Skip to main content
NCBI home page
Poultry Science logoLink to Poultry Science
. 2023 Apr 5;102(7):102682. doi: 10.1016/j.psj.2023.102682

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

Chunxia Cai *,†, Lujie Zhang *,†, Xinxin Liu *,†, Jianzeng Li *,†, Yanchao Ma *,†, Ruirui Jiang *,†, Zhuanjian Li *,†, Guoxi Li *,†, Yadong Tian *,†, Xiangtao Kang *,†,‡, Ruili Han *,†,‡,1
PMCID: PMC10172705  PMID: 37120872

Abstract

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.

Key words: valgus-varus deformity, carcass composition, meat quality, muscle atrophy, transcriptomics

INTRODUCTION

Chicken is an ideal source of meat for consumers due to its high protein content, low total fat content, and low cholesterol (John et al., 2016). With the increase in consumer demand to promote the development of the industry, not only has chicken become the second-largest consumption category, but the broiler industry has also become the second-largest livestock industry in China (Xin et al., 2015). Commercial broilers produced under intensive conditions are characterized by high growth efficiency and feed conversion rate, which meets consumers’ demand for chicken meat, but the leg disease problems of commercial broilers raised under intensive conditions are causing huge losses to the farming industry and are also affecting animal welfare (Hartcher and Lum, 2019; Baxter et al., 2021).

Valgus-varus deformity (VVD) is a difficult leg problem in fast-growing broilers. VVD is typically characterized by varus (inward angulation) or valgus (outward angulation) at a certain angle of the tibial tarsus and tarsometatarsus (Julian, 1984). It greatly impairs the walking ability of broilers, and in severe cases even leads to death by starvation and dehydration (Shim et al., 2012). In the growth and development of biological organisms, the tissue mass of bone and muscle are tightly correlated (Bonewald et al., 2013). Muscle and bone are developmentally homologous; they originate from mesenchymal stem cells in the mesoderm and ectoderm. In addition, their anatomical proximity to each other facilitates the communication of mechanical signals and chemicals between the two (Tagliaferri et al., 2015). Skeletal muscle fibers are mainly composed of fast and slow muscle fibers (Hakamata et al., 2018), myofibers account for 75% to 90% of the volume of skeletal muscle (Lefaucheur, 2010), the characteristics and types of muscle fibers determine the growth and development of poultry and meat quality (Joo et al., 2013). Previously, we performed clinical evaluation of bone morphology, bone quality, serum indicators, and growth performance (Guo et al., 2019). The metabolic status of tissues with age in VVD broilers and its relationship with inflammatory response were investigated (Li et al., 2022). We also analyzed the transcriptome of spleen tissues from normal broilers and VVD broilers to screen for candidate target genes associated with VVD (Tang et al., 2020). However, there are few studies on meat quality, the pathological status of skeletal muscle, and muscle-related transcriptome in broilers.

Muscle atrophy is mainly characterized morphologically by a decrease in muscle fiber diameter and muscle weight, accompanied by a shift from slow muscle fibers to fast muscle fibers (Ebert et al., 2010; Mo et al., 2022). In addition, it has been shown that the degree of skeletal muscle atrophy is positively correlated with the degree of myocyte apoptosis, accompanied by an upregulation of the expression of apoptosis-related factors (Bax, Bcl-2) (Primeau et al., 2002; Tews, 2002; Jin et al., 2004).

This study was conducted to determine meat quality, slaughter performance, and more comprehensive growth performance in normal and VVD Cobb broilers, the joint molecular biology, morphological methods, and transcriptomic data analysis of the leg muscles concluded that VVD causes muscle atrophy in the gastrocnemius muscle, laying the foundation for subsequent studies on VVD.

MATERIALS AND METHODS

Experimental Animals and Animal Management

Animal experiments were designed and conducted in accordance with the Regulations on the Administration of Laboratory Animals and were approved by the Animal Protection and Utilization Committee of Henan Agricultural University. The Cobb broilers used in the experiment were provided by a farm in Zhongmu, Henan Province, and all broilers used in the experiment were fed with the same diets and feeding management methods. Cage dimensions were 0.8 m long, 0.8 m wide, and 0.35 m high. Stocking density was 30 birds when 1 to 9 days old and 12 birds aging 10 to 35 days old. Lighting was maintained at 60 lux 24L (0–5 d), 8 lux 17L (6–25 d), and 60 lux 24L (26–35 d). All broilers were fed uniform diets containing 0.9% Ca, 0.45% P, 3,030 kcal/kg ME, and 22% CP at age 1 to 10 d; 0.84% Ca, 0.41% P, 3,108 kcal/kg ME, and 20.0% CP at age 11 to 22 d; 0.76% Ca, 0.38% P, 3,180 kcal/kg ME, and 19.0% CP at age 23 to 35 d. Fourteen broilers of normal (♂) and VVD (♂) were selected for euthanasia after clinical examination at 35 d for subsequent studies on carcass composition and meat quality.

Sampling and Measurement

Measurements of body size were made on d 35 (Tyasi et al., 2017). The body weight (BW) of the broilers was recorded after 12 h of fasting, then euthanized and the dressed weight (DW) was recorded after the initial treatment. The trachea, esophagus, crop, intestine, spleen, pancreas, bile, and reproductive organs were removed to measure the half-eviscerated weight (HEW), then the heart, liver, glandular stomach, myogastric, lung, abdominal fat, head, and feet were removed to measure the eviscerated weight (EW). Subsequently, gastrocnemius, pectoral, and leg muscles were artificially removed and weighed. Dressed yield, half-eviscerated yield, and eviscerated yield were calculated as the percentage of body weight.

