Transcriptome analyses of potential regulators of pre- and post-ovulatory follicles in the pigeon (Columba livia).

To identify the dominant genes controlling follicular maturation, ovulation and regression for pigeon, we used RNA-seq to explore the gene expression profiles of pre- and post-ovulatory follicles of pigeon. We obtained total of 4.73million (96% of the raw data) high-quality clean reads, which could be aligned with 20282 genes. Gene expression profile analysis identified 1461 differentially expressed genes (DEGs) between the pre- (P4) and post-ovulatory follicles (P5). Of these, 843 genes were upregulated, and 618 genes were down-regulated. Furthermore, many DEGs were significantly enriched in some pathways closely related to follicle maturation, ovulation and regression, such as ECM-receptor interaction, vascular smooth muscle contraction, progesterone-mediated oocyte maturation, phagosome. Importantly, the DGEs in ECM-receptor interaction pathway included COL1A1 , COL1A2 , COL4A1 , COL4A2 , ITGA11 , ITGB3 and SDC3 , in the progesterone-mediated oocyte maturation pathway involved CDK1 , CDC25A , CCNB3 , CDC20 and Plk1 , and in the vascular smooth muscle contraction covered CALD1 , KCNMA1 , KCNMB1 , CACNA1 , ACTA2 , MYH10 , MYL3 , MYL6 , MYL9 , closely related to promoting follicular maturation and ovulation in pre-ovulatory follicles. Moreover, it seems that the lysosomal cathepsin family has a decisive role in the regression of early stage of post-ovulatory follicle. Taken together, these data enrich the research of molecular mechanisms of pigeon follicular activities at the transcriptional level and provide novel insight of breeding-related physiology for birds.

Transcriptome analyses to reveal the dynamic change mechanism of pigeon magnum during one egg-laying cycle.

We analyzed the transcriptome of pigeon magnum in three stages (C1: pre-ovulation, C2: post-ovulation, C3: 5-6 days after ovulation) to elucidate the molecular and cellular events associated with morphological changes during the laying cycle. We observed that C1 was highly developed, apoptosis rate was highest in C2, and C3 attained the smallest size. Through RNA-sequencing, we obtained 54,764,938 (97.2%) high-quality clean reads that aligned to 20,767 genes. Gene expression profile analysis showed the greatest difference between C1 and C3; 3966 differentially expressed genes (DEGs) were identified, of which 2250 genes were upregulated and 1716 genes were downregulated in C1. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that protein processing and transport activities were prominent in C1, and upregulated genes included those related to signal recognition particle (SRP), signal recognition particle receptor (SRPR), translocon, GRP78, RRBP1, TRAP, TRAM1, and OST. Egg white protein-related gene expression was highest, with OVALY being the most highly expressed. In C2, apoptosis-related gene expression was higher than in C1, and fatty acid metabolism was active, which may be correlated with magnum tissue regression. Collagen- and laminin-related gene expression was prominent in C1 and C3, indicating roles in egg white protein generation and magnum reconstruction. PR gene expression was highest and exhibited drastic change in the three groups, indicating that PR and its regulation may be involved in changes in magnum morphology and function. Through the identification and functional analysis of DEGs and other crucial genes, this may contribute to understand the egg white protein production, magnum tissue regression, and magnum regeneration mechanisms.

Changes in morphology and miRNAs expression in small intestines of Shaoxing ducks in response to high temperature.

