Cryopreservation of sperm is a key strategy for preserving and transmitting the genetic traits of valuable or endangered breeds and is particularly applied in goats to improve sperm quality and performance. This study was conducted to evaluate the effect of supplementing cryopreservation extenders with the intracellular compounds of Lactobacillus plantarum (ICL) on goat sperm quality. The intracellular compounds were added to the semen extender at concentrations of 20, 40, 60, 80 and 100µL/mL, while the control group received only the basic extender without any supplementation. Following freezing and thawing, sperm quality parameters were assessed. Among the 10 intracellular compounds identified in L. plantarum, the highest proportions corresponded to 1-benzenesulfonyl-1H-pyrrole and benzenesulfonyl azide (CAS). Treatment with ICL20 significantly increased total motility compared with the control group (p < 0.05). Both ICL20 and ICL40 significantly improved progressive motility compared with the control and ICL100 groups. The mean amplitude of lateral head displacement (ALH, µm) was significantly higher in ICL20 and ICL40 compared with the control and ICL100. Moreover, ICL20 showed significantly higher beat/cross frequency (BCF, Hz) than the control, ICL80 and ICL100 groups. Treatments ICL20 and ICL40 also exhibited significantly greater viability compared with the control and ICL100. Malondialdehyde (MDA) concentration was significantly reduced in ICL20 and ICL40 compared with the control, ICL80 and ICL100. Sperm chromatin dispersion (SCD) analysis revealed that ICL20 had a significantly higher halo-to-core ratio compared with all other treatments. Overall, the findings indicate that supplementation of goat semen extenders with ICL, particularly at concentrations of 20 and 40µL/mL, improves sperm quality during cryopreservation and thawing.

Supplementation of Baicalin (BC) in extender improves structural and functional characteristics, total antioxidant capacity, and in vivo fertility of ram semen.

Background: Cucumis sativus (cucumber) is a widely consumed fruit known for its high water content and antioxidant properties. Despite its numerous health benefits, limited research has been conducted on its potential effects on male reproductive health. This study investigated the impact of cucumber fruit juice on male reproductive hormones and semen quality parameters. Methods: Male Wistar rats were divided into three groups: a control group and two experimental groups receiving 10 ml/kg and 20 ml/kg of Cucumis sativus fruit juice daily for four weeks. Serum samples were analyzed for follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone levels using enzyme-linked immunosorbent assay (ELISA). Semen samples were evaluated for sperm motility, viability, morphology, and abnormal sperm cell count. Results: The administration of cucumber juice did not significantly affect FSH, LH, or testosterone levels (p &gt; 0.05), indicating that the fruit juice does not interfere with endocrine regulation of male reproduction. However, sperm quality parameters showed significant improvements. Sperm motility increased notably in the experimental groups, with the highest motility observed in the 20 ml/kg group. Sperm viability and normal morphology were also significantly enhanced compared to the control (p &lt; 0.05). The percentage of sperm abnormalities, including head defects, mid-piece distortions, and cytoplasmic droplets, was significantly reduced, particularly at the higher dose. These findings suggest that cucumber juice may improve sperm function without altering hormonal balance. Conclusion: The results indicate that Cucumis sativus fruit juice has a beneficial effect on sperm quality, enhancing motility, viability, and morphology while reducing sperm abnormalities. These improvements may be attributed to the antioxidant and hydrating properties of cucumber, which help to reduce oxidative stress and support testicular function. However, the absence of significant hormonal changes suggests that its effects on male fertility are primarily local rather than systemic.

ProAKAP4 as a predictor of semen freezability in Asturcon ponies.

