Valgus-varus deformity induced abnormal tissue metabolism, inflammatory damage and apoptosis in broilers

ABSTRACT 1. This study explored the tissue metabolic status and the relationship with inflammation in valgus-valgus deformity (VVD) broilers with increasing age. 2. Tissue and blood from VVD and healthy broilers were collected at two, four and five weeks old. A fully automated biochemical analyser, real-time PCR, HE staining and enzyme-linked immunosorbent assay were used to detect tissue metabolic indexes, mRNA levels of inflammation and apoptosis cytokines in immune organs, histological changes and serum inflammation and immune-related protein contents in VVD broilers. 3. The results showed that VVD increased the levels of total protein, albumin, alanine aminotransferase at five weeks of age, aspartate aminotransferase, urea and creatine kinase in blood at two weeks of age. It upregulated the gene expression of inflammatory factors IL-1β, IL-6, IL-8, TNF-α, NF-κB and TGF-β and apoptotic factors FAS, Bcl-2, caspase-3 and 9 in immune organs; increased levels of serum proteins TNF-α, IL-1β and IL-6 and decreased levels of serum immunoglobulins IgY and CD3+. 4. In addition, with increasing age, IL-10 gene expression gradually increased in the BF and decreased in the spleen. 5. In conclusion, VVD broilers have disorders of liver and kidney metabolism, inflammation and apoptosis of immune organs and increased levels of serum inflammatory factor proteins.


Introduction
Due to intensive selection and breeding of broilers, leg disease has become an important factor endangering the growth and development, economic efficiency and animal welfare of broilers (Güz et al. 2019). The number of poultry with various leg problems has been estimated to be about 12.5 billion per year worldwide (Almeida Paz et al. 2010). For example, in the UK about 27.6% of poultry have locomotor impairment and 3.3% of them are barely able to walk (Knowles et al. 2008). Common broiler leg diseases include femoral head necrosis (FHN), tibial dyschondroplasia (TD) and valgus-varus deformity (VVD; Guo et al. 2019). The FHN occurs mainly in the late stages of breeding. The typical anatomical feature of FHN is that the cartilage of the entire femoral head resembles a cap, which often falls off (Liu et al. 2021). TD is characterised by avascular and non-mineralised cartilage tissue in the proximal growth plate of the tibial tarsal joint . VVD is a leg deformity of complex aetiology (Guo et al. 2019;van der Pol et al. 2017), initially known as 'leg twist' (Shim et al. 2012). This is mainly characterised by valgus (outward angulation) or varus (inward angulation) at a certain angle of the tibial tarsus and tarsometatarsus, bilaterally or unilaterally . The deformed walking posture leads to damage and abnormal development of the skeletal structures of the legs (Guo et al. 2019), which, in, turn affects the overall physiological and biochemical status and reduces immune function and increases vulnerability to external environmental factors, such as bacterial infections.
Blood biochemical indicators can indicate the overall physiological and biochemical status of the body (Rehman et al. 2017). Inflammation is a defensive response of the body caused by the stimulation of multiple inflammatory factors. The thymus, spleen and bursa of Fasciola (BF) play an important role in the response to inflammation. Cytokines are intermediate hubs of the inflammatory response, being, in the main, pro-inflammatory factors TNF-α, IL-1β, IL-6, IL-8 and anti-inflammatory factors IL-10 and TGF-β (Crusz and Balkwill 2015). The mitochondrial apoptosis pathway and the death receptor apoptosis pathway are common apoptotic pathways; pathway-related proteins include BAX, Bcl-2, FAS, FASL, caspase-3, caspase-8 and caspase-9 (Yin et al. 2020). NF-κB is a nuclear transcription factor that plays an important role in the regulation of inflammatory cytokines and the induction of apoptosis (Lawrence et al. 2001).
Cobb broilers were the first breed with independent intellectual property rights in China, accounting for 32.46% of the broiler market in 2020 (Source: China Animal Husbandry Association). The following trial analysed blood biochemical parameters, expression of BF, spleen and thymus inflammatory and apoptotic cytokines and serum levels of inflammatory-related proteins in Cobb VVD broilers at two, four and five weeks old. and Use Committee of Henan Agricultural University, and were approved by the Animal Care Committee of the College of Animal Science and Technology, Henan Agricultural University, China (18-0120).

