Coenzyme Q10 improves the quality and in vitro fertility of post-thawed buffalo (Bubalus bubalis) semen via its antioxidative effect.

This study aimed to determine the effects of Coenzyme Q10 (CoQ10) in the freezing medium on functional and oxidative stress parameters and in vitro fertilization (IVF) rate of buffalo sperm. Collected samples were relocated to the laboratory for initial evaluation, gentle dilution in extenders, cooling (4°C, 2 h), equilibration (4°C, 4 h), packaging (straws, 0.5 mL), programmable freezing, and thawing (37°C, 30 s). Statistical analysis depicted that adding CoQ10 (100 µM) in a freezing medium caused a significant augmentation in total motility (%), average path, and straight-line velocities (µm/sec) of buffalo sperm than control. Adding CoQ10 (100 µM) improved sperm progressive motility, rapid velocity, and functional parameters (%) compared to the control and 10 µM of CoQ10. Moreover, CoQ10 in a freezing medium caused a significant augmentation in seminal plasma catalase (U/mL) and glutathione reductase (GSH; nmol/109 ) at 100 µM than control and other treatments. CoQ10 inclusion (100 µM) ameliorates seminal plasma superoxide dismutase (U/mL), glutathione-S-transferase (GST; nmol/mL/min) fructose (µg/mL), and ATP (nmol/million) than control. Furthermore, CoQ10 at 100 µM improved seminal plasma glutathione peroxidase (µM) levels than control, 10 µM, and 20 µM. Lastly, hydrogen peroxide (H2 O2; nM) production was significantly lower at 100 µM than at control and 10 µM. CoQ10 (100 µM) caused a significant augmentation in the un-capacitated pattern followed by a reduction in the capacitated pattern, and apoptosis-like changes (%) than control, and other treatments, whereas viability was increased than control and other treatments. CoQ10 (100 µM) significantly improved the IVF rate in comparison with control, CoQ10 at 10 µM, and 20 µM groups. In conclusion, the addition of CoQ10 (100 µM) in the freezing medium can improve the quality and in vitro fertility of post-thawed buffalo semen via its antioxidative effect. Further studies are needed to evaluate the effect of CoQ10 on the in vivo fertility of buffalo bull semen.

Determination of biochemical and histopathological changes on testicular and epididymis tissues induced by exposure to insecticide Imidacloprid during postnatal development in rats.

BACKGROUND: Imidacloprid is a neonicotinoid insecticide belonging to the chloronicotinyl nitroguanidine chemical family. Toxicity of IMD for mammals in scientific studies has shown high mutagenic, immunotoxic, teratogenic and neurotoxic effects. The present study was designed to assess the toxic effects of imidacloprid (IMD) on the testicular and epididymis tissues as well as testosterone levels of neonatal male rats. METHODS: Neonatal male rats from postnatal day (PND) 1 to PND 26 were consecutively administered with different concentrations of IMD (1, 5 and 10 mg/kg) subcutaneously. The effect of IMD on body and organ weight, lipid profile, histopathological alterations, oxidative stress and altered testosterone levels were assessed in the testis and plasma. RESULTS: The results of body weight gain showed a significant difference in group 4 (10 mg/kg) animals as compared to the control. A significant increase in total cholesterol and triglycerides, while a decrease in high-density lipoprotein concentrations was evident. Similarly, a significant decrease in concentrations of antioxidant enzymes (CAT and SOD) among all the IMD-treated groups was evident, when compared to the control. Increased production of ROS was also noticed in the highest-dose treatment group. Further, we observed that IMD-treated rats indicated histopathological changes in the testis and epididymis along with a significant decrease in the plasma testosterone concentrations among IMI-treated groups in contrast to the control. Histological examination of the testis of IMD-treated neonatal male rats also showed decreased spermatogenesis in the treated groups when compared to the control. Furthermore, an increase in lumen diameter and a decrease in epithelial height of seminiferous tubules were also observed in IMD-treated rats in comparison with the control. CONCLUSION: It is concluded that sub-chronic exposure to IMD in neonatal male rats may induce histopathological changes in reproductive tissues and damage normal testicular functions via inducing oxidative stress, decrease in body weight, disturbing normal blood lipid profile and testosterone concentration. IMD exposure can induce pathophysiological effects calls for further evaluation of this widely used insecticide.

Relaxin (a regulatory-peptide) enhances cryotolerance, fertility potential and plasma reproductive hormones of Nili Ravi buffalo (Bubalus bubalis) during low breeding season.

