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ST6GAL2, a member of the sialoglycosyltransferase family, primarily localizes within the cellular Golgi apparatus. However, the role of the ST6GAL2 gene in skeletal muscle growth and development remains elusive. In this study, the impact of the ST6GAL2 gene on the proliferation, differentiation, and apoptosis of primary chicken myoblasts at the cellular level was investigated. Quantitative fluorescent PCR was used to measure the expression levels of genes. Subsequently, using gene knockout mice, we assessed its effects on skeletal muscle growth and development in vivo. Our findings reveal that the ST6GAL2 gene promotes the expression of cell cycle and proliferation-related genes, including CCNB2 and PCNA, and apoptosis-related genes, such as Fas and Caspase-9. At the individual level, double knockout of ST6GAL2 inhibited the formation of both fast and slow muscle fibers in the quadriceps, extensor digitorum longus, and tibial anterior muscle, while promoting their formation in the gastrocnemius and soleus. These results collectively demonstrate that the ST6GAL2 gene facilitates the proliferation, apoptosis, and fusion processes of primary chicken myoblasts. Additionally, it promotes the enlargement of cross-sectional muscle fiber areas and regulates the formation of fast and slow muscle fibers at the individual level, albeit inhibiting muscle fusion. This study provides valuable insights into the role of the ST6GAL2 gene in promoting proliferation of skeletal muscle.

Abstract Haplotype‐resolved 3D chromatin architecture related to allelic differences in avian skeletal muscle development has not been addressed so far, although chicken husbandry for meat consumption has been prevalent feature of cultures on every continent for more than thousands of years. Here, high‐resolution Hi‐C diploid maps (1.2‐kb maximum resolution) are generated for skeletal muscle tissues in chicken across three developmental stages (embryonic day 15 to day 30 post‐hatching). The sequence features governing spatial arrangement of chromosomes and characterize homolog pairing in the nucleus, are identified. Multi‐scale characterization of chromatin reorganization between stages from myogenesis in the fetus to myofiber hypertrophy after hatching show concordant changes in transcriptional regulation by relevant signaling pathways. Further interrogation of parent‐of‐origin‐specific chromatin conformation supported that genomic imprinting is absent in birds. This study also reveals promoter‐enhancer interaction (PEI) differences between broiler and layer haplotypes in skeletal muscle development‐related genes are related to genetic variation between breeds, however, only a minority of breed‐specific variations likely contribute to phenotypic divergence in skeletal muscle potentially via allelic PEI rewiring. Beyond defining the haplotype‐specific 3D chromatin architecture in chicken, this study provides a rich resource for investigating allelic regulatory divergence among chicken breeds.

Abstract The vaginal microbiome plays an essential role in the reproductive health of human females. As infertility increases worldwide, understanding the roles that the vaginal microbiome may have in infertility and in vitro fertilization (IVF) treatment outcomes is critical. To determine the vaginal microbiome composition of 1411 individuals (1255 undergoing embryo transplantation) and their associations with reproductive outcomes, clinical and biochemical features are measured, and vaginal samples are 16S rRNA sequenced. Our results suggest that both too high and too low abundance of Lactobacillus is not beneficial for pregnancy; a moderate abundance is more beneficial. A moderate abundance of Lactobacillus crispatus and Lactobacillus iners (~80%) (with a pregnancy rate of I‐B: 54.35% and III‐B: 57.73%) is found beneficial for pregnancy outcomes compared with a higher abundance (>90%) of Lactobacillus (I‐A: 44.81% and III‐A: 51.06%, respectively). The community state type (CST) IV‐B (contains a high to moderate relative abundance of Gardnerella vaginalis) shows a similar pregnant ratio (48.09%) with I‐A and III‐A, and the pregnant women in this CST have a higher abundance of Lactobacillus species. Metagenome analysis of 71 samples shows that nonpregnant women are detected with more antibiotic‐resistance genes, and Proteobacteria and Firmicutes are the main hosts. The inherent differences within and between women in different infertility groups suggest that vaginal microbes might be used to detect infertility and potentially improve IVF outcomes.

Abstract MicroRNAs (miRNAs) play a crucial role as transcription regulators in various aspects of follicular development, including steroidogenesis, ovulation, apoptosis, and gene regulation in poultry. However, there is a paucity of studies examining the specific impact of miRNAs on ovarian granulosa cells (GCs) across multiple grades in laying hens. Consequently, this study aims to investigate the roles of miRNAs in chicken GCs. By constructing miRNA expression profiles of GCs at 10 different time points, encompassing 4 pre-hierarchical, 5 preovulatory, and 1 postovulatory follicles stage, we identified highly expressed miRNAs involved in GC differentiation (miR-148a-3p, miR-143-3p), apoptosis (let7 family, miR-363-3p, miR-30c-5p, etc.), and autophagy (miR-128-3p, miR-21-5p). Furthermore, we discovered 48 developmentally dynamic miRNAs (DDMs) that target 295 dynamic differentially expressed genes (DDGs) associated with follicular development and selection (such as oocyte meiosis, progesterone-mediated oocyte maturation, Wnt signaling pathway, TGF-β signaling pathway) as well as follicular regression (including autophagy and cellular senescence). These findings contribute to a more comprehensive understanding of the intricate mechanisms underlying follicle recruitment, selection, and degeneration, aiming to enhance poultry’s reproductive capacity.

Forest musk deer is the most important animal for natural musk production, and the musk composition changes periodically during musk secretion, accompanied by variation in the com-position of deer-symbiotic bacteria. GC-MS and 16S rRNA sequencing were conducted in this study, the dynamic changes to correlated chemical composition and the microbiota across musk secretion periods (prime musk secretion period, vigorous musk secretion period and late musk secretion period) were investigated by integrating its serum testosterone level in different mating states. Results showed that the testosterone level, musk composition and microbiota changed with annual cycle of musk secretion and affected by its mating state. Muscone and the testosterone level peaked at vigorous musk secretion period, and the microbiota of this stage was distinct from the other 2 periods. Actinobacteria, Firmicutes and Proteobacteria were dominant bacteria across musk secretion period. PICRUSt analysis demonstrated that bacteria were ubiquitous in musk pod and involved in the metabolism of antibiotics and terpenoids in musk. “Carbohydrates and amino acids,” “fatty acids and CoA” and “secretion of metabolites” were enriched at 3 periods, respectively. Pseudomonas, Corynebacterium, Clostridium, Sulfuricurvum were potential biomarkers across musk secretion. This study provides a more comprehensive understanding of genetic mechanism during musk secretion, emphasizing the importance of Actinobacteria and Corynebacterium in the synthesis of muscone and etiocholanone during musk secretion, which required further validation.

Aging is a gradual process of natural change that occurs after reaching sexual maturity. It is also a known risk factor for many chronic diseases. Recent research has shown that senolytics can extend the lifespans and health spans of model organisms, and they have also been demonstrated effective in treating age-related diseases. In this study, we conducted a high-throughput screening of 156 drugs that targeted the PI3K/AKT/mTOR pathway to identify potential senolytic medications. Among these drugs, PF-04691502 was selected for further investigation to understand its molecular mechanism of action. Our findings indicate that PF-04691502, a dual inhibitor of PI3K/AKT and mTOR, specifically eliminates senescent cells. It reduces the expression levels of key markers of cellular senescence, such as SA-β-Gal, senescence-associated secretory phenotypes (SASPs) and p16INK4a. Additionally, PF-04691502 inhibits the phosphorylation of S6K and AKT, leading to the apoptosis of senescent cells. These results suggest that PF-04691502 holds promise as a new senolytic drug. This paper provides important insights into the potential application of PF-04691502 in the study of cell senescence.

Abstract The storage of lipids as energy in adipose tissue (AT) has been conserved over the course of evolution. However, substantial differences in ATs physiological activities were reported among species. Hence, establishing the mechanisms shaping evolutionarily divergence in ATs transcriptomes could provide a deeper understanding of AT regulation and its roles in obesity-related diseases. While previous studies performed anatomical, physiological and morphological comparisons between ATs across different species, little is currently understood at the molecular phenotypic levels. Here, we characterized transcriptional and lipidomic profiles of available subcutaneous and visceral ATs samples across 15 vertebrate species, spanning more than 300 million years of evolution, including placental mammals, birds and reptiles. We provide detailed descriptions of the datasets produced in this study and report gene expression and lipid profiles across samples. We demonstrate these data are robust and reveal the AT transcriptome and lipidome vary greater among species than within the same species. These datasets may serve as a resource for future studies on the functional differences among ATs in vertebrate species.