Cryopreservation is an essential component of assisted reproductive technologies in dogs. However, post-thaw semen quality is often compromised by cryo-induced oxidative stress, membrane disruption, and DNA damage, which ultimately reduce fertility outcomes. Conventional egg yolk (EY)-based extenders provide partial protection during freezing but pose biosecurity rick and exhibit batch-to-batch variability. Gum arabic (GA), a plant-derived polysaccharide with cryoprotective and antioxidant properties, has emerged as a potential alternative. The present study evaluated the comparative efficacy of EY and GA extenders, with and without microencapsulation and α-lipoic acid (ALA) supplementation, on post-thaw semen quality in dogs. Semen was collected from six sexually mature Labrador retrievers, with three ejaculates obtained from each dog. Semen samples were cryopreserved in straws under three treatment conditions: extender alone (Treatment 1), extender with microencapsulation (Treatment 2), and extender with microencapsulation plus ALA (Treatment 3). Post-thaw evaluation included sperm motility, viability, acrosomal integrity, hypo-osmotic swelling test (HOST), DNA fragmentation index (DFI), mitochondrial membrane potential (MMP), and total antioxidant capacity (TAC). The results indicated that microencapsulation and ALA supplementation improved several post-thaw sperm quality parameters. Treatment 3 showed the highest values of sperm viability, plasma membrane integrity, acrosomal integrity, MMP, and total antioxidant capacity in both extenders. Sperm motility and DFI did not differ significantly among treatments, although numerically lower DFI values were observed in GA-based treatments. In conclusion, the combined use of GA extender, sperm microencapsulation, and ALA supplementation may enhance the preservation of canine sperm functional integrity during cryopreservation. Further studies evaluating fertility outcomes are required to confirm the practical applicability of these approaches in canine reproductive technologies.

Are healthy and unhealthy diets associated with sperm DNA fragmentation and chromatin deprotamination in healthy men of reproductive age? A cross-sectional analysis conducted using healthy male volunteers (n = 200), aged 18-40 years, participating in the Led-Fertyl study. Adherence to five dietary patterns were estimated using a-priori indices: Mediterranean diet (Mediterranean Diet Adherence Screener; [MEDAS]); healthy and unhealthy plant-based diets; EAT-Lancet diet and Western diet. Dietary intake was assessed using a validated 143-item Food Frequency Questionnaire. Double-strand DNA fragmentation in spermatozoa evaluated by the Neutral Comet assay and chromatin deprotamination evaluated through chromomycin A3 (CMA3) staining by flow cytometry, were considered the main outcomes. No association between adherence to MEDAS, healthy and unhealthy plant-based, EAT-Lancet or Western dietary patterns with sperm DNA fragmentation was observed. Furthermore, adherence to MEDAS, healthy plant-based, EAT-Lancet or Western dietary patterns did not differ in sperm chromatin deprotamination. Yet, adherence to the unhealthy plant-based diet was non-linearly associated with increased chromatin deprotamination in all statistical models. Compared with the lowest tertile, participants in the highest unhealthy plant-based tertile had greater sperm chromatin deprotamination (β = 12.4, 95% CI 2.7 to 22.1, P = 0.026), suggesting a higher occurrence of abnormalities in sperm chromatin condensation. To the best of our knowledge, this study is the first to epidemiologically investigate the relationship between dietary patterns and sperm chromatin integrity, highlighting that adherence to unhealthy plant-based diets may lead to sperm chromatin abnormalities. These findings underscore the potential effect of specific dietary patterns on molecular sperm quality parameters and support further research into dietary strategies for optimizing sperm integrity and improving male fertility.

Spermatogenesis is worsened by reactive oxygen species (ROS) and antioxidants could reduce ROS induced sperm damage. This prospective cohort study evaluated the potential therapeutic effects of a combination of vitamins, minerals and antioxidants on the sperm quality parameters of infertile men with idiopathic low spermatogenesis. Seminal fluid analysis tests were performed before treatment, 3 and 6 months after treatment. The treatment resulted in a significant improvement in the rate (%) of sperm motility from 16.95±6.93 to 23.11±8.87, after 3 months and reached 23.68±8.73 after 6 months (p=0.0006) whereas a non-significant increase in sperm count (from 13.05±8.1 to 15.79±7.9 after 3 months and 15.26±10.3 million/ml after 6 months (p=0.1650). Morphology and agglutination showed little changes. A positive correlation between sperm count and motility was observed after 3 months of treatment (r = 0.594; p=0.007). The combination of vitamins and antioxidants improved the sperm motility significantly and to a lesser extent the sperm count, however, sperm morphology and agglutination remained relatively unchanged. Using antioxidants is safe and can improve semen parameters.

