Search Results (283)

(1) Background: Accurate assessment of sperm morphology is essential for evaluating the quality of cryopreserved canine semen used in artificial insemination and for improving cryopreservation protocols. This study compared six staining techniques, Eosin, Eosin–Nigrosin, Diff-Quick^®, Hemacolor^®, Spermac^®, and Formol-citrate Bengal Rose, for light-microscopic evaluation of frozen–thawed canine spermatozoa. (2) Methods: Semen from ten dogs was thawed, divided into four aliquots, and either left untreated or exposed to thermal stress at 6 °C, 18 °C, or 37 °C for two hours to induce morphological variation. A total of 360 slides and 960 evaluations were performed immediately after staining and again after 24 h, 7 days, and 3 months to assess staining quality and stability over time. (3) Results: Eosin produced stable staining for up to three months and was the most economical method, though its initial detail recognition was lower than that of Spermac^® and Formol-citrate Bengal Rose. Eosin–Nigrosin showed reduced contrast and detail. Diff-Quick^® provided better contrast than Eosin–Nigrosin, while Hemacolor^® maintained consistent quality regardless of stress treatment or storage duration. Spermac^® yielded the highest initial morphological detail but deteriorated during storage. Formol-citrate Bengal Rose combined high detail recognition with stable staining throughout the study. Cryopreservation increased looped tails, and incubation at 37 °C markedly elevated pathological sperm and rudimentary tails. (4) Conclusions: All six staining methods were suitable for evaluating and archiving frozen–thawed canine semen. Formol-citrate Bengal Rose and Spermac^® offered the best detail, while Eosin provided a cost-effective option with excellent long-term stability. Full article

Sodium butyrate (NaBu), a short-chain fatty acid and histone deacetylase inhibitor, has been reported to influence protein acetylation and cellular function; however, its effects on boar spermatozoa remain poorly understood. This study evaluated the effects of NaBu on sperm function and global protein acetylation in fresh after 24 h storage and frozen–thawed boar spermatozoa. Semen samples collected from boars (n = 4), with three ejaculates per boar, were supplemented with 0, 0.5, 0.75, or 1 mM NaBu, stored for 24 h at 17 °C, and subsequently cryopreserved. Sperm motility, mitochondrial membrane potential, membrane integrity, apoptosis-like changes, and chromatin status were assessed using CASA, flow cytometry, and fluorescence microscopy, whereas global protein acetylation was assessed by Western blotting. In fresh semen after 24 h storage, NaBu did not significantly affect the evaluated sperm functional parameters, whereas frozen–thawed spermatozoa showed significant changes in selected functional parameters, particularly total and progressive motility at 0.5 mM. Selected mitochondrial membrane potential parameters were also affected in frozen–thawed samples, while membrane integrity, apoptosis-like changes, and chromatin status remained largely unaffected. NaBu did not significantly alter global protein acetylation levels in either fresh after 24 h storage or frozen–thawed spermatozoa. Considerable inter-individual variability between boars was observed. These findings indicate that NaBu may affect selected in vitro functional properties of frozen–thawed boar spermatozoa; however, the observed functional changes were not associated with detectable statistically significant changes in global protein acetylation under the conditions tested. Further studies are needed to determine whether specific acetylated proteins, metabolic pathways, or stress-response mechanisms are involved. Full article

Sperm cryopreservation is important for livestock breeding and germplasm conservation, but freeze–thaw injury can impair ram sperm quality through oxidative stress, membrane damage, and metabolic disturbance. This study evaluated the concentration-dependent effects of kaempferol supplementation on the cryopreservation quality of semen from Yuansheng Aite dairy rams. Qualified ejaculates were pooled and randomly allocated to five equally spaced kaempferol treatment groups: 0, 25, 50, 75, and 100 μg/mL. Post-thaw sperm motility, oxidative stress status, ATP-related energy metabolism, acrosome integrity, and multi-omics profiles were evaluated. Data were analyzed using appropriate parametric or non-parametric tests after assessment of normality and homogeneity of variance. Orthogonal polynomial analysis was performed to evaluate linear and nonlinear dose–response patterns across the tested kaempferol concentrations. Kaempferol supplementation significantly affected PM, VCL, and VAP, while RPM, LIN, WOB, and VSL were not significantly affected. No significant linear effect was observed for the motility parameters, whereas VCL exhibited a significant quadratic response to kaempferol concentration. Based on the observed overall responses of sperm motility, antioxidant capacity, oxidative stress markers, ATP content, and acrosome integrity, 25 μg/mL kaempferol showed the most favorable overall profile among the tested concentrations and was selected for subsequent mechanistic analyses. Proteomic and metabolomic analyses suggested that the protective effects of kaempferol may be associated with pathways related to focal adhesion, cytoskeletal organization, oxidative phosphorylation-related energy metabolism, and central carbon metabolism. These findings indicate that moderate kaempferol supplementation may improve the post-thaw quality of Yuansheng Aite dairy ram semen, although further fertility-oriented studies are needed to confirm its practical reproductive benefits. Full article