Meat quality measurements were performed on the right breast muscle and leg muscle. The pH values and meat color values of the pectoralis major and leg muscles were measured 45 min (pH 45) after slaughter and 24 h after storage in a refrigerator at 4℃ (pH 24) using a pH-STAR pH meter (Germany, Matthaus) and an OPTO-STAR meat color meter (Germany, Matthaus). Pectoral muscles and leg muscles of similar mass and size were selected with a sampler, weighed, and put into a drip loss tube, stored in a refrigerator at 4℃ for 24 h. After removing the meat, the surface water of the meat samples was blotted out with filter paper and then weighed to calculate the drip loss. Cooking loss values were calculated as a percentage of weight loss during cooking. Shear force was measured using a C-LM3B muscle tenderness meter (Beijing Tianxiang Feiwei Instrumentation Co., Ltd, Beijing, China). The water content was determined by the direct drying method (GB/T 9695.15-2008), crude protein (CP) was determined by the Kjeldahl method (GB 50095-2010), and crude fat (CF) was determined by the Soxhlet extraction method (GB/T 9695.7-2008).

Pathological Examination

The gastrocnemius from 5 VVD broilers (♂) and 5 normal broilers (♂) were collected and preserved in 4% paraformaldehyde. The specimens were stained with hematoxylin-eosin, and the sections were scanned using a fully automated digital section scanning microscope (Motic BA600mot), and muscle tissue necrosis using the mcaudi digital section assistant system after scanning was completed. Five fields of view were randomly selected for each section under a 400× microscope, and each selected muscle fiber was measured using Image J.

RNA Extraction and RNA-seq Analysis

Total RNA was extracted from leg muscle samples of 3 VVD broilers (♂) and 3 normal broilers (♂) by using TRIzol reagent (Invitrogen, Carlsbad, CA). The NanoPhotometer1 spectrophotometer and RNA Nano6000 Assay Kit from the Agilent Bioanalyzer 2100 system (Agilent Technologies, Beijing, CA) were used to detect the concentration and integrity of total RNA. Qualified RNA samples were used to prepare libraries, library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, MA), and library quality was assessed using the Agilent Bioanalyzer 2100 system. The libraries meeting the quality standards were sequenced using the Illumina Hiseq NovaSeq 6000 platform from Novegene Bioinformatics Technology Co., Ltd. (Beijing, China), which generated paired-end reads of 150 bp. The RNA-seq sequencing data were deposited into NCBI (PRJNA913486) with accession numbers SRR22825284, SRR22825285, SRR22825286, SRR22825287, SRR22825288, and SRR22825289.

Functional Analysis of Differentially Expressed Genes

The analysis of differentially expressed genes (DEGs) was performed through the omicshare kidio bioinformatics cloud platform online software (https://www.omicshare.com). The DEGs sets obtained in the normal broilers and VVD broilers were used in this study and clustering analysis was performed using the fragments per kilobase of exon model per million mapped fragments (FPKM) values of these genes in each sample. Gene ontology (GO) entries and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways with P-value < 0.05 were considered significantly enriched. Protein–protein interaction (PPI) analysis of DEGs was predicted using the STRING database (https://string-db.org/, Organism: Gallus gallus) with a confidence interval set to 0.7. The Cytoscape (v3.6.1) software was used to visualize the protein interaction network map and identify core proteins by calculating the number of interactions between each network node.

Quantitative Real-Time PCR

A total of 6 ubiquitin–proteasome-related genes were selected from transcriptomic DEGs, and 12 leg muscle and cartilage samples from normal and VVD broilers were randomly selected for the test. Twelve gastrocnemius muscle samples were randomly selected from the normal broilers and VVD broilers for expression analysis of muscle atrophy-related genes. The reaction system volume was 10 µL containing 5 µL of SYBR1 Premix Ex TaqII (TaKaRa, Dalian, China), 3.2 µL of RNase-free water, 1 µL cDNA, and 0.4 µL of each primer. The qPCR was performed on a LightCycler 96 real-time fluorescent quantitative PCR instrument (Roche Applied Science, Indianapolis, IN), and the cycling conditions were as follows: 95°C for 3 min; 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 20 s; and 10 min extension at 72°C. The relative expression of the target gene was calculated by the 2−ΔΔCt method, and the chicken GAPDH gene was used as an internal reference gene. Three technical replicates were performed for each sample. The primer sequences are shown in Supplementary Table 1.

Statistical Analyses

The quantitative real-time PCR (qPCR) data were visualized using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA) and expressed as mean ± SEM. All carcass composition data and meat quality indicators were analyzed using 1-way ANOVA followed by SPSS version 24.0 software (SPSS Inc., Chicago, IL) and expressed as mean ± SEM. P < 0.05 indicates a significant difference (indicated by *), P < 0.01 indicates a highly significant difference (indicated by **), and P > 0.05 indicates no significant difference.

RESULTS

Effects of Broiler VVD on Carcass Characteristics

Compared with normal broilers, the slaughter indexes of VVD broilers including BW, DW, HEW, and EW were highly significant (P < 0.01), and the percentage of the half-eviscerated yield (PHEY) and percentage of the eviscerated yield (PEY) of normal broilers were significantly higher than those of VVD broilers (P < 0.05), but the percentage of dressed yield (PDY) not reach a significant level (P > 0.05). Body size traits including shank length, shank circumference, breast depth, breast width, body slope length, fossil bone length, and pelvis width were significantly higher in normal broilers than in VVD broilers (P < 0.01) (Table 1).

Table 1.

Comparison of carcass characteristics between the normal broilers and VVD broilers.

Trait Normal broilers (n = 14) Leg disorder broilers (n = 14) P-value
Body weight (g) 2,211.86A ± 78.58 953.14B ± 52.00 <0.001
Dressed weight (g) 1,931.79A ± 78.02 834.64B ± 46.66 <0.001
Half-eviscerated weight (g) 1,801.05A ± 75.41 754.78B ± 43.08 <0.001
Eviscerated weight (g) 1,535.79A ± 65.02 632.64B ± 37.57 <0.001
Percentage of dressed yield (%) 87.19 ± 0.56 87.47 ± 0.38 0.685
Percentage of the half-eviscerated yield (%) 81.23a ± 0.77 79.04b ± 0.68 0.042
Percentage of the eviscerated yield (%) 69.24A ± 0.75 65.15B ± 1.47 <0.001
Shank length (mm) 74.55A ± 1.03 63.29B ± 1.31 <0.001
Shank circumference (mm) 64.50A ± 1.02 53.93B ± 1.30 <0.001
Breast depth (mm) 74.09A ± 1.44 54.30B ± 1.56 <0.001
Breast width (mm) 85.39A ± 1.70 60.78B ± 1.92 <0.001
Fossil bone length (mm) 96.29A ± 2.19 79.74B ± 1.78 <0.001
Body slope length (mm) 156.47A ± 2.34 125.68B ± 2.79 <0.001
Pelvis width (mm) 85.62A ± 1.11 68.52B ± 1.34 <0.001
Open in a new tab

Notes: Data represent means ± SEM.