This study investigated the beneficial effects of relaxin on cryotolerance of buffalo spermatozoa and reproductive hormones during low breeding season. Collected semen was diluted in five aliquots with relaxin addition (0.25 mg/mL, 0.50 mg/mL, 0.75 mg/mL, 1 mg/mL, and control). After gentle dilution (37°C), cooling (4°C, 2 h), equilibration (4°C, 4 h), and packaging (straws, polyvinyl French, 0.5 mL), frozen (cell freezer), and thawed (37°C, 30 s) for analysis. Blood samples were collected at different time intervals i.e., -60, -30 and 0 min (pre-dose) and 30, 60, 90, 120 and 150 min (post-dose) from a jugular vein. This study manifest that adding relaxin (1 mg/ mL) in freezing medium ameliorates sperm motility, functionality (%), and seminal plasma total antioxidant capacity (TAC, µM/L) than control during low breeding season. Furthermore, we found that relaxin supplementation at 1 mg/mL significantly improves seminal plasma ATP concentrations (nmol/million) than control, 0.25 mg/mL, and 0.50 mg/mL, and fertility (control, and 0.75 mg/mL). Further, relaxin injection significantly improves plasma T, LH and IGF-1 levels (150 and 120 min vs. -60, and - 30), and FSH, KP, and GnRH concentrations (150 min vs. -60), during low breeding season. Taken together, this study revealed that relaxin ameliorates motility, functionality, and fertility of buffalo spermatozoa. Moreover, relaxin injection (1 mg/mL) improves essential reproductive hormones levels in buffalo signifying its importance in the field of reproductive physiology. Further studies are required to determine the exact mechanism of action of relaxin in enhancing semen quality, fertility and reproductive hormones.

Analysis of sperm chromatin packaging and reproductive biomarker to evaluate the consequence of advanced male age.

In this study, the semen parameters, sperm chromatin integrity, antioxidant enzyme levels, and reproductive hormone levels of subfertile male subjects from Pakistan were assessed in relation to their age. Data on the demographic characteristics of the 750 study participants, including their general health, body mass index (BMI), and reproductive status, were collected from subfertile men from Pakistan. Semen and blood were collected to determine standard semen parameters, sperm chromatin dispersion (Halosperm-SCD), sperm chromatin integrity using toluidine blue (TB) staining, sperm chromatin maturity using chromomycin A3 (CMA3+) staining, and reproductive hormone (FSH, LH, prolactin and testosterone levels). The patients were divided into three groups according to their age: Group 1 included male subjects aged 30 years or less (n = 90), Group 2 included male subjects between the ages of 31 and 40 years (n = 330), and Group 3 included male subjects over 40 years of age (n = 330). Conventional semen parameters, reactive oxygen species (ROS), superoxide dismutase (SOD), guaiacol peroxidase (GPX), catalase (CAT), and lipid peroxidation (MDA) did not statistically (p > 0.05) differ with increasing male age or between different age groups. When compared to younger men (<30 years), sperm SCD (23.2 ± 0.88%) was significantly (p = 0.01) lower as compared to male patients aged >40 years (26.6 ± 0.6%). The concentration of LH, FSH, and testosterone levels were comparable between the groups (p > 0.05), while a significant (p = 0.04) increase in sperm chromatin immaturity CMA3+ (30 ± 0.71%) was observed in the old age group (>40 years) compared to the <30-year group (26.6 ± 1.03%). A positive association was observed between advanced male age and sperm chromatin dispersion (SCD) (r = 0.124, p = 0.001) and decondensation (CMA3+) (r = 0.1, p = 0.009). Despite potential limitations, this study has been carried out with extensive information on the potential risk of male age on sperm integrity. The present study demonstrated the impact of male age on male reproductive health, as these patients had a higher percentage of sperm chromatin damage (SCD) in their semen. Sperm DNA damage assessment will help in the evaluation and diagnosis of the underlying cause of poor fertility and can help clinicians in selecting the right treatment options. Male age is one of the factors that have an impact on the decline in male fertility. As a result, it is preferable for patients receiving assisted reproductive technology to be younger.

Sulforaphane inclusion in a freezing medium augments post-thaw motility, functional and biochemical features, and fertility potential of buffalo (Bubalus bubalis) spermatozoa.

