Effects of dietary tryptophan supplementation on body temperature, hormone, and cytokine levels in broilers exposed to acute heat stress

The present study was conducted to investigate the effects of tryptophan (Trp) supplementation on rectal temperature, hormone, and cytokine levels in broilers subjected to acute heat stress. A total of 300 18-day-old female Arbor Acres broilers were randomly allocated to five dietary treatment groups with six replicates per treatment group and ten birds per replicate. Broilers were fed a basal diet and in the thermoneutral conditions (TN, 23 ± 1 °C) was considered as the TN group. Broilers were fed a basal diet and exposed to acute heat stress (HS, 34 ± 1 °C) was regarded as the HS group, and other broilers exposed to acute heat stress (34 ± 1°C) were fed a basal diet supplemented with 0.09%, 0.18%, and 0.27% Trp. Results indicated that acute heat stress increased the rectal temperature (P < 0.05), enhanced the concentrations of corticosterone (CORT), dopamine (DA), adrenaline (Adr), adrenocorticotropic hormone (ACTH), and corticotropin-releasing hormone (CRH) in serum (P < 0.05), and elevated the levels of serum tryptophan hydroxylase (TPH)1, tryptophan 2, 3-dioxygenase (TDO), indoleamine 2, 3-dioxygenase (IDO), and kynurenic acid (P < 0.05), compared with the TN group. Meanwhile, acute heat stress increased the levels of serum Trp, hypothalamic Trp, 5-hydroxytryptophan (5-HT), and interleukin-22 (P < 0.05) relative to the TN group. However, compared with the heat stress group, Trp supplementation decreased the rectal temperature of heat-stressed broilers and dietary 0.09% Trp supplementation decreased the levels of serum CRH and TDO (P < 0.05), increased the levels of serum Trp and IL-22 (P < 0.05) in heat-stressed broilers. In addition, dietary supplemented with 0.18% Trp reduced the levels of serum DA, Adr, noradrenaline (NA), CRH, TDO, IDO, kynurenic acid, IL-1β, and hypothalamic 5-HIAA/5-HT (P < 0.05), increased the levels of serum Trp, 5-HT, and IL-22, and upregulated the concentrations of hypothalamic Trp and 5-HT in heat-stressed broilers (P < 0.05). Moreover, dietary 0.27% Trp supplementation decreased the levels of serum DA, CRH, TDO, and hypothalamic 5-HIAA/5-HT (P < 0.05), and upregulated the levels of serum Trp, 5-HT, IL-22, hypothalamic Trp and 5-HT in heat-stressed broilers (P < 0.05). Taken together, dietary 0.18% Trp supplementation may be the optimal level for broilers reared under acute heat stress.


Introduction
Heat stress (HS) has been known to adversely affect poultry production and welfare, and reduce economic profit in the poultry industry. Due to having many feathers and Sweat glands absent throughout the skin, poultry is susceptible to heat stress (Gonzalez-Rivas et al. 2020), and commercial broilers are more sensitive to heat stress than local broilers (Yalcin et al. 2001). Emerging evidence has demonstrated that heat stress increases rectal temperature, reduces feed intake, decreases immune function, contributes to electrolyte imbalance and endocrine disorders, and decreases reproduction in poultry (Sohail et al. 2010;Nawab et al. 2018;Li et al. 2020). Elevated body temperature is the simplest parameter to identify heat-stressed broilers (Zheng et al. 2021). Heat stress activates the hypothalamic-pituitary-adrenal (HPA) axis and results in an increased concentration of cortisol corticosterone, and adrenocorticotropin in circulation (Zhang et al. 2017;He et al. 2019). Indeed, the over-activation of the HPA axis can lead to immunity and infection response Qiufen Li and Hua Zhou contributed equally to this work.
* Guanhong Li liguanh@163.com (Chrousos 1995;Wilder 1995;Sohail et al. 2012). Compared to chronic heat stress, the intensity of these effects is relatively strong in acute heat stress (Gonzalez-Rivas et al. 2020). Studies have demonstrated that acute heat stress increases heterophil/lymphocyte ratio, affects metabolic characteristics, and causes oxidative damage in broilers (Lin et al. 2006;Mujahid et al. 2007; Monson et al. 2019). Notably, acute heat exposure in the pre-slaughter period has negative effects on broilers (Kang and Shim 2021). Advanced techniques, equipment, and management conditions have been used in poultry breeding, which has in part mitigated the effect of high temperature on poultry. Moreover, nutrition manipulation has become an important method for alleviating heat stress in poultry (Abdel-Moneim et al. 2021).
In addition to being involved in protein synthesis in the body, tryptophan (Trp) is also engaged in multiple physiological functions. Trp is considered a functional amino acid, that is important for maintenance, reproduction, and immunity (Hoglund et al. 2019;Sun et al. 2020;Mund et al. 2020). There are three main pathways of Trp metabolism: the serotonin-synthesis pathway, the kynurenine pathway, and the indole pathway ( Fig. 1) . Serotonin (5-hydroxytryptamine, 5-HT) is one of the products of Trp metabolism formed by hydroxylation and decarboxylation. Numerous studies have shown that serotonin plays an important role in regulating physiological functions and the neuroendocrine stress response in humans and animals (Bai et al. 2017;Calefi et al. 2019). In addition, Trp supplementation improves daily weight gain, feed utilization ratio, antioxidant status, and immune function in broilers (Biefer et al. 2017;Mund et al. 2020). Meanwhile, Trp supplementation has been reported to alleviate acute heat stress-induced impairment in pigs and steers (Koopmans et al. 2005;Madoka et al. 2018).
However, there is scarce information regarding the effect of Trp supplementation on the physiological and immunological parameters in broilers exposed to acute heat stress. Therefore, we hypothesized that dietary Trp supplementation could reduce stress-related hormone secretion of broilers, reduce the inflammatory response, and then alleviate acute heat stress in broilers by increasing the level of 5-HT. Therefore, the current study was conducted to assess the effects of dietary Trp supplementation on acute heat stress in broilers by analyzing the body temperature, serum hormone cytokines levels.