All data were analyzed using 1-way ANOVA.

a,b,A,B

The values show a statistically significant difference; uppercase characters for P < 0.01; lowercase characters for P < 0.05.

Effect of VVD on Skeletal Muscle Quality in Broilers

To further determine whether VVD in broilers has an impact on skeletal muscle quality, we compared meat quality-related indicators. In comparison to the normal broilers, the meat color, cooking loss, water, CP, and shear force of breast muscle of VVD broilers reached a significant difference level (P < 0.05). The meat color, cooking loss, water, CF, and CP of leg muscle also reached a significant difference level (P < 0.05), whereas the drip loss and pH value of both were not significantly different (P > 0.05) (Table 2).

Table 2.

Comparison of meat quality between the normal broilers and VVD broilers.

Trait Breast muscle
 Leg muscle
Normal broilers (n = 14) Leg disorder broilers (n = 14) P-value Normal broilers (n = 14) Leg disorder broilers (n = 14) P-value
Color 45 min 74.65a ± 1.59 79.76b ± 1.79 0.046 63.44A ± 1.58 69.83B ± 2.02 <0.001
Color 24 h 80.77A ± 1.53 86.11B ± 0.95 <0.001 73.41A ± 0.83 79.93B ± 1.62 <0.001
pH 45 min 6.39 ± 0.07 6.40 ± 0.06 0.849 6.73 ± 0.05 6.84 ± 0.07 0.235
pH 24 h 6.10 ± 0.04 6.17 ± 0.03 0.278 6.51 ± 0.03 6.50 ± 0.06 0.910
Cooking loss (%) 17.49A ± 0.99 12.69B ± 1.12 <0.001 10.72A ± 0.85 4.75B ± 0.43 <0.001
Drip loss (%) 1.92 ± 0.17 1.85 ± 0.16 0.766 1.53 ± 0.12 1.52 ± 0.16 0.954
Water (%) 72.66A ± 0.28 73.93B ± 0.12 0.001 74.40a ± 0.23 73.34b ± 0.47 0.046
Crude fat (%) 3.29 ± 0.22 2.88 ± 0.22 0.196 4.32A ± 0.21 3.66B ± 0.24 <0.001
Crude protein (%) 24.68A ± 0.21 23.46B ± 0.19 <0.001 20.87A ± 0.17 19.71B ± 0.23 <0.001
Shear force (kg/f) 6.35A ± 0.43 3.89B ± 0.41 <0.001 12.96A ± 0.51 4.50B ± 0.47 <0.001
Open in a new tab

Notes: Data represent means ± SEM.

All data were analyzed using 1-way ANOVA.

a,b,A,B

The values show a statistically significant difference; uppercase characters for P < 0.01; lowercase characters for P < 0.05.

Morphological Evaluation of Skeletal Muscle

The weights of the leg muscle, gastrocnemius muscle, and breast muscle of normal broilers showed significantly higher than those of VVD broilers by morphological observation and weighing (P < 0.01) (Figure 1).

Figure 1.

Figure 1

Open in a new tab

Lower skeletal muscle weights in broilers affected by VVD. (A–C) Representative morphological images of skeletal muscle of normal broilers and VVD broilers under the same conditions. (D–F) The quantitative analysis of skeletal muscle weight of broilers in normal and VVD (n = 14). Data represent mean means ± SEM. Two-tailed unpaired t-test. (**P < 0.01, *P < 0.05.)

Histopathological and Biological Evaluation of Gastrocnemius Muscle

Compared with the normal broilers, VVD broilers’ muscle pathological sections had blurred cell borders, the disappearance of the transverse lines, and myocyte necrosis (Figures 2A–D). Histological studies showed that the area and diameter of individual myocytes in VVD broilers were smaller compared with those in normal broilers (P < 0.01) (Figures 2E and 2F). It was also found that myasthenic marker genes, fast muscle marker genes, and apoptosis-related factors were significantly highly expressed in the gastrocnemius muscle of the VVD broilers (P < 0.01) (Figures 3A–D). The above study indicates that muscle atrophy occurred in the gastrocnemius muscle of broilers with VVD.

Figure 2.

Figure 2

Open in a new tab

Histopathological analysis of gastrocnemius muscle. (A) Transverse section of the gastrocnemius muscle in a VVD broiler (HE*40). The direction indicated by the coordinate flag is the necrotic muscle fiber. (B) Transverse section of the gastrocnemius muscle in a normal broiler (HE*40). (C) Longitudinal section of the gastrocnemius muscle of a VVD broiler (HE*40). (D) Longitudinal section of the gastrocnemius muscle of a normal broiler (HE*40), the direction of the arrow indicates the skeletal muscle transverse pattern. (E) The gastrocnemius fiber area of VVD broilers is smaller than that of normal broilers. (F) The gastrocnemius fiber area of VVD broilers is smaller than that of normal broilers. Data represent mean means ± SEM. (**P < 0.01, *P < 0.05.)

Figure 3.

Figure 3

Open in a new tab

Biological analysis of gastrocnemius muscle. (A) Expression of gastrocnemius muscle atrophy marker genes. (B) Expression of apoptosis-related genes in gastrocnemius muscle. (C and D) Expression of gastrocnemius muscle fiber type marker genes. Data represent mean means ± SEM. (**P < 0.01, *P < 0.05.)

