Dietary Val enhanced fish growth
We have, firstly, verified the importance of the essential amino acid Val for the largemouth bass. Diets supplemented with Val augmented the FBW, PWG, and SGR, suggesting that dietary Val stimulated the growth of largemouth bass. Similar observations have been reported in Nile tilapia [5], red drum [7], and golden pompano [6]. The quadratic regression analysis for SGR showed that the dietary Val requirement of the largemouth bass (37.99-162.92 g) was 21.50 g Val/kg of the diet, the equivalent of 46.64 g/kg of dietary protein. This is higher than that reported in Nile tilapia (35.39 g/kg) [5] and red drum (33.60 g/kg) [7] but comparable to that reported for the golden pompano (46.22–46.91 g/kg) [6]. These disparities could be ascribed to differences in fish size and age, growth environment, and growth stages. In addition, the SGR was positively associated with FI (r = 0.937, P = 0.006) and FE (r = 0.973, P = 0.001) (Table 6) and FE was positively related to PER (r = 0.914, P = 0.011), PPV (r = 0.945, P = 0.004), LPV (r = 0.904, P = 0.013), and APV (r = 0.996, P = 0.000) (Table 6). These results suggested that the effects of Val on growth may be due to increased FI and FE and the improved FE was partly due to increased nutrient retention.
Dietary Val improved intestinal antioxidant capacity via Nrf2 signaling pathway after infection with A. veronii
A. veronii is an opportunistic pathogen that has been related to mortality in a number of species (mostly fish) [47]. The SR after challenge reflects the fish disease resistance [48]. Here, dietary Val significantly improved the SR after infection with A. veronii, which suggested that dietary Val might improve disease resistance. The intestine is one of the target organs of A. veronii, which causes damage to the intestinal anatomy [49]. In this study, dietary Val alleviated intestinal injury after infection with A. veronii. The oxidative condition of intestinal epithelial cells is linked to their structural integrity. Although ROS are normally produced during intracellular physiological reactions, excess production accompanied by insufficient elimination results in protein oxidation and lipid peroxidation that cause structural damage to the cells and, in turn, to the intestinal epithelium. Previous studies showed that A. veronii infection led to ROS overproduction [15, 49–51]. The degree of protein oxidation and lipid peroxidation can be reflected by the PC and MDA concentrations, respectively [52]. Here, we found that dietary Val significantly reduced ROS, PC, and MDA levels after infection with A. veronii. Similar findings were seen in the gill and muscle of the grass carp [3, 22]. ROS was found to be positively associated with the MDA content (r = 0.976, P = 0.001) and PC content (r = 0.949, P = 0.004). These results indicated that dietary Val might alleviate lipid peroxidation and protein oxidation damage by decreasing ROS accumulation. The most damaging ROS are superoxide and hydroxyl radicals [53]. The ASA and AHR activities are used as a measure of the ability of the cell or tissue to scavenge superoxide and hydroxyl radicals, respectively [54]. We found that optimal levels of dietary Val increased the activities of both ASA and AHR which correlated negatively with the ROS levels (ASA r = − 0.935, P = 0.006 and AHR r = − 0.935, P = 0.006) suggesting that dietary Val suppressed ROS accumulation by improving the total capacities of scavenging superoxide and hydroxyl radicals. This result is consistent with reports on the gills of grass carp and serum of the golden pompano [6, 22]. The ROS removal also largely relies on the antioxidant system, including non-enzymatic antioxidants (GSH) and antioxidant enzymes (SOD, CAT, GST, GPx, and GR) [17, 55]. In this study, dietary appropriate Val increased the content of GSH and the activities of SOD, CAT, GST, GPx, GR in the intestine. The ROS concentration was negatively related to SOD (r = − 0.901, P = 0.014), CAT (r = − 0.805, P = 0.053), and GST (r = − 0.892, P = 0.017) activities, suggesting that dietary Val promoted ROS scavenging partly through the modulation of antioxidant enzymes. This finding is consistent with results on the grass carp, Nile tilapia, and golden pompano, which reported that dietary Val increased the activities of antioxidant enzymes in the gills and muscles of grass carp, liver of the Nile tilapia, and serum of the golden pompano [1, 3, 6, 22].
