With the extensive use of plastics and brominated flame retardants, polystyrene microplastics (Ps-MPs) and polybrominated diphenyl ethers (PBDEs) frequently co-occur in the environment, raising growing concerns about their combined reproductive hazards. However, the synergistic toxicity of Ps-MPs and PBDEs on female fertility and oocyte quality remains insufficiently characterized. In this study, we established a 28-day oral exposure model in female ICR mice to evaluate the effects of Ps-MPs, PBDE-47, and their co-exposure on ovarian function, oocyte meiotic competence, and reproductive outcomes. Both Ps-MPs and PBDE-47 alone reduced ovarian weight, decreased antral follicles, increased follicular atresia, and markedly lowered ovulation and litter size, whereas co-exposure produced the most severe impairments. At the oocyte level, exposure significantly reduced germinal vesicle breakdown and first polar body extrusion, increased abnormal spindle formation and erroneous kinetochore-microtubule attachments, and suppressed TPX2 expression and α-tubulin acetylation. Cortical F-actin polarization, spindle migration, and membrane localization of JUNO and ovastacin were also disrupted, indicating widespread defects in meiotic and membrane maturation. Mechanistically, Ps-MPs and PBDE-47 induced a decline in mitochondrial membrane potential, aberrant mitochondrial distribution, excessive lipid accumulation, and Ca²⁺ imbalance, accompanied by autophagosome accumulation, lysosomal dysfunction, elevated ROS, increased γ-H2AX signals, and enhanced Annexin V labeling, ultimately triggering DNA damage and apoptosis. All alterations were most pronounced under co-exposure. Collectively, Ps-MPs and PBDE-47 synergistically impair female fertility by converging on mitochondrial dysfunction, autophagy-lysosome imbalance, and oxidative stress-mediated DNA damage, leading to substantial reductions in oocyte quality. These findings provide key mechanistic evidence for evaluating reproductive risks associated with real-world mixtures of microplastics and persistent organic pollutants.

OBJECTIVE:This study aimed to develop a recombinant gonadotropin-releasing hormone (GnRH) vaccine and to investigate the effects of different immunization doses on immunocastration efficacy and growth performance in male goats; thererby providing an animal welfare-friendly alternative to surgical castration (SC). METHODS:Forty male goats were randomly assigned to four groups (n = 10 per group): the saline-injected control (NC), SC, low-dose vaccine (LD), and high-dose vaccine (HD). The vaccine was given in accordance with a regular immunization protocol. Anti-GnRH antibody titers, serum testosterone concentrations, libido scores, histological characteristics of testicular tissues, and growth performance were measured to assess immunocastration efficacy. RESULTS:Both LD and HD groups exhibited significantly higher anti-GnRH antibody titers than the NC group (p<0.01). Serum testosterone concentrations and libido scores were markedly reduced in the immunized groups (p<0.01), indicating effective suppression of reproductive function. Histological and quantitative analyses demonstrated pronounced testicular atrophy in immunocastrated goats, characterized by a significant reduction in seminiferous tubule diameter and spermatogenic cell number, as well as the absence of spermatozoa within the lumen. Although only limited time points showed significant differences between the two immunization doses, the overall immunocastration efficacy of the low- and high-dose regimens was largely comparable, with the low-dose group exhibiting slightly stronger suppression in certain parameters or time points. Additionally, immunocastrated goats had significantly higher body weight and weight gain compared to surgically castrated goats (p<0.01), while differences with the NC group were not significant (p>0.05). CONCLUSION:The recombinant GnRH vaccine effectively suppresses reproductive function in male goats. Compared with SC, immunocastration was associated with improved growth performance, while no significant differences were observed relative to intact animals. These findings support immunocastration as a non-invasive and animal-friendly alternative to SC in livestock production.

