.

Effect of fumigation height and time on cryopreservation of ram semen.

The cooling rate is a crucial factor in the process of freezing semen, influencing the overall freezing effectiveness. The height and time of fumigation can significantly impact the rate of cooling. Appropriate cooling rates can help minimize the formation of ice crystals in spermatozoa and reduce potential damage to them. Therefore, the aim of this study was to evaluate the effect of different fumigation heights and time for the cryopreservation of Hu ram semen. Experiments I-IV assessed the effect of semen cryopreservation by testing the post-thawed spermatozoa total motility (TM), progressive motility (PM) and kinetic parameters fumigated at distances of 2, 4, 6 and 8 cm for durations of 5, 10, 15 and 20 min, respectively. Based on the results of experiments I to IV, experiment V evaluated the effect of semen cryopreservation by testing the post-thawed spermatozoa TM, PM, kinetic parameters, plasma membrane integrity, acrosome integrity and reactive oxygen species (ROS) level fumigated at distances of 2, 4, 6 and 8 cm for duration of 20 min. The results indicated that fumigation at 2 cm for 20 min significantly (P < 0.05) improved spermatozoa TM, PM, mean angular displacement (MAD), plasma membrane integrity and acrosome integrity compared to other groups. Additionally, it significantly (P < 0.05) reduced spermatozoa ROS level compared to the 6 and 8 cm groups. In conclusion, fumigation for 20 min at a distance of 2 cm from the liquid nitrogen surface is the most suitable cooling method for the cryopreservation of Hu ram semen.

Glyphosate presence in human sperm: First report and positive correlation with oxidative stress in an infertile French population.

Environmental exposure to endocrine disruptors, such as pesticides, could contribute to a decline of human fertility. Glyphosate (GLY) is the main component of Glyphosate Based Herbicides (GBHs), which are the most commonly herbicides used in the world. Various animal model studies demonstrated its reprotoxicity. In Europe, GLY authorization in agriculture has been extended until 2034. Meanwhile the toxicity of GLY in humans is still in debate. The aims of our study were firstly to analyse the concentration of GLY and its main metabolite, amino-methyl-phosphonic acid (AMPA) by LC/MS-MS in the seminal and blood plasma in an infertile French men population (n=128). We secondly determined Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) using commercial colorimetric kits and some oxidative stress biomarkers including malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) by ELISA assays. We next analysed potential correlations between GLY and oxidative stress biomarkers concentration and sperm parameters (sperm concentration, progressive speed, anormal forms). Here, we detected for the first time GLY in the human seminal plasma in significant proportions and we showed that its concentration was four times higher than those observed in blood plasma. At the opposite, AMPA was undetectable. We also observed a strong positive correlation between plasma blood GLY concentrations and plasma seminal GLY and 8-OHdG concentrations, the latter reflecting DNA impact. In addition, TOS, Oxidative Stress Index (OSI) (TOS/TAS), MDA blood and seminal plasma concentrations were significantly higher in men with glyphosate in blood and seminal plasma, respectively. Taken together, our results suggest a negative impact of GLY on the human reproductive health and possibly on his progeny. A precaution principle should be applied at the time of the actual discussion of GLY and GBHs formulants uses in Europe by the authorities.

The use of sodium caseinate in the freezing of sheep semen.

The aim of this study was to assess the addition of 2% sodium caseinate in a commercial egg yolk-based medium in frozen ovine semen. Eight Dorper males were used for the study. The ejaculate was divided into two portions and frozen without (G1) or with the addition of 2% sodium caseinate (G2). Kinetic parameters were evaluated using CASA (computer-assisted sperm analysis), and membrane and acrosome integrity as well as oxidative stress were assessed using flow cytometry. After thawing, a thermoresistance test was conducted at time points T0 and T90. For the fertility test, 100 ewes were inseminated with semen from two rams selected based on in vitro parameters, one with good post-thaw quality (+70% total motility) and the other with low post-thaw quality (-55% total motility). For the fertility test, the females were divided into 4 groups for insemination: low-quality ram without caseinate (GBS = 25) and with caseinate (GBC = 25), and high-quality ram without caseinate (GAS = 25) and with caseinate (GAC = 25). Regarding the results of sperm kinetics, there was a statistically significant difference in the parameters of average path velocity (VAP) and curvilinear velocity (VCL) between the group frozen with BotuBov and the group with added caseinate. At time point T90, straight-line velocity maintained a trend (p < .06), with BotuBov® (BB group) being superior to caseinate this time, and in the linearity parameter, caseinate was superior to BotuBov®. Flow cytometry analysis showed no difference between any of the evaluated tests. In the fertility test, there was no statistically significant difference in the pregnancy rate between the BotuBOV® group (23%, 11/48) and the sodium caseinate group (BC group) (33%, 17/52), and no differences were observed in the male versus diluent interaction (p = .70). In conclusion, sodium caseinate supplementation did not influence sperm kinetic parameters and the fertility of sheep.

