Effects of fecal microbiota transplantation and fecal virome transplantation on LPS-induced intestinal injury in broilers.

The interesting roles and efficiencies of fecal microbiota transplantation (FMT) have attracted considerable attention and have been gradually evidenced in specific animal models. While the growing evidence that bacteriophages play roles in FMT efficacy has attracted considerable interest. In this study, we aimed to explore the effects of FMT and fecal virome transplantation (FVT) in improving inflammatory damage and ileal microbiota disorder in broilers. A total of 224 Arbor Acres broilers were selected at 1-day-old and randomly divided into the following 4 groups, with 56 broilers in each group: the CON group (the negative control group, sterile physiological saline injection + sterile phosphate-buffered saline (PBS) solution gavage), LPS group (the positive control group, lipopolysaccharide (LPS) injection + sterile PBS solution gavage), LPS + FMT group (LPS injection + FMT solution gavage), LPS + FVT group (LPS injection + FVT solution gavage). The results showed that: LPS injection significantly upregulated the mRNA expression levels of IFN-γ (P < 0.05) and IL-8 (P < 0.001) in ileal mucosa of broilers at 11th day of age (D11), while LPS + FMT and LPS + FVT did not; LPS injection significantly upregulated the mRNA expression of ZO-1 in ileal mucosa at D11 (P < 0.01), while LPS + FMT and LPS + FVT did not; at D11, compare to CON group, LPS injection and LPS + FMT significantly increased the relative abundance of virulence factor Rab2 interacting conserved protein A-related genes in broiler ileum contents (P < 0.05), while LPS + FVT had no significant difference with CON group (P > 0.05); at D11, LPS injection significantly downregulated the biosynthesis of antibiotics pathway (P < 0.05) in the ileal contents, while LPS + FVT did not. In conclusion, both FMT and FVT could promote the recovery of inflammation caused by LPS. Furthermore, FVT had shown less disadvantage stimulation on the broilers and could reduce the risk of transmission of pathogenic genes, compared to FMT.

Fecal virus transplantation has more moderate effect than fecal microbiota transplantation on changing gut microbial structure in broiler chickens.

Growing evidence of fecal microbiota transplantation (FMT) and fecal virus transplantation (FVT) provides a possibility to regulate animal health, whereas little is known about the impact of the 2 methods. This study aimed to investigate the effects of gut microbes on jejunal function in healthy broiler chickens, with the objective of establishing a theoretical basis for the application of FMT and FVT. Cecal feces from 28-day-old AA broilers were collected to prepare gavage juice for FMT and FVT. FMT for Group FM, FVT for group FV and PBS gavage for group CON, continuously treated for 6 days start at 5-day-old chicks. Samples were collected at d 11 and d 21. The results showed that the treatment d 2 and the overall fecal score in treatment groups were significantly lower than CON group (P < 0.05). The jejunum morphology showed that FMT increased crypt depth, decreased villus height, V/C (P < 0.05) and FVT increased villus height (P < 0.05) at d 11. At d 21, villus height and crypt depth significantly higher (P < 0.05) in group FM and group FV. The expression of Claudin1, Occludin, ZO2, and Muc2 in the FV group was significantly increased (P < 0.05) at 11-day-old. FMT increased the secretion of sIgA at 11-day-old, and this influence lasted up to 21-day-old (P < 0.05). At 11-day-old, the expression of b0+AT of basic amino acid transport carrier and chymotrypsin activity (P < 0.05) had a significant correlation. At 21 d of age, FVT significantly increased the expression of PepT1 and SGLT1 (P < 0.05). At 11-day-old, FM group showed significantly higher faith pd index (P = 0.004) and Shannon index (P = 0.037), and separated from FV and CON according to PCoA. Among differentiating bacteria, Bacteroides significantly enriched (P < 0.05) in group FM, which positively correlated with the expression of ZO2, Muc2, Occludin, and Claudin1; R_Ruminococcus, L_Ruminococcus, Butyricicoccuss significantly enriched (P < 0.05) in group CON, which significantly higher than processing groups, R_Ruminococcus and L_Ruminococcus negatively correlated with the expression of Occludin (P < 0.05), and R_Ruminococcus, Butyricicoccus negatively correlated with the expression of Claudin1 (P < 0.05). At 21-day-old, PCoA based on Bray-Curtis shows that microbes taxa of 3 groups are isolated with each other and treatment groups were significant different with CON group based on Unweighted UniFrac and weighted UniFrac. The expression of PepT1 was significantly negatively (P < 0.05) correlated with Ruminococcus, and the expression of sIgA was significantly negatively (P < 0.05) correlated with Parabacteroides. In conclusion, FMT regulated intestinal flora rapidly, while it had little effect on intestinal function and a higher potential damaging risk on jejunal. FVT regulated intestinal flora structure softer, improved tight junction expression, but the mechanism of action needs further exploration.

