DL-Methionyl-DL-Methionine/DL-Methionine Supplementation Alleviated the Adverse Effects of Dietary Low Fishmeal Levels on Growth and Intestinal Health of Micropterus salmoides.

DL-methionyl-DL-methionine (AQUAVI® Met-Met) (Met-Met) (0.10%, 0.20%, 0.30%, and 0.40%) or DL-methionine (DL-Met) (0.10%, 0.20%, 0.30%, and 0.40%) were added to a low-fishmeal diet in an attempt to reduce fishmeal in the diet of Micropterus salmoides (M. salmoides). The fish were randomly allocated into ten experimental groups (n = 100), each with 4 replicates of 25 fish (16.39 ± 0.01 g) each. Compared to 25% FM, 0.40% of DL-Met and 0.10% of Met-Met promoted growth, and 0.10% of Met-Met decreased FCR. Compared to 25% FM, the supplementation of Met-Met or DL-Met improved the intestinal antioxidant capacity by upregulating the NF-E2-related factor 2-mediated antioxidant factors and enzyme activities and nuclear factor kappa-B-mediated anti-inflammatory factors while downregulating the pro-inflammatory factors, thereby exerting anti-inflammatory effects. Moreover, 0.10% of the Met-Met diet affected the Firmicutes-to-Bacteroidota ratio, increased the levels of Proteobacteria, changed the composition of intestinal flora (Roseburia, Lachnospiraceae_NK4A136_group, and unclassified_Oscillospiraceae), and enhanced intestinal dominant bacteria (Caldicoprobacter, Pseudogracilibacillus, and Parasutterella), leading to improved gut health. In summary, the supplementation of DL-Met or Met-Met alleviated the adverse effect of fishmeal reduction (from 40 to 25%) on the growth performance and intestinal health of M. salmoides.

Effects of dietary supplementation of Bacillus subtilis DSM 32315 on growth, immune response and acute ammonia stress tolerance of Nile tilapia (Oreochromis niloticus) fed with high or low protein diets.

Aquatic animals have benefited from Bacillus subtilis-based probiotics over the past few decades. This study evaluated the effects of B. subtilis DSM 32315 probiotics as a feed additive on growth, immune response and resistance to acute ammonia challenge in Nile tilapia. Specifically, four supplemental levels (0%, 0.1%, 0.2%, and 0.3%) of B. subtilis probiotics were tested under two dietary protein levels (32% and 28%). Five replicate tanks were randomly allotted to each dietary treatment, with each tank containing 30 Nile tilapia. After 8 weeks of feeding, Nile tilapia in each tank were exposed to 43.61 mg/L of total ammonia nitrogen for 48 h. The results revealed that reducing protein levels from 32% to 28% did not affect growth performance or antioxidant capacity. However, the low protein diet tended to induce an inflammatory effect shown by increased expressions of TGF-ß and IFN-γ genes (P < 0.05) in the liver. The impact was alleviated by the probiotic supplementation. Compared with the non-supplemented group, 0.1% probiotic supplementation remarkably increased plasma lysozyme activity, total antioxidant capacity and complement C3 and interleukin-10 mRNA levels (P < 0.05) in the 28% protein diet, while higher supplementation of probiotics (0.3%) was shown to be beneficial for the high protein diet (32%). In both the dietary protein levels, 0.1% supplementation of probiotics promoted the antioxidant capacity of Nile tilapia before exposure to ammonia stress but higher probiotic supplementation (0.3%) proved to be necessary under ammonia stress as evidenced by higher fish survival rate. Results exhibited that supplementation with B. subtilis probiotics had a better effect on the intestinal morphology (villi height and width) regardless of protein levels. In conclusion, dietary supplementation of B. subtilis DSM 32315 probiotics at 0.1% in the low protein diet and up to 0.3% in the high protein diet showed beneficial effects on the growth, immunity, and antioxidant capacity of Nile tilapia. Under ammonia stress conditions, the higher supplementation of B. subtilis DSM 32315 probiotics at 0.3% improves stress tolerance of Nile tilapia despite the two dietary protein levels (32%; 28%).

DL-methionyl-DL-methionine as an efficient methionine source for promoting zootechnical performance and methionine-related pathways in the whiteleg shrimp (Penaeus vannamei).