Cryopreservation of bovine semen using extract of Cinnamomum zeylanicum.

BACKGROUND: Antioxidants minimise oxidative stress and enhance sperm quality in the process of cryopreservation. OBJECTIVE: To assess the impact of Cinnamomum zeylanicum extract as an additive during the post-dilution and post-thaw stages of Murrah buffalo semen cryopreservation. MATERIALS AND METHODS: The semen sample was diluted using Tris-Egg-Yolk-Citric-Acid-Fructose-Glycerol extender and subsequently divided into three groups: Group 1, TEYCAFG without any additives or controls (C); Group 2, TEYCAFG fortified with a 50 ug/mL aqueous extract of cinnamon (T1); and Group 3, TEYCAFG fortified with a 50 ug/mL ethanolic extract of cinnamon (T2). The evaluation included an assessment of progressive motility, live spermatozoa, sperm abnormalities, HOST, CMPT, and enzyme leakage (GOT and GPT) at both the post-dilution and post-thaw stages. RESULTS: The groups that received cinnamon supplementation demonstrated statistically significant improvements (p<0.05) in various parameters, including an increase in the progressive motility, live spermatozoa, and HOS-positive spermatozoa, as well as greater distance traveled by vanguard spermatozoa compared to the control group. Furthermore, the cinnamon-added groups exhibited a significant decrease (p<0.05) in the percentage of sperm abnormalities and lower enzyme leakage (GOT and GPT) in post-thawed semen. CONCLUSION: Aqueous extract of C. zeylanicum at a concentration of 50 µg/mL provides superior protection of sperm structures and functions as compared to both the ethanolic extract of C. zeylanicum at the same concentration and the control group. Doi.org/10.54680/fr24310110712.

Low concentrations of soybean lecithin nanoparticles had a positive impact on Holstein bulls' cryopreserved semen.

Spermatozoa can experience negative changes when subjected to freezing and thawing, including lowered motility, viability and acrosome response. Herein, the effects of different concentrations of soybean lecithin nanoparticles on cryopreserved Holstein bull semen were examined. Semen was collected, cryopreserved and utilized for sperm kinetic parameter analysis following dilution, equilibration and thawing with 0.5% soybean lecithin (E1), the control extender, and 0.75% (E2), 0.5% (E3), 0.25% (E4) and 0.125% (E5) of lecithin nanoparticles. Results revealed that following dilution, the progressive motility (PM) at E3, E4 and E5 of lecithin nanoparticles was higher (p < .05) than it was for E2. After equilibration, compared to the E1, E2, and E3 values, the PM, vitality, normal morphology, membrane integrity and intact acrosome values at the E5 were consistently greater (p < .05). Comparing the percentages of intact acrosome and membrane integrity at E2 and E3 to E4 and E5, a substantial decrease (p < .05) was seen. Following thawing, the percentage of PM improved at E2 and E5, even though their mean PM values were similar (p > .05) compared to E1, E3 and E4. Vigour and progression parameters of sperm (DAP, DCL, DSL, VAP, VCL, VSL and STR) at E5 were higher (p < .05) than those at E1, E2, E3 and E4. In conclusion, the cryopreserved sperm from Holstein bulls revealed outstanding properties both after equilibration and after thawing with 0.125% lecithin nanoparticles, and they were sensitive to high dosages.

Comparison of different cryoprotectants for freezing donkey (Equus asinus) semen.