Skeletal muscle development directly determines growth efficiency and meat quality in livestock. Circular RNAs (circRNAs) and their encoded micropeptides are emerging regulators of this process, yet their roles in goats remain poorly understood. Through integrated ribosome profiling (Ribo-seq) and circRNA-seq of goat skeletal muscle satellite cells (SMSCs), we identified a MAPK1-derived protein-coding circRNA, circMAPK1 (novel-circ-0039015). Functional analyses revealed that circMAPK1 overexpression suppressed SMSCs proliferation and myogenic differentiation. Mechanistically, circMAPK1 encodes a 110-amino-acid micropeptide (circMAPK1-110aa) that binds Mitogen-Activated Protein Kinase Kinase 1 (MAP2K1) and reduces MAPK1/3 phosphorylation, thereby attenuating Mitogen-Activated Protein Kinase (MAPK) signaling. Importantly, pharmacological activation of the MAPK pathway with C16-PAF partially restored muscle cell growth and differentiation. These findings establish circMAPK1 as a negative regulator of goat skeletal muscle development and highlight its encoded micropeptides as a potential molecular target. By uncovering a new regulatory mechanism of muscle growth, this study provides valuable insights for breeding strategies aimed at enhancing meat yield and quality in goats.

Circadian rhythms are endogenous oscillations with a period of approximately 24 h. They enable organisms to anticipate and adapt to daily environmental changes, such as light and temperature. As the largest metabolic and motor organ in the body, skeletal muscle plays a decisive role in determining meat production efficiency in ruminants. Skeletal muscle development is largely governed by the proliferation and myogenic differentiation capacity of skeletal muscle satellite cells (SMSCs). More than 2,300 genes in skeletal muscle exhibit circadian oscillatory expression and are extensively involved in myogenesis, transcriptional regulation, and metabolic processes. The rhythmic expression of these genes is modulated by external factors including the photoperiod, feeding behavior, gut microbiota, and physical activity. Disruption of the endogenous circadian timing system can inhibit SMSC proliferation and myogenic differentiation, thereby impairing normal muscle development. Therefore, this review focuses on key management aspects of ruminant production—such as environmental control, nutritional regulation, and exercise management—and systematically elaborates on how these husbandry strategies may influence SMSC fate by modulating the circadian clock, along with the underlying molecular mechanisms.

Climate change endangers global biodiversity at an unprecedented pace, necessitating urgent understanding of rapid adaptive mechanisms. Notably, the alpine treeline ecotone has experienced marked environmental changes, characterized by an average upward elevation shift of 0.4 m/year. This trend raises critical questions about the evolutionary trajectory and genetic resilience of endemic species, and whether treeline upward migration benefits or harms treeline-endemic animals remains unclear. This study investigates the genomic basis of temporal adaptation and genetic response in the climate-sensitive Gansu pika (Ochotona cansus Lyon, 1907), an alpine treeline endemic species in the Hengduan Mountains in China. We integrated whole-genome sequencing (WGS) and DNA methylation data from the same 200-square-meter plot population (2015: WGS n = 3, methylation n = 1; 2016: both n = 6; 2022: both n = 7). Kinship analyses demonstrate that all sequenced individuals pertain to a single population and exclude direct kinship among these individuals. Principal component analysis demonstrated significant overlap among individuals collected between two periods, coupled with stable levels of heterozygosity and nucleotide diversity. However, a significant reduction in Tajima’s D was observed within the 2022 sample cohort when compared with that of 2015 and 2016. Concurrently, runs of homozygosity (ROH) analysis showed a decrease in inbreeding in the 2022 samples. Furthermore, the overall effective population size of this species increased over approximately 15 generations, collectively indicating enhanced genetic health. DNA methylation analysis detected localized hypomethylation within gene bodies against a globally stable background, suggesting epigenetic remodeling. Integrative cross-omics analysis revealed key genes (GLIS3 and FZD4) that are subject to both positive selection and significantly altered epigenetic regulation. These genes are functionally implicated in energy metabolism and tissue homeostasis, respectively, and may underpin the species’ adaptive response. This study demonstrates rapid adaptive evolution coinciding with a period of marked treeline upward shift in the Gansu pika, providing insights into potential persistence mechanisms of alpine endemics during environmental change.