Methionine (MET) supplementation is a current strategy to achieve shrimp requirement. Notwithstanding, the efficiency of the precisely formulated feeds can be diminished since shrimps are slow eaters and masticate feed externally that results in nutrient leaching. In this regard, a methionine dipeptide (DL-methionyl DL-methionine) benefits the feed industry by reducing MET water solubility while increasing its bioavailability. Therefore, the effects of feeding whiteleg shrimp (Penaeus vannamei) with increasing levels of methionine dipeptide were evaluated on zootechnical performance and methionine-, immune- and antioxidant-related pathways. A 74 d growth trial was conducted by feeding a control diet and four diets supplemented with AQUAVI® Met-Met at 0·08, 0·12, 0·24 and 0·32% of DM. Diet digestibility, body amino acids (AA) composition and nitrogen metabolites, metabolic enzymes, oxidative status and gene expression were evaluated. It can be concluded that graded dietary increase of methionine dipeptide up to 0·24 % for 74 d translated in significant gains on the growth performance, feed efficiency, nutrient and nitrogen gain and shrimp survival. Moreover, it was showed that Met-Met dietary spare leads to an improvement of free-AA pool and nitrogen metabolites concentration and reduces the signs of oxidative stress. Finally, in a closer look to the MET-related pathways passive to be altered by Met-Met spare, a clear modulation of the described antioxidant and cell proliferation routes was detected.

Are the Main Methionine Sources Equivalent? A Focus on DL-Methionine and DL-Methionine Hydroxy Analog Reveals Differences on Rainbow Trout Hepatic Cell Lines Functions.

The replacement of fishmeal by plant proteins in aquafeeds imposes the use of synthetic methionine (MET) sources to balance the amino acid composition of alternative diets and so to meet the metabolic needs of fish of agronomic interest such as rainbow trout (RT-Oncorhynchus mykiss). Nonetheless, debates still exist to determine if one MET source is more efficiently used than another by fish. To address this question, the use of fish cell lines appeared a convenient strategy, since it allowed to perfectly control cell growing conditions notably by fully depleting MET from the media and studying which MET source is capable to restore cell growth/proliferation and metabolism when supplemented back. Thus, results of cell proliferation assays, Western blots, RT-qPCR and liquid chromatography analyses from two RT liver-derived cell lines revealed a better absorption and metabolization of DL-MET than DL-Methionine Hydroxy Analog (MHA) with the activation of the mechanistic Target Of Rapamycin (mTOR) pathway for DL-MET and the activation of integrated stress response (ISR) pathway for MHA. Altogether, the results clearly allow to conclude that both synthetic MET sources are not biologically equivalent, suggesting similar in vivo effects in RT liver and, therefore, questioning the MHA efficiencies in other RT tissues.

Metabolic and nutritional responses of Nile tilapia juveniles to dietary methionine sources.

Commercial diets for tilapia juveniles contain high levels of plant protein sources. Soybean meal has been utilised due to its high protein content; however, soy-based diets are limited in methionine (Met) and require its supplementation to fulfil fish requirements. dl-Methinone (dl-Met) and Ca bis-methionine hydroxyl analogue (MHA-Ca) are synthetic Met sources supplemented in aquafeeds, which may differ in biological efficiency due to structural differences. The present study evaluated the effect of both methionine sources on metabolism and growth of Nile tilapia. A growth trial was performed using three isonitrogenous and isoenergetic diets, containing plant ingredients as protein sources: DLM and MHA diets were supplemented on equimolar levels of Met, while REF diet was not supplemented. Hepatic free Met and one-carbon metabolites were determined in fish fed for 57 d. Metabolism of dl-Met and MHA was analysed by an in vivo time-course trial using 14C-labelled tracers. Only dl-Met supplementation significantly increased final body weight and improved feed conversion and protein efficiency ratios compared with the REF diet. Our findings indicate that Met in DLM fed fish follows the transsulphuration pathway, while in fish fed MHA and REF diets it is remethylated. The in vivo trial revealed that 14C-dl-Met is absorbed faster and more retained than 14C-MHA, resulting in a greater availability of free Met in the tissues when fish is fed with DLM diet. Our study indicates that dietary dl-Met supplementation improves growth performance and N retention, and that Met absorption and utilisation are influenced by the dietary source in tilapia juveniles.

Optimal Levels of Fish Meal and Methionine in Diets for Juvenile Litopenaeus vannamei to Support Maximum Growth Performance with Economic Efficiency.

