The giant panda serves as a clear example of the critical role zoological institutions play in conservation programs for endangered wildlife. It is the bear species whose reproductive biology is best understood, and for which the greatest advances in reproductive technologies have been achieved. This review describes various procedures for collecting and cryopreserving giant panda sperm and examines how their morpho-functional characteristics appear to be associated with their reproductive biology. The influence of different factors, such as season and age, on sperm parameters is also addressed. Compared with other bear species, giant panda ejaculates display morpho-functional traits characterized by low pleiomorphy, high concentration, and enhanced kinematic variables, consistent with those typically observed in species with a high degree of polyandry. Giant panda sperm show notable resistance to freeze-thaw procedures. While morphometric characteristics have been considered a key factor in the osmotic stress occurring during cryopreservation, other molecular mechanisms may also contribute to cryoresistance. In the present review, we describe preliminary observations on the identification of aquaporin 3 and androgen receptors in giant panda spermatozoa and discuss their putative roles in modulating the sperm response to the freeze-thaw process.

Bull fertility is a multifactorial trait influenced by molecular determinants beyond conventional semen parameters. Seminal plasma extracellular vesicles (EVs) mediate intercellular communication and may modulate sperm function through the transfer of regulatory cargo, including microRNAs (miRNAs). This study investigated (i) differences in miRNA profiles of spermatozoa and seminal plasma EVs from proven- and low-fertility Holstein-Friesian bulls, and (ii) the functional and molecular effects of co-incubating low-fertility spermatozoa with EVs derived from proven- or low-fertility bulls. Extracellular vesicles were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis, Western blotting, and transmission electron microscopy. Sperm kinetics were assessed by computer-assisted sperm analysis. Small RNA sequencing was performed to profile miRNA cargo in spermatozoa and EVs. Although conventional semen parameters did not significantly differ between fertility groups, EV concentration was significantly higher in proven-fertility bulls. Co-incubation of low-fertility spermatozoa with seminal plasma EVs from proven-fertility bulls was associated with a distinct kinematic profile compared with untreated spermatozoa and spermatozoa incubated with EVs from low-fertility bulls. miRNA profiling identified 16 differentially expressed miRNAs between proven- and low-fertility spermatozoa, whereas EV miRNA cargo did not differ significantly between groups. Notably, incubation alone was associated with a larger number of miRNA changes, while co-incubation with proven-fertility EVs was associated with fewer changes in the sperm miRNA profile. These findings suggest that the observed effects of seminal plasma EVs on low-fertility bull spermatozoa cannot be explained by fertility-specific EV miRNA cargo and may involve other EV-associated mechanisms not investigated in the present study.

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.

Heat stress (HS) severely impairs reproductive performance and testicular function in livestock. This study aimed to evaluate the comparative efficacy of conventional black seed oil (BSO) and its nano-emulsion (BSO-NE) in mitigating summer thermal stress in Ossimi rams. Fifteen mature rams were divided into three groups (n = 5 each) and treated orally daily for 8 weeks during summer: a water-treated control, a BSO group (0.5 mL/kg body weight (BW)), and a BSO-NE group (0.05 mL/kg BW). Testicular ultrasonography, semen characteristics, and blood markers were monitored weekly, following a two-week winter baseline assessment in February. Results showed that BSO-NE significantly decreased (P < 0.05) testicular Doppler indices: specifically, the resistive index (RI) from weeks 2-4, pulsatility index (PI) at weeks 2 and 7, and systolic/diastolic ratio (S/D) at week 1, while producing the highest systemic concentrations (P < 0.05) of nitric oxide (NO) and testosterone from weeks 1-8. Consequently, BSO-NE significantly increased (P < 0.05) sperm motility, viability, normal morphology, and acrosome integrity from weeks 2-8, alongside increasing sperm cell concentrations from weeks 6-8. Furthermore, BSO-NE exhibited the most pronounced upregulation (P < 0.05) in total antioxidant capacity (TAC) and superoxide dismutase (SOD) from weeks 3-8, catalase (CAT) and glutathione peroxidase (GPx) from weeks 4-8 relative to the other cohorts. To summarize, oral supplementation of BSO-NE effectively counteracts seasonal HS in Ossimi rams, thereby maintaining reproductive performance through improved testicular blood flow and enhanced sperm quality.