Acer truncatum leaves extract modulates gut microbiota, improves antioxidant capacity, and alleviates lipopolysaccharide-induced inflammation in broilers.

This study investigated the appropriate way of dietary Acer truncatum leaves (ATL) addition, the effect of disease prevention and its mechanism of action. In experiment 1, 192 Arbor Acres broilers were assigned to 4 treatment groups, fed with basal diets containing 2% bran, replacing it with primary and fermented ATL, and additional 0.3% ATL extract to the basal diet for 42 d, respectively. In experiment 2, 144 broilers were assigned to 3 treatment groups for 21-d trial: (1) C-N group, basal diets, and injected with 0.9% (w/v) sterile saline; (2) C-L group, basal diets, and injected with lipopolysaccharide (LPS); (3) T-L group, ATL diets and injected with LPS. In experiment 1, ATL significantly decreased the index of abdominal fat at 42 d (P < 0.05). ATL extract had a better ability to improve antioxidant capacity and reduce inflammatory levels among all treatment groups, which significantly decreased the content of MDA in the liver and ileum mucosa at 21 d, and increased the expression of IL-10 and Occludin in jejunal mucosa at 42 d (P < 0.05). In experiment 2, ATL significantly increased the level of T-AOC in the liver, decreased the expression of NF-κB in the jejunal mucosa and ileum mucosa (P < 0.05), and restored LPS-induced the changed level of CAT in jejunal mucosa, the expression of IL-6, Claudin-1, and ZO-1 in jejunal mucosa and IL-1ß in ileum mucosa (P < 0.05). Analysis of gut microbiota indicated that ATL enhanced the abundances of Bacteroidota and reduced the proportion of Firmicutes (P < 0.05), and the changed levels of T-AOC in body, IL-1ß, IL-6, IL-10, and NF-κB in jejunum mucosa and propionic acid in cecal were associated with gut microbiota. Collectively, our data showed that the extract of ATL had a better antioxidant and anti-inflammatory effects than primality and fermented. Extraction of ATL modulated intestinal microbiota, and had a protective effect on oxidative stress, inflammation, and intestinal barrier function in broilers challenged with LPS.

Effects of high-dose folic acid on protein metabolism in breast muscle and performance of broilers.