This work investigated the optimal levels of fish meal (FML) and dietary methionine (Met) required for maximum growth performance of juvenile Litopenaeus vannamei with economic efficiency. Four sets of diets were prepared to contain 0.00, 6.00, 12.00 and 18.00% FML. Each set was supplemented with DL-methionyl-DL-methionine (DL-Met-Met) to result in a total dietary Met (Met + Cys) content of 0.58 (1.05), 0.69 (1.16), and 0.82% (1.29%), on a fed basis. Shrimp of 1.00 ± 0.08 g were stocked in 60 outdoor tanks of 1 m3 with 100 shrimp/m2, allowing five replications per dietary group. Shrimp in all the groups were fed 10 times daily for 70 days. In a subsequent trial, dietary protein and amino acid digestibility of four FML groups, but only at high dietary Met levels (~0.82%), were evaluated in 40 60 L indoor tanks (11 replicates per diet) for 93 days with 70 shrimp/m2. Final shrimp survival (92.85 ± 4.82%, mean ± standard deviation), weekly weight gain (1.17 ± 0.08 g), apparent feed intake (13.3 ± 0.5 g of feed per stocked shrimp), and feed conversion ratio (1.18 ± 0.06) were unaffected by dietary FML level and Met content. Gained yield was adversely affected when FML was reduced from 18 and 12% (1156 and 1167 g/m2, respectively) to 0 (1090 g/m2), but no change was observed at 6% (1121 g/m2). A significant interaction was detected between FML level and dietary Met. Under 0 and 6% FML conditions, higher levels of total dietary Met, 0.69 and 0.82%, respectively, were required to maximize shrimp BW. In comparison, at 12 and 18% FML, a dietary Met content of only 0.58% was sufficient. Overall, results indicated the use of FML can be minimized or completely eliminated without major detrimental effects on feed digestibility or shrimp growth performance, as long as proper supplementation of Met is carried out. Diets with 0 FML or with only 6% delivered the highest profit and return on investment compared to diets with higher levels.

Critical transporters of methionine and methionine hydroxyl analogue supplements across the intestine: What we know so far and what can be learned to advance animal nutrition.

DL-methionine (DL-Met) and its analogue DL-2-hydroxy-4-(methylthio) butanoic acid (DL-methionine hydroxyl analogue or DL-MHA) have been used as nutritional supplements in the diets of farmed raised animals. Knowledge of the intestinal transport mechanisms involved in these products is important for developing dietary strategies. This review provides updated information of the expression, function, and transport kinetics in the intestine of known Met-linked transporters along with putative MHA-linked transporters. As a neutral amino acid (AA), the transport of DL-Met is facilitated by multiple apical sodium-dependent/-independent high-/low-affinity transporters such as ASCT2, B0AT1 and rBAT/b0,+AT. The basolateral transport largely relies on the rate-limiting uniporter LAT4, while the presence of the basolateral antiporter y+LAT1 is probably necessary for exchanging intracellular cationic AAs and Met in the blood. In contrast, the intestinal transport kinetics of DL-MHA have been scarcely studied. DL-MHA transport is generally accepted to be mediated simply by the proton-dependent monocarboxylate transporter MCT1. However, in-depth mechanistic studies have indicated that DL-MHA transport is also achieved through apical sodium monocarboxylate transporters (SMCTs). In any case, reliance on either a proton or sodium gradient would thus require energy input for both Met and MHA transport. This expanding knowledge of the specific transporters involved now allows us to assess the effect of dietary ingredients on the expression and function of these transporters. Potentially, the resulting information could be furthered with selective breeding to reduce overall feed costs.

Characterization of the segmental transport mechanisms of DL-methionine hydroxy analogue along the intestinal tract of rainbow trout with an additional comparison to DL-methionine.

