**3. Discussion**

Reactive oxygen species (ROS), such as singlet oxygen, hydrogen peroxide, and hydroxyl radicals, are highly reactive molecules produced by mitochondria [19]. Under high ambient temperature conditions, ROS generation increases in various body tissues of chickens as the heat load elevates [3], and consequently oxidizes and impairs lipids, proteins, and DNA [20]. Because chicken muscle has a high polyunsaturated fatty acid content, making it more sensitive to oxidative deterioration [21], the oxidation of such lipids negatively influences their industrial values (i.e., growth performance, meat yield, and meat quality) [4–14].

Under HT conditions, chickens have been known to show lower growth rate and feed e fficiency, accompanied by decreasing meat yield [4,5]. In this study, although the HT environment did not a ffect the final body weight, body weight gain, or feed conversion ratio of the broiler chickens, it significantly depressed their feed intake and increased their average body temperature. These results concur with those of a previous study, which examined the e ffects of a similar HT environment [13]. In addition, the HT environment a ffected the weights of the breast muscles, breast tender muscles, livers, hearts, and abdominal fat tissue. In particular, the yield of breast muscle was significantly decreased by the HT environment. Furthermore, the plasma lipid peroxidation level was higher in the chickens kept under the HT conditions than those kept in the thermoneutral environment. Therefore, these results sugges<sup>t</sup> that the HT environment used in this study could realistically induce the negative e ffects commonly observed in broiler chickens kept in heat stress-inducing environments.

In this study, most of the plasma amino acid concentrations were una ffected by HT, with the exception of the serine, tyrosine, and glutamine concentrations. This was particularly notable in the chickens fed the basal diet, in which the HT environment significantly decreased the plasma glutamine concentration. This concurred with the results of a previous study, which reported that chickens kept under HT conditions had lower plasma glutamine concentrations, in addition to poorer performance and carcass characteristics [22]. Although glutamine is a non-essential amino acid, it has been reported to promote enterocyte proliferation and survival, and regulate intestinal barrier function under a variety of stress conditions [23]. Furthermore, intestinal morphology and permeability were found to be disrupted under HT conditions, with this being accompanied by an increase in the plasma endotoxin concentration in broiler chickens [24]. These results sugges<sup>t</sup> that glutamine is utilized to maintain intestinal integrity in broiler chickens under heat-stress conditions.

In addition, glutamine is known to be a precursor for the synthesis of purine and pyrimidine nucleotides, which are essential for DNA synthesis and the proliferation of cells [25]. In rats, GC-MS and liquid chromatography–mass spectrometry-based metabolomics analysis of the plasma indicated that short-term chronic heat exposure (37 ◦C for 48 h) altered pyrimidine and purine degradation [26]. To investigate the effects of an HT environment on metabolite profiles in the plasma of chickens, we performed untargeted GC-MS/MS-based metabolomics analysis. This analysis identified 172 metabolites in the plasma, of which 23 were significantly affected by the HT environment. The HT environment particularly affected the plasma levels of purine- (inosine, hypoxanthine, xanthine, xanthosine monophosphate, and uric acid) and pyrimidine-related (uracil, dihydrouracil, and thymine) metabolites. The changes in these metabolites are illustrated in Figure 2, with a general increase in concentration in response to the HT environment being apparent.

**Figure 2.** Integrated overview of the metabolic changes induced by either a cyclic high ambient temperature or by feeding orotic acid. Red arrows indicate high ambient temperature-induced changes in the metabolites, and blue arrows indicate orotic acid-induced changes. The metabolic scheme was based on information gathered from the KEGG PATHWAY Database (http://www.genome.jp/kegg/ pathway.html).

One possible explanation for the changes in the metabolite concentrations observed may be the higher oxidative stress levels in the broiler chickens kept under the HT conditions. Uric acid is known to act as an antioxidant in the plasma [27], and is thought to stabilize ascorbic acid [28]. However, the concentration of ascorbic acid was lower in the plasma of the chickens kept in the HT environment than in the chickens kept under thermoneutral conditions. Another possibility is that these changes were linked the higher energy expenditure of the broiler chickens kept at HT. When broiler chickens are exposed to environmental temperatures above 31 ◦C, they increase heat production [29], suggesting an increase in adenosine triphosphate (ATP) production. Adenylate kinase, which acts as a regulator for phosphate nucleotide levels inside cells, converts two adenosine diphosphate (ADP) molecules to one ATP molecule and one adenosine monophosphate (AMP) molecule [30]. Since the concentration of AMP is maintained at a much lower level than that of either ADP or ATP [31], the generated AMP may be catabolized to form hypoxanthine, xanthine, or uric acid.

