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The effects of radiofrequency electromagnetic radiation (RF-EMR) on semen quality have been in the spotlight in recent years, though research results to date have been contradictory. The effects of RF-EMR amongst others depend upon frequency, and there is currently no literature concerning the influence of 5G frequencies on both DNA integrity and spermatozoa vitality in males. The aim of this study was to investigate the effect of 5G RF-EMR on sperm membrane integrity, mitochondrial potential, and DNA integrity of in vitro exposed semen of breeding boars. The study included semen samples of eight breeding boars of the Pietren breed and four breeding boars of the German Landrace breed, from 1.5 to 3.5 years in age. Freshly diluted semen of each boar was divided into a control (n = 12) and experimental group (n = 12). The samples of the experimental group were exposed for 2 hours to continuous RF-EMR at a single frequency (700 MHz, 2500 MHz and 3500 MHz) and an electromagnetic field strength of 10 V/m using a transverse gigahertz electromagnetic cell. Sperm DNA fragmentation was assessed using a Halomax® kit and sperm membrane integrity and mitochondrial potential was assessed using a PI⁄SYBR-14 LIVE⁄DEAD viability kit with JC-1. A significantly higher proportion of spermatozoa with DNA fragmentation was found in exposed semen samples for all frequencies compared to the control group. The highest DNA damage was recorded in semen samples exposed to 5G RF-EMR at 2500 MHz (p < 0.01) and 3500 MHz (p < 0.05) vs. control semen samples. A significantly higher proportion of spermatozoa with damaged cell membrane and good mitochondrial potential was recorded in semen samples exposed with 3500 MHz. In vitro exposure of breading boar semen to 5G RF-EMR significantly increases the proportion of DNA fragmentation. The harmful effect of 5G RF-EMR on the proportion of spermatozoa with damaged DNA was frequency dependent. The 3500 MHz frequency displayed the most harmful effects due to significant impacts on DNA integrity and spermatozoa vitality indicators. • 5G RF-EMR significantly increases the proportion of boar's spermatozoa with damaged DNA. • The proportion of spermatozoa with damaged DNA depends on the used frequency. • Sperm damaged cell membrane and/or low mitochondrial potential increases after 3500 MHz exposure. [ABSTRACT FROM AUTHOR]

Biobanks hold a pivotal role in facilitating translational and clinical research endeavors. However, the effects of prolonged storage on frozen blood samples analytes are not well defined yet. The aim of this study was to investigate the long‐term stability of the quality of DNA, RNA, and endocrine markers within blood samples amassed from the biobank over the past 11 years. The results show that the overall quality and integrity of DNA remained not significantly influenced. However, RNA integrity and purity displayed substantial deterioration as storage duration increased, to ensure high‐quality RNA for downstream analyses, advised to prioritize using blood samples stored within 3 years. Furthermore, the study examined the influence of storage time on endocrine markers. Through repeated measures ANOVA and linear regression analyses, it was evident that storage duration significantly influenced the levels of endocrine markers. This insight aids researchers in selecting appropriate markers for their investigations and augments the precision and dependability of results when dealing with long‐term stored samples. [ABSTRACT FROM AUTHOR]

The molecular mechanism responsible for sperm DNA fragmentation is not fully understood. Therefore, identifying genes related to the response to DNA damage is an important area of research. Recently, the role of long non-coding RNAs (LncRNAs), especially DNA damage-sensitive RNA1 (DDSR1) in male infertility has been highlighted. In this research, a protein-protein interaction network (PPIN) was constructed using the STRING database, and functional classification was conducted using webgestalt servers. Subsequently, a group of 40 males with a high degree of sperm DNA fragmentation (DFI ≥ 25%) was compared to a control group of 20 healthy males with a normal sperm DNA fragmentation rate (DFI < 25%). To assess gene expression, real-time polymerase chain reaction (PCR) analysis was performed on DNA samples obtained from both healthy and infertile males. Our findings revealed that infertile men with an abnormal DFI index showed significantly lower expression levels of the long noncoding RNA DDSR1, as well as the genes BRCA1, MRE11A, RAD51, and NBN, compared to the control group. Pathway analysis of the network proteins using Reactome indicated involvement in crucial cellular processes such as the cell cycle, DNA repair, meiosis, reproduction, and extension of telomeres. In conclusion, the downregulation of LncRNA and genes associated with the DNA damage response in males with an abnormal DFI suggests that these factors may contribute to the development of sperm DNA fragmentation and could potentially serve as diagnostic markers for further investigation in therapeutic interventions in the future. [ABSTRACT FROM AUTHOR]

DNA extraction was successfully performed on all samples with variable concentrations. Fifteen liver samples were subjected to the extraction technique, with five liver samples utilized from each fixative, this comparison study aimed to assess DNA integrity across different fixatives. DNA was successfully extracted from all samples regardless of their varying quantities. Each sample yielded at least one motif with a length of 100 base pairs. All 15 samples produced 100 bp amplicon for the IRBP gene. All 15 samples generated a 200 bp amplicon from the NBF, ZF, and BF. The success proportion dropped with growing amplicon length, by which 66.6% (10/15) of 300 bp amplicons were successfully produced from NBF and ZF, showing significant differences at P

Simple Summary: Spermatozoa cryopreservation technology has been widely used to preserve high-quality male spermatozoa and has to some extent solved the problem of spermatozoa supply in yellow catfish (Pelteobagrus fulvidraco). However, cryopreservation can still cause cellular damage and affect spermatozoa viability and fertility. Therefore, it is necessary to study cryo-injury, which is beneficial for further optimizing the spermatozoa cryopreservation protocol. In this experiment, we investigated the effects of adding or not adding 10% DMSO on spermatozoa motility, ultrastructural damage, oxidative damage, and DNA damage of thawed yellow catfish. Using 10% DMSO as a cryoprotectant can improve spermatozoa motility (75.64 ± 1.12), significantly alleviate injuries to organelles and membrane structures, improve SOD (7.99 × 104 ± 5.24 nmol/mgprot) and T-AOC (3.7 × 105 ± 0.06 nmol/mgprot) enzyme activity, reduce MDA (1.09 ± 0.45 nmol/mgprot) enzyme activity, and mitigate the degree of oxidative damage. It also greatly reduces the DNA fragmentation rate (59 ± 1.56%). The research results provide data support for the optimization of the cryopreservation protocol for the spermatozoa of yellow catfish, which is beneficial for the preservation of the germplasm resources of yellow catfish and the effective utilization of resources during large-scale production. Spermatozoa cryopreservation protocols have been established for yellow catfish (Pelteobagrus fulvidraco), but cryopreservation can still cause cellular damage and affect spermatozoa viability and fertility. Therefore, the aim of this paper was to evaluate the effects of adding or not adding cryoprotectants during low-temperature storage on the ultrastructural damage, oxidative damage, and DNA damage of thawed yellow catfish spermatozoa. The mixed semen of three male yellow catfish was divided into a fresh spermatozoa group, a frozen spermatozoa group (DMSO+) with a cryoprotectant (10% DMSO), and a frozen spermatozoa group without a cryoprotectant (DMSO−). Ultrastructural of the spermatozoa after thawing were observed under an electron microscope and the spermatozoa were assayed for SOD, MDA, and T-AOC enzyme activities, as well as for DNA integrity. In terms of movement parameters, compared with DMSO−, the addition of DMSO has significantly improved sperm motility, curve line velocity (VCL), and straight line velocity (VSL). The ultrastructural results showed that although thawed spermatozoa exhibited increased damage than fresh spermatozoa, 10% DMSO effectively reduced the damage to the plasma membrane, mitochondria, and flagellum of spermatozoa by cryopreservation, and most of the spermatozoa were preserved with intact structure. The results of oxidative damage showed that compared with frozen spermatozoa, 10% DMSO significantly increased the activities of SOD and T-AOC enzymes and clearly reduced the activity of the MDA enzyme. The antioxidant capacity of spermatozoa was improved, lipid peroxidation was reduced, and the oxidative damage caused by cryopreservation was mitigated. The DNA integrity of spermatozoa showed that 10% DMSO clearly reduced the DNA fragmentation rate. In conclusion, 10% DMSO can effectively reduce the ultrastructural damage, oxidative damage, and DNA damage of yellow catfish spermatozoa during cryopreservation; it can also further optimize the cryopreservation protocol for yellow catfish spermatozoa. Meanwhile, it also provides a theoretical basis for the future optimization of the cryopreservation protocols. [ABSTRACT FROM AUTHOR]