Sulforaphane is a natural and highly effective antioxidant safeguarding the reproductive system, and alleviate oxidative stress. This study was designed in order to elaborate L-sulforaphane effect on semen quality, biochemical parameters, and fertility of buffalo (Bubalus bubalis) spermatozoa. Semen was collected from five buffalo bull with artificial vagina (42 °C) three times and evaluated for volume, consistency (color), motility, and sperm concentration. After critical examination, semen was diluted (50 × 106 spermatozoa per ml, 37 °C) in extenders with (2 µM, 5 µM, 10 µM, and, 20 µM) or without (control) sulforaphane, cooled (from 37 to 4 °C), equilibrated (4 °C), filled (straws, 4 °C), and cryopreserved (LN2, -196 °C). Data analysis exhibited that sulforaphane addition in extender augments total motility (%, 10 µM, and 20 µM than control), progressive motility (%), and rapid velocity (%, 20 µM than control), and velocity parameters (average path velocity, µm/s, straight line velocity, µm/s and curved linear velocity, µm/s, 20 µM than control, and 2 µM). Moreover, sulforaphane augments functional features (membrane functionality, mitochondrial potential, and acrosome integrity) of buffalo sperm (20 µM than control). Sulforaphane preserves biochemical features of seminal plasma of buffalo i.e., Calcium (µM), and total antioxidant capacity (µM/L), followed by reduction in lactate dehydrogenase (IU/L), reactive oxygen species (104 RLU/20 min/ 25 million), and lipid peroxidation (µM/ml) in 20 µM than control. Lastly, sulforaphane augments fertility rate of buffalo sperm at 20 µM than control, and 2 µM. Conclusively the existing study revealed that adding L-sulforaphane (20 µM) in a freezing medium augments motilities, kinematics, functional parameters, and fertility rate of buffalo spermatozoa. Correspondingly, sperm favorable biochemical features were also augmented with sulforaphane followed by reduction in oxidative stress parameters. Further studies are highly recommended to define the particular mechanism of action of sulforaphane in augmenting buffalo post-thawed semen quality, and in vitro fertility potential.

Analyzing the Differential Impact of Semen Preparation Methods on the Outcomes of Assisted Reproductive Techniques.

Sperm separation plays a critical role in assisted reproductive technology. Based on migration, density gradient centrifugation and filtration, a properly selected sperm could help in increasing assisted reproductive outcomes in teratozoospermia (TZs). The current study aimed to assess the prognostic value of four sperm selection techniques: density gradient centrifugation (DGC), swim-up (SU), DGC-SU and DGC followed by magnetic-activated cell sorting (DGC-MACS). These were evaluated using spermatozoa functional parameters. A total of 385 infertile couples underwent the procedure of intracytoplasmic sperm injection (ICSI), with an isolated teratozoospermia in the male partner. Semen samples were prepared by using one of the mentioned sperm preparation techniques. The improvements in the percentage of normal mature spermatozoa, rate of fertilization, cleavage, pregnancy and the number of live births were assessed. The normal morphology, spermatozoa DNA fragmentation (SDF) and chromatin maturity checked by using chromomycin A3 (CMA3) with DGC-MACS preparation were better compared to the other three methods. Embryo cleavage, clinical pregnancy and implantation were better improved in the DGC-MACS than in the other tested techniques. The DGC-MACS technique helped in the selection of an increased percentage of normal viable and mature sperm with intact chromatin integrity in patients with teratozoospermia.

Mutation pattern of cancer suppressor p53 gene in factory workers exposed to polypropylene and impact on their reproductive hormones.

BACKGROUND: Polypropylene is a thermoplastic polymer playing the role of an endocrine disruptor that interferes with the union, emission, transport or elimination of normal hormones. Epidemiological information indicated the relation of endocrine-disturbing chemicals with prostate cancer, testis tumor and diminished fertility. p53 is a key tumor silencer gene. The present study aimed to evaluate luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels and the risk of p53 mutations as a result of exposure to polypropylene in non-tumorous adult male factory workers. METHODS: In total, 150 (controls = 35, workers = 115) subjects were recruited. Groups were maintained according to the tenure of exposure G1 (1-5 years), G2 (6-10 years), G3 (11-15 years) and G4 (16-20 years). Concentrations of LH and FSH were determined through an enzyme-linked immunosorbent assay. Genotyping analysis was performed by polymerase chain reaction based gel electrophoresis followed by DNA sequencing. The structural and functional impact of the mutation on the p53 structure was evaluated using 50-ns molecular dynamics (MD) simulations and protein-DNA docking. RESULTS: Mean plasma LH levels were significantly decreased in G1 (p > 0.05) as well as the G2, G3 and G4 (p > 0.001) groups. Similarly, FSH levels were significant decrease in G1 (p > 0.05), G2 (p > 0.01), G3 (p > 0.001) and G4 (p > 0.001) compared to the control group. Sequencing results found three variants i.e. g.13450 T>G, g.13430C>T and g.13737G>A. One of them was predicted to be disease-causing others are polymorphisms. MD simulation of missense mutation R273H showed no structural impact on the protein structure in MD simulation, but it resulted in weaker binding of p53 with the DNA that might lower the gene expression of cell cycle regulatory proteins. CONCLUSIONS: These findings predict decreased fertility and risk of malignancies in the future. The spectrum of p53 mutations as a result of polypropylene exposure in the Pakistani population has not been investigated before. Further studies and meta-analyses are required to elucidate the role of different plasticizers in reproduction and cancer-causing risk factors in a larger population.

Determination of possible contraceptive potential of methanolic leaf extract of Mentha longifolia L. in adult male rats: a biochemical and histological study.

