*Article* **Effects of Heat Stress on Gut-Microbial Metabolites, Gastrointestinal Peptides, Glycolipid Metabolism, and Performance of Broilers**

**Guangju Wang, Xiumei Li, Ying Zhou, Jinghai Feng and Minhong Zhang \***

State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 82101185163@caas.cn (G.W.); llxiumei93@163.com (X.L.); 15624955881@163.com (Y.Z.); fjh6289@126.com (J.F.)

**\*** Correspondence: zmh66@126.com; Tel.: +86-10-6289-5517

**Simple Summary:** In the summer, heat stress is a main factor that causes poor performance in broilers. Broilers are more susceptible to high temperature environments than mammals due to their lack of sweat glands and being covered in feathers. Heat stress can alter the regulation of glycolipid metabolism, which is manifested by unstable levels of blood glucose, insulin, total cholesterol, and triglyceride. Heat stress also affects the structure of gut microbes and gastrointestinal peptides. However, the relationship among microbiota, gastrointestinal peptides, glycolipid metabolism, and production performance under heat stress is still unclear. Moreover, exploring these mechanisms can help in the development of strategies that alleviate the negative effects of performance by heat stress. Our results suggest that the poor production performance of broilers under heat stress may be related to short chain fatty acids fermented by intestinal microbiota involved in regulating metabolic disorders.

**Abstract:** This paper investigated the effects of heat stress on gut-microbial metabolites, gastrointestinal peptides, glycolipid metabolism, and performance of broilers. Thus, 132 male Arbor Acres broilers, 28-days-old, were randomly distributed to undergo two treatments: thermoneutral control (TC, 21 ◦C) and high temperature (HT, 31 ◦C). The results showed that the average daily gain (ADG), average daily feed intake (ADFI), and gastric inhibitory polypeptide (GIP) concentration in the jejunum significantly decreased the core temperature, feed conversion ratio (FCR), and ghrelin of the hypothalamus, and cholecystokinin (CCK) in jejunum, and serum significantly increased in the HT group (*p* < 0.05). Exploration of the structure of cecal microbes was accomplished by sequencing 16S rRNA genes. The sequencing results showed that the proportion of *Christensenellaceae* and *Lachnospiraceae* decreased significantly whereas the proportion of *Peptococcaceae* increased at the family level (*p* < 0.05). *Ruminococcus* and *Clostridium* abundances significantly increased at the genus level. Furthermore, the content of acetate in the HT group significantly increased. Biochemical parameters showed that the blood glucose concentration of the HT group significantly decreased, and the TG (serum triglycerides), TC (total cholesterol), insulin concentration, and the insulin resistance index significantly increased. Nonesterified fatty acid (NEFA) in the HT group decreased significantly. In conclusion, the results of this paper suggest that the poor production performance of broilers under heat stress may be related to short-chain fatty acids (SCFAs) fermented by intestinal microbiota involved in regulating metabolic disorders.

**Keywords:** heat stress; microbial metabolites; gastrointestinal peptides; glycolipid metabolism

#### **1. Introduction**

Chicken meat is considered an important source of dietary protein worldwide. As per the Food and Agriculture Organization (FAO), total global chicken production was 118.0 million tons in 2019, accounting for a large proportion of the whole meat production.

**Citation:** Wang, G.; Li, X.; Zhou, Y.; Feng, J.; Zhang, M. Effects of Heat Stress on Gut-Microbial Metabolites, Gastrointestinal Peptides, Glycolipid Metabolism, and Performance of Broilers. *Animals* **2021**, *11*, 1286. https://doi.org/10.3390/ani11051286

Academic Editor: Natalie Morgan

Received: 31 March 2021 Accepted: 28 April 2021 Published: 30 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Broilers are severely affected by heat stress; it can cause slow growth rate, low feed intake, and decreased immunity, leading to economic losses [1,2]. Studies have shown that heat stress leads to glucolipid metabolism disorder [3–5]. Heat stress accelerates decomposition of glycogen and inhibits glycogen production [6]. When ambient temperature is higher than the tolerable limit of broilers, the energy produced cannot meet the needs, resulting in insulin resistance and glucolipid metabolism disorders [7]. Previous studies investigated the impacts of heat stress on broilers, which was found to cause blood glucose instability [3,8,9], decrease insulin sensitivity [10], serum cholesterol, and triglycerides [11], increase body fat in the abdomen, and decrease plasma non-esterified fatty acid (NEFA) concentrations [12]. Recently, several studies have shown that heat stress affects the metabolites and structures of the intestinal microflora [13,14] and gastrointestinal peptide [15] in broilers. However, to date, there are no specific studies on the effects of heat stress on broiler microflora and its metabolites, gastrointestinal peptide, glycolipid metabolism, and the association among the three.

Multiple studies were conducted on the associations among bacterial metabolites, gastrointestinal peptides, and glucose and lipid metabolism on metabolic disorders in mice and humans. Intestinal microbiota are involved in metabolic processes and energy homeostasis [16]. Previous studies have shown that heat stress can alter the gut microbiota of mice [17]. The Firmicutes to Bacteroidetes ratio decreased significantly under 30 ◦C [18]. Short-chain fatty acids (SCFAs) are metabolites formed by gut microbes from complex dietary carbohydrates. Researchers found that SCFAs stimulated mouse L cells to produce glucagon-like peptide 1 (GLP-1), peptide YY (PYY), and other intestinal anorexia hormones, reducing appetite [19]. GLP-1 and GIP are related to insulin secretion, and approximately 70% of β-cell insulin secretion is controlled by GIP and GLP-1 [20]. Insulin secretion disorders lead to metabolic disorders, such as diabetes and obesity [21]. As mentioned above, heat stress causes metabolic disorders in broilers. However, the association between metabolic disorder and microbiota under heat stress is still unclear in mice and humans.

Thus, the purpose of this study was to investigate the effects of heat stress on the microbiota and its metabolites, gastrointestinal peptides, and glycolipid metabolism in broilers, and explore the relationship among them to provide a scientific basis for reconstructing the intestinal flora, to alleviate the decline in production performance caused by heat stress.

#### **2. Materials and Methods**

#### *2.1. Animals and Experimental Design*

A total 132 male, one day old, Arbor Acres broilers were purchased from commercial hatcheries and housed in three-layer (8400 cm<sup>2</sup> per layer) metal cages at ambient temperatures that decreased with age. All birds had free access to feed and water (room temperature) ad libitum. The broilers were fed on crumble diets (Table 1). Then, birds were divided into a thermoneutral control group (TC, 21 ◦C) or a high temperature group (HT, 31 ◦C), with six biological replicates per group, 11 birds per replicate, at 21 days old. Birds were transferred to the environmental temperature control chamber, while maintaining a temperature of 21 ◦C, a humidity of 60%, for 7 days. The experiment started at 28 days old. Temperature control was adjusted by an artificial environmental control chamber developed by the Institute of Animal Science of the Chinese Academy of Agricultural Sciences (CAAS). There was no significant difference in the initial body weight of broilers in the two groups. The temperature of the two groups was constant and humidity remained at 60% until the end of the experiment, which lasted for 21 days. The lighting program was continuous throughout the experimental (fluorescent light, 40 W). To reduce stress, irrelevant personnel were prohibited from entering the artificial environment control chamber. This program was approved by the Experiment Animal Welfare and Ethical at the Institute of Animal Science of CAAS.


**Table 1.** Composition and nutrient levels of the basal diet.

(1) Premix provided the following per kg of the diet: VA 10,000 IU, VD3 3400 IU, VE 16 IU, VK3 2.0 mg, VB1 2.0 mg, VB2 6.4 mg, VB6 2.0 mg, VB12 0.012 mg, pantothenic acid calcium 10 mg, nicotinic acid 26 mg, folic acid 1 mg, biotin 0.1 mg, choline 500 mg, Zn(ZnSO4·7H2O) 40 mg, Fe(FeSO4·7H2O) 80 mg, Cu(CuSO4·5H2O) 8 mg, Mn(MnSO4·H2O) 80 mg, I(KI) 0.35 mg, Se(Na2SeO3) 0.15 mg. (2) Calculated values.