Purpose: Circulating cell-free DNA (cfDNA) is a promising biomarker for predicting treatment response and disease outcomes in Breast Cancer (BC) patients undergoing neoadjuvant chemotherapy (NAC). To determine if cfDNA originates from tumors, matching tumor and cfDNA gene mutations are necessary, often requiring tumor DNA sequencing. We assessed plasma cfDNA integrity by measuring concentrations and ratios of larger-to-smaller Alu DNA fractions as a potential biomarker, eliminating the need for prior tumor sequencing. Methods: We included patients with localized and/or locally advanced BC receiving standard NAC alone or in combination with immunotherapy and/or anti-HER2 targeted therapy. Blood samples were collected before treatment, every 2 weeks during treatment, and before surgery. Results: Of the 38 evaluated patients, only 28 completed the protocol and underwent surgery after NAC. Seven patients (25%) achieved a pathologic complete response (pCR). We found that cfDNA integrity (cfDNAI) levels at 15 days after starting NAC were significantly higher in patients who achieved pCR (p = 0.045) and correlated significantly with Disease-Free Survival (DFS) in univariate analysis (p = 0.0371). Conclusions: Evaluation of cfDNAI 2 weeks after NAC initiation appears to be an early biomarker for tumor pCR and DFS. Measuring Alu fragments of different lengths may replace techniques requiring prior tumor sequencing to measure ctDNA, reducing costs and complexity of cfDNA serial measurements in BC patients undergoing NAC. [ABSTRACT FROM AUTHOR]

Abstract Biobanks hold a pivotal role in facilitating translational and clinical research endeavors. However, the effects of prolonged storage on frozen blood samples analytes are not well defined yet. The aim of this study was to investigate the long‐term stability of the quality of DNA, RNA, and endocrine markers within blood samples amassed from the biobank over the past 11 years. The results show that the overall quality and integrity of DNA remained not significantly influenced. However, RNA integrity and purity displayed substantial deterioration as storage duration increased, to ensure high‐quality RNA for downstream analyses, advised to prioritize using blood samples stored within 3 years. Furthermore, the study examined the influence of storage time on endocrine markers. Through repeated measures ANOVA and linear regression analyses, it was evident that storage duration significantly influenced the levels of endocrine markers. This insight aids researchers in selecting appropriate markers for their investigations and augments the precision and dependability of results when dealing with long‐term stored samples.

Background: Male sterility results from high testicular temperatures, which affect mammalian spermatogenesis. High testicular temperatures affect sperm motility, morphology and fertility according to their magnitude and duration. Aim: The aim of the current study is to examine the effects of heat-induced oxidative stress and cinnamaldehyde on Wistar rat testicular structure and function. Settings and Design: The rats used in this experiment were Wistar albino rats. Materials and Methods: This research has six animals per group. Male Wistar albino rats of 2.5–3 months old and 275–300 g. (I) control, (II) heat stress (HS) in a closed chamber at 41°C for 14 days and (III) HS with cinnamaldehyde (CA) 50 mg/kg body weight for 14 days. (IV) CA alone. After the study, the animals were euthanised, and test samples were taken for sperm count, morphology, haematoxylin and eosin stain for normal cellular morphology, antioxidants and DNA integrity assessments. Statistical Analysis Used: The data were analysed statistically using one- and two-way ANOVA tests for comparisons between groups. Results: The stress group had significantly lower sperm counts and poor sperm morphology. The stress group’s antioxidant capacity is much lower than that of the control group. Animals under stress have fragmented DNA. Treatment with cinnamaldehyde increased overall antioxidant capacity and seminal parameters, and rats behaved most like controls. Conclusion: CA restores malondialdehyde levels, total antioxidant capacity, sperm characteristics and mitigates testicular damage in rats exposed to experimental HS.

Context: Seasonal microclimatic fluctuations can cause changes in sperm quality even in dairy bulls bred under temperate climate. These changes can vary between sires of different age and affect sperm freezability. Aims: We aimed to evaluate the modulating effect of bull age and equilibration time before freezing on the seasonal pattern of sperm viability and DNA integrity post-thaw. Methods: In the frame of systematic sperm quality control, we assessed the integrity of sperm plasma membrane and acrosome (PMAI) in 15,496 cryopreserved bovine batches, and the percentage of sperm with high DNA fragmentation index (%DFI) after 0 h and 3 h incubation at 38°C post-thaw (3 h) in 3422 batches. Semen was equilibrated for 24 h before freezing if collected on Monday or Wednesday and 72 h if produced on Friday. We investigated the effect of season, bull age, equilibration, and temperature-humidity index (THI) on the day of semen collection on sperm traits using mixed-effects linear models. Key results: PMAI and %DFI (0 h and 3 h) deteriorated with increasing THI. The effect of THI on %DFI was detected with a 30-day time lag. Seasonal fluctuations of sperm quality were similar between young, mature, and older sires. Prolonged equilibration did not affect PMAI but was linked to elevated %DFI (3 h) in summer. Conclusions: Extending equilibration from 24 to 72 h is compatible with commercial standards of bovine sperm quality post-thaw; however, it could interfere with the seasonal pattern of the latter. Implications: Systematic monitoring of bovine sperm quality enables the prompt detection of stress factors related to microclimate and semen processing. Multiple correspondence analysis (MCA) plot for the categorical variables of season of semen collection and age class of 262 individual bulls. Points represent the score of each bull for the quantitative variables (percentage of sperm with intact plasma membrane and acrosome and percentage of sperm with high DNA fragmentation index (immediately after thawing and after 3 h of incubation at 38°C) in different seasons of the year. Photograph by Eleni Malama. This article belongs to the Collection Dedication to Jim Cummins. [ABSTRACT FROM AUTHOR]

This experiment was designed to investigate the impact of curcumin‐olive oil nanocomposite (CONC) supplementation on uteroplacental hemodynamics and ultrasonographic measurements as well as maternal oxidative status in midgestating goats. Twelve synchronized pregnant goats (85.58 ± 1.08 days of gestation; mean ± SD) were uniformly assigned to two groups (n = 6/group); the first group received daily oral supplementation of CONC (3 mg/kg body weight; nanocurcumin [NC] group) for 32 days, and the second group was offered physiological saline (control) following the NC group timeline. The goats of both groups were examined at 3‐day intervals for middle uterine (MUA) and umbilical (UMA) arteries hemodynamics (pulsatility index [PI], resistive index [RI], systole/diastole [S/D] and blood flow rate [BFR]) and diameters, uteroplacental thickness (UPT), placentomes' diameter (PD) and echogenicity, steroid hormones (progesterone and estradiol 17β), oxidative biomarkers (total antioxidant capacity [TAC], catalase [CAT], malondialdehyde [MDA]), nitric oxide (NO) and blood cells DNA integrity. The UPT (p = 0.012) and PD (p = 0.021) values were higher in the NC group than in their counterparts' control group (D11–32). There were increases in diameter (p = 0.021 and p = 0.012) and decreases (p = 0.021, p = 0.016 and p = 0.041 [MUA]; p = 0.015, p = 0.023 and p = 0.011 [UMA] respectively) in Doppler indices (PI, RI and S/D) of the MUA and UMA in the NC group compared to the control group (D14–32). On D20–32 (MUA) and D14–32 (UMA), the NC goats had higher BFR than the control group (p = 0.021, 0.018 respectively). The means of blood cells with fragmented DNA were lower (p = 0.022) in the NC group than in the control group on Days 8 and 21 postsupplementation. There were increases in CAT and NO (D20–32; p = 0.022 and p = 0.004 respectively), and TAC (D17–32; p = 0.007) levels in the NC goats compared to the control ones. The NC group had lower (p = 0.029) concentrations of MDA than the control group on Day 20 postsupplementation onward. In conclusion, oral supplementation of CONC improved uteroplacental blood flow and the antioxidant capacity of midgestating goats. [ABSTRACT FROM AUTHOR]

