Effects of dietary stinging nettle (Urtica dioica) on hormone stress and selected serum biochemical parameters of broilers subjected to chronic heat stress

Abstract Background Heat stress is one of the most critical problems confronting the poultry industry. Stinging nettle (SN) is a medicinal plant with potent antioxidant properties. Objective The goal of this study was to evaluate the effects of dietary SN at two different levels (2 and 4%) on the serum levels of cortisol and some selected parameters of broilers exposed to chronic heat stress. Methods A total of 240 broiler chickens were randomly assigned to six dietary groups as follows: (1) control: fed the basal diet; (2) HS: heat‐stressed broiler fed the basal diet; (3) HS‐SN2: heat‐stressed broiler fed 2% dietary SN; (4) HS‐SN4: heat‐stressed broilers fed 4% SN; (5) SN2: no heat‐stressed broilers fed the basal diet supplemented with 2% SN; (6) SN4: no heat‐stressed broilers fed the basal diet supplemented with 4% SN. Diet supplementation with SN was performed from days 14 to 35 and chronic heat stress was induced from days 22 to 29. The serum parameters were evaluated on days 14, 21, 29 and 35. Results HS had higher serum levels of cortisol, total cholesterol (TC), aspartate aminotransferase, alanine aminotransferase and creatine kinase (CK) compared to the other treatments. HS‐SN4 had significantly lower cortisol, TC, alanine aminotransferase and CK compared to HS and HS‐SN2. Conclusions The inclusion of 4% SN powder in the broilers’ diet alleviated the negative effects of heat stress by decreasing cortisol, TC and tissue damage indices. It seems that dietary SN could be used as a feed additive in the poultry diet for improving the health status and defence mechanisms of the birds under stressful conditions.


INTRODUCTION
Heat stress is one of the most prevalent environmental stressors affecting growth and impeding efficient poultry production (Souza-Junior et al., 2019). It is an imbalance between the amount of heat production and heat loss in the bird's body (Zhang et al., 2012;Saracila et al., 2021). Broiler chickens are more sensitive to high ambient temperatures due to the rapid body metabolic rate and absence of sweat glands (Lara & Rostagno, 2013). Heat stress causes impairment in the welfare and health status of broilers. Furthermore, it leads to increased mortality and reduced body weight, feed intake and feed efficiency in the broilers (Olfati et al., 2018). Exposure to high environmental temperature leads to an increase in the respiratory rate and alters the homeostasis and acid-base balance of the blood, leading to increases in the blood pH and incidence of respiratory alkalosis in broilers (Beckford et al., 2020). Exposure to chronic heat stress activates the hypothalamic-pituitary-adrenal axis, leading to an increased production of cortisol in broilers chickens (Quinteiro-Filho et al., 2017).
Therefore, blood biochemical assessment is a valuable tool to monitor the health situation and determine the severity of the heat stress in heat-stressed (HS) broilers (Dapeng et al., 2020).
Blood biochemical parameters are reliable indicators of the wellbeing of birds and reflect the nutritional, physiological and pathological status of birds' body (Kraus et al., 2021). Thus, some serum indices provide clinical information for diagnosis and characterization of avian diseases (Hosseinian and Hasanzade, 2021;Li et al., 2017). As mentioned below, high ambient temperature causes several changes in the physiological status and blood biochemical profile in broilers (Wasti et al., 2020). Several researchers stated that some serum parameters are employed to determine oxidative stress and metabolic status of the broilers exposed to heat stress (Hosseini-Vashan & Raei-Moghadam, 2019). It has been confirmed that the serum activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes were higher in broilers exposed to heat stress than the birds at optimum ambient temperature (Chand et al., 2018). Heat stress causes marked changes in serum levels of cholesterol, total protein (TP) and uric acid (UA) (Chand et al., 2018). Assessing the serum level of cortisol is another valuable indicator in various stressful conditions including heat stress in birds (Dapeng et al., 2020). Attenuating the adverse effects of heat stress is a valuable objective of the poultry industry. Therefore, various studies have applied different dietary antioxidants to ameliorate the harmful effects of heat stress in broilers (Khan et al., 2012). Various antioxidant compounds and antioxidantrich plants were added as natural feed additives to the diet to improve health status, antioxidant property and productive performance of birds under exposure to stressful conditions (Hosseinian & Ansari, 2021;Luo et al., 2018). Stinging nettle (SN) (Urtica dioica) is a medicinal plant that is employed as a phytogenic feed additive in the poultry diet (De Vico et al., 2018).
Urtica dioica L. (Urticaceae), commonly known as nettle or SN, is a perennial plant utilized as a traditional medicinal plant in numerous countries in the world (Mueen & Parasuraman, 2016). SN contains a high amount of terpenoids, phenylpropanoids, flavonol glyco-sides, carotenoids, tocopherol and ascorbic acid, which contribute to the anti-inflammatory, antioxidant, anti-viral and analgesic activities of nettle's leaves (Kregiel et al., 2018). Fresh leaves of SN or their extracts were added to broilers' diet in some parts of Europe as a feed additive (Loetscher et al., 2013). Kregiel et al. (2018)

