Research Note: Study on the in-situ preservation of pigeons based on the level of endangerment of genetic resources.

The large-scale and intensive development of the meat pigeon breeding industry have resulted in the replacement of a large number of low-performance local breeds by a few breeds with excellent production performance. However, due to the characteristics of pigeon species that are monogamous, for which the W chromosome cannot be recovered and for which semen cannot be cryopreserved, the preservation of pigeon species is still mainly based on in-situ preservation. In this study, pigeons were classified into 6 classes of endangerment based on the criteria of the 100-year inbreeding coefficient of poultry populations in the "Assessment of Endangered Poultry Genetic Resources" (NY/T 2996-2016). The results show that when the generation interval was 1.5 yr, the number of ideal populations with the same gene frequency variance or the same heterozygosity decay rate of pigeons in class 1 to 5 was ≤149, 150 to 204, 205 to 316, 317 to 649 and ≥650. In random-reserved breeding, when the generation interval was 1.5 yr, the number of male (female) pigeons corresponding to class 1 to 5 was ≤74, 75 to 102, 103 to 157, 158 to 324 and ≥325. In family-equal-reserved breeding, when the generation interval was 1.5 yr, the number of male (female) pigeons corresponding to class 1 to 5 was ≤36, 37 to 50, 51 to 78, 79 to 162 and ≥163. When the generation interval was 1.5 yr, the inbreeding increments corresponding to class 1 to 5 were ≥0.00335, 0.00244 to 0.00334, 0.00159 to 0.00243, 0.00078 to 0.00158 and ≤0.00077; with the same population size, the inbreeding coefficient and inbreeding increment decreased with the increase of generation interval; the population effective content, inbreeding coefficient and inbreeding increment of family-equal-reserved pigeons were lower than those of random-reserved pigeons. The results of this study have certain reference value for analyzing the status quo of local and endangered species, constructing live gene banks and breeding farms of poultry genetic resources, and rescuing endangered species.

Enhancing Growth and Gut Health in Squabs: The Impact of Fermented Mixed Feed.

The purpose of this study was to evaluate the effect of fermented mixed feed (FMF) (soybean meal-rapeseed meal-corn bran (6:3:1, m/m/m)) on the growth performance, intestinal microbial communities, and metabolomes of squabs. One hundred and eighty 1-day-old squabs were randomly allocated to two groups, each containing six replicates of fifteen squabs cared for by 60 pairs of breeding pigeons secreting crop milk. Each pair of breeding pigeons cared for three squabs. The control group was fed a basal diet, while the experimental group was fed the basal diet containing 5% FMF. The results showed that daily weight gain, carcass weight, villus height, and the mRNA level of ZO-1 in the ileum were increased in the birds fed FMF compared to the control squabs (p < 0.05). Greater abundances of beneficial bacteria such as Lactobacillus, Bifidobacteria, and Bacillus as well as fewer harmful bacteria (i.e., Enterococcus, Veillonella, and Corynebacterium) in the ilea of squabs fed FMF. Six differential metabolites were identified in the FMF-treated squabs; one metabolite was increased (ω-salicoyisalicin) and five were decreased (3-benzoyloxy-6-oxo-12-ursen-28-oic acid, estradiol-17-phenylpropionate, aminotriazole, phosphatidyl ethanolamine (22:6/0:0), and 1-arachidonoylglycerophosphoinositol). Positive correlations were observed between the abundance of Lactobacillus and villus height. Overall, FMF treatment improved both growth and intestinal health in pigeons, suggesting potential benefits for pigeon production.

Genome-wide association study of growth traits and validation of key mutations (MSTN c.C861T) associated with the muscle mass of meat pigeons.

Selective breeding of meat pigeons is primarily based on growth traits, especially muscle mass (MM). Identification of functional genes and molecular markers of growth and slaughter traits through a genome-wide association study (GWAS) will help to elucidate the underlying molecular mechanisms and provide a theoretical basis for the selective breeding of meat pigeons. The phenotypic data of body weight (BW) and body size (BS) of 556 meat pigeons at 52 and 80 weeks of age were collected. In total, 160 434 high-quality single nucleotide polymorphism sites were obtained by restriction site-associated DNA sequencing. The GWAS analysis revealed that MSTN, IGF2BP3 and NCAPG/LCORL were important candidate genes affecting the growth traits of meat pigeons. IGF2BP3 and NCAPG/LCORL were highly correlated to BW and BS, which are related to overall growth and development, while MSTN was associated with pectoral thickness and BW. Phenotypic association validation with the use of two meat pigeon populations found that the MSTN mutation c.C861T determines the MM. These results provide new insights into the genetic mechanisms underlying phenotypic variations of growth traits and MM in commercial meat pigeons. The identified markers and genes provide a theoretical basis for the selective breeding of meat pigeons.