Background: Lifestyle and environmental pollution harm male fertility. Perfluoroalkyl substances (PFAS) are bio-accumulates in the environment as well as in several human tissues, and one of the most common PFAS is perfluorooctanoic acid (PFOA). Therefore, this study aimed to evaluate the in vitro effects of PFOA with hydrophobic and waterproofing properties on motility and bio-functional sperm parameters. Methods: To accomplish this, 50 healthy men with normozoospermia and not exposed to high doses of PFAS were enrolled. Their spermatozoa were incubated for 3 h with increasing concentrations of PFOA (0, 0.01, 0.1, and 1 mM) to evaluate its effects. In particular, we evaluated the effects of PFOA on total and progressive sperm motility and, by flow cytometry, on the following bio-functional sperm parameters: degree of chromatin compactness, viability, early and late apoptosis, mitochondrial membrane potential, the degree of lipoperoxidation, and concentrations of mitochondrial superoxide anion. Results: The results showed that PFOA decreased both total and progressive sperm motility, impaired chromatin compactness, and increased sperm lipid peroxidation and mitochondrial superoxide anion levels. Conclusions: This study showed that PFOA alters several sperm parameters and thus it may play a negative role in male fertility. [ABSTRACT FROM AUTHOR]

Background: Male sterility results from high testicular temperatures, which affect mammalian spermatogenesis. High testicular temperatures affect sperm motility, morphology and fertility according to their magnitude and duration. Aim: The aim of the current study is to examine the effects of heat-induced oxidative stress and cinnamaldehyde on Wistar rat testicular structure and function. Settings and Design: The rats used in this experiment were Wistar albino rats. Materials and Methods: This research has six animals per group. Male Wistar albino rats of 2.5-3 months old and 275-300 g. (I) control, (II) heat stress (HS) in a closed chamber at 41°C for 14 days and (III) HS with cinnamaldehyde (CA) 50 mg/kg body weight for 14 days. (IV) CA alone. After the study, the animals were euthanised, and test samples were taken for sperm count, morphology, haematoxylin and eosin stain for normal cellular morphology, antioxidants and DNA integrity assessments. Statistical Analysis Used: The data were analysed statistically using one- and two-way ANOVA tests for comparisons between groups. Results: The stress group had significantly lower sperm counts and poor sperm morphology. The stress group's antioxidant capacity is much lower than that of the control group. Animals under stress have fragmented DNA. Treatment with cinnamaldehyde increased overall antioxidant capacity and seminal parameters, and rats behaved most like controls. Conclusion: CA restores malondialdehyde levels, total antioxidant capacity, sperm characteristics and mitigates testicular damage in rats exposed to experimental HS. [ABSTRACT FROM AUTHOR]

ABSTRACT The high quality of soybean seeds is essential for the success of the crop, as it determines the field performance of seedlings. Seed physiological attributes are associated with genotype stability; thus, the maintenance of genome integrity is crucial to ensure seed quality. To evaluate the quality of soybean seed lots at physiological and molecular levels, a completely randomized design was carried out in four replications, with 12 soybean seed lots (cv. BMX Potência RR). Seeds were evaluated through standard germination and vigor tests. Genomic DNA was extracted from the root meristem of seeds from different lots with the Kasvi Spin 50 ® extraction kit. The analysis was performed through electrophoresis and visualized under ultraviolet (UV) light. The Comet assay was also applied to investigate DNA damage. Soybean seed lots exhibited differences in germination and vigor, as determined by the tests of accelerated aging, field emergence, emergence speed index, shoot length, root length, shoot fresh weight, root fresh mass, and shoot dry mass. In seeds with low germination and vigor, DNA damage and genotoxicity were observed. DNA from samples with high germination and vigor observed by the field emergence and accelerated aging tests was more preserved than DNA extracted from seeds with low physiological quality.

Silver nanoparticles possess valuable physical, chemical, and biological properties, rendering them widely applied as bioactive agents in the industry. Nonetheless, their influence on the natural environment and on living organisms remains unclear. Therefore, this study aims to investigate the impact of polymer composites containing silver nanoparticles on sperm cells. The nanosilver polymer composites were chemically synthesized, employing sodium alginate as the stabilizer. The reducing agents employed were solutions comprising sodium borohydride and xylose. The concentration of silver nanoparticles obtained after synthesis was 100 parts per million. The examined biological species were rabbit sperm cells. The impact of nanosilver on the sperm was assessed through the elucidation of the toxicity profile, comet test, and analysis of morphological characteristics of the animal cells. The results of the study demonstrate a twofold impact of polymer composites infused with silver nanoparticles on domestic rabbit sperm when obtained through chemical synthesis using two reducing agents (xylose and sodium borohydride) at a 10 ppm concentration. The comet test showed no harmful effect on the DNA integrity of rabbit sperm by the tested compounds. Twenty-four-hour exposure of rabbit spermatozoa to silver nanoparticles, obtained by reducing xylose and borohydride, induced significant secondary changes in the morphological structure of male reproductive cells. These findings indicate the potential reproductive toxicity of silver nanoparticles. [ABSTRACT FROM AUTHOR]

Objective: This study evaluated the effects of temperature and storage time on the quality and DNA integrity of freeze-dried sperm from individuals with normozoospermia. Methods: Normal sperm samples from 15 men aged 24 to 40 years were studied. Each sample was divided into six groups: fresh, freezing (frozen in liquid nitrogen), freeze-dried then preserved at room temperature for 1 month (FD-1m-RT), freeze-dried then preserved at room temperature for 2 months (FD-2m-RT), freeze-dried then preserved at 4 °C for 1 month (FD-1m-4 °C), and freeze-dried then preserved at 4 °C for 2 months (FD-2m-4 °C). The morphology, progressive motility, vitality, and DNA integrity of the sperm were evaluated in all groups. Results: In all freeze-dried groups, sperm cells were immotile after rehydration. The freeze-dried groups also showed significantly less sperm vitality than the fresh and frozen groups. Significantly more morphological sperm abnormalities were found in the freeze-dried groups, but freeze-drying did not lead to a significantly higher DNA fragmentation index (DFI). The DFI was significantly higher in the FD-2m-RT group than in the other freeze-dried groups. Conclusion: The freeze-drying method preserved the integrity of sperm DNA. The temperature and duration of storage were also identified as factors that influenced the DFI. Accordingly, more research is needed on ways to improve sperm quality in the freeze-drying process. [ABSTRACT FROM AUTHOR]

Simple Summary: Salicornia ramosissima was studied as a potential feed material for juvenile European seabass in aquaculture. This halophyte was incorporated in three different levels (2.5, 5 and 10%) into the fish diet for two months. The results showed no negative impact on the fish's growth, and there was an improvement in antioxidant activity in specific tissues, particularly through the GSH-related defense subsystem. A genotoxic trigger was also identified in the first month, which was considered a protective mechanism, and in the second month, a clear protection of DNA integrity was observed, especially at higher supplementation levels. Overall, the findings suggest that supplemented diets with S. ramosissima have functional benefits, offering a potential way to enhance aquaculture practices and utilize a novel, low-value raw material. The identification of novel feed materials as a source of functional ingredients is a topical priority in the finfish aquaculture sector. Due to the agrotechnical practices associated and phytochemical profiling, halophytes emerge as a new source of feedstuff for aquafeeds, with the potential to boost productivity and environmental sustainability. Therefore, the present study aimed to assess the potential of Salicornia ramosissima incorporation (2.5, 5, and 10%), for 2 months, in the diet of juvenile European seabass, seeking antioxidant (in the liver, gills, and blood) and genoprotective (DNA and chromosomal integrity in blood) benefits. Halophyte inclusion showed no impairments on growth performance. Moreover, a tissue-specific antioxidant improvement was apparent, namely through the GSH-related defense subsystem, but revealing multiple and complex mechanisms. A genotoxic trigger (regarded as a pro-genoprotective mechanism) was identified in the first month of supplementation. A clear protection of DNA integrity was detected in the second month, for all the supplementation levels (and the most prominent melioration at 10%). Overall, these results pointed out a functionality of S. ramosissima-supplemented diets and a promising way to improve aquaculture practices, also unraveling a complementary novel, low-value raw material, and a path to its valorization. [ABSTRACT FROM AUTHOR]