DEGs Between Normal and VVD Groups

A total of 6 cDNA libraries were constructed using total RNA from the leg muscle of 3 VVD broilers (♂) and 3 normal broilers (♂). After quality control, clean data were localized to the chicken reference genome (Galgal 6.0). Supplementary Table 2 demonstrates that the percentage of mapped reads ranged from 86.94% to 91.37%. A total of 2,281 known genes and 6,780 novel genes were identified in the transcriptome data in this study. To investigate the mechanism of VVD leg disease in broilers, a total of 736 DEGs were analyzed in this study (P-value < 0.05 and log2|fold-change|≥1). In 6 VVD broilers and normal broilers, 337 genes were up-regulated and 399 genes were down-regulated in expression (Figure 4). In this study, we used the differential gene sets obtained from the normal and VVD groups to perform cluster analysis using the FPKM values of these genes in each sample (Figure 5). GO annotation, KEGG functional enrichment analysis, and protein interaction analysis were performed on 736 DEGs. GO enrichment indicated that these DEGs were mainly involved in the multicellular organismal process, multicellular organismal development, and anatomical structure development (Figure 6). KEGG analysis shows that DEGs are significantly enriched in proteasome, and lysosomes (Figure 7). Protein interactions were analyzed and the results showed that protein interactions were higher for genes encoding proteasome-related pathways, and members of the ubiquitin-specific processing (Figure 8). The DEGs related to these signaling pathways are likely to be responsible for the atrophy that occurs in the muscles.

Figure 4.

Figure 4

Open in a new tab

Volcano plot showing DE-mRNAs in VVD broilers and normal broilers. Green and red colors indicate significant up- and downregulation of gene expression in the VVD broilers, respectively.

Figure 5.

Figure 5

Open in a new tab

Cluster analyses of DEGs. Red means a relatively higher level of expression, blue means a relatively lower level of expression.

Figure 6.

Figure 6

Open in a new tab

The top 20 gene ontology (GO) terms of the DEGs. The Y-axis indicates the detailed terms and the X-axis indicates the gene ratio.

Figure 7.

Figure 7

Open in a new tab

The top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the DEGs. The Y-axis indicates the name of the KEGG pathway and the X-axis indicates the gene ratio. The size of the dots represents the number of genes, and the color of the dots represents −log10 (P-value).

Figure 8.

Figure 8

Open in a new tab

Protein interaction network of the DEGs.

Quantitative Real-Time PCR Analysis

To further investigate the role played by ubiquitin–proteasome system (UPS) in the leg muscle and cartilage, 6 genes related to ubiquitin–proteasome were randomly selected for qPCR, results showed that the expression of UPS-related genes was significantly higher in both normal broilers than in VVD broilers in the leg muscle (P < 0.05) (Figure 9A), whereas the opposite was true in the cartilage (Figure 9B).

Figure 9.

Figure 9

Open in a new tab

Expression levels of ubiquitin–proteasome-related genes in different tissues. (A) Expression of ubiquitin–proteasome-related genes in the leg muscle. (B) Expression levels of ubiquitin–proteasome-related genes in cartilage. The expression level was calculated using the comparative cycle threshold (2−ΔΔCt) value method. The data were expressed as the mean ± SEM. (**P < 0.01, *P < 0.05.)

DISCUSSION

Slaughter performance is an important indicator that affects the economic efficiency of the broiler industry and evaluates the measurement of the meat production performance of livestock and poultry (Li et al., 2017; Jin et al., 2019). Compared to the normal broilers, the EW, FEW, breast muscle weight, leg muscle weight, shank length, shank circumference, breast depth, breast width, body slope length, fossil bone length, and pelvis width in broilers affected by VVD had reached highly significant difference levels (P < 0.01). It showed that the leg problems of broilers reduced slaughter performance.

The quality of chicken meat directly affects the economic efficiency of broiler production, and the common indexes used to assess the quality of meat are meat color, tenderness, etc. (Mir et al., 2017). In this study, the cooking loss and shear force of VVD broilers were significantly lower than those of the normal broilers (P < 0.01), indicating that the meat water-holding capacity of VVD broilers was better, and the meat of normal broilers was chewy. Meat color is the most intuitive indicator for consumer evaluation of meat quality, whereas being either too light or too dark will be judged as poor quality meat (Carvalho et al., 2017). It was observed that the color of the skeletal muscle of VVD broilers is darker than normal broilers, and the color of meat becomes darker and brownish with the increase of oxygen exposure time in the air (Mancini and Hunt, 2005). Protein and fat are important indicators for meat quality studies. The level of intramuscular fat content reflects the ability of muscle fat deposition, and a suitable fat content can improve the juiciness, tenderness, palatability, and overall acceptability of meat to some extent (Madeira et al., 2016). In this study, the CF content of the leg muscle of normal chicken was significantly higher than that of VVD broilers (P < 0.01). The CP content of both breast muscle and leg muscle was significantly higher in normal broilers than in VVD broilers (P < 0.01). The physiology of skeletal muscle atrophy is manifested by a decrease in CP content. Therefore, it is assumed that VVD has a negative effect on meat quality and may have caused muscle atrophy. A study on tibial dyschondroplasia also found that tibial dyschondroplasia adversely affected both leg and breast meat quality in broilers (Cao et al., 2020).

Skeletal muscle plays a crucial role in regulating metabolism, locomotor capacity, and health in the animal body. This study found that the skeletal muscle mass of VVD broilers decreased compared with normal broilers (P < 0.01), a reduction in muscle mass can result in reduced locomotor capacity and increased morbidity and mortality (Isoyama et al., 2014; Gibson et al., 2015; Chen et al., 2019). In addition, compared with normal broilers, the muscle fiber diameter and area of VVD broilers decreased (P < 0.01). The morphology of skeletal muscle atrophy is characterized by a decrease in muscle weight and a reduction in the cross-sectional area of muscle fibers, and the present study on the morphology of the gastrocnemius muscle is consistent with the manifestation of muscle atrophy.