Materials and Methods
Experimental procedures were carried out in accordance with the Laboratory Animal Welfare and Ethics Censorship. The animal use and care protocol were reviewed and approved by the Laboratory Animal Ethics Committee of Jiangxi Agricultural University (JXAULL-2021-036).

Experimental design and diets
One-day-old female Arbor Acres broiler chicks were reared under standard environmental conditions and were fed a starter diet for 18 days. A total of 300 chicks with uniform body weight were then chosen and randomly allocated to five treatment groups. Each group had six replicate pens of ten chicks per pen. The five treatment groups were as follows: the thermoneutral conditions (TN) group, heat stress (HS) group, HS + 0.09%Trp group, HS + 0.18%Trp group, and HS + 0.27%Trp group. After a 3-day adaptation period (19 to 21 days of age, 23 ± 1°C), chicks in the TN group were continuously kept in the thermoneutral environment (23 ± Fig. 1 Pathways of Trp metabolism. 3H-KYN: 3-hydroxykynurenine; 5-HT: 5-hydroxytryptamine; 5-HIAA: 5-hydroxyindole acetic acid; IDO: indoleamine 2, 3-dioxygenase; KAT: kynurenine aminotransferase; KMO: kynurenine 3-monooxygenase; NAD: nicotinamide adenine dinucleotide; TDO: tryptophan 2, 3-dioxygenase; TPH: tryptophan hydroxylase 1°C). Chicks in the other four groups were subjected to acute heat stress (34 ± 1°C) for 24 h from 7:30 am to 7:30 am the next day. Relative humidity was kept at 65-70% among all groups during the entire trial period. The temperature was controlled with the electronic automatic temperature control system (Xianke generator equipment wholesale department, Nanchang, China). The humidity was controlled with an air humidifier (SCK0A45, Xiaomi, Beijing, China). The temperature and relative humidity were recorded by a digital Thermo hygrometer (8866,Deli,Ningbo,China). Chicks in the TN and HS groups were fed a basal diet. Chicks in the HS+0.09%Trp group, HS+0.18%Trp group, and HS+0.27%Trp group were fed a basal diet supplemented with 0.09%, 0.18%, and 0.27% Trp, respectively. The basal diet was formulated to meet the nutrient requirements recommended by the National Research Council (NRC, 1994) ( Table 1). L-Glutamic acid was added to the basal diet instead of L-Trp to make all diets isonitrogenous (Taherkhani et al. 2005;Taherkhani et al. 2008;Lee et al. 2020). Broilers were fed three times daily (7:30, 13:30, and 19:30), and allowed ad libitum access to feed and water. The equipment was cleaned and disinfected with 1% peracetic acid before feeding, and the broilers' house was cleaned every day.

Sample collection
Rectal temperature was measured with a mercury thermometer one hour before the end of heat stress treatment. After heat stress for 24 h, blood samples were collected from the wing vein, centrifuged at 3000 g for 10 min at 4°C to obtain serum, and stored at -20°C for further analysis. After that, the selected broilers were euthanized by cervical dislocation. Furthermore, hypothalamic samples were collected and immediately frozen at -80 °C until further analysis.

