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. 2024 Apr 20;103(7):103790. doi: 10.1016/j.psj.2024.103790

High-energy and high-amino acid diet enhances production performance and antioxidant capacity in yellow-feathered broilers under heat stress

Huihua Mao *, Jinglong Chen ‡, Jinbi Zhang *, Xu Zhang *, Shiyong Xu *, Ling Zhang †,1
PMCID: PMC11091689  PMID: 38713989

Abstract

This study investigated the ameliorating effects of high-energy and high-amino acid (HEHA) diets on heat stress (HS) in yellow-feathered broilers. Broilers aged 35 d were randomly assigned to 3 groups: control and HS groups fed the basic normal diet, and the HEHA group fed the HEHA diet (basal diet + 100 kcal/kg AME + 15 % DAAs). The HS and HEHA groups were exposed to cyclic HS (30 ± 1 to 34 ± 1 ℃) for 2 wk, while the control group was maintained at 26 ± 1 ℃. The results indicated that the HEHA diet significantly alleviated HS-induced feed intake and body weight loss. HEHA feeding mitigated the increase in body temperature during HS. Compared with observations in the HS group, the HEHA diet reduced the levels of ALT, Alb, and corticosterone in the serum and downregulated the gene expression of HSP27 and HSP60 in the liver. Moreover, the HEHA group showed higher GSH-px activity in the serum and SOD and GSH-Px activity in the jejunal mucosa than that of the HS group. HEHA supplementation also reduced MDA levels in the liver. In conclusion, the HEHA diet improved the production performance of broilers under HS by increasing their antioxidant capacities. These findings suggest an effective strategy to combat HS in poultry production.

Key words: yellow-feathered broiler, heat stress, metabolic energy, amino acid, production performance

INTRODUCTION

With the aggravation of global warming, the effects of temperature on animal husbandry have become increasingly important. Heat stress (HS) occurs when the heat produced by an animal exceeds its ability to dissipate it into the surrounding environment. Poultry is particularly sensitive to HS (Vandana et al., 2021; Shi et al., 2023). In modern intensive farming, excessive stocking densities, hot and humid summer environments, inadequate ventilation measures, and even power outages in chicken houses caused by power shortages can lead to HS (Goel, 2021; Wang et al., 2022). The impact of HS on poultry is multifaceted. It can lead to a 20 to 40% decrease in feed intake and body weight gain (Abdel-Moneim et al., 2021), increase the levels of free radicals, which cause oxidative stress (Habashy et al., 2019), and decrease poultry product (chicken meat and egg) quality (Barrett et al., 2019) . Prolonged exposure of poultry to HS not only affects productive performance, but also significantly increases the incidence of respiratory and gastrointestinal diseases, which are associated with considerable losses in production. In addition to improving the living environment and using anti-stress drugs, adjusting the nutritional balance of feed to alleviate HS symptoms in broilers is advantageous because it is cost-effective.

Dietary energy and crude protein levels are the main factors that affect the growth performance of broilers (Zinn et al., 1996; Zhang et al., 2021; Zhang et al., 2022; Korver, 2023). During HS, poultry birds reduce their feed intake to mitigate the heat production associated with digestion, absorption, and nutrient metabolism; consequently, leading to a decrease in energy availability and inadequate protein intake (Lara and Rostagno, 2013). To compensate, birds are typically fed high-energy diets. Higher dietary energy levels have been reported to enhance the production performance of birds under constant and cyclic HS conditions (Wasti et al., 2020). Furthermore, given that the crude protein content in feed is adjusted according to energy levels, high-energy diets generally have a higher crude protein content (Dale and Fuller, 1980). Moreover, proteins exhibit higher calorific increments than carbohydrates and fats (Fuller and Rendon, 1977), and an increase in dietary crude protein content can result in an increase in diet-induced thermogenesis, thereby exacerbating HS. The requirement for crude protein in animals is equivalent to that for amino acids. Therefore, reducing dietary crude protein levels and adding appropriate proportions of synthetic amino acids based on the “ideal protein” theory to achieve amino acid balance can better meet the nutritional needs of poultry birds and reduce heat production during protein digestion, absorption, and metabolism (Baker, 2009). In this study, we aimed to investigate the effect of a high-energy and high-amino acid (HEHA) diet on the production performance and antioxidant capacity of yellow-feathered broilers under HS. These findings obtained contribute to enhancing our understanding of the effects of nutrient regulation on poultry production.