Mentha longifolia L (ML) is locally used as an herbal contraceptive. We aim to assess the toxicity profile and validate the antifertility effects of its leaves in male rats. Adult male Sprague Dawley rats (n = 10 animals/group) were divided into four groups. The Control group received .9% saline, while groups II, III, and IV received 50, 75, and 100 mg/kg/day ML doses for 28 days. ML induced a dose-dependent decrease in percent fertility as well as the number of pups born in ML preexposed pairs, with maximum change observed in the ML3 group (60%, 3.75 ± 1.25) as compared to control (100%, 8.75 ± .48) group. A significant (P < .01) decrease in the rate of daily sperm production was observed in the ML3 group (.86 ± .16) in contrast to the control (2.93 ± .05). The levels of catalase and superoxide dismutase declined significantly in treatment groups (ML1-P < .001, ML2-P < .05, and ML3-P < .01). A significant increase in the production of reactive oxygen species (P < .001) was found in all treatment groups. The concentrations of LH (P < .001), FSH (P < .001), and testosterone (P < .001) were significantly reduced in a dose-dependent manner among ML-treated groups. Decreases in spermatogonial populations, mature spermatids, seminiferous tubule diameter, lumen diameter, and epithelial height were noticed at higher doses. The reversibility study depicted a reduction in both pregnancy outcomes and litter size. Besides contraceptive effects, M. longifolia L usage is associated with oxidative stress; therefore, it is important to consume this herb appropriately and its excessive usage should be prohibited.

Assessment of Geographical Distribution of Emerging Zoonotic Toxoplasma gondii Infection in Women Patients Using Geographical Information System (GIS) in Various Regions of Khyber Pakhtunkhwa (KP) Province, Pakistan.

Toxoplasmosis is a zoonotic parasitic disease caused by T. gondii, an obligate intracellular apcomplexan zoonotic parasite that is geographically worldwide in distribution. The parasite infects humans and all warm-blooded animals and is highly prevalent in various geographical regions of the world, including Pakistan. The current study addressee prevalence of Toxoplasma infection in women in various geographical regions, mapping of endemic division and t district of Khyber Pakhtunkhwa province through geographical information system (GIS) in order to locate endemic regions, monitor seasonal and annual increase in prevalence of infection in women patients. Setting: Tertiary hospitals and basic health care centers located in 7 divisions and 24 districts of Khyber Pakhtunkhwa (KP) province of Pakistan. During the current study, 3586 women patients from 7 divisions and 24 districts were clinically examined and screened for prevalence of T. gondii infection. Participants were screened for Toxoplasma infection using ICT and latex agglutination test (LAT) as initial screening assay, while iELISA (IgM, IgG) was used as confirmatory assay. Mapping of the studied region was developed by using ArcGIS 10.5. Spatial analyst tools were applied by using Kriging/Co-kriging techniques, followed by IDW (Inverse Distance Weight) techniques. Overall prevalence of T. gondii infection was found in 881 (24.56%) patients. A significant (<0.05) variation was found in prevalence of infection in different divisions and districts of the province. Prevalence of infection was significantly (<0.05) high 129 (30.07%) in Kohat Division, followed by 177 (29.06%), 80 (27.87%), 287 (26.72%), 81 (21.21%), 47 (21.07%), and 80 (13.71%) cases in Hazara Division, D.I Khan Division, Malakand Division, Mardan Division, Bannu Division, and Peshawar Division. Among various districts, a significant variation (<0.05) was found in prevalence of infection. Prevalence of infection was significantly (<0.05) high 49 (44.95%) in district Karak, while low (16 (10.81%) in district Nowshera. No significant (>0.05) seasonal and annual variation was found in prevalence of Toxoplasma infection. LAT, ICT and ELISA assays were evaluated for prevalence of infection, which significantly (<0.05) detected T. gondii antibodies. LAT, ICT and ELISA assays significantly (<0.05) detected infection, while no significant (>0.05) difference was found between positivity of LAT and ICT assays. A significant difference (<0.05) was found in positivity of Toxoplasma-specific (IgM), (IgG) and (IgM, IgG) immunoglobulin by ICT and ELISA assay. The current study provides comprehensive information about geographical distribution, seasonal and annual variation of Toxoplasmosis infection in various regions of Khyber Pakhtunkhwa province of Pakistan. Infection of T. gondii in women shows an alarming situation of disease transmission from infected animals in the studied region, which is not only a serious and potential threat for adverse pregnancy outcomes, but also cause socioeconomic burden and challenges for various public and animal health organizations in Pakistan and across the country.

Expression variation of OGG1 and HPRT gene and DNA damage in arsenic exposed industrial workers.