Benzalkonium chlorides (BACs), a class of quaternary ammonium compounds (QACs) widely used as disinfectants and preservatives, have increasingly replaced triclosan and triclocarban in personal care products following their global restriction. However, the potential reproductive toxicity of BACs remains largely unexplored. Notably, we investigated the effects of BACs exposure on porcine oocyte maturation and fertilization competence, as well as the underlying mitochondrial mechanisms. COCs were cultured under in vitro maturation (IVM) conditions for 44 h with 0.2-50.0 μg/mL BACs. BACs exposure markedly inhibited cumulus expansion, reduced first polar body extrusion, and impaired fertilization and early embryonic development in a dose-dependent manner. Cytological analysis revealed disrupted spindle assembly, decreased microtubule stability, and disorganized F-actin structures. BACs also caused abnormal cortical granule distribution, diminished Ovastacin expression, and reduced sperm-binding capacity. Mechanistically, BACs induced mitochondrial depolarization, reduced mitochondrial abundance, elevated mitochondrial ROS, and disrupted Ca²⁺ homeostasis, leading to oxidative stress, DNA damage, apoptosis, and autophagy-lysosomal imbalance. Further, BACs exposure suppressed the mitochondrial SIRT3-SOD2 antioxidant axis, while pharmacological activation of SIRT3 by UBCS039 or ROS scavenging with Mito-TEMPO partially rescued mitochondrial dysfunction and oocyte maturation defects. Collectively, these findings demonstrate that BACs exposure compromises oocyte quality and developmental competence via mitochondrial oxidative stress and SIRT3-SOD2 axis inhibition, providing new insights into the reproductive risks associated with emerging disinfectant contaminants.

Non-coding regulatory variation drives complex traits, domestication, and evolutionary adaptation, yet the sheep genome lacks high-resolution functional annotation. Here we present SheepEpimap, a multi-tissue regulatory atlas harmonizing 516 CUT&Tag histone modifications, ATAC-seq, and RNA-seq datasets across 43 adult tissues in sheep. We annotated 2.93 million cis-regulatory elements, yielding 557,441 enhancer-gene pairs and 145,407 variants with allele-specific effects. By training a sequence-to-function deep-learning model, we decoded the base-pair syntax of chromatin accessibility, annotated transcription factor motif instances genome-wide, and constructed 12,210 tissue-specific gene regulatory networks (GRNs). Integrating this resource with multi-tissue expression quantitative trait loci, selection sweeps, and genome-wide association studies prioritized non-coding variants driving domestication and complex traits. Finally, cross-species analysis revealed that sequence-conserved, tissue-matched enhancers were significantly enriched in the heritability of complex traits and diseases in humans. In summary, SheepEpimap (https://genome.ucsc.edu/s/mengzhu/SheepEpimap) provides an open-access foundational ecosystem for sheep functional genomics, precision breeding, and comparative biology.

Skeletal muscle development is crucial for goat meat production. While most research focuses on transcriptional regulation, translational control is often overlooked. This study integrated transcriptomic data to analyze the translational landscape during myogenic differentiation of goat skeletal muscle satellite cells (SMSCs). We found that differentiation pathways were activated at both levels, with enhancement at translation. Furthermore, we identified 25 novel lncORFs and 36 circORFs with coding potential. Among these, LncORF32653 and LncORF98488 encoded micropeptides promoting SMSCs proliferation and differentiation. We also identified circUSP25, encoding circUSP25-177aa, which inhibited proliferation but promoted differentiation. Thus, lncORF32653-53aa, lncORF98488-98aa, and circUSP25-177aa are key regulators of myogenesis, revealing the potential of RNAs annotated as non-coding to encode functional micropeptides.

