Black-bone Chicken Research Articles

Study on the Differences in Fecal Metabolites and Microbial Diversity of Jiangshan Black-Bone Chickens with Different Earlobe Colors.

The differences in earlobe color of Jiangshan black-bone chickens have been reported to be caused by the combined effects of melanin and collagen. In this study, we conducted LC-MS untargeted metabolomics and 16S rDNA diversity sequencing on the cecal contents of two types of earlobes: peacock green (Blue and Green group) and dark reddish-purple (Black group). The metabolomic sequencing identified a total of 747 differential metabolites (DMs), in which the metabolites were primarily enriched in tyrosine and tryptophan metabolism pathways between peacock green and dark reddish-purple earlobes. There were 15 different bacterial taxa among the three groups of earlobes at the genus level, and correlation analysis between metabolites and microbes revealed that the DMs between peacock green and dark reddish-purple earlobes were positively correlated with the different bacterial taxa. In short, there are differences in gut microbiota and metabolites between Jiangshan black-bone chickens with peacock green earlobes and those with dark reddish-purple earlobes. Our results suggest that the bacterial phyla Firmicutes and Bacteroidota may influence melanin synthesis by affecting tryptophan metabolism, induced by 5-Methoxyindoleacetate, and tyrosine metabolism, induced by maleylacetoacetic acid and maleic acid, leading to differences in earlobe color.

Effects of Different Levels of Green Tea Powder on Performance, Antioxidant Activity, Egg Mass, Quality, and Cecal Microflora of Chickens.

This study was conducted to investigate the effects of different levels of Green Tea Powder on the performance, egg quality, serum immune and antioxidant indices, and cecal microflora of 300-day-old Chishui black-bone chickens during the peak laying period. A total of 360 Chishui black-bone chickens were selected as the experimental animals. They were randomly allocated into four groups: the control group (CON), trial group I (T1), trial group II (T2), and trial group III (T3), each group with six replicates and 15 hens in each replicate. The control group was fed a basal diet, and the experimental groups were fed a basal diet supplemented with 0.8%, 1.6%, and 2.4% Green Tea Powder, respectively. The accommodation period was 14 d, and the experimental period was 60 d. The statistical software SPSS was used to perform a one-way analysis of variance (ANOVA) on the experimental data, and Duncan's method was used to perform multiple comparisons among groups. The results showed the following: compared with those of the control group, the average daily gain of the laying hens significantly decreased in the 1.6% Green Tea Powder group (p < 0.05); adding Green Tea Powder significantly reduced the content of malondialdehyde in the serum (p < 0.05), and the addition of 0.8% tea leaves significantly increased the immunoglobulin M and immunoglobulin A contents (p < 0.05); the egg yolk weight, eggshell thickness, eggshell strength, and yolk color of the laying hens significantly decreased in the 1.6% Green Tea Powder group (p < 0.05), and the addition of Green Tea Powder at the level of 2.4% significantly increased the percentage of umami, essential, and total amino acids (p < 0.05); and the structure of intestinal microorganisms was improved, and the abundance of Bacteroidetes and Bacteroidaceae significantly increased, while the abundance of Firmicutes and Lachnospiraceae significantly decreased (p < 0.05).

Analysis of genetic diversity and genetic structure of indigenous chicken populations in Guizhou province based on genome-wide single nucleotide polymorphism markers

Guizhou province in China is rich in indigenous chicken breeds, playing an essential role in the genetic improvement of modern chickens. Genetic diversity has decreased in recent decades due to accelerated breeding processes and changing conservation priorities. To determine the genetic diversity and population structure of Guizhou indigenous chicken breeds, we used 55K genotyping arrays to conduct population genetic analysis on 233 individuals from 8 Guizhou indigenous breeds and 263 individuals from 9 Guizhou indigenous chicken populations. We evaluated the genetic diversity parameter (heterozygosity, proportion of polymorphic markers, and nucleotide diversity), linkage disequilibrium (LD), population structure, and genetic differentiation (FST and genetics distance). Genetic diversity results indicated that the genetic diversity of chicken breeds in Guizhou province is relatively affluent. Among Guizhou breeds, Baiyi black-bone and Guizhou yellow chicken displayed the lowest genetic diversity, as the 2 breeds exhibit lower PN and heterozygosity, the extent of linkage disequilibrium is higher. According to the LD pattern, Guizhou indigenous breeds can be divided into 3 categories. Population structure analysis showed a certain degree of genetic differentiation among local chickens in Guizhou. We argue that Chishui black-bone and Puan black-bone chickens are 2 different geographical regional groups of the same breed. In principal component analysis, individuals from the 2 groups clustered together, and the phylogenetic tree results showed that the 2 groups clustered together to form a branch independent of other breeds, and they displayed an identical pattern of ancestral lineage composition. The research results will provide a reference for protecting local chicken genetic resources in Guizhou Province and promote the protection and utilization of genetic resources.