#### *2.2. Sample and Data Collection*

During the experiment, we used an 0.01 g sensitive electronic body weight scale (manufacturer: Mettler Toledo, PL2002) to record the initial feed weight, final feed weight, and body weight of the bird replicates, then calculated the average feed intake, average body weight, average daily weight gain, and feed efficiency for each replicate. This experiment used a rectal probe thermometer to measure the body core temperature of one bird replicate, which was randomly selected from each replicate, and measured four times a day during the experiment. At the end of the experiment, one bird, after 12 h fasting, was randomly selected from each replicate for insulin and blood glucose determination. Feed then continued for two hours, and one bird was randomly selected from each replicate to collect samples. The blood was immediately collected through the wing vein into heparinized tubes and centrifuged at 10,000× *g* for 4 min at 4 ◦C. Then the plasma was collected and stored at −20 ◦C until the analysis of the concentrations of PYY, ghrelin, CCK, GIP, and GLP-1. Immediately after the blood samples were obtained, the birds were humanely sacrificed, and the tissues of the cecum contents, intestinal mucosa, pancreas, and hypothalamus were collected. The samples were placed in a cryopreservation tube and stored in a −80 ◦C refrigerator.

#### *2.3. Determination of Gastrointestinal Peptides*

The concentrations of CCK, Ghrelin, GLP-1, GIP, and PYY in the intestinal mucosa and serum were determined by the enzyme-linked immunosorbent assay (ELISA). The intestinal mucosa needed to be grinded, weighed, diluted with PBS buffer at 1:9, and centrifuged for 20 min (2000–3000 rpm). The supernatant was then carefully taken for testing. The standard wells were set up, the samples added (diluent and enzyme label reagent in sequence), and were incubated for 60 min. The ELISA plate was washed with a washing solution for more than five times, and the color reagent and stop solution were added. Finally, an enzyme-labeled instrument was used to measure the absorbance of each well (OD value). The contents of each peptide in the sample were calculated through the standard curve.

#### *2.4. DNA Extraction and PCR Amplification*

Microbial community genomic DNA was extracted from cecal content samples using the E.Z.N.A.® soil DNA Kit (Omega BioTek, Norcross, GA, USA) according to the manufacturer's instructions. The DNA extract was checked on 1% agarose gel, and DNA concentration and purity were determined with NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The hypervariable region V3-V4 of the bacterial 16S rRNA gene was amplified with primer pairs 338F (50 -ACTCCTACGGGAGG-CAGCAG-3 0 ) and 806R (50 -GGACTACHVGGGTWTCTAAT -30 ) by an ABI GeneAmp® 9700 PCR thermocycler (Applied Biosystems, Carlsbad, CA, USA). The PCR amplification of 16S rRNA gene was performed as follows: initial denaturation at 95 ◦C for 3 min, followed by 27 cycles of denaturing at 95 ◦C for 30 s, annealing at 55 ◦C for 30 s and extension at 72 ◦C for 45 s, and single extension at 72 ◦C for 10 min, and end at 10 ◦C. The PCR mixtures contain 5 × TransStart FastPfu buffer 4 µL, 2.5 mM dNTPs 2 µL, forward primer (5 µM) 0.8 µL, reverse primer (5 µM) 0.8 µL, TransStart FastPfu DNA Polymerase 0.4 µL, template DNA 10 ng, and finally ddH2O up to 20 µL. PCR reactions were performed in triplicate. The PCR product was extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA), according to manufacturer's instructions, and quantified using Quantus™ Fluorometer (Promega, Madison, WI, USA).

#### *2.5. Illumina MiSeq Sequencing*

Purified amplicons were pooled in equimolar and paired-end sequences on an Illumina MiSeq PE300 platform/NovaSeq PE250 platform (Illumina, San Diego, CA, USA), according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).

#### *2.6. Processing of Sequencing Data*

The raw 16S rRNA gene sequencing reads were demultiplexed, quality-filtered by fastp version 0.20.0 [22] and merged by FLASH version 1.2.7 [23] with the following criteria: (i) the 300 bp reads were truncated at any site receiving an average quality score of <20 over a 50 bp sliding window, and the truncated reads shorter than 50 bp were discarded. Reads containing ambiguous characters were also discarded. (ii) Only overlapping sequences longer than 10 bp were assembled according to their overlapped sequences. The maximum mismatch ratio of overlap region was 0.2. Reads that could not be assembled were discarded. (iii) Samples were distinguished according to the barcode and primers, and the sequence direction was adjusted, exact barcode matched, and two nucleotides mismatched in the primer matching. Operational taxonomic units (OTUs) with 97% similarity cutoff [24,25] were clustered using UPARSE version 7.1 [24], and chimeric sequences were identified and removed. The taxonomy of each OTU representative sequence was analyzed by RDP Classifier version 2.2 [26] against the 16S rRNA database using confidence threshold of 0.7.

#### *2.7. Determination of SCFAs*

We accurately weighed 1 g of cecal content, added 5 mL of ultra-pure water, shook, and mixed for 30 min, overnight at 4 ◦C, then centrifuged at 10,000 rpm for 10 min, and transferred the supernatant to a 10 mL cuvette. We added 4 mL of ultra-pure water to the precipitation, shook and mixed for 30 min, then centrifuged at 10,000 rpm to transfer the supernatant into a 10 mL cuvette for constant volume. We transferred the liquid in the colorimetric tube to a 10 mL centrifuge tube, centrifuged at 12,000 rpm for 15 min, and then transferred the supernatant to a 2 mL centrifuge tube, according to V:V = 9:1 (900 µL supernatant + 100 µL 25% metaphosphoric acid), mixed well, and let it stand at room temperature for 3–4 h centrifugation, 45 um microporous membrane (nylon series) filtration. We added the machine bottle (more than 600 µL) to be tested. Chromatographic conditions: db-ffap column, 30 m \* 250 µm \* 0.25 µm; carrier gas: high purity nitrogen (99.999%), flow rate: 0.8 mL/min; auxiliary gas: high purity hydrogen (99.999%), detector FID temperature: 280 ◦C, injection port temperature: 250 ◦C, split ratio: 50:1, injection

volume: 1 µL; temperature programming: initial temperature: 60 ◦C, the temperature rose to 220 ◦C at the rate of 20 ◦C/min, and held for 1 min.

#### *2.8. Determination of Serum Biochemical Parameters*

The concentrations of blood glucose, serum insulin, triglycerides, total cholesterol, and NEFA were measured using the kits provided by Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). The insulin resistance index was calculated using a formula as previously described: insulin resistance index = insulin/(22.5e-lnglucose) [27].

#### *2.9. Statistical Analysis*

All statistical analyses were conducted using GraphPad Prism 8 software (GraphPad Software, Inc. La Jolla, CA, USA). An independent sample t-test (unpaired Student's *t* test and Mann–Whitney test) was used for the comparison of the two treatments. Replicate served as the experimental unit. The confidence interval was 95%, and *p* < 0.05 indicated a significant difference in the treatment effect; values were expressed as the mean ± SEM.

#### **3. Results**

#### *3.1. Effect of High Temperature on Performance*

The effect of heat stress on performance is shown in Table 2. Compared with the TC group, ADG and ADFI in the HT group decreased significantly (*p* < 0.05). Compared with the TC group, the core temperature of birds and FCR in the HT group significantly increased (*p* < 0.05).



Values are means ± SEM. TC, thermoneutral control group; HT, high temperature group; IABW, initial average body weight (29 d); FABW, final average body weight (43 d); ADG, average daily gain; ADFI, average daily feed intake; FCR, feed conversion rate; CT, core temperature. a,b Means within the same line with different superscript differ significantly.(*p* < 0.05).

#### *3.2. Effects of High Temperature on Gastrointestinal Peptide*

The effects of heat stress on the neuropeptides of gut and hypothalamus concentrations in six-week-old chickens were examined and the results are presented in Figures 1–3. Compared with the TC group, GIP concentration in the jejunum of the HT group significantly decreased (*p* < 0.05). The concentration of CCK in the jejunum and serum and ghrelin of the hypothalamus in the HT group was significantly higher than that in the TC group (*p* < 0.05). The GLP-1 in the ileum, PYY in the pancreas, and ghrelin in the jejunum in the HT group were not significantly different from that of the TC group.

in the HT group were not significantly different from that of the TC group.

in the HT group were not significantly different from that of the TC group.

