1. Introduction

Heat stress (HS) remains one of the main concerns in the poultry industry worldwide, mainly in the tropics, due to the high temperatures throughout the year. Broilers are homeothermic animals that are primarily affected by heat stress, leading to decreased growth performance, decreased immune response, and an imbalance of the oxidant/antioxidant mechanism [1-2]. The immunosuppressive effect of HS has been reported, demonstrating that the neuroendocrine system plays a significant role in birds' proper physiological functioning and homeostasis during HS. During the early stages of the temperature challenge, the sympathoadrenal medullary axis activates and regulates homeostasis. The adrenal medulla experiences a hormonal rise in epinephrine, norepinephrine, and glucocorticoids due to the impulse transmitted by sympathetic nerves in response to elevated temperature. As a result, muscle and liver glycogen levels drop while respiratory rates rise [3]. The activation of the hypothalamic-pituitary-adrenal axis intensifies with prolonged stress exposure. The immunosuppressive effect of HS has been documented, indicating that the neuroendocrine system is crucial for the physiological functioning and homeostasis of birds during HS. In the initial phases of the temperature challenge, the sympathoadrenal medullary axis is activated to regulate homeostasis. The adrenal medulla undergoes an increase in catecholamines (epinephrine and norepinephrine) and glucocorticoids as a result of impulses from sympathetic nerves in reaction to elevated temperature. Consequently, there is a reduction in muscle and liver glycogen levels, accompanied by a rise in respiratory rates [3]. In response to stress, the pituitary gland secretes adrenocorticotropic hormone (ACTH), which is induced by corticotropin-releasing hormone (CRH). ACTH stimulates corticosteroid release and synthesis in the adrenal glands [4]. Corticosterone increases are frequently followed by increases in heterophiles, increasing the heterophile-lymphocyte ratio [5]. Poultry exposed to high temperatures shows a drop in immune system components [6]. Additionally, it was noted that poultry under heat stress showed increased levels of serum inflammatory cytokines like tumor necrosis factor-α, interleukin-1, and interleukin-6 [7-8].

Therefore, alternatives for improving broiler immunity and other strategies to alleviate the adverse effects of HS are needed in the poultry industry. In this sense, supplementing poultry rations with bioactive compounds, such as curcumin, can increase antioxidant activity, contributing to decreased symptoms of HS.

Curcumin is the most critical bioactive component originating from turmeric, extracted from the rhizome of Curcuma longa L., belonging to the Zingiberaceae family [9]. A potent antioxidant and various other pharmacological effects are attributed to curcumin, including antibacterial, antiviral, lipid-lowering, antidiabetic, and anti-inflammatory properties [9]. The unique biological properties of turmeric have sparked extensive research into its health benefits, particularly focusing on curcumin, its active component [10]. The existing literature regarding the impact of curcumin supplementation on the immunity of heat-stressed challenge chickens is notably sparse. The current study aimed to assess the effects of varying dietary concentrations of curcumin, a natural antioxidant, on the immune responses of challenge chickens subjected to HS.

Therefore, alternatives for improving broiler immunity and other strategies to alleviate the adverse effects of HS are needed in the poultry industry. In this sense, supplementing poultry rations with bioactive compounds, such as curcumin, can increase antioxidant activity, contributing to decreased symptoms of HS.

Curcumin is the most critical bioactive component originating from turmeric, extracted from the rhizome of Curcuma longa L., belonging to the Zingiberaceae family [9]. A potent antioxidant and various other pharmacological effects are attributed to curcumin, including antibacterial, antiviral, lipid-lowering, antidiabetic, and anti-inflammatory properties [9]. The unique biological properties of turmeric have sparked extensive research into its health benefits, particularly focusing on curcumin, its active component [10]. The existing literature regarding the impact of curcumin supplementation on the immunity of heat-stressed challenge chickens is notably sparse. The current study aimed to assess the effects of varying dietary concentrations of curcumin, a natural antioxidant, on the immune responses of challenge chickens subjected to HS.

2. Materials and methods

The experiment was carried out at the Animal and Poultry Research Centre (El-Bostan Farm), part of the Animal and Poultry Production Department, Faculty of Agriculture, Damanhour University, Egypt. All procedures in the current investigation were authorized by the local Ethics Committee on Animal Use (DUFA-2024-16).