The enrichment analysis identified that the HT environment induced changes in purine metabolism, ammonia recycling, pyrimidine metabolism, homocysteine degradation, glutamate metabolism, the urea cycle, β-alanine metabolism, glycine and serine metabolism, and aspartate metabolism. Seven of these pathways (excluding homocysteine degradation and aspartate metabolism) concur with the findings of a previous study on the effects of short-term chronic heat exposure on the plasma metabolomic profiles of chicks [15]. Furthermore, the plasma homocysteine concentration is considered an oxidative stress marker [32]. In Japanese quail, HT increased both the homocysteine concentration and the lipid peroxidation level in the plasma [33,34], and it has been suggested that chickens kept under HT conditions have higher plasma homocysteine concentrations. Furthermore, the untargeted GC-MS/MS-based metabolomics analysis indicated that the HT environment decreased the plasma level of aspartic acid, which is a precursor for the synthesis of pyrimidines. These results sugges<sup>t</sup> that although purine and pyrimidine synthesis pathways in chickens were enhanced in response to the HT conditions, the levels of related metabolites (e.g., glutamine, aspartic acid, and ascorbic acid) may have been disrupted.

It is noteworthy that the HT environment changed not only pyrimidine metabolism, but also aspartate metabolism and β-alanine metabolism, because both pyrimidine metabolism and aspartate metabolism are closely related to β-alanine metabolism. β-alanine is one of constituents of carnosine (β-alanyl-l-histidine) and anserine (β-alanyl-1-methyl-l-histidine), which are known to be present at high concentrations in the breast muscle of chickens and to exert antioxidant activity [35]. In this study, we found that the chickens fed the basal diet and kept at a HT had the lowest carnosine content in the breast muscle of the three treatment groups. This low carnosine content may explain the higher lipid peroxidation level in the breast muscle samples from this group.

An HT-induced decrease in the muscle carnosine content was also observed by Yang et al. [36], while Tomonaga et al. [15] reported that the constituents of carnosine (β-alanine and histidine) in chicks decreased in response to short-term chronic heat exposure. In this study, although the plasma dihydrouracil concentration was the highest, the plasma level of β-alanine was the lowest in the chickens fed the basal diet and kept under the HT conditions. Therefore, these results sugges<sup>t</sup> that the conversion of dihydrouracil to β-alanine may be disrupted in chickens kept under HT conditions.

This study also evaluated the e ffects of feeding orotic acid to chickens, and found that it did not affect growth performance, as indicated by the final body weight, body weight gain, feed conversion ratio, feed intake, and tissue weights. However, orotic acid did ameliorate the HT-induced increases in lipid peroxidation level in the plasma of the chickens. This amelioration may have partially been due to the antioxidative activity of orotic acid [37], with the plasma level of orotic acid decreasing under the HT conditions and increasing in response to the orotic acid supplementation. The GC-MS/MS-based metabolomics analysis of the plasma indicated that 12 metabolites were a ffected by orotic acid supplementation, some of which have been reported to show antioxidative activity (e.g., uridine and 3-hydroxyanthranilic acid) [38,39] and appeared to be increased by orotic acid supplementation. Furthermore, the high-performance liquid chromatography analysis revealed that orotic acid significantly a ffected the plasma concentrations of aspartic acid, glutamic acid, and tyrosine. Of these three amino acids, aspartic acid and glutamic acid appeared to be increased by the orotic acid supplementation. Aspartic acid is converted into glutamic acid via the citric acid cycle [40], and glutamic acid is a component amino acid of glutathione, a well-known natural antioxidant [41,42]. These results sugges<sup>t</sup> that alterations in the concentrations of these metabolites and amino acids may contribute to the amelioration of the HT environment-induced increase in the plasma lipid peroxidation level. Furthermore, orotic acid supplementation significantly a ffected the carnosine content of the breast muscle of the chickens. As previously mentioned, carnosine plays a role as an antioxidant [35], and the reduction of the lipid peroxidation level in the breast muscle tissue may therefore be linked to the orotic acid-induced increase in the carnosine content.