Abstract Using an adult female miniature pig model with diet-induced weight gain/weight loss, we investigated the regulatory mechanisms of three-dimensional (3D) genome architecture in adipose tissues (ATs) associated with obesity. We generated 249 high-resolution in situ Hi-C chromatin contact maps of subcutaneous AT and three visceral ATs, analyzing transcriptomic and chromatin architectural changes under different nutritional treatments. We find that chromatin architecture remodeling underpins transcriptomic divergence in ATs, potentially linked to metabolic risks in obesity development. Analysis of chromatin architecture among subcutaneous ATs of different mammals suggests the presence of transcriptional regulatory divergence that could explain phenotypic, physiological, and functional differences in ATs. Regulatory element conservation analysis in pigs and humans reveals similarities in the regulatory circuitry of genes responsible for the obesity phenotype and identified non-conserved elements in species-specific gene sets that underpin AT specialization. This work provides a data-rich tool for discovering obesity-related regulatory elements in humans and pigs.

Abstract There have been many reports on the genetic mechanism in rhesus macaques (RMs) for environmental adaptation to high altitudes, but the synergistic involvement of gut microbiota in this adaptation remains unclear. Here we performed fecal metagenomic and metabolomic studies on samples from high- and low-altitude populations to assess the synergistic role of gut microbiota in the adaptation of RMs to high-altitude environments. Microbiota taxonomic annotation yielded 7471 microbiota species. There were 37 bacterial species whose abundance was significantly enriched in the high-altitude populations, 16 of which were previously reported to be related to the host’s dietary digestion and energy metabolism. Further functional gene enrichment found a stronger potential for gut microbiota to synthesize energy substrate acetyl-CoA using CO2 and energy substrate pyruvate using oxaloacetate, as well as a stronger potential to transform acetyl-CoA to energy substrate acetate in high-altitude populations. Interestingly, there were no apparent differences between low-altitude and high-altitude populations in terms of genes enriched in the main pathways by which the microbiota consumed the three energy substrates, and none of the three energy substrates were detected in the fecal metabolites. These results strongly suggest that gut microbiota plays an important energy compensatory role that helps RMs to adapt to high-altitude environments. Further functional enrichment after metabolite source analysis indicated the abundance of metabolites related to the degradation of exogenous toxins was also significantly higher in high-altitude populations, which suggested a contributory role of gut microbiota to the degradation of exogenous toxins in wild RMs adapted to high-altitude environments.

Measurement(s) Gene expression profiles of specific chicken skeletal muscles • cross section area of chicken skeletal muscles and type I muscle fiber quantity Technology Type(s) RNA sequence • Histological Procedure Factor Type(s) different skeletal muscles Sample Characteristic - Organism Gallus gallus Sample Characteristic - Environment farm

Enterocytozoon bieneusi is a zoonotic pathogen with a wide range of animal host. There are only few reports of E. bieneusi infection in wild Chinese rhesus macaques. Here, we determined the prevalence of E. bieneusi in nine wild rhesus macaque populations and assessed their zoonotic potential by performed genotype of ITS gene. A total of 324 fecal samples of rhesus macaque were collected in nine geographical populations from five Chinese provinces (Sichuan, Chongqing, Qinghai, Tibet and Hainan). 38 samples (11.7%) were found to be infected with E. bieneusi, and 11 genotypes were identified including three known genotypes (D, EbpC and SCC-2) and eight novel genotypes named Mul6∼13. Genotype D (63.2%) was the most prevalent, being observed in seven populations except XZ-2 and QH, and other genotypes were identified only in a single area. According to the phylogenetic analysis, Mul6∼9, Mul11∼13 and zoonotic genotype D were clustered into Group 1, indicating that these genotypes may be potentially zoonotic. Among nine populations, population SC-3 had the highest infection rate (26.3%), and the lowest was the wild QH population without infection, but the difference of infection rate among the nine populations is not significant. It is concluded that, rhesus macaque populations are generally infected E. bieneusi in many areas of China, and there may be a risk of cross infection with E. bieneusi in some areas found having zoonotic genotypes, and these areas should be paid more attention to prevent.

ABSTRACT: DNA N(6)-methyladenine (DNA-6mA) is a new epigenetic mark in eukaryotes, the distribution and functions of which in genomic DNA remain unknown. Although recent studies have suggested that 6mA is present in multiple model organisms and is dynamically regulated during development, the genomic features of 6mA in avian species have yet to be elucidated. 6mA immunoprecipitation sequencing approach was used to analysis the distribution and function of 6mA in the muscle genomic DNA during embryonic chicken development. 6mA immunoprecipitation sequencing was combined with transcriptomic sequencing to reveal the role of 6mA in the regulation of gene expression and to explore possible pathways by which 6mA is involved in muscle development. We here provide evidence that 6mA modification exists widely throughout the chicken genome, and show preliminary data regarding genome-wide distribution of this epigenetic mark. Gene expression was shown to be inhibited by 6mA modification in promoter regions. In addition, the promoters of some genes related to development were modified by 6mA, indicating that 6mA may be involved in embryonic chicken development. Furthermore, 6mA may participate in muscle development and immune function by regulating HSPB8 and OASL expression. Our study improves our understanding of the distribution and function of 6mA modification in higher organisms and provide new information about differences between mammals and other vertebrates. These findings demonstrate an epigenetic role for 6mA in gene expression and potential involvement in chicken muscle development. Furthermore, the results suggest a potential epigenetic role for 6mA in avian embryonic development.

The gut microbiota plays a critical role in the growth, development, nutritional digestion, and overall health of chickens. Furthermore, certain probiotics isolated from poultry intestines have demonstrated the potential to enhance immune function and production performance in chickens. To investigate the differences in gut microbiota among chickens from various geographical environments and different breeds of broiler and laying hens, we conducted 16S rRNA sequencing on the fecal microbiota of 140 Chinese native chickens and ten Roman layers. In addition, we isolated and screened the potential probiotics to examine their biological characteristics, genome profiles, and functionality in animals. Our findings revealed the significant variations in gut microbiota composition and structure between Tibetan chickens (ZJ), which reside in high-altitude regions, and Meihua chickens (MH) and Xuhai chickens (XH), which inhabit low-altitude regions. Specifically, Cupriavidus and Candidatus_Bacilloplasma were identified as unique microbial communities in high and low altitudes, respectively. Notably, among regions with similar altitudes, Luning chickens (LN) exhibited the lowest α diversity, accompanied by a remarkably high relative abundance of Firmicutes and Lactobacillus. Conversely, Wugu chickens (WGs) and Yaoshan chickens (YSs) displayed similar gut microbiota profiles. Furthermore, distinctive gut microbiota patterns were observed between the different breeds of broilers and laying hens. Commercial Roman layers (LMs) exhibited significantly lower alpha diversity compared to native chickens, and broilers and laying hens predominantly harbored Firmicutes, Bacteroidota, and Proteobacteria. Of particular interest, the probiotics Lactobacillus agilis MH1 and Lactobacillus salivarius ZJ1, derived from chicken feces, exhibited favorable genomic profiles, and demonstrated anti-colitis effects and immunomodulatory functions. These findings provide a crucial theoretical foundation for native chicken research and offer insights for the future development and formulation of chicken-derived probiotics.

Abstract Liver development is a complex process that is regulated by a series of signaling pathways. Three-dimensional (3D) chromatin architecture plays an important role in transcriptional regulation; nonetheless, its dynamics and role in the rapid transition of core liver functions during development and obesity-induced metabolic stress remain largely unexplored. To investigate the dynamic chromatin architecture during liver development and under metabolic stress, we generated high-resolution maps of chromatin architecture for porcine livers across six major developmental stages (from embryonic day 38 to the adult stage) and under a high-fat diet-induced obesity. The characteristically loose chromatin architecture supports a highly plastic genome organization during early liver development, which fundamentally contributes to the rapid functional transitions in the liver after birth. We reveal the multi-scale reorganization of chromatin architecture and its influence on transcriptional regulation of critical signaling processes during liver development, and show its close association with transition in hepatic functions (i.e., from hematopoiesis in the fetus to metabolism and immunity after birth). The limited changes in chromatin structure help explain the observed metabolic adaptation to excessive energy intake in pigs. These results provide a global overview of chromatin architecture dynamics associated with the transition of physiological liver functions between prenatal development and postnatal maturation, and a foundational resource that allows for future in-depth functional characterization.