*Animals* **2021**, *11*, x FOR PEER REVIEW 6 of 14

significantly decreased (*p* < 0.05). The concentration of CCK in the jejunum and serum and ghrelin of the hypothalamus in the HT group was significantly higher than that in the TC group (*p* < 0.05). The GLP-1 in the ileum, PYY in the pancreas, and ghrelin in the jejunum

significantly decreased (*p* < 0.05). The concentration of CCK in the jejunum and serum and ghrelin of the hypothalamus in the HT group was significantly higher than that in the TC group (*p* < 0.05). The GLP-1 in the ileum, PYY in the pancreas, and ghrelin in the jejunum

**Figure 1.** Effects of high temperatures on CCK, ghrelin, and PYY concentrations in the hypothalamus. (**A**), cholecystokinin; (**B**), ghrelin; (**C**), peptide YY. a,b, Means with different letters within columns indicates significant differences (*p* < 0.05). **Figure 1.** Effects of high temperatures on CCK, ghrelin, and PYY concentrations in the hypothalamus. (**A**), cholecystokinin; (**B**), ghrelin; (**C**), peptide YY. a,b, Means with different letters within columns indicates significant differences (*p* < 0.05). **Figure 1.** Effects of high temperatures on CCK, ghrelin, and PYY concentrations in the hypothalamus. (**A**), cholecystokinin; (**B**), ghrelin; (**C**), peptide YY. a,b, Means with different letters within columns indicates significant differences (*p* < 0.05).

**Figure 2.** Effects of high temperature on concentrations of CCK, ghrelin, GIP in jejunum, GLP-1 in ileum and PYY in pancreas. (**A**), cholecystokinin; (**B**), ghrelin; (**C**), glucagon-like peptide-1; (**D**), glucose-dependent insulinotropic polypeptide; (**E**), peptide YY. a,b, Means with different letters within columns indicates significant differences (*p* < 0.05). **Figure 2.** Effects of high temperature on concentrations of CCK, ghrelin, GIP in jejunum, GLP-1 in ileum and PYY in pancreas. (**A**), cholecystokinin; (**B**), ghrelin; (**C**), glucagon-like peptide-1; (**D**), glucose-dependent insulinotropic polypeptide; (**E**), peptide YY. a,b, Means with different letters within columns indicates significant differences (*p* < 0.05). **Figure 2.** Effects of high temperature on concentrations of CCK, ghrelin, GIP in jejunum, GLP-1 in ileum and PYY in pancreas. (**A**), cholecystokinin; (**B**), ghrelin; (**C**), glucagon-like peptide-1; (**D**), glucose-dependent insulinotropic polypeptide; (**E**), peptide YY. a,b, Means with different letters within columns indicates significant differences (*p* < 0.05).

**Figure 3.** Effects of high temperature on concentration of CCK, ghrelin, GLP-1, GIP and PYY in serum. (**A**), cholecystokinin; (**B**), ghrelin; (**C**), glucagon-like peptide-1; (**D**), glucose-dependent insulinotropic polypeptide; (**E**), peptide YY. a,b Means with different letters within columns indicates significant differences (*p* < 0.05). **Figure 3.** Effects of high temperature on concentration of CCK, ghrelin, GLP-1, GIP and PYY in serum. (**A**), cholecystokinin; (**B**), ghrelin; (**C**), glucagon-like peptide-1; (**D**), glucose-dependent insulinotropic polypeptide; (**E**), peptide YY. a,b Means with different letters within columns indicates significant differences (*p* < 0.05).

#### *3.3. Effects of High Temperature on Cecal Microbial Composition 3.3. Effects of High Temperature on Cecal Microbial Composition*

mal environment significantly increased the proportion of them.

**Table 3.** Effects of the thermal environment on cecum digesta microbiota composition.

**Level Species Name Treatments SEM** *<sup>p</sup>* **Value TC HT** 

phylum *Bacteroides* (%) 5.69 5.18 0.74 >0.05

family *Ruminococcaceae* (%) 51.32 51.14 1.25 >0.05

*Firmicutes* (%) 89.33 87.49 2.18 >0.05

*Actinomycetes* (%) 3.78 6.50 1.12 >0.05

*Lachnospiraceae* (%) 18.68 a 10.15 b 0.80 <0.05 *Christensenellaceae* (%) 1.20 a 0.51 b 0.07 <0.05

The intestinal microbial community of broilers is mainly concentrated in the cecum; it is the main place where microorganisms participate in the regulation of body metabolism. Therefore, we analyzed the composition of the caecal flora of two groups of broilers at the levels of phylum, family*,* and genus. The compositions of cecal microbiota at phylum, family*,* and genus levels are provided in Table 3. At the phylum level, the two groups of flora were mainly composed of *Firmicutes*, *Bacteroides*, and *Actinomycetes*; however, there was no significant difference in abundance. At the family level, the dominant bacteria were mainly *Ruminococcaceae*; the proportion of *Christensenellaceae* and *Lachnospiraceae* decreased significantly, and increased the proportion of *Peptostreptococcaceae* (*p* < 0.05). It may show that heat stress significantly affects the dominant intestinal flora at the family level. At the genus level, the dominant bacteria were mainly *Faecalibacterium*, *Romboutsia*. *Ruminococcus* and *Clostridium* abundances significantly increased, indicating that the ther-The intestinal microbial community of broilers is mainly concentrated in the cecum; it is the main place where microorganisms participate in the regulation of body metabolism. Therefore, we analyzed the composition of the caecal flora of two groups of broilers at the levels of phylum, family, and genus. The compositions of cecal microbiota at phylum, family, and genus levels are provided in Table 3. At the phylum level, the two groups of flora were mainly composed of *Firmicutes*, *Bacteroides*, and *Actinomycetes*; however, there was no significant difference in abundance. At the family level, the dominant bacteria were mainly *Ruminococcaceae*; the proportion of *Christensenellaceae* and *Lachnospiraceae* decreased significantly, and increased the proportion of *Peptostreptococcaceae* (*p* < 0.05). It may show that heat stress significantly affects the dominant intestinal flora at the family level. At the genus level, the dominant bacteria were mainly *Faecalibacterium*, *Romboutsia*. *Ruminococcus* and *Clostridium* abundances significantly increased, indicating that the thermal environment significantly increased the proportion of them.


**Table 3.** Effects of the thermal environment on cecum digesta microbiota composition.

Values are means <sup>±</sup> SEM. TC, thermoneutral control group; HT, high temperature group. a,b Means within the same line with different superscript differ significantly (*p* < 0.05).

#### *3.4. Effects of High Temperature on SCFAs Concentration*

The concentration of short chain fatty acids in cecal contents of the two treatment groups is shown in Table 4. Compared with the TC group, the content of acetate in the HT group significantly increased, but there was no significant difference in propionic acids and butyric acids.


**Table 4.** Effects of thermal environment on the content of SCFAs in the cecum.

Values are means ± SEM. SCFAs, short chain fatty acids; TC, thermoneutral control group; HT, high temperature group. a,b Means within the same line with different superscript differ significantly. (*p* < 0.05).

#### *3.5. Effects of High Temperatures on Serum Parameters*

The blood glucose, insulin, insulin resistance index, TG, TC, and NEFA concentrations are shown in Figure 4. Compared with the TC group, the blood glucose and NEFA concentrations in the HT group significantly reduced, the insulin, TC, and TG concentrations significantly increased. Compared with the TC group, the insulin resistance index of broilers under heat stress increased significantly, resulting in severe insulin resistance.

**Figure 4.** Effect of high temperature on blood glucose, insulin, insulin resistance index, TC, TG, and NEFA in serum. (**A**), blood glucose; (**B**), insulin; (**C**), insulin resistance index; (**D**), total cholesterol; (**E**), triglyceride; (**F**), non-esterified fatty acid. a,b means with different letters within columns indicates significant differences (*p* < 0.05). **Figure 4.** Effect of high temperature on blood glucose, insulin, insulin resistance index, TC, TG, and NEFA in serum. (**A**), blood glucose; (**B**), insulin; (**C**), insulin resistance index; (**D**), total cholesterol; (**E**), triglyceride; (**F**), non-esterified fatty acid. a,b means with different letters within columns indicates significant differences (*p* < 0.05).