Animal management and collection of broilers
VVD and health Cobb broilers were obtained from a commercial facility (total = 60 000) in Hebi. The broilers were raised in cages for 37 d. There were three chicken houses in the facility, each with four rows arranged as three layers, with 62 chicken cages per layer. Cage dimensions were 1 m long x 0.8 m wide x 0.35 m high. The cages housed 80 birds from 1-14 d old, then 15-20 birds from 15 to 37 d old. Water and food were provided ad libitum. Starting from fledging, VVD broilers were checked and collected daily in the morning, midday and evening for 37 d. Both VVD and healthy broilers at three-time points (two, four and five weeks old) were used for the follow-up experiments.

Clinical anatomy and observation
Eight severely deformed broilers (VVD group) and eight healthy broilers (normal group) were randomly selected from each of the two, four and five weeks old VVD broilers, to give a total of 48 broilers. The BF, spleen and thymus were collected. The entire leg bone was removed together with the hip joint and the femoral head was observed for necrosis and the proximal tibia was observed for non-vascularised and unmineralised white cartilage.

Blood biochemical indicators
A total of 96 broilers with clear VVD symptoms were randomly selected, of which 16 were two weeks old, 33 were four weeks old and 47 were five weeks old. Another 96 healthy broilers were used as controls for the weekly VVD broilers. Using pro-coagulation tubes, 5 ml blood samples were collected from each of the VVD and healthy broilers. The samples were left at room temperature for 30 min, then centrifuged at 3500 rpm for 5-10 min to collect the serum, which was analysed for alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), urea (UREA), creatine kinase (CK) and blood glucose (GLU), using an automatic biochemical analyser (Hitachi 7020).

DNA isolation and gender identification
The DNA from the blood of all broilers for this experiment will be extracted using Trizol reagent. After diluting the DNA to a concentration of 200 ng/μl, the PCR reaction was performed at an annealing temperature of 55.3°C in 10 μl of PCR reaction system (5 μl Taq mix, 1 µl DNA, 0.5 µl each of forward and reverse primers and 3 μl DNase/RNase-free water). 1% agarose gel electrophoresis was used to detect the length and number of bands. Primers and gene information are shown in Figure 2a.

RNA isolation and RT-qPCR analysis
The total RNA was extracted from the BF, spleen and thymus of 48 VVD and healthy broilers using Trizol reagent. The dried total RNA was resuspended in 30 μl DNase/RNase-free water. The concentration of total RNA was measured using a NanoDrop 2000/2000c spectrophotometer (Thermo, Waltham, MA) at 260/280 and 260/230 ratios, whereby 1 μl total RNA was added to 3 μl 5x gDNA digester mix. The mixture was diluted with sufficient ddH2O to give a total volume of 15 μl, which was then incubated at 42°C for 2 min. Next, 5 μl 4x Hifair ® III SuperMix plus was added so as to make a final volume of 20 μl. Reaction conditions were 25°C for 5 min, 55°C for 15 min, 85°C for 5 min; and reverse transcription into cDNA, which was diluted three times and stored at −20°C. The primers for inflammation and apoptosis genes are shown in Table 1. The 10 µl qPCR system consisted of 1 µl cDNA, 0.5 µl each of forward and reverse primers, 5 µl SYBR qPCR Master Mix and 3 ml DNase/RNase-free water. Reaction conditions were set to 40 cycles of 95°C for 30s, 95°C for 10s, 60°C for 30s; 95°C for 15s, 60°C for 1 min and 95°C for 15s. Three technical replicates per sample and expression levels of the BF, spleen and thymus associated genes were calculated by the 2 −ΔΔCt method.