Porcine oocyte in vitro maturation (IVM) remains suboptimal because current culture systems do not fully recapitulate the follicular environment present in vivo. Erythropoietin (EPO), a cytoprotective factor reported in the ovarian follicular compartment, may contribute to the regulation of oocyte maturation; but its role during porcine IVM remains unclear. This study investigated the presence and localization of EPO in porcine ovarian follicles and evaluated whether EPO supplementation during IVM improves oocyte developmental competence as assessed by parthenogenetic activation. EPO immunoreactivity was detected in porcine oocytes and surrounding follicular cells across multiple follicular stages. Porcine cumulus-oocyte complexes were then cultured in IVM medium supplemented with 0, 10, 100, or 1000 mU/mL EPO for 42 h. Nuclear maturation rates did not differ among groups, whereas 100 mU/mL EPO significantly increased blastocyst formation following parthenogenetic activation. EPO treatment was also associated with increased mitochondrial membrane potential-related fluorescence and MitoTracker fluorescence, reduced intracellular reactive oxygen species levels, increased BCL-2 expression, and reduced CASP3-related apoptotic signals. These findings suggest that EPO supplementation during porcine oocyte IVM enhances PA-assessed developmental competence and is associated with mitochondrial-related changes, reduced oxidative stress, and attenuated apoptosis-associated signals.

The practical application of sex-sorted semen in pig production remains limited because conventional flow cytometric sorting requires specialized equipment and relatively low throughput. The H4:L4 single-chain variable fragment (scFv) is an antibody developed in a previous study that preferentially recognizes an epitope on the plasma membrane of Y-chromosome-bearing sperm from Duroc boars, thereby providing a knowledge base for evaluating its broader utility in other pig breeds. This study evaluated H4:L4 single-chain variable fragment (scFv)-coated magnetic microbeads as an immunomagnetic platform for boar sperm sex sorting. Antibody binding was assessed in different pig breeds, followed by evaluation of antibody concentration, sperm separation efficiency, sex-ratio enrichment, and post-sorting sperm quality. Breed-associated binding response in H4:L4 scFv were observed, with the strongest response in Duroc sperm and detectable binding in Landrace, Large White, Hampshire, and Native Black pigs. ELISA confirmed concentration-associated binding, and 4 mg/mL was selected as the optimal coating concentration because it achieved sex-ratio enrichment comparable to 8 mg/mL while reducing antibody use. Following immunomagnetic separation, the supernatant fraction was enriched with X-chromosome bearing sperm, whereas the eluted fraction was enriched with Y-chromosome-bearing sperm. Imaging flow cytometry showed 75.9-77.0% X chromosome-bearing sperm in the supernatant and 76.3-78.6% Y-bearing sperm in the eluted fraction. qPCR supported these findings, showing increased BMP15 expression in the supernatant and increased UTY expression in the eluted fraction. Sperm quality was preserved in the X-enriched fraction, with motility, membrane integrity, mitochondrial membrane potential, and oxidative status comparable to conventional semen, whereas reduced sperm quality was observed in the Y-enriched fraction. These findings provide in vitro proof of concept that H4:L4 scFv-coated magnetic microbeads can produce X- and Y-chromosome-enriched fractions from Hampshire boar semen. However, the study did not evaluate fertilization, artificial insemination, pregnancy, farrowing outcomes, or offspring sex ratio. Consequently, the observed sperm enrichment cannot be extrapolated directly to fertility or piglet sex ratio, and controlled biological validation is required before practical application can be considered.

