24. Investigating the Influence of Dietary N-carbamylglutamate and Melatonin on Liver Hemodynamics, Amino Acid Concentration, and Transcriptomics in Heat-stressed Finishing Lambs

Abstract Heat stress in livestock alters blood circulation resulting in metabolic disturbances. N-carbamylglutamate (NCG), a precursor of L-arginine, promotes nitric oxide synthesis while melatonin (MEL) exhibits vasodilatory and vasoconstrictive effects, influencing hemodynamic functions. The liver, as central organ of amino acid (AA) regulation and nutrient partitioning will assume adaptive responses during heat stress consisting in alterations of transcriptomic dynamics related to cellular stress and metabolic processes. This study aimed to evaluate the effect of NCG and MEL paired supplementation on body weight (BW), hepatic hemodynamics, plasma AA concentration, and transcriptomics on heat stressed finishing lambs. Twenty-one dorper rams (4 months old) were used to test the hypothesis that NCG and MEL supplementation will increase liver blood flow (LBF) and plasma AA via upregulation of genes involved in angiogenesis. Lambs were assigned to treatment groups in a 2 × 2 factorial design, consisting of a cooled condition without supplementation (CL-CON; n = 5), heat stress condition without supplementation (HS-CON; n = 6), and cool or heat stressed conditions supplemented with 1.4 g/h/d of NCG and 8mg/h/d of MEL (CL-NCGMEL; n = 5; HS-NCGMEL; n = 5). After 35d, plasma samples were collected while LBF was assessed at the 10th intercostal space using Doppler ultrasonography. On day 42 post-supplementation, lambs were slaughtered, liver weight recorded, and a sample was collected for transcriptomics. Data were analyzed by the MIXED procedure of SAS with BW, LBF, and AA as dependent variables. The model also included the main effects of environment, treatment, and their interactions. For transcriptomics, gene expression was quantified with salmon, and differential genes were found using the R package DEseq2. Genes with a Benjamini-Hochberg-adjusted P-value ≤ 0.05 (BH P-value) were considered significant. An environment by treatment interaction was observed for BW (P = 0.0411) where HS-NCGMEL lambs had increased BW compared to HS-CON (40.79±1.65 kg vs. 35.25±1.8 kg), while no difference was observed between HS-NCGMEL and CL-CON (P > 0.05) demonstrating a mitigating effect of the supplementation. The liver weight of the HS-MEL lambs was also increased when compared with HS-CON (764.30±61.22 g vs. 552.04±65.43 g; P = 0.0449). No significant differences were found in liver blood flow (P > 0.05). Although, treatment effects were observed for AA (P ≤ 0.05); where NCGMEL lambs had decreased arginine (133.29±11.43 mM vs. 168.06±10.45 mM) and glutamine (370.03±27.43 mM vs. 444.41±24.29 mM) concentrations compared to CON lambs. Lastly, the cationic amino acid transporter 3-like protein, a sodium-independent transporter for cationic AA like L-arginine, was significantly upregulated in HS-NCGMEL vs. CL-CON (log2FC = 8.7; BH adj. P = 0.01). Additionally, Forkhead Box S1 (FOXS1), associated with endothelium-dependent vasodilation, was significantly upregulated in in HS-NCGMEL vs. CL-CON (log2FC =21.97; BH adj. P < 0.001). The significant transcriptomic upregulation along with AA utilization suggest metabolic adjustments and adaptive responses in supplemented heat stressed lambs.