Abstract Background Protamination and condensation of sperm chromatin as well as DNA integrity play an essential role during fertilization and embryo development. In some mammals, like pigs, ejaculates are emitted in three separate fractions: pre-sperm, sperm-rich (SRF) and post sperm-rich (PSRF). These fractions are known to vary in volume, sperm concentration and quality, as well as in the origin and composition of seminal plasma (SP), with differences being also observed within the SRF one. Yet, whether disparities in the DNA integrity and chromatin condensation and protamination of their sperm exist has not been interrogated. Results This study determined chromatin protamination (Chromomycin A3 test, CMA3), condensation (Dibromobimane test, DBB), and DNA integrity (Comet assay) in the pig sperm contained in the first 10 mL of the SRF (SRF-P1), the remaining portion of the sperm-rich fraction (SRF-P2), and the post sperm-rich fraction (PSRF). While chromatin protamination was found to be similar between the different ejaculate fractions (P > 0.05), chromatin condensation was seen to be greater in SRF-P1 and SRF-P2 than in the PSRF (P = 0.018 and P = 0.004, respectively). Regarding DNA integrity, no differences between fractions were observed (P > 0.05). As the SRF-P1 has the highest sperm concentration and ejaculate fractions are known to differ in antioxidant composition, the oxidative stress index (OSi) in SP, calculated as total oxidant activity divided by total antioxidant capacity, was tested and confirmed to be higher in the SRF-P1 than in SRF-P2 and PSRF (0.42 ± 0.06 vs. 0.23 ± 0.09 and 0.08 ± 0.00, respectively; P

We evaluated the effects of feeding 60% dried corn distillers’ grains plus solubles (DDGS) or the equivalent sulfur as CaSO4 on sperm characteristics and transcript abundance. Thirty-six half-sibling Angus bulls (256 ± 8.5 d; initial BW = 320 ± 2.7 kg) were assigned to one of three treatments: (1) 60% concentrate as corn (CON); (2) 60% DDGS as corn replacement (60DDGS); and (3) CON diet + equivalent sulfur of 60DDGS added as CaSO4 (SULF). The acrosome/cell membrane integrity, mitochondrial energy potential, oxidation status, DNA integrity, and zinc signatures were analyzed via flow cytometry. Sperm-specific gene expression was assessed via RNA sequencing. The flow cytometry data were analyzed using PROC MIXED in SAS to determine the effects of treatment. Pairwise comparisons based on edgeR were used to identify differentially expressed genes. The percentage of polarized mitochondria tended to be greater (p = 0.08) for SULF compared with CON and 60DDGS. Protamine 1 was upregulated (p < 0.01; FDR = 0.10) in 60DDGS compared to CON. Zinc signature 1 in 60DDGS and SULF was reduced (p = 0.03) compared to CON. This study suggests that feeding bulls diets containing 60% DDGS had little effect on DNA integrity and gene expression.

We evaluated the effects of feeding 60% dried corn distillers' grains plus solubles (DDGS) or the equivalent sulfur as CaSO4 on sperm characteristics and transcript abundance. Thirty-six half-sibling Angus bulls (256 ± 8.5 d; initial BW = 320 ± 2.7 kg) were assigned to one of three treatments: (1) 60% concentrate as corn (CON); (2) 60% DDGS as corn replacement (60DDGS); and (3) CON diet + equivalent sulfur of 60DDGS added as CaSO4 (SULF). The acrosome/cell membrane integrity, mitochondrial energy potential, oxidation status, DNA integrity, and zinc signatures were analyzed via flow cytometry. Sperm-specific gene expression was assessed via RNA sequencing. The flow cytometry data were analyzed using PROC MIXED in SAS to determine the effects of treatment. Pairwise comparisons based on edgeR were used to identify differentially expressed genes. The percentage of polarized mitochondria tended to be greater (p = 0.08) for SULF compared with CON and 60DDGS. Protamine 1 was upregulated (p < 0.01; FDR = 0.10) in 60DDGS compared to CON. Zinc signature 1 in 60DDGS and SULF was reduced (p = 0.03) compared to CON. This study suggests that feeding bulls diets containing 60% DDGS had little effect on DNA integrity and gene expression. [ABSTRACT FROM AUTHOR]

Background: Protamination and condensation of sperm chromatin as well as DNA integrity play an essential role during fertilization and embryo development. In some mammals, like pigs, ejaculates are emitted in three separate fractions: pre-sperm, sperm-rich (SRF) and post sperm-rich (PSRF). These fractions are known to vary in volume, sperm concentration and quality, as well as in the origin and composition of seminal plasma (SP), with differences being also observed within the SRF one. Yet, whether disparities in the DNA integrity and chromatin condensation and protamination of their sperm exist has not been interrogated. Results: This study determined chromatin protamination (Chromomycin A3 test, CMA3), condensation (Dibromobimane test, DBB), and DNA integrity (Comet assay) in the pig sperm contained in the first 10 mL of the SRF (SRF-P1), the remaining portion of the sperm-rich fraction (SRF-P2), and the post sperm-rich fraction (PSRF). While chromatin protamination was found to be similar between the different ejaculate fractions (P > 0.05), chromatin condensation was seen to be greater in SRF-P1 and SRF-P2 than in the PSRF (P = 0.018 and P = 0.004, respectively). Regarding DNA integrity, no differences between fractions were observed (P > 0.05). As the SRF-P1 has the highest sperm concentration and ejaculate fractions are known to differ in antioxidant composition, the oxidative stress index (OSi) in SP, calculated as total oxidant activity divided by total antioxidant capacity, was tested and confirmed to be higher in the SRF-P1 than in SRF-P2 and PSRF (0.42 ± 0.06 vs. 0.23 ± 0.09 and 0.08 ± 0.00, respectively; P < 0.01); this index, in addition, was observed to be correlated to the sperm concentration of each fraction (Rs = 0.973; P < 0.001). Conclusion: While sperm DNA integrity was not found to differ between ejaculate fractions, SRF-P1 and SRF-P2 were observed to exhibit greater chromatin condensation than the PSRF. This could be related to the OSi of each fraction. [ABSTRACT FROM AUTHOR]

The evaluation of sperm DNA integrity is recommended in the sixth edition of the 2021 World Health Organization guidelines. Oxidative stress has been identified as a crucial factor leading to genome decay, lipid peroxidation, and nucleoprotein oxidation. This double-blind, placebo-controlled clinical trial aimed to assess the effect of oral antioxidant treatment (Fertilis), which contains L-carnitine and some micronutrients, in the improvement of conventional sperm parameters, sperm DNA integrity and in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) outcomes. A total of 263 participants were enrolled and randomly divided into two groups: 131 participants received the antioxidant treatment, while 132 participants received a placebo. The male partners in both groups underwent the antioxidant treatment or the placebo for a duration of three months. For each participant, we performed a hormonal test, an infectious test, a spermogram, a TUNEL assay for sperm DNA fragmentation, a toluidine blue staining for sperm DNA decondensation, and an IVF/ICSI procedure. Sperm characteristics analysis (volume, count, motility, and vitality), sperm DNA fragmentation, and sperm DNA decondensation were assessed and compared to the results preceding the antioxidant treatment. The study outcome revealed a significant decrease in the DNA fragmentation index and a significant increase in sperm motility after 3 months of treatment (p = 0.01 and p = 0.02, respectively). Additionally, a significant improvement in clinical pregnancy rate (p = 0.01) and life birth rate (p = 0.031) was observed. No significant changes were observed in conventional sperm parameters (volume, count, and vitality) or sperm DNA decondensation (SDI). Antioxidant therapy has a beneficial impact on achieving pregnancy, whether through spontaneous conception or assisted reproductive procedures (ART). [ABSTRACT FROM AUTHOR]