Abstract Background The three-dimensional (3D) architecture of the genome has a highly ordered and hierarchical nature, which influences the regulation of essential nuclear processes at the basis of gene expression, such as gene transcription. While the hierarchical organization of heterochromatin and euchromatin can underlie differences in gene expression that determine evolutionary differences among species, the way 3D genome architecture is affected by evolutionary forces within major lineages remains unclear. Here, we report a comprehensive comparison of 3D genomes, using high resolution Hi-C data in fibroblast cells of fish, chickens, and 10 mammalian species. Results This analysis shows a correlation between genome size and chromosome length that affects chromosome territory (CT) organization in the upper hierarchy of genome architecture, whereas lower hierarchical features, including local transcriptional availability of DNA, are selected through the evolution of vertebrates. Furthermore, conservation of topologically associating domains (TADs) appears strongly associated with the modularity of expression profiles across species. Additionally, LINE and SINE transposable elements likely contribute to heterochromatin and euchromatin organization, respectively, during the evolution of genome architecture. Conclusions Our analysis uncovers organizational features that appear to determine the conservation and transcriptional regulation of functional genes across species. These findings can guide ongoing investigations of genome evolution by extending our understanding of the mechanisms shaping genome architecture.

Animal gut microbiota plays an indispensable role in host adaptation to different altitude environments. At present, little is known about the mechanism of animal gut microbiota in host adaptation to high altitude environments. Here, we selected wild macaques, humans, and dogs with different levels of kinship and intimate relationships in high altitude and low altitude environments, and analyzed the response of their gut microbiota to the host diet and altitude environments. Alpha diversity analysis found that at high altitude, the gut microbiota diversity of wild macaques with more complex diet in the wild environments is much higher than that of humans and dogs with simpler diet (p

FilaminC (Flnc) is a member of the actin binding protein family, which is preferentially expressed in the cardiac and skeletal muscle tissues. Although it is known to interact with proteins associated with myofibrillar myopathy, its unique role in skeletal muscle remains largely unknown. In this study, we identify the biological functions of Flnc in vitro and in vivo using chicken primary myoblast cells and animal models, respectively. From the results, we observe that the growth rate and mass of the skeletal muscle of fast-growing chickens (broilers) were significantly higher than those in slow-growing chickens (layers). Furthermore, we find that the expression of Flnc in the skeletal muscle of broilers was higher than that in the layers. Our results indicated that Flnc was highly expressed in the skeletal muscle, especially in the skeletal muscle of broilers than in layers. This suggests that Flnc plays a positive regulatory role in myoblast development. Flnc knockdown resulted in muscle atrophy, whereas the overexpression of Flnc promotes muscle hypertrophy in vivo in an animal model. We also found that Flnc interacted with dishevelled-2 (Dvl2), activated the wnt/β-catenin signaling pathway, and controlled skeletal muscle development. Flnc also antagonized the LC3-mediated autophagy system by decreasing Dvl2 ubiquitination. Moreover, Flnc knockdown activated and significantly increased mitophagy. In summary, these results indicate that the absence of Flnc induces autophagy or mitophagy and regulates muscle atrophy.

Abstract Background A growing body of evidence has revealed that the mammalian genome is organized into hierarchical layers that are closely correlated with and may even be causally linked with variations in gene expression. Recent studies have characterized chromatin organization in various porcine tissues and cell types and compared them among species and during the early development of pigs. However, how chromatin organization differs among pig breeds is poorly understood. Results In this study, we investigated the 3D genome organization and performed transcriptome characterization of two adipose depots (upper layer of backfat [ULB] and greater omentum [GOM]) in wild boars and Bama pigs; the latter is a typical indigenous pig in China. We found that over 95% of the A/B compartments and topologically associating domains (TADs) are stable between wild boars and Bama pigs. In contrast, more than 70% of promoter-enhancer interactions (PEIs) are dynamic and widespread, involving over a thousand genes. Alterations in chromatin structure are associated with changes in the expression of genes that are involved in widespread biological functions such as basic cellular functions, endocrine function, energy metabolism and the immune response. Approximately 95% and 97% of the genes associated with reorganized A/B compartments and PEIs in the two pig breeds differed between GOM and ULB, respectively. Conclusions We reported 3D genome organization in adipose depots from different pig breeds. In a comparison of Bama pigs and wild boar, large-scale compartments and TADs were mostly conserved, while fine-scale PEIs were extensively reorganized. The chromatin architecture in these two pig breeds was reorganized in an adipose depot-specific manner. These results contribute to determining the regulatory mechanism of phenotypic differences between Bama pigs and wild boar.

The domestic chicken Gallus gallus domesticus is a classic model for the study of folliculogenesis. Here the authors integrate multi-omics analyses characterizing the dynamic transcriptome and chromatin architecture in granulosa cells during chicken folliculogenesis.

Nitrogen (N), phosphorus (P), and potassium (K) exert various effects on strawberry (Fragaria ananassa Duchesne) yields. In this study, we employed an orthogonal experimental design (T1-T9) with three fertilization treatments (N, P, and K) at three levels to identify an optimal fertilization scheme for strawberry cultivation. The effects of fertilizer combinations the rhizosphere soil microbial community were also explored by using bacterial full-length 16S rRNA and fungal ITS (internal transcribed spacer) sequencing (30 samples for each analysis). The results showed that the average plant height and leaf area of the fertilized groups were 24.6% and 41.6% higher than those of the non-fertilized group (T0). After 60 d of planting, the sucrase activity in the T6 group increased by 76.67% compared to the T0 group, with phosphate fertilizer exerting a more significant impact on sucrase activity. The T6 treatment group had the highest alpha diversity index among bacterial and fungal microorganisms, and had a different microbial community structure compared with the control group. The most abundant bacterial taxa in the strawberry rhizosphere soil were Proteobacteria, Bacteroidota, and Acidobacteriota, and the most abundant fungal phyla were Monoblepharomycota, Glomeromycota, and Mucoromycota. Application of the optimal combined fertilizer treatment (T6) significantly increased the abundance of Proteobacteria and altered the abundance of Gemmatimonas compared to other treatment groups. Notably, Gemmatimonas abundance positively correlated with strawberry plant height and soil N, P, and K levels. These findings indicated that the relative abundance of beneficial bacteria could be enhanced by the application of an optimal fertilizer ratio, ultimately improving strawberry agronomic traits.

ABSTRACT: MicroRNAs (miRNAs) are involved in regulating the circadian clock. In our previous work, miR-218-5p was found to be a circadian miRNA in the chicken uterus, but its role in the eggshell formation process was not clear. In the present study, we found that the expression levels of miR-218-5p and two 2 predicted target genes carbonic anhydrase 2 (CA2) and neuronal PAS domain protein 2 (NPAS2) were oscillated in the chicken uterus. The results of dual-luciferase reporter gene assays in the present study demonstrated that miR-218-5p directly targeted the 3’ untranslated regions of CA2 and NPAS2. miR-218-5p showed an opposite expression profile to CA2 within a 24 h cycle in the chicken uterus. Moreover, over-expression of miR-218-5p reduced the mRNA and protein expression of CA2, while miR-218-5p knockdown increased CA2 mRNA and protein expression. Overexpression of CA2 also significantly increased the activity of carbonic anhydrase Ⅱ (P < 0.05), whereas knockdown of CA2 decreased the activity of carbonic anhydrase Ⅱ. miR-218-5p influenced carbonic anhydrase activity via regulating the expression of CA2. These results demonstrated that clock-controlled miR-218-5p regulates carbonic anhydrase activity in the chicken uterus by targeting CA2 during eggshell formation.

Abstract Background miRNAs regulate circadian patterns by modulating the biological clocks of animals. In our previous study, we found that the clock gene exhibited a cosine expression pattern in the fallopian tube of chicken uterus. Clock-controlled miRNAs are present in mammals and Drosophila; however, whether there are clock-controlled miRNAs in the chicken uterus and, if so, how they regulate egg-laying rhythms is unclear. In this study, we selected 18 layer hens with similar ovipositional rhythmicity (each of three birds were sacrificed for study per 4 h throughout 24 h); their transcriptomes were scanned to identify the circadian miRNAs and to explore regulatory mechanisms within the uterus of chickens. Results We identified six circadian miRNAs that are mainly associated with several biological processes including ion trans-membrane transportation, response to calcium ion, and enrichment of calcium signaling pathways. Verification of the experimental results revealed that miR-449c-5p exhibited a cosine expression pattern in the chicken uterus. Ca2+-transporting ATPase 4 (ATP2B4) in the plasma membrane is the predicted target gene of circadian miR-449c-5p and is highly enriched in the calcium signaling pathway. We speculated that clock-controlled miR-449c-5p regulated Ca2+ transportation during eggshell calcification in the chicken uterus by targeting ATP2B4. ATP2B4 mRNA and protein were rhythmically expressed in the chicken uterus, and dual-luciferase reporter gene assays confirmed that ATP2B4 was directly targeted by miR-449c-5p. The expression of miR-449c-5p showed an opposite trend to that of ATP2B4 within a 24 h cycle in the chicken uterus; it inhibited mRNA and protein expression of ATP2B4 in the uterine tubular gland cells. In addition, overexpression of ATP2B4 significantly decreased intracellular Ca2+ concentration (P

