Molecular evolution of the ATP-binding cassette subfamily G member 2 gene subfamily and its paralogs in birds.

BACKGROUND: ATP-binding cassette (ABC) transporters are involved in the active transportation of various endogenous or exogenous substances. Two ABCG2 gene subfamily members have been identified in birds. A detailed comparative study of the ABCG2 and ABCG2-like genes aid our understanding of their evolutionary history at the molecular level and provide a theoretical reference for studying the specific functions of ABCG2 and ABCG2-like genes in birds. RESULTS: We first identified 77 ABCG2/ABCG2-like gene sequences in the genomes of 41 birds. Further analysis showed that both the nucleic acid and amino acid sequences of ABCG2 and ABCG2-like genes were highly conserved and exhibited high homology in birds. However, significant differences in the N-terminal structure were found between the ABCG2 and ABCG2-like amino acid sequences. A selective pressure analysis showed that the ABCG2 and ABCG2-like genes were affected by purifying selection during the process of bird evolution. CONCLUSIONS: We believe that multiple members of the ABCG2 gene subfamily exist on chromosome 4 in the ancestors of birds. Over the long course of evolution, only the ABCG2 gene was retained on chromosome 4 in birds. The ABCG2-like gene on chromosome 6 might have originated from chromosome replication or fusion. The structural differences between the N terminus of ABCG2 protein and those of ABCG2-like proteins might lead to functional differences between the corresponding genes.

A 14-bp insertion in endothelin receptor B-like (EDNRB2) is associated with white plumage in Chinese geese.

BACKGROUND: Gang goose is a native species with gray plumage in Sichuan, China. As a result of overhunting, the number of gray Gang geese has decreased dramatically. To keep the species from extinction, conservation work for Gang geese was undertaken. In the process of pure breeding of gray Gang geese, approximately 2% of the offspring of each generation were white. This study aims to explain the genetic mechanism of this phenomenon and provide reliable molecular markers for goose-related plumage color breeding. RESULTS: We used the method of pooled whole genome sequencing and Fst (fixation statistics) to identify the differentiation degree of alleles between gray Gang geese and white Gang geese from their offspring. In this way, EDNRB2, a key gene that affects the migration of melanoblasts, was identified. Then, the transcriptome was sequenced for the two geese plumage color populations, and the DEGs (differentially expressed genes) were analyzed. The results indicated that EDNRB2, as a possible candidate gene, had a significantly differential mRNA expression. In addition, a 14-bp insertion (NW_013185915.1: g. 750,748-750,735 insertion. CACAGGTGAGCTCT) in exon 3 of EDNRB2 was analyzed and found to have a significant association between gray geese and Chinese white breeds (P = 0.00), while this mutation was not found in European geese. Meanwhile, the insertion was homozygous in all the white geese we detected and heterozygous in gray geese, indicating that this mutation is recessive. Furthermore, this 14-bp insertion leads to a frameshift mutation in the EDNRB2 coding region and nonsense-mediated mRNA decay. CONCLUSION: Our study strongly suggests that the 14-bp insertion in exon 3 of the EDNRB2 gene is associated with the white plumage phenotype in Chinese geese. This study is the first to investigate the relationship between EDNRB2 and white plumage in geese.

Supplementing Ryegrass Ameliorates Commercial Diet-Induced Gut Microbial Dysbiosis-Associated Spleen Dysfunctions by Gut-Microbiota-Spleen Axis.