The aim of this study was to evaluate two cryoprotectants, dimethylformamide (DMF) and methylformamide (MF) in two concentrations (5 and 7 %) in vitro in donkey semen using a rapid freezing technique and the effect on pregnancy rates in mares. Twenty-four ejaculates from 8 jacks (n = 8; r = 3) were divided into 4 extenders: BotuSemen Gold with 5 % or 7 % MF and 5 % or 7 % DMF, all containing 11 % lactose, 20 % egg-yolk and 0.5 % Equex. Post-thaw evaluations included: sperm motility, membrane function and acrosome status. A linear mixed effect model was used to test the effect of different freezing media on semen parameters. No differences were observed between the 4 freezing media used, for any of the seminal parameters (P > 0.05). However, samples with 5 % DMF showed the highest percentages of sperm with acrosomes and functional membranes (DMF: 5 %: 53.67 ± 22.01; 7 %: 33.92 ± 23.4; MF: 5 %: 44.5 ± 20.46; 7 %: 38.75 ± 27.4) (Data: mean ± SD; P > 0.05). Hence, thirty mares were inseminated: 15 with 5 % DMF and 15 with 7 % DMF. The pregnancy rate was 46 % (7/15) and 0 % (0/15) using the extender with 5 % or 7 % DMF, respectively (P = 0.003). To conclude, the use of 5 % or 7 % of MF or DMF did not affect the in vitro parameters. Despite the lack of differences in vitro with the two DMF concentrations, in vivo results only showed pregnancies when using 5 % DMF. Thus, the results of this study demonstrate the importance of accompanying in vitro semen evaluations with studies that evaluate post-insemination pregnancy rates.

Alpha-lipoic acid improves cryopreservation of rooster semen by reducing oxidative stress.

Inhibiting oxidative stress is key for ensuring sperm motility during semen cryopreservation. The aim of this study was to investigate the effect of adding alpha-lipoic acid (ALA) as an extender in rooster semen cryopreservation. Different concentrations of ALA were added to the frozen diluent of rooster semen; subsequently, computer-aided semen analysis was used to determine membrane functional integrity, acrosome integrity, antioxidant capacity (based on T-AOC, GSH-Px, SOD, CAT, and MDA contents), and mitochondrial integrity. The frozen sperm ultrastructure was observed using transmission electron microscopy. The results showed that the addition of different concentrations of ALA partially to greatly improved the quality of frozen sperm; in particular, 8 µg/mL ALA significantly improved multiple parameters of sperm quality, including sperm motility and antioxidant enzyme activity, after freeze-thaw. The results of this study provide empirical and theoretical support for effective rooster semen cryopreservation and can inform the development of new protective agents in the field of livestock reproduction.

Exposure to potentially toxic elements (PTEs) and the risk of male infertility- A Systematic review and meta-analysis.

BACKGROUND: Infertility has been defined as a failure to conceive for at least 12 months of regular unprotected sexual intercourse. The male factors are responsible for about 50 % of cases. Various factors such as endocrine, immunological, genetic, exposure to toxicants, and idiopathic factors are involved in male infertility. Recently, the role of PTEs in reproductive performance has been explored by various studies. OBJECTIVES: Current systematic review and meta-analysis have been carried out to compile and statistically analyze the findings of relevant studies and reach some conclusion. METHODOLOGY: A literature search was done according to the Preferred Reporting Items for Systematic Reviews and Meta Analyses (PRISMA) guidelines in three scientific literature databases; PubMed, Google Scholar, and Science Direct. Meta-analysis was performed using Review Manager 5.4 software. The study's protocol was registered in PROSPERO (CRD42023465776). RESULTS: Meta-analysis of lead in the blood of infertile cases and healthy controls indicated a significant association with male infertility, observed standard mean difference (SMD) was 0.67 at 95 % confidence interval (CI) (0.07, 1.28), and p = 0.03. In the case of lead analysis in semen, the values are as follows: SMD = 1.19 at 95 % CI (0.42, 1.96) with p = 0.002. Significant association appears for cadmium in semen with SMD 0.92 at 95 % CI (0.54, 1.29) and p < 0.00001. No significant association was observed for arsenic, barium, and mercury in blood. CONCLUSION: Most of the studies focus on the detection of PTE in semen samples followed by blood as sample type. Lead and cadmium exposure is significantly associated with male infertility. However, non-significant results for arsenic, barium, and mercury are observed.

Cryopreservation of human semen by inherently-controlled icing probability: Or how the surface profile of superhydrophobic carbon soot coatings and the sperm volume affect the outcome of slow freezing?

