Sperm capacitation, a crucial process for successful fertilization, is modulated by reactive oxygen species (ROS) and involves molecular changes including increased membrane fluidity, cholesterol depletion, and protein tyrosine phosphorylation. Although an effective equine in vitro fertilization protocol has recently been described, its requirement for prolonged capacitation limits applicability to high-quality ejaculates. Because stallion spermatozoa possess endogenous antioxidant systems that may mitigate ROS accumulation and delay ROS-mediated capacitation, this study investigated whether inhibition of catalase with 3-amino-1,2,4-triazole (3-AT), superoxide dismutase 1 with LCS1, γ-glutamylcysteine synthetase with buthionine sulfoximine (BSO), or their combination (INH), could enhance endogenous ROS and promote capacitation. Sperm were incubated for 24 h under control, capacitating, or inhibitor-treated capacitating conditions (3-AT, LCS1, BSO, or their combination [INH]). Samples were evaluated using CASA and flow cytometry. Incubation increased intracellular calcium in all samples, but sperm under capacitating conditions showed lower calcium levels than control after 22 h (100.8 ± 2.4 vs 112.7 ± 4.81 AU respectively; p ≤ 0.01), an effect further enhanced by 3-AT (p ≤ 0.05) and BSO (p ≤ 0.01). After 22 h, BSO concentrations above 500 μM increased membrane lipid disorder (CTR: 70.81 ± 5.2 vs 500 μM BSO: 104.85 ± 5.1 AU, p ≤ 0.01), and hyperactivation-associated motility kinematics (TM, VCL, ALH, BCF; all p ≤ 0.05). When data were pooled across time points, INH increased tyrosine phosphorylation compared with control (31.55 ± 2.1% vs 25.24 ± 1.8% respectively; p ≤ 0.01). In conclusion, inhibition of antioxidant systems-predominantly the glutathione system-may drive stallion sperm capacitation and reduce the time required for IVF protocols, although further studies are required to determine its impact on in vitro fertility.

Antisperm antibodies (ASA) have been implicated in reproductive failure in cows. Present study investigated 58 crossbred cows, of which 38 (normal-cows, NC, n = 16; repeat-breeder-cows, RBC, n = 22) were used for ASA analyses and fertility evaluation, while subsequent bacteriological component included additional 20 cows. ASA levels were assessed from maiden to conceptional-estrus using mixed-antiglobulin reaction (MAR-IgG and MAR-IgA) tests and ELISA, and candidate spermatozoal-bacterial protein pairs were assessed for sequence and three-dimensional structural similarity. MAR-IgG and MAR-IgA levels were significantly lower during conceptional-estrus than during maiden-estrus in both NC and RBC cows, whereas differences between NC and RBC at same estrus stage were less pronounced, indicating that ASA levels were closely associated with conception status than with fertility classification. Receiver operating characteristic analysis based on cow-level measurements showed that MAR-IgG and MAR-IgA discriminated conceptional from non-conceptional status, with area under the curve values of 0.799 and 0.806, respectively. Head-bound spermatozoa represented predominant antibody binding, suggesting that sperm head antigens constitute major ASA targets. Bacillus spp., Escherichia coli and Staphylococcus spp. were predominant bacterial isolates recovered from the genital tract. Sequence and structural analyses of previously identified cross-reactive sperm and bacterial proteins revealed substantial peptide-level similarity and structural-overlap, and structural-superimposition produced low root mean square deviation values, indicating close structural similarity between sperm and bacterial proteins. Collectively, these findings indicate that lower ASA levels are associated with successful conception and support molecular mimicry between genital bacterial and sperm proteins as a plausible mechanism contributing to reproductive immunoinfertility in cows.

