Dietary supplementation with Celecoxib to prevent the welfare problem of tibial dyschondroplasia in broiler chickens

Highlights

• TD is an avian welfare problem cause lameness in fast-growing broiler chickens.

• Therapeutic role of Celecoxib investigated in thiram-induced TD chickens.

• The expression level of COX-1/2, MMP-13 and ColX genes were investigated.

• Celecoxib plays a therapeutic role in the welfare problem of TD.

• Celecoxib supplementation prevented chondrocytes of thiram induced TD chickens from damage.

Abstract

The pathogenicity of tibial dyschondroplasia (TD) is unknown; therefore, it has not been completely evacuated. In this study, we planned to find the possible therapeutic role of Celecoxib supplementation in thiram-induced TD chickens.

Three hundred sixty (n = 360) broiler chickens were divided into 3 groups as control (C), thiram (T) and Celecoxib supplementary group (ST) for day 1 (8 day old), 2 (9 day old), 4 (11 day old) and 6 (13 day old). Thiram 100 mg·kg⁻¹ was given in group (T) along with basal diet to induce TD. Supplementation of Celecoxib at 40 mg·kg⁻¹ was given in group (ST) along with the basal diet and thiram.

Clinically it was observed that broiler chickens of group T had more difficulty in locomotion than group ST. Histopathological results revealed that damaged chondrocytes increased from day 1 to 6 in group T. However, Celecoxib supplementation to thiram induced TD chickens reduced the tibial lesions significantly. Cyclooxygenase-1 (COX-1) gene was highly expressed at different stages (P < 0.01) while its protein expression was seen at a lower level. Cyclooxygenase-2 (COX-2) gene and protein expression were also changed at different stages. Expression of matrix metalloproteinase-13 (MMP-13) gene were observed high on day 1 (P < 0.01), which insignificantly changed on day 2 and 4. MMP-13 protein expression found reduced parallel to its gene expression (P < 0.01), on day 2. Type X collagen (ColX) gene expression was upregulated (P < 0.01).

After adding Celecoxib in the diet, both COX-1 and COX-2 gene (P < 0.01) and their protein expression expressed similar to the control group, and to some extent observed parallel to MMP-13 gene (P < 0.01) & protein expression.

In conclusion, changes in the expression of COX-1/2, MMP-13 and ColX might be related to TD occurrence. The dietary supplementation of Celecoxib plays a therapeutic role to overcome the welfare problem of TD.

Introduction

The increase in demand for meat products is related to human population and income growth, which has led to an increasing level of animal production and intensive production practices. This production environment has raised societal concerns regarding the current animal welfare standards (Webster, 2001; Jahejo and Tian, 2021). There are many welfare problems which should be addressed, but the most important welfare problem in the poultry industry is tibial dyschondroplasia (TD). TD is a disease of broiler chickens in which rigid mass accumulated in the growth plate of broiler chickens which makes difficult for them in the movement and standing. The disease is very common in fast-growing broiler chickens, and the incidence can be as high as 40 to 60% in the flock. It causes huge economic losses to the poultry industry worldwide. Since TD was first proposed in 1965, and poultry disease experts, pathologists and bone biology experts have been conducting in-depth research till date to evacuate this welfare problem. However, the clear pathogenesis of TD is still unclear (Jahejo and Tian, 2021). Thus, any development regarding TD treatment and mechanisms is of utmost importance. Moreover, researchers have reported that a vascularization and un-mineralization in the growth plate is the cause of TD (Rath et al., 2007; Mehmood et al., 2019).

To study the treatment and prevention of TD, several methods of induced TD have been used (Rath et al., 1998; Rath et al., 2004; Rath et al., 2005). Tetramethyl thiuram disulfide (thiram) is a pesticide, use in the broiler chickens to cause TD for experimental purposes, which has a similar clinical picture with naturally occurring TD (Tian et al., 2013; Mehmood et al., 2019). Thiram is recommended as an ideal pesticide for the investigation of therapeutic compounds for TD treatment (Rath et al. (2005); Li et al. (2007)), researchers have developed a broiler TD model using thiram, which shows consistent results as naturally occurring TD, symptoms.

Our previous studies have reported that the immunoregulatory function of erythrocytes can be inhibited in the early stage of TD and promoted in the recovery stage by modulating the expression of prostaglandins (PGs) related genes which may involve in endochondral ossification (Niu et al., 2019; Jahejo et al., 2020c; Jahejo et al., 2020d). Moreover, chondrocytes damaged due to apoptosis is also the reason for TD occurrence (Wang et al., 2018; Jahejo et al., 2020). Although, researchers are more interested in the cyclooxygenase (COX)-2-dependent biological responses because inducible COX (cyclooxygenase) isozyme is involved in various diseases (Murakami et al., 2000; Nørregaard et al., 2015). Cyclooxygenase (COX), officially known as prostaglandin-endoperoxide synthase (PTGS), plays a critical role in a wide range of biological processes. PGs primary products of COX-2 activated many intracellular pathways by autocrine and paracrine modes and induced cellular proliferation, antiapoptotic activity and angiogenesis (He et al., 2016). Therefore, COX is a key enzyme in the cyclooxygenase pathway, which can transform arachidonic acid into PGs and can be used as non-steroidal anti-inflammatory drugs (NSAIDs) Inhibited. COX has two isozymes: COX-1 and COX-2. COX-1 expressed in normal tissues and considered to be important for maintaining prostaglandin levels within the physiological range; while COX-2 is generally not expressed in normal tissues, However, under pathological conditions, it can be expressed at high levels and produce a large number of prostaglandins (Rouzer and Marnett, 2009), which participate in pathological processes. Prostaglandins function through specific G protein-coupled receptors on the cell surface, and their actions have two sides. Prostaglandins maintain normal physiological functions within the physiological range, and high levels of prostaglandins are related to pathological processes. Prostaglandin increases the value and differentiation of growth plate chondrocytes (O'Keefe et al., 1992), matrix degradation and reconstruction (Eisenbarth et al., 1974; Raisz and Koolemans-Beynen, 1974), angiogenesis, calcification (Roos et al., 1974), bone resorption (Tashjian Jr et al., 1974). The differential expression of COX-1/2 affects the normal metabolism of prostaglandins and may change the normal developmental regulation of growth plates.

Previously, the microarray studies screened out COX-1, COX-2, MMP-13 and ColX as differentially expressed genes (DEGs) in the chondrocytes. However, the role of those DEGs in the TD chickens at an early stage is not clear yet. In this experiment, we attempt to explore the role of the cyclooxygenase pathway in the early development of TD and added celecoxib (COX-2 specific inhibitor) in the fed of broiler chickens to evaluate this welfare problem of TD. Immunohistochemistry and quantitative PCR were used to quantify the genes and proteins level at an early stage of TD.