Environmental temperature plays an important role in poultry industry along with rising of the global temperatures. When the environmental temperature exceeds the upper limit of comfort zone of broilers by 27℃, the imbalance between heat dissipation and heat production of the body will lead to heat stress . Hence, the broiler were exposed at 36℃ for 8 hours per day and lasted for 1 week to mimic a heat wave in the study. The broiler in heat stress group showed shortness of breath, wing spreading, lethargy and increased water consumption at this study. The results are consistent with those reported by Jastrebski et al. (2017) . Previous studies have shown that HSP70 is one of the most conservative proteins in evolution and very stable at heat stress, and can be used as stress indicator protein . Heat stress can significantly increase the expression of HSP70 in broiler cardiomyocytes ; moreover, the content of serum CORT is the most important index of heat stress in poultry . Hence, the content of HSP70 and CORT in the serum was used to judge that the broilers had heat stress, and they in heat stress group were significantly higher than that of the control group in the study. In the course of our experiment, the broilers showed a typical heat stress response in the early stages of thermal exposure.
Heat stress is linked to compromised productivity through the change of biochemistry and immunity in blood . It's important to note that immune system is one of the main targets of heat stress-induced negative effects in the organism . The spleen is the biggest peripheral immune organ, and thymus is central lymphoid organs in the immune system of poultry . Hence, we selected spleen and thymus as the target tissues to analyze the mechanism of change of ROS production for heat-stressed birds in the study. Previous studies have confirmed that heat stress affected the absolute weight and organ index of immune organs to a certain extent . In the current study, the absolute weight and index of thymus and spleen all decreased after heat stress; this indicate in the immune system is inhibited to some extent, which is consistent with previous research results [6, 34]. The results showed that heat stress could inhibit the development of thymus and spleen and cause damage to the immune organs.
Cytokines are important information molecules of the immune system in the immune response process of the body . Recent studies have shown that IL-6 and TNF-α, as proinflammatory factors, play important roles in both immune responses and inflammationin, which can activate immune response and inflammation [36, 37]. IL-10 is an important anti-inflammatory cytokine and can inhibit production of inflammatory cytokines by a variety of inflammatory cells . Studies have shown that IL-10 can inhibit the activation, synthesis and release of proinflammatory factors such as IL-6 and TNF-α through various mechanisms, so as to achieve the purpose of inhibiting the occurrence of inflammation . Our present study showed that the levels of IL-6 and TNF-α in serum for heat stressed broilers increased, but the IL-10 level had no significant change. Moreover, heat stress can increase the level of IL-6 and TNF–α, and decrease the level of IL-10 in thymus and spleen of broilers. The results confirmed that heat stress can increase the levels of inflammatory cytokines and decrease the levels of anti-inflammatory cytokines in serum,the thymus, and spleen of broilers.
Besides these cytokines, immunoglobulin also plays an important role in immune regulation. Existing research has demonstrated that IgM and IgG are respectively the immunoglobulin in poultry, which play a leading role in anti-infection . The relative expression of IgM and IgG mRNA in the spleen and thymus of heat stressed broilers increased significantly in the study. These results agree with previous studies reported by Honda et al. (2015) . This is an indication that heat stress caused infection to the immune organs; further enhanced the immune response of spleen in broilers. Hence, heat stress can lead to inflammatory response and immunoglobulin expression increase in thymus and spleen.
Heat stress affects mainly poultry performance by inducing the oxidative stress . Previous studies had confirmed that MDA content can reflect the degree of oxidative damage . In this study, we found that the MDA content of thymus and spleen increased significantly under heat stress, which indicated that heat stress caused oxidative damage of thymus and spleen in broiler. Existing research has demonstrated that hyperthermia causes oxidative stress damage to the cell by generating augmented ROS , while SOD and GSH-PX can remove ROS in the body . In this experiment, the contents of SOD, GSH-PX and T-AOC were decreased, while the ROS in thymus and spleen of broiler increased significantly after heat stress, which indicated that heat stress could reduce the antioxidant capacity and lead to the increase of ROS level in thymus and spleen, and causes oxidative injury in the cell of the thymus and spleen.
To explore the mechanism of change of ROS levels in the thymus and spleen of heat-stressed birds, the expression of several transporters in the thymus and spleen associated with ROS production was detected. ABCG2 often serves as an important marker for cells in oxidative stress  and is capable of protecting cells from ROS-mediated cell damage . It has been found that ABCG2 can reduce the ROS levels to alleviate the oxidative stress of cells  and improve the content and activity of SOD, the content of glutathione and the activity of glutathione reductase in cells . Our study found that the expression of ABCG2 mRNA and proteins in thymus and spleen of heat stressed broiler was significantly increased. The data showed that ABCG2 expression manifested as a compensatory increase to protect the thymus and spleen from heat-induced oxidative stress by reducing intracellular ROS or exuding other harmful substances under heat stress. However, more in-depth research is needed in the future to articulate the anti-injury mechanism of ABCG2 in heat stress. Moreover, SVCT-2 widely exists in various tissues and organs, which mainly participates in the absorption of vitamin C in the active metabolism tissues in the body, so as to protect these tissues from oxidative damage . Previous studies have indicated that supplementation with vitamin C could mitigate heat-related damage and enhance heat resistance in animals . It has been shown that SVCT-2 gene knockout can reduce the content of Vc in cells and tissues, increase cell oxidative damage and death, and finally lead to embryo death . Our studies found that the expression of SVCT-2 mRNA in thymus increased, while it is no significant different in the spleen of the heat-stressed broiler. However, the expression of SVCT-2 proteins in thymus and spleen of heat stressed broiler was significantly increased. The result indicated that SVCT-2 proteins are stimulated to possibly transfer more Vc and to compensatory to protect against cellular damage of thymus and spleen from the heat-generated stress; however, further research is needed to elucidate the exact mechanism.
Ca2+ is a second messenger that mediates cell apoptosis and oxidative stress. A previous study showed that Ca2+ dysregulation can give rise to neurodegenerative diseases through oxygenated stress damage . The MCU has the function of mediating Ca2+ influx and plays an important role in the regulation of Ca2+ concentration in cells and mitochondria . Since MCU is associated with Ca2+ concentration, while mitochondrial Ca2+ overload induces a large number of ROS production in cells . Our study showed that the expression of MCU in thymus and spleen of heat stressed broiler was significantly increased compared with the control group. This indicated that under the condition of heat stress, Ca2+ overload of mitochondria in thymus and spleen cells of broiler maybe affect ATP synthesis, further stimulated ROS production, and finally led to apoptosis, thus affecting immune function.