**1. Introduction**

Ambient temperatures above the thermoneutral zone cause environmental heat stress. Chickens are more vulnerable to heat stress than other domestic animals, because they lack sweat glands and have higher body temperatures [1,2]. Under such high ambient temperature (HT) conditions, the generation of reactive oxygen species increases in various body tissues as the heat load increases [3]. This results in a range of physiological changes that can severely depress growth performance and meat yield [4,5] and reduce meat quality, accompanied by increasing lipid peroxidation levels [6,7] and drip loss [8,9], decreasing the share force value [10,11] and changing meat color [12–14].

Gas chromatography–mass spectrometry (GC-MS)-based, untargeted metabolomics analysis has been used to comprehensively characterize the e ffects of HT on the physiology of chickens [15], and has found that the plasma levels of 38 metabolites changed significantly when chickens were exposed to chronic heat (38 ◦C) for 4 days. These altered metabolites indicated that such heat exposure a ffected 35 metabolic processes, including the sulfur amino acid metabolic pathway, the kynurenine pathway in tryptophan metabolism, and nucleic acid metabolism [15]. In agreemen<sup>t</sup> with this observation, dietary supplementation with either a sulfur amino acid (methionine) or tryptophan, which is mainly metabolized to kynurenine, has been reported to alleviate the negative e ffects of HT [16,17]. However, the involvement of nucleic acids and their metabolites in the metabolic changes that occur in chickens kept in HT environments remains unclear.

Orotic acid, which is found in high concentrations in cow's milk, is a key intermediate in the pyrimidine biosynthesis pathway [18]. Pyrimidine nucleotides are important constituents of RNA and of the phospholipids present in cell membranes. Orotic acid can enter the de novo synthesis pathway for pyrimidines beyond the rate-limiting step, and thereby improve throughput. In chickens, the plasma level of orotic acid was found to significantly decrease, by approximately 60%, in response to short-term chronic heat exposure [15], suggesting that the plasma orotic acid concentration may be important under HT conditions.

In this study, we evaluated the e ffects of prolonged heat exposure and feeding orotic acid at 0.7% of the diet on the growth performance, plasma and muscle lipid peroxidation levels, and plasma and muscle metabolite concentrations of broiler chickens. To mimic realistic HT conditions, a cyclic HT environment (35 ± 1 ◦C for 8 h/day) was used. In addition, a food-grade orotic acid (Lactoserum; Matsumoto Trading Co., Ltd., Tokyo, Japan), which contains more than 98% of orotic acid monohydrate, was used for dietary supplementation.