Arsenic exposure alters redox balance, induces DNA damage, and deregulates many genes. OGG1 gene involved in base repair mechanism, for excision of 8-oxoguanine (8-oxoG) from DNA formed as a result of accumulation of ROS in cell. HPRT gene encode transferase enzymes involved in purine recycling mechanism. The main focus of the study was to evaluate the expression variation in HPRT, OGG1 gene expression, and DNA damage of industrial workers. Blood samples of 300 occupational workers were collected from welding, brick kiln, furniture, pesticide, and paint industry (n = 60/industry) to evaluate the expression variation in HPRT, OGG1 gene expression, and DNA damage in blood cells by comet assay along with age and gender matched 300 control individuals. Blood arsenic content was higher (P<0.001) in an industrial group compared to the control. OGG1 and HPRT expression were (P<0.05) downregulated in exposed workers compared to controls. Spearman correlation analysis showed a significant positive correlation between HPRT vs OGG1 (P< 0.0001) in exposed workers compared to controls. Altered expression of both genes was observed between workers with <25years and >25years of age as well as between workers with <10years and >10year exposure. Reduced expression (P<0.05) of both genes and a high extent of DNA damage was evident in exposed smokers compared to respective non-smokers. DNA fragmentation was higher (P<0.05) in the furniture, welding and brick kiln group compared to control, and other industries. The present study suggests that altered expression of OGG1 and HPRT gene induce oxidative stress, showed a negative impact on the recycling of purines leading to DNA damage which increase the vulnerability of workers to carcinogenicity.

The influence of paternal overweight on sperm chromatin integrity, fertilization rate and pregnancy outcome among males attending fertility clinic for IVF/ICSI treatment.

BACKGROUND: Low and middle-income countries are facing a rapid increase in obesity and overweight burden, particularly in urban settings. Being overweight in men is associated with infertility and a higher risk to have a low sperm count or no sperm in their ejaculate. Despite potential limitations, this is one of few studies conducted to determine the potential risk of paternal overweight on sperm standard parameters, sperm chromatin integrity and assisted conception outcome including fertilization, embryo quality, cleavage rate, reduce blastocyst development, implantation, and cumulative live birth rate (CLBR). METHODS: A cross-sectional study of 750 infertile couples undergoing assisted reproduction technique at a single reproductive medicine center of Salma Kafeel Medical Centre Islamabad. Sperm from men undergoing ART were analyzed for chromatin integrity using sperm chromatin dispersion assay (SCD), Chromomycin A3 staining (CMA3), and toluidine blue (TB) staining, while other semen parameters were assessed on same day includes; standard semen parameters, reactive oxygen species (ROS), sperm deformity index (SDI), teratozoospermic index (TZI), and hypo-osmatic swelling test (HOST). Paternal body mass index (BMI) < 24.5-20 kg/m2 served as the reference group, while the male patients with BMI > 24.5-30 kg/m2 were considered to be overweight. RESULTS: In the analysis of the percentage of spermatozoa with chromatin maturity (CMA3) and chromatin integrity (TB) was reduced significantly in overweight men (p < 0.01) compared with a reference group. Increase in paternal BMI correlate with the increase in sperm chromatin damage (SCD r = 0.282, TB r = 0.144, p < 0.05), immaturity (CMA3, r = 0.79, p < 0.05) and oxidative stress (ROS) (r = 0.282, p < 0.001). Peri-fertilization effects were increased in oocytes fertilization in couples with overweight men (FR = 67%) compared with normal-weight men (FR = 74.8%), similarly, after univariant regression paternal weight remain predictor of sperm chromatin maturity, successful fertilization and CLBR. In the embryo, developmental stage number of the embryo in cleavage was higher in normal weight men, while day 3 (D3) embryos, percent good quality embryo D3, and blastocyst formation rate were compared able between the groups. The paternal overweight group had significant (p < 0.001) increased neonatal birth weight (2952.14 ± 53.64gm; within normal range) when compared with the reference group (2577.24 ± 30.94gm) following assisted reproductive technology (ART). CLBR was higher (p < 0.05) in normal weight men compared to couples with overweight male partners. CLBR per embryo transfer and per 2PN was a statistically significant (p < 0.05) difference between the two groups. An inverse association was observed in the linear regression model between paternal BMI with fertilization rate and CLBR. CONCLUSION: The present study demonstrated the impact of paternal overweight on male reproductive health, as these patients had a higher percentage of immature sperm (CMA3) with impaired chromatin integrity (SCD, TB) in their semen and had decreased fertilization rate, CLBR following assisted reproductive treatments. The present study supports that paternal overweight should be regarded as one of the predictors for fertilization, CLBR and useful for counseling, to consider body mass index not only in women but also for men, in those couples opting for ART treatment, and warrant a poor reproductive outcome in overweight men.

The ameliorating effects of crocetin on frozen-thawed quality, and fertility via attenuating oxidative status of bubaline spermatozoa.