Ursolic acid (UA) is a natural pentacyclic triterpene present in many fruits, vegetables and medicinal plants. We tested whether adding UA to the freezing extender improves the quality of frozen-thawed goat sperm. Ejaculates from six bucks were pooled at each collection, and the pooled sample was split into six equal aliquots diluted in Andromed® extender either without UA (UA-0, control) or with 0.5 µM (UA-1), 2 µM (UA-2), 5 µM (UA-3), 15 µM (UA-4) or 30 µM (UA-5). Samples were frozen using a standard protocol and thawed at 37 °C for 30 s. Post-thaw sperm quality was assessed by computer-assisted sperm analysis (CASA) for motility, the hypo-osmotic swelling test for membrane integrity, and flow cytometry for acrosome status (FITC-PSA/PI), phosphatidylserine externalization (Annexin V-FITC), mitochondrial membrane potential (Rhodamine 123) and intracellular reactive oxygen species (H₂DCFDA). Adding UA changed progressive motility (PR) and mitochondrial activity (MA), and both peaked at 0.5 µM. PR at 0.5 µM was significantly higher than at 15-30 µM, and MA at 0.5-2 µM was significantly higher than at 30 µM; however, no UA concentration differed significantly from the control. Total motility, membrane integrity (HOST), acrosome integrity, phosphatidylserine externalization and intracellular ROS did not differ among the treatments. In short, the response was dose-dependent: sperm frozen with 0.5 µM UA retained the best PR and MA, the high concentrations were harmful, and an improvement over the control itself could not be demonstrated. Confirming a fertility benefit will require in vitro fertilization and in vivo insemination trials.

The seminal microbiota is an integral component of fertility, both through its role in maintaining the health of the male reproductive tract and its contribution to the microbiota of the female reproductive tract. Many factors affect the dynamics of the seminal microbiota, such as the age and breed of the animal, and the husbandry conditions under which it is kept. Animal breeders introduce semen storage, representing an additional potential source of contamination. It is during this storage that the seminal microbiota can have an additional influence on sperm quality, permitting some bacteria to flourish whilst others are suppressed. Factors such as type of semen extender and temperature of storage exert an impact on the microbiota, but the predominant influence is the inclusion of antibiotics in the semen extender. Apart from a direct effect on sperm quality, these antibiotics may also alter the microbiota of the female reproductive tract, thus impacting on fertility. These factors should be borne in mind when deciding to include antibiotics in insemination doses. Alternatives to antibiotics do exist, such as physical separation of the sperm component from bacteria in the ejaculate, for example, with colloid centrifugation, and should be incorporated into prudent animal breeding strategies from a One Health perspective.

The voltage- and pH-gated Slo3 potassium channel is exclusively expressed in mammalian spermatozoa. Its sensitivity to both voltage and alkalization plays a crucial role in sperm fertility, which is tightly coupled to the capacitation process. Here, we show that sperm-enriched divalent cation Zn2+ undergoes dynamic alteration in spermatozoa during capacitation. We also found that intracellular Zn2+ regulates alkalinization-induced hyperpolarization in mouse spermatozoa, which is mediated by the Slo3 channel. Further examination of zinc regulation in mouse Slo3 (mSlo3) revealed that, in the Xenopus oocyte expression system, intracellular zinc directly inhibits mouse Slo3 currents in a dose-dependent manner at micromolar concentrations, with exceptionally slow dissociation. By combining MD simulations and electrophysiology, we also identified amino acid residues contributing to the Zn2+ slow dissociation from Slo3 channels. Our studies uncover the importance of intracellular zinc dynamics and its regulatory role in ion channels during sperm capacitation.

Cancer and its treatment can reduce fertility in men of reproductive age and can affect the ability to have children. It is still unclear how much of this effect is caused by the disease itself and how much by the treatment. In this study, we compared semen quality in men with cancer before and after treatment with that of healthy Swiss men. We found that some patients, especially those with testicular cancer, already had lower semen quality before starting the treatment. After treatment, semen quality often further decreased and more men had no sperm in their ejaculate. This was particularly seen in patients who received treatments known to strongly affect fertility. These findings show that both cancer and cancer treatment can impair male fertility. This highlights the importance of early counselling and offering sperm freezing before treatment, as well as providing long-term reproductive follow-up after cancer care.

