Runting and stunting syndrome (RSS) is a condition in which a number of the birds in a flock are considerably smaller owing to delayed growth (Zavala, 2006), and RSS chickens are easily observed in sex-linked dwarf (SLD) chickens. It is known that the SLD chickens are caused by the mutation of growth hormone receptor (GHR) gene and characterized by shorter shanks, and lower body weight (Ouyang et al., 2012). But the RSS affected SLD chickens are smaller and lower body weight than SLD chickens. RSS has been reported in poultry flocks around the world, including in the Netherlands (Kouwenhoven et al., 1978) and the UK (Bracewell and Wyeth, 1981). RSS causes economic hardship in the poultry industry through reduced uniformity, increased mortality, decreased body weight, poor feed conversion rate, and numerous secondary diseases. The first clinical signs may be observed as early as three days of age, although they are most commonly observed in 6- to 12-day-old chicks and may occur until 3 weeks of age. The chicks are frequently pale and may exhibit distention of the abdomen. In addition to smaller size and reduced body weight, other clinical signs were also observed in RSS chickens include poor feather development, listlessness, and diarrhea. The comb develops slowly, which is associated with impaired hearing and vision.

Why are RSS chickens easily observed in SLD chickens? Our study demonstrated the occurrence of mitochondrial abnormalities in the 7-week-old chicken's livers of RSS chickens and SLD chickens. RSS chicken's mitochondria dysfunction is more seriousness than SLD chickens, and we observed the abnormal nuclear genes related to mitochondrial function in 7-week-old RSS chickens and normal SLD chickens by expression patterns ( Table 2 ), and found these genes involved in oxidative phosphorylation (OXPHOS) and other metabolic pathways.

The chicken mitochondrial genome is a 16.775-kb circle of double-stranded DNA (Desjardins and Morais, 1990; Zavala, 2006) that encodes only 13 proteins, 2 rRNAs, and 22 tRNAs, which is very similar to the human mitochondrial genome (Desjardins and Morais, 1990). It has been reported that mutation or deletion of mitochondrial DNA (mtDNA) can induce mitochondrial disorders in humans (Zeviani et al., 1989; Moraes et al., 1991). As over 99% of mitochondrial proteins are encoded by the nuclear genome, mitochondrial disorders can also be caused by mutations or/and abnormal expression of nuclear-encoded mitochondrial proteins (Bourgeron et al., 1995). In this study, we detected no mtDNA mutations in RSS chickens and SLD chickens.

Very little is currently known about mitochondrial diseases of chickens, and the etiology of RSS in chickens has plagued researchers for several decades. OXPHOS is the metabolic pathway in which cells use enzymes to oxidize nutrients, thereby releasing energy that is used to reform adenosine triphosphate (ATP). This pathway is a highly efficient way of generating energy, compared to alternative fermentation processes such as anaerobic glycolysis. OXPHOS also produces reactive oxygen species (ROS) which lead to the propagation of free radicals, damaging cells, and contributing to disease and possibly aging. The enzymes responsible for this metabolic pathway are also the targets of many drugs that inhibit their activities.

In this study, we demonstrated that mitochondrial membranes are destroyed in RSS chicken hepatocytes using transmission electron microscopy (TEM). Activity assays revealed that a series of complexes proteins (complex I, complex II, complex III, and complex IV) showed decreased enzyme activities in RSS chickens and SLD chickens, suggesting that the OXPHOS function was compromised.

The signs of RSS are usually observed in meat-type chickens between 3 and 6 weeks of age. Previous work has demonstrated that certain strains of birds appear to be more susceptible to the effects of RSS than others, and that male birds are more severely affected than females (Zavala, 2006). However, it is interesting to note that RSS-resistant broiler strains have stronger immunological responses against bacterial and viral infections than RSS-susceptible strains (Rebel et al., 2006). Some researchers have suggested that the poor growth and retarded feathering consistently observed in RSS-affected birds are due to a chronic infection. However, we found that SLD chickens were more susceptible to the effects of RSS and exhibited weaker immunological responses than normal chickens. Therefore, we studied 7-week-old yellow-feather broilers with RSS like features. Of note, No indication of viral was also detected in the liver tissues of the RSS chickens by viruses detection as previously described (Zavala, 2006; Zhang et al., 2015).