The restoration of initial functionality of human spermatozoa subjected to cryopreservation is challenging, because the deleterious intracellular icing and the occurrence of osmotic shocks due to prolonged exposure to increased concentrations of intracellular solutes are oppositely dependent on the cooling rate. This longstanding problem could be overcome if using superhydrophobic soot coatings delaying the heat transfer rate, reducing the ice formation probability and triggering balanced and timely dehydration of the cells, but the effect of their surface profile and sperm volume on the success rate of slow freezing is unclear. Here, we show for the first time that the two-factor freezing injury is entirely avoidable by tailoring the solid-to-gas voids (pores) fraction in the soot, leading to increased nucleation free energy barrier, presumable incipiency of ice crystals with controllable shape and size and hence, fully (100 %) recovered post-thaw sperm motility. It is demonstrated that the reason for such a unique scientific result is the selection of soot coatings with appropriate morphochemical features, hypothetically (not directly proven yet) inducing equilibrium among the solution composition and ice crystals formation, retarding the undesirable compression of liquid-filled "slush ice" channels surrounding the cytoplasm and impeding the ice recrystallization. The novel insights introduced in this article open endless horizon for customizing and revolutionizing the technical protocols in cryobiology.

Effects of apigenin and trans-ferulic acid on microscopic and oxidative stress parameters in the semen of water buffalo bulls during cryopreservation.

Cryopreservation involves exposing sperm to stressful conditions that affect cell viability. The high quality of the Azerbaijani water buffalo's by-products, such as buffalo milk, makes it a species of significant importance. Our focus is on protecting its genetic resources by preserving its sperm, as their numbers will decrease in the coming years and they are at risk of extinction. This study's goal was to ascertain how apigenin (A) and trans-ferulic acid (t-FA) affected the semen quality of Azari water buffalo bulls under cryopreservation. Pooled buffalo sperm (n = 35 ejaculations) were diluted in a Tris-based diluent also containing varying amounts of apigenin (0.2, 0.4, 0.6, and 0.8 mM) and trans-ferulic acid (2.5, 5, 10 and 20 mM). Following a freeze-thaw procedure, samples were assayed for total antioxidant capacity (TAC), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione activity (GSH), glutathione peroxidase (GPx), progressive motility and total motility, motility properties, plasma membrane functionality, and viability. Sixty days after insemination, the rectal examination was performed on 38 buffaloes that had undergone sexual breeding to confirm pregnancy. The results of the study show that the addition of A-0.2, A-0.4, and t-FA-10 to buffalo semen increases the percentage of intact plasma membrane, motility, and sperm viability, as well as the levels of GSH, GPx, CAT. and TAC. In addition, there is a decrease in MDA and DNA damage after cryopreservation. Furthermore, the results show that 0.4 mM apigenin significantly increases conception rates compared to the control group. The base extender of Tris supplemented with A (0.4 and 0.2 mM) and t-FA (10 mM) improves the antioxidant indices of both frozen and thawed buffalo sperm, which in turn improves post-thawing sperm quality and in vivo fertility improves buffalo sperm.

Effects of organic and inorganic selenium supplementation on frozen-thawed ram semen at two cooling periods.

The aim of this study was to evaluate the effects of different selenium compounds on the sperm quality of cryopreserved ram semen. Ejaculates from four rams, collected using an artificial vagina heated to 38 °C, were individually evaluated. The approved ejaculates were pooled and diluted (1:1 v:v) in Tris-egg yolk extender (20%, v/v) and separated into two control groups, one cooled for 2 h and the other for 4 h. The pooled ejaculates at the two cooling periods were supplemented with two doses (0.5 and 1 µg/mL) of organic selenium (ORG), and inorganic selenium (SeNa), each. The samples were packed in 0.25 ml straws, at a concentration of 400 × 106 sperms/mL and stored in liquid nitrogen. The straws were thawed in a water bath at 37 °C for 20 s, and the samples were subjected to sperm kinetics evaluation by Computer Assisted Semen Analysis software. Sperm membrane integrity, acrosome morphology, and mitochondrial potential were assessed. In addition, oxidative stress markers reactive oxygen species (ROS), ferric reducing antioxidant power (FRAP), thiobarbituric acid reactive species (TBARS), and glutathione peroxidase (GPx) enzyme activity) were also evaluated. No significant improvement was observed in the ram semen quality at the two cooling times. Supplementation of the freezing extender with 0.5 µg/mL ORG, subjected to 4 h cooling period, increased the sperm motility when compared with the control group at the same cooling time. In addition, the 0.5 µg/mL SeNa group, under the 2 h cooling period, showed an increase in sperm motility when compared to the control group at the same cooling period. Considering the importance of sperm motility as a fertility parameter, our study indicates that supplementation with ORG and SeNa can help improve the total motility of the cryopreserved ram semen.