Background: Circulating cell-free DNA (cfDNA) concentration and integrity as noninvasive biomarkers play an important role in cancer diagnosis, prognosis and therapy monitoring. However, few studies have been conducted on the combination of plasma cfDNA concentration, integrity and tumor markers (CEA, CA125, NSE and CYFRA21-1) for cancer detection. Thus, the purpose of this study was to investigate the diagnostic value of combining plasma cfDNA concentration, integrity and tumor markers in early detection of non-small cell lung cancer (NSCLC). Methods: Plasma cfDNA concentration from 50 healthy controls and 84 NSCLC patients were assessed by quantitative real-time PCR of ALU repeated sequence. Plasma cfDNA integrity was calculated as the ratio of long to short fragments (ALU115/60). Results: Plasma cfDNA concentration (ALU60 and ALU115) and integrity ALU115/60 were significantly higher in NSCLC patients with stage III/IV than in healthy controls (p = 0.0002, p

Purpose: We aimed to examine the longitudinal, intra-personal changes in DNA fragmentation index (DFI) over time. Methods: Men who performed at least two DFI measurements (using sperm chromatin structure assay (SCSA) between 2003 and 2019 were included in this study and allocated to groups by time between DFI tests: < 1 year, 1–3 years, 3–5 years, and > 5 years. An analysis of DFI change over time according to age groups was additionally performed. Regression models were developed to predict changes in DFI with time. Results: Overall, 225 patients had two or more DFI measurements done at least a month apart (mean of 586.7± 710.0 days). The < 1 year (n = 124) and 1–3 years (n = 68) groups demonstrated decreased DFI levels, while an increase in DFI was shown in 3–5 years (n = 21) and more than 5 years (n = 12) groups − 7.1 ± 14.9%, − 4.5 ± 13.4%, + 3.2 ± 8.4%, and + 10.8 ± 18.0%, respectively, p < 0.001). This trend was similarly shown in age subgroups of under 40 years and 40–50 years at baseline DFI. Linear regression models showed that the factors predictive of DFI increase are baseline DFI and > 3 years between DFI tests. Conclusion: This study shows that DFI, in men being investigated for infertility, initially decreases in the first 3 years of follow-up, and then increases over time with the highest increase occurring after 5 years interval (an average increase of 10.8%). Testing infertile men's DFI levels at first evaluation may contribute to personalized consult regarding future reproductive outcomes. [ABSTRACT FROM AUTHOR]

Rice genotypes with possible tolerance to biotic and abiotic stresses are interested in genomic studies for their use in breeding programs. Colombian Plant Germplasm Bank (CPGB) has preserved local rice genotypes without genomic analysis yet. Considering the need to develop genomic studies with these genotypes and minimize costs in the genotyping process, in this research, five CTAB DNA extraction protocols (P1-P5) were evaluated and compared with a commercial kit. Four local rice accessions from the CPGB were used, and DNA concentration (fluorescence and absorbance), quality (absorbance, integrity, and enzymatic digestion), and cost analysis were determined. A selection index (SI) was established to choose the best DNA protocol. Finally, the selected protocol was validated in 14 local rice genotypes through Illumina sequencing. The six evaluated DNA extraction protocols presented significant differences in quantification and quality variables. From the SI established, the protocol selected was the P4 (SI = 147.7); this protocol allows obtaining DNA with high concentrations and quality levels at a 50% lower cost than the commercial kit. The sequences generated from the DNA obtained with the P4 protocol presented 7 million reads with high levels of quality (Q30) and length (> 50 pb) per evaluated accession; On average, 98.9% of these sequences mapped to the rice genome and identified up to 15,436 SNP markers without missing data. These results confirm that the DNA from rice local genotypes obtained with the P4 CTAB protocol has the requirements for routine use in genomic studies based on genotyping processes that require Illumina sequencing. [ABSTRACT FROM AUTHOR]

According to the United Nations' Food and Agriculture Organization, the quantities of pesticide used around the world have increased regularly since the 1990s. Given that pesticides may be classified as carcinogenic, mutagenic, neurotoxic, or toxic for reproduction, some have endocrine-disrupting properties that might be associated with a decline in sperm parameters in general and sperm DNA integrity in particular. These days, a sperm analysis is not enough to determine the etiology of male infertility. Genome integrity analysis is a key step in clarifying a large proportion of cases of male infertility. The objective of the present retrospective study was to assess the impact of self-reported pesticide exposure on sperm parameters and sperm DNA integrity in men consulting for infertility. In a retrospective study, a population of 671 men living in the Picardy region of France were assessed in a conventional sperm parameter analysis, Shorr staining, a DNA fragmentation assay (terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling), and chromatin decondensation with aniline blue staining. The exposed and the non-exposed groups did not differ significantly in some of the conventional sperm parameters (including volume, sperm count, and percent typical forms). However, vitality, progressive motility, and non-progressive motility were significantly lower in the exposed group. Levels of DNA fragmentation and chromatin decondensation were moderately higher in the exposed group. [ABSTRACT FROM AUTHOR]

Background: Sperm is more than just a mere transporter of paternal genetic information, although serving purposes other than conception. DNA integrity (DI) is regarded as a crucial aspect of semen quality and has great significance in predicting male fertility. Washing sperm is a form of preparation of sperm which is essential before processing intra-uterine-insemination since it removes chemicals from semen that might cause adversarial reactions in the uterus. During the process of washing, sperm gets separated from seminal fluid, which gradually improves the fertilizing capability of sperm, and numerous existing methods have faced complications with the determination of effective integrity in washing methods. The objective of the present study was to find the DI of the sperms through three washing methods that, include the density gradient (DG) method, swim up (SU) method, and magnetically activated cell sorting (MACS) method after the cryo-preservation (CP) for 3 months. Methodology: Semen samples were obtained from 40 oligoasthenospermic patients with progressive motility

Despite there have been many experiments conducted about antioxidants, the best sole or combination use of antioxidants to include as a standard ingredient to freezing extenders is yet to be found. This study was designed to investigate the different doses of methionine (2.5 and 5 mM), cysteine (1 and 2 mM), and butylated hydroxytoluene (BHT) (1 and 2 mM) for ram semen cryopreservation on post-thaw and post-incubation (6 h) time points over spermatological parameters. Semen samples were collected from Kivircik rams via electro-ejaculator in breeding season. After essential spermatological evaluations, appropriate samples were pooled then split into 7 equal aliquots to create study groups (antioxidant free control, 2.5 mM methionine, 5 mM methionine, 1 mM cysteine, 2 mM cysteine, 1 mM BHT, and 2 mM BHT). Semen samples were put into French straws (0.25 mL), and freezing procedure (two-step) was conducted via a programmable gamete freezer. At both time points, motility, HOST, PSA-FITC, and TUNEL assays were made to discover the impacts of cryopreservation and incubation process over sperm cells. Antioxidant supplemented groups yielded better results compared to the control groups in terms of various spermatological parameters not only at post-thaw time point but after incubation for 6 h of time. The study demonstrated that supplementing sperm freezing extenders with previous antioxidants may create new approaches to cryopreservation procedures, and through increasing success rate of freezing, fertility results may increase to better results in near future. [ABSTRACT FROM AUTHOR]

Type I diabetes is a prominent human pathology with increasing incidence in the population; however, its cause is still unknown. This disease promotes detrimental effects on reproduction, such as lower sperm motility and DNA integrity. Hence, the investigation of the underlying mechanisms of this metabolic disturbance in reproduction and its transgenerational consequences is of the utmost importance. The zebrafish is a useful model for this research considering its high homology with human genes as well as its fast generation and regeneration abilities. Therefore, we aimed to investigate sperm quality and genes relevant to diabetes in the spermatozoa of Tg(ins:nfsb-mCherry) zebrafish, a model for type I diabetes. Diabetic Tg(ins:nfsb-mCherry) males showed significantly higher expression of transcripts for insulin a (insa) and glucose transporter (slc2a2) compared to controls. Sperm obtained from the same treatment group showed significantly lower sperm motility, plasma membrane viability, and DNA integrity compared to that from the control group. Upon sperm cryopreservation, sperm freezability was reduced, which could be a consequence of poor initial sperm quality. Altogether, the data showed similar detrimental effects related to type I diabetes in zebrafish spermatozoa at the cellular and molecular levels. Therefore, our study validates the zebrafish model for type I diabetes research in germ cells. [ABSTRACT FROM AUTHOR]