MATERIALS AND METHODS

Experimental animals and groups

Selected 180 male 35-day-old Fast-type Yellow-feathered Broilers with similar weights were purchased from Jiangsu Lihua Animal Husbandry Co., Ltd. (Jiangsu, China) and randomly divided into 3 groups with 6 replicates per group. The groups comprised the normal-temperature control, high-temperature control, and high-energy and high-amino acid groups (i.e., CON, HS, and HEHA groups, respectively). Birds in the CON and HS groups were fed a basal diet, whereas those in the HEHA group were fed the HEHA diet, which involved reduced amounts of corn and soybean meal in the basal diet and supplementation with 1.7% soybean oil, 0.2% lysine, 0.12% methionine, 0.1% threonine, 0.02% tryptophan, 0.16% arginine, 0.06% valine, and 0.04% isoleucine. The Basic amino acid balance pattern is: Lys: Met + Cys: Thr: Trp: Arg: Val: Ile = 100: 78: 64: 16: 100: 88: 79. This resulted in 100-kcal/kg metabolizable energy (AME) and 15% digestible amino acids (DAA) levels increases in dietary, as well as the crude protein levels and digestible amino acid pattern in this modified diet remained unchanged. The experiment was conducted with a 1-wk pre-trial period and a 2-wk treatment period.

After the pretrail period (7-d pre-feeding), the birds in the HS and the HEHA groups were exposed to cyclic heat stress treatment from d 8 as follows: 00:00 to 06:00 and 20:00 to 00:00, 30 ± 1 ℃ for 10 h; 06:00 to 10:00 and 16:00 to 20:00, 32 ± 1 ℃ for 8 h; and 10:00 to 16:00 at 34 ± 1 ℃ for 6 h. This HS treatment is shown in Figure 1. The control birds were maintained at a constant temperature of 26 ± 1 ℃. Furthermore, the treatment lasted for 2 wk. Changes in feed intake, body weight, and rectal temperature were recorded during the study period.

Figure 1.

Figure 1

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Cyclic heat stress program setting.

Breeding Management

The breeding management experiment, which was performed using 3-layer caging, lasted 21 d. The birds had free access to water and food and 24-h light exposure. Further, their feces were cleaned regularly. To ensure a stocking density of approximately 0.1 m2/chicken, chicken cages with size 120 × 80 cm were used. The temperature was controlled using an animal nutrition metabolism environmental control chamber (Beijing Koolan Technology Co., Ltd., Beijing, China) and in this chamber, humidity was maintained between 50% and 70%. Other management measures were performed in accordance with the feeding standards for yellow-feathered broilers. On d 8 of the experiment, the cyclic HS treatment was commenced for the HS and the HEHA groups, while the CON birds were maintained at a constant temperature of 26 ± 1 ℃. The treatment lasted for 2 wk, and changes in feed intake, body weight, and rectal temperature were recorded during this period. The basal diet (28–56 d) was meet the Nutrition Research Council (1994) and the Feeding Standard of Chicken (NY/T 33-2004, China) requirements as appropriate. The composition and nutritional levels of the diets are listed in Table 1.

Table 1.

Composition of the basal diets.