Current study was conducted to appraise the cryoprotective influence of crocetin on quality, oxidative status, and fertility potential of bubaline spermatozoa. Collected semen from four bulls was diluted in five aliquots with (10 µM, 5 µM, 2 µM, 1 µM, and control [0 µM] supplementation of crocetin). After gentle dilution (37 °C), cooling (4 °C, in 2 h), equilibration (4 °C, for 4 h) and packaging of samples was done in straws (polyvinyl French, 0.5 ml), and then frozen (programmable cell freezer). This study established that crocetin supplementation significantly (p < 0.05) improves CASA (Computer Assisted Sperm motion Analyzer) total motility (%), rapid velocity (%), average-path, and curved-line velocities (µm/sec, 10 µM vs. control), and progressive motility (%), straight-line velocity (µm/sec), total antioxidant capacity (TAC, µMol/l), ATP concentrations (nmol/million), and fertility potential (%) (10 µM vs. control, and 1 µM), and mitochondrial potential (%) of buffalo spermatozoa (5, and 10 µM vs. control). Crocetin supplementation significantly (p < 0.05) alleviates DNA fragmentation, seminal plasma ROS (104 RLU/20/25 million, RLU = Relative light unit) levels, and lipid peroxidation (LPO, µMol/ml) in buffalo spermatozoa (10 µM vs. control). In a nutshell, crocetin supplementation improves post-thaw quality by means of motility parameters, motion kinematics, TAC, and ATP concentrations, and fertility potential, and abolished DNA fragmentation parameters, seminal plasma ROS, and LPO concentrations of buffalo spermatozoa. The exact mechanism by which crocetin acts are not fully elucidated; however, it is probable to speculate that the reduction in ROS, and LPO recorded in this study may be related to scavenging ability of this antioxidant during cryopreservation.

Biochemical and reproductive biomarker analysis to study the consequences of heavy metal burden on health profile of male brick kiln workers.

The present study aims to assess the effect of a heavy metal burden on general health, biochemical parameters, an antioxidant enzyme, and reproductive hormone parameters in adult male brick kiln workers from Pakistan. The study participants (n = 546) provided demographic data including general health as well as body mass index. Blood was collected to quantitatively assess hematological, biochemical, and reproductive hormone parameters as well as heavy metal concentrations using both atomic absorption spectroscopy (AAS) and particle-induced X-ray emission (PIXE). The data showed that 10% of the brick kiln workers were underweight and 10% obese (P = 0.059), with workers also reporting multiple health issues. Heavy metal concentrations utilizing AAS revealed significantly (p = 0.000) higher levels of cadmium, chromium, and nickel, while PIXE detected more than permissible levels of Si, P, S, Cl, K, Ca, Zn, Ti (p = 0.052), Mn (p = 0.017), Fe (p = 0.055), Co (p = 0.011), Ni (p = 0.045), and Cu (p = 0.003), in the blood of kiln workers. Moreover, a significant increase in platelet count (P = 0.010), a decrease in sodium dismutase levels (p = 0.006), a major increase in reactive oxygen species (p = 0.001), and a reduction in protein content (p = 0.013) were evident. A significant increase in cortisol levels (p = 0.000) among the workers group was also observed. The concentration of LH and FSH increased significantly (p = 0.000), while that of testosterone decreased (p = 0.000) in the worker group compared with controls. A significant inverse relationship was found between cortisol, LH (r = - 0.380), and FSH (r = - 0.946), while a positive correlation between cortisol and testosterone was also evident (r = 0.164). The study concludes that increased heavy metal burden in the blood of brick kiln workers exposes them to the development of general and reproductive health problems due to compromised antioxidant enzyme levels, increased oxidative stress conditions, and a disturbing reproductive axis.

Prebiotic potential of enzymatically prepared resistant starch in reshaping gut microbiota and their respond to body physiology.

The increase in consumer demand for high-quality food products has led to growth in the use of new technologies and ingredients. Resistant starch (RS) is a recently recognised source of fibre and has received much attention for its potential health benefits and functional properties. However, knowledge about the fate of RS in modulating complex intestinal communities, the microbial members involved in its degradation, enhancement of microbial metabolites, and its functional role in body physiology is still limited. For this purpose, the current study was designed to ratify the physiological and functional health benefits of enzymatically prepared resistant starch (EM-RSIII) from maize flour. To approve the beneficial health effects as prebiotic, EM-RSIII was supplemented in rat diets. After 21 days of the experiment, EM-RSIII fed rats showed a significant reduction in body weight gain, fecal pH, glycemic response, serum lipid profile, insulin level and reshaping gut microbiota, and enhancing short-chain fatty acid compared to control. The count of butyrate-producing and starch utilizing bacteria, such as Lactobacillus, Enterococcus, and Pediococcus genus in rat's gut, elevated after the consumption of medium and high doses of EM-RSIII, while the E. coli completely suppressed in high EM-RSIII fed rats. Short-chain fatty acids precisely increased in feces of EM-RSIII feed rats. Correlation analysis demonstrated that the effect of butyrate on functional and physiological alteration on the body had been investigated during the current study. Conclusively, the present study demonstrated the unprecedented effect of utilising EM-RSIII as a diet on body physiology and redesigning gut microorganisms.