LPS, an endotoxin derived from Gram-negative bacteria, disrupts granulosa cell (GCs) function, and impairs ovarian activity. This study aimed to investigate the role of miR-130b-3p targeting PPARG expression in LPS-exposed goat GCs. Results indicated that LPS decreased PPARG expression, while increased the level of miR-130b-3p which binds to the 3'UTR of PPARG mRNA. PPARG knockdown and miR-130b-3p overexpression further aggravated the reductions in PPARG expression and cell viability under LPS stimulation, while PPARG overexpression and miR-130b-3p inhibition effectively alleviated these LPS-induced inhibitory effects. LPS decreased the mRNA abundances of HSD3B, CYP19A1, CDK4, PCNA, SOD, and CAT, as well as the protein levels of HSD3B and CDK4; Conversely, it increased the mRNA abundances of IL-1β, TNF-α, Caspase-1, and GSDMD, as well as IL-1β protein and intracellular ROS levels. These LPS-induced alterations were further intensified by si-PPARG or miR-130b-3p mimics, as evidenced by more pronounced changes in HSD3B, CYP19A1, CDK4, SOD, CAT, IL-1β, TNF-α, and Caspase-1 and GSDMD mRNA abundances, as well as HSD3B (except siRNA) and CDK4 protein, and ROS levels. In contrast, PPARG overexpression or miR-130b-3p inhibition alleviated LPS-induced changes in HSD3B, CYP19A1, CDK4, PCNA, SOD, and CAT, IL-1β, TNF-α, Caspase-1 and GSDMD at the mRNA abundances, as well as HSD3B and CDK4 protein, and ROS levels. In conclusion, miR-130b-3p modulates steroidogenesis-related markers, cell viability, inflammation, oxidative stress, and pyroptosis-associated responses by modulating PPARG expression in goat GCs exposed to LPS.

Immunocastration (active immunization targeting GnRH) inhibits testosterone secretion, controls boar taint, and reduces aggressive behavior while avoiding surgical castration. Commercial two-dose regimens such as Improvac®/Improvest® are now established. This review covers the immunological mechanisms of GnRH immunocastration, vaccine preparation and delivery strategies, and effects on endocrine suppression, growth performance, carcass quality, and animal welfare. Sources of efficacy variation are summarized: antibody titer thresholds and individual non-response, the decisive effect of first-immunization age and inter-dose interval, and breed-specific lipid metabolism backgrounds. Current research still lacks key data on immunization schedule optimization, standardized carcass-quality thresholds, and field implementation constraints. A "schedule window-antibody threshold-carcass threshold" framework is proposed to support the precision application of immunocastration.

Understanding stage-specific regulation of placental and fetal metabolic signaling pathways is essential to elucidate mechanisms supporting fetal growth and developmental programming in dairy cattle. This study evaluated differences in protein abundance between mid-gestation and late-gestation in placentome and fetal liver in lactating Holstein cows. In placentome tissue, late-gestation was characterized by a marked decrease in phosphorylated ribosomal protein S6 (p-RPS6; P = 0.01), suggesting reduced signaling through the mTOR-RPS6 axis, which is involved in the regulation of protein synthesis. In contrast, proteins associated with cellular stress response and detoxification were significantly elevated, including heat shock protein 70 (HSP70; P = 0.01) and glutathione S-transferase Mu 1 (GSTM1; P = 0.03), suggesting enhanced oxidative stress defense and protein-folding capacity as gestation advances. In fetal liver, late-gestation exhibited a pronounced reduction in insulin signaling, evidenced by lower AKT abundance (P = 0.01). Additionally, the ratio of phosphorylated to total mTOR (P = 0.03) and p-RPS6 abundance (P = 0.01) were decreased, further supporting attenuation of anabolic signaling pathways. Conversely, total eukaryotic elongation factor 2 (eEF2) abundance increased (P = 0.01), potentially reflecting a compensatory mechanism to sustain translational capacity despite reduced upstream signaling. Collectively, these data indicate a shift from anabolic growth signaling toward enhanced stress adaptation and selective translational control in late-gestation. The coordinated downregulation of insulin/mTOR signaling alongside increased stress-response proteins highlights tissue-specific adaptations that may optimize fetal development under changing metabolic demands.