Non-Targeted Metabolomics of Serum Reveals Biomarkers Associated with Body Weight in Wumeng Black-Bone Chickens.

Growth performance is an important economic trait of broilers but the related serum metabolomics remains unclear. In this study, we utilized non-targeted metabolomics using ultra-high-performance liquid phase tandem mass spectrometry (UHPLC-MS/MS) to establish metabolite profiling in the serum of Chinese Wumeng black-bone chickens. The biomarker metabolites in serum associated with growth performance of chickens were identified by comparing the serum metabolome differences between chickens that significantly differed in their weights at 160 days of age when fed identical diets. A total of 766 metabolites were identified including 13 differential metabolite classes such as lipids and lipid-like molecules, organic acids and their derivatives, and organoheterocyclic compounds. The results of difference analysis using a partial least squares discriminant analysis (PLS-DA) model indicated that the low-body-weight group could be differentiated based on inflammatory markers including prostaglandin a2, kynurenic acid and fatty acid esters of hydroxy fatty acids (FAHFA), and inflammation-related metabolic pathways including tryptophan and arachidonic acid metabolism. In contrast, the sera of high-body-weight chickens were enriched for riboflavin and 2-isopropylmalic acid and for metabolic pathways including riboflavin metabolism, acetyl group transfer into mitochondria, and the tricarboxylic acid (TCA) cycle. These results provide new insights into the practical application of improving the growth performance of local chickens.

Dynamic Transcriptome Profile Analysis of Mechanisms Related to Melanin Deposition in Chicken Muscle Development.

The pectoral muscle is an important component of skeletal muscle. The blackness of pectoral muscles can directly affect the economic value of black-boned chickens. Although the genes associated with melanogenesis in mammals and birds have been thoroughly investigated, only little is known about the key genes involved in muscle hyperpigmentation during embryonic development. Here, we analyzed melanin deposition patterns in the pectoral muscle of Yugan black-boned chickens and compared differentially expressed genes (DEGs) between the muscles of Wenchang (non-black-boned chickens) and Yugan black-boned chickens on embryonic days 9, 13, 17, and 21. Melanin pigments were found to gradually accumulate in the muscle fibers over time. Using RNA-seq, there were 40, 97, 169, and 94 genes were identified as DEGs, respectively, between Yugan black-boned chicken muscles and Wenchang chickens at embryonic day 9, 13, 17, and 21 stages (fold change ≥2.0, false discovery rate (FDR) < 0.05). Thirteen DEGs, such as MSTRG.720, EDNRB2, TYRP1, and DCT, were commonly identified among the time points observed. These DEGs were mainly involved in pigmentation, melanin biosynthetic and metabolic processes, and secondary metabolite biosynthetic processes. Pathway analysis of the DEGs revealed that they were mainly associated with melanogenesis and tyrosine metabolism. Moreover, weighted gene co-expression network analysis (WGCNA) was used to detect core modules and central genes related to melanogenesis in the muscles of black-boned chickens. A total of 24 modules were identified. Correlation analysis indicated that one of them (the orange module) was positively correlated with muscle pigmentation traits (r > 0.8 and p < 0.001). Correlations between gene expression and L* values of the breast muscle were investigated in Yugan and Taihe black-boned chickens after hatching. The results confirmed that EDNRB2, GPNMB, TRPM1, TYR, and DCT expression levels were significantly associated with L* values (p < 0.01) in black-boned chickens (p < 0.05). Our results suggest that EDNRB2, GPNMB, TRPM1, TYR, and DCT are the essential genes regulating melanin deposition in the breast muscle of black-boned chickens. MSTRG.720 is a potential candidate gene involved in melanin deposition in the breast muscles of Yugan black-boned chickens.