ABSTRACT: Fatty liver hemorrhagic syndrome (FLHS) is a chronic hepatic disease which occurs when there is a disorder in lipid metabolism. FLHS is often observed in caged laying hens and characterized by a decrease in egg production and dramatic increase of mortality. Salidroside (SDS) is an herbal drug which has shown numerous pharmacological activities, such as protecting mitochondrial function, attenuating cell apoptosis and inflammation, and promoting antioxidant defense system. We aimed to determine the therapeutic effects of SDS on FLHS in laying hens and investigate the underlying mechanisms through which SDS operates these functions. We constructed oleic acid (OA)-induced fatty liver model in vitro and high-fat diet-induced FLHS of laying hens in vivo. The results indicated that SDS inhibited OA-induced lipid accumulation in chicken primary hepatocytes, increased hepatocyte activity, elevated the mRNA expression of proliferation related genes PCNA, CDK2, and cyclinD1 and increased the protein levels of PCNA and CDK2 (P < 0.05), as well as decreased the cleavage levels of Caspase-9, Caspase-8, and Caspase-3 and apoptosis in hepatocytes (P < 0.05). Moreover, SDS promoted the phosphorylation levels of PDK1, AKT, and Gsk3-β, while inhibited the PI3K inhibitor (P < 0.05). Additionally, we found that high-fat diet-induced FLHS hens had heavier body weight, liver weight, and abdominal fat weight, and severe steatosis in histology, compared with the control group (Con). However, hens fed with SDS maintained lighter body weight, liver weight, and abdominal fat weight, as well as normal liver without hepatic steatosis. In addition, high-fat diet-induced FLHS hens had high levels of serum total cholesterol (TC), triglyceride (TG), alanine transaminase (ALT), and aspartate aminotransferase (AST) compared to the Con group, however, in the Model+SDS group, the levels of TC, TG, ALT, and AST decreased significantly, whereas the level of superoxide dismutase (SOD) increased significantly (P < 0.05). We also found that SDS significantly decreased the mRNA expression abundance of PPARγ, SCD, and FAS in the liver, as well as increased levels of PPARα and MTTP, and decreased the mRNA expression of TNF-α, IL-1β, IL-6, and IL-8 in the Model+SDS group (P < 0.05). In summary, this study showed that 0.3 mg/mL SDS attenuated ROS generation, inhibited lipid accumulation and hepatocyte apoptosis, and promoted hepatocyte proliferation by targeting the PI3K/AKT/Gsk3-β pathway in OA-induced fatty liver model in vitro, and 20 mg/kg SDS alleviated high-fat-diet-induced hepatic steatosis, oxidative stress, and inflammatory response in laying hens in vivo.

Slow lorises are small arboreal and nocturnal primates. Due to the illegal trade, a large number of slow lorises were confiscated into wildlife sanctuaries or rescue centers. The re-release has been considered a preferable approach for alleviating the captive pressure, but inappropriate and long-term confinement make it difficult to achieve this goal. In this study, we investigated and compared the fecal and oral microbiome of Bengal slow lorises (Nycticebus bengalensis) under long-term captivity (LC) and short-term captivity (SC) groups based on 16s rRNA high-throughput gene sequencing. The oral microbiome displayed higher Chao1 richness but lower Shannon and Simpson indices than the fecal microbiome. The Bengal slow lorises under long-term captivity had abundant pathogenic genera in both gut and oral microbiomes, such as Desulfovibrio, Actinomyces, Capnocytophaga, Neisseria, and Fusobacterium, while some specific bacterial taxa associated with intestinal balance were more enriched in the SC group. Due to the plant gum scarcity in the diet, both groups had a low abundance of Bifidobacterium. Function profile prediction indicated that the LC group was enriched with genetic information processing and metabolism pathways due to the stable food intake. The increased membrane transport and xenobiotic metabolism and degradation functions in the SC group could be explained by the function of the host microbiome in facilitating adaptation to changing environments and diets. The results demonstrated that the oral microbiome had the potential to be used as a regular surveillance tool. Also, current captive management should be improved to ensure reintroduction success.

ABSTRACT: Low oxygen levels and extremely cold weather in high-altitude environments requires more energy intake to maintain body temperature in animals. However, little is known about the characteristics of cecal and ileac microbiota in Tibetan chicken and how the high and low altitude environments affect the gut microbiota communities in Tibetan chicken. In the present study, In the present study, Tibetan chickens (Group HA, 3572 m, 578.5 Pa) and their introduced flatland counterparts (Group LA, 580 m, 894.6 Pa) in the cecum and ileum to identify the possible bacterial species that are helpful for their host in environmental adaption. High-throughput sequencing was used to sequence the V3 to V4 hypervariable regions of the bacterial 16S rRNA gene. By comparing the gut microbial diversity of HA chicken with that of LA, the results indicated that the microbial diversity of the cecum and ileum in group HA was significantly lower (P < 0.05) than those in group LA. The cecum microbiome maintained higher population diversity and richness than the ileum (P < 0.05). Four phyla Firmicutes, Bacterioidetes, Actinobacteria, and Proteobacteria were dominant in two groups. Interestingly, there were significant differences in abundance ratio among the four groups (P < 0.05). The predominant bacteria in HA and LA ileum belong to Proteobacteria and Firmicutes, whereas in cecum, Bacterioidetes and Actinobacteria were predominant in both groups (P < 0.05). Correlation analysis showed that Sporosarcina, Enterococcus, and Lactococcus were strongly related to air pressure, and Peptoclostridium and Ruminococcaceae_UCG-014 are related to altitude and gut microbiota of LA group was influenced by altitude, while HA group affected by air pressure. Meanwhile, the Ruminococcus-torques-group was negatively correlated with the relative abundance of Paenibacillus, and positive correlated with those of other microorganisms. Furthermore, HA has higher abundance of microbiota involved in energy and glycan biosynthesis metabolism pathway, while LA has higher abundance of microbiota involved in membrane transport, signal transduction, and xenobiotics biodegradation and metabolism. Generally, our results suggested that the composition and diversity of gut microbes changed after Tibetan chickens were introduced to the plain. Tibetan chicken may adapt to new environment via reshaping the gut microbiota. Gut microbes may contribute to the host adaption to high altitude environments by increasing host energy and glycan biosynthesis.

Sustainable meat production is important to providing safe and quality protein sources for humans worldwide. Intensive artificial selection and high energy input into the diet of many commercial animals for the last decade has significantly increased the daily gain of body weight and shortened the raising period, but unexpectedly decreased the meat quality. The gastrointestinal tract of animals harbors a diverse and complex microbial community that plays a vital role in the digestion and absorption of nutrients, immune system development, pathogen exclusion, and meat quality. Fatty acid composition and oxidative stress in adipose and muscle tissue influences meat quality in livestock and poultry. Recent studies showed that nutraceuticals are receiving increased attention, which could alter the intestinal microbiota and regulate the fat deposition and immunity of hosts to improve their meat quality. Understanding the microbiota composition, the functions of key bacteria, and the host-microbiota interaction is crucial for the development of knowledge-based strategies to improve both animal meat quality and host health. This paper reviews the microorganisms that affect the meat quality of livestock and poultry. A greater understanding of microbial changes that accompany beneficial dietary changes will lead to novel strategies to improve livestock and poultry meat product quality.

ABSTRACT During the entire growth process, gut microbiota continues to change and has a certain impact on the performance of broilers. Here, we used 16S rRNA gene sequencing to explore the dynamic changes in the fecal bacterial communities and functions in 120 broilers from 4 to 16 weeks of age. We found that the main phyla (Firmicutes, Fusobacteria, Proteobacteria, and Bacteroides) accounted for more than 93.5% of the total bacteria in the feces. The alpha diversity of the fecal microbiota showed a downward trend with time, and the beta diversity showed significant differences at various time points. Then, the study on the differences of microbiota between high-weight (HW) and low-weight (LW) broilers showed that there were differences in the diversity and composition of microbiota between high- and low-weight broilers. Furthermore, we identified 22 genera that may be related to the weight change of broilers. The analysis of flora function reveals their changes in metabolism, genetic information processing, and environmental information processing. Finally, combined with microbial function and cecal transcriptome results, we speculated that microorganisms may affect the immune level and energy metabolism level of broilers through their own carbohydrate metabolism and lipid metabolism and then affect body weight (BW). Our results will help to expand our understanding of intestinal microbiota and provide guidance for the production of high-quality broilers. IMPORTANCE The intestinal microbiota has a certain impact on the performance of broilers. However, the change of intestinal microbiota after 4 weeks of age is not clear, and the mechanism of the effect of microorganisms on the weight change of broilers needs more exploration. After 4 weeks of age, the alpha diversity of microorganisms in broiler feces decreased, and the dominant bacteria were Firmicutes, Fusobacteria, Proteobacteria, and Bacteroides. There were differences in microbiota diversity and composition between high- and low-weight broilers. Intestinal microorganisms may affect the immune level and energy metabolism level of broilers through their own carbohydrate metabolism and lipid metabolism and then affect the body weight. The results are helpful to increase the understanding of intestinal microbiota and provide reference for the production of high-quality broilers.