The type and composition of food strongly affect the variation and enrichment of the gut microbiota. The gut-microbiota-spleen axis has been developed, incorporating the spleen's function and maturation. However, how short-chain fatty-acid-producing gut microbiota can be considered to recover spleen function, particularly in spleens damaged by changed gut microbiota, is unknown in geese. Therefore, the gut microbial composition of the caecal chyme of geese was assessed by 16S rRNA microbial genes, and a Tax4Fun analysis identified the enrichment of KEGG orthologues involved in lipopolysaccharide production. The concentrations of LPS, reactive oxygen species, antioxidant/oxidant enzymes, and immunoglobulins were measured from serum samples and spleen tissues using ELISA kits. Quantitative reverse transcription PCR was employed to detect the Kelch-like-ECH-associated protein 1-Nuclear factor erythroid 2-related factor 2 (Keap1-Nrf2), B cell and T cell targeting markers, and anti-inflammatory/inflammatory cytokines from the spleen tissues of geese. The SCFAs were determined from the caecal chyme of geese by using gas chromatography. In this study, ryegrass-enriched gut microbiota such as Eggerthellaceae, Oscillospiraceae, Rikenellaceae, and Lachnospiraceae attenuated commercial diet-induced gut microbial alterations and spleen dysfunctions in geese. Ryegrass significantly improved the SCFAs (acetic, butyric, propionic, isovaleric, and valeric acids), AMPK pathway-activated Nrf2 redox signaling cascades, B cells (B220, CD19, and IgD), and T cells (CD3, CD4, CD8, and IL-2, with an exception of IL-17 and TGF-ß) to activate anti-inflammatory cytokines (IL-4 and IL-10) and immunoglobulins (IgA, IgG, and IgM) in geese. In conclusion, ryegrass-improved reprogramming of the gut microbiota restored the spleen functions by attenuating LPS-induced oxidative stress and systemic inflammation through the gut-microbiota-spleen axis in geese.

Artificial Pasture Grazing System Attenuates Lipopolysaccharide-Induced Gut Barrier Dysfunction, Liver Inflammation, and Metabolic Syndrome by Activating ALP-Dependent Keap1-Nrf2 Pathway.

INTRODUCTION: Geese can naturally obtain dietary fiber from pasture, which has anti-inflammatory and antioxidant properties. This study aimed to investigate the inhibitory impacts of pasture on ameliorating LPS-ROS-induced gut barrier dysfunction and liver inflammation in geese. Materials and methods. The lipopolysaccharides (LPS), alkaline phosphatase (ALP), reactive oxygen species (ROS), tight junction proteins, antioxidant enzymes, immunoglobulins, and metabolic syndrome were determined using ELISA kits. The Kelch-like-ECH-associated protein 1-Nuclear factor erythroid 2-related factor 2 (Keap1-Nrf2) and inflammatory cytokines were determined using the quantitative reverse transcription PCR (RT-qPCR) method. The intestinal morphology was examined using the Hematoxylin and Eosin (H&E) staining method in ileal tissues. Results. Pasture significantly influences nutrient absorption (p < 0.001) by ameliorating LPS and ROS-facilitated ileal permeability (p < 0.05) and systemic inflammation (p < 0.01). Herein, the gut permeability was paralleled by liver inflammation, which was significantly mimicked by ALP-dependent Nrf2 (p < 0.0001) and antioxidant enzyme activation (p < 0.05). Indeed, the correlation analysis of host markers signifies the importance of pasture in augmenting geese's health and production by averting gut and liver inflammation. Conclusions. Our results provide new insight into the mechanism of the pasture-induced ALP-dependent Nrf2 signaling pathway in limiting systemic inflammation in geese.

Evidence Supporting the Regulatory Relationships through a Paracrine Pathway between the Sternum and Pectoral Muscles in Ducks.

Muscles and bones are anatomically closely linked, and they can conduct communication by mechanical and chemical signals. However, the specific regulatory mechanism between the pectoral muscle and sternum in birds was largely unknown. The present study explored the potential relationship between them in ducks. The result of the sections showed that more nuclei in proliferate states were observed in the pectoral muscle fibers attached to the calcified sternum, than those attached to the un-calcified sternum. The RNA-seq identified 328 differentially expressed genes (DEGs) in the sternum between the calcified and un-calcified groups. Gene ontology (GO) showed that the DEGs were mainly enriched in pathways associated with calcification. In addition, DEGs in the muscles between the calcified and un-calcified sternum groups were mainly annotated to signal transduction receptor pathways. The expression patterns of genes encoding for secreted proteins, in bone (CXCL12, BMP7 and CTSK) and muscle (LGI1), were clustered with muscle development (MB) and bone calcification (KCNA1, OSTN, COL9A3, and DCN) related genes, respectively, indicating the regulatory relationships through a paracrine pathway existing between the sternum and pectoral muscles in ducks. Together, we demonstrated that the pectoral muscle development was affected by the sternal ossification states in ducks. The VEGFA, CXCL12, SPP1, NOG, and BMP7 were possibly the key genes to participate in the ossification of the duck sternum. We firstly listed evidence supporting the regulatory relationships through a paracrine pathway between the sternum and pectoral muscles in ducks, which provided scientific data for the study of the synergistic development of bone and skeletal muscle.

