Effects of different addition levels of CHM-JM113 on growth performance, antioxidant capacity, organ index, and intestinal health of AA broilers.

The purpose of the present study was to investigate the effects of different levels of a Chinese herbal medicine formulation combined with JM113 (CHM-JM113) on growth performance, antioxidant capacity, organ index, and intestinal health of AA broilers. The AA broiler chicks were randomly allocated to 5 treatments as follows: a basic diet for the control group, the basic diet supplemented with 0.25% CHM-JM113, 0.5% CHM-JM113, 1% CHM-JM113 and 2% CHM-JM113 for the treatment group, respectively. The results showed that the addition of CHM-JM113 to the diet significantly reduced the mortality (p < 0.01) and improved the European Broiler Index (EBI) (p < 0.05), whereas it had no significance on growth performance of AA broilers (p > 0.05). Comparing the control group, 0.5 and 1% CHM-JM113 group significantly improved the organ index of liver, spleen and bursa (p < 0.05). In terms of intestinal morphology and structure, the addition of different levels of CHM-JM113 increased VH and VH/CD ratio, decreased CD in the small intestine compared to the control group, with 1 and 2% of the additive dose being more effective (p < 0.05). Chinese herbal medicine and probiotics as natural antioxidants also significantly increased the content of SOD in serum of 21-day-old broilers (p < 0.01), and significantly decreased the content of MDA in serum (p < 0.01). At 42 days of age, the addition of 1 and 2% CHM-JM113 significantly increased the content of SOD (p < 0.01) and significantly decreased the content of MDA in the organism (p < 0.01), accompanied by a significant increase in T-AOC and CAT content. In the study of the effect of CHM-JM113 on intestinal immunity, compared with the control group, we found that 1% or 2% CHM-JM113 had a better effect on the expression of occludin and claudin-1 in the intestinal segments of broilers (p < 0.05). For the expression of GATA-3, 0.5% CHM-JM113 may have a better effect (p < 0.05). CHM-JM113 may be used as an antibiotic alternative in broiler production.

Integrated transcriptomic analysis on chicken ovary reveals CYP21A1 affects follicle granulosa cell development and steroid hormone synthesis.

Egg production is an economically important trait in poultry breeding and production. Follicular development was regulated by several hormones released and genes expressed in the granulosa cells, impacting the egg production and fecundity of hens. However, the molecular functions of these candidate genes that modulate these processes remain largely unknown. In the present study, bioinformatics analyses were performed to identify the candidate genes related to egg production in the ovarian tissue of White Leghorns with high egg production and Beijing You chicken with low egg production during sexual maturity and peak laying periods. The ovarian granulosa cells were used to assess the function of CYP21A1 by transfecting with CYP21A1-specific small interfering RNAs (siRNAs) and overexpression plasmids. We identified 514 differentially expressed genes (|Log2(fold change) | >1, P <0.05) between the 2 chicken breeds in both laying periods. Among these genes, CYP21A1, which is involved in the steroid hormone biosynthesis pathway was consistently upregulated in White Leghorns. Weighted gene co-expression network analysis (WGCNA) further suggested that CYP21A1 was a hub gene, which could positively respond to treatment with follicle stimulation hormone (FSH), affecting egg production. The interference of CYP21A1 significantly inhibited cell proliferation and promoted cell apoptosis. Overexpression of CYP21A1 promotes cell proliferation and inhibits cell apoptosis. Furthermore, the interference with CYP21A1 significantly downregulated the expression of STAR, CYP11A1, HSD3B1, and FSHR and also decreased the synthesis of progesterone (P4) and estradiol (E2) in granulosa cells. Overexpression of CYP21A1 increased the synthesis of P4 and estradiol E2 and the expression of steroid hormone synthesis-related genes in granulosa cells. Our findings provide new evidence for the biological role of CYP21A1 on granulosa cell proliferation, apoptosis, and steroid hormone synthesis, which lays the theoretical basis for improving egg production.

Extracellular vesicle-coupled miRNA profiles of chicken seminal plasma and their potential interaction with recipient cells.

