Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Animal Science
Non Technical Summary
Non-Technical Project SummaryThe United States is the top producer of hybrid striped bass (HSB) in the world. Fishmeal has traditionally been used as the major protein feedstuff in HSB diets, but this approach is not sustainable in aquaculture due to the increasingly limited sources and high costs of fishmeal. Thus, there has been growing interest in using plant proteins (mainly soybean meal) to replace fishmeal in aquafoods, but success has been variable. We found that glycine (the most abundant amino acid in the body of fish and fishmeal) is relatively low in plant proteins. Based on results of our preliminary study, we hypothesize that dietary glycine plays an important role in HSB growth by maximizing protein synthesis, anti-oxidative capacity, and creatine (muscle builder) production in their tissues. We will test this hypothesis by pursuing three specific objectives to determine: (1) tissue-specific glycine- and creatine-synthetic pathways in HSB; (2) roles of glutathione (the most abundant low-molecular weight anti-oxidant) and creatine in mediating the effect of dietary glycine to improve growth, anti-oxidative responses, intestinal integrity, metabolic health, and immunity in HSB; and (3) roles of the target of rapamycin (TOR; the master regulator of protein synthesis) and autophagy/proteasomes (intracellular machineries for protein degradation) in mediating the effect of dietary glycine to promote protein synthesis and inhibit proteolysis in tissues of HSB. We expect that: (1) in contrast to land mammals, skeletal muscle is the major site for creatine synthesis from glycine in HSB; (2) glycine activates the TOR cell signaling pathway to promote protein synthesis in skeletal muscle, while reducing intramuscular protein breakdown via the autophagy/proteasomes pathways, to promote muscle growth; and (3) supplementing 2% glycine to soybean meal (58%)-based diets can replace 45% fishmeal in HSB diets. Our findings will have a significant impact on U.S. aquaculture by generating new fundamental knowledge about the crucial role of glycine in improving the growth of HSB and providing a new nutritional method for the use of glycine to substantially reduce the inclusion level of fishmeal in soybean meal-based diets for the fish.
Animal Health Component
15%
Research Effort Categories
Basic
85%
Applied
15%
Developmental
(N/A)
Goals / Objectives
The overall goal of our research is to improve the growth and productivity of hybrid striped bass (HSB), while developing effective nutritional strategies to replace fishmeal in their diets. In the current project, we will pursue three specific objectives to determine: (1) tissue-specific glycine- and creatine-synthetic pathways in HSB; (2) roles of glutathione (the most abundant low-molecular weight anti-oxidant) and creatine in mediating the effect of dietary glycine to improve growth, anti-oxidative responses, intestinal integrity, metabolic health, and immunity in HSB; and (3) roles of the target of rapamycin (TOR; the master regulator of protein synthesis) and autophagy/proteasomes (intracellular machineries for protein degradation) in mediating the effect of dietary glycine to promote protein synthesis and inhibit proteolysis in tissues of HSB.
Project Methods
Juvenile HSB (Morone saxatilis ?) x white bass (M. chrysops ?) with a body weight (BW) of ~ 3 g will be obtained from Keo Fish Farms (Keo, AR), and maintained in our indoor water recirculation system (55 L water per tank) at 26°C. Water is circulated through mechanical and biological filters and changed regularly (30-50% every 3 days). Air is supplied to the water through air stones connected to air pumps. The dissolved oxygen in water is maintained above 6 mg/L, its salinity at 1 ppt, and the photoperiod of the housing facility at 12 h per day. Water quality parameters (pH, ammonia, nitrite, and nitrate) are monitored daily and maintained within acceptable limits. Upon arrival at Texas A&M University, fish will be fed twice daily with 60% fishmeal diet for 1 week during the period of adaptation. Thereafter, the fish will be used for our study and fed our experimental diets.Specific Aim 1: HSB will be fed a low (15%)-fishmeal, soybean meal (58%)-based practical diet. On Days 0, 30, 60, 120, 180, HSB will be euthanized to obtain the stomach, proximal intestine, liver, skeletal muscle, kidney, heart, gill, skin, spleen, pancreas, and brain for enzyme assays. There will be 8 fish per age group. Frozen tissue (0.5 g), which will be combined from several fish at young ages, will be homogenized in 2 ml of freshly prepared buffer