Besides, some studies have indicated that another manifestation of muscle atrophy is a shift in muscle fiber type from slow to fast muscle fiber types (Blaauw et al., 2013). The conversion of muscle fiber types can be determined by the expression of slow myofiber (SM) and fast red myofiber (FRM) in the tissue. The conversion of muscle fiber type can also be determined by the ratio of myogenin (MYOG) to myogenic differentiation (MYOD) (Ehlers et al., 2014). In this trial, we used 2 methods to detect muscle fiber type by qPCR and found that the expression of the fast muscle marker genes FRM and MYOG was significantly higher in the VVD broilers (P < 0.05). This suggests that VVD affects a shift in muscle fiber type from slow to fast muscle, which is consistent with the research on the change of muscle fiber type from slow muscle fiber to a fast muscle fiber in chicken disuse muscular atrophy (Dingboom et al., 2002). What's more, the expression of the muscle atrophy marker genes muscle RING finger-1 (MuRF-1) and Atrogin-1 was significantly higher in the VVD broilers (P < 0.01). All studies suggest that VVD affects skeletal muscle growth and development, but the exact mechanism is unknown, for this reason we performed a comparative analysis of the transcriptome of the Cobb broiler leg muscle in normal broilers and VVD broilers.

Muscle growth is controlled by different genes through different regulatory and biological process pathways. GO and KEGG enrichment analyses of 736 DEGs identified in normal and VVD broilers were performed with the aim of finding key genes and pathways that influence muscle growth and development. GO was enriched for multicellular biological processes, anatomical development, regulation of multicellular biological development, and proteasome complexes. KEGG enrichment results revealed enrichment in differential genes proteasome pathway, PPAR, ECM-receptor interaction, and lysosomal signaling pathway. Among them, the proteasome pathway was the most significantly enriched. The proteasome is the main mechanism used by cells to regulate specific proteins and remove misfolded proteins, and studies have shown that proteasome activity is important for muscle growth and maintenance of muscle fiber integrity and that deletion of proteasome complex components can lead to muscle fiber degeneration and muscle disease (Kitajima et al., 2014). Protein interaction analysis also revealed that genes encoding proteasome-related genes (PSMA6, PSMD7, PSMB4, PSMC1, etc.) and ubiquitin-related genes (USP14 and UBFD1) were in strong reciprocal relationships. The UPS is considered to be the major intracellular protein degradation system and its function is important for muscle homeostasis and health (Kitajima et al., 2020). Muscle protein is an important protein to maintain the normal physiological function of animal skeletal muscle, the balance of protein synthesis rate and degradation rate maintains the stability of skeletal muscle mass, when the protein synthesis rate is less than the protein degradation rate, it will lead to the reduction of skeletal muscle mass and muscle fiber area (Tipton and Wolfe, 2001; Kimball et al., 2002).

Muscle and bone have the same origin and are structurally adjacent, and as endocrine organs, they secrete related factors that act on each other (Bosco et al., 2021). Muscle secretory factor myostatin (MSTN) leads to increased bone resorption by inducing the formation of osteoclasts (Dankbar et al., 2015). Irisin acts in bone remodeling by increasing osteoblast survival and producing the bone local remodeling regulator sclerostin (Kim et al., 2018). Matrix metallopeptidase 2 (MMP2) positively contributes to the growth of osteoblasts and osteoclasts (Mosig et al., 2007). Beta-aminoisobutyric acid (BAIBA) protects the function of reactive oxygen species to prevent bone loss (Kitase et al., 2018). Fibroblast growth factor 2 (FGF2) has an osteogenic effect (Kirk et al., 2020). Insulin-like growth factor-1 (IGF-1) works as a systemic growth factor, stimulating osteogenic progenitor cells at their source to form bone, increasing osteocortical thickness and trabecular volume, and responsible for periosteal bone formation by osteoblasts under increased load (Gross et al., 2002). Bone secretory factor transforming growth factor-beta (TGF-β) enters the circulation from the bone matrix and in combination with MSTN causes muscle atrophy or reduces muscle function (Bravenboer et al., 2001; Gumucio et al., 2015). Receptor activator of nuclear factor-kappa B ligand (RANKL) inhibits muscle mass and function (Xiong et al., 2021). Osteocalcin (OCN) binds to G protein-coupled receptor family 6 group A after decarboxylation, induces muscle metabolism, improves glucose uptake, and has a positive effect on muscle mass and muscle function (Mohammad Rahimi et al., 2021). Fibroblast growth factor 9 (FGF9) inhibits calcium homeostasis in myotubes in isolated myocytes (Huang et al., 2018). Prostaglandin E2 (PGE2) acts on injured muscles and induces the proliferation of muscle specific stem cells to proliferate, which can promote muscle remodeling and repair (Zhang et al., 2015). The secretion of these secretory factors is jointly influenced by exercise. Exercise upregulates the muscle factor irisin, MMP2, BAIBA, IGF-1, FGF-2 to stimulate osteogenesis, whereas exercise down-regulates the level of MSTN to stimulate osteogenesis and inhibit bone resorption. Exercise up-regulates OCN, FGF9, PGE2 to improve muscle mass, and down-regulates the levels of TGF-β, RANKL to improve muscle mass.

The UPS not only has a major impact on muscle, but also plays a key role in bone growth and development. By inhibiting the specific catalytic β-subunit of the proteasome, the function of osteoblasts is enhanced and the number of osteoclasts is increased to promote bone formation in vitro and in vivo (Garrett et al., 2003). The UPS can promote the expression of osteogenic protein 2, which promotes the maturation and differentiation of osteoblasts through the Wnt signaling pathway. In addition, it can also promote bone formation by regulating the expression and degradation of the transcription factor runt-related transcription factor 2 (Edwards et al., 2008). To this end, we further investigated the expression of UPS genes in leg muscle and cartilage tissues, and found that the expression of UPS in leg muscle of broilers with VVD was significantly lower than that of normal broilers, whereas the expression in cartilage showed the opposite trend. Combined with previous researches on skeleton, we learned that VVD broilers had significantly reduced bone density and bone strength, abnormal cartilage growth and impaired calcification, and slow proliferation rate of chondrocytes. We hypothesize that UPS cause reduced bone density and bone strength, decreased muscle mass and muscle atrophy in VVD broilers through regulation of bone matrix and protein degradation.

