Negative energy balance affects perinatal ewe performance, rumen morphology, rumen flora structure, and placental function.

This study investigated the effects of negative energy balance (NEB) on perinatal ewes, with a focus on changes in growth performance, serum biochemical parameters, rumen fermentation, ruminal bacteria composition, placental phenotype-related indicators, and expression levels of genes related to placental function. Twenty ewes at 130 days of gestation were randomly allocated to either the positive energy balance (PEB) or NEB groups. In the experiment, ewes in the PEB group were fed the same amount as their intake during the pre-feeding baseline period, while ewes in the NEB group were restricted to 70% of their individual baseline feed intake. The experiment was conducted until 42 days postpartum, and five double-lamb ewes per group were selected for slaughter. The results demonstrated that NEB led to a significant decrease in body weight, carcass weight, and the birth and weaning weights of lambs (P < 0.05). Additionally, NEB caused alterations in serum biochemical parameters, such as increased non-esterified fatty acids and ß-hydroxybutyrate levels and decreased cholesterol and albumin levels (P < 0.05). Rumen fermentation and epithelial parameters were also affected, with a reduction in the concentrations of acetic acid, butyric acid, total acid and a decrease in the length of the rumen papilla (P < 0.05). Moreover, NEB induced changes in the structure and composition of ruminal bacteria, with significant differences in α-diversity indices and rumen microbial community composition (P < 0.05). Gene expression in rumen papilla and ewe placenta was also affected, impacting genes associated with glucose and amino acid transport, proliferation, apoptosis, and angiogenesis (P < 0.05). These findings screened the key microbiota in the rumen of ewes following NEB and highlighted the critical genes associated with rumen function. Furthermore, this study revealed the impact of NEB on placental function in ewes, providing a foundation for investigating how nutrition in ewes influences reproductive performance. This research demonstrates how nutrition regulates reproductive performance by considering the combined perspectives of rumen microbiota and placental function.

Circular RNA circUSP13 sponges miR-29c to promote differentiation and inhibit apoptosis of goat myoblasts by targeting IGF1.

Circular RNAs (circRNAs) are an indispensable element of post-transcriptional gene regulation, influencing a variety of biological processes including myogenic differentiation; however, little is known about the function of circRNA in goat myogenic differentiation. Using RNA-sequencing data from our laboratory, we explored the influences of circUSP13, as a candidate circRNA, on myoblast differentiation since its expression is higher in myoblasts of lamb (first day of age) than that of the fetus (75th day of pregnancy). In in vitro experiments, circUSP13 significantly promoted differentiation and inhibited apoptosis in goat primary myoblasts. Mechanistically, circUSP13 localized with miR-29c in the cytoplasm of goat myoblasts to regulate IGF1 expression. We further demonstrated that circUSP13 sponges miR-29c, promoting IGF1 expression that upregulated the expression of MyoG and MyHC. Thus, our results identified circUSP13 as a molecular marker for breeding programs of mutton production, as well as the circUSP13-miR-29c-IGF1 axis as a potential therapeutic target for combating muscle wasting.

FTO regulates myoblast proliferation by controlling CCND1 expression in an m6A-YTHDF2-dependent manner.

N6-Methyladenosine (m6A) modification is the most abundant chemical modification in mRNA, and it participates in various biological processes, such as cell differentiation and proliferation. However, little is known about the function of m6A demethylase fat mass and obesity-associated (FTO) in myoblast proliferation. Here, we demonstrated that knockdown of FTO can significantly inhibit myoblast proliferation and promote apoptosis. RNA sequencing analysis revealed that a lot of downregulated genes in FTO knockdown cells are associated with cell cycle and apoptosis. Furthermore, silencing FTO drastically decreased cyclin D1 (CCND1) expression through YTHDF2-mediated mRNA degradation, thereby delaying the progression of G1 phase, and leading to impaired myoblast proliferation. These findings unraveled that FTO regulates myoblast proliferation by controlling CCND1 expression in an m6A-YTHDF2-dependent manner, which highlights the critical roles of m6A modification in myoblast proliferation.

LncRNA LOC102176306 plays important roles in goat testicular development.