Sperm cryopreservation is an integral part of gene pool conservation programs for local poultry breeds. It is known that cryostress can cause significant changes in the expression profiles of microRNAs and their target genes—key players in spermatogenesis—in Gallus gallus domesticus. However, the transmission of these changes across generations remains poorly understood. The aim of this study was to evaluate the transgenerational epigenetic effects of rooster sperm cryopreservation on molecular genetics and histological parameters in the gonads of offspring (F1) during the embryonic (10 days) and postnatal (1 day) periods. The analysis included a comprehensive histomorphometric analysis of the gonads and a quantitative assessment of the expression of microRNAs (gga-miR-6701-3p, gga-miR-301a-5p) and their target genes (DMRT1, TGFB2), using qRT-PCR. Histological analysis of the gonads of 10-day-old embryos revealed early morphological abnormalities in the F1 (n = 10) offspring obtained from frozen–thawed semen (experimental group). It was found that day-old F1 chicks (n = 17) obtained from frozen semen had testes with a significantly reduced number of seminiferous tubules (−36%, p < 0.05) with an increased diameter (+22%, p < 0.05) and an increased number of undifferentiated gonocytes (+53%, p < 0.001) compared to chicks obtained from native semen (control group, n = 20). A decrease in the expression of DMRT1 and TGFB2 in the gonads of embryos (−48% and −29%, respectively, p < 0.05) and day-old chicks (−12% and −43%, p < 0.001 for TGFB2) was found, accompanied by an inversion of microRNA dynamics: miR-6701-3p was decreased and miR-301a-5p was increased. The obtained data provide important evidence of transgenerational effects in birds and contribute to the search for solutions to problems associated with maintaining sperm quality after cryopreservation, and indicate that cryopreservation does not simply reduce the level of molecular activity, but disrupts the ontogenetic regulatory program embedded in the genome. Full article

Cryopreservation of stallion semen is associated with oxidative stress (OS), which can impair sperm function and fertility. This study evaluated antioxidant activities in seminal plasma and sperm cytosols and investigated their relationships with selected sperm functional parameters following cryopreservation, with or without antioxidant supplementation. Semen was collected from ten fertile stallions and processed using a split-ejaculate design, including fresh semen and six freezing treatments: HF-20 extender alone; HF-20 supplemented with matcha, spirulina, horseradish, or quercetin; and a commercial extender (INRA Freeze). Total antioxidant capacity (FRAP) and enzymatic activities (superoxide dismutase, SOD; catalase, CAT; and glutathione reductase, GR) were measured in seminal plasma and sperm lysates. Linear regression analyses revealed significant associations between seminal plasma and fresh spermatozoa with respect to SOD and GR activities. In frozen-thawed semen, FRAP and CAT activities differed between samples cryopreserved with and without antioxidant supplementation. Significant correlations were observed among antioxidant activities, sperm kinetics, OS markers, and DNA fragmentation indices. Principal component analysis provided an exploratory overview of multidimensional patterns of covariation among sperm kinetics, redox balance, and nuclear fragmentation, explaining for 73% of the total variance. Overall, the results suggest complex associations between the antioxidant system and sperm quality and indicate that antioxidant supplementation of freezing extenders may modulate the redox status of stallion sperm after thawing. Full article