Determination of the serum hormone levels related to heat stress
The levels of corticosterone, dopamine, adrenaline, noradrenaline, adrenocorticotropic hormone, and corticotropin-releasing hormone in serum were determined using commercial kits (Shanghai Enzyme Link Biotechnology Co., Ltd, Shanghai, China) according to the manufacturer's instructions.

Determination of the serum cytokine levels
The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-10, IL-22, and interferon (IFN)-γ in serum were determined using commercial ELISA kits (Shanghai Enzyme Link Biotechnology Co., Ltd, Shanghai, China) according to the manufacturer's instructions.

Statistical analysis
Data were analyzed using SPSS statistical software (SPSS for Windows, version 25.0, Chicago, IL, USA). Data for the TN and HS groups were analyzed using independent Students t-test. In contrast, data for the HS, HS+0.09% Trp, HS+0.18% Trp, and HS+0.27% Trp groups were subjected to one-way analysis of variance (ANOVA) and Duncan's multiple comparisons. All data are expressed as the mean ± standard error of the mean (SEM). Differences were considered to be statistically significant at P < 0.05.

Rectal temperature
As shown in Fig. 2, heat stress increased the rectal temperature (P < 0.001), while Trp supplementation decreased the rectal temperature in broilers exposed to heat stress (P < 0.001).

The hormones related to heat stress
As presented in Fig. 3, heat stress increased the levels of corticosterone (P < 0.001), dopamine (P = 0.005), adrenaline (P = 0.007), adrenocorticotropic hormone (P < 0.001), and corticotropin-releasing hormone (P = 0.007) in serum. Compared with the HS group, the concentration of corticotropinreleasing hormone (P < 0.001) in the serum was decreased in all Trp groups, and the level of dopamine (P = 0.001) was reduced in 0.18% and 0.27% Trp groups. While, adrenaline (P = 0.006) and noradrenaline (P = 0.033) were only reduced in the 0.18% Trp group, on contrary, the levels of corticosterone (P = 0.097) and adrenocorticotropic hormone (P = 0.906) were not significantly affected by Trp treatment.

metabolic enzymes and metabolites
According to Fig. 4, heat stress increased the levels of TPH1 (P = 0.004), TDO (P < 0.001), and IDO (P = 0.033) in the serum. Compared with the HS group, the concentration of TDO (P < 0.001) was reduced in all Trp groups. While IDO (P < 0.001) was only reduced in the 0.18% Trp group. On contrary, the levels of TPH1 (P = 0.689) and TPH2 (P = 0.070) were not significantly affected by Trp treatment. As shown in Fig. 5, heat stress increased the concentration of kynurenic acid (P = 0.002) in the serum, while reduced the concentrations of Trp (P = 0.007) in the serum and Trp (P < 0.001), 5-HT (P = 0.001) in the hypothalamus. Compared with the HS group, the concentration of Trp (P = 0.002) in the serum was increased in all Trp groups. The levels of 5-HT in the serum (P < 0.001) and hypothalamus (P = 0.003), Trp in the hypothalamus (P = 0.015) were increased in 0.18% and 0.27% Trp groups, and the level of 5-HIAA/5-HT in the hypothalamus (P = 0.008) was decreased in 0.18% and 0.27% Trp groups, while the level of kynurenic acid in the serum (P = 0.014) was only reduced in the 0.18% Trp group. On contrary, the levels of 5-HIAA (P = 0.052) and 5-HIAA/5-HT (P = 0.063) in the serum, 5-HIAA in the hypothalamus (P = 0.877) were not significantly affected by Trp treatment.

Serum cytokine profile
As presented in Fig. 6, heat stress increased the level of IL-22 (P = 0.003) in the serum. Compared with the HS group, the level of IL-22 (P = 0.001) in the serum was increased in all Trp groups, while IL-1β (P = 0.010) was only reduced in the 0.18% Trp group. on contrary, the levels of TNF-α (P = 0.074), IL-10 (P = 0.183), and IFN-γ (P = 0.823) were not significantly affected by Trp treatment. , and corticotropin-releasing hormone (F) levels in broilers exposed to heat stress. TN: chicks fed with a basal diet raised under thermoneutral conditions; HS: chicks fed with a basal diet raised under acute heat stress conditions; HS + 0.09%Trp: chicks fed with a basal diet supplemented with Trp by 0.09% raised under acute heat stress conditions; HS + 0.18% Trp group: chicks fed with a basal diet supplemented with Trp by 0.18% raised under acute heat stress conditions; HS + 0.27% Trp group: chicks fed with a basal diet supplemented with Trp by 0.27% raised under acute heat stress conditions. The levels of serum hormones related to heat stress were determined using commercial ELISA kits. Mean values of the TN group and HS group with * differ significantly (P < 0.05). Mean values of the HS group, HS + 0.09%Trp group, HS + 0.18%Trp group, and HS + 0.27%Trp group without common superscript letters differ significantly (P < 0.05)