During summer days the extreme heat may cause damage to the integrity of animal intestinal barrier. Little information is available concerning morphological changes in the duck intestines in response to high temperature. And the molecular mechanisms underlying the pathogenesis of high temperature-induced intestinal injury remain undefined. MicroRNAs (miRNAs) are known to play key roles in post-transcriptional regulation of gene expression that influences various biological processes. The purpose of this study was to explore the changes in morphology and miRNA expression profiles of the three intestinal segments (duodenum, jejunum and ileum) of ducks in response to high temperature. Sixty female Shaoxing ducks (Anas platyrhynchos), 60 days old, were allocated in two groups, including control ducks kept at 25 °C, and ducks subjected to high ambient temperatures of 30-40 °C for 15 successive days, which mimicked the diurnal temperature variations experienced in hot seasons. Three ducks from each group were executed at the end of feeding experiment, and the samples of three intestinal segments were collected for morphological examination and Illumina deep sequencing analyses. Histopathological examination of the intestinal mucous membrane was performed with HE staining method. The results demonstrated that varying degrees of damage to each intestinal segment were found in heat-treated ducks, and there were more severe injuries in duodenum and jejunum than those in ileum. Illumina high-throughput sequencing and bioinformatic methods were employed in this study to identify the miRNA expression profile of three different intestinal tissues in control and heat-treated ducks. A total of 75,981,636, 88,345,563 and 100,179,422 raw reads were obtained from duodenum, jejunum and ileum, respectively, from which 74,797,633 clean reads in duodenal libraries, 86,406,445 clean reads in jejunal libraries, and 98,518,858 lean reads in ileal libraries were derived after quality control, respectively. And a total of 276 known and 182 novel miRNAs were identified in the three intestinal segments of ducks under control and heat-treated conditions. By comparing the same tissues in different conditions, 16, 18 and 15 miRNAs were found to be significantly differentially expressed between control and heat-treated ducks in duodenum, jejunum and ileum, respectively, of which 1 miRNA was expressed in both the duodenum and jejunum, 2 miRNAs were expressed in both the duodenum and ileum, and 3 miRNAs were found to be expressed in both the jejunum and ileum. In addition, two differentially expressed miRNAs in each comparison were randomly selected and validated by quantitative qRT-PCR. Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that the differentially expressed miRNAs may be involved in the high temperature-induced intestinal injury in ducks. Our work provides the comprehensive miRNA expression profiles of small intestines in the normal and heat-treated ducks. These findings suggest the involvement of specific molecular mechanisms of post-transcriptional regulation to explain the high temperature-induced changes in the duck small intestine.

MiR-144 affects fatty acid composition by regulating ELOVL6 expression in duck hepatocytes.

Lipid metabolism in duck is very important for both raisers and people's health. In our previous studies, we have detected that miR-144 is related to duck lipid metabolism and validated one of its target genes, elongation of very long chain fatty acids protein 6 (ELOVL6). In the present study, we isolated, cultured, and identified duck hepatocytes, and transfected with miR-144 mimics/inhibitor to mediate the miR-144 level. The qRT-PCR results showed that the ELOVL6 expression in duck hepatocytes was down/upregulated, respectively. The fat contents and each fatty-acid percent content of the hepatocytes and medium were also determined. When ELOVL6 expression suppressed (miR-144 mimics transfected), the palmitic acid (C16:0) content was significantly increased (P < 0.05); the oleic acid (C18:1, n-9), eicosenoic acid (C20:1, n-9), and eicosatrienoic acid (C20:3) contents were significantly reduced (P < 0.05). The myristic acid (C14:0) and palmitic acid (C16:0) contents were significantly reduced (P < 0.05), and the oleic acid (C18:1, n-9) content was significantly increased (P < 0.05) when ELOVL6 expression upregulated (miR-144 inhibitor transfected). It indicated that miR-144 could regulate some saturated fatty acids elongated to longer unsaturated fatty acids through controlling ELOVL6 expression. Whereas, miR-144/ELOVL6 appeared not associated with fat deposition in duck hepatocytes (P > 0.05). Our findings suggest that miR-144 might regulate the percentages of fatty acids in duck hepatocytes through affecting ELOVL6 expression.

Transcriptomic analysis of different stages of pigeon ovaries by RNA-sequencing.

The pigeon ovary is an ideal model for deciphering the molecular mechanism of folliculogenesis. While most analysis has focused on the influence of hormones and factors on ovarian follicle development in this model, changes occurring in the ovarian stroma can also be extremely informative. Here, we profiled the transcriptome of pigeon ovaries at pre-ovulation, post-ovulation, and 5-6 days after ovulation using RNA-sequencing to gain insights into the molecular and cellular events mediating ovary activity. We obtained 44,784,505 clean reads that aligned with 14,088 genes. Gene expression profile analysis identified 409 differentially expressed genes between pre- and post-ovulation; 96 genes were up-regulated genes while 313 genes were down-regulated. Gene ontology analysis of the down-regulated genes revealed significant enrichment in components of the immune response, immune system, antigen processing and presentation, receptor binding, and biological adhesion. Pathway analyses of the high-expression genes of the post-ovulation ovary identified enrichment in phagosomes, lysosomes, cytokine-cytokine receptor interactions, cell adhesion molecules, and the Toll-like receptor signaling. These data together suggest that post-ovulatory follicle regression and elimination occurs through an immune response. Mol. Reprod. Dev. 83: 640-648, 2016. © 2016 Wiley Periodicals, Inc.