Whole-genome resequencing reveals melanin deposition candidate genes of Luning chicken

BackgroundMelanin in the black-bone chicken’s body is considered the material basis for its medicinal effects and is an economically important trait. Therefore, improving the melanin content is a crucial focus in the breeding process of black-bone chickens. Luning chickens are black-bone chickens, with black beaks, skin, and meat. To investigate the genetic diversity and molecular mechanisms of melanin deposition in Luning chickens, we conducted whole-genome resequencing to analyze their breeding history and identify candidate genes influencing their black phenotype, along with transcriptome sequencing of dorsal skin tissues of male Luning chickens.ResultsPopulation structure analysis revealed that Luning chickens tend to cluster independently and are closely related to Tibetan chickens. Runs of homozygosity analysis suggested potential inbreeding in the Luning chicken and Tibetan chicken population. By combining genetic differentiation index (Fst) and nucleotide diversity (θπ) ratios, we pinpointed selected regions associated with melanin deposition. Gene annotation identified 540 genes with the highest Fst value in LOC101750371 and LOC121108313, located on the 68.24–68.58 Mb interval of chromosome Z. Combining genomic and transcriptomic data, we identified ATP5E, EDN3, and LOC101750371 as candidate genes influencing skin color traits in black-bone chickens.ConclusionsThis study characterized the evolutionary history of Luning chickens and preliminarily excavated candidate genes influencing the genetic mechanism of pigmentation in black-bone chickens, providing valuable insights for the study of animal melanin deposition.

Single-nucleus RNA sequencing reveals cell types, genes, and regulatory factors influencing melanogenesis in the breast muscle of Xuefeng black-bone chicken

The black-bone chicken, known for its high melanin content, holds significant economic value due to this unique trait. Particularly notable is the prominent melanin deposition observed in its breast muscle. However, the molecular mechanisms governing melanin synthesis and deposition in the breast muscle of black-bone chickens remain largely unknown. This study employed a single-nucleus transcriptome assay to identify genes associated with melanin deposition in the breast muscle of black-bone chickens, which are presumed to influence pigmentation levels. A comprehensive analysis of the nuclear transcriptome was conducted on the breast muscle of Xuefeng black-bone chickens, encompassing 18 distinct cell types, including melanocytes. Our findings revealed that STIMATE, LRRC7, ENSGALG00000049990, and GLDC play pivotal regulatory roles in melanin deposition within the breast muscle. Further exploration into the molecular mechanisms unveiled transcription factors and protein interactions suggesting that RARB, KLF15, and PRDM4 may be crucial regulators of melanin accumulation in the breast muscle. Additionally, HPGDS, GSTO1, and CYP1B1 may modulate melanin production and deposition in the breast muscle by influencing melanocyte metabolism. Our findings also suggest that melanocyte function in the breast muscle may be intertwined with intercellular signaling pathways such as PTPRK-WNT5A, NOTCH1-JAG1, IGF1R-IGF1, IDE-GCG, and ROR2-WNT5A. Leveraging advanced snRNA-seq technology, we generated a comprehensive single-cell nuclear transcriptome atlas of the breast muscle of Xuefeng black-bone chickens. This facilitated the identification of candidate genes, regulatory factors, and cellular signals potentially influencing melanin deposition and melanocyte function. Overall, our study provides crucial insights into the molecular basis of melanin deposition in chicken breast muscle, laying the groundwork for future breeding programs aimed at enhancing black-bone chicken cultivation.

Research Note: Metabolomics revealed the causes of the formation of chicken structural blue earlobes

The earlobes of chickens exhibit a range of colors, but there has been relatively little research on the formation of structural blue earlobes. Previous results showed that the structural color earlobes were related to the interplay between melanin and collagen in light reflection. To investigate the metabolic differences in these earlobe colors, we conducted nontargeted liquid chromatograph mass spectrometer (LC-MS) for metabolomic sequencing on structural blue (Green and Blue groups) and nonstructural color (Black group) earlobes tissue of Jiangshan black-bone chickens. The content detection in earlobe tissues of different groups shows that there were significant differences in melanin and collagen content between the Black and Green group. The metabolome identified a total of 6,102 mass spectroscopic peaks and ultimately identified 919 annotated metabolites. Variable importance in the projection (VIP) analysis identified the common differential expressed metabolites (DMs) "Tyr Thr Ala Glu" among the 3 groups. By combining those DMs with differentially expressed genes (DEGs) in our previous transcriptome data from the same sample, and associated with KEGG pathway analysis, multiple pathways related to melanogenesis and collagen metabolism were enriched across the 3 groups. By analyzing the metabolites and genes in these pathways, as well as the interaction network diagram of DEGs, we identified some key genes, Wnt Family Member 6 (WNT6), Transcription Factor 7 (TCF7), Proopiomelanocortin (POMC) and Calcium/Calmodulin Dependent Protein Kinase II Alpha (CAMK2A), and some key DMs like DG (11M3/9M5/0:0) and gentisic acid. The differential gene expression and metabolic levels affect the production of melanin and collagen, leading to differences in the content in melanin and the thickness of the collagen layer between earlobe colors, while the thickness of the collagen layer could affect light scattering, ultimately resulting in different colored earlobes in Jiangshan black-bone chickens.