Discussion
Heat stress is a critical challenge in poultry health and adversely affects poultry growth performance, productivity, and meat quality (Nawab et al. 2018;Gonzalez-Rivas et al. 2020;Li et al. 2020). Traditionally, blood pH, rectal temperature, and respiratory rate were considered indicators of the response to heat stress in broilers (Chen et al. 2013;Mutibvu et al. 2017;Shakeri et al. 2019). In the present study, the rectal temperature of broilers in the HS group was higher than that in the TN group, in agreement with previous studies (Zhang et al. 2018;Vesco et al. 2020). Acute heat stress activated the autonomic nervous system (ANS) and hypothalamic-pituitary-adrenal (HPA) axis and increased the levels of cortisol, catecholamine, and glucocorticoids (Kadim et al. 2006;Gonzalez-Rivas et al. 2020). Meanwhile, an increase of cortisol affected the HPA axis and stimulated the levels of adrenocorticotropic and corticotropin-releasing hormones, as well as induced dysfunctions to increase the risk of diseases (Luo et al. 2019). In the present work, heat stress increased the levels of cortisol, dopamine, adrenaline, adrenocorticotropic hormone, and corticotropin-releasing hormone in the serum of broilers. The results of rectal temperature and hormones suggested that the broilers were in the state Fig. 4 Effect of dietary Trp supplementation on the serum tryptophan hydroxylase 1 (A), tryptophan hydroxylase 2 (B), tryptophan 2,3-dioxygenase (C), and indoleamine 2,3-dioxygenase (D) levels in broilers exposed to heat stress. TN: chicks fed with a basal diet raised under thermoneutral conditions; HS: chicks fed with a basal diet raised under acute heat stress conditions; HS + 0.09%Trp: chicks fed with a basal diet supplemented with Trp by 0.09% raised under acute heat stress conditions; HS + 0.18% Trp group: chicks fed with a basal diet supplemented with Trp by 0.18% raised under acute heat stress conditions; HS + 0.27% Trp group: chicks fed with a basal diet supplemented with Trp by 0.27% raised under acute heat stress conditions. The levels of Trp metabolism enzymes were determined using commercial ELISA kits. Mean values of the TN group and HS group with * differ significantly (P < 0.05). Mean values of the HS group, HS + 0.09% Trp group, HS + 0.18% Trp group, and HS + 0.27% Trp group without common superscript letters differ significantly (p < 0.05) Tropical Animal Health and Production (2022) 54: 164 of heat stress. Notably, it is urgent to develop nutritional strategies to prevent the detrimental effects of heat stress.
Of note, the current study observed that Trp supplementation decreased the rectal temperature and the concentrations of dopamine, adrenaline, noradrenaline, and corticotropin-releasing hormone in the serum of broilers exposed to heat stress. Similarly, previous work indicated that intravenous administration of Trp-reduced rectal temperature in steers reared under heat stress (Madoka et al. 2018), and dietary supplementation of Trp improved the intestinal barrier and immune functions in other animals subjected to darkness, drive, and other stresses (Yue et al. and hypothalamus (F-I) of broilers exposed to heat stress. TN: chicks fed with a basal diet raised under thermoneutral conditions; HS: chicks fed with a basal diet raised under acute heat stress conditions; HS + 0.09%Trp: chicks fed with a basal diet supplemented with Trp by 0.09% raised under acute heat stress conditions; HS + 0.18% Trp group: chicks fed with a basal diet supplemented with Trp by 0.18% raised under acute heat stress conditions; HS + 0.27% Trp group: chicks fed with a basal diet supplemented with Trp by 0.27% raised under acute heat stress conditions. The levels of Trp metabolites were determined using commercial ELISA kits. Mean values of the TN group and HS group with * differ significantly (P < 0.05). Mean values of the HS group, HS + 0.09% Trp group, HS + 0.18% Trp group, and HS + 0.27% Trp group without common superscript letters differ significantly (P < 0.05) Tropical Animal Health and Production (2022) 54: 164 2017). Consistently, the effect of stress on these hormones was abolished by Trp treatment (Yue et al. 2017;Alhassan et al. 2018). These results indicated that Trp supplementation may promote healthy function in broilers exposed to heat stress.