Identification of differentially expressed known and novel miRNAs in broodiness of goose.

MicroRNAs (miRNAs) are endogenous small noncoding RNAs plays a critical role in posttranscriptional regulation of gene expression. Broodiness is observed in most avian species and influences egg production. Several genes are known to play an important role in regulating the progress of reproduction. The goose is one of the most important waterfowls. However, the involvement of miRNAs in the broodiness behavior of Anser cygnoides (Swan Goose) is unknown. High-throughput sequencing and bioinformatics analysis were used to identify the miRNAs involved in egg-laying and brooding behavior of geese in our study. The results showed 38 up-regulated and 14 down-regulated known miRNAs/miRNA*s with reads>1,000 in at least one group and a fold change of >2.0, compared with those of the egg-laying group (P<0.001). We also identified 114 and 94 novel miRNAs in the broody and egg-laying groups, respectively. Of these, 4 novel miRNAs were differentially expressed between the two groups. The study showed the expression of small RNAs in goose reproduction and identified known and novel miRNAs regulated in broodiness. The results reveal that these differentially expressed miRNAs may be involved in broodiness of A. cygnoides.

Expression pattern of L-FABP gene in different tissues and its regulation of fat metabolism-related genes in duck.

Liver fatty acid binding protein (L-FABP) is a member of intracellular lipid-binding proteins responsible for the transportation of fatty acids. The expression pattern of duck L-FABP mRNA was examined in this study by quantitative RT-PCR. The results showed that duck L-FABP gene was expressed in many tissues, including heart, lung, kidney, muscle, ovary, brain, intestine, stomach and adipocyte tissues, and highly expressed in liver. Several lipid metabolism-related genes were selected to detect the regulation of L-FABP in duck. The expression of L-FABP and lipoprotein lipase was promoted by oleic acid. The L-FABP knockdown decreased the expression levels of peroxisome proliferator-activated receptor α (PPARα), fatty acid synthase and lipoprotein lipase by 61.1, 42.3 and 53.7 %, respectively (P < 0.05), but had no influences on the mRNA levels of PPARγ and leptin receptor. L-FABP might function through the PPARα to regulate the fat metabolism-related gene expression and play important roles in lipid metabolism in duck hepatocytes.

Integration of LC-MS-Based and GC-MS-Based Metabolic Profiling to Reveal the Effects of Domestication and Boiling on the Composition of Duck Egg Yolks.

Egg yolks contain abundant lipids, proteins, and minerals that provide not only essential nutrients for embryonic development but also cheap sources of nutrients for consumers worldwide. Previous composition analyses of egg yolks primarily focused on nutrients such as lipids and minerals. However, few studies have reported the effects of domestication and heating on yolk composition and characteristics. The objective of this study was to investigate the impact of domestication and boiling on the metabolite contents of egg yolks via untargeted metabolomics using GC-MS and LC-MS. In this study, eggs were collected from Fenghua teals, captive mallards, and Shaoxing ducks. Twelve duck eggs (half raw and half cooked) were randomly selected from each variety, and the egg yolks were separated for metabolic profiling. The analysis identified 1205 compounds in the egg yolks. Domestication generated more differential metabolites than boiling, which indicated that the changes in the metabolome of duck egg yolk caused by domestication were greater than those caused by boiling. In a comparative analysis of domestic and mallard ducks, 48 overlapping differential metabolites were discovered. Among them, nine metabolites were upregulated in domesticated ducks, including monoolein, emodin, daidzein, genistein, and glycitein, which may be involved in lipid metabolism; some of them may also act as phytoestrogens (flavonoids). Another 39 metabolites, including imethylethanolamine, harmalan, mannitol, nornicotine, linoleic acid, diphenylamine, proline betaine, alloxanthin, and resolvin d1, were downregulated by domestication and were linked to immunity, anti-inflammatory, antibacterial, and antioxidant properties. Furthermore, four overlapping differential metabolites that included amino acids and dipeptides were discovered in paired comparisons of the raw and boiled samples. Our findings provided new insights into the molecular response of duck domestication and supported the use of metabolomics to examine the impact of boiling on the composition of egg yolks.

A novel SNP of liver-type fatty acid-binding protein gene in duck and its associations with the intramuscular fat.