Inflammatory cytokine content by ELISA assay
Eleven two-week-old, eleven four-week-old and twenty fiveweek-old VVD broilers were randomly selected. Similarly, 42 healthy broilers were used as controls for the weekly VVD broilers. Serum was collected as described above. Detection of the presence of inflammatory cytokines TNF-α, IL-1β, IL-6 and TGF-β and immune-related proteins IgM, IgY, CD3 + and CD4 + was performed using ELISA kits at two, four and five weeks old (Meimian China).

Statistical analysis
All data were subjected to analysis using t-test and one-way ANOVA in SPSS 24.0 (SPSS for Windows, Standard ver-sion24.0; SPSS). The data were visualised using GraphPad Prism 9.0. Confidence limits were

Identification of VVD in broilers and anatomical findings
A normal broiler's tibia and tarsal bones appear straight or slightly curved ( Figure 1a). Broilers that appear to have unilateral or bilateral limb deformities legs are defined as VVD (Figure 1b,c). In the VVD birds, dissection of the hip joint showed that the femoral head was not detached and showed no necrosis. Knee joint dissection showed no white non-mineralised cartilage masses in the proximal end of the cartilage (Figure 1d,e). Together, these findings indicate that the leg disease is not FHN or TD, but rather VVD.

Liver, kidney and stress biochemical indicators in blood
Genotyping of experimental broilers revealed that the number of male VVD broilers was higher than that of female VVD broilers at two, four and five weeks old ( Figure 2b). Analysis of liver showed that TP were significantly higher in VVD broilers than in healthy broilers at two, four and five weeks of age (P < 0.01), ALB in VVD broilers were all higher than those in healthy broilers, with some reaching significant levels. Males with VVD had higher ALT levels than healthy broilers and reached significant levels at five weeks (P < 0.01), while females with VVD had significantly higher ALT levels than healthy broilers, reaching significance at four and five weeks (P < 0.05). The AST levels in VVD broilers were significantly higher than in healthy broilers only at two weeks of age (P < 0.05). Analysis of serum kidney and stress-related indicators, UREA, CK and GLU showed that UREA and CK were significantly higher in VVD broilers than in healthy broilers at two weeks of age (P < 0.01), while the differences at four and five weeks were not significant. The GLU levels in VVD broilers reached significant levels at 2, 4 and 5 weeks in both female and male broilers (Tables 2 and 3).

Relative mRNA expression in BF
The expression of inflammation-related factors in the BF of VVD broilers is shown in Figure 3(a-c). At two weeks of age, IL-1β and IL-6 expression in the BF of VVD birds was significantly higher and IL-10 expression was significantly lower than in healthy broilers (P < 0.01; Figure 3a). However, as age increased, IL-1β and IL-6 expression was reduced by varying degrees at four and five weeks old, but these differences were not significant (Figure 3b,c). The expression of IL-10 gradually increased, with highly significant differences at five weeks (P < 0.01). Expression of IL-8 was elevated, peaking at four weeks (P < 0.01). The TGF-β expression was significantly lower than the healthy group at four weeks old (P < 0.01), while, at two and five weeks of age, the difference in TGF-β levels between the two groups was not significant. Neither TNF-α or NF-κB levels were significantly different throughout the entire breeding cycle Figure 3(a-c).

Relative mRNA expression in spleen
The expression of inflammation-related factors in the spleen of VVD broilers is shown in Figure 4(a-b). The expression of IL-1β and IL-6 in the spleen of VVD broilers was significantly higher than in healthy broilers at four weeks of age (P < 0.01). There was no difference at two weeks, but the expression was significantly lower at five weeks old (P < 0.01; Figure 4a,c). With increasing age, expression of IL-10 in the VVD broilers gradually decreased. Expression levels in two-and four-week-old VVD broilers were significantly higher than in healthy broilers (P < 0.01 and P < 0.05, respectively; Figure 4a, b). Expression levels of IL-8 in two-week-old VVD broilers and TGF-β in four-week-old VVD broilers were significantly lower than in healthy broilers (P < 0.01; Figure 4a, b). TNF-α and NF-κB were not significantly different throughout the breeding cycle.