Items Control group HEHA group
Ingredients
Corn (CP = 7.2%) 693.75 677.65
Soybean meal 43% 200 192
Corn gluten meal 60 50 50
Soybean oil 30.3 47.2
Limestone 9.6 9.6
CaHPO4 5.2 5.4
98% DL-Met 1.5 2.7
98.5% Lys 2.9 4.9
98% Thr 0.2 1.2
98% Trp 0 0.2
98.5% Arg 0.1 1.7
98% Val 0 0.6
98% Lle 0 0.4
Phytase 0.15 0.15
Choline chloride 1 1
Trace mineral premix 2 2
Vitamin premix 0.3 0.3
NaCl 3 3
Total 1000 1000
Nutrient levels
CP 17 17
EE 5.66 7.29
CF 2.18 2.11
CA 3.8 3.75
ME (MJ/kg) 13.18 13.6
Ca 0.55 0.55
TP 0.42 0.41
AP 0.22 0.22
Dlys 0.87 1
DMet/lysP 0.48 0.53
DM&C/LysP 0.78 0.78
DThr/LysP 0.64 0.64
DArg/Lysp 1 1
DTrp/lysp 0.16 0.16
DlIe/Lysp 0.79 0.79
DVal/lysp 0.88 0.88
Protein energy ratio 14.94 14.83
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CP, crud protein; EE, ether extract; CF, crude fiber; CA, crude ash; ME, metabolizable energy; TP, total phosphorus; AP, available phosphorus. The micronutrient premixes were provided as follows: Mn 60 mg, I 0.35 mg, Fe 25 mg, Cu 8 mg, Zn 50 mg per kg diet; Vitamin premix provides: per kg diet VA 4,000 IU, VD3 1,600 IU, VK3 1.5 mg, VB1 1.0 mg, VB2 3.0 mg, VB6 3.0 mg, VB12 0.005 mg, pantothenic acid 8.0 mg, niacin 20 mg, folic acid 20 mg; Micronutrient premixes were provided in the diet per kg: Mn 60 mg, I 0.35 mg, Fe 25 mg, Cu 8 mg, Zn 50 mg; Vitamin premix provides: per kg diet VA 4,000 IU, VD3 1,600 IU, VK3 1.5 mg, VB1 1.0 mg, VB2 3.0 mg, VB6 3.0 mg, VB12 0.005 mg, pantothenic acid 8.0 mg, niacin 20 mg, folic acid 20 mg; Nutrient level was calculated.

Sample Collection and Processing

At the end of the experimental period, one chicken from each replicate was selected based on average weight measurements. Then, blood samples were collected from the subcutaneous vein of the birds and allowed to clot in a coagulation tube for 2 h. Thereafter, sera samples were collected via centrifugation at 3,000 r/min for 10 min and used to measure blood biochemical and antioxidant-related indicators. Next, the birds were sacrificed, and their livers, spleens, thymus, bursa of Fabricius, and etc., were collected and weighed to calculate the organ indices (organ index = organ weight (g) / live weight (kg)). Further, liver and jejunal mucosal tissue samples were collected and stored in liquid nitrogen until antioxidant indicator level measurements.

Production Performance Measurement

Prior to housing and before and after HS treatment, the weights of the birds and their daily feed in-take were recorded. Thereafter, average daily gain (ADG), average daily feed intake (ADFI), feed-to-gain ratio (F/G), and mortality rate (Mortality) were calculated.

Rectal Temperature Measurement

At the d 1, 3, 7, 10, and 14 of the HS treatments, 1 chicken was randomly selected from each replicate for rectal temperature measurements. At the end of the experimental period, differences in rectal temperature between the groups were compared horizontally and vertically, and changes in rectal temperature over time within the same group were also noted.

Measurement of Blood Indices

A fully automated Mindray BS280 blood biochemistry analyzer (Mindray, Shenzhen, China) and an accompanying reagent kit were used to measure serum alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, albumin, glucose, uric acid, total cholesterol, triglycerides, low-density lipoprotein, potassium, sodium, chloride, and calcium levels.

Measurement of Antioxidant Indices

Serum, liver, and jejunal mucosa malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activities were determined using reagents obtained from Nanjing Jiancheng Biological Reagent Co., Ltd. (Nanjing, China). The assays were performed according to manufacturer's instructions.

Measurement of Corticosterone

Serum corticosterone levels were determined by Elisa kit purchased from Wuhan Eliret Biotechnology Co., LTD.