Effect of organic acids on growth performance, intestinal morphology, and immunity of broiler chickens with and without coccidial challenge.

A total of 360-day-old broiler chicks were allocated into six groups in 2 (Coccidial challenge or not) × 3 (dietary treatments) factorial design. Three dietary treatments including: basic diet, basic diet plus organic acids (OAs) in drinking water, and basic diet plus OAs in the feed with and without coccidial challenge. The OAs in water or feed improved (P < 0.01) average body weight (ABW), average body weight gain (ABWG), and feed conversion ratio (FCR) as compared with the control diet during starter, grower, and whole experimental period. Coccidial challenge decreased BW, ABWG, and average feed intake (AFI), as well as resulted in poor FCR during the starter and whole experimental period (P < 0.05). Though there was no interaction between OAs supplementation and coccidial challenge, the OAs supplementation improved the overall performance with and without coccidial challenge birds on 21 d and 35 d. IgG was found higher (P = 0.03) in broilers fed OAs in feed without the coccidial challenge group. On 18 d, OAs supplementation in feed increased TNF-γ (P = 0.006), whereas the coccidial challenge decreases TNF-γ (P = 0.01) and IL-10 (P = < .0001), and increases IgM (P = 0.03), IgG (P = 0.04) and IgA (P = 0.02). On 29 d, the coccidial challenge increases IgM and IgA. On 18 d, jejunal lesion score was found significantly higher in the coccidial challenged group as compared to OAs supplementation with coccidial challenged groups on 18 d (P < 0.0001) and 29 d (P = 0.03). Crypt depth was higher, and Villus-height to Crypt depth ratio was lower in the coccidial challenge group on 18 and 29 d. The Goblet cells were found higher in the non-coccidial challenge on 18 d. After 18 d, 16S rDNA gene sequence analysis of ileal chyme has shown that coccidial challenge decreases Lactobacillus_reuteri species as compared to the non-challenged group (P = 0.02). After 29, Cyanobacteria abundance reduced (P = 0.014) in the challenged group than the non-challenged group at the phylum level. At the genus level, Lactobacillus (P = 0.036) and unidentified Cyanobacteria (P = 0.01) were found higher in the non-challenged group than the coccidial challenge group. The results indicate that the OAs supplementation showed improved responses in a pattern similar to the non-challenged control group by neutralizing the negative effects of the coccidial challenge.

Limitation and Potential Effects of Different Levels of Aging Corn on Performance, Antioxidative Capacity, Intestinal Health, and Microbiota in Broiler Chickens.

Three-hundred and sixty-day-old male broilers underwent three treatments with six replicates of 20 birds per treatment. The experimental diets included NC: normal corn diet; ACL: lower level (39.6-41.24%) of AC; and ACH: a higher level (56.99-59.12%) of AC. During phase 1 (0-21 d), broilers fed on AC showed lower (p < 0.05) body weight (BW), body weight gain (BWG), and feed conversion ratio (FCR) as compared with the NC group. During phase 2 (22-42 d), the NC group and ACL group showed better (p < 0.05) BW, BWG, and FCR than the ACH group. The footpad lesion score (p = 0.05) and litter moisture percentage (p < 0.05) were found to be higher in the ACH group. During phase 1, the ACL group showed a lower level of malondialdehyde (MDA) contents (p < 0.05) in serum; moreover, catalase (CAT) (p < 0.05) and glutathione peroxidase (GSH-Px) activities (p < 0.05) were found lower in both AC-containing groups. During phase 2, CAT activity in serum was found higher (p < 0.05) in the ACH group. During phase 1, the NC group showed higher CAT (p = 0.05), GSH-Px (p < 0.05), and superoxide dismutase (SOD) activity (p = 0.03); however, it showed lower MDA (p < 0.05) and total-antioxidative capability (T-AOC) (p < 0.05) in the liver. During phase 1, in breast muscle, CAT, SOD, and T-AOC were higher (p < 0.05) in the NC group. During phase 1, total cholesterol and high-density lipoprotein were found to be lower (p < 0.05) in the ACL group. Similarly, triglyceride and low-density lipoprotein were found to be lower (p < 0.05) in the ACL group than the ACH group. During phase 1, villus height was found to be higher (p < 0.05) in the ACH group. Moreover, the goblet cell (GC) was found to be higher (p < 0.05) in the NC group than the ACL group. During phase 2, GC was found to be higher (p < 0.05) in the ACL group. In ileal digesta, during phase 1, acetic acid, propionic acid, and butyric acid (BA) levels were found to be higher (p < 0.05) in the ACL group. In cecal digesta, BA was significantly lower (p < 0.05) in the NC group.