TDO expression is under the control of glucocorticoids and glucagon (Castro-Portuguez and Sytphin 2020). During inflammation, the expression of IDO increases and stimulates the kynurenine metabolic pathway (Breda et al. 2016;Sun et al. 2020). Our results found that heat stress enhanced the level of glucocorticoids and was associated with an increase in TDO and IDO concentrations in the serum, which, in turn, results in increased kynurenine formation. An increase in kynurenine activated the aryl hydrocarbon receptor, suppressed T cell proliferation, and induced the apoptosis of T/B cells (Fuertig et al. 2016;Platten et al. 2019). Importantly, the current study found that Trp supplementation decreased the rate of Trp catabolism through the kynurenine pathway by reducing the levels of IDO, TDO, and kynurenine in the serum of broilers subjected to heat stress. 5-HT is a type of neurotransmitter involved in the HPA axis with hypothalamic corticotropin-releasing hormone (Dinan 1996;Winberg et al. 1997) and participates in the body behaviors and the neuroendocrine system in response to stress. 5-HIAA is an indicator reflecting 5-HT conversion serotonin activity in the nervous system (Hierden et al. 2004). In this study, heat stress elevated serum 5-HIAA/5-HT and decreased hypothalamic 5-HT level significantly, which was consistent with previous work (Calefi et al. 2019). Emerging evidence has reported that increasing the 5-HT level is beneficial for alleviating heat stress in animals (Koopmans et al. 2006;Shen et al. 2012a;Shen et al. 2012b). Notably, dietary supplementation with Trp increased the concentration of 5-HT in the serum and hypothalamus, which alleviated high-density feeding-induced stress (Liu et al. 2015). In the present study, Trp supplementation, especially at the 0.18% and 0.27 levels increased the rate of Trp catabolism through the 5-HT pathway by enhancing the Fig. 6 Effect of dietary Trp supplementation on the serum tumor necrosis factor (TNF)-α (A), interleukin (IL)-1β (B), IL-10 (C), IL-22 (D), and interferon (IFN)-γ (E) levels of broilers exposed to heat stress. TN: chicks fed with a basal diet raised under thermoneutral conditions; HS: chicks fed with a basal diet raised under acute heat stress conditions; HS + 0.09%Trp: chicks fed with a basal diet supplemented with Trp by 0.09% raised under acute heat stress conditions; HS + 0.18% Trp group: chicks fed with a basal diet supple-mented with Trp by 0.18% raised under acute heat stress conditions; HS + 0.27% Trp group: chicks fed with a basal diet supplemented with Trp by 0.27% raised under acute heat stress conditions. The levels of serum cytokines were determined using commercial ELISA kits. Mean values of the TN group and HS group with * differ significantly (P < 0.05). Mean values of the HS group, HS + 0.09%Trp group, HS + 0.18%Trp group, and HS + 0.27%Trp group without common superscript letters differ significantly (P < 0.05) Tropical Animal Health and Production (2022) 54: 164 concentrations of Trp and 5-HT in serum and hypothalamus and decreasing the 5-HIAA/5-HT in the hypothalamus.
In addition, a previous study reported that heat stress damaged intestinal barrier function by increasing the levels of TNF-α and IL-1β (Siddiqui et al. 2020). Similarly, the current work found that heat stress reduced the serum IL-22 level in broilers. IL-22 is engaged in the homeostasis balance between immunity and microorganisms, with the involvement of antimicrobial peptide release based on microbial abundance and diversity (Zelante et al. 2013;Behnsen et al. 2014). Notably, Trp plays a critical role in the production of IL-22 in the intestine (Lee et al. 2011;Gao et al. 2018). Consistently, in this study, the level of IL-22 in the serum was increased in all Trp groups compared with the HS group, while IL-1β was only reduced in the 0.18% Trp group. These findings suggested that Trp supplementation may alleviate heat stress in broilers, and the optimal level was 0.18%.

Conclusions
In summary, Trp supplementation could decrease rectal temperature, suppress the kynurenine metabolic pathway, increase 5-HT synthesis, and enhance anti-inflammatory ability in heat-stressed broilers. Moreover, 0.18% Trp supplementation may be more beneficial to heat-stress broilers. This study demonstrates that the supplementation of Trp in diets might be an effective nutritional strategy to protect against acute heat stress impairment. Data availability Data generated or analyzed during this study are included in the current article.

Declarations
Statement of animal rights Experimental procedures were carried out in accordance with the Laboratory Animal Welfare and Ethics Censorship. The animal use and care protocol were reviewed and approved by the Laboratory Animal Ethics Committee of Jiangxi Agricultural University (JXAULL-2021-036).

Conflicts of interest
The authors declare no conflicts of interest.