Lactobacillus salivarius SNK-6 Regulates Liver Lipid Metabolism Partly via the miR-130a-5p/MBOAT2 Pathway in a NAFLD Model of Laying Hens.

Lactobacillus spp., as probiotics, have shown efficacy in alleviating nonalcoholic fatty liver disease (NAFLD). Here, we screened a new probiotic strain, Lactobacillus salivarius SNK-6 (L. salivarius SNK-6), which was isolated from the ileum of healthy Xinyang black-feather laying hens in China. We investigated the beneficial activity of L. salivarius SNK-6 in a NAFLD model in laying hens and found that L. salivarius SNK-6 inhibited liver fat deposition and decreased serum triglyceride levels and activity of aspartate transaminase and alanine transaminase. MBOAT2 (membrane-bound O-acyltransferase domain containing 2) was directly targeted by miR-130a-5p, which was downregulated in the liver of NAFLD laying hens but reversed after L. salivarius SNK-6 treatment. Downregulation of MBOAT2, L. salivarius SNK-6 supplementation in vivo, and L. salivarius SNK-6 cell culture treatment in vitro suppressed the mRNA expression of genes involved in the PPAR/SREBP pathway. In addition, 250 metabolites were identified in the supernatants of L. salivarius SNK-6 culture media, and most of them participated in metabolic pathways, including amino acid, carbohydrate, and lipid metabolism. Targeted metabolomic analysis revealed that acetate, butyrate, and propionate were the most abundant short-chain fatty acids, while cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, and tauroursodeoxycholic acid were the four most-enriched bile acids among L. salivarius SNK-6 metabolites. This may have contributed to the reparative effect of L. salivarius SNK-6 in the NAFLD chicken model. Our study suggested that L. salivarius SNK-6 alleviated liver damage partly via the miR-130a-5p/MBOAT2 signaling pathway.

The miR-216/miR-217 Cluster Regulates Lipid Metabolism in Laying Hens With Fatty Liver Syndrome via PPAR/SREBP Signaling Pathway.

Fatty liver syndrome (FLS), a common metabolic disease in laying hens, caused by excessive hepatic fat deposition is a bottleneck in the poultry industry. However, no specific therapeutic methods have been developed. Evidence suggests that microRNAs (miRNAs) are essential for liver lipid metabolism and homeostasis, providing strong evidence for targeting miRNAs as a potential treatment option for liver diseases. However, the roles of miRNAs in the pathogenesis of FLS remain unclear. In present study, RNA-sequencing was performed to discern the expression patterns of miRNAs in normal and fatty livers of laying hens. In total, 12 dysregulated miRNAs (2 down-regulated and 10 up-regulated) were detected between the normal and fatty livers. Functional enrichment analysis showed the potential impacts of the dysregulated miRNAs on lipid metabolism. Notably, miR-216a/b and miR-217-5p, which belong to the miR-216/miR-217 cluster, were up-regulated in the sera and livers of FLS chickens, as well as free fatty acid (FFA)-induced LMH cells. Oil-red O staining revealed that up-regulation of the miR-216/miR-217 cluster induced lipid accumulation in FFA-induced LMH cells. Furthermore, the dual luciferase gene reporter assay and RT-qPCR analysis demonstrated that 3-hydroxyacyl-CoA dehydratase 2, F-box protein 8, and transmembrane 9 superfamily member 3 (TM9SF3) were directly targeted by miR-216a/b and miR-217-5p, respectively, and suppressed in the fatty livers of laying hens. Moreover, overexpression of the miR-216/miR-217 cluster or reduction in TM9SF3 levels led to activation of the proliferator-activated receptor/sterol regulatory-element binding protein (PPAR/SREBP) pathway. Overall, these results demonstrate that the miR-216/miR-217 cluster regulates lipid metabolism in laying hens with FLS, which should prove helpful in the development of new interventional strategies.

Whole-genome sequencing reveals the artificial selection and local environmental adaptability of pigeons (Columba livia).