Total RNA Isolation and qPCR

Total RNA from liver was isolated using TRIzol (Invitrogen, Carlsbad, CA) method. The concentration and purity of total RNA were determined using Nanodrop (ND 2000, Thermo, Waltham, MA). Sample selection was based on the criteria that the OD260/OD280 ratio of total RNA should fall within the range of 1.8 to 2.0, and the OD260/OD230 ratio should be between 2.0 and 2.2. The cDNA synthesized by using NovoScript Plus All-in-one 1st Strand cDNA Synthesis SuperMix (Novoprotein:E047-01B), the Reverse Transcription Reaction System was showed in Supplementary Table 1. qRT-PCR was carried out using NovoStart Fast SYBR qPCR SuperMix (Novoprotein:E301-01A), the specific steps are as follows: the first step is predenaturation, 95℃, 30 s, the number of cycles is 1; The second step is denaturation and annealing/extension stage, denaturation 5 s at 95℃, annealing/extension 30 s at 60℃, PCR reaction cycle 40 times, reaction system is showed in Supplementary Table 2. Primers were designed by Primer 5, synthesized by Servicebio (Wuhan, China) and the Primer information was showed in Supplementary Table 3. Results were analyzed on an ABI Step One Plus instrument (Applied Biosystems, Carlsbad, CA) using the method of 2−△△Ct.

Statistical Analysis

All data were recorded and processed using Microsoft Excel 2013 (Microsoft, Redmond, WA). Further, analysis of variance analysis was performed using One-way ANOVA in SPSS software v20.0 (IBM, Armonk, NY). The results were expressed as the mean ± standard deviation, and statistical significance was set at P < 0.05.

RESULTS

Effect of the HEHA Diet on Rectal Temperature in the Heat-Stressed Yellow Broilers

Figure 2 shows the effects of the HEHA diet on rectal temperature in the heat-stressed yellow broilers. From this figure, it is evident that under HS, the rectal temperature of the broilers significantly increased. Notably, feeding with the HEHA diet resulted in no significant differences between the HS and HEHA groups on the first day of the HS treatment. However, on d 7, 10, and 14, the rectal temperatures of the birds in the HEHA group were significantly lower than those of the birds in the HS group (P < 0.05).

Figure 2.

Figure 2

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The effects of the HEHA diet on rectal temperature in the heat-stressed yellow broilers. Birds were fed basal diet and HEHA diet under thermoneutral or chronic heat stress. The rectal temperature was detected at d 1, 3, 7, 10, 14 after HS exposure, and one chicken was randomly selected from each replicate for rectal temperature measurements.

Effect of the HEHA Diet on Production Performance in the Heat-Stressed Yellow Broilers

From Table 2, which shows the effects of the HEHA diet on the production performance of the heat-stressed yellow broilers, it is evident that exposure to HS for 14 d significantly reduced the body weights of the broilers, whereas the HEHA diet tended to alleviate the HS-induced weight loss (P = 0.07). Additionally, relative to the HS group, the HEHA group showed significantly improved ADG and ADFI (P < 0.05) as well as a significantly reduced F/G (P < 0.05). Our results also indicated that to a certain extent, the HEHA group showed a lower mortality rate (5%) compared with the 10% mortality rate observed for the HS group.

Table 2.

Effect of high energy and high amino acid diet on growth performance of yellow-feathered broilers under heat stress.

Item CON HS HEHA P-value
Average weight (g) 35d 1,151.7 ± 9.8 1,130 ± 26.8 1,141.7 ± 31.25 0.33
42d 1,771.4 ± 46.1a 1,556.7 ± 128b 1,600 ± 67.5b 0.002
56d 2,586.9 ± 116.3a 2,081.7 ± 170.8b 2,266.7 ± 106.8b 0.001
Average daily gain (g) 58.25 ± 6.15a 37.47 ± 6.21b 47.63 ± 10.47a 0.001
Average daily feed intake (g) 171.52 ± 15.64a 135.24 ± 24.04c 147.84 ± 15.53b 0.013
Feed conversion ratio 2.95 ± 0.12a 3.61 ± 0.20b 3.12 ± 0.22a 0.001
Death rate 0 10 5 -
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Means followed by the different letters in the same row show a significantly difference (P < 0.05). Values are means ± SD of 6 birds in each group.

Effect of the HEHA Diet on Organ Indices in the Heat-Stressed Yellow Broilers

The effects of the HEHA diet on the organ indices of heat-stressed yellow broiler chickens, shown in Table 3, indicated that HS exposure had no significant effect on liver, abdominal fat, spleen, breast muscle, and bursa of Fabricius indices. However, compared to the CON group, the thymus organ index was significantly decreased (P < 0.05). Our results also indicated that the spleen and thymus indices of the birds in the HEHA group were significantly higher than those of the birds in the HS group (P < 0.05).