Protamines and DNA integrity as a biomarkers of sperm quality and assisted conception outcome.

Present research aim was to identify functional tests in semen associated with DNA damage and chromatin maturity (protamination) which predict the outcome in assisted reproduction. Couples were grouped according to male partner semen parameters, into normozoospermia (NZs), severe male factor (SMF) and mild male factor (MMF). DNA fragmentation index (DFI) in spermatozoa was analysed by sperms chromatin dispersion (SCD), sperm chromatin structure assay (SCSA) and acridine orange testing (AOT). Chromomycin A3 (CMA3) and toluidine blue (TB) staining to measure sperm chromatin maturity (CM). DFI and chromatin decondensation were significantly lower in N compared to male factor categories (MMF and SMF). Aneuploidy embryos were significantly higher in couples with male factor infertility (MMF and SMF). A positive correlation was observed between fertilization rate (FR) and live birth rate (LBR) with sperm concentration, motility, vitality, normal sperm morphology and negative correlation between sperm DFI and sperm CM. No correlation was observed between embryo aneuploidy and sperm DFI or CM. Lower percentage of spermatozoa chromatin integrity are associated with low fertilization and live birth rate. Male factor infertility, due to impaired semen parameters and chromatin defects could be regarded in future as an indication of IVF/ICSI, and predictor of assisted reproductive techniques outcome.

Assessment of antiandrogenic and antispermatogenic activity of Hedera nepalensis in adult male rats.

The use of medicinal plants for fertility regulation has been prevalent worldwide for many centuries. They possess natural substances having antiandrogenic properties and can be used as source of contraception. In the current study, methanolic leaf extract of Hedera nepalensis was evaluated for antiandrogenic and antispermatogenic activity in adult male rats through various reproductive parameters. Experimental findings showed significantly increased oxidative stress with reduced antioxidant activity at highest dose regimens in both in vitro and in vivo studies. Increased ROS generation and lipid peroxidation lead to DNA damage in rat sperm. In vivo determination of sperm parameters exhibited notable reduction in sperm motility, viability and DSP in dose-treated animals. Histopathological observations revealed reduced epithelial height and wider lumen having less number of spermatozoa in high-dose-treated groups. Additionally, a marked decline noted in Testosterone concentration in all extract treated groups, while plasma LH and FSH levels only in high-dose-treated groups were noted. The findings of the current study conclude that methanolic leaf extract of H. nepalensis has the potential to disturb male fertility by generating oxidative stress and hormonal imbalance leading to histological alterations and sperm DNA damage.

Association of endothelial nitric oxide synthase gene variants with preeclampsia.

BACKGROUND: Preeclampsia (PE) is a complex pregnancy hypertensive disorder with multifaceted etiology. The endothelial nitric oxide synthase (eNOS) gene and nitric oxide (NO) levels has been reported to be associated with PE predisposition in various populations. Therefore, present study was designed to investigate the role of NO levels and eNOS gene variants in preeclamptic women in Pakistan. METHODS: A total of 600 women were evaluated, 188 of PE with mild features, 112 of PE with severe features and 300 normotensive pregnant women. NO levels were detected by Greiss reaction method and genotyping following sequencing was conducted for eNOS gene variants. Further insilico studies were performed to get insights into the structural and functional impact of identifies mutation on eNOS protein as well as on protein regulation. RESULTS: Reduced concentrations of NO were reported in all PE groups (p < 0.05) as compared to controls. The frequency of c.894 T (p.298Asp) and g.-786C alleles were significantly associated with PE. In addition, novel homozygous variant g.2051G > A was also significantly associated with PE when compared to normotensive women. Dynamic simulation studies revealed that Glu298Asp mutation destabilize the protein molecule and decrease the overall stability of eNOS protein. Molecular docking analysis of mutant promoter with transcription factors STAT3 and STAT6 proposed changes in protein regulation upon these reported mutations in upstream region of the gene. CONCLUSION: Considering the results of current study, the functional alterations induced by these variants may influence the bioavailability of NO and represents a genetic risk factor for increased susceptibility to PE. However, large studies or meta-analysis are necessary to validate these findings.