Luteinizing hormone (LH) is a central regulator of testicular development, while the epigenetic mechanisms underlying LH-mediated regulation of immature Leydig cell (iLC) development and function warrant further investigation. In the present study, graded administration of a gonadotropin-releasing hormone agonist (GnRH-a) established differential serum LH levels in Shaziling boars. Reduced LH levels were associated with delayed testicular development and increased body weight, body length, and chest circumference. In vitro, LH treatment promoted G1-to-S phase progression and cell proliferation and increased adenosine triphosphate (ATP) content and total-protein-normalized insulin-like peptide 3 (INSL3) concentrations. Both in vivo and in vitro analyses showed that DNA methyltransferase 3 alpha (DNMT3A) expression was inversely associated with LH levels. Integrated analysis of reduced representation bisulfite sequencing (RRBS) and RNA sequencing (RNA-seq) identified 220 genes exhibiting both differential methylation and differential expression. These genes were enriched in pathways related to Leydig cell function, particularly the cyclic adenosine monophosphate (cAMP) signaling pathway. Functional validation showed that knockdown of the hub genes SLIT-ROBO Rho GTPase activating protein 3 (SRGAP3) and hyaluronan synthase 1 (HAS1) reduced ATP content, while HAS1 knockdown also inhibited cell proliferation. Collectively, these findings identify DNA methylation-associated transcriptional regulation as a potential epigenetic mechanism linking altered LH levels to iLC function.

Protamine 1 (PRM1) and protamine 2 (PRM2) are essential regulators of sperm chromatin condensation and genome integrity, and their dysregulation has been associated with impaired male fertility. However, their role in rams remains insufficiently characterized. This study investigated the relationship between protamine gene expression and semen quality in rams and explored their potential upstream regulatory mechanisms using gene regulatory network (GRN) analysis. Fifteen ejaculates from five rams were analyzed. Based on total sperm motility using computer-assisted sperm analysis (CASA), ejaculates were classified into a high-motility group (n = 8) and a low-motility group (n = 7). PRM1 and PRM2 expression levels were quantified by RT-qPCR. Following normality confirmation (p > 0.05), parametric tests were applied using the ejaculate as the biological experimental unit. Samples with reduced motility showed significantly lower expression of both protamines (p < 0.01). Moreover, progressive sperm motility was strongly correlated with both PRM1 (r = 0.71, p = 0.019) and PRM2 (r = 0.69, p = 0.03) transcript levels. Cross-species GRN inference using scGeneRAI and a reference human spermatogenesis dataset identified several hypothesis-generating candidate transcription factors, including HMGB4, HMGB1, H2AFZ, NKX6-1, and SMC3, consistently supported across multiple bootstrap resampling runs. These findings demonstrate a strong association between protamine expression and sperm motility in rams. While the identified candidate regulators provide a valuable framework for future species-specific validation, they also highlight promising candidate molecular biomarkers of male fertility in livestock.