Heavy Metal Resistance in Salmonella Typhimurium and Its Association With Disinfectant and Antibiotic Resistance.

Metals are widely used in animal feed for their growth-stimulating and antimicrobial effects, yet their use may potentially promote the proliferation of antibiotic resistance through co-selection. We studied the prevalence and associations of metal, antibiotic, and disinfectant resistances of 300 Salmonella Typhimurium isolates from pig meat, pig manure, chicken meat, poultry manure, and human stool from Sichuan, China. Seventy four percent of the 300 Salmonella Typhimurium isolates were considered resistant to Cu, almost 50% to Zn and Cr, over 25% to Mn and Cd, and almost 10% to Co. Most of the isolates carried at least one heavy metal resistance gene (HMRG). The Cr-Zn-Cd-resistance gene czcD was carried by 254 isolates and the Cu-resistance genes pcoR and pcoC by 196 and 179 isolates, respectively. Most of the isolates were resistant to at least one antibiotic and almost 80% were multidrug-resistant. The prevalence of resistance to six antibiotics was higher among the pig meat and manure isolates than among other isolates, and that of streptomycin and ampicillin were highest among the pig meat isolates and that of ciprofloxacin and ofloxacin among the pig manure isolates. From 55 to 79% of the isolates were considered resistant to disinfectants triclosan, trichloroisocyanuric acid, or benzalkonium chloride. The metal resistances and HMRGs were associated with resistance to antibiotics and disinfectants. Especially, Cu-resistance genes were associated with resistance to several antibiotics and disinfectants. The transfer of the Cr-Zn-Cd-resistance gene czcD, Cu-resistance gene pcoC, and Co-Ni-resistance gene cnrA into Escherichia coli and the increased Cu-resistance of the transconjugants implied that the resistance genes were located on conjugative plasmids. Thus, the excessive use of metals and disinfectants as feed additives and in animal care may have the potential to promote antibiotic resistance through co-selection and maintain and promote antibiotic resistance even in the absence of antibiotics.

Understanding the Genetic Domestication History of the Jianchang Duck by Genotyping and Sequencing of Genomic Genes Under Selection.

The Jianchang duck is mainly distributed in Southwest China, and has the characteristics of fast growth rate and strong abilities in lipid deposition in the liver. In order to investigate the effects of domestication process on formation of the unique characteristics of Jianchang duck, the whole genome of sixteen individuals and three pooling of Jianchang duck were re-sequenced, and genome data of 70 mallards and 83 domestic ducks from thirteen different places in China were obtained from NCBI. The population stratification and evolution analysis showed gene exchanges existed between the Jianchang and other domestic duck populations, as well as Jianchang ducks and mallards. Genomic comparison between mallards and Jianchang ducks showed genes, including CNTN1, CHRNA9, and SHANK2, which is involved in brain and nerve development, experienced strong positive selection in the process of Jianchang duck domestication. The genomic comparison between Jianchang and domestic duck populations showed that HSD17B12 and ESM1, which affect lipid metabolism, experienced strong positive selection during the domestication process. FST analysis among populations of Jianchang duck with different plumage colors indicated that MITF was related to the phenotype of a white feather, while MC1R was related to the phenotype of hemp feather. Our results provided a base for the domestication process of Jianchang duck and the genomic genes for unique traits.