Long ncRNAs regulate a complex array of fundamental biological processes, while its molecular regulatory mechanism in Leydig cells (LCs) remains unclear. In the present study, we established the lncRNA LOC102176306/miR-1197-3p/peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) regulatory network by bioinformatic prediction, and investigated its roles in goat LCs. We found that lncRNA LOC102176306 could efficiently bind to miR-1197-3p and regulate PPARGC1A expression in goat LCs. Downregulation of lncRNA LOC102176306 significantly supressed testosterone (T) synthesis and ATP production, decreased the activities of antioxidant enzymes and mitochondrial complex I and complex III, caused the loss of mitochondrial membrane potential, and inhibited the proliferation of goat LCs by decreasing PPARGC1A expression, while these effects could be restored by miR-1197-3p inhibitor treatment. In addition, miR-1197-3p mimics treatment significantly alleviated the positive effects of lncRNA LOC102176306 overexpression on T and ATP production, antioxidant capacity and proliferation of goat LCs. Taken together, lncRNA LOC102176306 functioned as a sponge for miR-1197-3p to maintain PPARGC1A expression, thereby affecting the steroidogenesis, cell proliferation and oxidative stress of goat LCs. These findings extend our understanding of the molecular mechanisms of T synthesis, cell proliferation and oxidative stress of LCs.

Analysis of DNA methylation profiles during sheep skeletal muscle development using whole-genome bisulfite sequencing.

BACKGROUND: DNA methylation is an epigenetic regulatory form that plays an important role in regulating the gene expression and the tissues development.. However, DNA methylation regulators involved in sheep muscle development remain unclear. To explore the functional importance of genome-scale DNA methylation during sheep muscle growth, this study systematically investigated the genome-wide DNA methylation profiles at key stages of Hu sheep developmental (fetus and adult) using deep whole-genome bisulfite sequencing (WGBS). RESULTS: Our study found that the expression levels of DNA methyltransferase (DNMT)-related genes were lower in fetal muscle than in the muscle of adults. The methylation levels in the CG context were higher than those in the CHG and CHH contexts, and methylation levels were highest in introns, followed by exons and downstream regions. Subsequently, we identified 48,491, 17, and 135 differentially methylated regions (DMRs) in the CG, CHG, and CHH sequence contexts and 11,522 differentially methylated genes (DMGs). The results of bisulfite sequencing PCR (BSP) correlated well with the WGBS-Seq data. Moreover, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation analysis revealed that some DMGs were involved in regulating skeletal muscle development and fatty acid metabolism. By combining the WGBS-Seq and previous RNA-Seq data, a total of 159 overlap genes were obtained between differentially expressed genes (DEGs) and DMGs (FPKM > 10 and fold change > 4). Finally, we found that 9 DMGs were likely to be involved in muscle growth and metabolism of Hu sheep. CONCLUSIONS: We systemically studied the global DNA methylation patterns of fetal and adult muscle development in Hu sheep, which provided new insights into a better understanding of the epigenetic regulation of sheep muscle development.

Long non-coding RNA LOC105611671 modulates fibroblast growth factor 9 (FGF9) expression by targeting oar-miR-26a to promote testosterone biosynthesis in Hu sheep.

Fibroblast growth factors (FGFs) play crucial roles in early gonadal development and germ cell maturation of mammals; FGF9 is involved in mammalian testis steroidogenesis. However, the upstream regulators of FGF9 in ovine testosterone biosynthesis remain unknown. Long non-coding RNAs (lncRNAs) are crucial regulators of multiple biological functions that act by altering gene expression. In the present study, we analysed the role of LOC105611671, a lncRNA upstream of FGF9, in Hu sheep steroidogenesis. We found that LOC105611671 expression increased significantly in Hu sheep testes during sexual maturation (P<0.05). Moreover, levels of FGF9 and testosterone were decreased by LOC105611671 knockdown in Hu sheep Leydig cells (LCs). Results of transient transfection and luciferase assays revealed that FGF9 is a functional target gene of oar-miR-26a in ovine LCs. Further functional validation experiments revealed that LOC105611671 regulates testosterone biosynthesis by targeting oar-miR-26a. Overall, the present study describes the expression profile of LOC105611671 during sexual maturation and demonstrates that LOC105611671 modulates FGF9 expression by targeting oar-miR-26a to promote testis steroidogenesis in Hu sheep. Our research provides a new theoretical basis for genetic and molecular research on testosterone biosynthesis in sheep.