Birds, diets and experimental groups
On day 1, 240 1-day-old male Arbor Acres broiler chicks were randomly placed in stainless-steel cages (200 cm length, 100 cm width, 80 cm height) with 10 birds/m 2 density, along with one feed trough (185 cm long, 8.5 cm wide and 5.3 cm deep) and six nipple drinkers in thermostatically controlled rooms. All the nutritional and environmental conditions were programmed based on A. Acres Broiler Management Guide (Aviagen, 2018).
During the experiment, all the broilers had unrestricted access to a commercial starter (1-10 days old), grower (11-25 days old) and finisher (

Heat stress challenge
On day 1, the ambient temperature was set as 34 ± 1 • C and continued for the first 3 days. Then, room temperature gradually decreased 1 • C every 2 days until reaching to 24 ± 1 • C and maintained at this temperature by controlling heat and ventilation until day 22. On day 22, the ambient temperature suddenly increased from 24 ± 1 to 38 ± 1 • C in the HS groups (HS, HS-SN2, and HS-SN4). The cyclic heat stress (38 ± 1 • C) was induced with a heater from days 22 to 29 and the relative humidity was kept at 50 ± 5% during this period (Altan et al., 2003). The HS groups were reared in the controlled room at 38 ± 1 • C for 8 h/D (between 09:00 AM and 5:00 PM), followed by 24 • C for the remain- heat stress period, the fans were kept active to maintain the homogeneity of the environmental temperature in room space. Afterwards, on day 29, the room temperature suddenly decreased from 38 ± 1 to 24 ± 1 • C in the HS groups, all the treatment groups were kept under ambient temperature (24 ± 1 • C) until 35 days of age ( Figure 1).

Blood biochemical assay
Blood sample (4 mL) was taken from the brachial vein from eight birds in each group on days 14, 21, 29 and 35. Following the blood coagulation at room temperature, the blood samples were centrifuged at 3000 × g for 10 min to obtain serum. Subsequently, sera were carefully collected and stored at −20 • C until biochemical analysis. The levels of serum TP, total cholesterol (TC) and UA were measured using an automated analyser with commercial clinical investigation kits (Biorexfars kit, Fars, Iran

Statistical analyses
Statistical analyses were carried out using SPSS (SPSS for Windows, Version 22, SPSS Inc., Chicago, Illinois). All the data obtained were subjected to one-way ANOVA followed by the post-hoc Tukey HSD test. The results were expressed as mean ± standard error (mean ± SE). Repeated measures ANOVA was employed for determining the effects of sampling time, differences between groups and interactions between time × treatments followed by Tukey's post-hoc comparison test. Values of p < 0.05 were considered statistically significant. There were no statistically significant differences in UA between the control group with HS-SN2, HS-SN4, SN2and SN4 groups on day 35 (Table 3; p > 0.05). The serum level of TP had no significant differences between the various treatments on days 29 and 35 (Table 3; p > 0.05).

RESULTS
As shown in Table 4, the serum activities of AST and CK had no significant differences among the various studied groups on days 14 and 21 (Table 4; p > 0.05). On days 29 and 35, AST, ALT and CK were sig-nificantly higher in HS than the other experiment treatments (Table 4; p < 0.05). HS-SN2 and HS-SN4 had significantly lower serum activities of ALT and CK compared with HS on days 29 and 35. At the different days of study, the least serum levels of AST, ALT and CK were seen in control, SN2 and SN4 groups (Table 4; p < 0.05).