The presence of EVs in seminal plasma (SPEVs) suggests their involvement on fertility via transmitting information between the original cells and recipient cells. SPEVs-coupled miRNAs have been shown to affect sperm motility, maturation, and capacitation in mammals, but rarely in poultry species. The present study aims to reveal the profile of SPEVs miRNAs and their potential effect on sperm storage and function in poultry. The SPEVs was successfully isolated from 4 different chicken breeds by ultracentrifugation and verified. Deep sequencing of SPEVs small RNA library of each breed identified 1077 miRNAs in total and 563 shared ones. The top 10 abundant miRNAs (such as miR-10-5p, miR-100-5p, and miR-10a-5p etc.) accounted for around 60% of total SPEVs miRNA reads and are highly conserved across species, predisposing their functional significance. Target genes prediction and functional enrichment analysis indicated that the most abundantly expressed miRNAs may regulate pathways like ubiquitin-mediated proteolysis, endocytosis, mitophagy, glycosphingolipid biosynthesis, fatty acid metabolism, and fatty acid elongation. The high abundant SPEVs-coupled miRNAs were found to target 107 and 64 functionally important mRNAs in the potential recipient cells, sperm and sperm storage tubules (SST) cells, respectively. The pathways that enriched by target mRNAs revealed that the SPEVs-coupled miRNA may rule the fertility by affecting the sperm maturation and regulating the female's immune response and lipid metabolism. In summary, this study presents the distinctive repertoire of SPEVs-coupled miRNAs, and extends our understanding about their potential roles in sperm maturation, capacitation, storage, and fertility, and may help to develop new therapeutic strategies for male infertility and sperm storage.

miR-140-3p promotes follicle granulosa cell proliferation and steroid hormone synthesis via targeting AMH in chickens.

Granulosa cells (GCs) are the ovary's most critical cells since they undergo cell differentiation and hormone synthesis changes closely associated with follicle development. While micro RNA 140-3p (miRNA-140-3p) has an apparent cell signaling role, particularly in cell proliferation, its biological role in chicken ovarian follicle growth and development remains elusive. This study explored miR-140-3p's effects on chicken GC proliferation and steroid hormone synthesis. MiR-140-3p dramatically increased GC proliferation, prevented apoptosis, increased progesterone synthesis, and enhanced gene expression related to steroid hormone synthesis. In addition, the anti-Müllerian hormone (AMH) gene was identified as a direct miR-140-3p target. MiR-140-3p abundance correlated negatively with AMH mRNA and protein levels in GCs. Our findings show that miR-140-3p influences chicken GC proliferation and steroid hormone synthesis by suppressing AMH expression.

An integrative analysis of lncRNAs and mRNAs highlights the potential roles of lncRNAs in the process of follicle selection in Taihang chickens.

Taihang chickens are a domestic breed distributed throughout Hebei province in the Taihang Mountains of China and are characterized by their high meat and egg quality. However, the relatively limited egg production by this breed constrains their more widespread commercial utilization. The follicle selection process is closely linked to oocyte development and ovulation, making it a key determinant of laying performance and fecundity in hens. To understand the biological basis for such follicle selection and to identify the associated regulatory pathways, we conducted a genome-wide analysis of long noncoding RNAs (lncRNAs) and mRNAs from the pre-hierarchical follicles and hierarchical follicles of Taihang laying hens. We identified 81 lncRNAs and 528 mRNAs that were differentially expressed during follicle selection, and integrated network analyses suggested that these RNAs were associated with the cell cycle, focal adhesion, oocyte meiosis, peroxisome proliferator-activated receptor signaling, and steroid hormone biosynthesis pathways. The identified lncRNAs were also predicted to influence a series of target genes in cis and trans, suggesting that they may be important regulators of ovarian follicular development. Overall, the present analysis of lncRNA and mRNA expression patterns associated with ovarian follicle development offers a new foundation for future studies of reproductive physiology in Taihang chickens, highlighting new opportunities to improve the laying performance of this important domestic breed.

Virulence factors and molecular characteristics of Shigella flexneri isolated from calves with diarrhea.

BACKGROUND: The natural hosts of Shigella are typically humans and other primates, but it has been shown that the host range of Shigella has expanded to many animals. Although Shigella is becoming a major threat to animals, there is limited information on the genetic background of local strains. The purpose of this study was to assess the presence of virulence factors and the molecular characteristics of S. flexneri isolated from calves with diarrhea. RESULTS: Fifty-four S. flexneri isolates from Gansun, Shanxi, Qinghai, Xinjiang and Tibet obtained during 2014 to 2016 possessed four typical biochemical characteristics of Shigella. The prevalences of ipaH, virA, ipaBCD, ial, sen, set1A, set1B and stx were 100 %, 100 %, 77.78 %, 79.63 %, 48.15 %, 48.15 and 0 %, respectively. Multilocus variable number tandem repeat analysis (MLVA) based on 8 variable number of tandem repeat (VNTR) loci discriminated the isolates into 39 different MLVA types (MTs), pulsed field gel electrophoresis (PFGE) based on NotI digestion divided the 54 isolates into 31 PFGE types (PTs), and multilocus sequence typing (MLST) based on 15 housekeeping genes differentiated the isolates into 7 MLST sequence types (STs). CONCLUSIONS: The findings from this study enrich our knowledge of the molecular characteristics of S. flexneri collected from calves with diarrhea, which will be important for addressing clinical and epidemiological issues regarding shigellosis.