Muskrat musk is considered to be a potential substitute for traditional musk. However, little is known about the similarity between muskrat musk and musk, and whether it is related to muskrat age. In this study, muskrat musk (MR1, MR2, and MR3) were from 1, 2, and 3-year-old muskrats, respectively, and white musk (WM) and brown musk (BM) were picked from male forest musk deer. The results indicated that muskrat musk had higher similarity to WM than BM. Further research showed that RM3 had the highest matched degree with WM. By significantly different metabolite analysis, we found that 52 metabolites continue to increase from 1- to 3-year-old muskrats. In total, 7 and 15 metabolites were significantly decreased in RM1 vs. RM2 and RM2 vs. RM3, respectively. Meanwhile, 30 and 17 signaling pathways were observed from increased and decreased metabolites, respectively. The increased metabolites mainly entailed enrichment in amino acid biosynthesis and metabolism, steroid hormone biosynthesis, and fatty acid biosynthesis. In conclusion, muskrat musk from three-year-old muskrat is a relatively good substitute for white musk, and the result also implies that these biological processes of amino acid biosynthesis and metabolism, steroid hormone biosynthesis, and fatty acid biosynthesis are beneficial to the secretion of muskrat musk.

A comprehensive transcriptomic survey of the pig could enable mechanistic understanding of tissue specialization and accelerate its use as a biomedical model. Here the authors characterize four distinct transcript types in 31 adult pig tissues to dissect their distinct structural and transcriptional features and uncover transcriptomic variability related to tissue physiology.

Quercetin, a naturally non-toxic flavonoid within the safe dose range with antioxidant, anti-apoptotic and anti-inflammatory properties, plays an important role in the treatment of aging-related diseases. Sirtuin 1 (SIRT1), a member of NAD+-dependent deacetylase enzyme family, is extensively explored as a potential therapeutic target for attenuating aging-induced disorders. SIRT1 possess beneficial effects against aging-related diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), Depression, Osteoporosis, Myocardial ischemia (M/I) and reperfusion (MI/R), Atherosclerosis (AS), and Diabetes. Previous studies have reported that aging increases tissue susceptibility, whereas, SIRT1 regulates cellular senescence and multiple aging-related cellular processes, including SIRT1/Keap1/Nrf2/HO-1 and SIRTI/PI3K/Akt/GSK-3β mediated oxidative stress, SIRT1/NF-κB and SIRT1/NLRP3 regulated inflammatory response, SIRT1/PGC1α/eIF2α/ATF4/CHOP and SIRT1/PKD1/CREB controlled phosphorylation, SIRT1-PINK1-Parkin mediated mitochondrial damage, SIRT1/FoxO mediated autophagy, and SIRT1/FoxG1/CREB/BDNF/Trkβ-catenin mediated neuroprotective effects. In this review, we summarized the role of SIRT1 in the improvement of the attenuation effect of quercetin on aging-related diseases and the relationship between relevant signaling pathways regulated by SIRT1. Moreover, the functional regulation of quercetin in aging-related markers such as oxidative stress, inflammatory response, mitochondrial function, autophagy and apoptosis through SIRT1 was discussed. Finally, the prospects of an extracellular vesicles (EVs) as quercetin loading and delivery, and SIRT1-mediated EVs as signal carriers for treating aging-related diseases, as well as discussed the ferroptosis alleviation effects of quercetin to protect against aging-related disease via activating SIRT1. Generally, SIRT1 may serve as a promising therapeutic target in the treatment of aging-related diseases via inhibiting oxidative stress, reducing inflammatory responses, and restoring mitochondrial dysfunction.

Tibetan chickens living at high altitudes show specific physiological adaptations to the extreme environmental conditions. However, the regulated base of how chickens adapt to high-altitude habitats remains largely unknown. In this study, we sequenced 96 transcriptomes (including 48 miRNA and 48 mRNA transcriptomes of heart, liver, lung, and brain) and resequenced 12 whole genomes of Tibetan chickens and Peng'xian yellow chickens. We found that several miRNAs show the locally optimal plastic changes that occurred in miRNAs of chickens, such as miR-10c-5p, miR-144-3p, miR-3536, and miR-499-5p. These miRNAs could have effects on early adaption to the high-altitude environment of chickens. In addition, the genes under selection between Tibetan chickens and Peng'xian yellow chickens were mainly related to oxygen transport and oxidative stress. The I-kappa B kinase/NF-kappa B signaling pathway is widely found for high-altitude adaptation in Tibetan chickens. The candidate differentially expressed miRNAs and selected genes identified in this study may be useful in current breeding efforts to develop improved breeds for the highlands.

ABSTRACT: Aged male chickens experience rapid declines in spermatogenesis, antioxidant capacity, immunity, and hormone synthesis. Vitamin E plays a significant role in reproduction, nervous system function, and disease resistance in animals. Quercetin also exerts many biological effects, such as antioxidant ability, immunostimulation, and protection of spermatozoal plasma membranes. This study evaluated the effects of combining dietary quercetin (Q) and vitamin E (VE) on sperm quality, antioxidant capacity, immunity, and expression of genes related to spermatogenesis, immunity, apoptosis, and inflammation in aged male chickens. A total of 120 Tianfu breeder male chickens (65 wk old) were randomly allotted to 4 treatments with 3 replicates (10 birds each). The birds were fed diets containing Q (0.4g/kg), VE (0.2g/kg), Q+VE (0.4g/kg + 0.2g/kg), and a basal diet for 11 wk. At the end of the experimental period, blood, semen, liver, testes, and spleen samples were collected from 2 birds per replicate. Serum hormones, antioxidant parameters, cytokines, and immunoglobulins were evaluated; and the mRNA expression of genes related to spermatogenesis, apoptosis, and inflammation are determined in the testes and liver tissues.The results showed that the combination quercetin and vitamin E significantly promoted the sperm count and motility, as well as elevated the levels of testosterone, follicle-stimulating hormone, and luteinizing hormone, antioxidant enzymes (Superoxide dismutase, Glutathione, and Total antioxidant capacity), and serum immunoglobulins (IgA and IgM) in the aged male chickens; also Q+VE showed protective effects on the liver against injury. In addition, Q+VE significantly increased the expression of genes related to spermatogenesis (AR, pgk2, Cyclin A1, and Cyclin A2), immunity (IFN-γ and IL-2), and anti-inflammatory cytokines (IL-10) (P < 0.05), whereas the expression of proinflammatory cytokines (IL-1β and IL-6) was decreased (P < 0.05). Taken together, these data indicate that the combination of quercetin and vitamin E improved reproductive characteristics such as spermatogenesis, sperm quality, and hormone regulation, as well as promoted antioxidant defense, hepatoprotective capacity, and immune response in aged male chickens without any detrimental effects.

In the present study, the synergistic effects of quercetin (Q) and vitamin E (E) on cecal microbiota composition and function, as well as the microbial metabolic profile in aged breeder hens were investigated. A total of 400 (65 weeks old) Tianfu breeder hens were randomly allotted to four experimental groups (four replicates per group). The birds were fed diets containing quercetin at 0.4 g/kg, vitamin E (0.2 g/kg), quercetin and vitamin E (QE; 0.4 g/kg and 0.2 g/kg), and a basal diet for a period of 10 wks. After the 10 week experimental period, the cecal contents of 8 aged breeder hens per group were sampled aseptically and subjected to high-throughput 16S rRNA gene sequencing and untargeted metabolomic analysis. The results showed that the relative abundances of phyla Bacteroidota, Firmicutes, and Actinobacteriota were the most prominent among all the dietary groups. Compared to the control group, the relative abundance of the families Bifidobacteriaceae, Lachnospiraceae, Tannerellaceae, Mathonobacteriaceae, Barnesiellaceae, and Prevotellaceae were enriched in the QE group; and Bacteroidaceae, Desulfovibrionaceae, Peptotostretococcaceae, and Fusobacteriaceae were enriched in the Q group, whereas those of Lactobacillaceae, Veillonellaceae, Ruminococcaceae, Akkermansiaceae, and Rikenellaceae were enriched in the E group compared to the control group. Untargeted metabolomics analyses revealed that Q, E, and QE modified the abundance of several metabolites in prominent pathways including ubiquinone and other terpenoid–quinone biosynthesis, regulation of actin cytoskeleton, insulin secretion, pancreatic secretion, nicotine addiction, and metabolism of xenobiotics by cytochrome P450. Furthermore, key cecal microbiota, significantly correlated with important metabolites, for example, (S)-equol positively correlated with Alistipes and Chlamydia in E_vs_C, and negatively correlated with Olsenella, Paraprevotella, and Mucispirillum but, a contrary trend was observed with Parabacteroides in QE_vs_C. This study establishes that the synergy of quercetin and vitamin E alters the cecal microbial composition and metabolite profile in aged breeder hens, which lays a foundation for chicken improvement programs.