Silver nanoparticles possess valuable physical, chemical, and biological properties, rendering them widely applied as bioactive agents in the industry. Nonetheless, their influence on the natural environment and on living organisms remains unclear. Therefore, this study aims to investigate the impact of polymer composites containing silver nanoparticles on sperm cells. The nanosilver polymer composites were chemically synthesized, employing sodium alginate as the stabilizer. The reducing agents employed were solutions comprising sodium borohydride and xylose. The concentration of silver nanoparticles obtained after synthesis was 100 parts per million. The examined biological species were rabbit sperm cells. The impact of nanosilver on the sperm was assessed through the elucidation of the toxicity profile, comet test, and analysis of morphological characteristics of the animal cells. The results of the study demonstrate a twofold impact of polymer composites infused with silver nanoparticles on domestic rabbit sperm when obtained through chemical synthesis using two reducing agents (xylose and sodium borohydride) at a 10 ppm concentration. The comet test showed no harmful effect on the DNA integrity of rabbit sperm by the tested compounds. Twenty-four-hour exposure of rabbit spermatozoa to silver nanoparticles, obtained by reducing xylose and borohydride, induced significant secondary changes in the morphological structure of male reproductive cells. These findings indicate the potential reproductive toxicity of silver nanoparticles.

Background: Semen cryopreservation could be enhanced by integration of biocompatible nanomaterials. Conditions in which nanoparticles are synthesized should be taken in consideration to avoid divergences in research results concerning beneficial or toxic effects. The objective of this study was to examine the potential of zinc oxide nanoparticles (ZnONPs) synthesized by simple co-precipitation method for providing protection to buffalo sperm against cryo-damage. Methods: ZnONPs were characterized by means of X-ray diffraction, high resolution transmission electron microscope (HR-TEM), zetasizer and Raman spectroscopy. Semen was collected from six healthy buffalo bulls using artificial vagina and pooled semen samples were diluted with tris-egg yolk-fructose extender only (control) or extender supplemented with 10, 25, 50, 100 and 200 µg/ml of ZnONPs. Results: Sperm motility, membrane integrity and acrosome defects were measured. DNA damage of spermatozoa was evaluated using comet assay. To determine antioxidative role of ZnONPs, malondialdehyde (MDA) level as a biomarker of lipid peroxidation was measured in seminal plasma. Sperm motility, viability index as well as membrane integrity increased with ZnONPs concentrations up to 100 μg/ml. Likewise, no adverse effect was reported on sperm DNA integrity with all concentrations. MDA decreased up to concentration of 100 μg/ml ZnONPs. Notably, adding 25, and 50 μg/ml ZnONPs to extender produced best significant positive effects on semen parameters compared to ZnONPs free group. Conclusion: The results confirm that low concentrations of ZnONPs fabricated by co-precipitation method using water as exchange media could effectively preserve the quality of buffalo bull frozen-thawed sperm by reduction of cryoinjury.

Human fertilization begins when a capacitated spermatozoon binds to the zona pellucida (ZP) surrounding a mature oocyte. Defective spermatozoa-ZP interaction contributes to male infertility and is a leading cause of reduced fertilization rates in assisted reproduction treatments (ARTs). Human ejaculate contains millions of spermatozoa with varying degrees of fertilization potential and genetic quality, of which only thousands of motile spermatozoa can bind to the ZP at the fertilization site. This observation suggests that human ZP selectively interacts with competitively superior spermatozoa characterized by high fertilizing capability and genetic integrity. However, direct evidence for ZPmediated sperm selection process is lacking. This study aims to demonstrate that spermatozoa-ZP interaction represents a crucial step in selecting fertilizationcompetent spermatozoa in humans. ZP-bound and unbound spermatozoa were respectively collected by a spermatozoa-ZP coincubation assay. The timecourse data demonstrated that ZP interacted with a small proportion of motile spermatozoa. Heat shock 70 kDa protein 2 (HSPA2) and sperm acrosome associated 3 (SPACA 3) are two protein markers associated with the sperm ZPbinding ability. Immunofluorescent staining indicated that the ZP-bound spermatozoa had significantly higher expression levels of HSPA2 and SPACA3 than the unbound spermatozoa. ZP-bound spermatozoa had a significantly higher level of normal morphology, DNA integrity, chromatin integrity, protamination and global methylation when compared to the unbound spermatozoa. The results validated the possibility of applying spermatozoa-ZP interaction to select fertilization-competent spermatozoa in ART. This highly selective interaction might also provide diagnostic information regarding the fertilization potential and genetic qualities of spermatozoa independent of those derived from the standard semen analysis. [ABSTRACT FROM AUTHOR]

The experiment was conducted to evaluate the metabolic activity through MTT (3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) reduction assay of Indian red jungle fowl sperm during cryopreservation process. In addition, MTT test was compared with sperm motility and viability data. Sperm analysis was made at pre-dilution, postdilution, post-cooling, post-equilibration and post freeze-thawing stages. Pearson's correlation coefficient showed significant (P<0.01) positive association of MTT reduction rate with motility (r=0.85) and viability (r=0.84). A significant (P<0.05) decline was observed in motility, viability and metabolic status after each stage of cryopreservation. The highest (P<0.05) stage-specific percent decrease in motility and viability were observed after freeze-thawing. Nevertheless, stage-specific percent decrease in metabolic status differ (P<0.05) after dilution (5.93 ± 0.12), cooling (8.41 ± 0.03), equilibration (19.1 ± 0.04) and freeze-thawing (30.9 ± 0.08). The total decrease in motility, viability and metabolic activity after the complete freeze-thawing cycle were calculated as 56%, 47% and 52%, respectively. It is concluded that MTT reduction assay is simple and reliable method to evaluate mitochondrial redox activity in sperm of this threatened species. [ABSTRACT FROM AUTHOR]

This study aimed to develop a protocol for the cryopreservation of Pseudoplatystoma corruscans semen. For this, mature males were hormonally induced with a single dose of carp pituitary extract (5 mg/kg body weight). Semen was collected and evaluated. Two cryoprotectants were tested to compose the diluents: dimethyl acetamide (DMA) and dimethyl sulfoxide (Me2SO), in two concentrations (8% and 10%), + 5.0% glucose + 10% egg yolk. The semen was diluted in a 1: 4 ratio (semen: extender), packed in 0.5 mL straws and frozen in a dry shipper container in liquid nitrogen vapors. After thawing, sperm kinetics, sperm morphology and DNA integrity of cryopreserved sperm were evaluated. Pseudoplatystoma corruscans males produced semen with sperm motility > 80%. After thawing, all treatments provided semen with total sperm motility > 40%, with no significant difference (P < 0.05) between them, as well as between the other sperm kinetic parameters evaluated. The treatments with DMA provided a smaller fragmentation of the DNA of the gametes. Sperm malformations were identified in both fresh and cryopreserved semen, with a slight increase in these malformations being identified in sperm from thawed P corruscans semen samples. [ABSTRACT FROM AUTHOR]

Polyphenols are a diverse and widely distributed class of secondary metabolites, which possess numerous beneficial properties including a modulation of glucose and lipid metabolism. This placebo-controlled human intervention study was performed to explore effects of polyphenol-rich beverage (PRB) uptake on lipid metabolism, as well as DNA integrity. In this case, 36 healthy men were randomly divided to consume either 750 mL of a PRB (containing 51% chokeberry, cranberry, and pomegranate) or a placebo drink daily for eight weeks. Only PRB consumption was found to decrease fat and protein intakes significantly compared to the preceding one-week washout period. During the intervention with PRB an increased fat-free mass was shown after four weeks, whereas a significant elevation in body weight and leptin was observed in placebo group. Blood lipids were not significantly altered after PRB consumption, while triglyceride levels increased after placebo drink intake. In platelets, a significant inhibition of phosphodiesterase (PDE) activity was observed, more pronounced in test group. Consuming the PRB decreased total DNA strand breaks in whole blood as well as H2O2-induced breaks in isolated lymphocytes. Overall, our study suggested beneficial effects on lipid metabolism by reduced energy intake, modulation of biomarkers such as PDE activity and improved DNA integrity associated with PRB consumption. [ABSTRACT FROM AUTHOR]