#### **4. Discussion 4. Discussion**

Thermal environment severely impairs the performance [28,29], changes the composition of gut microbiota [30], and affects glycolipid metabolism [31] of broilers. However, previous studies have not been able to account for the associations among them. The present study clearly established a significant outcome of a thermal environment on performance, gut microbiota, gastrointestinal peptides, and glycolipid metabolism in broilers, Thermal environment severely impairs the performance [28,29], changes the composition of gut microbiota [30], and affects glycolipid metabolism [31] of broilers. However, previous studies have not been able to account for the associations among them. The present study clearly established a significant outcome of a thermal environment on performance, gut microbiota, gastrointestinal peptides, and glycolipid metabolism in broilers, and developed a preliminary understanding of the relationship among them.

and developed a preliminary understanding of the relationship among them. Significant elevations were detected in the core temperatures of the birds, meaning that heat stress was produced. Previous literature demonstrated that heat stress could negatively impact ADFI, ADG, and FCR in broilers [2,29,32–34]. The present study showed that ADFI, ADG, and FCR were seriously affected by high temperatures of 31 °C compared with the 21 °C group, which agreed with the results of previous studies [2,35]. These results are widely accepted, they imply that heat exposure will directly impair productive performance of birds. In general, the reason for the decline in productive performance is largely due to the lower feed intake; birds reduce feed intake to minimize excess metabolic energy production to keep themselves cool [36]. Another important reason for the weight gain reduction observed in birds experiencing heat stress would be metabolism disorder. Previous studies have shown that thermal environment can disrupt metabolic homeostasis, accelerating protein catabolism [37], increasing abdominal fat deposition [38], changing blood glucose levels, decreasing insulin sensitivity, and causing a negative energy balance [39]. Birds suffering from heat stress may choose to use more energy to resist the damage caused by high temperatures rather than to grow or reproduce. Significant elevations were detected in the core temperatures of the birds, meaning that heat stress was produced. Previous literature demonstrated that heat stress could negatively impact ADFI, ADG, and FCR in broilers [2,29,32–34]. The present study showed that ADFI, ADG, and FCR were seriously affected by high temperatures of 31 ◦C compared with the 21 ◦C group, which agreed with the results of previous studies [2,35]. These results are widely accepted, they imply that heat exposure will directly impair productive performance of birds. In general, the reason for the decline in productive performance is largely due to the lower feed intake; birds reduce feed intake to minimize excess metabolic energy production to keep themselves cool [36]. Another important reason for the weight gain reduction observed in birds experiencing heat stress would be metabolism disorder. Previous studies have shown that thermal environment can disrupt metabolic homeostasis, accelerating protein catabolism [37], increasing abdominal fat deposition [38], changing blood glucose levels, decreasing insulin sensitivity, and causing a negative energy balance [39]. Birds suffering from heat stress may choose to use more energy to resist the damage caused by high temperatures rather than to grow or reproduce. Therefore, the underlying cause of poultry production performance degradation may be metabolic disorder. The current study

observed that heat stress led to elevated levels of insulin, serum triglycerides, and total cholesterol, and a reduction in serum blood glucose and NEFA. Changes in blood glucose in broilers exposed to high temperatures showed mixed results, as there was evidence for it to increase [3], decrease, or remain unchanged [9]. Numerous factors influence blood glucose and the reasons for the differences include different physiological statuses and experimental designs. The results of elevated levels of insulin [40], serum triglycerides, total cholesterol, and reduced NEFA [41] in this study are consistent with previously reported studies. It is possible that insulin levels were elevated due to compensatory secretions to maintain glucose tolerance. The increased basal insulin levels may explain the lack of an increase in basal NEFA levels because insulin is a potent antilipolytic hormone. We also calculated that the insulin resistance index increased significantly, meaning insulin resistance occurs under heat stress. In summary, these observations imply that heat stress alters carbohydrate and lipid metabolism, leading to metabolic disorder.

Gut microbes have previously been susceptible to heat stress in both mice and livestock. The present study found that heat stress changed the colony composition at the family level, reduced the proportions of *Christensenellaceae* and *Lachnospiraceae*, and increased the proportion of *Peptococcaceae*. It was reported that *Christensenellaceae* is related to healthy glucose metabolism and reduces the risk of obesity [42]. A previous study reported gut dysbiosis in mice that underwent chronic water avoidance stress; in particular, the abundance of *Lachnospiraceae* declined [43]. Similar results were obtained in this paper. The increase of *Peptococcaceae* was mainly related to histopathological infection, indicating that heat stress increased the threat factors of gut health. At the genus level, heat stress increased the proportion of *Ruminococcus* and *Clostridium*. In addition, our results showed that heat stress increased the content of acetate in cecal contents. Since *Ruminococcus* and *Clostridium* are the main acetate-producing microorganisms, the increase of acetate content in cecal contents may be associated to the increase of *Ruminococcus* and *Clostridium*.

Gastrointestinal peptides are mainly secreted by intestinal endocrine cells, including ghrelin, CCK, GIP, PYY, GLP-1, which are directly involved in the regulation of gastrointestinal peristalsis, sensation, and secretion. They have the dual functions of promoting the secretion of neurotransmitters and hormones, and regulating feeding behavior, nutrient absorption, energy metabolism, intestinal peristalsis, and emptying [44]. The results we obtained show that heat stress significantly increased CCK concentration in the jejunum and circulation—similar to the results obtained by a previous study [45]. CCK proved to be an anorexic hormone in various poultry studies, which can gather short-term post-eating satiety signals and inhibit broiler feeding [46]. It was reported that exogenous injection of CCK could significantly reduce the feed intake of poultry [47]. Therefore, the significant decrease in feed intake of broilers in the HT group may be due to the large amount of CCK secreted by the intestines. Several studies have shown that a certain dose of ghrelin is injected into the cerebral ventricle of chicks, and the results are found to strongly inhibit the feed intake of chicks [48,49]. In the HT group, a significant increase in hypothalamic ghrelin levels was observed, which is consistent with the results of a previous study [36]. There were no significant changes in other peptides except for ghrelin in the hypothalamus; no significant changes in PYY, ghrelin, and GLP-1 were observed in the intestinal tract and circulation. This is consistent with previous research [50]. It was found that heat stress significantly reduced the plasma GIP concentration of poultry and pigs [51]. The present study found that heat stress significantly reduced the GIP concentration in the jejunum of broilers, and the serum GIP concentration had a decreasing trend. This is supported by previous results. The decreased GIP concentration may be caused by the decreased number of intestinal K cells or the decreased GIP expression in K cells. Another possible explanation for this is that blood glucose was significantly reduced because GIP secretion is glucose-dependent [52].

The gut microbiota participating in host metabolism is frequently reported on [53–55]. According to various studies, such a process is associated with bacterial metabolites, such as SCFAs [56,57]. In the current study, the proportion of acetate-producing bacteria, such

as *Ruminococcus* and *Clostridium,* increased, leading to an increase in acetate production. Studies have found that oral prebiotics may affect the secretion of related gastrointestinal hormones, such as GLP-1 and ghrelin, through the short-chain fatty acids produced by microbiota fermentation [55]. SCFAs have also been shown to cross the blood–brain barrier, directly into the brain, to control feed intake [58]. Furthermore, it was reported that SCFAs can stimulate mouse intestinal L cells to secrete PYY, CCK, and ghrelin, by activating G protein coupled receptors, such as Gpr41 [59]. A recent study suggested that SCFAs inhibit the secretion of GIP by activating the FFAR3 receptor of K cells [60]. This evidence implies that the increased CCK and decreased GIP relate to the SCFAs in the current study. Therefore, it is not surprising that the increase in CCK secretion by L cells and the decrease in GIP secretion by K cells may be due to the increased acetate content in the cecal contents.

#### **5. Conclusions**

This paper argues that the poor production performance of broilers under heat stress may be related to SCFAs fermented by gut microbiota involvement in regulating metabolic disorders. Our data will provide valuable insight for future studies on humans in tropical environments who suffer from metabolic disorders, such as diabetes and obesity.

**Author Contributions:** Conceptualization, G.W. and M.Z.; methodology, G.W., X.L., and M.Z.; software, G.W.; validation, G.W., X.L., and J.F.; formal analysis, G.W., Y.Z., and X.L.; investigation, G.W., Y.Z., and X.L.; resources, G.W.; data curation, G.W., X.L.; writing—original draft preparation, G.W.; writing—review and editing, G.W., X.L., J.F., and M.Z.; visualization, G.W.; supervision, M.Z.; project administration, J.F.; funding acquisition, M.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the National Key Research and Development Program of China (2016YFD0500509). This research was also supported by the Science and Technology Innovation Project of the Chinese Academy of Agricultural Sciences (ASTIP-IAS09).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Ethics Committee of Experiment Animal Welfare and Ethical at the Institute of Animal Science of CAAS (NO:IAS2021-75; 8 April 2021).

**Data Availability Statement:** Not Applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Mohammed M. Qaid 1,2,\* and Maged A. Al-Garadi 1,2,\***


Tel.: +966-509-844-024 (M.M.Q.); +966-533429411 (M.A.A.-G.)

**Simple Summary:** Broilers must be reared under thermoneutral conditions and comfort zones; therefore, any deviation from the neutral thermal zone causes stress and a consequent disturbance in the turnover or the metabolism of nutrients. This review addressed the biosynthesis of amino acids and/or protein metabolism under normal conditions and heat stress conditions. In addition, hormonal responses to stress and the role of endocrine hormones in protein metabolism have been reviewed. In addition, the aim of this review is to summarize the studies related to the assessment of heat stress, the physiological stress regulation mechanism, and the nutritional strategies for the prevention of heat stress in poultry.