CONCLUSION

We combined carcass traits, meat quality indicators and histopathological changes in gastrocnemius muscle of Cobb broilers and found that the presence of broiler leg problems (VVD) adversely affected slaughter traits and meat quality of Cobb broilers, leading to gastrocnemius muscle atrophy. DEGs of the leg muscle transcriptome yielded pathways associated with protein degradation and muscle atrophy, and muscle growth and development are inextricably linked to bone growth and development. The role of these pathways on muscle and skeleton needs to be further explored, the next step will be to localize these key pathways and key genes in detail at the cellular and protein levels, with a view to providing a basis for studying the mechanisms of VVD leg muscle development.

ACKNOWLEDGMENTS

This study was supported by the Scientific Studio of Zhongyuan Scholars (no. 30601985); Natural Science Foundation of Henan Province (no. 222300420458); National Natural Science Foundation of China (no. 32172720); Key Research Project of the Shennong Laboratory (SN01-2022-05).

DISCLOSURES

We declare that we have no conflict of interest.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2023.102682.

Appendix. Supplementary materials

mmc1.docx (16.3KB, docx)

REFERENCES

  1. Baxter M., Richmond A., Lavery U., O'Connell N.E. A comparison of fast growing broiler chickens with a slower-growing breed type reared on Higher Welfare commercial farms. PLoS One. 2021;16 doi: 10.1371/journal.pone.0259333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blaauw B., Schiaffino S., Reggiani C. Mechanisms modulating skeletal muscle phenotype. Compr. Physiol. 2013;3:1645–1687. doi: 10.1002/cphy.c130009. [DOI] [PubMed] [Google Scholar]
  3. Bonewald L.F., Kiel D.P., Clemens T.L., Esser K., Orwoll E.S., O'Keefe R.J., Fielding R.A. Forum on bone and skeletal muscle interactions: summary of the proceedings of an ASBMR workshop. J. Bone Miner. Res. 2013;28:1857–1865. doi: 10.1002/jbmr.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bosco F., Musolino V., Gliozzi M., Nucera S., Carresi C., Zito M.C., Scarano F., Scicchitano M., Reale F., Ruga S., Maiuolo J., Macrì R., Guarnieri L., Coppoletta A.R., Mollace R., Muscoli C., Palma E., Mollace V. The muscle to bone axis (and viceversa): an encrypted language affecting tissues and organs and yet to be codified? Pharmacol. Res. 2021;165 doi: 10.1016/j.phrs.2021.105427. [DOI] [PubMed] [Google Scholar]
  5. Bravenboer N., Engelbregt M.J., Visser N.A., Popp-Snijders C., Lips P. The effect of exercise on systemic and bone concentrations of growth factors in rats. J. Orthop. Res. 2001;19:945–949. doi: 10.1016/S0736-0266(01)00026-2. [DOI] [PubMed] [Google Scholar]
  6. Cao Q.Q., Kong A.A., Tao K.S., Zheng S.H., Tong C., Wang X.B., Tong Z.X., Rehman M.U., Huang S.C. Characterization of growth performance, meat quality and serum biochemical parameters in chickens suffering from tibial dyschondroplasia. Livestock. Sci. 2020;233 [Google Scholar]
  7. Carvalho R.H., Honorato D.C.B., Guarnieri P.D., Soares A.L., Pedrão M.R., Oba A., Paião F.G., Ida E.I., Shimokomaki M. In-transit development of color abnormalities in turkey breast meat during winter season. J. Anim. Sci. Technol. 2017;59:30. doi: 10.1186/s40781-017-0157-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen F., Wu P., Shen M., He M., Chen L., Qiu C., Shi H., Zhang T., Wang J., Xie K., Dai G., Wang J., Zhang G. Transcriptome analysis of differentially expressed genes related to the growth and development of the Jinghai yellow chicken. Genes (Basel) 2019;10:539. doi: 10.3390/genes10070539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dankbar B., Fennen M., Brunert D., Hayer S., Frank S., Wehmeyer C., Beckmann D., Paruzel P., Bertrand J., Redlich K., Koers-Wunrau C., Stratis A., Korb-Pap A., Pap T. Myostatin is a direct regulator of osteoclast differentiation and its inhibition reduces inflammatory joint destruction in mice. Nat. Med. 2015;21:1085–1090. doi: 10.1038/nm.3917. [DOI] [PubMed] [Google Scholar]
  10. Dingboom E.G., van Oudheusden H., Eizema K., Weijs W.A. Changes in fibre type composition of gluteus medius and semitendinosus muscles of Dutch Warmblood foals and the effect of exercise during the first year postpartum. Equine Vet. J. 2002;34:177–183. doi: 10.2746/042516402776767312. [DOI] [PubMed] [Google Scholar]
  11. Ebert S.M., Monteys A.M., Fox D.K., Bongers K.S., Shields B.E., Malmberg S.E., Davidson B.L., Suneja M., Adams C.M. The transcription factor ATF4 promotes skeletal myofiber atrophy during fasting. Mol. Endocrinol. 2010;24:790–799. doi: 10.1210/me.2009-0345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Edwards C.M., Zhuang J., Mundy G.R. The pathogenesis of the bone disease of multiple myeloma. Bone. 2008;42:1007–1013. doi: 10.1016/j.bone.2008.01.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ehlers M.L., Celona B., Black B.L. NFATc1 controls skeletal muscle fiber type and is a negative regulator of MyoD activity. Cell Rep. 2014;8:1639–1648. doi: 10.1016/j.celrep.2014.08.035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Garrett I.R., Chen D., Gutierrez G., Zhao M., Escobedo A., Rossini G., Harris S.E., Gallwitz W., Kim K.B., Hu S., Crews C.M., Mundy G.R. Selective inhibitors of the osteoblast proteasome stimulate bone formation in vivo and in vitro. J. Clin. Invest. 2003;111:1771–1782. doi: 10.1172/JCI16198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gibson D., Burden S., Strauss B., Todd C., Lal S. The role of computed tomography in evaluating body composition and the influence of reduced muscle mass on clinical outcome in abdominal malignancy: a systematic review. Eur. J. Clin. Nutr. 2015;69:1079–1086. doi: 10.1038/ejcn.2015.32. [DOI] [PubMed] [Google Scholar]