Objective: This study aimed to analyze the molecular mechanism of heterosis in East Friesian sheep×Hu sheep (EH) hybrid sheep and Suffolk×EH (SHE) hybrid sheep (Ovis aries).Methods: In this research, the growth performance data of Hu sheep (H), EH and SHE from birth to 8 months of age were analyzed. Three 8-month-old sheep of each of the three strains (9 sheep in total) were chosen and their longissimus dorsi muscles were collected for transcriptome sequencing. We verified the expression of seven differentially expressed genes (DEGs) by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR).Results: The results showed: (1) body weight and chest circumference of EH were significantly greater than H (p<0.05), except at 4 months of age. Body weight and chest circumference of SHE was significantly higher than EH (p<0.05), except at 6 months of age. (2) 310 DEGs were screened in the EH and H, gene ontology and Kyoto encyclopedia of genes and genomes showed DEGs were mainly concentrate on the categories of actin cytoskeleton, calcium binding, cGMP-PKG and mitogen-activated protein kinase (MAPK) signaling pathway, which correlating the development of skeletal muscle and energy metabolism. 329 DEGs were screened in the SHE and EH. DEGs were mainly enriched in extracellular matrix-receptor interactions and cell adhesion molecules. (3) Protein–protein interaction screening yielded five (MYL2, TNNI1, TNNI3, MYH11, TNNC1) and three (SOX10, COL2A1, MPZ) pivotal DEGs regulating muscle development in EH and SHE. (4) RT-qPCR test results were consistent with transcriptome sequencing.Conclusion: This study provides candidate genes for improving sheep growth traits. It provides a theoretical basis for analyzing the mechanism of muscle development in crossbred sheep.

In briefSteroid hormone secretion by granulosa cells in the ovary is regulated by lipid synthesis and metabolism. This study found that miR-novel-216 affects granulosa cell proliferation and free fatty acid content by regulating TPD52 expression, thereby increasing the reproductive hormone secretion and promoting polytocous trait in goats.AbstractGranulosa cells in the ovaries of livestock are crucial for secreting steroid hormones that regulate follicular development, with lipid synthesis and metabolism playing key roles in this process. The molecular mechanisms behind steroid hormone secretion regulated by fatty acid metabolism in goat granulosa cells have been unclear. Our previous transcriptome analysis of Yunshang black goat ovaries revealed that miR-novel-216, which had lower expression in high-fertility goats, might regulate granulosa cell function. We further investigated the role of miR-novel-216 by isolating and culturing goat granulosa cells in vitro and found that it inhibits cell proliferation, lipid accumulation and progesterone synthesis in goat granulosa cells. The qTar and miRanda analyses predicted TPD52 as a target of miR-novel-216, confirmed by dual luciferase and transfection assays. Previous studies have shown that progesterone synthesis in granulosa cells is closely related to free fatty acid composition. We investigated the effect of in vitro construction of TPD52 overexpression and interference plasmids on the free fatty acid content of goat granulosa cells using mass spectrometry sequencing. The results showed that the overexpression of TPD52 in goat granulosa cells significantly increased the free fatty acid content and promoted granulosa cell proliferation, lipid accumulation and progesterone synthesis, whereas the opposite was true for inhibition of TPD52. It was shown that miR-novel-216 affects granulosa cell proliferation and free fatty acid levels by regulating the expression of TPD52, which increases reproductive hormone secretion and promotes polytocous trait in goats. This provides a foundation for developing breeding strategies to improve goat fertility.