2.1. Bird management

From day one to 35 days of age, chicks were kept in galvanized wire cages with standard dimensions of 65×65×45 cm in a semi-open house with two exhaust fans to maintain normal ventilation. Over the experiment period, food and water were given freely. All experimental chicks were brooded at 33 °C upon arrival and gradually decreased from 30 to 28 °C during the second week. Consistent heat augmentation was executed by utilising digital heaters to keep the optimal temperature. Throughout the trial, the relative humidity was about 60±5%. Chicks were subjected to a standard lighting schedule according to commercial broiler strain guidelines, 23 hours of light/1h darkness during the first week and 20 h light / 2h darkness from the second week until the end of the experiment. A systematic immunisation initiative against Newcastle disease and Gumboro disease was implemented. Thus, the chicks were vaccinated against Gumboro at the age of 12 days through drinking water. The Hatchner B, Lasota, and Colon 30 vaccines were administered at 7, 18, and 28 days of age, respectively.

2.2. Diet and experimental design

For this experiment, 120-7-day unsexed Ross 308 chicks were randomly divided into four treatment groups with five replications of six chickens per replication. The 1^st group was fed a basal diet (control group), the 2^nd, 3^rd, and 4^th groups were fed basal diets supplied with different levels of curcumin (100, 200, and 400 mg/kg diet), respectively (Table 1). The experiment lasted for 35 days. From 14 to 35 days of age, all experimental birds were exposed to 6 hours per day (10:00 to 16:00) of continuous heat stress at 35±1^oC and 24±2^oC for the remaining time and relative humidity 60±5%.

Table 1. Composition and calculated analysis of the basal diet.

2.8. Serum cytokine analysis

The contents of interleukin-2 (IL-2), IL-6, IL-10, and interferon-gamma (IFN-γ) were measured using chicken ELISA Kits (R&D Systems, Minneapolis, MN, USA).

2.9. Statistical analysis

The model expressed in equation 1 was used for analysis in a one-way analysis of variance (ANOVA) with the GLM technique [25]

Yij = µ+Ti+eij                (1)

Where Yij is an observation, μ is the overall mean, Ti is the effect of treatment (i = 1,2, …, .4), and eij is the experimental random error. Prior to statistical analysis, the percentage data of the attributes that were analysed underwent square root or arc sine transformation, and the bacterial counts were converted using a Log transformation. The difference among means was determined using Student-Newman-Keuls. Means differences at p≤0.05 were tested. 

3. Results and discussion

3.1.   Growth performance

Meat-type chickens subjected to chronic heat stress (CHS) had their live body weight (LBW) affected by various dietary doses of curcumin, as shown in Table 2. The LBW on the seventh day of age exhibited no significant variation across the experimental groups. However, heat-stressed meat-type chickens fed a baseline diet supplemented with varying amounts of curcumin had a significant increase in LBW at 35 days (P≤0.002) of age with respect to the control. Generally, the LBW of birds subjected to varying curcumin dosages was markedly greater with respect to the control group at 35 days of age. The current study's findings revealed that varying levels of curcumin enhanced the LBW of broiler chicks exposed to CHS with no significant difference between different levels of curcumin by 18.9, 17.2 and 23.0% for (100, 200, and 400 mg curcumin/kg diets), respectively with respect to the control at 35 days of age. The birds fed varying doses of curcumin consumed 1.62, 2.04,2.98 % more feed and grew 20.61, 18.63, and 25% higher with respect to the control. Furthermore, for the entire experimental duration, the percentages of improvement of Feed Conversion Ratio (FCR) were 15.54, 13.99, and 17.62 % for (100, 200, and 400 mg curcumin/kg diets), respectively, with respect to the control group. The observed enhancement in FCR can be linked to the increase in BWG noted during this experiment. In the current study, a progressive enhancement in performance with an elevated amount of curcumin under HS conditions. The observed improvement may result from essential oil compounds in curcumin exhibiting significant antimicrobial activity, which inhibits bacterial growth. This action supports intestinal flora performance, enhancing digestion and energy utilization, ultimately contributing to improved growth.

Furthermore, curcumin supplementation enhances the production of bile acids and the activities of lipase, amylase, and proteases, which are crucial for metabolism and promote expedited digestion [26]. The small intestine's structure is crucial for nutrient digestion and absorption [27]. Furthermore, the inclusion of phenolic compounds, such as curcumin, has the potential to reduce gut inflammation, enhance nutrient digestibility, and improve metabolic activity [28-30]. Additionally, these substances have the potential to alter the gastrointestinal system's architecture and functioning [31].

