Genome-wide association study identifies SNPs for growth performance and serum indicators in Valgus-varus deformity broilers (Gallus gallus) using ddGBS sequencing.

BACKGROUND: Valgus-varus deformity (VVD) is a lateral or middle deviation of the tibiotarsus or tarsometatarsus, which is associated with compromised growth, worse bone quality and abnormal changes in serum indicators in broilers. To investigate the genetic basis of VVD, a genome wide association study (GWAS) was performed to identify candidate genes and pathways that are responsible for VVD leg disease, serum indicators and growth performance in broilers. RESULTS: In total, VVD phenotype, seven serum indicators and three growth traits were measured for 126 VVD broilers (case group) and 122 sound broilers (control group) based on a high throughput genome wide genotyping-by-sequencing (GBS) method. After quality control 233 samples (113 sound broilers and 120 VVD birds) and 256,599 single nucleotide polymorphisms (SNPs) markers were used for further analysis. As a result, a total of 5 SNPs were detected suggestively significantly associated with VVD and 70 candidate genes were identified that included or adjacent to these significant SNPs. In addition, 43 SNPs located on Chr24 (0.22 Mb - 1.79 Mb) were genome-wide significantly associated with serum alkaline phosphatase (ALP) and 38 candidate genes were identified. Functional enrichment analysis showed that these genes are involved in two Gene Ontology (GO) terms related to bone development (cartilage development and cartilage condensation) and two pathways related to skeletal development (Toll-like receptor signaling pathway and p53 signaling pathway). BARX2 (BARX homeobox 2) and Panx3 (Pannexin 3) related to skeleton diseases and bone quality were obtained according to functional analysis. According to the integration of GWAS with transcriptome analysis, HYLS1 (HYLS1 centriolar and ciliogenesis associated) was an important susceptibility gene. CONCLUSIONS: The results provide some reference for understanding the relationship between metabolic mechanism of ALP and pathogenesis of VVD, which will provide a theoretical basis for disease-resistant breeding of chicken leg soundness.

The effect of salidroside on the bone and cartilage properties in broilers.

Leg disorders frequently occur in fast-growing broiler chickens, constituting severe health and welfare problems. Although salidroside (SAL) promotes osteogenesis and inhibits apoptosis of chondrocytes in rats, it remains to be determined whether SAL can effectively improve bone growth in broilers. The present study was designed to investigate the effects of dietary SAL supplementation on bone and cartilage characteristics in broiler chickens. Ninety-six Arbor Acres broiler chickens were randomly divided into 4 groups: control, low-dose SAL, medium-dose SAL, and high-dose SAL groups. The broiler chickens were raised until 42 d of age, with samples of bone and cartilage collected for biomechanical testing and bone metabolism index detection. The results showed that SAL significantly increased the vertical external diameter, cross-sectional moment of inertia, and cross-sectional area of the femur and tibia. Additionally, SAL enhanced bone mineral density and strength, as evidenced by significant increases in stiffness, Young's modulus, ultimate load, and fracture work of the femur and tibia. Furthermore, SAL influenced the relative content of phosphate, carbonate, and amide I in cortical bone. Moreover, SAL upregulated the expression of osteogenic genes (Collagen-1, RUNX2, BMP2, and ALP) in a dose-dependent manner and maintained the homeostasis of the extracellular matrix (ECM) of chondrocytes. These results indicated that SAL promoted leg health in broilers by improving bone and cartilage quality and enhancing chondrocyte activity.

Plasma exosome-derived miR-455-5p targets RPS6KB1 to regulate cartilage homeostasis in valgus-varus deformity (Gallus gallus).

Valgus-varus deformity (VVD) is a common long bone deformity in broilers. Imbalance in cartilage homeostasis is the main feature of leg disease. Exosomes act as an important intercellular communication vector that regulates chondrogenesis by encapsulating specific nucleic acids and proteins. However, the exact mechanism of how plasma exosomal miRNAs regulate cartilage homeostasis in VVD broilers remains unclear. This study first demonstrated the structural disorder, growth retardation, and reduced proliferative capacity of VVD cartilage in vitro and in vivo. Subsequently, VVD and Normal broiler plasma exosomes were collected for miRNA sequencing. Cartilage-specific miR-455-5p was extraordinarily emphasized by performing bioinformatics analysis on differential miRNA target genes and further validated by tissue expression profiling. PKH67 fluorescently labeled plasma exosomes were shown to be taken up by chondrocytes, deliver miR-455-5p, inhibit chondrocyte proliferation, and disrupt their homeostasis, and these effects could be inhibited by the miR-inhibitors. Mechanistically, MiR-455-5p targets Ribosomal Protein S6 Kinase B1 (RPS6KB1) to inhibit RPS6 phosphorylation and reduce the synthesis of key proteins for cartilage proliferation, which in turn inhibits cartilage proliferation and disrupts its homeostasis. In conclusion, the present study identified abnormalities in VVD cartilage tissue and clarified the specific mechanism by which plasma exosome-derived miR-455-5p regulates cartilage homeostasis.

