Use of plant-based ingredients (PBI) to feed farmed fish is limited because of anti-nutritional factors and limiting amino acids especially lysine (Lys) and methionine (Met) which are the first limiting indispensable amino acids (IDAA) in plant sources. As one of the strategies to increase the limited use of PBI, we hypothesized that “interchangeable imbalanced-balanced feeding strategy” of IDAA with limited and complementary diets would increase the utilization efficiency of limiting amino acids. This feeding strategy of nutrient intake may in fact be similar to the situation in nature, where animals acquire food from different sources containing complementary nutrients. Diverse food sources may satisfy the nutritional requirement of animals in nature. This study addressed the effect of dietary level of limiting IDAA in relation to alternative feed sources and feeding strategies in Atlantic salmon juveniles. Four sub-objectives were addressed to accept or reject the hypothesis of: the essentiality and interaction of dietary lysine and methionine supplements in Atlantic salmon alevins (Chapter 2); the effect of dietary methionine concentrations, and evaluation of feeding strategies alternating methionine delivery with imbalanced IDAA (insufficient or enriched in methionine) and complete IDAA diets (Chapter 3); investigation of the efficiency of methionine under an alternative feeding strategy for practical diet and to determine the maximum use of soybean meals in Atlantic salmon starter diet (Chapter 4); and examination of the effect of dietary methionine deficiency on the activity of digestive enzymes that are critical in early life stages of fish (Chapter 5).
In chapter 2, fish alevins (160±4 mg) at the swim-up stage were randomly distributed into eighteen tanks at a density of 44 fish/tank (3 replicates) at 15°C in a semi-recirculation system. A casein-gelatin (CG) based, semi-purified diet was formulated (control) and four free amino acid (AA) diets that replaced 58% of the protein in the CG diet with an AA mixture were prepared either devoid or supplemented with lysine and/or methionine (designated as: +L+M, -L+M, +L-M, and -L-M, respectively). Fish were fed one of five experimental diets at 1-3% of biomass for 13 weeks. A commercial diet (CD) was also used as a reference diet. Depleted levels of dietary methionine in Atlantic salmon alevins have a striking impact on growth with severe cataract development. Feeding activity and protein efficiency ratio in the fish fed methionine deficient diet decreased indicating anorexia. The pool of indispensable free AA in muscle was decreased when dietary AA was insufficient. The increased 24 h postprandial hepatosomatic index (HSI) in AA imbalanced groups might be related to enhanced deposition of glycogen and lipids from protein degradation. Postprandial metabolism of fatty acid synthase (FAS) gene expression was 3-fold higher compared to the control group. These results indicate that deficiency of dietary Met severely impact growth, free amino acid pools in muscles, and bilateral cataract development with metabolic changes (HSI and FAS gene expression) in Atlantic salmon alevins.
To further investigate how the mechanisms of methionine deficiency affect metabolic processes, we designed “interchangeable imbalanced-balanced feeding strategy” with a cycling provision of dietary methionine (at hourly or daily intervals) to improve growth by up-regulating the efficiency of methionine utilization in protein synthesis and transmethylation (chapter 3). Salmon alevins (265±3 mg) were randomly distributed into 24 tanks at a density of 50 fish/tank (3 replicates) at 15°C in a semi-recirculation system. Casein-gelatin based semi-purified diets were formulated by replacing 58% of the protein content with the free amino acid mixture. Experimental diets were supplemented with L-methionine at levels of 0, 1.9, 5.8, and 17.4 g/kg (designated as M0, M1/3, M1, and M3, respectively). An additional diet without glycine addressed methionine toxicity (M3-G). Fish were fed three meals a day with one of the five experimental diets. Alternative feeding groups included: AF1-sequentially fed M0 for 2 d followed by M1 for 1d; AF2 fed M0 for 2 d followed by M3 for 1 d; AF3 fed M0 in two meals followed by one meal of M3. Weight gain was correlated with an increase of dietary methionine levels after 9 weeks of the feeding trial, while M0 fed fish were significantly smaller. Anorexia and mild cataracts were observed in the M0, M1/3, and AF1 groups and paralleled the increasing trend in feed consumption time. AF2 fed fish showed the most voracious feeding behavior. The increased HSI at 8 h postprandial fish fed M0 may indicate an enhanced deposition of glycogen and lipids from protein degradation. From these results, it can be suggested that a cycling provision of dietary methionine may increase growth performance by increasing the palatability in long-term feeding trial experiments.