Selenium (Se) is one of the essential trace elements for ruminants. The impact of varying dietary Se levels on rumen epithelium and microbiota remains inadequately studied. This study used multi-omics techniques to investigate the coordinated response of rumen microbiota and epithelium to dietary glucosamine selenium (GASe) supplementation. Sixty female Wanlin White goats (19.90 ± 2.00 kg) at 4 months of age were randomly divided into 4 groups with 15 replications of 1 goat each. Goats in the control (CON), and the low (L), medium (M) and high does (H) groups were supplemented GASe at 0, 0.6, 1.2 and 2.4 mg Se/kg, respectively. After a 60-d feeding trial, 5 goats per group were randomly selected for slaughter and sampling. The results showed that average daily gain, rumen papilla length, and glutathione peroxidase (GPx) activity in the L group were significantly higher than those in the CON and H groups (quadratic; P < 0.05). Microbial analysis revealed that unclassified_Christensenellaceae (quadratic) had higher (P < 0.05) abundances in the L group and showed significant positive correlations with rumen papilla length and GPx activity (P < 0.05). In contrast, the abundances of pathogenic Campylobacter were highest in the H group (linear or quadratic; P < 0.05). Weighted gene co-expression network analysis (WGCNA) identified the gene module MEturquoise and the compound module ME6 as significantly associated with papilla length and GPx activity (P < 0.05). Genes in the MEturquoise module were enriched in ribosome, oxidative phosphorylation, RNA polymerase, and nucleotide sugar biosynthesis pathways, while the ME6 module was mainly associated with amino acid and nucleotide metabolism. Multi-omics analysis revealed that metabolic responses bridged transcriptional regulation and microbial dynamics. Additionally, 2 microbiota-derived substances, actinorhodin and dimethylpropiothetin, were significantly associated with papilla length and GPx activity (P < 0.05). In conclusion, an appropriate amount of GASe can improve goat growth performance, promote rumen epithelial growth and health by enhancing cellular metabolism and increasing beneficial bacteria abundance. However, excessive GASe supplementation may reduce rumen epithelial antioxidative and metabolic capacities, and promote pathogenic bacteria proliferation. Furthermore, this study demonstrates that metabolites mediate the coordinated response of the rumen microbiota and host to dietary GASe supplementation. Supplementation of 0.598 to 0.832 mg Se/kg GASe in the diet is recommended for goats.

Excessive fat deposition compromises the health of companion animals and the carcass quality of food-producing livestock. Follicle-stimulating hormone (FSH) has been demonstrated to play a critical regulatory role in fat deposition, with its function dependent on binding to its cognate receptor (FSHR) in target organs. In this study, female Sprague-Dawley (SD) rats were immunized with subunit vaccines targeting FSHβ and FSHR, respectively, and obesity was induced by a high-fat diet (HFD) to investigate the effects of these vaccines on adipose deposition in female mammals. The results revealed that active immunization against FSHβ and FSHR effectively suppressed HFD-induced obesity and the elevated serum triglyceride levels. Histological observations found that FSHβ and FSHR immunity decreased adipocyte hypertrophy and increased the cross-sectional area of skeletal muscle fibers caused by HFD, partially ameliorated HFD-associated hepatic sinusoidal spaces and vacuolated steatosis in the cytoplasm. RT-qPCR results indicated that FSHβ and FSHR immunization inhibited lipid synthesis by downregulating adipogenic-related genes, including C/ebpα, Creb, Pparγ, Lpl, and Perilipin. These findings suggest that both vaccines can mitigate HFD-induced adipose deposition in rats, with the FSHR vaccine exhibiting more pronounced effects. This study provides a novel strategy to mitigate pet health deterioration caused by excessive obesity and the decline in carcass quality of food-producing livestock.

OBJECTIVE:Post-pubertal male goats exhibit undesirable behaviors (fighting, mounting) and reduced growth due to high testosterone, while traditional castration causes stress. Immunocastration offers a humane alternative. This study aimed to develop effective immunocastration vaccines. METHODS:The gonadotropin-releasing hormone (GnRH) octamer vaccine (G8), luteinizing hormone receptor (LHR) and GnRH octamer tandem vaccine (LG) were developed and synthesized for this study. Forty 3-month-old male goats were randomly divided into four groups, and subjected to different treatments: surgical castration (SC group), immunization with the G8 vaccine (G8 group), immunization with the LG vaccine (LG group), or left intact (non-castration [NC] group). After the first immunization, serum antibodies and testosterone levels, as well as body weight, body size, and scrotal size, were measured at various time points. Testicular size and slaughter rate were measured at the time of slaughter, 20 weeks after the first immunization. RESULTS:Both vaccines effectively elicited the corresponding antibodies in male goats; the testosterone levels in the G8 and LG groups were significantly reduced compared to the NC group (p<0.01). Four weeks after the first immunization, this trend persisted throughout the experiment; the testicular organ index and size of the G8 group were significantly (p<0.05) smaller than those of NC group at slaughter. In comparison to the NC group, the seminiferous tubule diameter in the G8 and LG groups was significantly reduced (p<0.01), accompanied by a notable decrease in Leydig Cells and various stages of spermatogenic cells. Additionally, the weight gain of goats in the SC group was significantly lower than that of other groups two weeks after the first immunization (p<0.05). CONCLUSION:The two immunocastration vaccines developed in this study effectively inhibit the testicular development and spermatogenesis in male goats, leading to a reduction in testosterone levels.