Carcass composition, meat quality, leg muscle status, and its mRNA expression profile in broilers affected by valgus-varus deformity.

Valgus-varus deformity (VVD) is a common leg disease in commercial broilers, which seriously affects animal welfare and causes economic losses. Up to now, most of the studies on VVD have been on skeleton, whereas there are fewer studies on VVD muscle. In this study, carcass composition and meat quality of 35-day-old normal and VVD Cobb broilers assess the effect of VVD on broiler growth. Molecular biology, morphology, and RNA sequencing (RNA-seq) were used to study the difference between normal and VVD gastrocnemius muscle. In comparison with the normal broilers, the breast muscle and leg muscle of the VVD broilers had lower shear force, notably lower crude protein, lower water content, cooking loss, and deeper meat color (P < 0.05). The morphological results showed that the weight of skeletal muscle was significantly higher in the normal broilers than that in the VVD broilers (P < 0.01), the diameter and area of myofibrils in the affected VVD were smaller than in the normal broilers (P < 0.01). Quantitative real-time PCR (qPCR) of gastrocnemius muscle revealed that the expression of myasthenic marker genes, fast myofiber marker genes, and apoptosis-related factors were significantly higher in the VVD broilers than in the normal broilers (P < 0.01). In total, 736 differentially expressed genes (DEGs) were identified firstly in the normal and VVD leg muscle by RNA-seq. Gene ontology (GO) enrichment indicated that these DEGs were mainly involved in the multicellular organismal process and anatomical structure development. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEGs are significantly enriched in proteasome. Protein interaction analysis obtained that DEGs with high interaction were proteasome-related coding genes and ubiquitin-related genes, these DEGs were closely associated with muscle atrophy. These show that VVD has an adverse effect on growth characteristics, slaughter characteristics, and meat quality in broilers, which may cause leg muscle atrophy. This study provides some reference values and basis for studying the pathogenesis of VVD in broilers.

Integration Analysis of circRNA-miRNA-mRNA and Identification of Critical Networks in Valgus-Varus Deformity (Gallus gallus).

Valgus-valgus deformity (VVD) is a common leg deformity in broilers with inward or outward deviation of the tibiotarsus and tarsometatarsus. The competing endogenous RNA (ceRNA) network plays an essential role in the study of leg disease. However, its role in the etiology and pathogenesis of VVD remains unclear. Here, based on case (VVD) and control (normal) group design, we performed analyses of differentially expressed circRNAs (DEcircRNAs), differentially expressed miRNAs (DEmiRNAs) and differentially expressed mRNAs (DEmRNAs). Transcriptome data derived 86 DEcircRNAs, 13 DEmiRNAs and 410 DEmRNAs. Functional analysis showed that DEmRNAs were significantly enriched in cell cycle, apoptosis, ECM-receptor interaction, FoxO signaling pathway and protein processing synthesis. DEcirc/miRNA-associated DEmRNAs were associated with skeletal and muscle growth and development pathways, including mTOR, Wnt, and VEGF signaling pathways. Subsequently, a circRNA-miRNA-mRNA regulatory network was constructed based on the ceRNA hypothesis, including 8 circRNAs, 6 miRNAs, and 31 mRNAs, which were significantly enriched in the skeletal developmental pathway. Finally, two key mRNAs (CDC20 and CTNNB1) and their regulatory axes were screened by the PPI network and cytohubba. The expression levels of CDC20 and CTNNB1 in cartilage and seven other tissues were also quantified by qPCR. In conclusion, we analyzed the functions of DEmRNA, DEcircRNA and DEmiRNA and constructed the hub ceRNA regulatory axis, and obtained two hub genes, CDC20 and CTNNB1. The study more deeply explored the etiology and pathogenesis of VVD and lays the foundation for further study of the role of the ceRNA network on skeletal development.

Multi-Stage Transcriptome Analysis Revealed the Growth Mechanism of Feathers and Hair Follicles during Induction Molting by Fasting in the Late Stage of Egg Laying.

