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Selenium is a well-known essential element with important roles in human reproductive health mainly due to its antioxidant character. This study aimed to investigate the potential role of selenoproteins on gut microbiota and male reproductive health. A new assay for the absolute quantification of selenoproteins in testicular tissue based on two dimensional chromatography with inductively coupled plasma mass spectrometry was performed for the first time. The gut microbiota profile was obtained by 16S rRNA gene sequencing. Numerous associations were found between testicular selenoproteins and gut microbiota (e.g. Mucispirillum , related with sperm activity and testosterone, was associated with glutathione peroxidase (GPx) and selenoalbumin (SeAlb), while Escherichia/Shigella , related to sex hormones, correlated with GPx, selenoprotein P (SelP) and SeAlb). The effects of Se-supplementation on testicular selenoproteins only occur in conventional mice, suggesting a potential selenoproteins-microbiota interplay that underlies testicular function. The selenoproteins GPx and SelP have been quantified for the first time in the testicles, and the novel identification of SeAlb, a protein with nonspecifically incorporated Se, is also reported. These findings demonstrate the significant impact of Se-supplementation on gut microbiota and male reproductive health. In addition, the analytical methodology applied here in selenoprotein quantification in testicular tissue opens new possibilities to evaluate their role in gut microbiota and reproductive health axis.

Mate competition encourages individuals to modulate characters involved in mating success. Adult Iberian red deer ( Cervus elaphus hispanicus ) males show a dark ventral patch (DVP) that plays a central role in mating rivalry, whose size and chemical compounds varied according to the level of male-male competition within the population. In the pigmentation of the DVP appears, after urinary excretion, a molecule called DOPEG originating from the metabolism of norepinephrine, leading us to investigate whether differential expression mechanisms of key genes dopamine B-hydroxylase (DBH) and tyrosine hydroxylase (TH), encoding enzymes catalyzing the process can be sensitive to different competitive population situations and responsible for the plastic development of the DVP in red deer. We found that social environment with higher intrasexual competition, where male invest more in sexual traits, was associated with increased levels of DBH and TH transcripts, while Dopamine showed reversed values. We found alternative splicing for the TH gene, although differences between social environments appeared just related to expression levels. Our results support the internal cause of trait modulation based on differential gene expression in relation to the conditions of intrasexual competition in social environment. We propose the quantification of DBH transcripts as a molecular biomarker of male red deer reproductive activity.

Sexual selection favors male traits that increase their ability to monopolize the breeding access to several females. Deer antlers are cranial appendages that regenerate annually in males. Throughout life, the phenology of antler growth advances and antler mass increases until the stag reaches, between 8 and 10 years old, maximum body mass and highest reproductive success. The molecular mechanisms of antler development are of great interest in both evolutionary and regenerative medicine studies. To minimize errors in the assessment of gene expression levels by qRT-PCR, we analyzed the stability of a panel of eight candidate reference genes and concluded that qRT-PCR normalization to three stable genes is strongly convenient in experiments performed in red deer antler blood. To validate our proposal, we compared the expression level of three genes linked to red deer antler growth (ANXA2, APOD and TPM1) in fifteen male red deer classified as young (up to 4 years old) and adults (4–6 years old). Our data confirms that B2M, ACTB and RPLP0 are valuable reference genes for future gene expression studies in red deer antler blood, which would provide increased insight into the effects of intrinsic factors that determine antler development in red deer.

Sexual signals in animals encompass a variety of forms including visual, acoustic, and chemical signals that are fundamental for intra- and interspecific communication, including sexual selection processes. Among these, odor signals play a critical role. Chemical compounds involved in sexual signaling vary in nature, with lipids and proteins being particularly important. In the male Iberian red deer (Cervus elaphus hispanicus), the dark ventral patch has been identified as a key sexual signal during mating competition, associated with specific chemical compounds deposited during urination. Hair plays a significant role in this signaling due to its ability to retain compounds on the scales of the hair cuticle. In this study, we used a shotgun proteomic approach to investigate whether the ventral patch hair retains proteins on its surface that could reveal metabolic and cell-signaling adaptations potentially related to reproductive activity. Characterization of the origin and functionality of the proteins found in the hair of the dark ventral patch of male red deer, and their relationship to distinct metabolic pathways, provides an initial understanding of the potential role of these compounds in chemical communication in red deer intrasexual competition for mates.