Table 3.

Effect of high energy and high amino acid diet on organ index of yellow-feathered broilers under heat stress.

Index CON HS HEHA P-value
Liver 22.66 ± 2.60 21.31 ± 6.21 22.55 ± 10.47 0.46
Abdominal fat 21.18 ± 3.20 27.61 ± 7.96 24.09 ± 7.98 0.53
Spleen 1.20 ± 0.36 0.94 ± 0.14 1.11 ± 0.32 0.33
Breast 49.87 ± 4.39 52.30 ± 6.29 48.72 ± 7.77 0.61
Bursal 0.43 ± 0.28 0.29 ± 0.10 0.35 ± 0.13 0.35
Thymus 2.17 ± 0.33a 0.8 ± 0.27c 1.61 ± 0.29b 0.001
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Means followed by the different letters in the same row show a significantly difference (P < 0.05). Values are means ± SD of 6 birds in each group.

Effect of the HEHA Diet on Blood Biochemistry in the Heat-Stressed Yellow Broilers

From Table 4, which shows the effects of the HEHA diet on the blood biochemistry of the heat-stressed yellow broilers, it is evident that, compared to the CON group, the HS group showed significantly elevated and reduced levels of aspartate aminotransferase and albumin levels, respectively (P < 0.05). Further, the HEHA diet significantly improved changes in aspartate aminotransferase and albumin levels caused by HS exposure (P < 0.05). We also observed that the HS treatment reduced blood potassium ion level, but significantly increased sodium and calcium ion levels (P < 0.05), and compared with the HS group, the HEHA group showed a significantly higher potassium ion level (P < 0.05).

Table 4.

Effect of high energy and high amino acid diet on Serum Index of yellow-feathered broilers under heat stress.

Index CON HS HEHA P-value
AST (U/L) 3.58 ± 0.79 3.83 ± 1.19 4.63 ± 1.06 0.56
ALT (U/L) 218.12 ± 21.72c 315.25 ± 69.00a 263.75 ± 46.74b 0.003
Alb (g/L) 14.83 ± 1.12a 13.04 ± 1.62c 14.14 ± 1.25b 0.035
Glu (mmol/L) 14.02 ± 1.11 14.53 ± 1.90 13.23 ± 1.76 0.23
UA (μmol/L) 336.17 ± 95.27 261.83 ± 110.62 275.00 ± 57.29 0.15
T-CHOL (mmol/L) 2.98 ± 0.33 2.94 ± 0.48 2.99 ± 0.46 0.95
TG (mmol/L) 1.39 ± 0.46 1.01 ± 0.43 1.32 ± 0.56 0.13
HDL-c (mmol/L) 1.74 ± 0.18 1.78 ± 0.13 1.86 ± 0.08 0.23
LDL-c (mmol/L) 1.17 ± 0.20 1.14 ± 0.25 1.16 ± 0.16 0.54
LDH (U/L) 1,187.42 ± 297.27 1,198.00 ± 310.80 1,254.50 ± 229.92 0.82
K (mmol/L) 3.99 ± 0.69a 3.43 ± 0.29c 3.74 ± 0.44b 0.004
Na (mmol/L) 148.75 ± 1.23b 152.28 ± 2.89a 151.71 ± 0.81a 0.001
Cl (mmol/L) 108.69 ± 2.25 109.60 ± 1.19 110.20 ± 0.51 0.2
Ca (mmol/L) 2.66 ± 0.08b 2.71 ± 0.06a 2.74 ± 0.06a 0.045
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ALT, alanine aminotransferase; AST, aspartate aminotransferase; Alb, albumin; Glu, glucose; T-Chol, total cholesterol; TG, triglycerides, HDL-c, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol; LDH, lactate dehydrogenase. Means followed by the different letters in the same row show a significantly difference (P < 0.05). Values are means ± SD of 6 birds in each group.