**Abstract:** This review examined the influence of environmental heat stress, a concern facing modern broiler producers, on protein metabolism and broiler performance, as well as the physiological mechanisms that activate and control or minimize the detrimental impacts of stress. In addition, available scientific papers that focused on amino acids (AA) digestibility under stress conditions were analyzed. Furthermore, AA supplementation, a good strategy to enhance broiler thermotolerance, amelioration, or stress control, by keeping stress at optimal levels rather than its elimination, plays an important role in the success of poultry breeding. Poultry maintain homeothermy, and their response to heat stress is mainly due to elevated ambient temperature and the failure of effective heat loss, which causes a considerable negative economic impact on the poultry industry worldwide. Reduced feed intake, typically observed during heat stress, was the primary driver for meat production loss. However, accumulating evidence indicates that heat stress influences poultry metabolism and endocrine profiles independently of reduced feed intake. In conclusion, high ambient temperatures significantly reduced dietary AA intake, which in turn reduced protein deposition and growth in broilers. Further studies are required to determine the quantity of the AA needed in warm and hot climates and to introduce genetic tools for animal breeding associated with the heat stress in chickens.

**Keywords:** amino acids; broiler; heat stress; heat tolerance; protein metabolism

#### **1. Introduction**

Poultry meat is an essential source of dietary protein, and the industry has developed high grade poultry because of improved farming techniques, automation equipment, and comprehensive and balanced feeding, and other new technologies [1]. In the past decade, broiler production has increased rapidly in tropical and subtropical areas and is expected to sustain robust growth in the future. However, according to a review [2], high environmental temperatures are one of the greatest challenges of poultry and live stock performance, leading to the decline in production efficiency in these countries. Furthermore, modern commercial broilers are more sensitive to heat stress than previous generations due to their higher performance, growth rate, and feed conversion efficiency [3]. Indeed, commercial poultry strains can reach a high production yield, but their body metabolism,

**Citation:** Qaid, M.M.; Al-Garadi, M.A. Protein and Amino Acid Metabolism in Poultry during and after Heat Stress: A Review. *Animals* **2021**, *11*, 1167. https://doi.org/ 10.3390/ani11041167

Academic Editor: Natalie Morgan

Received: 11 March 2021 Accepted: 12 April 2021 Published: 19 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

being comparatively accelerated, has poor thermoregulation and is poorly adapted to the living environments compared to native backyard chickens [1]. The higher growth rate of broilers has several consequences, such as higher feed consumption and metabolism and elevated production of internal heat. To reduce the heat load and avoid heat-induced mortality in birds, heat loss and/or lowering heat production could be achieved through reduced feed consumption, resulting in a depressed growth rate and lower final body weight, reduced breast meat weight, and lower egg quality, size, and rate in hens. In addition, the decreased feed consumption could result in nutrient deficiency, such as proteins, AA, and energy [4–6].

Among the environmental factors, heat stress negatively affects feed consumption, body weight gain [7], and carcass characteristics [2]. Additionally, HS may cause oxidative stress in the body and develop many free radicals, stimulating membrane lipid peroxidation, and hence the degradation of DNA and protein membranes [8,9]. In poultry, mitochondrial superoxide production as oxidative stress was observed on exposure to HS [9]. Besides the role of AA as protein and peptide components, some AA (e.g., glutamine, cysteine, leucine, arginine, tryptophan, and proline) are involved in the regulation of metabolic pathways, thereby affecting growth, protein accumulation, maintenance, immunity, and health [10].

The ambient temperature impacts the protein turnover rate in broiler skeletal muscle [11]. Not only protein anabolism, but also protein catabolism are energetically expensive. The growth depression of heat-exposed chickens showed lower protein gain and retention. High protein sources have beneficial effects through the improved growth of heat-stressed broilers [12]. They indicated that protein synthesis in broilers was more affected than protein breakdown with HS, resulting in reduced protein deposition in the skeletal muscles. A review [11] showed that the high ambient temperatures and dietary protein consumption affected muscle protein turnover in broilers. In broiler chickens, HS alters muscle protein and AA metabolism and accelerates liver gluconeogenesis for energy supply [13]. Dietary approaches, such as modifications of energy and protein content of the diet, are the most practical and preferred ways to alleviate heat distress in poultry and enhance broiler performance under these conditions [14,15]. Improving the overall equilibrium of the dietary AA was more effective than increasing total protein consumption [16].

Limited studies are available that address the effects of HS on protein metabolism in broilers. Therefore, it is necessary to detect mechanisms or methods that allow producers to effectively reduce the detrimental influences of environmental HS on broilers, in particular on protein metabolism via protein accretion or degradation of muscle. The present review will focus on the effects of environmental heat stress on protein metabolism and broiler performance, as well as the physiological mechanisms and nutritional strategies that mitigate the negative effects of heat stress, particularly the role of AA in reducing HS in stressed broilers.

#### **2. Amino Acid and/or Protein Metabolism**

AA are required for most biological activities. The AA transport into the apical membrane and out of the lateral basal membrane of enterocytes. Their transport relies on sodiumdependent symporters, proton-motive forces, antiporters, and the gradient of other AA. The metabolic fate of absorbed AA mainly depends on nutrient availability [17]. AA moving through catabolic pathways ultimately serve as precursors of gluconeogenesis [13] and contribute to 40% of the total AA loss in fasted animals. Proteins are synthesized from free AA, which become available either from dietary (the end product of digestion) or from metabolic origins as the result of AA biosynthesis within the body. These AA, either circulating via the blood or accumulating within tissues, form pools. The AA concentrations within these pools are based on the equilibrium between gains and losses [18]. Dietary AA are used to build protein for muscle growth, membrane glycoproteins, and enzymes involved in numerous biochemical processes, and act as precursors for the synthesis of DNA/RNA [10]. The AA catabolize in the liver to integrate into protein, which supplies

peripheral tissues [13]. Protein turnover refers to the equilibrium between the anabolism and catabolism of protein. The metabolic utilization of AA is equally diverse. Anabolism or protein synthesis facilitates dietary AA to fuse into proteins, or biosynthesize in the body tissues. Catabolism occurs through the breakdown of proteins to build amino groups that produce urea or further protein. In addition, to produces carbon skeleton molecules for glucose production (glucogenesis) or fatty acids (lipogenesis), carbon dioxide, and the release of energy. The role of endocrine hormones in protein metabolism is shown in Table 1.

**Table 1.** Role of endocrine hormones in protein metabolism and summary of potential methods for assessing stress.


\* abbreviations: ACTH: adrenocorticotropin, CS: corticosterone, CRH: Corticotropin-releasing hormone, T3: triiodothyronine, and T4: thyroxine, adapted from [19,20].

> Nitrogen excretion can be used to determine protein balance through the measurement of nitrogen losses during protein catabolism or recycling [18]. Endogenous or dietary proteins hydrolyze the previous absorption. The tissue proteins of birds are renewed frequently with the liberation of endogenous AA. Furthermore, there are many metabolic reactions converting metabolites into nonessential AA [18].

> Recently, AA are applied not only as signaling molecules of the cell and the protein phosphorylation cascade, but also as regulators of gene expression. Moreover, AA are fundamental precursors for hormone synthesis and other nitrogenous elements that have considerable biological significance. Normal levels of AA and their metabolites, such as glutathione, polyamines, taurine, nitric oxide, serotonin, and thyroid hormones are needed for their functions. Nevertheless, elevated levels of AA and their metabolites, such as ammonia, asymmetric dimethylarginine, and homocysteine are considered pathogenic for the body, and lead to oxidative stress, and cause diseases and disorders of the cardiovascular and neurological systems. Therefore, an ideal balance of AA in the feed and bloodstream is crucial for the homeostasis of the body. AA not only have a role as the building blocks of polypeptides and proteins, but also regulate the fundamental metabolic routes that are essential for growth, maintenance, immunity, and reproduction. These functional AA include glutamine, leucine, proline, arginine, cysteine, and tryptophan [10].