To meet human needs, domestic pigeons (Columba livia) with various phenotypes have been bred to provide genetic material for our research on artificial selection and local environmental adaptation. Seven pigeon breeds were resequenced and can be divided into commercial varieties (Euro-pigeon, Shiqi, Shen King, Taishen, and Silver King), ornamental varieties (High Fliers), and local varieties (Tarim pigeon). Phylogenetic analysis based on population resequencing showed that one group contained local breeds and ornamental pigeons from China, whereas all commercial varieties were clustered together. It is revealed that the traditional Chinese ornamental pigeon is a branch of Tarim pigeon. Runs of homozygosity (ROH) and linkage disequilibrium (LD) analyses revealed significant differences in the genetic diversity of the three types of pigeons. Genome sweep analysis revealed that the selected genes of commercial breeds were related to body size, reproduction, and plumage color. The genomic imprinting genes left by the ornamental pigeon breeds were mostly related to special human facial features and muscular dystrophy. The Tarim pigeon has evolved genes related to chemical ion transport, photoreceptors, oxidative stress, organ development, and olfaction in order to adapt to local environmental stress. This research provides a molecular basis for pigeon genetic resource evaluation and genetic improvement and suggests that the understanding of adaptive evolution should integrate the effects of various natural environmental characteristics.

Comparative Genome and Transcriptome Integration Studies Reveal the Mechanism of Pectoral Muscle Development and Function in Pigeons.

Pigeon breed resources provide a genetic model for the study of phenomics. The pectoral muscles play a key role for the meat production performance of the meat pigeon and the athletic ability of the High flyers. Euro-pigeons and Silver King pigeons are commercial varieties that exhibit good meat production performance. In contrast to the domestication direction of meat pigeons, the traditional Chinese ornamental pigeon breed, High flyers, has a small and light body. Here, we investigate the molecular mechanism of the pectoral muscle development and function of pigeons using whole-genome and RNA sequencing data. The selective sweep analysis (F ST and log2 (θπ ratio)) revealed 293 and 403 positive selection genes in Euro-pigeons and Silver King, respectively, of which 65 genes were shared. With the Silver King and Euro-pigeon as the control group, the High flyers were selected for 427 and 566 genes respectively. There were 673 differentially expressed genes in the breast muscle transcriptome between the commercial meat pigeons and ornamental pigeons. Pigeon genome selection signal combined with the breast muscle transcriptome revealed that six genes (SLC16A10, S100B, SYNE1, HECW2, CASQ2 and LOC110363470) from commercial varieties of pigeons and five genes (INSC, CALCB, ZBTB21, B2M and LOC110356506) from Chinese traditional ornamental pigeons were positively selected which were involved in pathways related to muscle development and function. This study provides new insights into the selection of different directions and the genetic mechanism related to muscle development in pigeons.

Genomic analysis of GBS data reveals genes associated with facial pigmentation in Xinyang blue-shelled layers.

Facial pigmentation is an important economic trait of chickens, especially for laying hens, which will affect the carcass appearance of eliminated layers. Therefore, identifying the genomic regions and exploring the function of this region that contributes to understanding the variation of skin color traits is significant for breeding. In the study, 291 pure-line Xinyang blue-shelled laying hens were selected, of which 75 were dark-faced chickens and 216 were white-faced chickens. The population was sequenced and typed by GBS genotyping technology. The obtained high-quality SNPs and pigmentation phenotypes were analyzed by a genome-wide association study (GWAS) and a F ST scan. Based on the two analytical methods, we identified a same genomic region (10.70-11.60 Mb) on chromosome 20 with 68 significant SNPs ( - log⁡ 10 ( P ) > 6 ), mapped to 10 known genes, including NPEPL1, EDN3, GNAS, C20orf85, VAPB, BMP7, TUBB1, ELMO2, DDX27, and NCOA5, which are associated with dermal hyperpigmentation.

Integrating miRNA and mRNA expression profiles in plasma of laying hens associated with heat stress.