The GC-MS/MS-based metabolomics analysis indicated that orotic acid a ffected the plasma levels of pyrimidine-related metabolites (orotic acid, uracil, uridine, 2'-deoxyuridine, and β-alanine). This e ffect was likely linked to the entry of orotic acid into the de novo synthesis pathway for pyrimidines beyond the rate-limiting step. Furthermore, the de novo synthesis pathways for both pyrimidines and purines require 5-phosphoribosyl-1-pyrophosphate (PRPP), and the synthesis of PRPP by PRPP synthetase is the rate-limiting step for both pathways. In rats, it has been reported that orotic acid stimulates hepatic purine biosynthesis [43]. These results sugges<sup>t</sup> that orotic acid may up-regulate the de novo synthesis of purines by providing additional PRPP. However, there was no significant interaction between the temperature and dietary treatments for the plasma metabolites. Further studies are required to understand the e ffects of orotic acid supplementation on the regulation of the de novo synthesis pathways for pyrimidines and purines in chickens.

The enrichment analysis identified changes in not only pyrimidine metabolism, but also in β-alanine metabolism, the malate–aspartate shuttle, and aspartate metabolism, in response to supplementing orotic acid. Of these, β-alanine metabolism and aspartate metabolism may be

particularly important for the alleviation of HT-induced negative effects, as they were also found to be altered by HT conditions. Orotic acid supplementation was found to significantly increase the plasma level of β-alanine and the carnosine content of the breast muscles of the chickens. In chicks, orally administered β-alanine increased the carnosine content of the brain and muscles in a dose-dependent manner [44]. Therefore, these results sugges<sup>t</sup> that orotic acid increases the muscle carnosine content by altering β-alanine metabolism, and thereby maintains the antioxidative capacity of broiler chickens under HT conditions. However, the mechanisms by which orotic acid supplementation increased the plasma level of β-alanine, even under HT conditions, remain unclear. Further research evaluating the effects of HT and orotic acid supplementation on the gene expression and activities of enzymes related to the conversion of dihydrouracil to β-alanine (i.e., dihydropyrimidinase and β-ureidopropionase) in chickens is necessary if we are to improve our insight into the reason for the HT-induced decrease in the muscle carnosine content and the alleviating effects of orotic acid supplementation.

As mentioned above, HT conditions severely increase lipid peroxidation levels [6,7], and consequently reduce meat quality (i.e., increase in drip loss, decrease in share force value, and change in meat color) [8–14]. The breast muscles of the chickens kept under cyclic HT conditions showed the highest lipid peroxidation levels, in conjunction with the lowest carnosine content, suggesting that their meat was of lower quality than that of the control chickens. In contrast, orotic acid increased the muscle carnosine content, possibly via pyrimidine metabolism and β-alanine metabolism, and consequently alleviated the HT environment-induced increase in the muscle lipid peroxidation levels. Cong et al. [45] reported that dietary supplementation of carnosine improved meat quality, antioxidant capacity, and lipid peroxidation status in broiler chickens. In addition, carnosine exerts antioxidant activity, even in cocked chicken meat [35]. These results sugges<sup>t</sup> that orotic acid supplementation may have a positive effect on either carnosine content in meat or the meat quality of chickens.

#### **4. Materials and Methods**

#### *4.1. Animals and Experimental Design*

All experimental protocols and procedures were reviewed and approved by the Animal Care and Use Committee of Kagoshima University (approval number A18010). One hundred 1-day-old male broiler chicks (Chunky strain ROSS 308) were obtained from a commercial hatchery (Kumiai Hina Center, Kagoshima, Japan). Chicks were housed in an electrically-heated battery brooder and provided with water and a commercial diet (23% crude protein, 12.8 MJ/kg; Nichiwa Sangyou Company, Hyogo, Japan) until they were 14 days old. On day 14, 32 chicks were randomly selected from the group of 100. These chicks were housed individually in wire-bottomed aluminum cages (50 × 40 × 60 cm) and fed the basal diet (Table 7) for 3 days until the start of the main experimental period. The chicks were then randomly allocated to one of four groups, with the main experimental factors being diet and ambient temperature, in a 2 × 2 factorial design. The dietary treatments consisted of the basal diet or the basal diet supplemented with 0.7% of Lactoserum (Matsumoto Trading Co., Ltd., Tokyo, Japan), a food grade orotic acid that contains more than 98% of orotic acid as monohydrate form (i.e., more than 87.9% of orotic acid), and the temperature treatments consisted of either a thermoneutral environment at 25 ± 1 ◦C or an HT environment at 35 ± 1 ◦C. The experiment was conducted in a temperature-controlled room with 24 h of light and 50–70% relative humidity. The chicks assigned to the HT treatment groups were kept at 35 ± 1 ◦C for 8 h every day to mimic a realistic summer environment. At 32 days old, all the chickens were weighed, anesthetized by carbon dioxide, and killed by cervical dislocation. The chickens were then dissected, and the weights of the breast muscles (pectoralis major muscle), breast tender muscles (pectoralis minor muscle), leg muscles (thigh and drumstick), livers, hearts, and abdominal fat tissue depots were recorded. Blood samples were collected in heparinized test tubes, centrifuged at 5900× *g* for 10 min at 4 ◦C to separate the plasma, and stored at −30 ◦C until analysis.