Spirulina supplementation improves lipid metabolism and autophagic activities in the liver and muscle of Hu lambs fed a high-energy diet.

The current study aimed to examine the effects of dietary spirulina supplementation in high-energy (HE) diets on fatty acid metabolism in sheep, and preliminarily explored the potential mechanisms underlying the associated autophagy-mediated regulation of lipid metabolism. In a 2 × 3 factorial design, including six treatment combinations of two metabolisable energy diets (10 and 11 MJ/kg DM), three spirulina supplementation levels (0, 1%, and 3%) were used. Serum alanineaminotransferase (ALT) (p = 0.003) and aspartatetransaminase (AST) (p = 0.002) activities increased, whereas total PUFA content (p < 0.001) decreased in the liver of lambs fed a HE diet. With the addition of spirulina, serum ALT (p = 0.037) and AST (p = 0.014) activities decreased, whereas EPA (p = 0.004), GLA (p = 0.019), n-6 PUFA (p = 0.005), and total PUFA contents (p = 0.019) increased. Moreover, the crude protein content in the Longissimus thoracis et lumborum (LTL) increased (p = 0.013), the expression of PPARα and PPARγ was up-regulated, while ELOVL2 was down-regulated in liver and LTL (p < 0.05). Spirulina supplementation increased mRNA expression levels of autophagy-associated genes, including that of Beclin-1, AMPK, and ULK1 (p < 0.05). In conclusion, spirulina supplementation in a HE diet exerted a protective effect on the liver, increased PUFA content, and modulated expression levels of autophagy-related genes in growing lambs.

The emerging role of N6-methyladenine RNA methylation in metal ion metabolism and metal-induced carcinogenesis.

N6-methyladenine (m6A) is the most common and abundant internal modification in eukaryotic mRNAs, which can regulate gene expression and perform important biological tasks. Metal ions participate in nucleotide biosynthesis and repair, signal transduction, energy generation, immune defense, and other important metabolic processes. However, long-term environmental and occupational exposure to metals through food, air, soil, water, and industry can result in toxicity, serious health problems, and cancer. Recent evidence indicates dynamic and reversible m6A modification modulates various metal ion metabolism, such as iron absorption, calcium uptake and transport. In turn, environmental heavy metal can alter m6A modification by directly affecting catalytic activity and expression level of methyltransferases and demethylases, or through reactive oxygen species, eventually disrupting normal biological function and leading to diseases. Therefore, m6A RNA methylation may play a bridging role in heavy metal pollution-induced carcinogenesis. This review discusses interaction among heavy metal, m6A, and metal ions metabolism, and their regulatory mechanism, focuses on the role of m6A methylation and heavy metal pollution in cancer. Finally, the role of nutritional therapy that targeting m6A methylation to prevent metal ion metabolism disorder-induced cancer is summarized.

Dietary spirulina supplementation modifies rumen development, fermentation and bacteria composition in Hu sheep when consuming high-fat dietary.

Introduction: This study aims to investigate the long-term effects of spirulina supplementation in a high-fat diet (HFD) on rumen morphology, rumen fermentation, and the composition of rumen microbiota in lambs. Spirulina is a blue-green microalgae that has been shown to have high nutritional value for livestock. Methods: Fifty-four lambs were randomly divided into three groups: a normal chow diet (NCD) group, a high-fat diet (HFD) group, and a high-fat diet supplemented with 3% spirulina (HFD+S) group. Rumen morphology, rumen fermentation, and rumen microbiota were analyzed at the end of the study. Results: Spirulina supplementation improved the concentration of volatile fatty acids and rumen papilla length. Additionally, there was a tendency for an increase in rumen weight and an upregulation of the genes Claudin-1, Claudin-4, and Occludin in the HFD+S group. Pyrosequencing of the 16S ribosomal RNA gene also showed that spirulina supplementation significantly changed the rumen microbiota composition in the HFD group, with a decrease in richness and diversity. Specifically, the relative abundance of Prevotella 9 and Megasphaera was significantly increased in the HFD group compared to the NCD group, while spirulina supplementation reversed these changes. Discussion: This study suggests that 3% spirulina supplementation can improve rumen development and fermentation, and effectively relieve rumen microbe disorders in lambs caused by a high-fat diet. However, further research is needed to confirm the findings and to examine the long-term effects of spirulina supplementation in different types of livestock and under different dietary conditions.