Preeclampsia (PE) is a complex pregnancy hypertensive disorder with multifaceted etiology characterized by increased hypertension and proteinuria after 20 weeks of gestation. The present study was directed to determine the role of eNOS in susceptibility to PE and the association of c.894G > T (p.(Glu298Asp), intron 4b/4a, g.-786 T > C and other possible variants of eNOS gene with preeclampsia in Pakistani population. Computational analysis of identified variants in the coding and non-coding region of the eNOS gene was also conducted to determine the change in gene regulation and further protein stability. A total of 600 women were evaluated, 188 with mild and 112 with PE with severe features PE with 300 normotensive pregnant women. NO levels and genotyping following sequencing was conducted for eNOS gene variants. Further insilico studies were performed to get insights into the structural and functional impact of identifies mutation on eNOS protein as well as on protein regulation. Data from the current study suggest that there might be other risk variants of the eNOS gene (g.2051G > A and g.1861G > A) and lower levels of serum NO that confers in an increased risk of PE. The detailed computational investigation further confirmed the deformities and changes in protein flexibility upon Glu298Asp. These structural alterations might be associated with preeclampsia. Variants in the promoter region of the eNOS gene further validate the change in gene regulation for the onset of disease. Identification of key structural and functional features in eNOS protein and gene regulatory region might be used for designing specific drugs for therapeutic purpose.

Evaluation of environmental effects of heavy metals on biochemical profile and oxidative stress among children at brick kiln sites.

The present study was designed to study the health risks among children living at brick kiln industries. A survey was conducted, questionnaires were filled out, and demographic data was collected from Punjab, Pakistan. Heavy metals burden and BMI were calculated, hematological and enzyme analysis, comet assay and hormonal ELISA were performed. The results showed decreased BMI, RBC count and hematocrit in the exposed group. Nickel, cadmium, zinc and chromium concentrations in whole blood were detected among exposed children. Antioxidant enzymes and growth hormone concentration decreased, while reactive oxygen species and cortisol level increased in the exposed group. The comet assay findings showed decreased percentage DNA in the head and increased in the tail region among exposed group. It was concluded that children living at brick kiln sites experienced decreased BMI, altered antioxidant enzymes status and hormone levels and cellular DNA damage that pose a major threat on child health.

Therapeutic effects of chitosan-embedded vitamin C, E nanoparticles against cisplatin-induced gametogenic and androgenic toxicity in adult male rats.

Cisplatin, an anticancer drug used in treating various types of cancers, can cause reproductive toxicities during chemotherapy. Keeping this in view, the present study was designed to investigate the possible protective effects of normal vitamin C and E and vitamin C and E nanoparticles (embedded in chitosan) against cisplatin-induced reproductive toxicities. Vitamins C, E, and their nanoparticles in this regard proved to be an effective therapy. The work aimed to treat cisplatin-induced reproductive toxicities through vitamin C and E and their nanoparticles. Cisplatin exposure caused significant reduction in the weight, testosterone level, and changed lipid profile. Similarly, cisplatin induced significant widespread testicular atrophy and testicular lesions as evidenced by the gaps in the epithelium and loss of differentiating germ cells. Vitamin C and E and their nanoparticles rescued the weight, testosterone level, and testicular disturbances, which is associated with improved histological view of testicular tissues. The current study highlights evidence that designing a medication of vitamin C and E nanoparticles is useful in mitigating cisplatin-induced reproductive toxicity in cancerous male patients underlying chemotherapy.

Improving the quality and in vitro fertilization rate of frozen-thawed semen of buffalo (Bubalus bubalis) bulls with the inclusion of vitamin B12 in the cryopreservation medium.

This study was designed to evaluate the effects of vitamin B12 in cryopreservation medium on frozen-thawed semen of buffalo (Bubalus Bubalis) bulls. Semen from five bulls (fertility-proven) were diluted in five aliquots not supplemented (control), or supplemented with 1, 2, 4, or 5 mg/mL of vitamin B12 and evaluated using the Computer Assisted Sperm motion Analysis, antioxidant enzymes, lipid peroxidation (LPO), reactive oxygen species (ROS), ATP concentrations, and in vitro fertilization rate (%). Sperm progressive motility, rapid velocity (%), mitochondrial potential, and acrosome integrity were greater (P < 0.05) with supplementation of 4, and 5 mg/mL vitamin B12 than the control sample. Similarly, compared with the control, samples with 5 mg/mL vitamin B12 supplementation had markedly greater average-path, straight-line, and curved-line velocities (µm/sec). Semen samples supplemented with 2, 4 and 5 mg/mL vitamin B12 had greater concentrations of GPx (U/mL) and SOD (U/mL), whereas LPO (µM/mL) was less (P < 0.05) compared with the control sample. Seminal plasma ROS concentrations (104/25 × 106) were less in the 5 mg/mL vitamin B12 supplemented than control sample. Semen samples supplemented with 5 mg/mL of vitamin B12 had greater concentrations of ATP than control and the 1 mg/mL vitamin B12 supplemented sample. Semen samples supplemented with 5 mg/mL of vitamin B12 had greater plasmalemma and DNA integrities (%) than the control sample. In summary, vitamin B12 supplementation augments semen quality, as evidenced by values for CASA variables, antioxidant enzymes, and ATP concentrations, which may occur as a consequence of inhibition in LPO and ROS production by buffalo spermatozoa.