This current study aimed to determine the impact of Mitoquinone (MitoQ) on the quality of frozen–thawed donkey semen. Ejaculates were collected from six male donkeys (Equus asinus), and ejaculates were polled and aliquoted into 12 samples. Samples were diluted with TRIS–egg yolk glycerol extender that reached 200 million sperm/mL. After centrifugation, the pellet was diluted at 1:15 with TRIS–egg yolk glycerol extender and divided into the five main groups containing MitoQ with different concentrations: 0 nmol/mL (control; MitoQ0), 100 nmol/mL (MitoQ1), 150 nmol/mL (MitoQ2), 200 nmol/mL (MitoQ3), and 250 nmol/mL (MitoQ4). After thawing, semen quality was evaluated using CASA kinematic parameters, fluorescence microscopy, and biochemical markers such as alanine and aspartate aminotransferase levels (ALT and AST). Malondialdehyde (MDA) and catalase (CAT) levels were also measured. MitoQ1 with 100 nmol/mL significantly increased total motility (p = 0.04), progressive motility (p = 0.04), viability (p = 0.03), kinetic parameters (p = 0.04), linearity (p = 0.02), and straightness (p = 0.04). A significant decline in MDA (p = 0.02), ALT (p = 0.03), and AST (p = 0.03) levels in MitoQ1 with 100 nmol/mL was found, with an elevation of CAT levels (p = 0.02) compared to other concentrations and the control in TRIS–egg yolk glycerol extender. Different concentrations of MitoQ did not affect acrosome and DNA integrity. In conclusion, the addition of MitoQ during cryopreservation has a positive effect on sperm motility, viability, and kinetic parameters, especially at a concentration of 100 nmol/mL when used with a TRIS–egg yolk glycerol extender for frozen–thawed donkey sperm. Full article

Cryopreservation of gametes is crucial for conserving genetic diversity in livestock and endangered species, but the process can significantly impair sperm quality due to oxidative stress. Our aim was to evaluate the impacts of coenzyme Q10 (CoQ10) supplementation on the in vitro quality of cooled and cryopreserved goat semen. Semen samples collected from six mature Saanen bucks were pooled then diluted with AndroMed^® semen extender to a final concentration of 800 × 10⁶ sperm/mL. Diluted semen was supplemented with 0, 1, 2, 5, 10, and 20 µM CoQ10. Extended semen was either cooled at 4 °C for 72 h or cryopreserved using a Styrofoam box in which the straws were arranged on the freezing rack and placed 4 cm over the liquid nitrogen (LN₂) for 10 min then stored in a LN₂ tank for one-week before being thawed at 37 °C for 30 sec. Sperm quality, including total and progressive motility, sperm kinematics, live sperm %, and sperm membrane integrity, was assessed at 0 h (fresh semen), and 24, 48, and 72 h post-cooling. For post-thaw sperm, we evaluated the same parameters plus acrosome integrity, mitochondrial activity, lipid peroxidation, and sperm ultrastructural changes using scanning electron microscopy (SEM). The pooled semen sample was considered the experimental unit for all treatments. Cooled semen data were analyzed using a General Linear Model (GLM) with univariate analysis, followed by Tukey’s test for multiple comparisons. In contrast, data from frozen–thawed semen were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s test. CoQ10 supplementation at 10 and 20 µM significantly (p < 0.05) improved sperm motility, viability, and membrane integrity in cooled and frozen–thawed semen in comparison with the control group (0 µM CoQ10). Moreover, the same concentrations significantly (p < 0.05) enhanced acrosome integrity, mitochondrial activity, and reduced the percentages of sperm with lipid peroxidation in frozen–thawed semen. Furthermore, 10 and 20 µM CoQ10 significantly mitigated the ultrastructural defects in frozen–thawed spermatozoa. In conclusion, CoQ10 supplementation during the cooling and cryopreservation of dairy goat semen significantly improved sperm quality. Among the tested concentrations, 10 and 20 µM exhibited the most favorable outcomes. Full article