  16. Gross T.S., Srinivasan S., Liu C.C., Clemens T.L., Bain S.D. Noninvasive loading of the murine tibia: an in vivo model for the study of mechanotransduction. J. Bone Miner. Res. 2002;17:493–501. doi: 10.1359/jbmr.2002.17.3.493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gumucio J.P., Sugg K.B., Mendias C.L. TGF-β superfamily signaling in muscle and tendon adaptation to resistance exercise. Exerc. Sport Sci. Rev. 2015;43:93–99. doi: 10.1249/JES.0000000000000041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Guo Y., Tang H., Wang X., Li W., Wang Y., Yan F., Kang X., Li Z., Han R. Clinical assessment of growth performance, bone morphometry, bone quality, and serum indicators in broilers affected by valgus-varus deformity. Poult. Sci. 2019;98:4433–4440. doi: 10.3382/ps/pez269. [DOI] [PubMed] [Google Scholar]
  19. Hakamata Y., Watanabe K., Amo T., Toyomizu M., Kikusato M. Characterization of mitochondrial content and respiratory capacities of broiler chicken skeletal muscles with different muscle fiber compositions. J Poult Sci. 2018;55:210–216. doi: 10.2141/jpsa.0170141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hartcher K.M., Lum H.K. Genetic selection of broilers and welfare consequences: a review. Worlds Poult. Sci. J. 2019;76:154–167. [Google Scholar]
  21. Huang J., Wang K., McCormick L., Brotto L., Dallas S., Brotto M. Fibroblast Growth Factor 9 (FGF9) is expressed in an osteocyte-like “Mini-bone” cell line and inhibits C2C12 myogenesis via overexpression of myostatin. FASEB J. 2018;32:lb491. [Google Scholar]
  22. Isoyama N., Qureshi A., Avesani C., Lindholm B., Bàràny P., Heimbürger O., Cederholm T., Stenvinkel P., Carrero J.J. Comparative associations of muscle mass and muscle strength with mortality in dialysis patients. Clin. J. Am. Soc. Nephrol. 2014;9:1720–1728. doi: 10.2215/CJN.10261013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jin S., Fan X., Yang L., He T., Xu Y., Chen X., Liu P., Geng Z. Effects of rearing systems on growth performance, carcass yield, meat quality, lymphoid organ indices, and serum biochemistry of Wannan yellow chickens. Anim. Sci. J. 2019;90:887–893. doi: 10.1111/asj.13220. [DOI] [PubMed] [Google Scholar]
  24. Jin X., Qu N., He Y., Guo Y., Wang C., Lu C., Li C., Wei Z., Chen J. Cell apoptosis in atrophic skeletal muscle induced by immoblization in rabbits–an experimental study using TUNEL. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2004;21:628–630. 635. [PubMed] [Google Scholar]
  25. John K.A., Maalouf J., Barsness C.B., Yuan K., Cogswell M.E., Gunn J.P. Do lower calorie or lower fat foods have more sodium than their regular counterparts? Nutrients. 2016;8:511. doi: 10.3390/nu8080511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Joo S.T., Kim G.D., Hwang Y.H., Ryu Y.C. Control of fresh meat quality through manipulation of muscle fiber characteristics. Meat Sci. 2013;95:828–836. doi: 10.1016/j.meatsci.2013.04.044. [DOI] [PubMed] [Google Scholar]
  27. Julian R.J. Valgus-varus deformity of the intertarsal joint in broiler chickens. Can. Vet. J. 1984;25:254–258. [PMC free article] [PubMed] [Google Scholar]
  28. Kim H., Wrann C.D., Jedrychowski M., Vidoni S., Kitase Y., Nagano K., Zhou C., Chou J., Parkman V.A., Novick S.J., Strutzenberg T.S., Pascal B.D., Le P.T., Brooks D.J., Roche A.M., Gerber K.K., Mattheis L., Chen W., Tu H., Bouxsein M.L., Griffin P.R., Baron R., Rosen C.J., Bonewald L.F., Spiegelman B.M. Irisin mediates effects on bone and fat via αV integrin receptors. Cell. 2018;175:1756–1768.e1717. doi: 10.1016/j.cell.2018.10.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kimball S.R., Farrell P.A., Jefferson L.S. Invited review: role of insulin in translational control of protein synthesis in skeletal muscle by amino acids or exercise. J. Appl. Physiol. (1985) 2002;93:1168–1180. doi: 10.1152/japplphysiol.00221.2002. [DOI] [PubMed] [Google Scholar]
  30. Kirk B., Feehan J., Lombardi G., Duque G. Muscle, bone, and fat crosstalk: the biological role of myokines, osteokines, and adipokines. Curr. Osteoporos. Rep. 2020;18:388–400. doi: 10.1007/s11914-020-00599-y. [DOI] [PubMed] [Google Scholar]
  31. Kitajima Y., Tashiro Y., Suzuki N., Warita H., Kato M., Tateyama M., Ando R., Izumi R., Yamazaki M., Abe M., Sakimura K., Ito H., Urushitani M., Nagatomi R., Takahashi R., Aoki M. Proteasome dysfunction induces muscle growth defects and protein aggregation. J. Cell Sci. 2014;127:5204–5217. doi: 10.1242/jcs.150961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kitajima Y., Yoshioka K., Suzuki N. The ubiquitin-proteasome system in regulation of the skeletal muscle homeostasis and atrophy: from basic science to disorders. J. Physiol. Sci. 2020;70:40. doi: 10.1186/s12576-020-00768-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kitase Y., Vallejo J.A., Gutheil W., Vemula H., Jähn K., Yi J., Zhou J., Brotto M., Bonewald L.F. β-aminoisobutyric acid, l-BAIBA, is a muscle-derived osteocyte survival factor. Cell Rep. 2018;22:1531–1544. doi: 10.1016/j.celrep.2018.01.041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lefaucheur L. A second look into fibre typing—relation to meat quality. Meat Sci. 2010;84:257–270. doi: 10.1016/j.meatsci.2009.05.004. [DOI] [PubMed] [Google Scholar]