Liver-type fatty acid-binding protein (L-FABP) is a member of intracellular lipid-binding proteins involved in the transportation of fatty acids. We detected the polymorphisms of duck L-FABP gene and its association with the intramuscular fat (IMF) and other fat-related traits. The complete sequence of duck L-FABP gene (four exons and three introns, 2,542 bp) was obtained in this study. The polymorphism of L-FABP gene was examined with direct DNA sequencing method in 231 individuals from different breeds, and a novel single nucleotide polymorphism in the exon 3 was detected. The polymorphism was shown to be associated with the contents of C16:0, C18:3 and the total IMF in pectoral muscle. The content of C16:0 in genotype CC was significantly higher than CT (P < 0.01) and TT (P < 0.01), and the genotype CT was higher than TT (P < 0.01). The content of C18:3 in genotype TT was significantly higher than CC and CT (P < 0.01), whereas the genotype CC and CT had no significant difference (P > 0.05). The content of IMF in genotype CC was significantly higher than CT (P < 0.01). However, no significant difference was detected between genotype CC and TT or genotype CT and TT (P > 0.05).

Dietary supplementation with fermented plant product modulates production performance, egg quality, intestinal mucosal barrier, and cecal microbiota in laying hens.

Fermented plant product (FPP) is a kind of functional complex containing probiotics and a variety of bioactive substances, which has multiple physiological functions. However, there is no systematic appraisal of FPP as a feed additive for laying hens. This study was conducted to evaluate the utilization of FPP in laying hens. A total of 120 healthy 34-week-old Xianju layers with similar body weight and egg production were randomly allocated into two dietary treatments with four replicates per treatment and 15 birds per replicate for 8 weeks. The dietary treatments included the basal diet without FPP (CON group) and CON diet supplemented with 500 mg/kg of FPP (FPP group). Compared with the CON group, the egg production and egg mass were significantly increased in the FPP group from 38 to 42 and 34 to 42 weeks of age (P < 0.05). Birds fed with the diet containing 500 mg/kg FPP had higher albumen height (P < 0.01) and Haugh unit (P < 0.05) than those of the controls. FPP supplementation significantly increased the villus height (VH) and crypt depth (CD) in the jejunum of laying hens (P < 0.01), as well as the ratio of VH to CD (P < 0.05). The mRNA expression of tight junctions showed that dietary supplementation with FPP significantly increased the expression levels of Occludin (P < 0.01) and ZO-1 (P < 0.05) in jejunum of hens compared to the control group. In addition, dietary supplementation with FPP influenced cecal microbiota of laying hens, which was characterized by the changes in the microbial community composition, including the increased abundances of Firmicutes, Faecalibacterium, Oscillospira, Clostridium, Ruminococcus, and Coprococcus, along with the decreased abundance of Bacteroidetes, Proteobacteria, Phascolarctobacterium, Odoribacter, Desulfovibrio, and Mucispirillum. Spearman's correlation analysis revealed that bacteria such as Faecalibacterium, Ruminococcus, Coprococcus, and Blautia were significantly and positively correlated with the intestinal barrier markers (P < 0.05), with extremely significant correlations between Ruminococcus and ZO-1, and Coprococcus and Occludin (P < 0.01), whereas Desulfovibrio had a negative correlation with the expression of Occludin (P < 0.05). As it can be concluded, FPP supplementation increased the egg production, egg mass, albumen height, and Haugh unit of laying hens, and improved intestinal health by ameliorating intestinal barrier function, which may be partially attributed to the regulation of cecal microbiota. Our findings suggest that FPP has the potential to be used as a feed additive to promote the performance of layers.

Effects of Coated Sodium Butyrate and Polysaccharides From Cordyceps cicadae on Intestinal Tissue Morphology and Ileal Microbiome of Squabs.

