**2. Results**

The final body weight, body weight gain, and feed conversion ratio were not a ffected by either the HT or orotic acid supplementation, whereas the feed intake was significantly depressed in the chickens kept under the HT conditions (Table 1). In addition, the body temperatures of the chickens kept in the HT environment were significantly increased compared with those of the chickens kept under thermoneutral conditions. However, orotic acid supplementation alleviated the increase in body temperature under the HT conditions.

Although the weights of the leg muscles were not a ffected by the rearing temperature, the weights of the breast muscles, breast tender muscles, livers, and hearts were lower in the chickens kept under the HT conditions (Table 2). The yield of the breast muscles (the ratio of breast muscles weight to body weight) was the highest in the chickens supplemented with orotic acid and kept in the thermoneutral environment compared to the other three groups of chickens (Table S1). However, the yield of neither the breast tender muscles nor the leg muscles were di fferent among these four groups. Furthermore, the weight of the abdominal fat tissue was increased by the HT environment.

The malondialdehyde (MDA) concentration, which serves as an index for the lipid peroxidation level, was significantly a ffected by either temperature or orotic acid supplementation. The plasma MDA concentration of chickens fed a control diet and kept under the HT condition was significantly increased compared with the chickens kept in the thermoneutral environment (Figure 1). However, under the HT condition, the dietary supplementation of orotic acid decreased the plasma MDA concentration compared to the control diet. On the other hand, the HT condition did not significantly increase the muscle MDA concentration of chickens fed a control diet. Furthermore, dietary supplementation of orotic acid did not alleviate that of chickens under the HT condition.

**Figure 1.** Effects of a cyclic high ambient temperature and feeding orotic acid on the plasma (**A**) and muscle (**B**) malondialdehyde (MDA) concentrations of broiler chickens. Results are expressed as mean ± standard error of the mean (SEM) (*n* = 8). Means with the same superscript letter within columns are not significantly different at *p* < 0.05. T: the effect of high ambient temperature; O: the effect of feeding orotic acid; T × O: the statistical interaction between high ambient temperature and feeding orotic acid.

Table 3 shows the plasma-free amino acid concentrations of the chickens kept under thermoneutral or HT conditions. There were significant effects of ambient temperature on the plasma serine, glutamine, and tyrosine concentrations, while orotic acid supplementation significantly affected the plasma aspartic acid, glutamic acid, and tyrosine concentrations. The two-way ANOVA revealed no significant interaction between the HT and orotic acid supplementation on these 18 plasma-free amino acids.

Untargeted gas chromatography–triple quadrupole mass spectrometry (GC-MS/MS)-based metabolomics analysis identified a total of 172 metabolites in the plasma of the chickens (Table 4 and Table S2). Of these metabolites, 23 were significantly affected by HT, of which 11 were significantly increased and 12 were decreased (Table 4). On the other hand, 12 were affected by orotic acid supplementation, of which 11 were significantly increased, and 1 was decreased. However, the two-way ANOVA revealed no significant interaction between the temperature and dietary treatments for the plasma metabolites.

The enrichment analysis indicated that nine and four metabolic pathways were affected by the HT environment and the orotic acid supplementation, respectively (Table 5).

The free amino acid and carnosine concentrations in the broiler chickens' breast muscles are shown in Table 6. The HT treatment significantly affected the serine, glutamine, arginine, methionine, and phenylalanine concentrations, while the orotic acid supplementation significantly affected the histidine, methionine, and carnosine concentrations. The two-way ANOVA revealed no significant interaction between the temperature and dietary treatments for the concentrations of the 18 free amino acids and carnosine in the muscle.


**Table 1.** Effects of a cyclic high ambient temperature and feeding orotic acid on the growth performance parameters of broiler chickens.

Results are expressed as mean ± standard error of the mean (SEM) (*n* = 8). Means with the same superscript letter within rows are not significantly different at *p* < 0.05. T: the effect of temperature; O: the effect of feeding orotic acid; T × O: the statistical interaction between temperature and feeding orotic acid; N.S.: not significant.

**Table 3.** Effects of a cyclic high ambient temperature and feeding orotic acid on plasma-free amino acids of broiler chickens (μM).




Results are expressed as mean ± standard error of the mean (SEM) (*n* = 8). Means with the same superscript letter within rows are not significantly different at *p* < 0.05. T: the effect of temperature; O: the effect of feeding orotic acid; T × O: the statistical interaction between temperature and feeding orotic acid; N.S.: not significant.

**Table 4.** Effects of a cyclic high ambient temperature and feeding orotic acid on plasma metabolites of broiler chickens.




The relative quantities of the metabolites were means ± SEM (*n* = 8) and expressed as percentage of an arbitrary control set to 100%. Means with the same superscript letter within rows are not significantly different at *p* < 0.05. T: the effect of temperature; O: the effect of feeding orotic acid; T × O: the statistical interaction between temperature and feeding orotic acid; N.S.: not significant.




temperature; O: the effect of feeding orotic acid; T × O: the statistical interaction between temperature and feeding orotic acid; N.S.: not significant.


**Metabolism Name** *p***-Value** Urea cycle <0.05 β-Alanine metabolism <0.05 Glycine and serine metabolism <0.05 Aspartate metabolism <0.05

**Metabolic pathway a**ff**ected by orotic acid**

Pyrimidine metabolism <0.01 β-Alanine metabolism <0.01 Malate–aspartate shuttle <0.01 Aspartate metabolism <0.05 **Table 6.** Effects of a cyclic high ambient temperature and feeding orotic acid on free amino acids and carnosine in the breast muscle of broiler chickens (mg/100 g).