Relative mRNA expression in thymus
The expression of inflammation-related factors in the thymus of VVD broilers is shown in Figure 5(a-c). Throughout the breeding cycle, the VVD broilers had lower increases in IL-1β, IL-6 and IL-8, with significant differences in IL-8 at two weeks of age (P < 0.05), IL-6 at four weeks (P < 0.01) and IL-1β at five weeks (P < 0.01) (Figure 5a-c). Expression of IL-10 in the VVD broilers was significantly lower than in the healthy b2roilers at 4 weeks (P < 0.01) (Figure 5b). Unlike in the BF and spleen, the expression of TNF-α, TGF-β and NF-κB in the thymus of Note: Values are mean ± SE. ** P < 0.01; * P < 0.05.

Figure 2. Sex of VVD broilers and healthy broilers.
the VVD broilers was significantly higher than in the healthy broilers at 5 weeks of age (P < 0.01; Figure 5c).

Apoptosis gene expression
Expression of anti-apoptotic gene Bcl-2 was significantly higher in spleen and thymus at four-weeks-old and BF in the five-week-old VVD broilers than in the healthy broilers (P < 0.01 or P < 0.05; Figure 6a). The death receptor apoptosis pathway protein FAS and its ligand FASL were significantly elevated (P < 0.01), as well as its downstream apoptosis initiation protein caspase-3 and apoptosis execution protein caspase-9 (P < 0.01 or P < 0.05; Figure 6b). The rest of the apoptotic gene expressions in different periods and organs were not significantly different ( Figure S1).

Histologische analyse
This study examined pathological sections and HE staining of the two-week-old BF, four-week-old spleen and fiveweek-old thymus. The results showed that the mucosal layer and lymph nodes of the two-week-old BF were intact in both VVD and healthy broilers. There are more lymphocytes and macrophages in the cortex and medullary regions of VVD broilers than in healthy broilers (Figure 7a,d).
The central artery and spleen nodules of the four-week -old spleen of VVD and healthy broilers are clearly visible and there are a large number of lymphocytes inside the spleen nodules. The VVD broilers had more lymphocytes in the white pulp region of healthy broilers, with dense cells and deeper colouration (Figure 7b,e). Immune cells in the thymic cortical and medullary regions of five-weekold VVD broilers were structurally disturbed and irregularly arranged, with lymphopenia in the cortical region. Medullary epithelial cells in the medullary region was significantly more numerous than in healthy broilers (Figure 7c,f).

Serum inflammatory and immune factors
Serum inflammatory cytokines TGF-α, IL-1β, IL-6, TGF-β and immunoglobulins IgM, IgY, CD3 + and CD4 + were detected by ELISA. Levels of TNF-α, IL-1β and IL-6 were significantly higher in the VVD broilers than in the healthy broilers at 2, 4 and 5 weeks of age (P < 0.01; Figure 8a-c). TGF-β levels were significantly lower in VVD broilers than in healthy broilers at four weeks of age (P < 0.05); however, there was no significant difference at two and five weeks (Figure 8d).
Immunoglobulin IgM in the serum of VVD broilers and healthy broilers was not significantly different at two, four and five weeks ( Figure S2). While levels of IgY were significantly lower in VVD broilers than in healthy broilers at two and five weeks (P < 0.01), there was no significant difference at four weeks of age (Figure 8e). Cellular immunity-related cytokine CD3 + levels were significantly lower in VVD broilers than in healthy broilers at four and five weeks (P < 0.01), while there was no significant difference at two weeks ( Figure 8f). The levels of CD4 + in the serum of VVD broilers and healthy broilers was not significantly different at two, four or five weeks of age ( Figure S2).