ABSTRACT: Circular RNAs (circRNAs) has been reported in various tissues of animals and associated with multiple biological processes. From our previous sequencing data, we found a novel circRNA, circCCDC91 which was generated from exon 2 to 8 of the CCDC91 gene. We observed that circCCDC91 was differentially expressed in the chicken breast muscle among 4 different embryonic developmental time points (embryonic day 10 [E10], E13, E16, and E19). Therefore, we assumed that circCCDC91 have a potential function in chicken skeletal muscle development. In this study, we firstly verify the annular structure and expression pattern of circCCDC91, and further investigate on whether circCCDC91 could promote chicken skeletal development. Mechanistically, circCCDC91 could absorb miR-15a, miR-15b-5p, and miR-15c-5p to modulate the expression of Insulin receptor substrate1 (IRS1), as well as activate insulin-1ike growth factor 1-phosphatidylinositol 3-kinase/AKT (IGF1-PI3K/AKT) signaling pathway. In addition, circCCDC91 could rescue skeletal muscle atrophy by activating IGF1-PI3K/AKT pathway. Taken together, the findings in this study revealed that the newly identified circCCDC91 promotes myoblasts proliferation and differentiation, and alleviates skeletal muscle atrophy by directly binding to miR-15 family via activating IGF1-PI3K/AKT signaling pathway in chicken.

Enterocytozoon bieneusi is an obligate intracellular parasite and the most common pathogen of microsporidiosis in humans and animals. In this study, a total of 198 fecal samples were collected from 11 captive populations of Rhesus macaque in Chinese zoos, to investigate the prevalence and analyze the zoonotic potential of E. bieneusi by genotype of the Internal Transcribed Spacer (ITS) gene on the rRNA of E. bieneusi via nested PCR. Results showed that the average infection rate of E. bieneusi in the 11 populations was 13.6%, and the highest infection rate was 56.5% in the population of Xinjiang Tianshan Zoo. Seven genotypes were identified including 2 known genotypes (D and CM1) and 5 novel genotypes (Mul1, Mul2, Mul3, Mul4 and Mul5). Phylogenetic analysis revealed that the novel genotypes Mul2, Mul3, Mul4 and Mul5 belonged to Group 1 showed the zoonotic potential．These findings extend the distribution of E. bieneusi genotypes and provide baseline data for controlling E. bieneusi infection.

Avian leucosis (AL) is a disease characterized by tumors and is caused by the avian leukosis virus (ALV). Because of the high variability of viruses and complex pathogenic mechanisms, screening and breeding J subgroup of ALV (ALV-J) resistant avian breeds is one of the strategies for prevention and treatment of AL, thus screening of significant immune markers is needed to promote the development of disease-resistant breeds. In this study, data-independent acquisition (DIA) technology was used to detect the DEPs of three breeds of chicken according to different comparison to investigate the potential markers. Results showed special DEPs for spleen development of each breed were detected, such as PCNT, DDB2, and ZNF62. These DEPs were involved in intestinal immune network used in production of IgA signaling pathways and related to immune response which can be used as potential markers for spleen development in different breeds. The DEPs such as RAB44 and TPN involved in viral myocarditis, transcriptional misregulation in cancer, and tuberculosis can be used as potential markers of spleen immune response after ALV-J infection in chickens. Pair-wise analysis was performed for the three breeds after the infection of ALV-J. The proteins such as RFX1, TAF10, and VH1 were differently expressed between three breeds. These DEPs involved in antigen processing and expression, acute myelogenous leukemia, and viral carcinogenesis can be used as potential immune markers after ALV-J infection of different genetic backgrounds. The screening of potential markers at protein level provides a strong theoretical research basis for disease resistance breeding in poultry.

During sexual maturation and ovulatory cycle in chickens, ovaries undergo dynamic morphological and functional changes. The aim of this study was to evaluate the integrated proteome and metabolome analyses of chicken ovaries to characterize the changes in protein and metabolite profiles during sexual maturity. The ovary of Rohman layers before (125 days of age) and after (139 days of age) sexual maturation were collected for proteome and metabolome sequencing. The results showed that a total of 680 differentially expressed proteins (DEPs) and 1,046 differential metabolites (DMs) were identified in the chicken ovary during sexual maturity. Among the DEPs, 595 proteins were up-regulated and 85 were down-regulated, whereas 519 metabolites were up-regulated and 527 were down-regulated. KEGG pathway enrichment analysis showed that DEPs were significantly enriched in glycerolipid metabolism, calcium signaling pathway, folate biosynthesis, fat digestion and absorption, NF-kB signaling pathway, and PPAR signaling pathway. However, DMs were significantly enriched in the metabolism pathways, PPAR signalling pathway, glycerolipid metabolism, ferroptosis, biosynthesis of amino acids, and biosynthesis of unsaturated fatty acids. The results of the integrated analyses of DEPs and DMs revealed that the PPAR signaling pathway and glycerolipid metabolism were the most significantly enriched pathways. Among the identified DEPs, lipoprotein lipase (LPL) was upregulated in sexually mature chicken ovaries and was significantly enriched in the glycerolipid metabolism pathway, which may partially explain the possible reasons for steroidogenesis and lipid reserves responsible for oocyte maturation and ovarian follicle development during sexual maturity in chickens. The results further revealed that LPL silencing decreased the content of lipid droplets (LDs), as well as the mRNA expression of lipid metabolism-related genes including; sterol regulatory element binding protein-1 (SREBP-1) and fatty acid synthase (FASN); and steroidogenesis-related genes such as; cytochrome P450 11A1 (CYP11A1) and steroidogenic acute regulatory (StAR). The present study revealed that upregulation of LPL in the chicken ovary during sexual maturity promotes granulosa cell (GC) lipid metabolism and steroidogenesis. These findings provide a theoretical support for further studies to elucidate the mechanism of lipid metabolism to regulate the function of avian GCs during sexual maturity in chickens.

The current study aims to investigate the effects of the synergy between quercetin and vitamin E in aged hen’s diet on hatchability and antioxidant levels of the embryo and newly hatched chicks from prolonged storage eggs. A total of 400 breeder laying hens of 65 weeks of age were selected and randomly divided into 4 groups. Birds were fed a basal diet alone (Control), and basal diets supplemented with quercetin (Q) (0.4 g/kg) and vitamin E (VE) (0.2 g/kg) alone and their combination (0.4 g/kg Q + 0.2 g/kg VE) for 14 weeks, respectively, to determine their effects on yolk antioxidant status, fertility, embryonic mortality, hatchability, antioxidant status of embryonic tissues, as well as the antioxidant status of the newly hatched chicks. The results showed that the hen’s dietary Q + VE increased the yolk weight, as well as increased the antioxidant status of the egg yolk (p < 0.05). Compared with the control group, the supplementation of Q + VE significantly increased the hatchability of set-fertile eggs and decreased early embryonic mortality in eggs stored for 7 and 14 days, respectively (p < 0.05), and also improved the antioxidant capacity of the embryos obtained from eggs stored for 14 days (before incubation) (p < 0.05). Moreover, Q + VE increased the levels of SOD, GSH-Px, T-AOC, T-SOD, and CAT in the liver, heart, and pectoral muscle of the embryo, 1-day-old and 14-day-old chicks (p < 0.05), as well as upregulated the antioxidant related genes (GPx-1, GPx-2, GPx-4, DIO-1, and SOD-1) in the liver of the embryo, 1-day-old and 14-day-old chicks hatched from 14-days storage eggs (p < 0.05). Meanwhile, the MDA levels were decreased by the Q + VE in the embryo and post-hatched chicks (p < 0.05). In conclusion, these findings suggested that maternal dietary Q + VE exerts beneficial synergistic effects on the antioxidant capacity of the egg yolk, embryo, and chicks during prolong egg storage, therefore, Q + VE could be used as a dietary measure to enhance hatchability and chick quality in poultry production.