Attaining the optimal feed conversion ratio is the unaltered goal for poultry breeding, meat yield is one of the vital reference indexes for that. Folic acid is involved in protein metabolism by acting as a transmitter of one carbon unit, and the detail mechanism for the high-dose folic acid on growth of broiler skeletal muscle is still unclarified. The present study was conducted to investigate the effect and regulatory mechanism of folic acid on deposition and metabolism of protein in broiler breast muscle. A total of 196 one-day-old AA broilers were randomly assigned to 2 treatment groups. The chicks were fed corn-soybean diet with folic acid levels of 1.3 mg/kg (CON) or 13 mg/kg (FA), respectively. The results showed that high dose of folic acid significantly increased the body weight gain, average daily gain, average daily feed intake, and feed conversion ratio of broilers during 1 to 42 d. Compared with control group, folic acid statistically augmented the breast muscle ratio of broilers at 42 d, abdominal fat percentage was also decreased in FA group. Folic acid significantly increased the gene expression of folate receptor (FR) in duodenum and jejunum at 21 d, and its relative expression in jejunum of broilers at 42 d. Furthermore, relative expression of myogenin in broiler breast muscle was upregulated in folic acid group. Folic acid supplementation significantly enhanced the protein expression of phosphorylated serine/threonine kinase (AKT) and ribosomal protein S6 kinase 1 (S6K1) in the breast muscle of broilers at 21 d and 42 d. In conclusion, the results proved that high-dose folic acid activated the AKT/mammalian target of rapamycin (mTOR) pathway and increased the activity of phosphorylation of S6K1, thereby regulating the protein deposition in breast muscle. Meanwhile, the gene expression of the myogenic determinant factor was upregulated by folic acid and then promoted the growth of breast muscle. Consequently, the growth performance, meat production and feeding efficiency were improved of broilers by adding folic acid at 13 mg/kg.

Effects of in ovo feeding and dietary addition oils on growth performance and immune function of broiler chickens.

This study aimed to investigate the effects of in ovo feeding (IOF) and dietary addition (DA) oils on growth, development and immune function of broiler chickens. In experiment 1, a total of 500 eggs were randomly assigned to 3 treatments: non-injected group (CON) with 100 eggs; soybean oil injected group (SO) with 200 eggs and linseed oil injected group (LO) with 200 eggs. Results showed that there were no detrimental effects of IOF of oils on embryonic development. In experiment 2, a two factor experimental design was adopted. After hatching, 120 chicks which came from each oil-injected group were divided into 2 treatments with 6 replicates, and chickens were fed soybean oil diet and linseed oil diet, respectively. The results showed that DA linseed oil increased final body weight (FBW) of broilers at d 21 post hatch, IOF of linseed oil decreased average daily feed intake (ADFI) and feed conversion ratio (FCR) of broilers from d 1 to 21 (P < 0.05), while the plasma leptin level of 21-day-old broilers was increased by IOF or DA linseed oil (P < 0.05). Main effect analysis showed that DA linseed oil increased the spleen index and mRNA expression of IFN-γ in spleen of broilers at 7 d of age (P < 0.05). IOF of linseed oil upregulated the mRNA expression of IFN-γ in the spleen of chicks at 1 d and mRNA expression of IL-2 and IL-4 in spleen of broilers at 21 d (P < 0.05), and the interaction effect showed that IOF and DA linseed oil synergically increased the expression of IL-2 and IL-4 in spleen of broilers at 21 d. Compared with SO group, LO increased the Shannon index of hatching-day cecum microflora (P < 0.05). Principal co-ordinates analysis (PcoA) showed that LO group clearly separated from CON and SO groups. Finally, Spearman correlation analysis also manifested that Alkalicoccus was significantly correlated with spleen index and mRNA expression of IL-2, and Phreatobacter was significantly correlated with the mRNA expression of IL-2 and IFN-γ in spleen, Acinetobacter had a positive correlation with thymus index (P < 0.05). In conclusion, IOF of linseed oil reduced the ADFI and FCR of broilers and increased the species diversity and changed the structure of cecal microflora of chicken embryos at the 19th day of incubation (E19). Immune function of broilers spleen was also regulated by IOF and DA linseed oil.

Gut microbiota mediates the protective role of Lactobacillus plantarum in ameliorating deoxynivalenol-induced apoptosis and intestinal inflammation of broiler chickens.