Freeze–thaw procedures impair sperm morphology and function, affecting viability, motility, redox balance, and subcellular organization. Although antioxidants may mitigate these effects, their interaction with donor-specific variability remains unclear. We combined quantitative holotomography with conventional physiological assessments within a multivariate framework based on principal component analysis (PCA) and nested cross-validated Linear Discriminant Analysis (LDA) to evaluate donor-specific responses to antioxidant-supplemented cryopreservation. Spermatozoa from ten stallions was analyzed before and after freezing under five conditions: fresh semen; frozen semen with INRA Freeze, frozen semen with HF-20, and HF-20 supplemented with matcha, spirulina, horseradish, or quercetin. For each condition, sperm kinetics, mitochondrial activity, oxidative stress, DNA integrity, and three-dimensional volumetric measurements of whole-cell and subcellular compartments derived from holotomography were integrated into a single dataset. LDA achieved 0.734 cross-validated accuracy for stallion classification, revealing strong donor-specific signatures. In contrast, classification by antioxidant treatment was near chance (0.248). Fresh semen was clearly distinct from all cryopreserved groups. Holotomography showed reduced whole-cell and post-acrosomal/midpiece volumes after freezing, while nuclear volume was unchanged. Antioxidant supplementation produced minor, inconsistent effects, with partial midpiece preservation in some donors but no global pattern. Overall, inter-stallion variability dominates post-thaw sperm phenotype. Antioxidant effects were detectable but modest, supporting individualized strategies to optimize equine semen cryopreservation protocols. Full article

Background/Objectives: Testicular cancer accounts for approximately 1% of all male malignancies, with an incidence ranging from 1 to 10 per 100,000 men and it predominantly affects young individuals, with nearly 60% of cases diagnosed between 15 and 35 years of age. In recent decades, the incidence of testicular cancer has markedly increased, paralleling a global rise in male infertility rates. Although chemotherapy is known to adversely affect fertility, the extent to which the tumor itself and its different histological subtypes impact semen quality remains incompletely understood. The aim of this study was to evaluate semen parameters in men diagnosed with testicular cancer prior to oncological treatment and to assess the possible association between tumor histology and semen quality. Methods: This retrospective study included data from 284 men diagnosed with testicular cancer who underwent semen cryopreservation prior to surgery, chemotherapy, or radiotherapy. Data were collected between January 2016 and June 2022 at the Maternal and Child Department of the University of Naples Federico II. Histopathological classification was available for 278 patients and revealed the following distribution: 59% (165/278) classic seminoma, 14.7% (41/278) seminomatous mixed germ cell tumors, 13.3% (37/278) non-seminomatous mixed germ cell tumors, and 12.6% (35/278) non-seminomatous germ cell tumors. Results: No significant association was observed between tumor histology and abnormal semen parameters. According to World Health Organization (WHO) reference values, semen parameters in patients with testicular cancer were predominantly distributed between the 5th and 25th percentiles. Microscopic semen analysis revealed significantly lower sperm concentration, total motility, and normal morphology in cancer patients (p < 0.001; p < 0.001; and p < 0.002, respectively). Logistic regression analysis showed a significant association between age and testicular cancer risk (p < 0.001), with a negative coefficient indicating that the likelihood of developing the disease decreases with increasing age. Additionally, patients with seminoma were significantly older than those with non-seminomatous tumors: on average, 4.07 years older than those with pure non-seminoma (p = 0.007) and 5.60 years older than those with mixed non-seminoma (p < 0.001). No statistically significant age differences were observed among non-seminomatous subtypes. Conclusions: These findings underscore the importance of systematic semen evaluation in young men diagnosed with testicular cancer and highlight the critical role of fertility preservation strategies in the comprehensive management of these patients. Full article

Cryopreserved bovine semen represents an accessible source of genetic material due to its widespread use in assisted reproductive technologies and the conservation of genetically valuable animals. However, DNA extraction from spermatozoa within this type of sample remains challenging due to the high protein content and the complex structure of the ejaculate, which can affect DNA yield and quality. The aim of this study was to identify and validate an efficient method for obtaining high-quality DNA from spermatozoa present in cryopreserved bovine semen for molecular diagnostic applications. Five DNA extraction protocols were evaluated: TRIzol™, MagMax™ Nucleic Acid Purification Kit, Rapid DNA™ Fecal/Soil Microbe Kit, a conventional phenol–chloroform protocol, and a modified phenol–chloroform–isoamyl alcohol protocol. All extracted genetic material was assessed by spectrophotometry (concentration and purity), and DNA integrity was evaluated by agarose gel electrophoresis. Statistical analysis revealed significant differences in DNA concentration among extraction methods (Friedman test, χ² = 22.0, df = 4, p = 0.0002). Post hoc comparisons indicated that the modified phenol–chloroform–isoamyl alcohol protocol yielded significantly higher DNA concentrations compared to selected methods. This protocol showed the highest DNA concentration (1006.2 ± 829.4 ng/μL) and favorable purity values, and enabled consistent amplification in both conventional PCR and qPCR assays targeting the β-actin gene and Tritrichomonas foetus, respectively. These findings suggest that the modified protocol represents a suitable and promising approach for extracting genomic DNA from spermatozoa in cryopreserved bovine semen, with potential applications in molecular diagnostics and reproductive biotechnology. Full article