This experiment was conducted to investigate the effects of dietary supplementation with different levels of coated sodium butyrate (CSB) and polysaccharides extracted from Cordyceps cicadae (CCP) on growth performance, intestinal tissue morphology and ileum microbiome in squabs. A total of 420 1-day-old squabs were randomly divided into seven groups with 5 replicates each and 12 squabs per replicate. The squabs were fed basal diet (control group) and basal diet supplemented with different levels of CSB (275, 550, and 1,100 mg/kg, groups CSB-275, CSB-550, CSB-1100) and CCP (27.5, 55, and 110 mg/kg, groups CCP-27.5, CCP-55, and CCP-110), respectively. The experiment was conducted for 28 days. The results revealed that the final BW and average daily gain concentration were higher (P < 0.05) in squabs of CSB-275 and CCP-110 groups than those in the CON group. Comparing with control group, the squabs in the groups CSB-275, CSB-550, and CCP-55 obtained higher villus height/crypt depth (VH/CD) of the duodenum and higher VH of the jejunum (P < 0.05). Operational taxonomic units in the groups CSB-550 and CCP-27.5 were also increased (P < 0.05). Regarding the relative abundance of flora, the Actinobacteria abundance in the groups CSB-550, CSB-1100, and CCP-55 were higher than in control group (P < 0.05), and the Aeriscardovia abundance of CSB-275, CSB-550, CSB-1100, and CCP-110 were elevated (P < 0.05). However, the Enterococcus abundance in CSB-275, CSB-550, CSB-1100, and CCP-27.5 decreased (P < 0.05). In summary, results obtained in the present study indicate that CSB and CCP can improve growth performance, intestinal microbial balance and gut health of squabs.

Genome-Wide Association Studies and Haplotype-Sharing Analysis Targeting the Egg Production Traits in Shaoxing Duck.

Age at first egg (AFE) and egg number (EN) are economically important traits related to egg production, as they directly influence the benefits of the poultry industry, but the molecular genetic research that affects those traits in laying ducks is still sparse. Our objective was to identify the genomic regions and candidate genes associated with AFE, egg production at 43 weeks (EP43w), and egg production at 66 weeks (EP66w) in a Shaoxing duck population using genome-wide association studies (GWASs) and haplotype-sharing analysis. Single-nucleotide polymorphism (SNP)-based genetic parameter estimates showed that the heritability was 0.15, 0.20, and 0.22 for AFE, EP43w, and EP66w, respectively. Subsequently, three univariate GWASs for AFE, EP43w, and EP66w were carried out independently. Twenty-four SNPs located on chromosome 25 within a 0.01-Mb region that spans from 4.511 to 4.521 Mb were associated with AFE. There are two CIs that affect EP43w, i.e., twenty-five SNPs were in strong linkage disequilibrium region spanning from 3.186 to 3.247 Mb on chromosome 25, a region spanning from 4.442 to 4.446 Mb on chromosome 25, and two interesting genes, ACAD8 and THYN1, that may affect EP43w in laying ducks. There are also two CIs that affect EP66w, i.e., a 2.412-Mb region that spans from 127.497 to 129.910 Mb on chromosome 2 and a 0.355-Mb region that spans from 4.481 to 4.837 Mb on chromosome 29, and CA2 and GAMT may be the putative candidate genes. Our study also found some haplotypes significantly associated with these three traits based on haplotype-sharing analysis. Overall, this study was the first publication of GWAS on egg production in laying ducks, and our findings will be helpful to provide some candidate genes and haplotypes to improve egg production performance based on breeding in laying duck. Additionally, we learned from a method called bootstrap test to verify the reliability of a GWAS with small experimental samples that users can access at https://github.com/xuwenwu24/Bootstrap-test.

Genome-wide DNA methylation differences between conservation and breeding populations of Shaoxing ducks.

The genome-wide DNA methylation assay was used to analyze the difference in methylation between the breeding and conservation populations of Shaoxing ducks. The methylation level of the breeding population was higher than that of the two conservation populations, and the proportion of CG methylation sites was the largest in the three populations, most of the methylation sites were located in the exon region. There were 1247 different methylation regions in the two populations (group A and B), and 927 different methylation regions in the two groups (group A and group C). The differential methylation regions of the three groups were evenly distributed in the gene and intergene regions. GO and KEGG enrichment analysis showed that the differentially expressed genes in the A and B groups were mainly involved in synaptic and cell connections and the signaling pathways were significantly enriched in cAMP and oxytocin signaling pathways. The results showed that the group C was significantly enriched in eight signaling pathways, including the cAMP signaling pathway and long-term enhancement, compared to the group A. There were thirty-five differentially methylated genes, including CACNA1C, GRIA1, GRIA2, GABBR2, PDE10A, BRAF, GRM5, CPEB3, FMn2, GABRB2, PTK2, and CNTN1. These genes were involved in the development and ovulation of ovaries and follicles and were closely related to the excellent production performance of the breeding population. In addition, ATP2B1, ATP2B2, and other genes related to eggshell quality were identified, which can be used as molecular markers to improve eggshell quality in the future.