High temperature is one of the most common environmental stressors plaguing animal husbandry worldwide. Little is known about the regulatory roles of miRNAs in response to heat stress in laying hens. To systematically identify heat stress-responsive miRNAs and their targets in laying hens, the differential expression of miRNAs and mRNAs was compared under heat stress and normal temperature. We identified 16 miRNAs and 502 genes that were significantly changed in heat-stressed laying hens. By comparing the differentially expressed genes (DEGs) and the putative targets of the altered miRNAs based on bioinformatics prediction, 82 coordinated genes were identified. Gene ontology classification analyses of the 82 putative target genes showed that the biological category 'cellular response to stress' was prominently annotated. Notably, the response-related gene autophagy-related protein 9A was most likely controlled by the upregulated miRNAs gga-miR-92-5p, gga-miR-1618-5p, gga-miR-1737, and gga-miR-6557 in response to heat stress. Analysis of DEGs also revealed an increase in lipid metabolism in heat-stressed laying hens. Some of these genes were negatively correlated with the altered miRNAs, suggesting that they are potential targets of the miRNAs. Taken together, our results advance our understanding of the regulatory mechanism of heat-stress-induced injury in laying hens, specifically with regard to miRNAs.

Heat stress mediates changes in fecal microbiome and functional pathways of laying hens.

Chicken gastrointestinal microbiota plays important roles in health, productivity, and disease. However, knowledge of the relationship between heat stress and the gut microbial ecosystem of poultry, especially laying hens, is still limited. Here, we aimed to provide important knowledge for heat stress intervention in the egg industry. We performed high-throughput sequencing metagenomics on fecal contents to unravel the microbial taxa and functional capacity of the gut microbiome of caged laying hens under heat stress. Results showed that the fecal microbial communities of laying hens were dominated by Firmicutes, Bacteroidetes, and Proteobacteria phyla. The Firmicutes were significantly decreased, and Bacteroidetes were increased in the fecal microbiota under heat stress. Functional prediction of these changes in microbiota revealed that metabolism-related pathways, including cysteine and methionine metabolism and benzoate degradation, were more abundant. Conversely, retinol metabolism and phenylpropanoid biosynthesis were decreased by heat stress, suggesting differences in metabolism between layers in different temperature environments. Clear contributions were identified between active taxa (genus level) and metabolic pathways, which were associated with the liver and intestinal dysfunction in layers. These data revealed that heat stress induced a significant taxonomic perturbation in the gut microbiome of caged laying hens. This was related to the negative effects of heat stress in poultry and provided important basic knowledge for heat stress intervention.

Identifying Genetic Differences Between Dongxiang Blue-Shelled and White Leghorn Chickens Using Sequencing Data.

The Dongxiang Blue-shelled chicken is one of the most valuable Chinese indigenous poultry breeds. However, compared to the Italian native White Leghorn, although this Chinese breed possesses numerous favorable characteristics, it also exhibits lower growth performance and fertility. Here, we utilized genotyping sequencing data obtained via genome reduction on a sequencing platform to detect 100,114 single nucleotide polymorphisms and perform further biological analysis and functional annotation. We employed cross-population extended haplotype homozygosity, eigenvector decomposition combined with genome-wide association studies (EigenGWAS), and efficient mixed-model association expedited methods to detect areas of the genome that are potential selected regions (PSR) in both chicken breeds, and performed gene ontology (GO) enrichment and quantitative trait loci (QTL) analyses annotating using the Kyoto Encyclopedia of Genes and Genomes. The results of this study revealed a total of 2424 outlier loci (p-value <0.01), of which 2144 occur in the White Leghorn breed and 280 occur in the Dongxiang Blue-shelled chicken. These correspond to 327 and 94 PSRs containing 297 and 54 genes, respectively. The most significantly selected genes in Blue-shelled chicken are TMEM141 and CLIC3, while the SLCO1B3 gene, related to eggshell color, was identified via EigenGWAS. We show that the White Leghorn genes JARID2, RBMS3, GPC3, TRIB2, ROBO1, SAMSN1, OSBP2, and IGFALS are involved in immunity, reproduction, and growth, and thus might represent footprints of the selection process. In contrast, we identified six significantly enriched pathways in the Dongxiang Blue-shelled chicken that are related to amino acid and lipid metabolism as well as signal transduction. Our results also reveal the presence of a GO term associated with cell metabolism that occurs mainly in the White Leghorn breed, while the most significant QTL regions mapped to the Chicken QTL Database (GG_4.0) for the Dongxiang Blue-shelled breed are predominantly related to lesions, bone mineral content, and other related traits compared to tibia length and body weight (i.e., at 14, 28, 42, and 70 d) in the White Leghorn. The results of this study highlight differences in growth, immunity, and egg quality traits between the two breeds, and provide a foundation for the exploration of their genetic mechanisms.