The activities of enzymes are closely associated with their mRNA levels [56]. Here, we found that optimal levels of dietary Val significantly elevated the mRNA levels of SOD1, CAT, GST, GPx, and GR. Furthermore, the mRNA levels of SOD1 (r = 0.861, P = 0.028), CAT (r = 0.966, P = 0.002), and GPx (r = 0.840, P = 0.036) were positively correlated with the activities of their respective enzymes. Glutamate cysteine ligase is an enzyme responsible for the synthesis of GSH, a process that is dependent on the GCLC catalytic subunit [20]. We observed that dietary Val significantly enhanced the mRNA levels of GCLC. Nrf2 regulates the transcription of various antioxidant enzymes in the nucleus, a process that can be inhibited by Nrf2 binding to the cytoplasmic Keap1, preventing translocation to the nucleus [19, 20]. The activation of the Nrf2 signaling pathway was positively linked to the nuclear Nrf2 protein level, which could be enhanced either by increasing the transcription of Nrf2 or by decreasing the level of Keap1 [57, 58]. Here, we found that appropriate levels of dietary Val significantly augmented both the Nrf2 mRNA and the level of the protein product in the nucleus while decreasing Keap1 mRNA. Furthermore, the Nrf2 mRNA level was positively associated with the mRNA levels of CAT (r = 0.962, P = 0.002), GR (r = 0.810, P = 0.051), and GCLC (r = 0.925, P = 0.008). These results demonstrated that dietary Val improved intestinal antioxidant capacity by up-regulated the antioxidant enzyme-related genes transcription via the Nrf2 signaling pathway.
Dietary Val ameliorated ERS-mediated apoptosis through modulation of the PERK/eIF2α signaling pathway in intestines after infection with A. veronii
Apoptosis may result from ROS-induced oxidative damage which, if excessive, may seriously damage the structural integrity of the intestine [24]. Apoptosis is characterized by DNA fragmentation [59]. Here, we found that dietary Val reduced the ladder-like pattern of DNA fragments after infection with A. veronii. This is consistent with results on grass carp gills and chicken sperm [22, 60]. These results demonstrated that Val is able to protect against A. veronii-induced apoptosis in the intestine.
The ER is a vital subcellular organelle that regulates protein synthesis, ensures the correct folding of polypeptide chains into proteins, and maintains intracellular calcium homeostasis [61]. It has been observed that bacterial infection can impair ER homeostasis and eventually lead to ERS in fish [26–29]. The ERS is mediated by three major pathways: the PERK/eIF2α pathway, the IRE1α/XBP1 pathway, and the ATF6 pathway. PERK is responsible for phosphorylating the translation initiation factor eIF2α at the Ser51 site, resulting in the translation of ATF4 by the phosphorylated eIF2α [30, 31]. CHOP, a key downstream target of ATF4, can trigger apoptosis during ERS [30, 31]. Here, we found that dietary Val significantly reduced the mRNA levels of GRP78, ATF6, IRE1, PERK, eIF2α, ATF4, and CHOP, as well as the protein levels of p-PERK and p-eIF2α, caspase 9, and caspase 3. These results indicated that Val ameliorated A. veronii-induced apoptosis through modulation of the PERK/eIF2α signaling pathway. Feng et al. also reported that dietary Val significantly decreased caspase 9 mRNA in the grass carp gill [22]. To the best of our knowledge, the present study is the first report showing that dietary Val protects the intestine against infection-induced apoptosis through inhibiting the PERK/eIF2α signaling pathway. We also found that ROS levels were positively correlated with the mRNAs of GRP78 (r = 0.794, P = 0.059), ATF6 (r = 0.883, P = 0.020), CHOP (r = 0.864, P = 0.026), and caspase 3 (r = 0.853, P = 0.031), which suggested that dietary Val ameliorated ERS- mediated apoptosis by reducing the expression of related genes expression, which was partly due to decreased ROS accumulation. The decreased ROS levels induced by Val may be a consequence of the contribution of Val to Gln synthesis. Val is a substrate for Gln synthesis [62]. A previous study showed that Gln significantly decreased ROS levels in fish [63]. However, the mechanisms responsible for these effects require further investigation.