Exploring the Prevalence and Distribution Patterns of Antibiotic Resistance Genes in Bovine Gut Microbiota Using a Metagenomic Approach.

Antibiotic resistance genes (ARGs) have become recognized contaminants and pose a high public health risk. The animal gut microbiota is a reservoir of ARGs, but the knowledge of the origin and dissemination of ARGs remains unclear. In this study, we provide a comprehensive profile of ARGs and mobile genetic elements in the gut microbiota from 30 bovines to study the impact of modern antibiotics on resistance. A total of 42 ARG types were detected by annotating the metagenomic sequencing data from Comprehensive Antibiotic Resistance Database (CARD). We found that the diversity and abundance of ARGs in individual yaks were significantly lower than those in dairy and beef cattle (p < 0.0001). The results of heat map and single nucleotide polymorphism clustering suggest that ARGs from dairy and beef cattle are more similar, whereas those from yaks cluster separately. The long-term use of antibiotics may contribute to this difference, suggesting that antibiotic consumption is the main cause of ARG prevalence. Furthermore, abundant insertions were also found in this study, signifying a strong potential for horizontal transfer of ARGs among microbes, especially pathogens.

Effect of chronic cyclic heat stress on the intestinal morphology, oxidative status and cecal bacterial communities in broilers.

The objective of this study was to examine the effects of chronic cyclic heat stress (HS) on the intestinal morphology, oxidative stress and cecal bacterial communities of broilers. One-day-old Arbor Acres (AA) male broilers (n = 100) were acclimated for 3 weeks and then randomly allocated into two groups, normal control (NC) group (22 ± 1 °C, 24 h/day) and HS group (32 ± 1 °C, 10 h/day lasted for 2 weeks). At 35 d of age, intestinal segments (duodenum, jejunum and ileum) and cecal digesta were collected for detection. HS affected intestinal morphology, inducing epithelial cell abscission, inflammatory cell infiltration, and lamina propria edema. Compared with the NC group, HS significantly decreased (P < 0.01) villus height (VH) and the VH-to-crypt depth (CD) ratio (VCR), increased (P < 0.05) CD in the duodenum and ileum, but had no effect on the VH in the jejunum. Moreover, HS induced oxidative stress with antioxidant enzymes activity decreasing (P < 0.05) while malondialdehyde (MDA) content increasing (P < 0.05) in small intestine. Pearson's correlation analysis indicated that MDA content was negatively correlated with VH (P < 0.05). The result of 16S rRNA sequencing showed that HS exposure impacted cecal microbiota alpha diversity (phylogenetic diversity whole-tree index) and beta diversity. Based on principal coordinate analysis (PCoA) plots for weighted UniFrac metrics and unweighted pair group method with arithmetic mean (UPGMA), there were 8 discriminative features at the genus level (linear discriminant analysis score > 2). Parabacteroides, Saccharimonas, Romboutsia and Weissella were reduced, while Anaerofustis, Pseudonocardia, Rikenella and Tyzzerella were enriched in heat-stressed broilers. Collectively, these results indicated that chronic cyclic HS induced oxidative stress that caused damage to intestinal villus-crypt structures, and then altered the cecal microflora profile.

Naturally occurring genetic mutations in the 5'-upstream regulatory region of bovine FSHB generate a novel cis-regulatory element that affects its expression.