Discussion
Broiler chickens with normal standing tibial tarsal adduction or abduction exceeding 10° are defined as valgus and varus (Leterrier and Nys 1992;González-Cerón et al. 2015). VVD has a high heritability, with some broilers being born with twisted legs (Bihan-Duval et al. 1997;Leterrier and Nys 1992). The study rarely found displacement of the gastrocnemius tendon and there was no evidence of TD or FHN.
This study found that TP and ALB in the serum of VVD broilers were consistently higher than in healthy broilers (P < 0.01), indicating damaged liver and decreased immune function in VVD broilers (Hu et al. 2014). VVD broilers had significantly higher levels of AST than healthy broilers at two and five-weeks of age, presumably because of low and irregular feeding for a long period; and abnormal liver metabolism in the stages of breeding. Urea is not only the end product of protein metabolism but also one of the main indicators of proper kidney function, reflecting early glomerular injury and a decrease in glomerular filtration function. Research shows that the lower the serum urea level, the higher the nitrogen utilisation rate (Coma et al. 1995). The current results suggested that early stage VVD may cause glomerular damage and reduced tubular collecting duct filtration in broilers. High-density farming increases stress in broilers (Nasr et al. 2021). Serum CK of VVD broilers was significantly higher than in healthy broilers at two weeks of age (P < 0.01). It could be speculated that, because of highdensity breeding at one to two weeks of age, early onset VVD broilers can experience greater stress. At three to five weeks of age, flock density gradually decreased and the differences in serum CK of VVD broilers became not significant. Although blood GLU level is an indicator of stress, these differences were not significant at any sampling age.
2The thymus, spleen and BF are the principal immune organs of the inflammatory response. Under normal circumstances, the boundaries of the white pulp of the spleen are clear, with a distinct trabeculate structure, a central artery and its spleen nodules intact (Zhao et al. 2020). The mucosal layer and lymph nodes of the bursa are normal and there are no significant vacuoles and inflammatory cell infiltrates (Shah et al. 2020;Farouk et al. 2022). The thymus cortex and medulla are clearly demarcated, the immune cells are arranged and diffuse Hassall bodies are visible (Yang et al. 2020;Farouk et al. 2022). This experiment found that at two-weeks old VVD broilers, there were more lymphocytes and macrophages in the bursal, cortical and medullary regions, and, at four weeks of age, more immune cells and more disordered trabecula in the white pulp of the spleen were observed. At five weeks of age, the thymus immune cells were structurally disordered, irregularly arranged and more medullary epithelial cell occurs. This indicated pathological changes and inflammatory states in the immune organs of VVD broilers.
Studies have shown that cytokines IL-1β, IL-6, IL-8 and TNF-α are the main factors that cause inflammation of the BF and spleen in the early and middle infection phases (Xu et al. 2019;Fan et al. 2020;Shah et al. 2020). IL-1β is mainly produced by monocytes and dendritic cells and increases the expression of adhesion molecules and promotes the proliferation of B cells (Dinarello 2009). IL-6 is mainly produced by mononuclear macrophages and TH 2 cells and mostly plays a 'monitor' role in acute inflammatory responses and tissue damage (Tanaka et al. 2014). IL-8 is mainly produced by mononuclear macrophages and T lymphocytes and induces neutrophil activation, recruitment and migration. Neutrophils are key cells in the management of infections and play an important role in tissue damage caused by sepsis or other microbial infections (Russo et al. 2014). In the current study, IL-1β, IL-6 and IL-8 were differentially highly expressed at two and four weeks of age (P < 0.01 or P < 0.05), which indicated that VVD broilers have varying degrees of inflammation during the early and middle stage of breeding.  Studies have shown that caspase-1 mice are more susceptible to collagen-induced arthritis (Ippagunta et al. 2010), caspase-1 promotes the maturation of IL-1β (Dinarello 2009) and that caspase-1 and IL-1β deficiency reduces cartilage destruction in mouse models with joint inflammation and chronic arthritis (Joosten et al. 2009;Vande Walle et al. 2014). The current study found increased expression of caspase-1 and IL-1β in the five-week thymus of VVD broilers, and hence it was hypothesised that the VVD broiler cartilage was more susceptible to destruction. Anti-inflammatory factors such as IL-10 and TGF-β play an important role in inhibiting excessive inflammation (Moore et al. 2001). The protein content of IL-10 and TGF-β in the supernatant of spleen cells of osteoporotic mice was significantly lowered (Song et al. 2009). In the current experiment, the expression of IL-10 in VVD broilers gradually increased in the BF and gradually decreased in the spleen during the entire breeding cycle. TGF-β has significantly increased in the five-week-old thymus, which countered the high expression of the proinflammatory factors.