In chapter 4, the study aimed to investigate the possibility of an increasing substitution of fish meal protein by plant sources on growth in juvenile Atlantic salmon. The fish also tested “interchangeable imbalanced-balanced feeding strategy” with practical feed ingredients as a fish meal replacer: SBM3010 (Schillinger Genetics) and Profine VF (Solae), which are genetically improved soybean meal, and soy protein concentrate, respectively. Sardine meal as a major protein source (control) was substituted with a mixture of PBI (50:50) at the level of 25, 50, and 75% (designated as SP25, SP50, and SP75, respectively). SP diets were divided into 3 batches and reformulated to include three different concentrations of methionine at level 0, the level equal to control (requirement), and 3-fold higher than requirement (M0, M1, and M3, respectively). The diets were designated as SP25-M0, SP25-M1, SP25-M3, SP50-M0, SP50-M1, SP50-M3, SP75-M0, SP75-M1, and SP75-M3. Five mono-feed groups were fed control diet or one of SP diets (SP25-M1, SP50-M1, and SP75-M1). Three duo-feed groups included: AF1-sequentially fed SP25-M0 for 2 d followed by SP25-M3 for 1d; AF2 fed SP50-M0 for 2 d followed by SP50-M3 for 1 d; AF3 fed SP75-M0 for 2 d followed by SP75-M3 for 1 d. Salmon alevins (639±24 mg) were randomly distributed into 24 tanks at a density of 35 fish/tank (3 replicates) at 15°C in a semi-recirculation system. Fish were fed diets with one of 8 dietary regimes for 9 weeks. Compromised growth was observed in AF50, AF75, and SP75-M1. Interestingly there were no differences in growth performance between control and SP50-M1 groups. No differences were observed in the results of HSI, visceral somatic index (VSI), and pH levels of stomach and rectum among dietary treatments. Feeding ad libitum during the last 3 days toward the end of 9 weeks showed no differences of feed intake among treatments. There are trends of decrease and increase in the level of hydroxyproline and methionine, respectively, in muscle of fish in duo-feed regime in comparison to mono-feed regime. A major finding of this study is that cycling provision of methionine in combination with a balanced or imbalanced dietary methionine seems to increase the efficiency of its retention and utilization, as shown in the level of methionine in muscle.
It was hypothesized in chapter 5 that Met deficiency may be directly responsible for the synthesis/secretion and consequent activity of digestive enzymes particularly critical in early life stage of fish. The studies described in chapter 2 and 3 were designated as experiment 1 and 3. In experiment 1, dietary lysine and methionine supplements were reflected in the level of these amino acids in muscle. Methionine deficiency was responsible for a decreased specific growth rate and compromised proteolytic enzyme activity including pepsin, trypsin, chymotrypsin, aminopeptidase, and alkaline phosphatase. However, Lys deficiency has not shown such a trend. To address possible impact of fish size on enzyme activity in experiment 1, additional feeding experiment (experiment 2) was conducted with 2 factorial designs (fish size x feeding ration). Following the last meal of commercial diet, activity of pepsin, trypsin, and chymotrypsin were analyzed at 4, 8, 16, 24, and 48 h in the gastro-intestinal tract. It was found that fish size was a significant factor in pepsin activity at 4, and 16 h, and feeding ration and their interaction influenced the chymotrypsin activity at 8 h postprandial. At other time points activities of proteases has not influenced by any of the examined factors. Overall enzyme activity was not significantly changed due to fish size or feeding ration. In experiment 3, the relation between dietary methionine and enzyme activity after 9 weeks of feeding trial indicated that the fish fed a diet with low methionine content (M0, and M1/3) showed depressed activity in pancreatic (trypsin and chymotrypsin) and intestinal brush border (aminopeptidase, and alkaline phosphatase) enzymes. Those fed diet with alternating feeding schedule also exhibited decreased activity of these enzymes, although overall AF2 and AF3 were fed the same amount of dietary methionine on the long term basis as the control. Only the M0 group (unsupplemented with methionine), however, had significantly lower weight gain. These results for the first time in fish nutrition research indicate that dietary methionine is general physiological response of digestive system, in particular leading to depressed function of pancreas.