The expression of proinflammatory (IL-1β, IFN-γ, TNF-α) and regulatory (IL-10, TGF-β, IL-4) cytokines, as well as the transcription factor FoxP3, was quantified in the liver and hepatic lymph node (HLN) of sheep primoinfected and reinfected with Fasciola hepatica at early (4, 8 and 16 days post-infection [dpi]) and late (100 dpi) stages. The liver exerted a Th2 immune response at very early stages after the primoinfection with F. hepatica that induced the downregulation of IFN-γ, followed by a Th1/Th2/Treg response although the late stages were characterised by the expression of Th1/Th2 immune mediators. Contrarily, in reinfected sheep a robust mixed Th1/Th2/Treg immune response was found at very early stages meanwhile at late stages we observed a Th2/Treg immune response overcoming the expression of Th1 immune mediators. However, the HLN displayed a completely different Th1/Th2/Treg expression profile compared to the liver. Primoinfections with F. hepatica in HLN induced a mixed Th1/Th2/Treg environment from early stages, establishing a Th2 immune response at a late stage. However, the reinfected sheep exerted a Th2 immune response at early stages led by the IL-4 expression in opposition to the Th1/Th2/Treg found in the liver, meanwhile at late stages the HLN of reinfected sheep exerted a mixed Th1/Th2/Treg immune response. This is the first work publishing the expression of immune mediators in the liver and HLN from reinfected sheep with F. hepatica. The study of the immune responses exerted by the natural host in the target organs directly implied in the development of F. hepatica are crucial to better understand the immunopathogenesis of the fasciolosis being a key factor to develop effective vaccines.

Gene expression for Th1/Th2 cytokines (IL-4 and IFN-ɣ), regulatory cytokines (TGF-β and IL-10) and the transcriptional factor FoxP3 was analyzed in the liver and hepatic lymph nodes (HLN) from sheep immunized with partially protective and non-protective vaccine candidates and challenged with Fasciola hepatica. FoxP3 T cells were also evaluated by immunohistochemistry (IHQ). The most remarkable difference between the partially protected vaccinated (V1) group and the non-protected vaccinated (V2) group was a more severe expansion of FoxP3 T cells recorded by IHQ in both the liver and HLN of the V2 group as compared to the V1 group, whereas no differences were found between the V2 group and the infected control (IC) group. Similar results were recorded for FoxP3 gene expression although significant differences among V1 and V2 groups were only significant in the HLN, while FoxP3 gene expression was very similar in the V2 and IC groups both in the liver and HLN. No significant differences for the remaining cytokines were recorded between the V1 and V2 groups, but in the liver the V2 group shows significant increases of IFN-ɣ and IL-10 as compared to the uninfected control (UC) group whereas the V1 group did not. The lower expansion of FoxP3 T cells and lower increase of IFN-ɣ and IL-10 in the partially protected vaccinated group may be related with lower hepatic lesions and fluke burdens recorded in this group as compared to the other two infected groups. The most relevant change in regulatory cytokine gene expression was the significant increase of TGF-β in the liver of IC, V1 and V2 groups as compared to the UC group, which could be related to hepatic lesions.

Seed germination and early seedling development have been studied in the recalcitrant species using targeted transcriptional, hormonal, and sugar analysis. Embryos and seedlings were collected at eight morphologically defined developmental stages, S0-S7. A typical triphasic water uptake curve was observed throughout development, accompanied by a decrease in sucrose and an increase in glucose and fructose. Low levels of abscisic acid (ABA) and high levels of gibberellins (GAs) were observed in mature seeds. Post-germination, indole-3-acetic acid (IAA), increased, whereas GA remained high, a pattern commonly observed during growth and development. The abundance of transcripts from ABA-related genes was positively correlated with the changes in the content of the phytohormone. Transcripts of the drought-related genes and were more abundant at S0, then decreased in parallel with increasing water content. Transcripts for , and were abundant at S0, supporting the occurrence of an active metabolism in recalcitrant seeds at the time of shedding. The importance of ROS during germination is manifest in the high transcript levels for Sod and Gst, found in mature seeds. The results presented herein help distinguish recalcitrant (e.g., ) seeds from their orthodox counterparts. Our results indicate that recalcitrance is established during seed development but not manifest until germination (S1-S3). Post-germination the patterns are quite similar for both orthodox and recalcitrant seeds.