OBJECTIVE:Goat milk contains a high concentration of caseins which are beneficial to human health. Understanding the regulatory mechanisms of casein synthesis in goats contribute to improving milk quality. Epithelial-specific E74-like factor 5 (ELF5) is important in milk protein synthesis, although the molecular mechanism by which ELF5 regulates casein synthesis remains unclear. This study aims to investigate the regulatory roles of ELF5 on casein production in goat mammary epithelial cells (GMECs). METHODS:Primary GMECs were isolated from goats and characterized using immunofluorescence and oil red O staining. The goat ELF5 gene overexpression vector and small interfering RNA were transfected into GMECs, respectively. Cell viability was accessed using cell counting kit-8 assay, and cell apoptosis was detected by flow cytometry. Genes involved in cell proliferation, apoptosis, and casein synthesis were examined by quantitative realtime polymerase chain reaction and Western blot. After co-treatment of Janus kinase 2 (JAK2) or signal transducer and activator of transcription 5 (STAT5) inhibitors, casein gene expression was examined by using Western blot. The interaction between ELF5 and STAT5 was verified by co-immunoprecipitation assay. RESULTS:ELF5 enhances cell viability and the expression of genes associated with proliferation, while simultaneously inhibiting apoptosis and the expression of apoptosis-related genes. Then, ELF5 upregulates the expression of αS1-, αS2-, β- and κ-casein, in addition to enhancing JAK2/STAT5 signaling pathway. The inhibition assay of JAK2 and STAT5 activity reveals that ELF5 regulates casein synthesis via JAK2/STAT5 signaling pathway. CONCLUSION:ELF5 upregulates casein synthesis through the activation of the JAK2/STAT5 signaling pathway, offering a strategy for manipulating ELF5 to increase casein content and improve the protein quality of goat milk.

Phospholipase A (PLA) in goat semen aggregates with egg yolk in semen diluent, leading to sperm death. The aim of this study is to address the issue of sperm death caused by the interaction between PLA and egg yolk, and to explore the protective effect and metabolic regulation mechanism of the combination of dexamethasone (DXMS) and azithromycin (AZM) on goat sperm under low temperature conditions. At a low temperature of 4 °C, different concentrations of DXMS were added to semen diluents containing 30 μg/mL AZM to detect the quality of goat sperm. The optimal concentration of DXMS was determined to be 20 μg/mL. On the 5th day of storage, antioxidant capacity, total cholesterol (TC) levels, energy metabolism, and metabolomics analysis were performed on the sperm of the 20 μg/mL DXMS group. The results showed that there was no aggregation caused by the interaction between PLA and egg yolk in the group containing 30 μg/mL AZM at 4 °C. 20 μg/mL DXMS significantly improved sperm motility, plasma membrane integrity, acrosome integrity, glutathione peroxidase (GPX) (P < 0.05), catalase (CAT) (P < 0.01), and superoxide dismutase (SOD) activity (P < 0.01). The content of reactive oxygen species (ROS) and Fe2+ significantly decreased (P < 0.01), while the content of ATP (P < 0.01) and TC (P < 0.05) significantly increased. Through metabolomics analysis, a total of 56 differential metabolites (P < 0.05) were screened, including 5a, 6-Anhydrotetracycline, Betamethasone, and 11-Dehydrocorticosterone, mainly enriched in 8 metabolic pathways (P < 0.05), including steroid hormone biosynthesis, glycerophospholipid metabolism, and choline metabolism in cancer. Among them, 5 metabolic pathways are related to lipid metabolism. The results indicate that AZM effectively inhibits the aggregation of PLA and yolk, and the combination of AZM and DXMS enhances the preservation quality of goat sperm during low-temperature preservation by regulating lipid metabolism.