Further, we revealed that the mRNA of the insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) was upregulated in the RSS chicken's liver tissue. IGF2BP3 plays an important role in the formation and growth of many tumors and is typically expressed in embryonic tissue, muscles, and the placenta. In fact, the expression of IGF2BP3 is low in the liver tissues of normal chickens. IGF2BP3 is an oncofetal protein with high expression during embryogenesis, low expression in adult tissues, and re-expression in malignant tissues (Palanichamy et al., 2016a). Another study revealed that IGF2BP3, as a human ontogenic master switch, restricts megakaryocyte development by modulating a lineage-specific P-TEFb activation mechanism, revealing potential strategies toward enhancing platelet production (Elagib et al., 2017). In this study, the expression of IGF2BP3 in RSS chickens was examined by qRT-PCR. We found that IGF2BP3 expression was increased in the livers of RSS chickens and SLD chickens. This raises the question of the nature of the association between enhanced IGF2BP3 expression and RSS in chickens. Minchenko et al. demonstrated that the IGF2BP3 gene is expressed in hypoxic tissues, indicating its participation in the regulation of metabolic and proliferative processes via IGF/INS receptors (Minchenko et al., 2015). In this study, we found that both the livers of RSS and SLD chickens exhibited signs of mitochondrial dysfunction, and the livers exhibited black or yellow in color. This suggests that the livers of RSS chickens are hypoxic. This would explain why IGF2BP3 expression was increased in the livers of the RSS chickens. The RCR results further demonstrated that the livers of RSS chickens were hypoxic compared with SLD chickens. The abnormal expression of IGF2BP3 confirmed that oxidative respiratory dysfunction occurs in the mitochondria of both RSS chickens and SLD chickens. Under hypoxic conditions, NADH and FADH₂ cannot participate in respiratory chain regeneration, thereby interrupting the OXPHOS and preventing the normal synthesis of ATP. Our results also demonstrated that the ATP production was reduced in the livers of RSS chickens.

Moreover, we also revealed that the amounts of several components of the OXPHOS pathway in RSS chickens. NDUFA8, NDUFA9, NDUFB1, NDUFB5, NDUFB6, NDUFB8, NDUFB9, NDUFB10, and NDUFS4 are downregulated in female RSS chickens compared to SLD chickens. However, the expression of these genes of female RSS chickens is contrary to that of male RSS chickens, which may indicate that the regulatory mechanisms of male and female RSS chickens are different. In particular, succinate dehydrogenase complex subunit A (SDHA) was abnormal expression in the livers of RSS chickens compared with those of SLD chickens. SDHA is anchored to the inner mitochondrial membrane by one hydrophobic subunit of SDHC (Sun et al., 2005). The catalytic subunit of SDHA is hydrophilic and extends into the mitochondrial matrix (Lancaster and Simon, 2002). SDHA contains a covalently attached flavin adenine dinucleotide (FAD) cofactor and the succinate-binding site. Flavination is important for succinate oxidation (Robinson et al., 1994). A previous study confirmed that flavination defects interfere with SDHA activity, likely reducing the overall growth rates in vitro and in vivo that have been observed in experimental models of SDHA deficiency (Guzy et al., 2008). Another study demonstrated that knockdown of the SDHA/B genes in HeLa cells led to the accumulation of succinate (Xiao et al., 2012). Konieczna et al. found that partial silencing of SDHA with specific siRNAs resulted in either less efficient or delayed entrance of the cells to the S phase (Konieczna et al., 2015). However, the cells remained viable. The results of this study revealed that SDHA was abnormal expression in the livers of RSS chickens compared with SLD chickens, but no mutation was detected. Lower OXPHOS activity will lead to insufficient energy supply, which appears to be one of the reasons for the development of RSS in chickens.

Decreased OXPHOS activities, along with higher ROS production in RSS and SLD chickens, are indicative of damaged mitochondrial function and efficiency. The vacuolar hydropic degeneration may induce the enlargement of hepatocytes and compress adjacent organelles, leading to damage of the nuclei and mitochondria of liver cells. This phenomenon was observed not only in the RSS chickens but also in SLD chickens with GHR mutation. Previous studies have confirmed that growth hormone (GH) stimulates cellular oxygen consumption in CHO cells transfected with cDNA coding for the full-length GHR (Perret-Vivancos et al., 2006a). GH affects mitochondrial metabolism by indirectly regulating the activities of OXPHOS enzyme complexes (Brown-Borg et al., 2012). We found that the abnormal interaction between GH and GHR can influence mitochondrial oxidation and respiratory functions. The five multimeric enzyme complexes that drive OXPHOS and cellular respiration are contained within the inner mitochondrial membrane (Barrientos et al., 2009). Determining the activities of individual enzymes in the respiratory chain complex provides an important diagnostic indicator of mitochondrial disease (Frazier and Thorburn, 2012). In mitochondrial disease, the activities of single or multiple complexes are reduced in the affected tissue (El-Hattab and Scaglia, 2013). Activity assays typically reveal decreased activities of complex I, complex III, and complex IV in liver tissues (El-Hattab and Scaglia, 2013). In this study, we observed decreased enzyme activities of complex I, complex II, complex III, and complex IV in RSS and SLD chickens.