This study aimed to evaluate the effects of Diplotaenia turcica root extract, an endemic plant species of Türkiye, on sperm quality parameters and testicular antioxidant activity in rats with streptozotocin-induced type 2 diabetes. A total of 42 male Wistar rats were randomly and equally allocated into six groups: Control, Diabetes, Diabetes + Diplotaenia turcica ethanol extract, Diabetes + Diplotaenia turcica aqueous extract, Diplotaenia turcica ethanol extract, and Diplotaenia turcica aqueous extract. The control group was fed a standard pellet diet for two weeks and received 10 mL/kg of normal saline orally. In the Diabetes, Diabetes + Diplotaenia turcica ethanol extract, and Diabetes + Diplotaenia turcica aqueous extract groups, type 2 diabetes was induced by feeding a high-fat diet for two weeks, followed by an intraperitoneal injection of streptozotocin dissolved in citrate buffer. The Diplotaenia turcica treatment groups received 150 mg/kg of the respective extract orally for two weeks, according to the experimental design. At the end of the experimental period, blood and sperm samples were collected under anesthesia. In the diabetic group, significant decreases in sperm count and motility were observed, along with a significant increase in abnormal sperm morphology (p&lt;0.001). In addition, malondialdehyde levels in testicular tissue were elevated, whereas glutathione levels and catalase activity were significantly reduced (p&lt;0.05). In the diabetic groups treated with Diplotaenia turcica, improvements in sperm parameters and significant increases in antioxidant enzyme activities were observed (p&lt;0.05). These findings suggest that Diplotaenia turcica root extract may serve as a potential protective agent against diabetes induced sperm abnormalities and testicular oxidative damage.

The aim of the study was to characterize the electrophoretic profiles of alpaca seminal plasma (SP) proteins and establish their association with sperm quality parameters at different cryopreservation stages. Sperm quality was assessed in raw, cooled, and thawed semen from 128 ejaculates collected from 16 Huacaya alpacas, and SP proteins were analysed by SDS-PAGE in raw samples. Statistical associations were determined using Spearman's rank correlation (p ≤ 0.05). Twenty-three protein bands were identified: 21 bands ranging from 9.23 to 138.38 kDa, and 2 below 6.5 kDa. Notably, the 21.03 kDa protein was absent in six males, five of whom also lacked the 18.88 kDa band. These individuals exhibited superior post-thaw sperm quality, particularly higher motility. The 21.03 kDa protein showed a negative correlation (p ≤ 0.05) with sperm motility and membrane function in raw, cooled, and thawed semen, and a positive correlation with acrosome integrity in thawed semen. Similarly, the 18.88 kDa protein showed a negative correlation with sperm motility and membrane function, but a positive correlation with acrosome integrity in thawed semen (p ≤ 0.05). In conclusion, these findings suggest that specific SP proteins may serve as potential biomarkers for sperm quality and cryotolerance in alpacas, reflecting individual variability in response to cryopreservation.

This study evaluated the efficiency of an scFv antibody-based sex-sorting method using HL magnetic microbeads for the separation of X- and Y-chromosome-bearing sperm in Landrace boar semen, as well as its effects on sperm quality parameters. A dose-dependent plateau in H4L4 antibody coupling efficiency was observed, with 2 mg identified as the optimal concentration, consistently achieving approximately 50% sperm-binding efficiency across boars. Sex ratio analysis confirmed effective discrimination between X- and Y-bearing sperm, with the X-enriched fraction showing a high proportion of X-sperm (77-81%) and the Y-enriched fraction exhibiting high Y-sperm purity (>83%). CASA revealed a significant effect of the sex-sorting process on sperm kinematics (p < 0.001). X-enriched sperm maintained physiological motility, with total and progressive motility comparable to CON, whereas the Y-enriched fraction showed a marked decline in kinematic performance. Flow cytometric analysis demonstrated that membrane integrity remained high in CON and X-enriched semen (>81%), while a significant reduction in viability was observed in the Y-enriched fraction (50.85%; p < 0.001). Consistently, mitochondrial membrane potential analysis indicated pronounced physiological stress in Y-enriched sperm, with significantly reduced mitochondrial activity compared to CON and X-enriched fractions. No significant differences were detected among individual boars (p > 0.05). In conclusion, this study demonstrates that HL magnetic bead-based sperm sexing effectively separates X- and Y-sperm in chilled Landrace boar semen, while preserving the quality of X-enriched sperm.