  35. Li J., Ma Y., Zhang L., Cai C., Guo Y., Zhang Z., Li D., Tian Y., Kang X., Han R., Jiang R. Valgus-varus deformity induced abnormal tissue metabolism, inflammatory damage and apoptosis in broilers. Br. Poult. Sci. 2022;64:1–10. doi: 10.1080/00071668.2022.2121640. [DOI] [PubMed] [Google Scholar]
  36. Li Y.P., Wang Z.Y., Yang H.M., Xu L., Xie Y.J., Jin S.L., Sheng D.F. Effects of dietary fiber on growth performance, slaughter performance, serum biochemical parameters, and nutrient utilization in geese. Poult. Sci. 2017;96:1250–1256. doi: 10.3382/ps/pew385. [DOI] [PubMed] [Google Scholar]
  37. Madeira M., Pires V., Alfaia C., Lopes P., Martins S., Pinto R., Prates J. Restriction of dietary protein does not promote hepatic lipogenesis in lean or fatty pigs. Br. J. Nutr. 2016;1:1–13. doi: 10.1017/S0007114516000453. [DOI] [PubMed] [Google Scholar]
  38. Mancini R.A., Hunt M.C. Current research in meat color. Meat Sci. 2005;71:100–121. doi: 10.1016/j.meatsci.2005.03.003. [DOI] [PubMed] [Google Scholar]
  39. Mir N.A., Rafiq A., Kumar F., Singh V., Shukla V. Determinants of broiler chicken meat quality and factors affecting them: a review. J. Food Sci. Technol. 2017;54:2997–3009. doi: 10.1007/s13197-017-2789-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Mo J., Wang Z., Liu Q., Li Z., Nie Q. Construction and analysis of disuse atrophy model of the gastrocnemius muscle in chicken. Int. J. Mol. Sci. 2022;23:6892. doi: 10.3390/ijms23136892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Mohammad Rahimi G.R., Niyazi A., Alaee S. The effect of exercise training on osteocalcin, adipocytokines, and insulin resistance: a systematic review and meta-analysis of randomized controlled trials. Osteoporos. Int. 2021;32:213–224. doi: 10.1007/s00198-020-05592-w. [DOI] [PubMed] [Google Scholar]
  42. Mosig R.A., Dowling O., DiFeo A., Ramirez M.C., Parker I.C., Abe E., Diouri J., Aqeel A.A., Wylie J.D., Oblander S.A., Madri J., Bianco P., Apte S.S., Zaidi M., Doty S.B., Majeska R.J., Schaffler M.B., Martignetti J.A. Loss of MMP-2 disrupts skeletal and craniofacial development and results in decreased bone mineralization, joint erosion and defects in osteoblast and osteoclast growth. Hum. Mol. Genet. 2007;16:1113–1123. doi: 10.1093/hmg/ddm060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Primeau A.J., Adhihetty P.J., Hood D.A. Apoptosis in heart and skeletal muscle. Can. J. Appl. Physiol. 2002;27:349–395. doi: 10.1139/h02-020. [DOI] [PubMed] [Google Scholar]
  44. Shim M.Y., Karnuah A.B., Anthony N.B., Pesti G.M., Aggrey S.E. The effects of broiler chicken growth rate on valgus, varus, and tibial dyschondroplasia. Poult. Sci. 2012;91:62–65. doi: 10.3382/ps.2011-01599. [DOI] [PubMed] [Google Scholar]
  45. Tagliaferri C., Wittrant Y., Davicco M.J., Walrand S., Coxam V. Muscle and bone, two interconnected tissues. Ageing Res. Rev. 2015;21:55–70. doi: 10.1016/j.arr.2015.03.002. [DOI] [PubMed] [Google Scholar]
  46. Tang H., Guo Y., Zhang Z., Li Z., Zhang Y., Li Y., Kang X., Han R. Integrative analysis of long non-coding RNA and mRNA in broilers with valgus-varus deformity. PLoS One. 2020;15 doi: 10.1371/journal.pone.0239450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tews D.S. Apoptosis and muscle fibre loss in neuromuscular disorders. Neuromuscul. Disord. 2002;12:613–622. doi: 10.1016/s0960-8966(02)00030-5. [DOI] [PubMed] [Google Scholar]
  48. Tipton K.D., Wolfe R.R. Exercise, protein metabolism, and muscle growth. Int. J. Sport Nutr. Exerc. Metab. 2001;11:109–132. doi: 10.1123/ijsnem.11.1.109. [DOI] [PubMed] [Google Scholar]
  49. Tyasi T., Qin N., Jing Y., Mu F., Zhu H., Liu D., Yuan S., Xu R. Assessment of relationship between body weight and body measurement traits of indigenous Chinese Dagu chickens using path analysis. India J. Anim. Res. 2017;51:588–593. [Google Scholar]
  50. Xin X., Zhang Y., Jimin W., Nuetah J.A. Effects of farm size on technical efficiency in China's broiler sector: a stochastic meta-frontier approach. Can. J. Agr. Econ. 2015;64:493–516. [Google Scholar]
  51. Xiong J., Le Y., Rao Y., Zhou L., Hu Y., Guo S., Sun Y. RANKL mediates muscle atrophy and dysfunction in a cigarette smoke-induced model of chronic obstructive pulmonary disease. Am. J. Respir. Cell Mol. Biol. 2021;64:617–628. doi: 10.1165/rcmb.2020-0449OC. [DOI] [PubMed] [Google Scholar]
  52. Zhang Y., Desai A., Yang S.Y., Bae K.B., Antczak M.I., Fink S.P., Tiwari S., Willis J.E., Williams N.S., Dawson D.M., Wald D., Chen W.D., Wang Z., Kasturi L., Larusch G.A., He L., Cominelli F., Di Martino L., Djuric Z., Milne G.L., Chance M., Sanabria J., Dealwis C., Mikkola D., Naidoo J., Wei S., Tai H.H., Gerson S.L., Ready J.M., Posner B., Willson J.K., Markowitz S.D. Tissue regeneration. Inhibition of the prostaglandin-degrading enzyme 15-PGDH potentiates tissue regeneration. Science. 2015;348:aaa2340. doi: 10.1126/science.aaa2340. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

mmc1.docx (16.3KB, docx)

Articles from Poultry Science are provided here courtesy of Elsevier

RESOURCES