In aged animals, the physiological functions of the gastrointestinal tract (GIT) are reduced. Dietary intervention is necessary to re-activate GIT functions. The objective of this study was to investigate the impacts of dietary combination of quercetin (Q) and vitamin E (VE) on the intestinal structure and barrier integrity in aged breeder chickens. A sum of 400 (65-wks-old) Tianfu breeder hens were randomly allotted into four (4) groups with four (4) replicates, and fed with basal diet; basal diet supplemented with 0.4g/kg of Q; basal diet supplemented with 0.2g/kg of VE; and basal diet supplemented with the combination of Q (0.4 g/kg) and VE (0.2 g/kg) for 14 weeks. At the end of the 14th week, serum and gut segments were collected from eight hens per group for analyses. The results showed that Q+VE exerted synergistic effects on intestinal morphology by promoting villi height and crypt depth (P < 0.05), as well as mitigated the intestinal inflammatory damage of the aged hens, but decreased the concentration of serum D-lactate and diamine oxidase; and increased the levels of secretory immunoglobulin A (sIgA) and Mucin-2 mRNA (P < 0.05). Furthermore, the mRNA expression of intestinal tight junction proteins including occludin, ZO1, and claudin-1 was increased by Q+VE (P < 0.05). Moreover, Q+VE decreased the mRNA expression of the pro-inflammatory genes (TNF-α, IL-6, and IL-1β), and increased the expression of anti-inflammatory genes (IL-10 and IL-4) (P < 0.05). These results were consistent with the mRNA expression of Bax and Bcl-2. In addition, Q+VE protected the small intestinal tract from oxidative damage by increasing the levels of superoxide dismutase, total antioxidant capacity, glutathione peroxidase, catalase (P < 0.05), and the mRNA expression of SOD1 and GPx-2. However, Q+VE decreased malondialdehyde levels in the intestine compared to the control (P < 0.05). These results indicated that dietary Q+VE improved intestinal function in aged breeder hens, by protecting the intestinal structure and integrity. Therefore, Q+VE could act as an anti-aging agent to elevate the physiological functions of the small intestine in chickens.

The reproductive tract of chickens is an important organ for egg formation. The vagina is in close contact with the external environment, which may lead to the invasion of a variety of pathogenic bacteria, affect the internal and external quality of eggs, and even increase mortality and cause economic loss. In recent years, probiotics as a substitute for antibiotics have brought economic benefits in livestock and poultry production. In the present study, we investigated the effects of vaginal administration of Bacteroides fragilis on the cloacal microbiota, vaginal transcriptome and metabolomics of chickens and evaluated the beneficial potential of B. fragilis. The results showed that B. fragilis treatment could affect the microbial composition of the cloaca. Transcriptome analysis found that the immune-related genes CCN3, HAS2, and RICTOR were upregulated, that the inflammatory genes EDNRB, TOX, and NKX2-3 were downregulated, and that DEGs were also enriched in the regulation of the inflammatory response, cellular metabolism, and synaptic response pathways. In addition, the differential metabolites were mainly related to steroid hormone biosynthesis, unsaturated fatty acid biosynthesis, and arachidonic acid metabolism, and we identified associations between specific differential metabolites and genes. Overall, this study provides a theoretical basis for the application of B. fragilis as a potential probiotic in livestock and poultry production.

Abstract Background The mammal intestinal microbiota is involved in various physiological processes and plays a key role in host environment adaption. However, for non-human primates (NHPs), little is known about their gut microbial community in high-altitude environments and even less about their adaption to such habitats. We characterised the gut microbial community of rhesus macaques from multiple high-altitude environments and compared it to those of low-altitude populations. Results We collected faecal samples of rhesus macaques from four high-altitude populations (above 3000 m) and three low-altitude populations (below 500 m). By calculating the alpha diversity index, we found that high-altitude populations exhibited a higher diversity. Statistical analysis of beta diversity indicated significant differences between high- and low-altitude populations. Significant differences were also detected at the phylum and family levels. At the phylum level, the high-altitude gut microbial community was dominated by Firmicutes (63.42%), while at low altitudes, it was dominated by Bacteroidetes (47.4%). At the family level, the high-altitude population was dominated by Ruminococcaceae (36.2%), while the low-altitude one was dominated by Prevotellaceae (39.6%). Some families, such as Christensenellaceae and Rikenellaceae, were consistently higher abundant in all high-altitude populations. We analysed the overlap of operational taxonomic units (OTUs) in high-altitude populations and determined their core OTUs (shared by all four high-altitude populations). However, when compared with the low-altitude core OTUs, only 65% were shared, suggesting a divergence in core OTUs. Function prediction indicated a significant difference in gene copy number of 35 level-2 pathways between high- and low-altitude populations; 29 of them were higher in high altitudes, especially in membrane transport and carbohydrate metabolism. Conclusions The gut microbial community of high-altitude rhesus macaques was significantly distinct from that of low-altitude populations in terms of diversity, composition and function. High-altitude populations were dominated by Firmicutes and Ruminococcace, while in low-altitude populations, Bacteroidetes and Prevotellaceae were dominant. The difference in gut microbiota between these two populations may be caused by differences in host diet, environmental temperature and oxygen pressure. These differentiated gut microbial microorganisms may play a critical role in the adaptive evolution of rhesus macaques to high-altitude environments.

The genetic structure and evolutionary relationship of Dulong chicken with other native Chinese species remained unclear. In this study, the mitochondrial control region was analyzed in total of 343 samples comprising 59 from Dulong chicken and 284 from 8 other Chinese local breeds revealed 51 mutation sites that defined 42 haplotypes. The maximum genetic variation was observed between the Shimian caoke and Pengxian yellow chickens. Phylogenetic analysis revealed that these local chickens mainly scatter in two southwestern clades. Dulong chickens have close relationship with other native chicken. Finding of this study suggests a single matrilineal lineage of indigenous Dulong chickens.

ABSTRACT: Circadian timing system controlled the rhythmic events, for example, ovulation and oviposition in chickens. However, how biological clock mediates eggshell formation remains obscure. Here, A 24-h mRNA transcriptome analysis was carried out in the uterus of 18 chickens with similar oviposition time points to identify the rhythmic genes and to reveal critical genes and biological pathways involved in the eggshell biomineralization. JTK_CYCLE analysis and real-time PCR revealed a total of 1,793 genes from the sequencing database with 23,513 genes (FPKM>1) were rhythmic genes regulating the rhythmic system and the expression of typical clock genes Per2, Cry1, Bmal1, Clock, Per3, and Rev-erbβ were rhythmically expressed, which suggested that endogenous clock in uterus might control the eggshell mineralization. Time of peak expression of the rhythmic genes was analyzed based on their acrophase. The main phases clustered at the periods from Zeitgeber time 0 (ZT0) to ZT4 (6:00–10:00) and from ZT10 to ZT14 (16:00-20:00). The rhythmic genes were annotated to the following Gene Ontology terms rhythmic process, lyase, ATP binding, cell membrane component. KEGG pathway enrichment analysis revealed the top 15 rhythmic genes were involved in vital biological pathways, including syndecan (1, 2, 3)-mediated signaling, post-translational regulation of adheres junction stability and disassembly, FoxO family signaling, TGF-β receptor and transport of small molecular pathways. 166 of total 1,235 genes (13.4%) were defined as rhythmic transfer factors (TFs) and they were investigated expression time distribution of cis-elements of circadian clock system D-box, E-box, B-site, and Y-Box within 24 h. Results indicated that rhythmic TFs at each phase are potential drivers of their circadian transcription activities. Compared with the control, the expression abundances of ion transport elements SCNN1G, CA2, SPP1, and ATP1B1 were significantly decreased after the interference of Bmal1 gene in synchronized uterine tubular gland cells. Clock genes changed their expression along with the eggshell formation, indicating that there is circadian clock in the uterus of chicken and it regulates the expression of eggshell formation genes.

ABSTRACT: Follicular atresia is an important cause of reproductive decline in egg-laying hens. Therefore, a better understanding of the regulation mechanism of follicle atresia in poultry is an important measure to maintain persistent high egg performance. However, how the role of the regulatory relationship between autophagy and apoptosis in the intrafollicular environment affects the follicular atresia of chickens is remain unclear. The objective of this study was to explore the regulatory molecular mechanisms in regard to follicular atresia. 20 white leghorn layers (32-wk-old) were equally divided into 2 groups. The control group was fed freely, and the experimental group induced follicular atretic by fasting for 5 d. The results showed that the expression of prolactin (PRL) levels was significantly higher in the fasted hens, while the levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) were lower. Most importantly, RNA sequencing, qPCR, and Western blotting detected significantly elevated levels of autophagy and apoptosis markers in atresia follicles. Interestingly, we found that fibromodulin (FMOD) levels was significantly lower in follicles from fasted hens and that this molecule had an important regulatory role in autophagy. FMOD silencing significantly promoted autophagy and apoptosis in granulosa cells, resulting in hormonal imbalance. FMOD was found to regulate autophagy via the transforming growth factor beta (TGF-β) signaling pathway. Our results suggest that the increase in autophagy and the imbalance in internal homeostasis cause granulosa cell apoptosis, leading to follicular atresia in the chicken ovary. This finding could provide further insight into broodiness in chicken and provide avenues for further improvements in poultry production.