Background and Aim: Cryopreservation is commonly used in canine reproductive biotechnology to preserve genetic material for long-term storage. However, the freeze–thaw process induces oxidative stress, mitochondrial dysfunction, and structural damage in spermatozoa, thereby reducing post-thaw sperm quality and fertility potential. Mitoquinone (MitoQ), a mitochondria-targeted antioxidant, has been suggested as a promising cryoprotective additive that mitigates mitochondrial reactive oxygen species and enhances cellular integrity. This study aimed to assess the dose-dependent effects of MitoQ supplementation on mitochondrial function, oxidative stress, and the post-thaw quality of cryopreserved canine spermatozoa to identify an optimal concentration for semen cryopreservation. Materials and Methods: Semen samples were collected from 10 healthy and fertile male dogs of various breeds. Each ejaculate was divided into four equal parts and cryopreserved with a Tris–fructose–egg yolk extender supplemented with different MitoQ concentrations (0, 100, 200, or 400 nM). After four weeks of storage in liquid nitrogen, the samples were thawed and evaluated in vitro. Post-thaw sperm quality was measured using computer-assisted sperm analysis to assess motility and kinematic parameters. Additional evaluations included sperm viability, plasma membrane integrity, acrosome integrity, lipid peroxidation levels, apoptotic status via flow cytometry, mitochondrial membrane potential, and intracellular hydrogen peroxide production. Data were analyzed using a mixed-effects model with dog as a random effect, and significance was set at p &lt; 0.05. Results: MitoQ supplementation had concentration-dependent effects on several sperm quality parameters. The 200 nM MitoQ group showed the most consistent improvements in post-thaw sperm quality. Total motility and curvilinear velocity were significantly higher at this concentration compared to the control and other treatment groups. Additionally, sperm treated with 200 nM MitoQ displayed improved plasma membrane and acrosome integrity, reduced lipid peroxidation, and a lower proportion of dead sperm cells. Trends toward increased viability and enhanced mitochondrial activity were also seen at this concentration. In contrast, 400 nM MitoQ supplementation was linked to decreased membrane integrity and increased oxidative stress markers, suggesting possible pro-oxidant effects at higher doses. Overall, the results indicate that moderate MitoQ supplementation supports mitochondrial redox balance and partially reduces cryo-induced damage in canine spermatozoa. Conclusion: MitoQ supplementation at 200 nM modestly but consistently enhanced several in vitro indicators of post-thaw sperm quality in dogs, such as motility, membrane stability, and oxidative stress markers. These findings highlight the narrow therapeutic window of mitochondria-targeted antioxidants and stress the importance of dose optimization during semen cryopreservation. Although the observed improvements suggest potential benefits for reproductive biotechnology, further research with fertility-related functional outcomes and larger sample sizes is needed to confirm the practical reproductive impact of MitoQ supplementation in canine semen cryopreservation. Keywords: canine sperm cryopreservation, mitoquinone, mitochondrial function, oxidative stress, post-thaw sperm quality, reactive oxygen species, sperm motility, sperm viability.

Sperm quality, reproductive hormones, and membrane-bound progesterone receptor expression in Chinese sturgeon (Acipenser sinensis).

Therapeutic Potential of Goat Whey Protein Hydrolysate and Vitamin-D3 in Combating D-Galactose-Induced Sperm Senescence in Male Mice.

Safety evaluation of ursolic acid: Absence of acute oral and reproductive toxicity in a rodent model.