Induced molting is a common method to obtain a new life in laying hens, in which periodic changes in feathers are the prominent feature. Nevertheless, its precise molecular mechanism remains unclear. In this study, feather and hair follicle samples were collected during fasting-induced physiological remodeling for hematoxylin-eosin staining, hormone changes and follicle traits, and transcriptome sequencing. Feather shedding was observed in F13 to R25, while newborns were observed in R3 to R32. Triiodothyronine and tetraiodothyronine were significantly elevated during feather shedding. The calcium content was significantly higher, and the ash content was significantly lower after the changeover. The determination of hair follicle traits revealed an increasing trend in pore density and a decrease in pore diameter after the resumption of feeding. According to RNA-seq results, several core genes were identified, including DSP, CDH1, PKP1, and PPCKB, which may have an impact on hair follicle growth. The focus was to discover that starvation may trigger changes in thyroid hormones, which in turn regulate feather molting through thyroid hormone synthesis, calcium signaling, and thyroid hormone signaling pathways. These data provide a valuable resource for the analysis of the molecular mechanisms underlying the cyclical growth of hair follicles in the skin during induced molting.

Identification and genetic analysis of major gene ST3GAL4 related to serum alkaline phosphatase in chicken.

Alkaline phosphatase (ALP) is a marker of osteoblast maturation and an important indicator of bone metabolism. The activity of ALP can reflect the bone metabolism and growth traits of animals, so the polymorphism affecting ALP expression deserves further study. In this study, we identified an SNP site in ST3GAL4 found by genome-wide association studies (GWAS) in previous studies, 8 SNPs were also identified by DNA sequencing. Interestingly, there were 4 SNPs (rs475471G > A, rs475533C > T, rs475621A > G, rs475647C > A) completely linked by haplotype analysis. Therefore, we selected a tag SNP rs475471G > A to further analyze the ALP level of different genotypes in Hubbard leg disease population and an F2 chicken resource population produced by Anka and Gushi chickens and carried out population genetic analysis in 18 chicken breeds. Association analysis showed that this QTL within ST3GAL4 was highly correlated with ALP level. The mutant individuals with genotype AA had the highest ALP level, followed by GA and GG carriers. The mutant individual carriers of AA and GA genotype had higher values for body weight (BW), chest width (CW), body slanting length (BSL), pelvis width (PW) at 4-week, the semi-evisceration weight (SEW), evisceration weight (EW) and Leg weight (LW) than GG genotypes. The amplification and typing of 4852 DNA samples from 18 different breeds showed GG genotype mainly existed in egg-type chickens and dual-type chickens, while the AA genotype was mainly distributed in commercial broilers and F2 resource population. The individual carriers of the AA genotype had the highest ALP and showed better growth performance. Besides, tissue expression analysis used Cobb broiler showed significant differences between different genotypes in the spleen and duodenum. Taken together, this was the first time to determine 9 SNPs within ST3GAL4 related to ALP in chickens, 4 of them were complete linkage with each other, which provides useful information on the mutation of ST3GAL4 and could predict the serum ALP level of chicken early and as an effective potential molecular breeding marker for chickens.

Identification of key miRNAs affecting broilers with valgus-varus deformity by RNA sequencing and analysis of miRNA-mRNA interactions.

Valgus-varus Deformity (VVD) leg disease often affects chickens raised in modern large-scale breeding operations. Losses due to VVD are costly to farmers, and the condition also causes significant suffering in affected birds. In this study, we profiled RNAs from the spleens of VVD (BS) and healthy (JS) broilers using high-throughput sequencing to identify miRNAs that might be involved in the development of the disease. Fifty differentially expressed miRNAs (DEMs) were found, of which 30 were up-regulated and 20 were down-regulated in VVD-affected birds (|log 2 Fold Change| ≥ 1 and q-value < 0.05). DEMs were matched with putative target genes and 864 target genes were found. Gene Ontology (GO) analyses of these target genes showed that they were significantly enriched in the "cytoplasm" term (q-value < 0.05), and most of the target genes were enriched in "cellular component". Kyoto encyclopedia of genes and genomes (KEGG) analysis showed that they were significantly enriched in 11 signaling pathways (P-value < 0.05), including metabolic pathways, 2-oxocarboxylic acid metabolism, regulation of actin cytoskeleton, purine metabolism, endocytosis and so on. And we found that they were enriched in immune-related pathways in which MAPK, Notch, JAK-Stat, Toll-like receptor, p53 and other single pathways were involved in the development of skeletal diseases. Differentially expressed mRNAs obtained from our previous study were used to construct an interaction network consisting of 16 DEMs and 21 differentially expressed mRNAs (|log 2 Fold Change| ≥ 1 and q-value < 0.05). We found that miR-12247-5p, miR-15c-5p, miR-15b-5p, FKBP5 and HSP90AB1 were at the center of network interaction. This study provides a foundation for further investigations of the pathogenesis and genetic mechanisms underlying VVD.