TNF-α, produced mainly by mononuclear macrophages and TH 1 cells, is the pro-inflammatory cytokine most quickly produced in the inflammatory response and a multifunctional cytokine that regulates inflammationrelated immune responses (Crusz and Balkwill 2015b). The expression of TNF-α is significantly higher in human encephalomyelitis (Ahmadvand Koohsari et al. 2021). The results showed that TNF-α expression was significantly elevated at five weeks in the thymus (P < 0.01). NF-κB is a key regulator of inflammation; it regulates TNF-α transcription and is activated by IL-6 and TNF-α (Vitkina et al. 2016;Wang et al. 2018). IL-6 can induce anti-apoptotic regulators such as Bcl-2 and BclXL, thereby protecting T cells from apoptosis (Dienz and Rincon 2009). TNF-α has been shown to induce the opening of mitochondrial permeability conversion pores, the loss of mitochondrial membrane potential, increased  expression of FAS/FASL and BAX and activation of caspase-3, −8 and −9 (Gao et al. 2013). The current results are consistent with these conclusions. At four weeks of age, IL-6 may induce increased expression of Bcl-2 in VVD spleen; NF-κB may regulate the transcription of TNF-α, leading to higher expression of FAS, caspase-3 and caspase-9. It was suspected that an increase in the inflammatory factor of VVD regulates apoptosis of thymic cells.
Cytokines and immunoglobulins in serum are direct indicators of the level of inflammation and immunity. It has been shown that pro-inflammatory factors have a negative impact on bone health, leading to various diseases such as osteoporosis and osteoarthritis (D'Amelio et al. 2008). Other studies have shown that serum levels of TNF-α and IL-6 protein were elevated and IL-10 expression was decreased in osteoporotic mice (Dar et al. 2018;Sapra et al. 2021). In addition, ELISA of paw tissue and serum from arthritic mice have revealed increased levels of IL-1β, IL-6, TNF-α (Mateen et al. 2016;Chen et al. 2021). The findings for serum VVD pro-inflammatory factors at two, four and five weeks of age were consistent with the results of other studies of skeletal diseases, which suggested that VVD causes abnormal skeletal metabolism and triggers inflammatory responses in broilers. IgY and IgM play a major role in humoral immunity. CD3 + and CD4 + are surface proteins of T cells, mainly involved in cellular immunity and antigen presentation. Studies have shown that serum IgY levels are significantly elevated in chicken-derived type II collagen-induced arthritis and osteoporosis (Song et al. 2009;Vierboom et al. 2010). CD3 + and CD4 + T cells were significantly higher in the peripheral blood of arthritic rats (Wang et al. 2007;Vierboom et al. 2010). The ELISA results showed a significant decrease in IgY in serum at two and five weeks and of CD3 + in serum from four-and five-week-old VVD broilers (P < 0.01). When the results of IgM and IgY levels were analysed together, IgM did not reach significant levels. It was speculated that humoral and cellular immunity are activated in VVD broilers.
In conclusion, VVD broilers showed disturbances in liver and kidney metabolism, inflammatory reactions in immune organs, apoptosis in the thymus and elevated levels of serum inflammatory and immune factors. This study is the first to investigate metabolism markers in VVD broiler tissues and the inflammation and apoptosis of immune organs throughout the breeding cycle, revealing physiological states and inflammatory conditions. However, the specific mechanisms linking the deformed leg to these clinical data need to be further investigated.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This study was supported by the Scientific Studio of Zhongyuan Scholars (NO. 30601985); Natural Science Foundation of Henan Province (NO. 222300420458); and Natural Science Foundation of Henan Province Youth Project (NO. 202300410203).