l-Argine regulates the proliferation, apoptosis and endocrine activity by alleviating oxidative stress in sheep endometrial epithelial cells.

l-arginine (L-Arg) is a semiessential amino acid that plays crucial roles in the reproductive performance of animals. This research aimed to investigate the effect of supplementing L-Arg on endometrial epithelial cells (EECs) of Hu sheep. In vivo, female Hu sheep were randomly divided into three groups: control group (n = 5), nutrient-restricted group (n = 5), and L-Arg supplemented nutrient-restricted group (n = 5). Then, the effect of L-Arg on ovine endometrial growth and antioxidant capacity was assessed. We found that L-Arg supplementation promoted the growth of endometrial ductal gland invaginations (DGI), and alleviated oxidative stress in nutrient-restricted sheep. In order to investigate its mechanism, a H2O2-induced EECs oxidative stress model was established, and roles of L-Arg in EECs oxidation resistance, proliferation, apoptosis and endocrine activity were studied in vitro. Our results showed that L-Arg markedly decreased the release of reactive oxygen species (ROS) and malonaldehyde (MDA), and enhanced the expression and activity of certain antioxidant enzymes in EECs challenged by the H2O2 (p < 0.05). Supplementation of L-Arg significantly reduced the effect of 200 µM H2O2 on the viability of EECs (p < 0.05). In addition, EECs treated with L-Arg significantly alleviated the G0/G1-phase cell cycle arrest, apoptosis, and the inhibition of endometrial growth factors expression caused by H2O2 (p < 0.05). Overall, the results demonstrate that L-Arg performs crucial roles in maintaining the proliferation of ovine EECs, endocrine activity and inhibiting apoptosis through reducing oxidative stress. This study offers a theoretical basis for using L-Arg to improve sheep the uterine function.

M6A RNA Methylation Mediates NOD1/NF-kB Signaling Activation in the Liver of Piglets Challenged with Lipopolysaccharide.

N6-methyladenosine (m6A) is the most abundant internal modification that widely participates in various immune and inflammatory responses; however, its regulatory mechanisms in the inflammation of liver induced by lipopolysaccharide in piglets remain largely unknown. In the present study, piglets were intraperitoneally injected with 80 µg/kg LPS or an equal dose of sterile saline. Results indicated that LPS administration increased activities of serum alanine aminotransferase (ALT), induced M1 macrophage polarization and promoted secretion of inflammatory cytokines, and finally led to hepatic lesions in piglets. The NOD1/NF-κB signaling pathway was activated in the livers of the LPS group. Moreover, the total m6A level was significantly elevated after LPS treatment. MeRIP-seq showed that 1166 and 1344 transcripts contained m6A methylation in control and LPS groups, respectively. The m6A methylation sites of these transcripts mainly distributes in the 5' untranslated region (5'UTR), the coding sequence (CDS), and the 3' untranslated region (3'UTR). Interestingly, these genes were mostly enriched in the NF-κB signaling pathway, and LPS treatment significantly changed the m6A modification in NOD1, RIPK2, NFKBIA, NFKBIB, and TNFAIP3 mRNAs. In addition, knockdown of METTL3 or overexpression of FTO both changed gene levels in the NOD1/NF-κB pathway, suggesting that activation of this pathway was regulated by m6A RNA methylation. Moreover, the alteration of m6A RNA methylation profile may be associated with the increase of reactive oxygen species (ROS), HIF-1α, and MAT2A. In conclusion, LPS activated the NOD1/NF-κB pathway at post-transcriptional regulation through changing m6A RNA methylation, and then promoted the overproduction of proinflammatory cytokines, ultimately resulting in liver inflammation and damage.