The present study investigates the effect of an ethanolic extract of Hygrophila auriculata seeds on the male reproductive organ weights, testicular lipid peroxidation and antioxidant potential, testosterone levels, and effects on sperm quality parameters in a streptozotocin (STZ)-induced diabetic male rat model, a widely used experimental model of diabetes-associated infertility. The extract was administered orally to STZ-induced diabetic rats to evaluate its effect on reproductive parameters in terms of evaluating sperm count, motility, viability, morphology and chromatin dispersal assay along with organ weight of male reproductive organs and serum testosterone levels. Antioxidant activity in testicular extract was assessed by measuring lipid peroxidation, glutathione peroxidase, superoxide dismutase, reduced glutathione and catalase assays. The results revealed that athough the male reproductive organ weights decreased significantly in diabetic and treatment groups as compared to the control, the semen quality parameters such as sperm count, motility and viability improved significantly (p&lt;0.05) in the treatment groups. The H. auriculata seed extract significantly (p&lt;0.05) reduced lipid peroxidation in the testicular tissue as compared to the untreated diabetic controls. H. auriculata seed extract was not observed to have ameliorative potential against sperm DNA damage. The ethanolic extract of H. auriculata seed significantly (p&lt;0.05) improved serum testosterone concentration as compared to the control and diabetic rat model groups implying an androgenic effect of the extract. The study provides evidence that the ethanolic extract of H. auriculata seeds protects testicular integrity and sperm quality by reducing lipid peroxidation levels in a diabetic rat model.

Nuts are rich in antioxidants and other bioactive compounds, and recent evidence suggests that their regular consumption may be associated with sperm quality. However, the current scientific evidence remains limited and inconsistent. The study aimed to evaluate the association between nut consumption and sperm quality parameters in healthy men of reproductive age. A cross-sectional analysis was conducted using the data from 222 young men enrolled in the Led-Fertyl study. Nut consumption was categorized as <3, ≥3 to <7, and ≥ 7 servings/week (1 serving=30g). The main outcomes were sperm quality parameters (sperm count, concentration, vitality, motility, and normal morphology). Multivariate linear and logistic regression models were fitted to analyze associations. Total sperm count (β=3.38; 95%CI: 1.59, 5.16) and concentration (β=1.17; 95%CI: 0.15, 2.19) were higher among participants in the highest category of nut consumption (≥7 servings/week) compared to those in the lowest (<3 servings/week). A similar association was observed when modeling nut consumption as continuous; each additional serving per day was associated with higher total sperm count and concentration (β=2.38; 95%CI: 1.03, 3.72 and β=0.83; 95%CI: 0.06, 1.59, respectively). A theoretical substitution of 1 serving/day of nuts with 1 serving/day of potato chips or pastries was associated with lower total sperm count and concentration. Furthermore, compared to participants in the lowest category of nut consumption, those in the highest were 75% less likely to have abnormal sperm motility (OR: 0.25; 95%CI: 0.07, 0.95) and 69% less likely to have seminogram abnormalities (OR: 0.31; 95%CI: 0.14, 0.68). Our findings suggest that regular nut consumption is associated with higher total sperm count and concentration in young, healthy men of reproductive age.

The first experiment assessed the effect of bovine herpesvirus 1 (BoHV-1) on thawed bull semen. Semen samples testing negative for BoHV-1 via Nested-PCR were incubated at 37 °C with viral concentrations of 0 (control), 10⁴, 10⁵, and 10⁶ TCID₅₀/mL. Sperm quality parameters were evaluated over an 8-hour period. The second experiment assessed the efficacy of two protocols for reducing the viral load (viral titration) in experimentally infected semen (10⁴ TCID₅₀/mL): Percoll gradient (PG) and magnetic nanoparticles coupled with anti-BoHV-1 antibodies (MNPs). Sperm kinematics were influenced by both bull and time, with bull × time interaction (P < 0.001). Membrane integrity and morphology were influenced only by time (P < 0.01), with no effect of viral infection (P > 0.05). In viral titration, all PG-treated samples tested negative. Samples treated with MNPs also yielded negative results after a 60 min-incubation. It is concluded that BoHV-1 had no direct effect on thawed bull sperm quality and that PG appears to be effective removing the viral load. Furthermore, MNP is a viable alternative for virus control in semen, with no apparent deleterious effects to sperm motility.