The expression of T regulatory cells (Foxp3), regulatory (interleukin [IL]-10 and transforming growth factor beta [TGF-β]) and proinflammatory (tumor necrosis factor alpha [TNF-α] and interleukin [IL]-1β) cytokines was quantified using real time polymerase chain reaction (qRT-PCR) in the liver of sheep during early stages of infection with Fasciola hepatica (1, 3, 9, and 18 days post-infection [dpi]). Portal fibrosis was also evaluated by Masson’s trichrome stain as well as the number of Foxp3+ cells by immunohistochemistry. Animals were divided into three groups: (a) group 1 was immunized with recombinant cathepsin L1 from F. hepatica (FhCL1) in Montanide adjuvant and infected; (b) group 2 was uniquely infected with F. hepatica; and (c) group 3 was the control group, unimmunized and uninfected. An overexpression of regulatory cytokines of groups 1 and 2 was found in all time points tested in comparison with group 3, particularly at 18 dpi. A significant increase of the number of Foxp3+ lymphocytes in groups 1 and 2 was found at 9 and 18 dpi relative to group 3. A progressive increase in portal fibrosis was found in groups 1 and 2 in comparison with group 3. In this regard, group 1 showed smaller areas of fibrosis than group 2. There was a significant positive correlation between Foxp3 and IL-10 expression (by immunohistochemistry and qRT-PCR) just as between portal fibrosis and TGF-β gene expression. The expression of proinflammatory cytokines increased gradually during the experience. These findings suggest the induction of a regulatory phenotype by the parasite that would allow its survival at early stages of the disease when it is more vulnerable.

Fasciola hepatica (F. hepatica) is a widely prevalent trematode responsible for parasitic infections and remains a major target for control interventions worldwide. Its ability to establish chronic infection relies on complex host-parasite interactions mediated by molecular mechanisms that regulate gene expression. Among these mechanisms, microRNAs (miRNAs), a class of non-coding RNAs, act as key post-transcriptional regulators and play important roles in immune modulation during infection. These molecules are increasingly investigated as potential diagnostic biomarkers and targets for novel vaccine strategies. However, accurate miRNA quantification by qRT-PCR requires stable endogenous normalizers to control for technical and biological variability, and such normalizers have not yet been validated for fasciolosis studies in ovine plasma. Therefore, this study aims to identify stable endogenous miRNA reference genes for RT-qPCR normalization in plasma samples from sheep experimentally infected with F. hepatica during the early stages of infection. We evaluated a panel of candidate miRNAs and ranked their expression stability using RefFinder, an integrative tool that combines several algorithms, including NormFinder, the ΔCt method, geNorm, and BestKeeper. Stability analysis identified hsa-let-7c-5p, bta-let-7f, and oar-miR-16b as the most stable miRNAs across all conditions. These miRNAs showed consistent expression across samples and time points, indicating minimal susceptibility to infection-related biological variation or technical bias. They are therefore suitable endogenous normalizers for plasma miRNA quantification in early fasciolosis. The geometric mean of these three miRNAs was employed for normalization to enable accurate and reproducible relative quantification of three target miRNAs whose expression patterns are associated with distinct stages of Fasciola infection. Temporal profiling revealed an early upregulation of oar‑miR‑133‑5p, a later peak of oar‑miR‑3957‑5p, and a decreasing trend of fhe‑miR‑novel‑11‑5p. Because we obtained these expression patterns after normalization with rigorously validated stable references, they are more likely to reflect true biological dynamics rather than technical variability. Together, these findings support the stage-specific diagnostic potential of the target miRNAs and further implicate their involvement in host-parasite interactions during infection. These findings highlight the value of validated miRNAs as endogenous reference genes of plasma-based studies in ovine fasciolosis. They also improve diagnostic sensitivity and specificity by establishing a robust methodological framework for biomarker discovery, as well as for subsequent functional analyses and large-scale evaluations.

Diclofenac (DCF) exposure is of great concern due to the ecotoxicological risk linked with a decline of vulture populations in Southeast Asia, but also because it can affect the reproduction and neurotoxicity in mammals. Otherwise, selenium (Se) is an antioxidant essential element with key roles in health and with antagonistic action against pollutants, but in some cases with a synergistic effect. To investigate the potential intertwined mechanisms between DCF, Se, and gut microbiota, gut metabolomic and gut microbiota profiles were determined in mice after DCF exposure and Se supplementation. Speciation of selenoproteins in plasma was carried out by isotopic dilution analysis to quantify the levels of selenoproteins. Significant differences in the levels of 79% of the gut metabolites were determined after DCF exposure. The most significant altered pathway in DCF and DCF-Se groups is the primary bile biosynthesis, being the only pathway altered in mice exposed to DCF, while in DCF-Se, the metabolism of galactose and linoleic acid is also altered. Moreover, specific associations between specific gut microbiota and metabolites were determined in the studied mice groups suggesting intertwined mechanisms. Selenium supplementation modulated the gut metabolic and microbiota profiles affected by DCF.