Effect of High Dietary Iron on Fat Deposition and Gut Microbiota in Chickens.

To meet the demand of consumers for chicken products, poultry breeders have made improvements to chickens. However, this has led to a new problem in the modern poultry industry, namely excessive fat deposition. This study aims to understand the effects of dietary iron supplementation on fat deposition and gut microbiota in chickens. In this study, we investigated the effects of iron on the growth performance, fat deposition, and gut microbiota of silky fowl black-bone chickens. A total of 75 7-week-old silky fowl black-bone chickens were randomly divided into three groups (five replicates per group, five chickens per replicate) and fed them for 28 days using a growing diet (control group), a growing diet + 10% tallow (high-fat diet group, HFD group), and a growing diet + 10% tallow + 500 mg/kg iron (HFDFe500 group), respectively. We detected the effects of iron on the growth performance, fat deposition, and gut microbiota of silky fowl black-bone chickens using the growth performance index test, oil red O staining, and HE staining, and found that the high-fat diet significantly increased liver and serum fat deposition and liver injury, while the addition of iron to the diet could reduce the fat deposition caused by the high-fat diet and alleviate liver injury. In addition, 16S rDNA sequencing was used to compare the relative abundance of gut microbiota in the cecal contents in different feeding groups. The results showed that the high-fat diet could induce gut microbiota imbalance in chickens, while the high-iron diet reversed the gut microbiota imbalance. PICRUSt functional prediction analysis showed that dietary iron supplementation affected amino acid metabolism, energy metabolism, cofactors, and vitamin metabolism pathways. In addition, correlation analysis showed that TG was significantly associated with Firmicutes and Actinobacteriota (p < 0.05). Overall, these results revealed high dietary iron (500 mg/kg) could reduce fat deposition and affect the gut microbiota of silky fowl black-bone chickens, suggesting that iron may regulate fat deposition by influencing the gut microbiota of chickens and provides a potential avenue that prevents excessive fat deposition in chickens by adding iron to the diet.

Identification of Metabolites in Muscles of Lueyang Black-Bone Chickens: A Comparative Analysis of Caged and Cage-Free Rearing Modes Using Untargeted Metabolomic Techniques.

The Lueyang black-bone chicken is a specific native chicken strain in China. This study aimed to investigate the effects of different rearing systems on the meat quality of Lueyang black-bone chickens. Six hundred Lueyang black-bone hens were randomly divided into two groups at 7 weeks of age and raised in cage and cage-free systems for 20 weeks. The carcass yield, meat quality, and total metabolites were measured in both the leg and breast muscles. By comparison, the carcass yield of hens in the cage-free (CF) group (1.26 ± 0.09 kg) was significantly lower than that in the caged rearing (CR) group (1.52 ± 0.15 kg). However, the shear force of leg muscles in the CF group (27.98 ± 2.43 N) was significantly greater than that in the CR group (24.15 ± 1.93 N). In addition, six samples from each group were randomly selected and their metabolites were detected by the non-targeted metabolomics technique. Among these metabolites, 408 and 354 significantly differentially abundant metabolites were identified in breast and leg muscles, which were mainly involved in glycerophospholipid metabolism, unsaturated fatty acid biosynthesis, arginine and proline metabolism, and nucleotide metabolism. We found that the levels of 19 phospholipids, mainly phosphatidylcholines and lysophosphatidylcholines, were significantly greater in the CF group than in the CR group. Additionally, the contents of eight unsaturated fatty acids, linoleic acid, and linolenic acid were dramatically greater in the CF group than in the caged group. The accumulation of 4-hydroxy-proline, glutamate, and adenosine 3'-monophosphate (AMP) was enhanced in the CF group. Moreover, many more volatile organic compounds were identified in the muscles of the cage-free group, enhancing the flavor of the chicken meat. In conclusion, the cage-free rearing mode facilitates the accumulation of nutrients and flavor substances in the chicken meat and is a better rearing system for Lueyang black-bone chickens.