Evidences in duck (Anas platyrhynchos) by transcriptome data for supporting the biliverdin was mainly synthesized by shell gland.

The blue-shelled egg not only plays a key role in helping birds to avoid predation as a result of crypsis and mimetism, but it also provides eggshell strength and filters solar radiation; moreover, it has an important economic trait for poultry. However, the source of biliverdin for blue-shelled egg remains unsolved in ducks. The current study detected the biliverdin content and localization of heme oxygenase 1 (HMOX1) in duck shell gland; moreover, RNA-seq analysis was performed in the shell gland of blue-shelled and white-shelled ducks. Results indicated that biliverdin is a primary pigment for blue-shelled egg in ducks, and the HMOX1 protein showed high expression in ciliated epithelial cells of shell gland between blue-shelled and white-shelled ducks. In the pathway of biliverdin synthesis, only 5-aminolevulinate synthase 1 expression level was significantly upregulated in blue-shelled ducks, and nuclear factor, erythroid 2 like 1 and period circadian clock 2 may be the essential elements in biliverdin synthesis of duck shell gland. Furthermore, some of the transporter genes, such as activator-Like and solute carrier family 13 member 5, may be involved in the formation of blue egg in duck. Results of the current study suggested that the biliverdin is most likely synthesized and secreted from epithelial cells of shell gland. In addition, ALAS1 may play a key role in the formation of blue egg in ducks.

Transcriptome reveals B lymphocyte apoptosis in duck embryonic bursa of Fabricius mediated by mitochondrial and Fas signaling pathways.

As a central immune organ unique to birds, the bursa of Fabricius (BF) provides a proper microenvironment for B-cell development. The bursal B-cells undergo rapid proliferation and differentiation at the embryonic stages, but 95% of them undergo apoptosis after hatching. Few studies have focused on the cause of bursal B-cells apoptosis at the embryonic stages in birds. To explore the cause, we compared the transcriptional profiles of three characteristic embryonic stages in duck, including embryonic day 14 (ED14), 22 (ED22) and 1 day after hatching (D1). Our results showed that the apoptotic B-cells were first observed at ED22 while there were no apoptotic B-cells at ED14. By performing enrichment analysis for DEGs and qRT-PCR, our results demonstrated that both mitochondrial and Fas signaling pathways mediated bursal B-cell apoptosis during the duck embryonic development. Further, protein-protein interactions (PPIs) and KEGG enrichment analysis together showed that BMP4, FoxO1 and IGF-1 may regulate bursal B-cells apoptosis. In addition, the DEGs showed two stage-specific expression patterns. By analyzing the genes of two expression patterns, the results indicated that B-cell false differentiation may be one of the reasons of apoptosis in the duck embryonic BF. Overall, these data demonstrated that from ED14-ED22, apoptosis of bursal B-cells was mediated by mitochondrial and Fas signaling pathways and could be regulated by BMP4, FoxO1 and IGF-1 in duck. One of the primary causes of bursal B-cell apoptosis may be false differentiation in B-cells.

Potential and limitation of a new defatted diatom microalgal biomass in replacing soybean meal and corn in diets for broiler chickens.

Three experiments were conducted to determine if defatted diatom Staurosira sp. biomass (DFA) (Cellana, Kailua-Kona, HI, USA) from biofuel production could replace a portion of soybean meal (SBM) and (or) corn in diets for broiler chicks. In experiment 1, 2-day-old chicks were fed diets with DFA at 0% (control), 7.5% replacing SBM, or 7.5 and 10% replacing SBM and corn. Chicks fed the DFA-containing diets had lower body weight gain (P < 0.05) than the controls in the starter period. Two follow-up experiments, experiments 2 and 3, indicated that supplementing the 7.5% DFA diet (replacing SBM) with amino acids, but not exogenous protease or electrolytes, restored growth performance of chicks to the control levels. Responses of plasma and liver biomarkers and gross examination of digestive tract showed no toxicity of DFA. In conclusion, DFA could substitute for 7.5% of SBM alone, or in combination with corn, in diets for broiler chicks when appropriate amino acids are added.