DISCUSSION
Heat stress is a major environmental stressor with a negative impact on poultry well-being and health status. Various studies have stated that heat stress causes oxidative stress, resulting in a change in the blood biochemical stress parameters (Habashy et al., 2017). Several nutritional methods have been assessed to attenuate the adverse effects of heat stress . Diet supplementation with medicinal plants is one of the most common solutions to alleviate heat stress in broilers (Chauhan et al., 2020;Sȃrȃcilȃ et al., 2020). Accordingly, the current research assessed the effects of dietary SN on some blood biochemical metabolites in broilers exposed to heat stress. This study revealed that dietary SN powder at a high dose (4%) minimizes the chronic heat stress in broilers by normalizing the serum values of cortisol, TC and UA (Table 3). Furthermore, dietary SN at 4% level decreased the serum activities of ALT, AST and CK enzymes as tissue damage indices in the birds exposed to chronic heat stress (Table 4) (Horváth & Babinszky, 2018). SN has significant antioxidant effects due to its phenolic and terpenoid compounds (Kregiel et al., 2018). Carvacrol and carvone are the main terpenoids in this herb with antioxidant and growth-promoting properties (Upton, 2013 (Tables 3 and 4).
In the current study, the concentrations of cortisol, TP, cholesterol, UA, ALT, AST and CK on days 14 and 21 had no statistically significant difference among the examined groups, implying no stress on the birds before exposure to heat stress ( Control: basal diet at normal temperature; HS: basal diet at cyclic heat stress; HS-SN2: basal diet supplemented by 2% stinging nettle at cyclic heat stress; HS-SN4: basal diet supplemented by 4% stinging nettle at cyclic heat stress; SN2; basal diet supplemented by 2% stinging nettle at normal temperature; SN4: basal diet supplemented by 4% stinging nettle at normal temperature. Different letters in the superscripts of the same columns indicate significant differences (p < 0.05).
of cortisol in broilers (Xu et al., 2018). In the current experiment, both dietary levels of SN in the HS-SN2 and HS-SN4 groups reduced the cortisol compared with the HS group on the basal diet on days 29 and 35; however, this effect was stranger at high dose (4%) of SN supplementation (Table 3; p < 0.05). Based on the current investigation, 4% dietary SN led to a significant decrease in the serum level of cortisol, indicating that this herb could attenuate the stress response induced by heat exposure in broilers.
Herein, the serum levels of TP had no significant differences among the various groups (Table 3; p > 0.05). Several studies have reported that the TP level increased in birds following inflammation or stressors (Kim et al., 2015;Shen et al., 2010). Furthermore, Sugiharto et al.
(2017) stated that heat stress resulted in an increase in the TP in broilers. Interestingly, no significant differences were was observed in the TP concentration between the different studied treatments (Table 3).
Similarly, some other researchers have found no significant alterations in the TP in birds exposed to heat stress (Kataria et al., 2008;Vosmerova et al., 2010).
In the studied birds, heat stress led to an increase in TC concentration in HS compared with the control group. These findings are in line with the results by Luo et al. (2018). They confirmed that heat stress decreased the activity of lipolytic enzymes and the rate of lipolysis in the body, which led to an increase in serum levels of triglyceride and TC. In the present experiment, the serum level of cholesterol decreased in the HS-SN2, HS-SN4 and SN-4 groups fed by supplemented diet with SN. The cholesterol reduction activity of SN could depend on its sterol and phenolic content (Kregiel et al., 2018). Moreover, SN reduces lipogenesis and lipid metabolism, resulting in a reduction in the serum content of TC (Avci et al., 2006). In agreement with the obtained findings from this study, Zhang et al. (2020) demonstrated that the inclusion of SN in the diet of the laying hen was associated with the decreased TC and triglycerides. Based on the results presented in TA B L E 4 The effects of stinging nettle on the serum activities levels of enzymes related to tissue damage in broilers exposed to chronic heat stress  In the present study, the serum level of UA was significantly higher in the HS group compared to the control group (Table 3; p < 0.05). UA is the end-metabolic product of protein metabolism in avian species and has been suggested as a potent scavenger of free radicals in broiler chickens (Azzam et al., 2019). Xie et al. (2015) reported that the circu- Based on presented results in Table 4, heat stress increased (p < 0.05) the serum activities of ALT, AST and CK in the HS group compared with the control group, on days 29 and 35. Chronic thermal exposure leads to the over-production of free radicals and subsequent oxidative damage in the various body tissues (Akbarian et al., 2016). The liver negatively affected by chronic heat stress in birds (Miao et al., 2020). The serum levels of ALT and AST enzymes are employed as reliable indices of liver damage and can reflect the degree of oxidative damage in the hepatic cells during exposure to chronic heat stress (Miao et al., 2020;Xie et al., 2015). Xie et al. (2015) reported that chronic heat stress increased the serum activities of AST, and CK enzymes, which is in agreement with the obtained findings of this study.
Furthermore, heat stress significantly increased the serum activity of CK, as a valuable indicator for muscular damage induced by chronic heat stress (Xie et al., 2015), which is consistent with the results in Table 4. The serum activities of ALT and CK decreased in the HS broilers fed by 2 and 4% dietary SN (HS-SN2 and HS-SN4, respectively) compared with HS (Table 4, p < 0.05). These results are in line with the previous studies stating that SN had hepato-protective property (Uyar et al., 2016). SN inhibits liver damage with its potent antioxidant activity (Yildizhan et al., 2020). In the current experiment, the effects of SN on alleviating the hepatic damage were revealed with the decreases in the serum level of ALT and CK in the broilers. The obtained results from the current study indicated that SN regulates the oxidative stress induced by heat stress in broilers and protects the hepatic cells from damage in the birds exposed to heat stress.

CONCLUSIONS
Heat stress exposure caused an elevation in circulating cortisol as a main stress indicator. Furthermore, the serum levels of TC, UA and activities of ALT, AST and CK increased in HS broilers. The supplemented diet with SN at a level of 4% decreased the serum levels of cortisol, cholesterol, AST, ALT and CK in the broilers exposed to chronic heat stress. Accordingly, it seems that dietary SN attenuates the negative effects of HS in broilers. In addition, it could be suggested that SN be applied as a feed additive for improving the health status of broilers under stressful conditions.

AUTHOR DECLARATION
All of the authors listed on the manuscript are employed by a government agency (Shiraz University) that has not a primary function other than research and/or education.

ANIMAL ETHICAL STATEMENT
All protocols used in the study were approved by the Iranian Animal Ethics Standards under the supervision of the Iranian Society for the prevention of cruelty to animals and Shiraz University Research Council (IACUC no: 4687/63).