ABSTRACT: Laying hens experience a rapid decline in egg production, egg quality, and immunity, usually at the end of the peak laying period. Quercetin, a known flavonoid, exerts biological activities, including phytoestrogenic, immunity, antibiotic, antioxidant, and anti-inflammatory properties. Vitamin E also shows egg production and immunoregulatory potential in animals. This study evaluated the capacity of dietary quercetin, vitamin E, and the combination of both, to promote egg production and egg quality, and to improve the immunity of aging breeder hens. We also elucidated how quercetin and vitamin E combination could synergistically affect egg production, egg quality, and immunity in aging breeder hens. A total of 400 Tianfu broiler breeders at the age of 52 wk were randomly allotted to 4 treatments with 4 replicates, 100 hens per treatment and 25 hens per replicate. They were fed diets containing quercetin at 0.4 g/kg, Vitamin E (200 mg/kg), quercetin and vitamin E (0.4 g/kg and 200 mg/kg), and a basal diet (control) for a period 10 wk. Daily feed intake and egg production rate were recorded, and weekly records were recorded on egg quality tests. At the end of the 10-wk experimental period, blood samples and immune organ (spleen) were collected from 2 birds per replicate, totaling 32 birds. Feed intake, immune organ index, serum cytokines, and immunoglobulins were evaluated, and the mRNA expression of genes related to immunity was determined from the spleen tissue. Generally, the results showed that separately or as a combination, supplemental quercetin and vitamin E significantly improved performance and egg quality (P < 0.05), and significantly increased serum immunoglobulins (IgA, IgM, and IgG) and cytokines (IFN-γ and IL-2) concentrations, as well as promoted immune organ development and index, and promoted the expression of splenic immune-related genes (IL-2 and INF-γ) (P < 0.05), compared with the control. It was confirmed in this study that the combination of quercetin and vitamin E exert synergistic effects on egg production, egg quality, and immune function in aging hens.

Background The formation of musk is a complex biophysical and biochemical process that change with the rut of male forest musk deer. We have reported that the mating status of male forest musk deer might result to the variations of chemical composition and microbiota of musk and its yields. Critical roles for microRNAs (miRNAs) of multi-tissues were profiled in our previous study; however, the role for miRNAs of the musk gland remains unclear in this species. Methods In this study, we used Illumina deep sequencing technology to sequence the small RNA transcriptome of unmated male (UM) and mated male (UM) of Chinese forest musk deer. Results We identified 1,652 known miRNAs and 45 novel miRNAs, of which there were 174 differentially expressed miRNAs between UM and MM. chi-miR-21-5p, ipu-miR-99b and bta-miR-26a were up-regulated in UM among the 10 most differentially expressed miRNAs. Functional enrichment of the target genes showed that monosaccharide biosynthetic process, protein targeting, cellular protein catabolic process enriched higher in MM. Meanwhile, structural molecule activity, secretion by cell, regulated exocytosis and circulatory system process enriched more in UM, hinting that the formation of musk in UM was mediated by target genes related to exocytosis. The miRNA-mRNA pairs such as miR-21: CHD7, miR143: HSD17B7, miR-141/200a: Noc2 might involve in musk gland development and musk secretion, which need to be verified in future study.

ABSTRACT: Body weight at the onset of egg production is a major factor influencing hen productivity, as suitable body weight is crucial to laying performance in laying hens. To better understand the association between body weight and microbial community membership and structure in different sites of the digestive and reproductive tracts in chickens, we performed 16S rRNA sequencing surveys and focused on how the microbiota may interact to influence body weight. Our results demonstrated that the microbial community and structure of the digestive and reproductive tracts differed between low and high body weight groups. In particular, we found that the species Pseudomonas viridiflava was negatively associated with body weight in the 3 digestive tract sites, while Bacteroides salanitronis was negatively associated with body weight in the 3 reproductive tract sites; and further in-depth studies are needed to explore their function. These findings will help extend our understanding of the influence of the bird digestive and reproductive tract microbiotas on body weight trait and provide future directions regarding the control of body weight in the production of laying hens.

A balanced, diverse gut microbiota is vital for animal health. The microbial population is shaped by multiple factors including genetic background and environment, but other determinants remain controversial. Numerous studies suggest that the dominant factor is genetic background while others emphasize the environmental factors. Here, we bred asexual hybridization queens (AHQs) of honeybees through nutritional crossbreeding (laid in Apis mellifera colony but bred in Apis cerana colony), sequenced their gut microbiome, and compared it with normally bred sister queens to determine the primary factor shaping the gut microbiota. Our results showed that the dominant genera in the gut microbiota of AHQs were Brevundimonas, Bombella, and Lactobacillus, and its microbial community was more related to A. mellifera queens. The AHQs had a moderate number of different bacterial species and diversity, but total bacterial numbers were low. There were more significant taxa identified in the comparison between AHQ and A. cerana queen according to LEfSe analysis results. The only genetic-specific taxon we figured out was Brevundimonas. The growth of core bacterial abundance showed different characteristics among different queen groups in the first week after emerging. Collectively, this study suggested that the genetic background played a more dominant role than environmental factors in shaping the gut microbiota of honeybee queen and the microbiota of midgut was more sensitive than that of rectum to this impact.

Aflatoxin B1 (AFB1) is a widely distributed contaminant in moldy corn, rice, soybean, and oil crops. Many studies have revealed its adverse effects, such as carcinogenicity, immunotoxicity, and hepatotoxicity, on the health of humans and animals. To investigate the immunotoxic effects on chicken immune organs induced by AFB1, we integrated RNA and small-RNA sequencing data of the spleen and the bursa of Fabricius to elucidate the response of the differentially expressed transcriptional profiles and related pathways. AFB1 consumption negatively influenced egg quality, but no obvious organ damage was observed compared to that of the control group. We identified 3918 upregulated and 2415 downregulated genes in the spleen and 231 upregulated and 65 downregulated genes in the bursa of Fabricius. We confirmed that several core genes related to immune and metabolic pathways were activated by AFB1. Furthermore, 42 and 19 differentially expressed miRNAs were found in the spleen and the bursa of Fabricius, respectively. Differentially expressed genes and target genes of differentially expressed miRNAs were mainly associated with cancer progression and immune response. The predicted mRNA–miRNA pathway network illustrated the potential regulatory mechanisms. The present study identified the transcriptional profiles and revealed potential mRNA–miRNA pathway crosstalk. This genetic regulatory network will facilitate the understanding of the immunotoxicity mechanisms of chicken immune organs induced by high concentrations of AFB1.

Skeletal muscle is a heterogeneous tissue that is essential for initiating movement and maintaining homeostasis. The genesis of skeletal muscle is an integrative process that lasts from embryonic development to postnatal stages, which is carried out under the modulation of many factors. Recent studies have shown that circular RNAs (circRNAs), a class of non-coding RNAs, are involved in myogenesis. However, more circRNAs and their mechanisms that may regulate skeletal muscle development remain to be explored. Through in-depth analysis of our previous RNA-Seq data, circFNDC3AL was found to be a potentially functional circRNA highly expressed during embryonic development of chicken skeletal muscle. Therefore, in this study, we investigated the effect of circFNDC3AL on skeletal muscle development in chickens and found that circFNDC3AL promoted chicken skeletal muscle satellite cell (SMSC) proliferation and differentiation. To gain a thorough understanding of the exact modulatory mechanisms of circFNDC3AL in chicken skeletal muscle development, we performed target miRNA analysis of circFNDC3AL and found that circFNDC3AL has a binding site for miR-204. Subsequently, we demonstrated that miR-204 inhibited chicken SMSC proliferation and differentiation, which showed the opposite functions of circFNDC3AL. Furthermore, we identified the miR-204 target gene B-cell CLL/lymphoma 9 (BCL9) and validated that miR-204 had an inhibitory effect on BCL9, while the negative effect could be relieved by circFNDC3AL. In addition, we verified that BCL9 performed the same positive functions on chicken SMSC proliferation and differentiation as circFNDC3AL, as opposed to miR-204. In conclusion, our study identified a circRNA circFNDC3AL that upregulates BCL9 expression to promote the proliferation and differentiation of chicken SMSCs by binding to miR-204.