Inefficient mitochondrial function was also observed upon measuring the RCR, ATP level, ΔΨm, and ROS production. As mitochondria are responsible for the majority of oxygen consumption, the RCR is an essential parameter for studying mitochondrial function (Frezza et al., 2007). Meanwhile, ΔΨm is essential for mitochondrial function, and mitochondrial dysfunction is normally accompanied by a decrease in ΔΨm (Javadov et al., 2006). Further, excessive ROS production can impair the activities of mitochondrial respiratory complexes and reduced ATP production (Sudheesh et al., 2013). Here, we observed alterations in the RCR, ATP level, ΔΨm, and ROS production in both RSS chickens and SLD chickens. However, the mitochondrial function was lowest in RSS chickens, indicating further mitochondrial dysfunction in RSS chickens compared with SLD chickens. This may further impair immunity, causing greater mortality in RSS chickens.

Mitochondria, the only organelles in animal cells that contain their own genome, exchange Ca²⁺ and ROS with the endoplasmic reticulum (Pacher and Hajnoczky, 2001; Zorov et al., 2014; Booth et al., 2016). An evolutionarily conserved machinery enables the complete and sequential fusion of the outer and inner mitochondrial membranes (Robinson et al., 1994). In this study, no inflammation was detected by H&E staining and no lymphocyte infiltration was observed. Vacuolation of hepatocytes was observed, and the cytoplasm was disappeared in RSS and SLD chickens. The hydropic degeneration in the hepatocytes was the main cause of the liver cell vacuoles. GHR mutation induces an abnormal interaction between GH and GHR. As GHR gene is associated with oxidative respiration (Perret-Vivancos et al., 2006b), this may further influences mitochondrial oxidation and respiratory functions. Here, the TEM results revealed that the inner and outer mitochondrial membranes of the RSS chickens exhibited varying degrees of damage or had even disappeared. Consequently, the five multimeric enzyme complexes within the inner mitochondrial membrane are unable to drive OXPHOS and cellular respiration. The GHR mutation, along with contributions from the additional factors mentioned above, may lead to the hindered growth and development in SLD chickens. If mitochondrial damage becomes more severe in SLD chickens, these may exhibit the signs of RSS. The methods reported in this study can also be used as a model for inner mitochondrial membrane GHR mutation.

Together, all of these changes in mitochondrial functions could account for mitochondria dysfunction, resulting in poor weight gain and retarded growth or stunting of chicks. Our findings revealed that the RSS in chicken is caused by mitochondria dysfunction. We didn't find mtDNA mutation in RSS and SLD chickens. The GHR mutation may cause mitochondria dysfunction in SLD chicken which is induced by GHR mutation, but RSS chicken's mitochondria dysfunction is more serious. We hypothesize that RSS chicken's mitochondrial disorders are caused by other nuclear gene mutations.

All procedures involving animals were approved by South China Agriculture University's Institutional Animal Care and Use Committee (approval number SCAU#0017), according to the regulations established by this committee. Animals involved in this study were humanely sacrificed as necessary.

HL and BH contributed equally to this manuscript. HL designed the study, and wrote the paper. BH carried out experiments, and analyzed data. QL and SH participated in the design of the experiment and data analysis. YLu, BZ, and YG participated in data collection and interpretation, and helped with performing some of the manuscripts' experiments. YLi, MS, and QN helped for useful discussion and language correction. XZ and DZ participated in the design, manuscript writing and final approval of the manuscript. All authors read and approved the final manuscript.

This work was supported by grants from the National Natural Science Fund Committee National Youth Project (Project grant no: 31401046), Guangdong Provincial Promotion Project on Preservation and Utilization of Local Breed of Livestock and Poultry and Guangdong Youth Talent Project.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.