#### **3. Biosynthesis of Amino Acids**

Birds and all vertebrates, dissimilar to plants and many bacteria, are unable to synthesize some AA, so these are termed essential AA and are required for tissue renewal through protein synthesis. Thus, essential AA must be supplemented in the diet. For protein synthesis, all AA are similarly essential owing to the absence of any AA interfere with the anabolic processes. However, nutritionally AA are classified into three groups [18]. Essential AA must be provided by feed and may be classified into two groups. One group is strictly essential because they cannot be synthesized, even from AA metabolic intermediates, such as glucogenic that yield intermediates of glycolysis pathway or ketogenic that yield intermediates of acetyl-CoA or acetoacetate. The transaminases of that group are absent, for instance methionine, lysine, tryptophan, threonine, and phenylalanine. The other group may be insufficiently synthesized from their precursors, for example,

glycine, leucine, isoleucine, valine, arginine, histidine, and proline. Semiessential AA may be synthesized from essential AA. Tyrosine and cysteine originate from phenylalanine and methionine, respectively. Cysteine is synthesized from serine (nonessential AA) and methionine (essential AA). Nonessential AA are easily synthesized from intermediary metabolites or similarly nonessential AA: alanine, serine, aspartic and glutamic acids in the former group; and asparagine and glutamine in the latter group [18].

#### **4. Effect of HS on Protein Metabolism or Turnover**

Heat as a stress factor affects protein metabolism during the postabsorptive stage as muscle breakdown and changes in the quantity of lean tissue may occur in different species [21]. In the muscle protein, the RNA/DNA synthesis capacity is reduced by HS [22]. During environmental hyperthermia, muscle tissue catabolism is increased due to increased plasma markers during muscle breakdown. In lactating cows, HS increases plasma urea nitrogen concentration [23]; however, whether this elevation stems from reduced plasma volume, increased protein degradation, or other reasons remains unknown. Thus, blood urea nitrogen (BUN) is used as an indicator of muscle catabolism or breakdown, because tissue degradation results in an increase in BUN [21]. A review [24] reviewed that uric acid excretion is increased in stressed poultry owing to corticosterone -driven gluconeogenesis. Other indicators or measures of protein breakdown (muscle catabolism) include increased plasma creatinine, Nt-methyl histidine, creatine, and creatine kinase (CK) concentrations. An increase in these markers has been detected during heat load in chickens, turkeys [25], cows [26], pigs [27], and humans [28]. The increased level of these parameters indicates enhanced muscle protein catabolism.

Insulin stimulates protein synthesis or accretion. However, during heat-load, increased muscle protein degradation causes the liver to utilize available AA as gluconeogenic substrates from the carbon skeleton through the gluconeogenesis pathway [19]. Under stress conditions, the corticoid hormones (CS, ACTH) suppress the synthesis of tissue proteins and boost proteolysis, as catabolic action is elevated in the blood stream. Glycerol produced from lipid degradation is one of the gluconeogenic substrates and accounts for 20% of the glucose production. Therefore, the other products of protein catabolism are used as substrates for glucose production. First, not only the heart and lung tissue proteins are enhanced by the catabolic transformations of protein composition, but also all tissues except for the nervous system. The muscle tissues (muscle protein) that have the highest body nitrogen content are more sensitive to corticosterone administration, resulting in decreased muscle mass and growth retardation in stressed chickens [29]. A study [30] found that feed deprivation reduced protein synthesis in the liver of starved chickens, as well as plasma albumin and total protein levels. A study [31] indicated that the depletion of plasma free AA, elevated blood uric acid concentration, reduced protein synthesis possibly reflected reduced N retention and more active protein catabolism in broilers challenged by very shortterm high temperatures. However, chronic exposure to HS decreased protein digestion, decreased feed digestibility, reduced protein breakdown, reduced protein synthesis in the muscles, and decreased most plasma free AA (especially branched-chain AA and sulfur) [32], whereas the serum levels of glutamic acid, aspartic acid, and phenylalanine increased [33]. It was found that protein synthesis and N deposition were depressed and proteolysis increased during HS [34]. AA catabolism was enhanced under chronic HS [13]; thus, all plasma free AA concentrations decreased, except for glutamic acid, aspartic acid, and phenylalanine. Based on these studies, protein breakdown may increase rapidly in very short-term HS, resulting in a decrease in protein synthesis and an increase in plasma uric acid levels, but then decrease protein breakdown and maintain uric acid levels around normal concentrations as the thermal stress continues.

Two previous studies, one on chickens and the other on turkeys, found that heat stress reduced uric acid levels in the blood, which could be attributed to a lower level of total protein as a result of hypotonic overhydration [25]. Although no sodium concentration was determined in these studies, water intoxication due to excessive water intake causes

overhydration when the amount of water intake exceeds that of water excretion in the kidney. As a result, the sodium level in the blood is diluted, resulting in hyponatremia. As a result, hyponatremia is the most common electrolyte disorder that must be carefully managed [35].

There is little knowledge about the renal function of broilers in hot climates, especially in terms of compensating for water and electrolyte loss. During acute heat exposure, there were variable changes in urinary electrolyte excretion in chickens. Reduced glomerular filtration rates (GFR), tubular sodium reabsorption rates, and filtered water amounts may help heat-acclimated birds reduce the metabolic heat load associated with active solute recovery from the glomerular ultrafiltrate. When heat-acclimated birds consume excessive water intake to support evaporative cooling, these changes in kidney function are thought to reduce urinary fluid and solute loss [36]. More research is needed, however, to better explain how various factors may contribute to this evidence.

In addition, a study [37] demonstrated increased uric acid levels in heat stressed chickens. Hence, the application of high-protein diets in HS broilers leads to increased blood plasma uric acid and relieved oxidative stress. Furthermore, the activity of enzymes during AA or protein metabolism under stress conditions has been analyzed. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are intracellular enzymes produced in the liver, skeletal muscles, and heart of poultry, and used as indicators of the liver, muscle, and heart damage [38].

The rate of protein accretion is always a constant balance between breakdown (proteinlysis) and synthesis (protein-genesis) [18]. The reduction in protein accretion under conditions of chronic HS is because the rate of protein-genesis is more greatly affected than the rate of proteolysis [33]. Protein synthesis was reduced more in the breast muscles than that in leg muscles; this may be related to higher oxidative metabolism of the leg muscles and increased glycolytic metabolism of the breast muscle [39]. Increasing dietary protein content from 20% to 25% at 32 ◦C did not affect the rate of protein synthesis but did increase muscle protein deposition, possibly by reducing protein breakdown [33]. The authors of [40] suggested that energy for protein synthesis at the molecular level may be limited at high temperatures; glucose supplementation improves the growth rate at high ambient temperatures. The effects of HS on protein turnover are controversial but may be related to the magnitude and duration of the heat load producing either a detrimental or therapeutic effect. Both HS and pair feeding reduced the muscle mass of rats; however, pair-fed animals had higher protein degradation, leading to a more severe loss of skeletal muscle that might be attributed to protein preservation triggered by heat exposure [41].

#### **5. Heat Shock Proteins**

Heat stress produces the over-expression of heat shock factors and heat shock proteins (HSP) in bird tissues. HSP regulate multiple molecular pathways in cells in response to stress conditions and change the homeostasis of cells and tissues [1]. HSP affect mediators of inflammation and infection. HSP are molecular chaperones during increased heat, and offer defense. HSP possess mediated responses to endotoxin stimulated synthesis of cytokine, and [42] reviewed that HSP 70 overlap with NFκB transcription, leading to the deactivation of the inflammatory response. Intestinal permeability offers new targets for HS remedy. A study [43] reported that when any living organisms are exposed to HS, the synthesis of most proteins is delayed; however, a group of highly conserved proteins, HSP, is rapidly synthesized. HS causes an increase in HSP synthesis, and are also known as stress proteins [44].

A study [45] indicated that HS and subsequent elevated HSP might inhibit muscle mass increase, even with unchanged feed intake. Glutamine seems to have a protective effect on heat-shocked skeletal myotubes by inhibiting protein degradation [46] and this effect might be mediated by HSPs (primarily HSP70 and HSP25/27), independently of glutamine metabolism based on a nonsufficient-metabolizable glutamine analog to mimic the HSP enhancing effect [47]. Additionally, a review [48] reported that increased HSPs

defend cells from damage and protect them from apoptosis. HSP 70 is the most common family of HSPs and considered the most conservative, and is plentiful in most living organisms and increases synthesis after cell stress [49]. Glutamine supplementation has been found to increase HSP expression and improve the stress response [47]. defend cells from damage and protect them from apoptosis. HSP 70 is the most common family of HSPs and considered the most conservative, and is plentiful in most living or‐ ganisms and increases synthesis after cell stress [49]. Glutamine supplementation has been found to increase HSP expression and improve the stress response [47].

