Abstract
Several amino acids (AA) are known to regulate key metabolic pathways that are crucial for immune responses. In particular, arginine (ARG) appears to have important roles regarding immune modulation since it is required for macrophage responses and lymphocyte development. Moreover, citrulline (CIT) is a precursor of arginine, and it was reported as an alternative to ARG for improving macrophage function in mammals. The present study aimed to explore the effects of dietary ARG and CIT supplementation on the gilthead seabream (Sparus aurata) immune status. Triplicate groups of fish (23.1 ± 0.4 g) were either fed a control diet (CTRL) with a balanced AA profile, or the CTRL diet supplemented with graded levels of ARG or CIT (i.e., 0.5 and 1% of feed; ARG1, CIT1, ARG2, and CIT2, respectively). After 2 and 4 weeks of feeding, fish were euthanized and blood was collected for blood smears, plasma for humoral immune parameters and shotgun proteomics, and head-kidney tissue for the measurement of health-related transcripts. A total of 94 proteins were identified in the plasma of all treatments. Among them, components of the complement system, apolipoproteins, as well as some glycoproteins were found to be highly abundant. After performing a PLS of the expressed proteins, differences between the two sampling points were observed. In this regard, component 1 (61%) was correlated with the effect of sampling time, whereas component 2 (18%) seemed associated to individual variability within diet. Gilthead seabream fed ARG2 and CIT2 at 4 weeks were more distant than fish fed all dietary treatments at 2 weeks and fish fed the CTRL diet at 4 weeks. Therefore, data suggest that the modulatory effects of AA supplementation at the proteome level were more effective after 4 weeks of feeding and at the higher inclusion level (i.e., 1% of feed). The bactericidal activity increased in fish fed the highest supplementation level of both AAs after 4 weeks. Peripheral monocyte numbers correlated positively with nitric oxide, which showed an increasing trend in a dose-dependent manner. The colony-stimulating factor 1 receptor tended to be up-regulated at the final sampling point regardless of dietary treatments. Data from this study point to an immunostimulatory effect of dietary ARG or CIT supplementation after 4 weeks of feeding in the gilthead seabream, particularly when supplemented at a 1% inclusion level.
Highlights
Feeds can have significant health implication in farmed fish and good practices in diet formulation are imperative, since it represents a significant expenditure to the aquaculture industry
No differences were observed in final body weight (FBW), daily growth index (DGI), relative growth rate (RGR), voluntary feed intake (VF), feed efficiency (FE), or feed conversion ratio (FCR) among the dietary treatments (Table 4)
The proportion of monocytes was observed to be higher in fish fed diets with the highest supplementation level (i.e., ARG2 and CIT2) compared to their respective counterparts fed the lower supplementation level (Figure 5)
Summary
Feeds can have significant health implication in farmed fish and good practices in diet formulation are imperative, since it represents a significant expenditure to the aquaculture industry. In recent years, the industry is willing to explore the so called “functional feeds,” fortified diet formulations that have added benefits besides meeting fish essential nutritional requirements, being both health and growth boosters [1, 2]. Amongst a wide range of candidate functional ingredients, little attention has been paid to the role of individual amino acids as potential immunomodulators in fish. In this regard, arginine (ARG) is one of the most versatile amino acids by serving as the precursor for the synthesis of protein, nitric oxide (NO), urea, polyamines, proline, glutamate, creatine and agmatine in terrestrial animals [3]. Polyamines are important for lymphocytes proliferation and differentiation, and NO is a strong bactericidal agent synthetized by activated phagocytes
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