Enhanced Expression of Serotonin Receptor 5-Hydroxytryptamine 2C is Associated with Increased Feather Damage in Dongxiang Blue-Shelled Layers.

The gene encoding the serotonin receptor 5-hydroxytraptamine 2C (HTR2C) has been implicated in behavioral phenotypes in a number of species. In previous studies, a mutation in the chicken HTR2C gene was found to be associated with feather condition, thereby suggesting a relationship between the gene and receiving feather pecking activity. The present study analyzed the chicken HTR2C gene at both the genomic make-up and expression level in Dongxiang blue-shelled layer. A significant association between the single nucleotide polymorphism (SNP) rs13640917 (C/T) and feather condition was confirmed in the Chinese local layer. Enhanced HTR2C gene expression (151.1-fold) that was associated with high feather damage indicated that the right cerebrum might be the critical region for HTR2C to participate in the regulation of receiving feather pecking behavior.

An Efficient Genotyping Method in Chicken Based on Genome Reducing and Sequencing.

Single nucleotide polymorphisms (SNPs) are essential for identifying the genetic mechanisms of complex traits. In the present study, we applied genotyping by genome reducing and sequencing (GGRS) method to construct a 252-plex sequencing library for SNP discovery and genotyping in chicken. The library was successfully sequenced on an Illumina HiSeq 2500 sequencer with a paired-end pattern; approximately 400 million raw reads were generated, and an average of approximately 1.4 million good reads per sample were generated. A total of 91,767 SNPs were identified after strict filtering, and all of the 252 samples and all of the chromosomes were well represented. Compared with the Illumina 60K chicken SNP chip data, approximately 34,131 more SNPs were identified using GGRS, and a higher SNP density was found using GGRS, which could be beneficial for downstream analysis. Using the GGRS method, more than 3528 samples can be sequenced simultaneously, and the cost is reduced to $18 per sample. To the best of our knowledge, this study describes the first report of such highly multiplexed sequencing in chicken, indicating potential applications for genome-wide association and genomic selection in chicken.

Genotyping by genome reducing and sequencing for outbred animals.

Next-generation sequencing (NGS) approaches are widely used in genome-wide genetic marker discovery and genotyping. However, current NGS approaches are not easy to apply to general outbred populations (human and some major farm animals) for SNP identification because of the high level of heterogeneity and phase ambiguity in the haplotype. Here, we reported a new method for SNP genotyping, called genotyping by genome reducing and sequencing (GGRS) to genotype outbred species. Through an improved procedure for library preparation and a marker discovery and genotyping pipeline, the GGRS approach can genotype outbred species cost-effectively and high-reproducibly. We also evaluated the efficiency and accuracy of our approach for high-density SNP discovery and genotyping in a large genome pig species (2.8 Gb), for which more than 70,000 single nucleotide polymorphisms (SNPs) can be identified for an expenditure of only $80 (USD)/sample.

Molecular characterization and identification of a novel polymorphism of 200 bp indel associated with age at first egg of the promoter region in chicken follicle-stimulating hormone receptor (FSHR) gene.

Follicle-stimulating hormone receptor (FSHR) plays an important role in animal follicular development. Polymorphisms in FSHR promoter region likely impact transcription and follicle growth and maturation. In this study, a fragment of ~1.9 kb of cFSHR promoter for Zang, Xianju, Lohmann Brown, Jining Bairi and Wenchang breeds (line) was obtained. Totally 49 variations were revealed, of which 39 are single nucleotide substitutions, one is nucleotide substitution of (TTG) to (CAC) and nine are indels. Polymorphism at -874 site (a 200 bp indel mutation) was identified, and their effects on egg production traits as well as gene expression were analyzed. At this site, allele I(+) was dominant in Lohmann Brown and Xinyang Brown (a synthetic egg-laying line) lines, but very rare in three Chinese indigenous chicken breeds, namely Jining Bairi, Wenchang, Zang and one synthetic boiler line (Luqin). In Xinyang Brown population, the polymorphism was associated with age at first egg (AFE) (P < 0.05) and its effect on egg number at 37 weeks of age (E37) and egg number at 57 weeks of age (E57) was not significantly different (P > 0.05). The cFSHR mRNA level was not significantly different between three genotypes in small white and small yellow follicles of Xinyang Brown hens, however, allele I(+) tends to increase cFSHR transcription.