Lactoferrin affects in vitro and in vivo fertilization and implantation in rats.

Lactoferrin (LF) is present in the oviduct, reduces in vitro gamete interaction, and affects sperm capacitation parameters in humans. Our aim was to investigate LF actions on further stages of the reproductive process in the Wistar rat model. Motile sperm were obtained from cauda epididymis to assess LF binding by direct immunofluorescence and LF effect on acrosome reaction (AR) using a Coomassie blue staining. After ovarian hyperstimulation of female rats, oocytes were surgically recovered and coincubated with motile sperm and different doses of LF to estimate the in vitro fertilization (IVF) rate. To evaluate the LF effect on pregnancy and embryo implantation, female rats (80 days old) were placed with males and received daily intraperitoneal injections of LF during one complete estrous cycle (pregnancy experiments) or during the first 8 gestational days (implantation experiments). The number of pregnant females and live born pups was recorded after labor. Moreover, the number of implantation sites was registered during the implantation period. LF was able to bind to the sperm head, midpiece, and tail. 10 and 100 µg/ml LF stimulated the AR but reduced the IVF rate. The administration of 100 and 200 mg/kg LF significantly decreased the number of implantation sites and the litter size, whereas 100 mg/kg LF declined the pregnancy rate. The results suggest that LF might interfere with the reproductive process, possibly interfering with gamete interaction or inducing a premature AR; nevertheless, the mechanisms involved are yet to be elucidated.

Di-2-ethylhexyl phthalate (DEHP) exposure induces sperm quality and functional defects in mice.

Di-2-ethylhexyl phthalate (DEHP) harms mammalian testis development, yet the specific mechanism of its effect on sperm quality and function is unclear. In this study, male mice were administrated DEHP (200 mg/kg/day) via intragastric (i.g.) injection for 35 days. The sperm quality and function of DEHP-exposed mice were evaluated. DEHP exposure reduced the relative testis weight and serum testosterone levels. In addition, sperm count and motility parameters decreased significantly, which led to reduced sperm fertility characterized by reduced acrosome reaction rate, sperm-egg binding capacity and blastocyte formation. DEHP exposure decreased anti-oxidant indicators and the expressions of Cat, Sod1, Prdx6 and Sirt1 in the testis. DEHP-exposure also resulted in decreased proliferating cell nuclear antigen (PCNA) expression in mice testis, as well as the dose-dependent inhibition of the proliferation of GC-1 and GC-2 cells. These phenotypes may be related to increased cell apoptosis characterized by BAX/BCL2 and P53 up-regulation. DEHP exposure resulted in the down-regulation of SIRT1 and p-AKT in mice testis and decreased levels of GC-1and GC-2 cells. DEHP co-incubation with sperm in vitro resulted in decreased tyrosine phosphorylation and progressive motility, as well as p-AKT expression in capacitated sperm. Differential sperm proteomics identified 495 differentially expressed proteins, including 257 proteins down-regulated in the DEHP-exposure group. Bioinformatics analysis showed that proteins involved in sperm-egg interaction and fertilization processes were significantly down-regulated. Pathway analysis demonstrated that the adhesion pathway was enriched in down-regulated proteins, while the pathway associated with ribosomes was enriched in up-regulated proteins. Conclusively, DEHP exposure impaired male fertility by affecting sperm quality and function, and a pathway mediating the DEHP-induced decline in sperm quality and function was identified. The study provides additional information for understanding the molecular mechanisms of DEHP exposure and its effects on male reproduction.

Use of antioxidants and time of cold storage: effects over viability parameters and enzymatic levels in semen of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) / Uso de antioxidantes e tempo de armazenamento a frio: efeitos sobre parâmetros de viabilidade e níveis enzimáticos no sêmen da truta arco-íris (Oncorhynchus mykiss, Walbaum, 1792)

The cold storage of milt implies potentials alterations in its quality because the storage generates as main process, free radicals that produce spermatozoa membrane lipids damage with the consequent motility and fertilising capacity disruptions. To decrease the damage generated by free radicals the cells have antioxidant defences (proteins, enzymes, and low molecular weight substances). The objective of the present study evaluated the time storage effect and different antioxidants prepared in spermatic diluents on sperm viability of O. mykiss milt stored at 4°C. The two-way ANOVA denoted that the time storage and antioxidant influence have significant effects separated or combined on viability parameters (sperm motility and viability, proteins concentrations and superoxide dismutase enzymatic activity in seminal plasma). In contrast, only the storage time affected the fertilising capacity and catalase enzymatic activity in seminal plasma. The resulting analysis can conclude that the antioxidant presence improves the viability of cold stored milt, especially the transport conditions and the antioxidants allow the fecundity despite motility decrease.