consisting of 300 mM sucrose, 5 mM HEPES (pH 7.4), 1 mM EDTA, 3 mM dithiothreitol, 0.5% (vol:vol) Triton X-100, and 0.1% (wt:vol) protease inhibitor (aprotinin, chymostatin, pepstatin A, and phenylmethylsulfonyl fluoride; 5 µg/ml each) for 2 min on ice. The whole homogenization mixture will be transferred to a tube. All tubes will be centrifuged at 600 × g for 10 min at 4°C. The supernatant fluid will be subjected to three cycles of freezing in liquid nitrogen and thawing in a 26°C water bath before use for determining the activities of the following enzymes: 4-hydroxyproline oxidase, 4-hydroxy-2-oxoglutarate aldolase, glutamate-pyruvate transaminase, alanine:glyoxylate transaminase, threonine dehydrogenase, serine hydroxymethyltransferase, arginine:glycine amidinotransferase (AGAT), guanidinoacetate N-methyltransferase (GAMT), S-adenosylmethionine synthetase, and creatine kinase. Furthermore, we will use (proximal intestine, liver, skeletal muscle, kidney, and skin) for incubation to directly determine the conversion of 4-hydroxyproline into glycine. The tissue extracts will be used for the HPLC analysis of glycine and other amino acids.Specific Aim 2: Beginning at ~5 g of BW, HSB will be fed a low (15%)-fishmeal, soybean meal (58%)-based practical diet supplemented with: 0% glycine, 2% glycine, 0.1% L-buthionine sulfoximine (BSO), 2% glycine + 0.1% BSO, 2% glycine + 0.1% BSO + 0.2% glutathione, 0.2% guanidine, 2% glycine + 0.2% guanidine, and 2% glycine + 0.2% guanidine + 1% creatine. BSO or glutathione will be included in the pelleted diet as a convenient way to administer the chemical to fish. An appropriate amount of alanine will be added to the diets other than the 2% glycine group, so that all diets will be isonitrogenous. There will be 8 tanks of fish (15 fish/tank) in each treatment group (a total of 960 fish; i.e., 8 groups x 8 tanks/group x 15 fish/tank = 960 fish for the trial). Fish will be fed their diets twice daily (8:00 AM and 8:00 PM) to apparent satiety for 8 weeks. Feed intakes of fish in each tank will be measured daily and their BWs be recorded every 4 weeks, respectively. At the end of the feeding period, the following procedures will be performed: (1) calculations of growth performance and feed efficiency, as well as measurements of the body composition of fish; (2) blood collection for analyses of amino acids, glutathione, and malonaldehyde (a product of lipid peroxidation); (3) analyses of intestinal morphology, expression of glycine transporters, and tight-junction proteins; (4) determination of intestinal amino acid transport using Ussing chambers and labeled amino acids; (5) analyses of concentrations of amino acids and glutathione, as well as the activities of glycine-synthetic enzymes in skeletal muscle, liver, kidney, and proximal intestine; (6) analyses of concentrations of creatine, creatine phosphate, and the activities of related enzymes in the skeletal muscle, liver, pancreas, kidney, and proximal intestine; and (7) immunological assessments of HSB challenged with 100 µg of trinitrophenyl-lipopolysaccharide.Specific Aim 3: Beginning at ~5 g of BW, HSB will be fed a low (15%)-fishmeal, soybean meal (58%)-based practical diet supplemented with: 0% glycine, 2% glycine, 10 ppm rapamycin, 2% glycine + 10 ppm rapamycin. Rapamycin (a specific inhibitor of TOR) will be included in the pelleted diet as a convenient way to administer the substance to fish. An appropriate amount of alanine will be added to the diets other than the 2% glycine group, so that all diets will be isonitrogenous. There will be 8 tanks of fish (15 fish/tank) in each treatment group (a total of 480 fish; i.e., 4 groups x 8 tanks/group x 15 fish/tank = 480 fish for the trial). Fish will be fed their diets twice daily (8:00 AM and 8:00 PM) to apparent satiety for 8 weeks. Feed intakes of fish in each tank will be recorded daily and BWs of fish will be measured every 4 weeks. At the end of the 8-week feeding period, the following procedures will be performed: (1) calculations of growth performance and feed efficiency, as well as measurements of the body composition of fish; (2) collection of blood and other tissues for analyses of amino acids and glutathione; (3) determination of protein synthesis in skeletal muscle, liver, kidney, and proximal intestine, using the [3H]phenylalanine flooding dose method; (4) analysis of TOR cell signaling proteins in skeletal muscle, liver, kidney, and proximal intestine, using western blot techniques. In addition, we will determine rates of proteolysis in the skeletal muscle, liver, kidney, and proximal intestine of HSB via the autophagy-lysosomal pathway and ubiquitin-dependent proteasomes.