**Table 7.** Composition and analysis of the basal diet.

1 Content per kg of the vitamin and mineral premix: vitamin A = 90 mg, vitamin D3 = 1 mg, DL-alpha-tocopherol acetate = 2000 mg, vitamin K3 = 229 mg, thiamin nitrate = 444 mg, riboflavin = 720 mg, calcium d-pantothenate = 2174 mg, nicotinamide = 7000 mg, pyridoxine hydrochloride = 700 mg, biotin = 30 mg, folic acid = 110 mg, cyanocobalamine = 2 mg, calcium iodinate = 108 mg, MgO = 198,991 mg, MnSO4 = 32,985 mg, ZnSO4 = 19,753 mg, FeSO4 = 43,523 mg, CuSO4 = 4019 mg, and choline chloride = 299,608 mg.

#### *4.2. Determination of MDA Concentration*

To evaluate the oxidative stress levels, the MDA concentrations in the breast muscles and plasma were determined colorimetrically, using the thiobarbituric acid reactive substances assay, as described by Yagi [46] and Ohkawa et al. [47].

#### *4.3. Determination of Free Amino Acid Concentrations*

The analysis of the free amino acid and carnosine concentrations in the plasma and breast muscle tissue samples from the chickens was performed using a pre-column technique with liquid chromatography, according to previously reported methods [48]. A liquid chromatography system with automated pre-column derivatization functionality was used in the analysis (Nexera X2; Shimadzu Corporation, Kyoto, Japan). A total of 21 compounds were measured in the analysis, including the following basic amino acids and associated molecules: alanine, anserine, arginine, asparagine, aspartic acid, carnosine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. The concentrations of the amino acids in the plasma are expressed in μmol/L, and those in the breast muscle are expressed in mg/100 g. In this study, alanine and anserine were not separated, and carnosine was not detected in the plasma.

#### *4.4. Sample Preparation for GC-MS*/*MS Analysis*

Fifty μL aliquots of plasma were suspended in 250 μL of methanol/chloroform/water (5:2:2), with 5 μL of 1 mg/mL 2-isopropylmalic acid as the internal standard. The samples were then mixed in a shaker at 1200 rpm at 37 ◦C for 30 min, and then centrifuged at 16,000× *g* at 4 ◦C for 5 min. Next, 225 μL of the supernatant was mixed with 200 μL of distilled water and vortex-mixed, followed by centrifugation at 16,000× *g* at 4 ◦C for 5 min. Subsequently, 250 μL of the supernatant was dried under a vacuum using a centrifugal evaporator (RD-400; Yamato Scientific, Tokyo, Japan), after cooling at −80 ◦C for 10 min. Methoxyamine hydrochloride in pyridine (20 mg/mL, 40 μL) was then added to the tubes, and they were vortex-mixed, then shaken at 1200× *g* at 30 ◦C for 90 min in the dark to allow oximation. N-methyl-N-trimethylsilyltrifluoroacetamine (20 μL) was then added to each tube, and the contents were vortex-mixed. To prepare trimethylsilyl derivatives, the tubes were shaken at 1200× *g* at 37 ◦C for 45 min in the dark.

#### *4.5. GC-MS*/*MS Analysis and Data Processing*

GC-MS/MS analysis was performed as previously described [49], using a GCMS-TQ8050 (Shimadzu Corporation, Kyoto, Japan). A 30 m × 0.25 mm (internal diameter) BPX-5 column (SGE, Melbourne, Australia) with a 0.25 μm film thickness was used, according to the method described in the Smart Metabolites Database (Shimadzu, Kyoto, Japan).

Data processing was performed using the Smart Metabolites Database (Shimadzu, Kyoto, Japan), MS-DIAL version 3.08 [50], and the MRMPROBS program version 2.42 [51]. Peaks were recorded for the 45−600 m/z mass range, and were automatically detected via MS-DIAL using the peak detection option of a minimum peak height of 2000. A data quality check was conducted using the thresholds of −10 < RI < 10, dot production > 0.8, and presence > 0.6, and the remaining data was then manually checked. Ultimately, 172 metabolites were identified in the plasma samples. The relative quantities of the metabolites were calculated using the peak areas of each metabolite relative to that of the internal standard (2-isopropylmalic acid), and expressed as a percentage of an arbitrary control set to 100%.