The highly compact chromatin and naturally fragmented RNA content of bovine sperm make it difficult to obtain sufficient total RNA for transcriptome sequencing. Bovine sperm studies have mostly adapted the somatic-cell protocol and used generic kits. To date, no commercial extraction kit is available for total RNA from sperm. These limitations prompted optimization of a sperm total RNA isolation protocol, tested using both fresh and cryopreserved sperm. Ejaculates were collected from two Girolando bulls (Bos taurus × Bos indicus) and were subsequently processed for cryopreservation. In total, eight RNA extraction protocols were tested, namely four TRIzol^®-based (GP) protocols and four spin-column (SC) methods. Across both fresh and cryopreserved sperm, Protocol D of SC protocols (SC-D) was the most suitable choice for total RNA sequencing. For fresh sperm, ~25–29 million filtered reads were obtained, and cryopreserved sperm yielded ~83–125 million filtered reads, indicating that cryopreserved sperm is also a good source of sperm RNA based on initial sequencing metrics. By pinpointing SC-D as the optimal balance in terms of yield and sequencing performance, and by identifying practical solutions for DNA carryover and storage-related duplication, the study’s results provide a streamlined protocol and quality control framework for total RNA sequencing of Girolando sperm. Full article

Background: Cryopreservation induces the production of excessive reactive oxygen species (ROS), which decreases sperm physiological functions. Phytochemicals with antioxidant properties, such as fisetin, have shown promising results in reducing oxidative stress (OS). Aim: We aimed to evaluate whether fisetin can counteract the OS exerted on sperm. Methodology: Fisetin (15 and 30 µM) was tested on normozoospermic semen samples that were either frozen in liquid nitrogen or treated with H₂O₂ to induce OS. Sperm motility, sperm viability, mitochondrial membrane potential, metabolic activity, ROS content, lipid peroxidation, reduced glutathione, ATP contents, and apoptosis were tested and compared to controls. Results: The protective effect of fisetin on human sperm was observed against OS-induced stress. Fisetin significantly improved sperm motility, viability, mitochondrial and metabolic activity, and ATP content by reducing OS and lipid peroxidation. Fisetin reduced necrotic cell death and improved sperm survival under H₂O₂-OS. Conclusions: Fisetin protects human sperm from OS, with 30 µM showing greater effectiveness, supporting its potential use in sperm preservation and OS conditions. Further studies are needed to optimize its concentration, elucidate its mechanism of action, and confirm its putative use as an additive in sperm cryoprotective media. Full article

Background and Objectives: The microbiome plays a pivotal role in male infertility, with distinct microbial species exerting both beneficial and deleterious effects on reproductive function. Sexually transmitted bacteria and several viruses, including human papillomavirus (HPV), have been identified in semen. This cross-sectional study aimed to examine the prevalence of single and co-infections of sexually transmitted bacteria (STB)—such as Chlamydia trachomatis, Mycoplasma spp., and Ureaplasma spp.—with various HPV subtypes in Greek male partners of infertile couples and to evaluate their potential impact on sperm parameters. In addition, the possible effect of cryopreservation on the maintenance of these pathogens was assessed. Materials and Methods: Eighty-two semen samples were initially collected from 82 individuals undergoing routine sperm analysis. In total, 80/82 (97.6%) participants proceeded to further analysis, as 2/82 (2.4%) were excluded due to poor DNA quality. Results: A total of 18/80 (22.5%) sperm samples tested positive for STB, with Ureaplasma spp. representing the most frequently detected pathogen. Co-infection of Ureaplasma spp. and Mycoplasma hominis was observed in 4/80 (5%) samples. Twelve samples (12/80, 15%) were positive for HPV, including low-risk (LR) and high-risk (HR) types, and HPV 16 was the predominant HR genotype. Notably, a co-infection of STB and HPV was not found in our specimens. STB-positive samples demonstrated significantly higher sperm concentration and improved progressive motility compared with STB-negative samples. HPV-positive samples exhibited lower sperm volume and concentration and increased non-progressive motility compared with HPV-negative samples. Following three months of cryopreservation, LR HPV and STB were no longer detectable, whereas HR HPV types remained detectable. Conclusions: These preliminary findings are interesting, as they could be useful for routine screening of HPV and STB in sperm samples preserved in sperm banks and highlight the need for future research. Full article