Effects of Allicin on the Immune Performance and Expression of Immune-related Genes in Muchuan Black-bone Chickens

Background: In response to China’s ban on medicinal feed additives with growth-promoting functions in livestock and poultry feed, there is an urgent need in modern animal husbandry for alternatives to antibiotics to sustain livestock health and improve feed conversion efficiency. Allicin, known for its safety, efficiency, lack of drug resistance and absence of residue, regulates the intestinal flora structure; inhibits the growth of harmful bacteria; improves intestinal health, immunity and stress resistance; and improves animal production. Methods: To investigate the impact of allicin on immune performance and immune-related gene expression in Muchuan black-bone chickens, diets were supplemented with varying allicin levels (50, 100, 200 mg/kg), designated as test groups A, B and C, respectively. Initially, growth performance was assessed. Subsequently, immunoglobulins were measured via enzyme-linked immunosorbent assay (ELISA) and colorimetry; immune titers for avian influenza and Newcastle disease were evaluated using hemagglutination and hemagglutination inhibition tests and the expression of immune genes was analyzed via real-time quantitative PCR. Finally, serum biochemical indices in each group were determined. Result: Compared to the control group, allicin supplementation resulted in increased average fasting body weight in the experimental group and significantly (P less than 0.05) higher thymus and musogastric indices in groups A and C, respectively. Serum levels of immunoglobulin M in groups A and B and immunoglobulin G levels were significantly higher in all experimental groups than in controls. Antibody titers against avian influenza H5 and H7 strains were higher in all test groups, with Newcastle disease antibody titers in groups B and C significantly higher than those in controls. The mRNA expression levels of IFN-ã were lower across all experimental groups, whereas the expression levels of TLR3 in groups B and C and TLR15 in group B were higher. Serum levels of triglycerides in group A were significantly higher and blood urea nitrogen levels were significantly lower in groups B and C than in controls. These findings indicate that dietary allicin (50-200 mg/kg) significantly improves growth performance, immunity and protein and lipid metabolism in Muchuan black-bone chickens, offering valuable data for the development and use of allicin as an antibiotic alternative.

Transcriptome and proteome revealed the differences in 3 colors of earlobe in Jiangshan Black-bone chicken

The earlobe is a featherless, exposed thickening located beneath the ear canal of chickens, which plays a visual signaling role in age, performance, mental vitality, reproduction, and other aspects. However, despite its importance, there have been few studies on the color differences and formation mechanisms of chicken earlobes, particularly the structurally blue earlobes characteristic of the Jiangshan black-bone chicken. In this study, we explored the physiological mechanisms that may influence the formation of differently colored earlobes using 3 types of earlobes from Jiangshan black-bone chickens: light peacock green (Green group), dark peacock green (Blue group), and dark reddish purple (Black group). All 3 earlobe colors exhibited positive melanin Masson-Fontana staining, and the thickness of collagen fibers in the dermis decreased in the order of Green, Blue, and Black groups. A total of 1,953 differentially expressed genes (DEGs) were detected in the 3 earlobes through mRNA sequencing, among which the GO term "collagen trimer" was significantly enriched in DEGs between groups. Additionally, 716 differentially expressed proteins (DEPs) were identified in the 3 earlobes using 4D-DIA proteomics, with the term "collagen fibril organization" being significantly enriched in DEPs between the Green and Black groups. Integrated analysis of transcriptome and proteome data revealed that 12 DEGs and DEPs were commonly differentially expressed between the Green and Black groups, including the gene LUM (corneal keratan sulfate proteoglycan), which was significantly enriched in the "collagen fibril organization" GO term. In conclusion, our study suggests that LUM plays a crucial role in the formation of peacock green earlobes in Jiangshan black-bone chickens. The high level of LUM in peacock green (Green and Blue groups) may affect collagen nanostructures, leading to a stronger effect of melanin-supported dermal collagen on the production of non-iridescent structural colors through coherent scattering, resulting in a bright structural blue color in Jiangshan black-bone chickens. In contrast, the low expression of LUM in dark reddish purple (Black group) reduces the reflection of non-iridescent structural colors, making the earlobe color appear almost black, similar to melanin.