Male infertility is rising worldwide. Spermatogenesis is energetically demanding and tightly coupled to mitochondrial quality control (MQC)-including biogenesis, fusion-fission dynamics, and mitophagy. Here we synthesize current evidence across developmental stages of germ cells, highlighting stage-specific shifts from glycolysis-supported ATP production to oxidative phosphorylation (OXPHOS)-dominated metabolism and remodeling of mitochondrial architecture into the midpiece sheath. We summarize genetic and functional data implicating OPA1/MFN1-2 in inner/outer membrane fusion, DRP1/FIS1/MFF in fission and sheath organization, and the roles of PINK1-Parkin-dependent and -independent mitophagy in mitochondrial quality control, while acknowledging that cytoplasm removal and acrosome maintenance during spermiogenesis may involve general autophagy rather than mitophagy specifically. Mitochondrial dysfunction-excess reactive oxygen species (ROS), mitochondrial membrane potential (MMP) collapse, dysregulated dynamics, or defective mitophagy-impairs spermatogenesis and sperm function. We also identify methodological gaps that limit translation, particularly the lack of standardized phenotyping and human genetic evidence. Clarifying MQC causality and druggability may support future diagnostic or therapeutic research in male infertility.

Sperm DNA integrity is a crucial determinant of fertility and early embryonic development. The Sperm Chromatin Dispersion (SCD) test provides a simple, practical, and cost-effective tool for assessing DNA fragmentation; however, the diagnostic reliability depends heavily on the staining technique used to visualize halos of dispersed chromatin. This study evaluated and optimized several commonly and widely used histochemical stains associated with a modified SCD protocol in bovine spermatozoa to improve accuracy and interpretation; hence providing new and useful data for scientists in the field. Frozen-thawed semen samples from five fertile bulls were processed using SCD procedures and stained with Toluidine Blue (TB), Giemsa (G), May Grünwald-Giemsa (MGG), Eosin Thiazine (ET), Feulgen & Rossenbeck (F&R), Hematoxylin & Eosin (H&E), Nuclear Fast Red (NFR), Eosin-Nigrosine (EN), and various modifications of the above stains. Most stains and their modifications (TB, G, ET, NFR, EN) failed to provide consistent or distinguishable halos, whilst specific modifications of MGG, F&R, and H&E produced reproducible halos. The MGG-modified protocol enabled halo visualization but introduced notable nonspecific background staining that reduced distinctiveness of halos. Specific modified protocols of F&R and H&E also visualized halos but reacted variably with agarose: F&R did not produce nonspecific background staining while H&E did. In conclusion, our results demonstrate that specific modifications of MGG, F&R, and H&E visualized halos and thus are suitable for SCD test.

A new nematode population of the genus Ficophagus was recovered from figs of Ficus amplissima from ICAR-Indian Agricultural Research Institute, New Delhi, India. It is described herein as Ficophagus siddiqii n. sp., distinguished among all known species of the genus by its slender, cylindrical female body and presence of four faint lateral incisures at mid-body, with the outermost pair being the most prominent; a short post-uterine sac (PUS; 5.0-10 μm), a secretory-excretory pore situated near the lips, amoeboid sperm, and three pairs of subventral papillae on the male tail. Conoid tail in both sexes; the male tail tip broadly rounded without mucro and female tail tip pointed or spike-like projection. Gubernaculum absent, rose-thorn-shaped spicules with relatively high condylus - rounded to truncate. This species is also distinguished by its biogeography and host plant, as it is collected from Ficus amplissima. Further, it is characterised by molecular sequences of 18S SSU, 28S D2-D3, Cytochrome c Oxidase Subunit I (COI), and Internal Transcribed Spacer (ITS). Molecular comparisons of the Indian population of Ficophagus siddiqii n. sp. with other Ficophagus species using D2-D3, SSU, COI, and ITS sequences revealed a clear separation of Ficophagus siddiqii n. sp. from other Ficophagus species.