The quality of mutton deteriorates during storage, yet the underlying mechanisms under different conditions remain to be fully elucidated. This study investigated the quality changes in Hu sheep biceps femoris during refrigerated (4 degrees C for 0, 2, 4, 6, and 8 days) and frozen (-20 degrees C for 0, 1, 3, 6, and 9 months) storage by analyzing meat quality, lipid oxidation, protein oxidation, redox parameters, volatile flavor substances and metabolites. Results showed that prolonged storage significantly decreased meat quality (P < 0.05), driven by intense lipid and protein oxidation. On day 8, the levels of malondialdehyde and protein carbonyl groups were 3.15 times and 1.86 times that on day 0 (P < 0.05). After 9 months of freezing, these two substances reached 7.68 times and 2.09 times the levels observed on day 0 (P < 0.05). This oxidative damage coincided with a compromised antioxidant capacity. Hexanal and dimethyl sulfide were identified as key volatile compounds in refrigerated and frozen storage. Differential metabolites were enriched in amino acid metabolism during refrigeration and lipid metabolism during frozen storage, showing significant correlations with key volatiles. These findings enhance the understanding of temperature and time effects on mutton quality during refrigerated and frozen storage.

Previous studies have confirmed that methylation regulates gene transcription in the hypothalamus-pituitary-gonadal axis during puberty initiation, but little is known about the regulation of DNA methylation on gene expression in the pineal gland. To screen pineal gland candidate genes related to the onset of goat puberty and regulated by genome methylation, we collected pineal glands from prepubertal and pubertal female goats, then, determined the DNA methylation profile by whole genome bisulfite sequencing and the transcriptome by RNA sequencing on Illumina HiSeqTM2500. We analyzed differentially expressed genes between the Pre group and Pub group using the DESeq2 software (version 1.20.0), and applied the Benjamini and Hochberg method for adjusting P-values. Genes with a P-value less than 0.05 and an absolute log2 fold change greater than 0 were considered differentially expressed genes. Results showed that there was no significant difference in the whole-genome methylation level of the pineal gland between prepubertal and pubertal goats, but the methylation pattern changed significantly, indicating that genomic DNA methylation of the pineal gland might play a role in regulating the initiation of goat puberty. Changes in DNA methylation patterns affected some pineal gland transcriptomes, while the transcriptional level of most genes remained unaffected by DNA methylation differences. Genes regulated by DNA methylation regulates genes primarily involved in metabolic processes, oxidative phosphorylation, and signaling pathways related to thermogenesis. Methylation significantly regulated the expression of genes such as ATP5F1D, CACNB2, and PTEN, while genes like LIN28B, GIP, OPN1SW, and DCC showed the most notable fold changes, which may indicate their involvement in the onset of puberty.