Chilled semen preservation supports artificial insemination (AI) programs.However, oxidative stress (OS) during cold storage can impair sperm function, particularly in older bucks with diminished antioxidant defence.This study evaluated the combined effects of sodium selenite supplementation and age on the maintenance of semen quality and cellular and sperm membrane integrity of Saanen buck spermatozoa in AndroMed chilled extender.Nine Saanen bucks were assigned to three age groups: G1 (18 months), G2 (36 months), and G3 (72 months).Semen was collected and diluted with AndroMed extender supplemented with sodium selenite at 0, 1, 5, 10, and 25ppm.Sperm quality parameters, including motility, viability, abnormalities, and plasma membrane integrity (PMI), were evaluated during liquid storage at 5C (0, 24, and 48-h).The results indicated that buck age significantly affected fresh semen quality, with G2 bucks exhibiting optimal parameters.Supplementation with 5ppm sodium selenite was the most effective in maintaining sperm motility, viability, and PMI during chilled storage across all age groups.Conversely, excessive sodium selenite supplementation (25ppm) had detrimental effects on chilled semen quality.Furthermore, the combination of G2 bucks and 5ppm sodium selenite supplementation in AndroMed extender can effectively maintain sperm quality during chilled storage.This approach may improve AI outcomes using chilled semen.

The sperm quality, freezability, and fertility of crossbred bulls exhibit significant unpredictability, which consequently impacts the breeding program. The present study documents the supplementation of humanin, a mitochondria-derived peptide, on crossbred bull’s sperm quality, freezability, antioxidant status and in-vitro fertility. For this objective a total of 24 ejaculates, 8 from each of the three Vrindavani crossbred bulls, were selected based on sperm progressive motility (≥ 70%) and concentration (≥ 500 million). The selected fresh semen samples were divided into four aliquots and diluted with Tris-Fructose-Egg-Yolk-Glycerol (TFEGY) extender supplemented with various concentrations of humanin peptide; control (Group-1) without supplementation; Group II with 2.5 µM; Group III supplemented with 5 µM and Group IV supplemented with 7.5 µM. Semen quality parameters, sperm kinematics, sperm plasma membrane integrity, mitochondrial membrane potential, sperm cryo-capacitation status, ejaculate freezability rate, antioxidant status and in-vitro fertility assays were done. At the pre-freeze stage, there was no significant effect on the percentage of individual progressive motility in any group. Supplementation with 5 µM humanin resulted in an 87.5% ejaculate recovery rate after cryopreservation. At the post-thaw stage, sperm quality parameters, sperm kinematics, sperm plasma membrane integrity, mitochondrial membrane potential, sperm cryo-capacitation status, antioxidant status and in-vitro fertility were significantly improved in Group III (5 µM humanin supplementation). In conclusion, our results demonstrate that humanin supplementation in crossbred bull semen enhances semen freezability and post-thaw ejaculate recovery rate.

Male infertility, often characterized by impaired sperm parameters, remains a significant clinical challenge. This study aimed to evaluate the efficacy and safety of hirudotherapy (medicinal leech therapy) in improving sperm quality parameters in men with idiopathic infertility. In this randomized controlled trial, 50 male volunteers aged 20-50 years were randomly assigned to either an experimental group receiving weekly leech therapy for 3 months (n = 25) or a control group receiving no intervention (n = 25). Semen parameters were assessed at baseline and after the intervention period. Evaluations included semen analysis according to WHO guidelines, sperm DNA fragmentation by TUNEL assay, protamine deficiency by chromomycin A3 staining, and assessments of sperm viability and morphology using flow cytometry and Diff-Quik staining. Statistical analyses were performed using SPSS version 21.0, with significance set at p < 0.05. Compared to the control group, leech therapy significantly improved several sperm quality parameters. Sperm concentration increased from 57.7 ± 17.1 to 63.4 ± 13.1 million/mL (p = 0.006). Total sperm count, progressive motility, and normal morphology also improved significantly (p < 0.05). DNA fragmentation decreased significantly in the experimental group compared to controls (mean difference: -3.9%; p = 0.04). Protamine deficiency also showed a significant reduction (p = 0.02). No adverse effects related to the intervention were observed. This randomized controlled trial suggests that hirudotherapy may improve sperm quality parameters, including concentration, motility, morphology, and DNA integrity, in men with idiopathic infertility. These findings support further investigation of leech therapy as a complementary approach to male infertility management. Trial Registration: IRCT20230502058045N.