The protection of Lactobacillus plantarum JM113 against deoxynivalenol (DON)-induced apoptosis and intestinal inflammation on the jejunum of broiler chickens and the potential roles of gut microbiota were determined. A total of 144 one-day-old male broilers (Arbor Acres) were randomly divided into 3 treatment groups consisting of 6 replicates with 8 birds per replicate, including the CON (basal diet), the DON (basal diet + 10 mg/kg DON), and the DL (basal diet + 10 mg/kg DON + 1 × 109 CFU/kg L. plantarum JM113). The DON-diet decreased (P < 0.05) the mRNA expression of mucosal defense proteins and mechanistic target of rapamycin pathway genes. Meanwhile, DON challenge significantly increased Bcl-2-associated X gene/B-cell lymphoma 2 gene (Bcl-2) in the jejunum (P < 0.05) and demonstrated proapoptosis status. In contrast, the DL group showed normal immunity-related gene expression of jejunal mucosa and manifested a superior antiapoptosis status. Adding L. plantarum JM113 significantly raised (P < 0.05) propionic acid, n-butyric acid, and total short-chain fatty acids concentrations in cecal contents of birds fed with DON diet. In addition, DON exposure altered bacterial community structure and disturbed the abundance of several bacterial phyla, families, and genera, leading to dysbiosis. Supplementation with JM113 shifted the gut microbiota composition to that of the CON group. Finally, Spearman correlation analysis suggested that most positive correlations with the mRNA expression of immunity-related and apoptosis-regulatory gene were observed within the phylum Bacteroidetes, and most negative correlations with the indicators were observed within the phylum Firmicutes. The mRNA expression of Bcl-2, TLR2, mTOR, Raptor, and RPS6KB1 (P < 0.05), which are regarded as important cell proliferation and antiapoptosis parameters, were significantly negatively associated with the relative abundances of norank_f__Erysipelotrichaceae, Subdoligranulum, and Anaeroplasma, whereas they had a strong positive correlation with Ruminococcaceae_UCG-004, Alistipes, and Ruminococcaceae_NK4A214_group. These results implied that L. plantarum JM113 supplementation could ameliorate DON-induced apoptosis and intestinal inflammation via manipulating the bacterial community composition and could be used as a potential candidate to attenuate intestinal impairments.

Effects of encapsulated essential oils and organic acids on laying performance, egg quality, intestinal morphology, barrier function, and microflora count of hens during the early laying period.

The objective of this study was to investigate the effect of encapsulated essential oils and organic acids (EOA) on the growth performance, egg quality, intestinal morphology and functions, and microbial count of laying hens from week 21 to 30. A total of five hundred and four 21-wk-old layers were randomly allotted into 4 groups consisting of 7 replicates with 18 birds per replicate. The birds were fed a basic diet (CON) or diets with EOA at 150 mg/kg, 300 mg/kg, and 450 mg/kg in the other 3 groups, respectively. Compared to the CON group, the addition of 150 mg/kg EOA significantly increased laying rate (P < 0.05) of hens from week 21 to 25. A linear increasing (linear, P < 0.01) in ileal villus height of laying hens fed EOA from 150 to 300 mg/kg was observed at week 30. At week 25, the supplementation of 300 mg/kg EOA significantly increased (P < 0.05) mRNA relative expression of aminopeptidase, sodium-glucose cotransporter 1, and Na+-independent neutral amino acid transporter in duodenum and glucose transporter 2 in jejunum of laying hens compared to the CON groups. Meanwhile, the relative expression of glucose transporter 2 mRNA in the jejunum was upregulated with increasing concentration of EOA in diets (linear, P < 0.05). Hens in EOA 300 group had higher mRNA relative expression of mucin-2 in ileum (P < 0.05) than hens in CON group. Additionally, the secretory immunoglobulin in ileum A were linear decreased (linear, P < 0.01) with the increasing supplement of EOA. Dietary supplementation with EOA tended to increase (P = 0.083) the counts of Bifidobacterium in cecal digesta at week 25 and 30. In conclusion, dietary with EOA may maintain intestinal tract morphology and promote digestive and absorptive capacities and barrier function, especially at 300 mg/kg. This study provided evidence of using EOA as a potential feed additive for laying hens.

Systematic analysis of the regulatory roles of microRNAs in postnatal maturation and metergasis of liver of breeder cocks.