Table 2. Effect of different dietary levels of curcumin supplementation on growth performance of broiler chickens.

Dietary supplementations	Leukocytic components
	WBC's (103/mm3)	Heterophils (%)	Lymphocyte (%)	H/L	Monocyte (%)
Control	18.71b	30.21	30.71b	0.983a	14.03b
100 mg Curcumin	19.21b	32.91	42.12a	0.782b	15.60a
200 mg Curcumin	21.10ab	30.81	45.71a	0.674b	16.10a
400 mg Curcumin	23.30a	33.22	47.71a	0.696b	16.30a
SEM	0.712	5.790	1.21	0.001	0.301
P value	0.006	0.067	0.003	0.207	0.002

^a,b Means in the same column followed by different letters are significantly different at (p ≤ 0.05);  SEM,   Standard error of mean;. WBC's = White blood cell.  

3.3 .Protein profile

Table 4 illustrates the impact of varying dietary amounts of curcumin supplementation on the protein profile of broiler chicks subjected to HS conditions. The findings indicated a significant increase in the serum total protein of meat-type chickens receiving curcumin supplementation under CHS with respect to the control. Treatment groups given 100, 200, and 400 mg curcumin/kg diets differed from the control group by 16.6%, 22.3%, and 15.5%, respectively, as compared to the control mean. The results demonstrated a non-significant impact of curcumin supplementation on albumin level (g/dl) at 35 days of age. Curcumin treatments significantly elevated the concentration of serum globulin (g/dl) in heat-stressed meat-type chickens with respect to the control at 35 days of age. The percentage increases were 43.04%, 53.6%, and 39.07% for diets containing 100 mg, 200 mg, and 400 mg of curcumin per kg, respectively, with respect to the control. The results demonstrated that A/G ratio values showed no statistically significant variations among the experimental groups. The findings suggest a notable improvement in total protein and globulin concentrations in heat-stressed broilers administered a basic diet augmented with differing levels of curcumin. This aligns with the observations of Muhammad and Al-Hassani [41], indicated that varying concentrations of turmeric root powder led to a substantial elevation in protein profile in broilers subjected to HS. The reduced A/G ratio observed in the current investigation may signify an enhancement in the immune competence of broilers administered decocted turmeric. Sugiharto et al. [38] proposed that a diminished A/G ratio is associated with enhanced immune competence and disease resistance in meat-type chickens.

Table 4. Effect of different levels of dietary curcumin supplementation on protein profile of broiler chicks under heat stress conditions.

Dietary supplementations	Total protein (g/dl)	Albumin (g/dl)	Globulin (g/dl)	A/G ratio
Control	4.51b	3	1.5b	1.99
100 mg Curcumin	5.26a	3.1	2.16a	1.44
200 mg Curcumin	5.52a	3.2	2.32a	1.38
400 mg Curcumin	5.21a	3.11	2.10a	1.48
SEM	0.236	0.125	0.189	0.054
P value	0.006	0.518	0.020	0.074

^a,b Means in the same column followed by different letters are significantly different at (p ≤ 0.05); SEM, Standard error of mean.

3.4. Immunological status

Table 5 presents data on serum α–globulin and β–globulin concentrations (µg/dl). Serum γ–globulin levels in heat-stressed broiler chickens receiving a control diet supplemented with 100 mg or 200 mg of curcumin per kg significantly exceeded those of the control group. Conversely, α-globulin and β-globulin exhibited no significant variation among the experimental groups. The serum immunoglobulin (Ig) fractions (IgG, IgM, and IgA) of meat-type chickens at the end of the experiment, influenced by varying dietary levels of curcumin, are presented in Table 5.

Serum levels of IgG, IgA, and IgM (mg/dl) were significantly elevated (P≤0.009) in response to varying dietary curcumin levels with respect to the control. Results indicated curcumin levels significantly affected serum IgM and IgA concentration (P≤0.006). The groups administered curcumin exhibited elevated serum levels of IgG by (16.6, 24.8, and 16.7), IgM by (30.9, 36.4, and 37.9), and IgA by (41.5, 49.4, and 56.7) in comparison to the control group.

^a,b Means in the same column followed by different letters are significantly different at (p ≤ 0.05); SEM, Standard error of mean; IgG, Immuno globulin G; IgM, Immuno globulin M; IgA, Immuno globulin A.

Data for lysosome activity (LA, %), bactericide activity (BA, %), lymphocyte transformation test (LTT, %), phagocytic index (PI, %), and phagocyte activity (PA, %) of broiler chicks at 35 days of age are shown in Table 6.