Effect of the HEHA Diet on Serum Corticosterone and Heat Shock Protein in the Heat-Stressed Yellow Broilers

Increased serum corticosterone and liver heat shock protein synthesis are markers of HS. Figure 3A showed that cyclic heat stress increased serum corticosterone levels, while HEHA diets significantly reduced corticosterone levels (P < 0.05). By examining the gene expression of liver heat shock protein, we found that the expressions of HSP27 and HSP60 in liver were significantly increased under heat stress exposure, and their expressions were significantly decreased by feeding HEHA diet (P < 0.05) (Figure 3B and 3C).

Figure 3.

Figure 3

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The level of serum corticosterone and the gene expression of heat Shock protein. (A) The level of serum corticosterone; (B) The Hsp 27 expression in liver; (C) The Hsp 60 expression in liver. The value P < 0.05 on the column chart indicates that the difference is significant (*), P < 0.01 indicates that there is a highly significant difference (**), and no P value indicates that there is no significant difference, n = 6.

Effect of the HEHA Diet on Antioxidant Capacity in the Heat-Stressed Yellow Broilers

As shown in Table 5, antioxidant enzyme activities were detected in the serum, liver, and jejunal mucosa of the birds. Relative to the HS group, the HEHA group showed significantly higher serum GSH-Px activity (P < 0.05), significantly lower liver MDA activity, (P < 0.05), and significantly higher jejunal mucosa SOD activity (P < 0.05).

Table 5.

Effect of high amino acid diet on antioxidant performance of yellow-feathered broilers under heat stress.

Index CON HS HEHA P-value
Serum MDA (nmol/mL) 8.56 ± 2.17 9.59 ± 1.22 9.64 ± 1.52 0.38
SOD (U/mL) 21.33 ± 6.43 26.74 ± 8.06 20.56 ± 10.28 0.45
GSH-Px (μg/mL) 90.01 ± 21.82b 72.13 ± 26.65b 115.23 ± 17.14a 0.02
Liver MDA (nmol/mg prot) 14.29 ± 4.44a 12.95 ± 6.63a 9.41 ± 3.08b 0.047
SOD (U/g prot) 22.61 ± 3.77 28.02 ± 9.34 27.52 ± 5.94 0.44
GSH-Px (μg/g prot) 54.12 ± 9.58 57.86 ± 14.64 60.03 ± 8.28 0.06
Jejunum MDA (nmol/mg prot) 2.11 ± 1.75 1.09 ± 1.55 1.18 ± 0.25 0.73
SOD (U/g prot) 23.24 ± 6.42b 19.61 ± 4.93b 45.27 ± 13.56a 0.001
GSH-Px (μg/g prot) 9.01 ± 8.09b 26.09 ± 13.26a 36.17 ± 21.61a 0.026
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Means followed by the different letters in the same row show a significantly difference (P < 0.05). Values are means ± SD of 6 birds in each group.