Male Infertility Oxidative System (MiOXSYS) has been proposed as a rapid and promising technology for the evaluation of sperm oxidative stress. In this case–control study, 134 men with normal sperm parameters (NSP) and 574 men with abnormal sperm parameters (ASP), according to the World Health Organization sperm assessment references values established in 2010, were enrolled. Conventional sperm parameters were evaluated in all patients. Sperm static oxido-reduction potential (sORP) was assessed using the MiOXSYS. Sperm DNA integrity was measured in 604 patients. To ensure that sperm concentration was not a confounding factor in the sORP index ratio, sperm and seminal fluid sORP from 57 randomly selected additional patients were also measured using the MiOXSYS. sORP index (mV/106 sperm/ mL) was higher in patients with ASP and seemed to correlate with conventional sperm parameters. Although receiver-operating characteristic analysis revealed that a sORP index cut-off of 0.79 could differentiate normal from ASP with 57.7% sensitivity and 73.1% specificity, these values are much lower than those found in the literature. These values also need to be higher to be applicable in a clinical setting. Furthermore, absolute sORP (mV) was not different in the presence or absence of spermatozoa. sORP index relationships with sperm parameters seem rather be due to sperm concentration, denominator of the sORP index ratio. The establishment of a reliable method using the absolute sORP value, independent of sperm concentration, needs to be addressed. Other oxidative stress biomarkers could be used to validate this method.

The identification of novel feed materials as a source of functional ingredients is a topical priority in the finfish aquaculture sector. Due to the agrotechnical practices associated and phytochemical profiling, halophytes emerge as a new source of feedstuff for aquafeeds, with the potential to boost productivity and environmental sustainability. Therefore, the present study aimed to assess the potential of Salicornia ramosissima incorporation (2.5, 5, and 10%), for 2 months, in the diet of juvenile European seabass, seeking antioxidant (in the liver, gills, and blood) and genoprotective (DNA and chromosomal integrity in blood) benefits. Halophyte inclusion showed no impairments on growth performance. Moreover, a tissue-specific antioxidant improvement was apparent, namely through the GSH-related defense subsystem, but revealing multiple and complex mechanisms. A genotoxic trigger (regarded as a pro-genoprotective mechanism) was identified in the first month of supplementation. A clear protection of DNA integrity was detected in the second month, for all the supplementation levels (and the most prominent melioration at 10%). Overall, these results pointed out a functionality of S. ramosissima-supplemented diets and a promising way to improve aquaculture practices, also unraveling a complementary novel, low-value raw material, and a path to its valorization.

The evaluation of sperm DNA integrity is recommended in the sixth edition of the 2021 World Health Organization guidelines. Oxidative stress has been identified as a crucial factor leading to genome decay, lipid peroxidation, and nucleoprotein oxidation. This double-blind, placebo-controlled clinical trial aimed to assess the effect of oral antioxidant treatment (Fertilis), which contains L-carnitine and some micronutrients, in the improvement of conventional sperm parameters, sperm DNA integrity and in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) outcomes. A total of 263 participants were enrolled and randomly divided into two groups: 131 participants received the antioxidant treatment, while 132 participants received a placebo. The male partners in both groups underwent the antioxidant treatment or the placebo for a duration of three months. For each participant, we performed a hormonal test, an infectious test, a spermogram, a TUNEL assay for sperm DNA fragmentation, a toluidine blue staining for sperm DNA decondensation, and an IVF/ICSI procedure. Sperm characteristics analysis (volume, count, motility, and vitality), sperm DNA fragmentation, and sperm DNA decondensation were assessed and compared to the results preceding the antioxidant treatment. The study outcome revealed a significant decrease in the DNA fragmentation index and a significant increase in sperm motility after 3 months of treatment (p = 0.01 and p = 0.02, respectively). Additionally, a significant improvement in clinical pregnancy rate (p = 0.01) and life birth rate (p = 0.031) was observed. No significant changes were observed in conventional sperm parameters (volume, count, and vitality) or sperm DNA decondensation (SDI). Antioxidant therapy has a beneficial impact on achieving pregnancy, whether through spontaneous conception or assisted reproductive procedures (ART).

Abstract Background Most plants encounter water stress at one or more different stages of their life cycle. The maintenance of genetic stability is the integral component of desiccation tolerance that defines the storage ability and long-term survival of seeds. Embryonic axes of desiccation-sensitive recalcitrant seeds of Acer pseudoplatnus L. were used to investigate the genotoxic effect of desiccation. Alkaline single-cell gel electrophoresis (comet assay) methodology was optimized and used to provide unique insights into the onset and repair of DNA strand breaks and 8-oxo-7,8-dihydroguanine (8-oxoG) formation during progressive steps of desiccation and rehydration. Results The loss of DNA integrity and impairment of damage repair were significant predictors of the viability of embryonic axes. In contrast to the comet assay, automated electrophoresis failed to detect changes in DNA integrity resulting from desiccation. Notably, no significant correlation was observed between hydroxyl radical (٠OH) production and 8-oxoG formation, although the former is regarded to play a major role in guanine oxidation. Conclusions The high-throughput comet assay represents a sensitive tool for monitoring discrete changes in DNA integrity and assessing the viability status in plant germplasm processed for long-term storage.

Background: Microfluidics (MF), an advanced sperm sorting technology results in the extraction of spermatozoa with higher DNA integrity and lower DNA damage compared to existing conventional sperm sorting methods. Aims: The aim of the present study is to assess the efficiency of MF and to isolate the best spermatozoa for intracytoplasmic sperm injection (ICSI) over the density gradient (DG) technique. Settings and Design: We recruited couples who choose the oocyte donation programme for this study to eliminate confounding factors associated with oocyte quality. Materials and Methods: Sperm was processed by MF (n = 180) and DG (n = 151). ICSI was performed and positive pregnancy, miscarriage and clinical pregnancy rates were compared. Statistical Analysis Used: All variables were analysed using Graph Pad Prism 5. The unpaired two-tailed t-test was used to assess the significance. A value of P < 0.05 was considered statistically significant. Results: There was no significant difference in pregnancy rates between the groups. However, a clear demarcation is seen in terms of clinical pregnancy rates, where the DG group achieved higher clinical pregnancies (91.7%) compared to the MF group (80.7%). Further, we compared miscarriage rates and biochemical pregnancies, and found a significantly higher miscarriage and biochemical pregnancy rate in the MF group (14.5% and 4%, respectively) compared to the DG group (6% and 1%, respectively). Conclusions: Based on the available literature, we anticipated a higher clinical pregnancy rate with MF compared with conventional processing. Our results show MF does not have any add-on positive effect on clinical pregnancy rate.

Human fertilization begins when a capacitated spermatozoon binds to the zona pellucida (ZP) surrounding a mature oocyte. Defective spermatozoa-ZP interaction contributes to male infertility and is a leading cause of reduced fertilization rates in assisted reproduction treatments (ARTs). Human ejaculate contains millions of spermatozoa with varying degrees of fertilization potential and genetic quality, of which only thousands of motile spermatozoa can bind to the ZP at the fertilization site. This observation suggests that human ZP selectively interacts with competitively superior spermatozoa characterized by high fertilizing capability and genetic integrity. However, direct evidence for ZP-mediated sperm selection process is lacking. This study aims to demonstrate that spermatozoa-ZP interaction represents a crucial step in selecting fertilization-competent spermatozoa in humans. ZP-bound and unbound spermatozoa were respectively collected by a spermatozoa-ZP coincubation assay. The time-course data demonstrated that ZP interacted with a small proportion of motile spermatozoa. Heat shock 70 kDa protein 2 (HSPA2) and sperm acrosome associated 3 (SPACA 3) are two protein markers associated with the sperm ZP-binding ability. Immunofluorescent staining indicated that the ZP-bound spermatozoa had significantly higher expression levels of HSPA2 and SPACA3 than the unbound spermatozoa. ZP-bound spermatozoa had a significantly higher level of normal morphology, DNA integrity, chromatin integrity, protamination and global methylation when compared to the unbound spermatozoa. The results validated the possibility of applying spermatozoa-ZP interaction to select fertilization-competent spermatozoa in ART. This highly selective interaction might also provide diagnostic information regarding the fertilization potential and genetic qualities of spermatozoa independent of those derived from the standard semen analysis.