γ-Linolenic Acid Prevents Lipid Metabolism Disorder in Palmitic Acid-Treated Alpha Mouse Liver-12 Cells by Balancing Autophagy and Apoptosis via the LKB1-AMPK-mTOR Pathway.

Excessive fat deposition is the main character in nonalcoholic fatty liver disease (NAFLD), while γ-linolenic acid (GLA) is a polyunsaturated fatty acid that can reduce lipid deposition. This study investigated the effect and regulatory mechanism of GLA (100 µM) on lipid metabolism in alpha mouse liver 12 (AML-12) cells treated by 400 µM palmitic acid (PA). GLA reduced lipid content and increased fatty acid ß oxidation, as indicated by decreasing triglyceride and cholesterol contents and increasing mRNA and protein expressions of CPT1α and PPARα. GLA relieved oxidative stress caused by PA, upregulated mRNA levels of superoxide dismutase and glutathione peroxidase, and decreased reactive oxygen species content. GLA reduced apoptosis, as indicated by decreases in the BAX/BCL2 expression level and apoptosis percentage. GLA activated autophagy, autophagosome-lysosome fusion, and LKB1-AMPK-mTOR signaling and upregulated mRNA and protein expressions of Beclin-1, autophagy-related 5, and liver kinase B1 (LKB1). These effects of GLA on lipid metabolism disorders of PA-treated hepatocytes were reversed by autophagy inhibitor 3MA and AMPK inhibitor compound C, confirming our conclusions. Overall, GLA can protect AML-12 cells from lipid metabolism disorder caused by PA via balancing autophagy and apoptosis mediated by the LKB1-AMPK-mTOR pathway. Consequently, GLA, as a dietary supplement, can help to prevent and treat NAFLD by regulating lipid metabolism and autophagy.

Roles of WNT6 in Sheep Endometrial Epithelial Cell Cycle Progression and Uterine Glands Organogenesis.

The uterus, as part of the female reproductive tract, is essential for embryo survival and in the maintenance of multiple pregnancies in domestic animals. This study was conducted to investigate the effects of WNT6 on Hu sheep endometrial epithelial cells (EECs) and uterine glands (UGs) in Hu sheep, with high prolificacy rates. In the present study, Hu sheep with different fecundity, over three consecutive pregnancies, were divided into two groups: high prolificacy rate group (HP, litter size = 3) and low prolificacy rate group (LP, litter size = 1). A comparative analysis of the endometrial morphology was performed by immunofluorescence. RNA-seq was used to analyze the gene's expression in endometrium of HP and LP Hu sheep, providing a candidate gene, which was investigated in EECs and organoid culture. Firstly, higher density of UGs was found in the HP Hu sheep groups (p < 0.05). The RNA-seq data revealed the importance of the WNT signaling pathway and WNT6 gene in Hu sheep endometrium. Functionally, WNT6 could promote the cell cycle progression of EECs via WNT/ß-catenin signal and enhance UGs organogenesis. Taken together, WNT6 is a crucial regulator for sheep endometrial development; this finding may offer a new insight into understanding the regulatory mechanism of sheep prolificacy.

INHBA transfection regulates proliferation, apoptosis and hormone synthesis in sheep granulosa cells.

Inhibin subunit beta A (INHBA) participates in the synthesis of inhibin A, activin A and activin AB. Here we investigated the effect and molecular mechanism of INHBA on proliferation, apoptosis and hormone synthesis in sheep granulosa cells (GCs) using in vitro transfection. We first noticed that INHBA expression increased with follicle diameter and was widely distributed in ovarian tissue. The proliferation rate of GCs was significantly increased and decreased with overexpression and silence of INHBA, respectively, compared with the negative controls. INHBA transfection affected GC proliferation and apoptosis, regulating the expression of many cell cycle-related and apoptosis-related genes. INHBA overexpression significantly decreased activin and estradiol secretion while increasing inhibin and progesterone secretion. The expression of follicle-stimulating hormone beta subunit was significantly decreased and increased with INHBA overexpression and knockdown, respectively. Notably, silence of INHBA inhibited the expression of many transforming growth factor beta-related genes. Overall, the functional molecule of INHBA gene may be associated with follicular development via regulating proliferation, apoptosis and folliculogenesis-related hormone secretion of sheep GCs. In addition, our findings may contribute to a better understanding of the law of follicular development and thus improve the reproductive performance of female animals.