O armazenamento a frio de leite implica potenciais alterações em sua qualidade, pois gera como processo principal radicais livres que provocam danos aos lipídios da membrana dos espermatozoides, com as consequentes alterações na motilidade e na capacidade de fertilização. Para diminuir os danos causados pelos radicais livres, as células têm defesas antioxidantes (proteínas, enzimas e substâncias de baixo peso molecular). O presente estudo avaliou o efeito do tempo de armazenamento e diferentes antioxidantes preparados em diluentes espermáticos no armazenamento de viabilidade de O. mykiss milt a 4°C. A ANOVA de duas vias denotou que o armazenamento no tempo e a influência antioxidante têm efeitos significativos separados ou combinados nos parâmetros de viabilidade (motilidade espermática, viabilidade espermática, concentrações de proteínas e atividade enzimática da superóxido dismutase no plasma seminal), enquanto apenas o tempo de armazenamento afetou a capacidade de fertilização e atividade enzimática da catalase no plasma seminal. A análise resultante pode concluir que a presença de antioxidante melhora a viabilidade do leite frio, especialmente as condições de transporte, e os antioxidantes permitem a fecundidade apesar da diminuição da motilidade.

Prenatal exposure to bisphenol S and bisphenol A differentially affects male reproductive system in the adult offspring.

Early exposure to bisphenol may result in adverse reproductive health in later life. The use of bisphenol S (BPS) has increased considerably after bisphenol A (BPA) is regulated worldwide. However, little is known about the fetal exposure to BPS compared with BPA and its effects on the reproductive system in the adult male offspring. Here, we investigated the effects of orally administered BPS and BPA (0.4, 4.0, 40.0 µg/kg bw/d) during gestation (gD4-21) on testicular development by evaluating the sperm DNA damage & methylation and testicular functions in the 90 d Wistar rats. Male offspring prenatally exposed to BPS (0.4 µg/kg) had higher plasma testosterone than BPA and control. The testis histology reveals thickened membrane by producing a wide interstitial gap between seminiferous tubules, increased testicular inflammation, oxidative stress, TIMP-1 expression, and decreased VCAM-1 expression. BPS promotes apoptosis by up-regulating IL-6, cleaved caspases, and a spike in sperm DNA fragmentation. Prenatal BPS exposure reduces sperm motility mediated via impaired PI3K-AKT signaling and increases testicular TEX11 expression in the offspring. Exposure of the fetus to BPS interferes developmental programming of the male reproductive system in the offspring. BPS could be an equally potent endocrine disruptor affecting male reproductive functions.

Impact of calcium and reactive oxygen species on human sperm function: Role of NOX5.

NOX5 is introduced as a new therapeutic target for infertility treatment. This study aimed to compare the basal and stimulated reactive oxygen species (ROS) production and sperm function in human teratozoospermic (n = 15) and normozoospermic (n = 17) semen samples following calcium overload and NOX5 activation. Washed spermatozoa incubated for 1 h under five various conditions: control group, adding a calcium ionophore A23187, phorbol myristate acetate (PMA), A23187 + PMA, and diphenylene iodonium (DPI) + A23187 + PMA. ROS generation was measured immediately after treatment for 30 min. Motility, viability, acrosome reaction, and apoptosis were evaluated after 1-h incubation. ROS production significantly increased when A23187 or PMA was added to the sperm medium. DPI had suppressive effects on ROS generation. Progressive and total motility significantly decreased following calcium elevation and NOX5 activation, which was somewhat returned by DPI. Necrotic and live cells in teratozoospermia was, respectively, higher and lower than normozoospermia samples. Incubation with A23187 significantly increased the percentage of early and late apoptosis. Teratozoosperm are more vulnerable than normal spermatozoa, and produce more basal and stimulated ROS. It seems that calcium overload induces apoptosis in spermatozoa and loss of viability through MPT pore opening and increased intracellular ROS.