We previously reported that numerous naturally occurring genetic mutations in the 5'-upstream regulatory region (5'-URR) of the bovine follicle-stimulating hormone beta-subunit gene (FSHB) were associated with reduced serum follicle-stimulating hormone (FSH) levels, poor-quality semen and low fertility in bulls. In addition, two different FSHB mRNA transcripts resulting from the linked mutations of genomic DNA were discovered in mutation-bearing bull pituitaries. Here, using electrophoretic mobility shift assay, we identified c.-1539_-1538delGGinsTTAACT mutations in the 5'-URR that generated a novel cis-regulatory element in bovine FSHB. Moreover, this novel element seemed to play a role in repressing FSHB transcription based on a promoter activity analysis in LßT2 gonadotrope cells. Quantitative assays of FSHB mRNA in the bovine pituitaries suggested that the levels of FSHB wild-type transcripts in the mutation-bearing bulls were significantly lower (P < 0.05) than in those of bulls without FSHB genetic mutations and that the levels of FSHB-mutated transcripts were significantly lower (P < 0.05) than that of wild-type transcripts in the mutation-bearing bulls. Altogether, our results suggest that decreased serum FSH levels and male fertility in bulls with the c.-1539_-1538delGGinsTTAACT mutation likely result from the alteration of cis-regulatory elements and induction of FSHB transcription.

Folic acid-functionalized mesoporous silica nanospheres hybridized with AIE luminogens for targeted cancer cell imaging.

Fluorescent nanoparticles (FNPs) have been found to be useful as visualization tools for biological sensing, probing, imaging, and monitoring. Applied to targeted cancer cell imaging, FNPs are highly desirable for early stage cancer diagnosis and treatment. However, the light emission from most of the FNPs reported is severely limited because of the aggregation-caused quenching (ACQ) effect. Herein, we present highly emissive inorganic-organic nanoparticles with core-shell structures for targeted cancer cell imaging. Coated with a folate-functionalized silica shell, 9,10-distyrylanthracene (DSA) fluorogens with aggregation-induced emission (AIE) properties served as the fluorescent core, affording folate-functionalized fluorescent silica nanoparticles (FFSNPs) with a high fluorescence quantum yield (up to 20%). The FFSNPs are of small size (diameter ~60 nm), monodispersed, stable in aqueous suspension, and pose little toxicity to living cells and thus can be utilized for targeted HeLa cell imaging. In addition, the FFSNPs are mesoporous and therefore can potentially be used as vehicles for controlled, externally activated release of anticancer drugs.

Microarray analysis of differentially expressed microRNAs in non-regressed and regressed bovine corpus luteum tissue; microRNA-378 may suppress luteal cell apoptosis by targeting the interferon gamma receptor 1 gene.

MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules that down-regulate the expression of target genes in a sequence-dependent manner. Recent studies indicated that miRNAs are mechanistically involved in the regulation of the mammalian corpus luteum (CL). However, few studies have profiled the different miRNA expression patterns in bovine non-regressed and regressed CL. In this study, miRNA microarray was employed to investigate the different miRNA expression patterns in bovine CL. Among the 13 differentially expressed miRNAs, seven were preferentially expressed in non-regressed CL, while six miRNAs were more highly expressed in regressed CL. Real-time RT-PCR was used to validate the microarray results. Mir-378 miRNA, known to be associated with apoptosis, was 8.54-fold (P < 0.01) up-regulated in non-regressed CL, and the interferon gamma receptor 1 (IFNGR1) gene, which potentially plays a role in apoptosis of the luteal cell, was predicted to be the target of mir-378. The results of real-time RT-PCR of mir-378 and western blot analysis of the IFNGR1 protein at different stages of CL development showed that mir-378 decreased the expression of IFNGR1 protein but not IFNGR1 mRNA. Taken together, our data support a direct role for miRNA in apoptosis of bovine CL.

[Genetic variation in the 5' flanking region of bovine TLR4 gene and correlation with mastitis].

Toll-like receptor 4 recognizes pathogen ligands and mediates signaling to initiate innate and adaptive immune responses. In this experiment, a 477 bp segment of the 5'-flanking region of TLR4 gene of Chinese Holstein, Sanhe cattle and Chinese simmental was amplified by polymerase chain reaction. After sequencing, a polymorphic site in amplified production of TLR4 was identified of having either a G or a C at position 245. This polymorphism in the three populations was detected by digesting the fragment with restriction endonuclease Msp I. Results showed that both alleles (A and B) were found in the three populations and the value of polymorphism information content indicated that this was a moderate polymorphism. Chi2 test indicated that the polymorphism locus in Sanhe cattle did not fit Hardy-Weinberg equilibrium (P< 0.05). In addition, the effect of the TLR4 polymorphism on somatic cell score was analyzed, and the results indicated that the somatic cell score were significantly affected by lactation month and the type of breeds(P<0.05), but not by different genotypes (P > 0.05).