FTO-mediated demethylation of GADD45B promotes myogenesis through the activation of p38 MAPK pathway.

N6-methyladenosine (m6A) modification plays a critical role in mammalian development. However, the role of m6A in the skeletal muscle development remains largely unknown. Here, we report a global m6A modification pattern of goat skeletal muscle at two key development stages and identified that the m6A modification regulated the expression of the growth arrest and DNA damage-inducible 45B (GADD45B) gene, which is involved in myogenic differentiation. We showed that GADD45B expression increased during myoblast differentiation, whereas the downregulation of GADD45B inhibits myogenic differentiation and mitochondrial biogenesis. Moreover, the expression of GADD45B regulates the expression of myogenic regulatory factors and peroxisome proliferator-activated receptor gamma coactivator 1 alpha by activating the p38 mitogen-activated protein kinase (MAPK) pathway. Conversely, the inactivation of p38 MAPK abolished the GADD45B-mediated myogenic differentiation. Furthermore, we found that the knockdown of fat mass and obesity-associated protein (FTO) increases GADD45B m6A modification and decreases the stability of GADD45B mRNA, which impairs myogenic differentiation. Our results indicate that the FTO-mediated m6A modification in GADD45B mRNA drives skeletal muscle differentiation by activating the p38 MAPK pathway, which provides a molecular mechanism for the regulation of myogenesis via RNA methylation.

Estradiol-17ß regulates proliferation and apoptosis of sheep endometrial epithelial cells by regulating the relative abundance of YAP1.

Yes-associated protein 1 (YAP1) transcription regulator of the Hippo protein kinase pathway, serves as a key regulator of tissue growth and organ size by regulating cell proliferation and apoptosis. Effects of YAP1 on proliferation and apoptosis of sheep endometrial epithelial cells (EEC) as a result of estradiol-17ß (E2) treatment, however, remain unclear. In the present study, the abundance of YAP1 protein in the uterine horn was greater than that in the uterine body or cervix. The YAP1 protein was primarily localized in the endometrial luminal and glandular epithelial cells of the uterine horn of ewes on day 2 of the estrous cycle. Compared with control samples, there was a lesser abundance of YAP1 mRNA transcript that was associated with a lesser proliferation and greater apoptosis of EEC. There were also lesser concentrations of epidermal growth factor and insulin-like growth factor 1 in the spent culture medium when there was a lesser abundance of YAP1 mRNA in EEC compared with those in the control group. When there was a greater abundance of YAP1 mRNA transcript, there were greater concentrations of epidermal growth factor and insulin-like growth factor 1 in the spent media. Furthermore, with estradiol-17ß treatment the abundance of YAP1 mRNA transcript was similar to that of the control samples. Taken together, estradiol-17ß may function as an essential regulator of EEC proliferation and apoptosis by modulation of concentrations of YAP1 protein in the sheep uterus. These results indicate there are molecular mechanisms of estradiol-17ß and YAP1 in EEC proliferation and apoptosis of ewes.

Effects of spirulina supplementation on lipid metabolism disorder, oxidative stress caused by high-energy dietary in Hu sheep.

The aim of this study was to investigate the effect of spirulina supplementation in a high-energy (HE) diet on lipid metabolism, oxidative status and immunity in Hu lambs. The lambs were assigned to two groups receiving either a standard diet (ST) or a HE diet. Each group was divided into three subgroups: no spirulina supplementation (control), 1% spirulina supplementation, or 3% spirulina supplementation. The body fat, serum cholesterol, triacylglycerol and oxidative stress increased in lambs fed the HE diet. However, 3% spirulina supplementation in the HE diet reduced above parameters and enhanced antioxidant capacity, including increased SOD activity and T-AOC content in serum and Longissimus thoracis et lumborum (LTL). Additionally, lambs receiving 3% spirulina supplementation showed an improvement in immunity-related parameters, including increased IgG concentration in serum and red and white blood cell counts. In conclusion, 3% spirulina supplementation in HE diet ameliorated lipid metabolic disorder and oxidative stress caused by a HE diet.