Analysis of genome and methylation changes in Chinese indigenous chickens over time provides insight into species conservation.

Conservation of natural resources is a vital and challenging task. Numerous animal genetic resources have been effectively conserved worldwide. However, the effectiveness of conservation programmes and the variation information of species have rarely been evaluated. Here, we performed whole-genome and whole-genome bisulfite sequencing of 90 Chinese indigenous chickens, which belonged to the Tibetan, Wenchang and Bian chicken breeds, and have been conserved under different conservation programmes. We observed that low genetic diversity and high DNA methylation variation occurs during ex situ in vivo conservation, while higher genetic diversity and differentiation occurs during in situ conservation. Further analyses revealed that most DNA methylation signatures are unique within ex situ in vivo conservation. Moreover, a high proportion of differentially methylated regions is found in genomic selection regions, suggesting a link between the effects of genomic variation and DNA methylation. Altogether our findings provide valuable information about genetic and DNA methylation variations during different conservation programmes, and hold practical relevance for species conservation.

Molecular cloning, expression, and regulation of goose leptin receptor gene in adipocytes.

A full-length cDNA encoding the goose (Anser anser) leptin receptor (LEPR) was cloned and sequenced. The goose LEPR gene encodes a 1,156-amino acid protein containing a signal peptide, a single transmembrane domain and specific motifs involving putative leptin-binding and signal transduction. The deduced goose LEPR protein shows more than 90% identity to duck and 75% identity to chicken and turkey. Quantitative real-time analysis reveals that the goose LEPR is predominantly expressed in brain. The expression of LEPR in goose adipocytes can be up-regulated by oleic acid in vitro. Moreover, the expression levels of genes, which have been demonstrated to be related to adipocyte differentiation, are down-regulated in LEPR-knockdown adipocytes, indicating LEPR's potential role in adipocyte differentiation in goose.

Effects of Compound Probiotics on Cecal Microbiome and Metabolome of Shaoxing Duck.

This experiment was conducted to investigate the effects of compound probiotics on intestinal microflora and metabolome of Shaoxing ducks. A total of 640 1-day-old Shaoxing ducks were randomly divided into two treatments with eight replicates and forty ducks for each replicate. The ducks were fed basal diet (Ctrl) and basal diet supplemented with 0.15% compound probiotics (MixP). The experiment lasted for 85 days. The results showed that the abundance of Bacteroidetes and Bacteroides in MixP was higher than that in Ctrl (P < 0.05). However, the abundance of Firmicutes and Oscillospira and Desulfovibrio in MixP was lower than that in Ctrl (P < 0.05). Concentrations of 71 metabolites differed significantly (P < 0.05) between the MixP and the Ctrl groups; for example, Pyridoxal (Vitamin B6), L-Arginine, and Betaine aldehyde were up-regulated (P < 0.05), and 7-oxocholesterol, 3-hydroxy-L-kynureni-ne, and N-acetyl-d-glucosamine were down-regulated (P < 0.05). KEGG was enriched in 15 metabolic pathways. The pathways of Vitamin B6 metabolism, Vascular smooth muscle contraction, Vitamin digestion and absorption, and Protein digestion and absorption were influenced by compound probiotics supplementation. Thus, supplementation of compound probiotics improved cecal heath through shifts in the cecal microbiome and metabolome.

High-temperature exposure alters the community structure and functional features of the intestinal microbiota in Shaoxing ducks (Anas platyrhynchos).

The gut microbiome is a complex ecosystem that contributes to host nutrition and health. However, our current knowledge of the relationship between ambient temperature and gut microbiota of poultry is still limited. The objective of the present study was to characterize the intestinal microbiota of ducks exposed to high ambient temperature. Sixty 60-day-old Shaoxing ducks were allocated to control and heat-treated groups. The ducks in the control group were kept at 25°C, and the ducks in the heat treatment group were raised at 30-40°C, which simulated the temperature change of day and night in summer. After 15 D, the intestinal contents of the duodenum, jejunum, and ileum were obtained from 6 ducks of each group. Genomic DNA was extracted and amplified based on the V4-V5 hypervariable region of 16S rRNA. The results showed that Firmicutes was the dominant bacterial phylum with the highest abundance in the contents of the small intestine of ducks, and the relative abundance of the phylum Firmicutes in all 3 intestinal segments was increased by high temperature. At the genus level, Lactobacillus was found to be the most dominant bacterial genus across 3 gut segments, and its abundance was increased in ducks under heat treatment. Compared with the corresponding intestine segment of control ducks, a total of 36 genera in the duodenum, 19 genera in the jejunum, and 6 genera in the ileum of heat-treated ducks were found to be significantly different in the abundance (linear discriminant analysis score >3.0, P < 0.05). Functional prediction of gut microbiota revealed that high temperature caused changes in the abundance of metabolism and transcription-related pathways. It is noteworthy that most of the altered pathways are related to metabolism. In conclusion, high temperature induced remarkable taxonomic changes in the gut microbiome of ducks, which might be related to the negative effects of high temperature in ducks. Our present study provided an important theoretical ground for high-temperature intervention.