DISCUSSION

Most studies on HS in poultry have shown that compared with that under neutral temperature conditions, feed intake is significantly decreased under HS conditions. It has also been reported that for every 1°C increase in temperature under cyclic HS, feed intake decreases by 1.5%, whereas under constant HS conditions, it decreases by 3.5% (Ain et al., 1996). The decrease in feed intake reduces the heat production associated with digestion, absorption, and nutrient metabolism and can also lead to decreased feed conversion efficiency. One study revealed that HS poultry fed the same amount of feed as neutral temperature-housed poultry showed growth reductions ranging from 60 % to 99 % owing to decreased feed intake (Ma et al., 2021; Teyssier et al., 2022). Therefore, a decrease in feed intake was primarily responsible for the decreased growth performance of HS poultry. The results of this study show that the HEHA diet effectively increase feed intake in broilers under HS conditions, thereby increasing ADG, reducing the F/G ratio, and improving organ indices. Additionally, rectal temperature measurements showed that the HEHA diet decreased rectal temperature and reduced endogenous heat production. Under neutral temperature conditions, increasing feed metabolizable energy can effectively improve production performance; however, whether high-energy feed intake can alleviate HS-induced damage is controversial. In this study, metabolizable energy was increased by increasing the feed lipid content. Furthermore, fat oxidation was associated with a lower heat increase than that of starch and protein oxidation. Zulkifli et al. observed that under HS, diets with a higher proportion of carbohydrates or fats result in better production performance than diets without fat supplementation of equal calorie levels (Hakim et al., 2022) . Ghazalah (2008) also recommended an increase in the metabolizable energy level of broiler diets to 3,300 kcal/kg and lipid content to 5% for chickens exposed to high temperatures. Owing to the higher heat increment associated with crude protein than with fat and starch, some researchers have proposed that it is possible to improve HS by reducing dietary crude protein level. However, numerous studies have shown that reducing dietary crude protein levels not only fails to improve performance under HS, but also significantly reduces daily gain and feed conversion efficiency (Liu et al., 2016; Awad et al., 2018; Wu et al., 2023). However, amino acids do not require enzymes for digestion and do not generate heat during the digestion process. Therefore, adding synthetic amino acids to the diet and balancing amino acid levels can effectively reduce HS-induced damage (Al et al., 2017). Maharjan et al. (2020) fed broilers under high temperature conditions with 120% digestible lysine and maintained the ratio of other amino acids to digestible lysine. They observed a significant increase in ADG and feed conversion efficiency; however, feed intake remained unchanged. In this study, the inclusion of the HEHA diet in broiler mitigated the HS-induced elevation of body temperature, which was potentially attributed to a reduction in endogenous heat production. On the one hand, the HEHA diet enhanced feed energy by augmenting soybean oil content, which exhibited lower caloric gain than starch. On the other hand, digestible amino acid levels were increased by 15% without altering the amino acid balance pattern, thereby minimizing excess heat generated during crude protein digestion. Additionally, an increase in metabolizable energy and digestible amino acid levels in the diet improved the nutritional status of broilers under HS conditions, thereby alleviating the negative effects of HS on their production performance.

Blood biochemical indices reflect the physiological and health statuses of the body. In this study, HS significantly elevated serum ALT and sodium levels, while reducing serum albumin and potassium levels. However, the inclusion of the HEHA diet effectively ameliorated blood biochemical index disorders induced by HS. Given that the liver is the largest metabolic and detoxification organ in broilers, it is highly susceptible to stressors that disrupt hepatic and systemic metabolic homeostasis (Emami et al., 2021). Ding et al. demonstrated that HS result in decreased liver weight, increased ALT levels, and liver damage (Ding et al., 2023). Previous studies have indicated that under HS conditions, reduced feed intake further affects protein metabolism in broilers. Ahmed-Farid et al. (2021) reported a significant reduction in blood albumin levels during HS, which was consistent with our findings. Moreover, HS can induce changes in the acid-base balance leading to respiratory alkalosis (Wang et al., 2018). The response of poultry to stress primarily involves the activation of the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system. Precious reports have shown that broilers experiencing HS exhibit activation of their hypothalamic-pituitary-adrenal axis resulting in increased plasma corticosterone levels (Hanafi et al., 2022). Our study revealed a significant elevation in serum corticosterone levels due to HS, which was mitigated by the thermal challenge. In conclusion, feeding broilers a HEHA diet effectively alleviated symptoms associated with liver injury as well as disturbances related to protein metabolism and electrolyte imbalances induced by HS exposure.

Heat shock proteins (HSPs) are highly conserved proteins synthesized and expressed by animals in response to HS and play a crucial role in maintaining normal physiological activities. Numerous studies have demonstrated that HS induces the expression of HSPs in poultry and that their expression is a marker of HS. Madkour et al. (2022) reported that exposure to 32 ± 2 ℃ significantly upregulated the gene expression of HSP70 and HSP90. Similarly, Emami et al. (2021) found that high-temperature exposure (8 h/d at 35℃) increased intestinal HSP27 and HSP70 expression in Cobb broilers. In this study, the expression of HSP27 and HSP60 in the liver was significantly elevated under HS compared with that in the control group, confirming the successful establishment of the model. After feeding the HEHA diet, their expression significantly decreased, reflecting a change in the HS state induced by dietary intervention. Pedrycz and Brzeski (2006) suggested that L-arginine can reduce the expression of HSP70 in the kidney cells of fetal mice, while another study showed an increase in muscle exercise-induced expressions of HSP70 and HSP60 upon supplementation with L-leucine, L-isoleucine, L-arginine or L-glutamine (Moura et al., 2017). However, it remains unclear whether the HEHA diet directly regulates the hepatic expression of HSPs or indirectly influences their levels.