Skin and hair pigmentation in animals involve intricate regulatory processes. Circular RNA-microRNA (circRNA-miRNA) networks play vital roles in various biological processes, although their involvement in pigmentation has been underexplored. This study focused on circKIF27 expression, which differs significantly in melanocytes isolated from white and brown Boer coat-colored skin, yet its function remains unclear. Here, we investigated the roles of circKIF27 in melanocytes. In situ hybridization assays demonstrated that circKIF27 is expressed in the cytoplasm of melanocytes. qRT-PCR results revealed differential expression levels of circKIF27 in various tissues of male and female goats. Functional analysis showed that circKIF27 overexpression in melanocytes significantly reduces melanin production (P<0.01) and inhibits cell proliferation (P<0.0001). Bioinformatics analysis identified a putative miR-129-5p binding site on circKIF27, and luciferase reporter assays confirmed their interaction. Overexpression of miR-129-5p in melanocytes enhances melanin production (P<0.01) and promotes cell proliferation (P<0.05). Further analysis revealed that TGIF2 possesses two potential miR-129-5p binding sites, and miR-129-5p overexpression in melanocytes significantly inhibits TGIF2 expression (P<0.0001), suggesting a targeted regulatory relationship between these two molecules. Silencing TGIF2 expression via siRNA-TGIF2 transfection leads to increased melanocyte proliferation (P<0.0001) and increased melanin production (P<0.01). These findings highlight the involvement of the circRNA-miRNA network in pigmentation, offering new insights into the molecular mechanisms underlying pigmentation and guiding animal hair color breeding strategies.

Cryopreservation-induced damage remains a major limitation in ram semen preservation, particularly impairing sperm motility and structural integrity. Punicalagin, a natural polyphenol with potent antioxidant properties, has demonstrated potential in mitigating oxidative stress. However, its role in ruminant sperm cryoprotection remains incompletely understood. This study investigated the effects of punicalagin on cryopreserved sheep sperm and explored its underlying molecular mechanisms. A comprehensive evaluation of cryodamage indicators was conducted before and after cryopreservation. The effects of varying punicalagin concentrations (0-20 μM) on sperm motility, antioxidant pathways, and mitochondrial function were assessed. Untargeted metabolomics was employed to characterize changes in the sperm metabolite profile, and the selective Nrf2 inhibitor (ML385) was employed to validate the core signaling pathway. Cryopreservation significantly reduced progressive motility, mitochondrial membrane potential (ΔΨm, MMP), and adenosine triphosphate (ATP) levels while increasing tail bending rate and acrosomal defects (P < 0.05). Supplementation with punicalagin (15 μM) significantly decreased reactive oxygen species (ROS) levels, enhanced superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities, restored ΔΨm, and downregulated apoptotic markers (Bax/Fas) (P < 0.05). Punicalagin activated the Nrf2/PGC-1α pathway, an effect abolished by ML385. Metabolomic analysis revealed punicalagin-regulated lipid metabolism and identified five differential metabolites, including 20-COOH-10,11-dihydro-LTB4, Acylcarnitine 12:2, Acylcarnitine 20:6, Pyridine, and Undecanoic acid, as metabolic biomarkers for cryodamage evaluation (AUC >0.9). These findings demonstrate that punicalagin enhances sperm cryotolerance through dual mechanisms involving antioxidant pathway activation and metabolic reprogramming, providing a novel strategy for improving ruminant sperm cryopreservation.

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) play key roles in regulating testosterone secretion and spermatogenesis in male mammals, respectively, and they maintain the fertility of male animals by binding to their corresponding receptors. We designed and prepared a recombinant LH receptor (LHR) subunit vaccine and a recombinant FSH receptor (FSHR) subunit vaccine and used male Sprague Dawley (SD) rats as a model to examine their effects on testicular development, spermatogenesis, and testosterone secretion in prepubertal and pubertal mammals. Both vaccines (LHR-DTT and FSHR-DTT) significantly decreased the serum testosterone level in prepubertal rats (p < 0.05) but had no effect on the testosterone secretion in pubertal rats; both vaccines decreased the number of cell layers in the seminiferous tubules and reduced spermatogenesis in prepubertal and pubertal rats. Subunit vaccine FSHR-DTT decreased the sperm density in the epididymis in both prepubertal and pubertal rats (p < 0.01) and lowered testicular index and sperm motility in pubertal rats (p < 0.05), whereas LHR-DTT only reduced the sperm density in the epididymis in pubertal rats (p < 0.05). These results indicate that the FSHR subunit vaccine may be a promising approach for immunocastration, but it still needs improvements in effectiveness.