Chirostoma humboldtianum is an endemic atherinopsid from Mexico that has been of high socio-cultural and economic importance since pre-Hispanic times. It was the first ictic species from Mexico described for science, and it is considered the basal species that gave rise to a nominal group known as white fish or silversides. The aim of this research was to analyze semen quality in relation to breeding fish size and its effect on sperm cryopreservation during an annual cycle. Sexually mature males were collected from January to December 2023, in San Felix dam, Tiacaque, Mexico State, Mexico. The water temperature was measured, and the photoperiod was obtained from the Instituto Nacional de Geografía y Estadística (INEGI). Males were classified into two groups of total length (TL) after analyzing the variation in TL through a size histogram: (G1) 9–13 cm and (G2) longer than 13 cm. Semen volume (µL), sperm concentration (cells µL⁻¹), and motility percentage (%) were determined in all individuals of each group. Likewise, eight straws were cryopreserved per month per group, each one with 10 µL of semen from a mixture of three randomly selected individuals and cryopreserved at −196 °C for 72 h. The post-thawing motility percentage was subsequently verified. Males produce semen continuously all year round, with two periods of higher volume, March and June–August, defining two reproductive periods. The beginning of the first one coincides with the increase in water temperature, from 13 ± 2 to 18 °C. Males with a length more than 13 cm had a higher semen production compared to smaller males (17.33 ± 7.34 and 12.52 ± 4.41 µL, respectively, p < 0.05). The largest semen volumes were registered in March and from June to August in both groups. However, G2 males presented with a larger semen volume. Both groups had a marked decrease in a similar manner in April–May and September to January (p < 0.05). Sperm concentration was similar throughout the year in both groups (p > 0.05) (G1 = 1.35 ± 0.36 × 10⁶ µL⁻¹) (G2 = 1.31 ± 0.35 × 10⁶ µL⁻¹). In addition, fresh sperm motility was high in both groups (G1 = 96 ± 3%, G2 = 97 ± 4%) (p > 0.05). The highest sperm concentrations were observed in March to July, through to October for both groups (p > 0.05), while post-thaw sperm motility decreased by about 50% (G1 = 45 ± 4%) (G2 = 46 ± 6%) during the annual cycle (p > 0.05). The largest post-thaw motility was observed in March and from July to September in G1 and in March and from June to October in G2. Analysis of semen quality throughout the annual cycle reveals aspects of the reproductive biology of C. humboldtianum, including two reproductive peaks and continuous semen production. Full article

The recovery and cryopreservation of epididymal spermatozoa enable genetic preservation in male dogs that die unexpectedly or require castration; however, sperm collection is typically performed immediately after surgery, and artificial insemination is often surgical. This study aimed to evaluate whether epididymides stored at 4–5 °C for 24 h prior to sperm recovery retain fertilizing capacity after cryopreservation and whether pregnancy can be achieved using endoscopically guided transcervical intrauterine insemination. Testes and epididymides from an eight-month-old German Shepherd dog were stored in physiological saline at 4–5 °C for 24 h following castration. Spermatozoa were recovered from the cauda epididymis using single-incision aspiration, evaluated, frozen according to the Uppsala method, and stored in liquid nitrogen for two months. After thawing, 62 × 10⁶ progressive motile spermatozoa were inseminated once into a bitch in heat using transcervical endoscopic guidance. Pregnancy was confirmed by ultrasonography, and nine healthy puppies were delivered at term. These findings demonstrate that 24 h refrigerated storage of the epididymis does not impair post-thaw fertilizing ability and that non-surgical transcervical intrauterine insemination represents an effective alternative to surgical techniques for the use of frozen–thawed epididymal semen in dogs. Full article