Oxidative stress is a direct manner of organ damage caused by HS and can also be an important sign of it. Numerous studies have shown that the exposure of animals to HS results in the disruption of their antioxidant systems. This is associated with a decrease in antioxidant enzyme activity, leading to excessive ROS production and damage to the body (Slimen et al., 2014). Our study showed that the HEHA diet significantly increased serum GSH-Px activity and intestinal mucosal SOD activity in heat-stressed yellow-feathered broilers. It also reduced liver MDA levels, improved HS-induced oxidative-reductive status disorder, and reduced liver damage. Methionine, the first limiting amino acid in poultry diets, is widely recognized as an endogenous antioxidant that effectively scavenges ROS and alleviates oxidative stress by promoting GSH synthesis (Upadhyayula et al., 2023). Wen et al. (2017) showed that a high-methionine diet significantly improved the total antioxidant capacity of chicken breast muscle, and de Freitas et al. (2021) reported that dietary methionine supplementation could effectively improve breast muscle antioxidant capacity and meat quality, thereby enhancing the total antioxidant capacity of broilers. In addition, arginine exhibits strong antioxidant properties. Ma et al. (2010) found that the supplementation of a finishing pig diet with 0.5 or 0.1 % Arg increased arginine concentration and decreased cortisol levels in serum, while enhancing antioxidative capacity and glutathione peroxidase activity in serum. Duan et al. (2015) also showed that dietary supplementation with 1.35% digestible arginine can improve T-AOC levels in the broiler breeder serum, egg yolk and serum, and liver and breast of one-day-old offspring. Furthermore, Wang et al. (2022) reported that a low-protein, high-amino acid diet increased serum peroxidase activity in heat-stressed chickens. Therefore, the increased antioxidant capacity induced by HEHA may be related to highs level of DAAs, especially methionine and arginine. Additionally, the indirect regulatory effects of HEHA should not be ignored. In summary, the protective effects of the HEHA diet against HS-induced oxidative damage may be mediated by synthetic amino acid supplementation.

CONCULSIONS

In this study, our results indicated that increasing dietary metabolizable energy level by 100 kcal/kg and digestible amino acid levels by 15%, while maintaining the crude protein level and digestible amino acid pattern via adding soybean oil and synthetic amino acid, not only increased feed intake, but also enhanced the antioxidant capacity of broilers, thereby improving HS-induced decline in productivity. Therefore, this study provides a theoretical basis for improving HS via precise nutritional regulation; however, further research is required to characterize the nutritional partitioning and requirements of chickens under HS conditions to establish efficient and economically feasible solutions to HS in the poultry industry.

Acknowledgments

ACKNOWLEDGMENTS

This work was supported by the Natural Science Foundation of Jiangsu Province of China (BK20220706), the construction of key industry colleges in the "14th Five-Year Plan" for universities in Nanjing City (Ning jiao High Education [2021] No.16), and Key Discipline of Animal Science in Jin Ling Institute of Technology.

Institutional Review Board Statement: The animal study protocol was approved by the Ethics Committee of Jinling Institute of Technology (20230015, 1st Jun).

Author Contributions: Huihua Mao: writing original draft and funding acquisition; Jinbi Zhang: review and editing and Investigation; Xu Zhang: Formal Analysis and Software; Shiyong Xu: Conceptualization and Data curation; Ling Zhang: Supervision and Project administration.

DISCLOSURES

Huihua Mao reports financial support was provided by Jinling Institute of Technology. Huihua Mao reports financial support was provided by Nanjing Government. Jinglong Chen reports financial support was provided by The Natural Science Foundation of Jiangsu Province. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2024.103790.

Appendix. Supplementary materials

mmc1.docx (111.2KB, docx)
mmc2.docx (17.3KB, docx)

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Associated Data

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Supplementary Materials

mmc1.docx (111.2KB, docx)
mmc2.docx (17.3KB, docx)

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