Paternity assessment: application on estimation of breeding value in body-weight at first egg trait of egg-laying duck (Anas platyrhynchos).

Paternity index was analyzed using five microsatellite loci among Chinese egg-laying ducks (Anas platyrhynchos). Based on the paternity relationship that was identified by paternity index analysis, the estimated breeding value (EBV) was calculated using BLUP (best linear unbiased predictor) method. Body weight at first egg (BWF) is the only considered trait in this study. In total, 12 sires, 31 dams and 77 daughters were involved in the EBV calculation. The results demonstrated that five microsatellite loci's polymorphism information content (PIC) ranged from 0.795 in locus AY493338 to 0.957 in locus AY493264 with average 0.899; the parent-offspring relationships were built by these microsatellites' genotype, 12 families of half sibling and 2 families of full sibling were involved, and the relationship error is smaller than 10(-7). The EBV results suggest that the average EBV was significantly higher in females (average EBV is 10.234 and 0.1045 for mother and daughter, respectively) than males (average EBV is just -26.44). The EBV results on BWF were in good agreement with the principle of GH (growth hormone) expression in poultry. These results show that paternity analyses of Chinese egg-laying ducks were basically resolved using the five microsatellite loci selected. The paternity relationships can apply in Chinese egg-laying duck breeding to quicken the improvement of genetic progress.

Different rearing conditions alter gut microbiota composition and host physiology in Shaoxing ducks.

Emerging evidences have linked the gut microbiota to poultry physiology. Gut microbiota composition in Shaoxing ducks were profiled under different rearing conditions: raised on the litter floor and the plastic mesh floor. A total of 46 and 39 luminal content samples from the duodenum, ileum, and cecum of the ducks reared under the two conditions were analyzed by 16S rRNA gene amplicon sequencing analysis. Proteobacteria (48.66%), Proteobacteria (33.38%), and Bacteroidetes (55.35%) were the dominant phyla in the duodenum, ileum, and cecum of the ducks reared on the litter floor respectively, while Firmicutes (30.80%), Firmicutes (66.62%) and Bacteroidetes (47.15%) were the topmost phyla in the duodenum, ileum, and cecum of the ducks reared on the plastic mesh floor. Physiologically, the height of villi and the ratio of villus height to crypt depth in the ileum and duodenum were significantly greater in the ducks reared on plastic mesh floor. Furthermore, our results demonstrate that the gut microbiota was significantly associated with the duck phenotypes, such as chest depth and serum estradiol levels (p < 0.05), which were altered by the different rearing conditions. Collectively, our results showed that the rearing floor types have an important effect on the gastrointestinal microbial composition of ducks.

Assignment of CCR7 gene to chicken chromosome 27 by radiation hybrid panel mapping.

The protein encoded by CC chemokine receptor 7 (CCR7) is a member of the G protein-coupled receptor family. This receptor was identified as a gene induced by the Epstein-Barr virus (EBV), and is thought to be a mediator of EBV effects on B lymphocytes. This receptor is expressed in various lymphoid tissues and activates B and T lymphocytes. It has been shown to control the migration of memory T cells to inflamed tissues, as well as stimulate dendritic cell maturation. To map the CCR7 gene in chicken chromosome, a 6 000 rads chicken-hamster radiation hybrid panel (ChickRH6) was used. PCR of samples from ChickRH6 revealed that the location of CCR7 gene is linked to the maker SEQ0347 (6 cR away) with LOD score of 16.6 and that the marker SEQ0347 is located on chromosome 27 at 27 cR of RH (radiation hydrid) map. We compared the corresponding human mRNA sequence with the predicted coding sequence of chicken CCR7 gene, and found that the assembled contig shared a high percentage of similarity with that of the human gene.