The aim of this study was to identify the unknown transport mechanism of the extensively used monocarboxylate methionine feed supplement DL-methionine hydroxy analogue (DL-MHA) in rainbow trout intestine. Transport across the pyloric caeca (PC), midgut (MG), and hindgut (HG) regions were kinetically studied in Na+- and H+-dependent manners. Gene expression of monocarboxylate (MCTs) and sodium monocarboxylate transporters (SMCTs) were assessed. Results demonstrated that DL-MHA transport from 0.2-20 mM was Na+-dependent and obeyed Michaelis-Menten kinetics with low affinity in PC & MG in apical/basal pH of 7.7/7.7. Changes in apical/basal pH (6.0/6.0, 6.0/7.7, and 7.7/8.7) had insignificant effects on kinetics. In contrast, HG flux kinetics were only obtained in pH 7.7/8.7 or in the presence of lactate with medium affinity. Additionally, DL-MHA transport from 0-150 µM demonstrated the presence of a Na+-dependent high-affinity transporter in PC & MG. Conclusively, two distinct carrier-mediated DL-MHA transport mechanisms along the trout gut were found: 1) in PC & MG: apical transport was regulated by Na+-requiring systems that possibly contained low- and high-affinity transporters, and basolateral transport was primarily achieved through a H+-independent transporter; 2) in HG: uptake was apically mediated by a Na+-dependent transporter with medium affinity, and basolateral exit was largely controlled by an H+-dependent transporter. Finally, two major methionine feed supplements, DL-MHA and DL-methionine (DL-Met) were compared to understand the differences in their bioefficacy. Flux rates of DL-MHA were only about 42.2-66.0% in PC and MG compared to DL-Met, suggesting intestinal transport of DL-MHA was lower than DL-Met.

SLC transporters ASCT2, B0 AT1-like, y+ LAT1, and LAT4-like associate with methionine electrogenic and radio-isotope flux kinetics in rainbow trout intestine.

Methionine (Met) is an important building block and metabolite for protein biosynthesis. However, the mechanism behind its absorption in the fish gut has not been elucidated. Here, we describe the fundamental properties of Met transport along trout gut at µmol/L and mmol/L concentration. Both electrogenic and unidirectional DL-[14 C]Met flux were employed to characterize Met transporters in Ussing chambers. Exploiting the differences in gene expression between diploid (2N) and triploid (3N) and intestinal segment as tools, allowed the association between gene and methionine transport. Specifically, three intestinal segments including pyloric caeca (PC), midgut (MG), and hindgut (HG) were assessed. Results at 0-150 µmol/L concentration demonstrated that the DL-Met was most likely transported by apical transporter ASCT2 (SLC1A5) and recycled by basolateral transporter y+ LAT1 (SLC7A7) due to five lines of observation: (1) lack of Na+ -independent kinetics, (2) low expression of B0 AT2-like gene, (3) Na+ -dependent, high-affinity (Km , µmol/L ranges) kinetics in DL-[14 C]Met flux, (4) association mRNA expression with the high-affinity kinetics and (5) electrogenic currents induced by Met. Results at 0.2-20 mmol/L concentration suggested that the DL-Met transport is likely transported by B0 AT1-like (SLC6A19-like) based on gene expression, Na+ -dependence and low-affinity kinetics (Km , mmol/L ranges). Similarly, genomic and gene expression analysis suggest that the basolateral exit of methionine was primarily through LAT4-like transporter (SLC43A2-like). Conclusively, DL-Met uptake in trout gut was most likely governed by Na+ -dependent apical transporters ASCT2 and B0 AT1-like and released through basolateral LAT4-like, with some recycling through y+ LAT1. A comparatively simpler model than that previously described in mammals.

Early feeding of rainbow trout (Oncorhynchus mykiss) with methionine-deficient diet over a 2 week period: consequences for liver mitochondria in juveniles.

Methionine is a key factor in modulating the cellular availability of the main biological methyl donor S-adenosylmethionine (SAM), which is required for all biological methylation reactions including DNA and histone methylation. As such, it represents a potential critical factor in nutritional programming. Here, we investigated whether early methionine restriction at first feeding could have long-term programmed metabolic consequences in rainbow trout. For this purpose, trout fry were fed with either a control diet (C) or a methionine-deficient diet (MD) for 2 weeks from the first exogenous feeding. Next, fish were subjected to a 5 month growth trial with a standard diet followed by a 2 week challenge (with the MD or C diet) to test the programming effect of the early methionine restriction. The results showed that, whatever the dietary treatment of fry, the 2 week challenge with the MD diet led to a general mitochondrial defect associated with an increase in endoplasmic reticulum stress, mitophagy and apoptosis, highlighting the existence of complex cross-talk between these different functions. Moreover, for the first time, we also observed that fish fed the MD diet at the first meal later exhibited an increase in several critical factors of mitophagy, hinting that the early nutritional stimulus with methionine deficiency resulted in long-term programming of this cell function. Together, these data extend our understanding of the role of dietary methionine and emphasize the potential for this amino acid in the application of new feeding strategies, such as nutritional programming, to optimize the nutrition and health of farmed fish.