A study [45] indicated that HS and subsequent elevated HSP might inhibit muscle mass increase, even with unchanged feed intake. Glutamine seems to have a protective effect on heat‐shocked skeletal myotubes by inhibiting protein degradation [46] and this effect might be mediated by HSPs (primarily HSP70 and HSP25/27), independently of glutamine metabolism based on a nonsufficient‐metabolizable glutamine analog to mimic the HSP enhancing effect [47]. Additionally, a review [48] reported that increased HSPs

to stress conditions and change the homeostasis of cells and tissues [1]. HSP affect medi‐ ators of inflammation and infection. HSP are molecular chaperones during increased heat, and offer defense. HSP possess mediated responses to endotoxin stimulated synthesis of cytokine, and [42] reviewed that HSP 70 overlap with NFκB transcription, leading to the deactivation of the inflammatory response. Intestinal permeability offers new targets for HS remedy. A study [43] reported that when any living organisms are exposed to HS, the synthesis of most proteins is delayed; however, a group of highly conserved proteins, HSP, is rapidly synthesized. HS causes an increase in HSP synthesis, and are also known

#### **6. Physiological Mechanism of Stress Regulation in Poultry 6. Physiological Mechanism of Stress Regulation in Poultry**

*Animals* **2021**, *11*, x 6 of 14

as stress proteins [44].

According to a review [50], physiological stress regulation mechanisms are classified into three stages: alarm reaction (neurogenic system), resistance or adaptation (endocrine system), and exhaustion. Under HS in fowls, heat generation and metabolizable energy (ME) intake are decreased, which might be owing to reduced thyroid hormones and corticosterone concentration since those endocrine hormones are related to protein turnover acceleration in muscle and thermogenesis [51]. During HS in birds, abnormal pathways occur, including gluconeogenesis, and as protein catabolism increases the efficiency of energy absorption is decreased because of the increased energy retention. Therefore, during periods of stress, it is possible for decreases in growth to be accompanied by increases in body fat deposition [52]. Poultry's normal body temperature is around 41–42 ◦C, and the thermoneutral temperature for maximum growth is between 18–21 ◦C [53]. The environmental temperature, the thermal neutral zone, and the influence of the ambient temperature on heat production and body temperature are shown in Figure 1. According to a review [50], physiological stress regulation mechanisms are classified into three stages: alarm reaction (neurogenic system), resistance or adaptation (endocrine system), and exhaustion. Under HS in fowls, heat generation and metabolizable energy (ME) intake are decreased, which might be owing to reduced thyroid hormones and cor‐ ticosterone concentration since those endocrine hormones are related to protein turnover acceleration in muscle and thermogenesis [51]. During HS in birds, abnormal pathways occur, including gluconeogenesis, and as protein catabolism increases the efficiency of energy absorption is decreased because of the increased energy retention. Therefore, dur‐ ing periods of stress, it is possible for decreases in growth to be accompanied by increases in body fat deposition [52]. Poultry's normal body temperature is around 41 °C–42 °C, and the thermoneutral temperature for maximum growth is between 18 °C–21 °C [53]. The environmental temperature, the thermal neutral zone, and the influence of the ambi‐ ent temperature on heat production and body temperature are shown in Figure 1.

**Figure 1.** Feed and water intake, and body temperature production related to ambient tempera‐ ture. Modified after [54]. **Figure 1.** Feed and water intake, and body temperature production related to ambient temperature. Modified after [54].

Poultry produce heat through muscular activity and metabolic processes. The optimum or ideal temperature for performance is 19–22 ◦C in laying hens, and 18–22 ◦C in broilers [55]. Heat produced in the body is lost through conduction, convection, radiation, evaporation, and fecal excretion. Heat loss falls into two main categories. First, sensible heat loss occurs through convection, conduction, and radiation when hens are in a comfortable environment of 21–25 ◦C, and show optimum growth rate, egg quality and size, quality of egg shell, egg production, and hatchability. Second, insensible heat loss occurs through panting (evaporative heat loss), and begins when the temperature reaches 26.67 ◦C [56]. In addition, birds can increase respiration rates up to 10× normal. Additionally, chickens diminish heat by raising and spreading their wings and separating themselves from others. HS has a negative impact on both physiological and behavioral activities. Monitoring these criteria during rearing is critical for identifying HS properties and taking appropriate actions to mitigate the effects of HS while developing high-quality poultry through physiological and management strategies such as heat stress acclimation and poultry housing facilities.

#### **7. Hormonal Responses to Stress and the Hormonal Control of Protein Metabolism**

and poultry housing facilities.

*Animals* **2021**, *11*, x 7 of 14

Poultry produce heat through muscular activity and metabolic processes. The opti‐ mum or ideal temperature for performance is 19 °C–22 °C in laying hens, and 18 °C–22 °C in broilers [55]. Heat produced in the body is lost through conduction, convection, radia‐ tion, evaporation, and fecal excretion. Heat loss falls into two main categories. First, sen‐ sible heat loss occurs through convection, conduction, and radiation when hens are in a comfortable environment of 21 °C–25 °C, and show optimum growth rate, egg quality and size, quality of egg shell, egg production, and hatchability. Second, insensible heat loss occurs through panting (evaporative heat loss), and begins when the temperature reaches 26.67 °C [56]. In addition, birds can increase respiration rates up to 10× normal. Addition‐ ally, chickens diminish heat by raising and spreading their wings and separating them‐ selves from others. HS has a negative impact on both physiological and behavioral activ‐ ities. Monitoring these criteria during rearing is critical for identifying HS properties and taking appropriate actions to mitigate the effects of HS while developing high‐quality poultry through physiological and management strategies such as heat stress acclimation

Hormone signaling plays a vital role in regulating homeostasis, which includes growth, metabolism, reproduction, and immunity. The overall responses to stress shown in Figure 2. Rapid endocrine responses are mediated by the sympathetic nervous system activation of the adrenal medulla (SA system). However, the long-term effects are due to the activation of the hypothalamic–pituitary–adrenal cortex axis (HPA axis) and the production of glucocorticoids for long time. **7. Hormonal Responses to Stress and the Hormonal Control of Protein Metabolism** Hormone signaling plays a vital role in regulating homeostasis, which includes growth, metabolism, reproduction, and immunity. The overall responses to stress shown in Figure 2. Rapid endocrine responses are mediated by the sympathetic nervous system activation of the adrenal medulla (SA system). However, the long‐term effects are due to the activation of the hypothalamic–pituitary–adrenal cortex axis (HPA axis) and the pro‐ duction of glucocorticoids for long time.

**Figure 2.** Scheme of overall responses to stress. **Figure 2.** Scheme of overall responses to stress.

The effects of stress on growth performance and reproduction through stress hormo‐ nal axis, the reproductive axis, and their interaction are shown in (Figures 3 and 4). The effects of HS on appetite and reproductive hormones are negative. Monitoring appetite and reproductive hormone regulation during rearing are critical for mitigating the nega‐ tive effects of HS and developing high‐quality poultry through hormonal strategies. The effects of stress on growth performance and reproduction through stress hormonal axis, the reproductive axis, and their interaction are shown in (Figures 3 and 4). The effects of HS on appetite and reproductive hormones are negative. Monitoring appetite and reproductive hormone regulation during rearing are critical for mitigating the negative effects of HS and developing high-quality poultry through hormonal strategies. *Animals* **2021**, *11*, x 8 of 14

**Figure 3.** Hypothalamic–pituitary–adrenal axis and its impact on animal reproduction (stress hormone axis include CRH: **Figure 3.** Hypothalamic–pituitary–adrenal axis and its impact on animal reproduction (stress hormone

Corticotropin‐releasing hormone; ACTH: Adrenocorticotropic hormone; corticosteroids contain glucocorticoids and min‐ eralocorticoids; POMC: pro‐opiomelanocortin; LUC‐NE: locus ceruleus neurons which secrete noradrenaline; reproduc‐ tive hormone axis includes GnRH Gonadotropin‐releasing hormone; LH: luteinizing hormone; gonad steroid: testos‐

**Figure 4.** Behavioral and physiological adjustments of chickens at high ambient temperatures and its effects on nutrient

terone, estradiol, progesterone; ⊥: inhibition. Modified after [20].

intake and utilization. Modified after [57].

axis include CRH: Corticotropin-releasing hormone; ACTH: Adrenocorticotropic hormone; corticosteroids contain glucocorticoids and mineralocorticoids; POMC: pro-opiomelanocortin; LUC-NE: locus ceruleus neurons which secrete noradrenaline; reproductive hormone axis includes GnRH Gonadotropin-releasing hormone; LH: luteinizing hormone; gonad steroid: testosterone, estradiol, progesterone; ⊥: inhibition. Modified after [20]. **Figure 3.** Hypothalamic–pituitary–adrenal axis and its impact on animal reproduction (stress hormone axis include CRH: Corticotropin‐releasing hormone; ACTH: Adrenocorticotropic hormone; corticosteroids contain glucocorticoids and min‐ eralocorticoids; POMC: pro‐opiomelanocortin; LUC‐NE: locus ceruleus neurons which secrete noradrenaline; reproduc‐ tive hormone axis includes GnRH Gonadotropin‐releasing hormone; LH: luteinizing hormone; gonad steroid: testos‐ terone, estradiol, progesterone; ⊥: inhibition. Modified after [20].