The liver function of chickens is intensively remodeled from birth to adult, which was validated by metabolomics research in the present study. In order to understand the roles of microRNAs (miRNA) in liver maturation and metergasis, miRNA expression profiles in livers of 20 male chicks aged one day and five adult cocks aged 35 weeks were determined. A total of 191 differentially expressed miRNAs with the criteria of P < 0.05 and fold changes either >1.5 or <0.67 and 32 differentially expressed miRNAs with the criteria of false discovery value (FDR) < 0.05 and fold changes either >1.5 or <0.67 were detected. Subsequently, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses of the targets revealed that candidate miRNAs may involve in the regulation of hepatic metabolism and immune functions, and some pathways including cell cycle which were implicated in postnatal liver development. Furthermore, 1211 differentially expressed mRNAs (messenger RNA) in livers between the postnatal and matured chickens were used to define the roles of differentially expressed miRNAs in regulating the expression of target genes. Our results revealed the first miRNA profile related to the adaption of mature liver functions after birth in breeder cock.

Effect of dietary Astragalus Polysaccharide supplements on testicular piRNA expression profiles of breeding cocks.

Astragalus Polysaccharide (APS), as the main active ingredient of Astragalus membranaceus, has extensive biological activities related to immune, metabolic, and anti-oxidative regulatory processes. Previous studies have proven that piRNAs could play important roles in genital gland. This study aimed to identify the differentially expressed piRNAs in chicken testes in response to dietary APS supplements and further evaluate the roles of these piRNAs related to the effect of dietary APS supplements on testicular changes. We generated piRNA expression profiles of testes from breeding cocks fed without or with extra APS. As results, there were 42 up-regulated and 86 down-regulated piRNAs in APS group, compared with the control group meeting the criteria of P<0.05 and fold change <0.67 or fold change >1.5. The potential targets were subsequently annotated against the Kyoto Encyclopedia of Genes and Genomes databases. The results revealed that dietary APS supplements could regulate tight junction pathways by regulating the piRNA expression profiles, which were related to the regulation of a better testicular condition for spermatogenesis. Our results provided a novel insight into the effect of dietary APS supplements on testicular piRNA expression profiles and its potential roles in testicular condition regulation.

Epigenetic regulation of pro-inflammatory cytokine genes in lipopolysaccharide -stimulated peripheral blood mononuclear cells from broilers.

Inflammatory response which can be mediated by inflammatory genes, can be induced by pathogenic microorganisms, be associated with enteric diseases and the loss of growth performance in broilers. The understanding of epigenetic regulation of inflammatory genes could help explain the response to infection of microorganisms and inhibit the reaction of inflammation in broilers. This study investigated the effect of histone acetylation by histone deacetylases (HDAC) inhibitors trichostain A (TSA) and DNA methylation by demethylation agent 5-Aza-2'-deoxycytidine (AZA) and methyl donor methionine (Met) and folic acid (FA) on the expression of pro-inflammatory cytokines in lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMC) from healthy broilers. The results showed that the mRNA expression of IL-1ß, IL-6 and TNF-α can be down-regulated by pre-treatment of TSA in LPS-stimulated broiler PBMC. The expression of pro-inflammatory cytokines related with the expression of HDAC7 and HDAC10 which can influence histone acetylation, and may also be affected by increasing the acetylation of non-histone proteins. The demethylation by AZA increased the expression of IL-6 and TNF-α in LPS-stimulated broiler PBMC. The addition of FA and Met decreased the expression of DNA methyltransferases (DNMT)1 and DNMT3a, while the Met also down-regulated the expression of IL-6 and TNF-α in LPS-stimulated cells. In addition, the Met administration increased the methylation of -191 CpG site (up-stream from transcription start site) of IL-6 and -419 CpG site of TNF-α. This study indicated that the expression of pro-inflammatory cytokines is regulated by protein acetylation. Demethylation also increased the expression of IL-6 and TNF-α, which can be regulated by Met through increasing the promoter methylation. These results may have implications for controlling inflammation by epigenetic regulation in broilers.