Investigating the impact of pigmentation variation of breast muscle on growth traits, melanin deposition, and gene expression in Xuefeng black-bone chickens

The blackness traits, considered an important economic factor in the black-bone chicken industry, still exhibits a common phenomenon of significant difference in blackness of breast muscle. To improve this phenomenon, this study compared growth traits, blackness traits, and transcriptome of breast muscles between the High Blackness Group (H group) and Low Blackness Group (L group) in the Xuefeng black-bone chickens. The results are as follows: 1) There was no significant difference in growth traits between the H group and the L group (P > 0.05). 2) The skin/breast muscle L values in the H group were significantly lower than those in the L group, while the breast muscle melanin content exhibited the opposite trend (P < 0.05). 3) A significant negative correlation was observed between breast muscle melanin content and skin/breast muscle L value (P < 0.05), and skin L value exhibiting a significant positive correlation with breast muscle L value (P < 0.05). 4) The breast muscle transcriptome comparison between the H group and L group revealed 831 and 405 DEGs in female and male chickens, respectively. This included 37 shared DEGs significantly enriched in melanosome, pigment granule, and the melanogenesis pathway. Seven candidate genes (DCT, PMEL, MLANA, TYRP1, OCA2, EDNRB2, and CALML4) may play a crucial role in the melanin production of breast muscle in Xuefeng black-bone chicken. The findings could accelerate the breeding process for achieving desired levels of breast muscle blackness and contribute to the exploration of the mechanisms underlying melanin production in black-bone chickens.

Analysis of the Difference of Intestinal Microbes in Muchuan Black-bone Chickens with Two Skin Colors

Background: Skin color is an important economic trait in black-bone chicken production and it has been identified that the gut microbiota is an important factor in animal health and physiology. However, it is unknown about whether the gut microbiota in black-bone chickens is connected to the coloration of chicken skin. Methods: To investigate the relationship between the gut microbiota and skin coloration, comparing the differentiation of the gut microbiota structure and abundances in black- (B group) and white-skinned (W group) Muchuan black-bone chicken. Here, we characterized the microbiota in the jejunal contents of both the B and W groups using the Illumina MiSeq platform, targeting the genomic V4 region of the 16S rRNA gene. Result: In terms of community richness, the ACE community richness index of the B group was significantly higher than that of the W group (p less than 0.05) and the proportion of unique OTUs was higher in group B than in group W. According to the species annotation and abundance information of all samples at the genus level, Parabacteroides, Faecalicoccus and Alistipes were enriched in group B, whereas Actinomyces, Elstera and Nosocomiicoccus were enriched in group W. In the evolutionary branch diagram, the relative abundances of Enterobacteriaceae (Gammaproteobacteria, Enterobacteriales) were significantly higher in group B than in group W (LDA Score greater than 4). Our data provide a basis for the hypothesis that the discriminating bacterial taxa in gut is associated with the feature variants for skin coloration of Muchuan black-bone chickens, which were fed in the same feed and stocking niches.

Characterization of Myosatellite Stem Cells from Black Bone Chicken and Effect of Different Animal Serums on Growth Performance

Isolation of muscle stem cells (myosatellite stem cells) of black bone chickens using enzymatic digestion, the pronase, and screening by low-speed centrifugation process combined with 40 μm cell strainer filtration showed that this method was able to separate chicken muscle stem cells efficiently. The results proved that the isolated cells were myosatellite stem cells expressing marker genes, c-met, Pax7, MyoD, and myogenin, by RT-PCR assay. Additionally, immunofluorescence staining with monoclonal antibodies confirmed the isolated cells were MSCs, with positive staining for canonical markers proteins: Pax7, Desmin, and actin. Evaluating the influence of fetal bovine serum (FBS), horse serum (HS), and chicken serum (CKS) supplements on MSC proliferation revealed that FBS emerged as the most effective despite its escalating cost and emerging need for alternatives. Notably, Chicken Serum (CKS) demonstrated potential as an alternative, closely following the cell proliferation rate achieved by FBS. Our findings affirm the efficacy of the devised isolation method and highlight the potential of using chicken serum in cell culture protocols. HIGHLIGHTS This research established a technique for isolating myosatellite stem cells from black bone chickens through enzymatic processes combined with gradient methods. Centrifugation aids in obtaining a purer isolation of these stem cells and allows for the evaluation of various animal serums’ effects. In cell cultures, the performance of chicken serum showed no significant difference compared to bovine serum. GRAPHICAL ABSTRACT

Identification and functional analysis of ovarian lncRNAs during different egg laying periods in Taihe Black-Bone Chickens.