*Animals* **2021**, *11*, x 8 of 14

**Figure 4.** Behavioral and physiological adjustments of chickens at high ambient temperatures and its effects on nutrient intake and utilization. Modified after [57]. **Figure 4.** Behavioral and physiological adjustments of chickens at high ambient temperatures and its effects on nutrient intake and utilization. Modified after [57].

#### **8. Assessment of Stress**

HS has a negative impact on production performance, intestinal health, body temperature, immune responses, appetite hormone regulation, and oxidative properties. It is critical to monitor these criteria during rearing in order to identify HS possessions and take timely action to mitigate the negative effects of high ambient temperature. Stress can be an assessment by three potential methods through behavioral/physiological, endocrine, and metabolic systems measurements. These have been suggested as possible indicators of animal well-being (Table 1) [20]. In addition, neuropeptide Y (NPY) expression is increased in heat-exposed chick brains. NPY has a hypothermic action through the body temperature and heat stress regulation in chicks [58].

#### **9. Nutritional Strategies for Preventing HS in Poultry**

Nutritional strategies targeted to alleviate and overcome the adverse effects of HS in domestic fowl [59], include preserving feed consumption, electrolytes, water balance, or even by adding vitamins (as ascorbic acid) and minerals [4,5]. Primary strategies in changing the diet formulation of broilers under constant or cycling high-temperature conditions include the suitable use of protein-rich ingredients (AA and crude protein) [12]. It is necessary to ensure the balance of certain AA, especially, the arginine: lysine ratio, and the supplementation synthetic methionine to correct any nutritional shortages [60]. A review [61] found that when protein is the source of energy, the heat increment or specific dynamic action is much greater than when fat or carbohydrate are the sources of energy. Consequently, there are concerns regarding diet-induced heat production related to protein in hyperthermic broilers. Some authors have mentioned the harmful effects of feeding high protein diets [62], leading to the recommendation of a reduced protein

diet to control further higher thermogenesis [63]. However, higher dietary crude protein (CP) can compensate reduced AA consumption in stressed broilers, thus it seems to be beneficial in hot conditions, resulting in an improved growth rate [64]. In addition, a review [65] reported that limited protein supplementation decreased water drinking under HS and limits broilers' performance. Therefore, the AA balance plays a chief role in the scientific conflict regarding the proteins needed for hyperthermic poultry, and it is necessary to determine the AA required for thermoneutrality. Additionally, the protein needed can change gradually after HS exposure, depending on the time exposed. Moreover, a study [66] found lower protein degradation with HS could be normalized with thyroxine supplementation.

Dietary supplementation with one or a mixture of functional AA (glutamine, leucine, proline, arginine, cysteine, and tryptophan) is possibly beneficial. First, for ameliorating or reducing health threats during different periods of the life cycle, such as the metabolic syndrome, fetal growth limitation, weaning-associated wasting syndrome and intestinal dysfunction, neonatal morbidity and mortality, diabetes, obesity, infertility and cardiovascular disease. Second, for improving or optimizing the efficiency of metabolic transformations to boost muscle development, meat and egg quality, and milk production, and reducing adiposity by inhibiting excess fat deposition. Thus, AA has important functions in both health and nutrition [10].

Dietary glutamine supplementation alleviates heat stress, resulting in improved performance and humoral immune response in poultry [67]. In addition, glutamine minimizes the HS effects in heat-stressed chickens in the first weeks of life [68]. Besides it plays several roles in the metabolism and homeostasis of tissues. A study [69] reported that glutamic acid and glutamine supplementation, as a conditionally essential AA in broilers under stress conditions, could be beneficial in improving the growth performance and health. For optimal broiler performance, the use of a high-fat diet (fat is less thermogenic than carbohydrates) with adequate levels of essential AA [70] has been suggested. However, high lysine or Arg:Lys ratio during HS did not reduce the adverse effects of heat stress or even improve the growth rate. Consequently, there is a further challenge to determine the best nutrient during feeding in many fowls during HS [6]. The addition of appropriate feed additives may be beneficial in improving intestinal absorption and minimizing the negative effects of HS. The addition of active substances during incubation is the most recent advancement. By instilling thermotolerance in newly hatched birds, these methods are expected to have an impact on the poultry industry. The physiology, production, and immunological response of broilers under heat stress are all affected by the feeding regimen, which should be tailored to the Ross-308 and Cobb-500 strains [71]. It is necessary to monitor nutritional strategies during nutrition applications in order to prevent HS and produce healthy and comfortable poultry with maintaining feed consumption, dietary adjustments, and appropriate diet formulation. For example, dietary protein-rich ingredients, AA balance, or dietary supplementation with one or a combination of functional AA are all important. Electrolytes, vitamins (such as ascorbic acid), and mineral drinking water supplementation, as well as acid–base balance, are also suggested.

#### **10. Effect of HS on Amino Acids**

The breakdown of dietary protein results in highly elevated heat generation than that of the catabolism of carbohydrates and fats in poultry under a thermoneutral zone (Table 2).

**Table 2.** The biochemical efficiency of absorbed nutrients for ATP and lipid synthesis; reviewed in [72].


367

A study [73] found that feeding broilers more protein than their nutrient requirements did not improve performance at 33 ◦C. Low protein diets, on the other hand, had a negative impact on broiler performance at high ambient temperatures [64]. A study [74] attributed these effects to lower feed consumption, decreased consumption of AA, and therefore poor body weight gain and feed efficiency. According to a review [75], HS reduced AA levels in the birds including citrulline in chicks' plasma and leucine in the embryonic brain and liver. As a result, oral L-citrulline increased thermotolerance and decreased body temperature in layer chicks. A review [72] reported that under HS conditions, broilers aged 21 d–49 d should be fed diets containing 90 to 100 percent of the National Research Council (NRC) [76] recommended levels of AA and protein in diets containing 13.4 MJ ME/kg. According to previous studies, nutritionists did not compensate for diminished consumption in the hot ambient temperature by elevating protein and AA levels. Therefore, the final impact on growth relies on optimal or ideal protein quantity. The ideal AA composition for the maintenance or production varies with ambient temperature and among species, which can be attributed to metabolic stress alterations (Table 3).

**Table 3.** Estimated ideal protein ratio for a starting hen, broiler, and pig, expressed as a lysine needed percentage [70].


NA<sup>1</sup> = not available.

The optimal or ideal AA for maintenance varies from the ideal AA for production. Birds require higher methionine and cystine, threonine, and fewer leucines than turkeys and pigs, relative to lysine. Some authors reported that greater lysine or Arg:Lys ratios in broiler diets have a beneficial impact, whereas others showed an adverse effect at HT on gain and breast yield [72]. Therefore, dietary AA influenced heat generation [77] and improved broiler performance under high temperatures while decreasing nitrogen excretion by 21% between 28 and 49 days of age [78].

#### **11. Future Perspectives and Conclusions**

In conclusion, this review discusses the impact and consequences of HS in poultry. In addition, previous work was summarized, and some recommendations for developing high-quality and comfortable poultry through physiological (including HSP regulation), hormonal, and nutritional strategies were provided. Although the influence of HS on protein metabolic conversions in poultry can be concluded from this review, the scientific and medical evidence is inconclusive. Thus, further molecular studies are necessary to determine efficient HS regulation strategies, to better clarify the mechanisms involved in HS tolerance, to understand the HSP family as a useful biomarker for detecting HS. Then, for improved production efficacy in poultry, it is necessary to manage heat stress optimally. Recently, researchers interested in exploring a new generation of genetic tools that are capable of clarifying the molecular pathways associated with the heat stress in chickens, are offering new perspectives for the use of these tools in animal breeding.

**Author Contributions:** Funding acquisition, M.A.A.-G.; project administration, M.A.A.-G.; writing original draft, M.M.Q.; writing—review and editing, M.M.Q. and M.A.A.-G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Deanship of Scientific Research at King Saud University, grant number IFKSURP-322.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors extend their appreciation to the Deputyship for Research and Innovation, "Ministry of Education" in Saudi Arabia for funding this research work through the project number IFKSURP-322. The authors extend their thanks to the RSSU at King Saud University for their technical support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