The analysis of variance indicates that LA % for birds receiving varying dietary levels of curcumin was significantly elevated (P≤0.003) with respect to the control. The serum LA% exhibited significant increases of 69.3%, 51.2%, and 46.8% for diets containing 100, 200, and 400 mg of curcumin/kg, respectively, with respect to the control at 35 days of age. BA in groups fed a control diet with varying dietary levels of curcumin was significantly elevated (P≤0.005) with respect to the control at 35 days of age. The serum BA% exhibited significant increases of 26.6%, 34.5%, and 29.9% for diets containing 100, 200, and 400 mg of curcumin per kg, respectively, with respect to the control at 35 days of age. Furthermore, LTT % for chickens subjected to HS and fed a basal diet with varying levels of curcumin showed a significant increase with respect to the control. The serum LTT% exhibited significant increases of 27.1%, 25.7%, and 31.7% for diets containing 100, 200, and 400 mg of curcumin/kg, respectively, with respect to the control at 35 days of age. These results indicated that treated broiler chicks with dietary curcumin significantly increased (P≤0.004) in their PA% with respect to the control. Thus, the percentages of significant increase in serum PA% were 20.4, 16.6, and 13.4 % for (100, 200, and 400 mg curcumin/kg diets), respectively, with respect to the control at 35 days of age. It can be seen that the meat-type chicken's PI concentration was significantly affected by curcumin levels at 35 days of age with respect to the control. Thus, the percentages of a significant increase in serum PI% were 52.3, 71.9, and 85.9% for (100, 200, and 400 mg curcumin/kg diets), respectively, with respect to the control at 35 days of age. Moreover, these previously mentioned parameters for treated groups (curcumin groups) did not represent any statistical change between each other.

Table 7 displays the serum IL-2, IL-6, IL-10, and INF levels of meat-type chickens at 35 days of age that were altered by varied amounts of curcumin in their diet. Serum IL-2 concentration for broilers exposed to HS and fed a basal diet with varying levels of curcumin significantly increased with respect to the control (P≤0.0001). Thus, the percentages of a significant increase in serum IL-2 were 94.1, 2.09, and 22.7% for (100, 200, and 400 mg curcumin/kg diets), respectively, with respect to the control at 35 days of age. Also, results indicated that curcumin levels significantly influenced serum IL-6 concentration (P≤0.002). The percentages of significant increase in serum IL-6 were 12.5%, 17.2%, and 16.7% for the 100, 200, and 400 mg curcumin/kg diets, respectively, with respect to the control at 35 days of age. Additionally, the findings indicated that curcumin levels significantly influenced serum IL-10 (P≤0.006). The percentages of significant increase in serum IL-10 were 11.3%, 21.5%, and 31.6% for diets containing 100, 200, and 400 mg curcumin/kg, respectively, with respect to the control at 35 days of age. Results indicated that curcumin levels significantly affected serum INF (P≤0.006) with respect to the control.  The percentages of significant increase in serum INF were 40.5%, 37.2%, and 43.3% for diets containing 100 mg, 200 mg, and 400 mg of curcumin per kg, respectively, with respect to the control at 35 days of age. 

Heat stress induces oxidative stress in broilers, resulting in cellular damage and compromised immune function. Curcumin is a potent antioxidant, effectively neutralizing free radicals and diminishing oxidative stress. This protective effect can improve overall immune function, including producing IL-2, IL-6, IL-10, and INF.

Thus, the percentages of significant diminish in E. coli were 40.20, 45.22, and 46.73% for (100, 200, and 400 mg curcumin/kg diets), respectively, with respect to the control at 35 days of age, concerning the values of E. coli. While the percentages of significant diminish in Proteus were 59.55, 49.34, and % for (100, 200, and 400 mg curcumin/kg diets), respectively, with respect to the control at 35 days of age, concerning the values of Proteus. This suggests that curcumin effectively inhibits this pathogenic bacterium, and reducing pathogenic bacteria like E. coli and Proteus is crucial for preventing infections and improving overall health and growth performance in broilers.

4. Conclusion

Based on the results of improved body weight gain and feed utilization, it can be inferred that broiler chicks grown under CHS perform better when fed curcumin, particularly at a dosage of 100 mg/kg diet. Indicative of an enhanced immunological condition, this enhancement was also noted in the chicks' blood biochemical indicators.