Introduction: Long non-coding RNA (lncRNA) refers to a category of non-coding RNA molecules exceeding 200 nucleotides in length, which exerts a regulatory role in the context of ovarian development. There is a paucity of research examining the involvement of lncRNA in the regulation of ovary development in Taihe Black-Bone Chickens. In order to further investigate the egg laying regulation mechanisms of Taihe Black-Bone Chickens at different periods, transcriptome analysis was conducted on the ovarian tissues at different laying periods. Methods: This study randomly selected ovarian tissues from 12 chickens for RNA-seq. Four chickens were selected for each period, including the early laying period (102days, Pre), the peak laying period (203days, Peak), and the late laying period (394days, Late). Based on our previous study of mRNA expression profiles in the same ovarian tissue, we identified three differentially expressed lncRNAs (DE lncRNAs) at different periods and searched for their cis- and trans-target genes to draw an lncRNA-mRNA network. Results and discussion: In three groups of ovarian tissues, we identified 136 DE lncRNAs, with 8 showing specific expression during the early laying period, 10 showing specific expression during the peak laying period, and 4 showing specific expression during the late laying period. The lncRNA-mRNA network revealed 16 pairs of lncRNA-target genes associated with 7 DE lncRNAs, and these 14 target genes were involved in the regulation of reproductive traits. Furthermore, these reproductive-related target genes were primarily associated with signaling pathways related to follicle and ovary development in Taihe Black-Bone Chickens, including cytokine-cytokine receptor interaction, TGF-beta signaling pathway, tyrosine metabolism, ECM-receptor interaction, focal adhesion, neuroactive ligand-receptor interaction, and cell adhesion molecules (CAMs). This study offers valuable insights for a comprehensive understanding of the influence of lncRNAs on poultry reproductive traits.

Whole-transcriptome sequencing reveals a melanin-related ceRNA regulatory network in the breast muscle of Xichuan black-bone chicken

The economic losses incurred due to reduced muscle pigmentation highlight the crucial role of melanin-based coloration in the meat of black-bone chickens. Melanogenesis in the breast muscle of black-bone chickens is currently poorly understood in terms of molecular mechanisms. This study employed whole-transcriptome sequencing to analyze black and white breast muscle samples from black-bone chickens, leading to the identification of 367 differentially expressed (DE) mRNAs, 48 DElncRNAs, 104 DEcircRNAs, and 112 DEmiRNAs involved in melanin deposition. Based on these findings, a competitive endogenous RNA (ceRNA) network was developed to better understand the complex mechanisms of melanin deposition. Furthermore, our analysis revealed key DEmRNAs (TYR, DCT, EDNRB, MLPH and OCA2) regulated by DEmiRNAs (gga-miR-140-5p, gga-miR-1682, gga-miR-3529, gga-miR-499-3p, novel-m0012-3p, gga-miR-200b-5p, gga-miR-203a, gga-miR-6651-5p, gga-miR-7455-3p, gga-miR-31-5p, miR-140-x, miR-455-x, novel-m0065-3p, gga-miR-29b-1-5p, miR-455-y, novel-m0085-3p, and gga-miR-196-1-3p). These DEmiRNAs competitively interacted with DElncRNAs including MSTRG.2609.2, MSTRG.4185.1, LOC112530666, LOC112533366, LOC771030, LOC107054724, LOC121107411, LOC100859072, LOC101750037, LOC121108550, LOC121109224, LOC121110876, and LOC101749016, as well as DEcircRNAs, such as novel_circ_000158, novel_circ_000623, novel_001518, and novel_circ_003596. The findings from this study provide insight into the mechanisms that regulate lncRNA, circRNA, miRNA, and mRNA expression in chicken melanin deposition.

A single-cell transcriptome atlas of Lueyang black-bone chicken skin

As the largest organ of the body, the skin participates in various physiological activities, such as barrier function, sensory function, and temperature regulation, thereby maintaining the balance between the body and the natural environment. To date, compositional and transcriptional profiles in chicken skin cells have not been reported. Here, we report detailed transcriptome analyses of cell populations present in the skin of a black-feather chicken and a white-feather chicken using single-cell RNA sequencing (scRNA-seq). By analyzing cluster-specific gene expression profiles, we identified 12 cell clusters, and their corresponding cell types were also characterized. Subsequently, we characterized the subpopulations of keratinocytes, myocytes, mesenchymal cells, fibroblasts, and melanocytes. It is worth noting that we have identified a subpopulation of keratinocytes involved in pigment granule capture and a subpopulation of melanocytes involved in pigment granule deposition, both of which have a higher cell abundance in black-feather chicken compared to white-feather chicken. Meanwhile, we also compared the cellular heterogeneity features of Lueyang black-bone chicken skin with different feather colors. In addition, we also screened out 12 genes those could be potential markers of melanocytes. Finally, we validated the specific expression of SGK1, WNT5A, CTSC, TYR, and LAPTM5 in black-feather chicken, which may be the key candidate genes determining the feather color differentiation of Lueyang black-bone chicken. In summary, this study first revealed the transcriptome characteristics of chicken skin cells via scRNA-seq technology. These datasets provide valuable information for the study of avian skin characteristics and have important implications for future poultry breeding.