Cryopreservation of ram semen induces structural and functional alterations that compromise sperm function. Given that seminal plasma contributes to sperm physiology regulation, increasing attention has focused on extracellular vesicles (EVs), which carry proteins involved in membrane organization and capacitation. These properties suggest that EVs may contribute to maintaining sperm stability during cryopreservation. Therefore, this study evaluated the effect of seminal plasma-derived EVs on post-thaw functional parameters of ram sperm. Semen was cryopreserved in the presence or absence of EVs isolated by ultracentrifugation previously characterized by nanoparticle tracking analysis, TEM, FTIR and Western blotting. Post-thaw sperm quality was assessed through viability, membrane lipid disorder, reactive oxygen species production, protein phosphorylation, acrosome status, intracellular calcium levels, and motility. Sperm cryopreserved with EV-supplemented extender showed reduced membrane lipid disorder and lower intracellular calcium levels compared to controls (p < 0.05). CASA analysis revealed no significant differences in total or progressive motility; however, EV supplementation was associated with a modified kinematic profile, characterized by increased velocity and trajectory-related parameters (p < 0.001), suggesting a potential effect on sperm movement patterns. No significant differences were observed in viability, ROS levels, protein phosphorylation (tyrosine residues or PKA), or acrosome status. These findings provide proof-of-concept evidence that supplementation with seminal plasma-derived EVs during ram semen cryopreservation is associated with changes in membrane lipid organization, intracellular calcium homeostasis, and selected sperm kinematic parameters. Further studies are required to determine whether these changes translate into improved fertilizing ability.

This study aimed to investigate how dietary taxifolin (TAX) supplementation affected the reproductive hormone profile, testicular function, antioxidant status, and semen characteristics in aged breeder roosters. Ninety-six Jingfen No. 6 roosters (63 weeks old) were allocated to four dietary treatments containing different TAX levels for six weeks: 0 (control), 200, 400, or 600 mg/kg TAX. Roosters in the 200 mg/kg group showed the most notable enhancements in reproductive performance, including significant elevations in semen volume and sperm motility at weeks 2, 4, and 6. TAX supplementation enhanced sperm kinematic parameters, particularly straightness, linearity, curvilinear velocity, and average path velocity, and the 200 mg/kg group consistently demonstrated better performance than the other treatments. Histological examination revealed that supplementation with 200 mg/kg TAX effectively improved testicular structure and spermatogenesis. In contrast, higher TAX concentrations (400-600 mg/kg) induced vacuolization and impaired tubular development. Plasma testosterone and follicle-stimulating hormone levels peaked in the 200 mg/kg group, whereas luteinizing hormone (LH) levels peaked in the 400 mg/kg group. TAX supplementation markedly enhanced the antioxidant capacity in the plasma, liver, seminal plasma, and testes. Further analysis of gene expression showed the dose-dependent upregulation of genes associated with antioxidant activity (SOD1, SOD2, CAT, GPX1, and GPX4) and stimulation of the nuclear factor erythroid 2-related factor 2/mitogen-activated protein kinase (MAPK/Nrf2) signaling pathway, particularly at 400-600 mg/kg TAX. Collectively, the results suggest that providing 200 mg/kg TAX optimally enhances multiple key metrics in aged breeder roosters, including semen quality, testicular health, reproductive hormone secretion, and antioxidant defense.

Male canine infertility is increasingly recognized in clinical practice, yet diagnostic workups are often limited to conventional semen parameters. Building on our previous characterization of testicular lesions in stray dogs from the 'Land of Fires' (Campania Region, Italy), this study extends the phenotyping of male reproductive dysfunction by integrating testicular tissue remodeling and redox balance with sperm output and nuclear quality. Testes from 40 stray male dogs were classified into four groups: Group A: testes with normal spermatogenesis; Group B: testes showing mild degeneration; Group C: testes showing moderate to severe degeneration; Group D: neoplastic testes. Testicular remodeling was assessed by immunofluorescence for vimentin and α-smooth muscle actin (α-SMA), while testicular oxidative status was examined by measuring hydrogen peroxide (H2O2) production and superoxide dismutase (SOD) activity. Subsequently, epididymal sperm concentration (ESC) was quantified, and Toluidine Blue (TB) staining was performed to evaluate sperm chromatin condensation through an automated whole-slide image analysis pipeline. Finally, sperm nuclear basic proteins (SNBPs) were profiled by AU-PAGE to evaluate protamine representation. Overall, the histopathological lesions were found to be associated with both a reduction in epididymal sperm output and a deterioration in sperm quality, as evidenced by the presence of nuclear and biochemical abnormalities. These results support a multi-level model linking testicular pathology to impaired spermatogenic function in dogs.