Pig breeding is mainly conducted through artificial insemination with liquid-stored semen. It is, therefore, crucial to ensure that sperm quality is over the standard thresholds, as reduced sperm motility, morphology or plasma membrane integrity are associated with reduced farrowing rates and litter sizes. This work aims to summarise the methods utilised in farms and research laboratories to evaluate sperm quality in pigs. The conventional spermiogram consists in the assessment of sperm concentration, motility and morphology, which are the most estimated variables in farms. Yet, while the determination of these sperm parameters is enough for farms to prepare seminal doses, other tests, usually carried out in specialised laboratories, may be required when boar studs exhibit a decreased reproductive performance. These methods include the evaluation of functional sperm parameters, such as plasma membrane integrity and fluidity, intracellular levels of calcium and reactive oxygen species, mitochondrial activity, and acrosome integrity, using fluorescent probes and flow cytometry. Furthermore, sperm chromatin condensation and DNA integrity, despite not being routinely assessed, may also help determine the causes of reduced fertilising capacity. Sperm DNA integrity can be evaluated through direct (Comet, transferase deoxynucleotide nick end labelling (TUNEL) and its in situ nick variant) or indirect tests (Sperm Chromatin Structure Assay, Sperm Chromatin Dispersion Test), whereas chromatin condensation can be determined with Chromomycin A3. Considering the high degree of chromatin packaging in pig sperm, which only have protamine 1, growing evidence suggests that complete decondensation of that chromatin is needed before DNA fragmentation through TUNEL or Comet can be examined.

Abstract This study aimed to develop a protocol for the cryopreservation of Pseudoplatystoma corruscans semen. For this, mature males were hormonally induced with a single dose of carp pituitary extract (5 mg/kg body weight). Semen was collected and evaluated. Two cryoprotectants were tested to compose the diluents: dimethyl acetamide (DMA) and dimethyl sulfoxide (Me2SO), in two concentrations (8% and 10%), + 5.0% glucose + 10% egg yolk. The semen was diluted in a 1: 4 ratio (semen: extender), packed in 0.5 mL straws and frozen in a dry shipper container in liquid nitrogen vapors. After thawing, sperm kinetics, sperm morphology and DNA integrity of cryopreserved sperm were evaluated. Pseudoplatystoma corruscans males produced semen with sperm motility > 80%. After thawing, all treatments provided semen with total sperm motility > 40%, with no significant difference (P < 0.05) between them, as well as between the other sperm kinetic parameters evaluated. The treatments with DMA provided a smaller fragmentation of the DNA of the gametes. Sperm malformations were identified in both fresh and cryopreserved semen, with a slight increase in these malformations being identified in sperm from thawed P. corruscans semen samples.

Abstract Introduction: The partnership between a research community engagement team (CE Team) and a community advisory board (CAB) formed the basis for bidirectional communication in developing resources for participant recruitment in a DNA integrity study. Engaging with a minoritized community, this partnership focused on respect, accessibility, and expanded engagement. Methods: A ten-member CAB, working in two groups defined by meeting time convenience, provided insight and feedback to the CE Team in the creation of recruitment and consent materials, via an iterative design process in which one CAB group reviewed and enhanced materials, and the second group tested and refined them further. The continuous analysis of CE Team notes from CAB meetings captured information needed both for materials refinement and implementation of CAB-suggested activities. Results: The partnership resulted in the co-creation of recruitment and consent materials that facilitated the enrollment of 191 individuals into the study. The CAB encouraged and assisted in expanded engagement inclusive of community leaders. This broader engagement provided information about the DNA integrity study to community decision-makers as well as responded to questions and concerns about the research. The bidirectional communication between the CAB and the CE Team encouraged the researchers to consider topics and research interests related to the current study but also responsive to community concerns. Conclusions: The CAB helped the CE Team develop a better understanding of the language of partnership and respect. In this way, the partnership opened doors for expanded community engagement and effective communication with potential study participants.

Background: Methylphenidate hydrochloride (MPH) is a psychostimulant widely used in the treatment of attention‐deficit hyperactive disorder (ADHD), as well as a performance enhancer, for at least 60 years. Despite the notable effectiveness as a psychostimulant, ADHD is a chronic disorder and has a two‐third chance of accompanying the individual throughout life. Long‐term use of MPH has been associated not only with an increase in the development of neurodegenerative diseases, but it also causes side effects on male fertility in experimental animals. Objectives: To investigate whether methylphenidate poses a risk to sperm DNA structure and to the quality of embryos conceived after treatment during adolescence in rats. Materials and methods: Wistar rats at 38 days of age were treated either with 5 mg/kg body weight of MPH, in a single daily dose for 30 days, via gavage or with distilled water‐only protocol. Levels of oxidative stress in testicular and epididymal tissues were evaluated. Sperm chromatin quality and acrosome integrity was assessed under flow cytometry. From 107 days of age, animals were mated with untreated females. The effects of the paternal contribution at two different embryo development moments—cleavage stage (2.5 days post coitum) and late gestation (20 days post coitum) —were analyzed. Results: MPH caused high levels of sperm DNA damage, which was reflected in 40% of decrease in early embryo quality and a lower number of live pups at 20 dpc. Discussion: The high level of fragmentation seen in the embryos sired from the MPH group is consistent with the poor chromatin structure of the sperm and does not seem to be a result of oxidative stress in the reproductive tissues. Conclusions: The results presented here suggest that the subchronic use of MPH during male prepubertal phase may cause long‐term subfertility and compromise embryo survival. [ABSTRACT FROM AUTHOR]

Background: The environment plays a key role in male infertility, changing the incidence in various populations, and pesticides are one of the most studied hazards. The use of the latter has never decreased, jeopardizing the safety of workers and the general population. Objective: Our purpose was to summarize the results of studies discussing the association between pesticides and male fertility. Methods: A comprehensive literature search was performed through MEDLINE via PubMed, Scopus, and Web of Science. Only human studies were considered. Semen parameters and DNA integrity were considered to evaluate the effect of pesticides on men. Results: A total of 64 studies that investigated their impact in terms of semen parameters (51 studies) and chromatin and DNA integrity (25 studies) were included. The most frequently affected parameters were total sperm count, sperm motility, and sperm morphology, although a reduction in ejaculate volume and concentration occur in several cases. A tangible worsening of semen quality was associated with organochlorines and organophosphates. Furthermore, pesticide exposure, especially pyrethroids, was related to a higher DNA fragmentation index and chromosome aneuploidy in most articles. Conclusion: The epidemiological evidence supports the association between pesticides and male fertility for workers and the exposed population in terms of semen quality, DNA fragmentation, and chromosome aneuploidy. [ABSTRACT FROM AUTHOR]

Spermatogonia cryopreservation is a method to preserve valuable genomes from both maternal and paternal origin. The damage associated with the application of this technology on post-thaw cell quality is important to assess, including at the epigenetic level. This study aimed to assess post-thawed spermatogonia quality by evaluating alterations in plasma membrane integrity, DNA integrity (fragmentation and apoptosis), lipid peroxidation (malondialdehyde levels) and epigenetic modifications (DNA methylation profile). We observed that plasma membrane integrity (fresh 78.98 % ± 5.66; cryopreserved 62.81 % ± 3.25; P = 0.003) and DNA integrity (fresh 32.95 % ± 2.28; cryopreserved 37.28 % ± 1.87; P = 0.0026) were affected by cryopreservation, while no difference in lipid peroxidation was observed (fresh 1.13 % ± 0.45; cryopreserved 0.91 % ± 0.96; P = 0.701). While global levels of DNA methylation were unaffected by cryopreservation (fresh 82.80 % ± 0.47; cryopreserved 83.32 % ± 0.81; P = 0.745), some differentially methylated cytosines (DMC) were observed in cryopreserved versus fresh spermatogonia (156 DMC). This study showed that spermatogonia cryopreserved according to our protocol provides a good supply of undamaged cells for several applications. The significance of the few detected DMCs deserves further attention since it may affect gamete differentiation and epigenetic profile., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)