Synergism of fermented feed and ginseng polysaccharide on growth performance, intestinal development, and immunity of Xuefeng black-bone chickens

Microbial fermented feed (MF) is considered a valuable strategy to bring advantages to livestock and is widely practiced. Oral supplementation of Ginseng polysaccharide (Gps) eliminated weight loss in chickens following vaccination. This study investigated the effects of the combined use of Gps and MF on growth performance and immune indices in Xuefeng black-bone chickens. A total of 400 Xuefeng black-bone chickens at the age of 1 day were randomly assigned to four groups. Normal feed group (Control group), ginseng polysaccharide (200 mg/kg) group (Gps group), microbially fermented feed (completely replace the normal feed) group (MF group), and microbially fermented feed and add ginseng polysaccharide just before use (MF + Gps group). Each group contained 5 pens per treatment and 20 birds per pen. The body weight and average daily gain in the Gps, MF, and MF + Gps groups increased significantly (P < 0.01), while the feed conversion ratio decreased significantly (P < 0.01). The combined use of MF and Gps showed a synergistic effect. There was no significant difference in villus height (cecal) between the experimental group and the Con group. The crypt depth of the three experimental groups exhibited a significantly lower value compared to the Control group (P < 0.05). The V/C ratio of the Gps group and MF + Gps was significantly increased (P < 0.05), but there was no significant difference in the MF group. Moreover, the diarrhea rate of the Gps and the MF + Gps groups was lower than that of the Con group, while that of the MF + Gps group decreased the mortality rate (P < 0.05). The serum tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) levels in the MF, Gps, and MF + Gps groups decreased significantly (P < 0.01), the serum immunoglobulin G (IgG) levels increased significantly (P < 0.01), while the combination of MF and Gps had a synergistic effect. The combined use of Gps and MF not only further improved growth performance and immune parameters, but also reduced the diarrhea rate and mortality.

Differential analysis of immunoglobulin gene expression pattern in chickens of distinct breeds and developmental periods.

Immunoglobulin is an essential component of the body's defense against pathogens, aiding in the recognition and clearance of foreign antigens. Research concerning immunoglobulin gene and its diversity of expression across different breeds within the same species is relatively scarce. In this study, we employed RACE (Rapid Amplification of cDNA Ends) technology, prepared DNA libraries, performed high-throughput sequencing, and conducted related bioinformatics analysis to analyze the differences in immunoglobulin gene diversity and expression at different periods in Hy-line brown hens, Lueyang black-bone chickens, and Beijing-You chickens. The study found that the composition of chicken immunoglobulin genes is relatively simple, with both the light chain and heavy chain having a functional V gene. Additionally, the mechanisms of immunoglobulin diversity generation tended to be consistent among different breeds and periods of chickens, primarily relying on abundant junctional diversity, somatic hypermutation (SHM), and gene conversion (GCV) to compensate for the limitations of low-level V(D)J recombination. As the age increased, the junctional diversity of IgH and IgL tended to diversify and showed similar expression patterns among different breeds. In the three chicken breeds, the predominant types of mutations observed in IGHV and IGLV SHM were A to G and G to A transitions. Specifically, IGLV exhibited a preference for A to G mutations, whereas IGHV displayed a bias toward G to A mutations. The regions at the junctions between framework regions (FR) and complementarity-determining regions (CDR) and within the CDR regions themselves are typically prone to mutations. The locations of GCV events in IGLV and IGHV do not show significant differences, and replacement segments are concentrated in the central regions of FR1, CDR, and FR2. Importantly, gene conversion events are not random occurrences. Additionally, our investigation revealed that CDRH3 in chickens of diverse breeds and periods the potential for diversification through the incorporation of cysteine. This study demonstrates that the diversity of immunoglobulin expression tends to converge among Hy-line brown hens, Lueyang black-bone chickens, and Beijing-You chickens, indicating that the immunoglobulin gene expression mechanisms in different breeds of chickens do not exhibit significant differences due to selective breeding.