Recipient Organization
UNIV OF IDAHO
875 PERIMETER DRIVE
MOSCOW,ID 83844-9803
Performing Department
Animal & Veterinary Science
Non Technical Summary
Within aquaculture, fishmeal (FM) is the primary source of protein for manufactured feeds and the industry currently uses the majority of FM and fish oil (FO) produced globally. Despite significant reductions in poultry, beef and swine feeds, global supplies of FM and FO remain insufficient to meet projected future demand. The reality of finite supplies has necessitated the search for alternatives to FM and FO for use in aquaculture feeds. Plant-based proteins offer a logical substitute since they are abundant, cost-effective and readily available. Research on alternative protein sources to replace FM for the culture of fish has been intensive and most often include the use of various forms and extractions of soybean meal (SBM). Unfortunately, detrimental effects on fish growth, feed efficiency and overall health have often been observed with SBM. Although considerable work has examined alternatives to FM and FO in aquafeeds for salmonids, evaluation strategies for aquaculture feed ingredients must also consider the fish consuming the aquaculture products. This points out the need to not only explore alternative feeds but also ways finfish and shellfish may be genetically selected to utilize alternative feed components thereby minimizing FM and FO in aquafeeds. Our overall goal is to increase soybean meal (SBM) inclusion levels in trout feeds by elucidating the resistance to the development of distal intestinal enteritis observed in a selective-bred strain of rainbow trout. We also intend to examine how this selected strain differentially utilizes essential, limiting amino acids and fatty acids. Characterizing strain selection and dietary strategies in rainbow trout that improve their production efficiency in the United Stateswill help tominimize the use of FM and FO in aquafeeds.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
GOAL 1: Assess oral tolerance and inflammatory responses to alternative proteinsOur overall goal is to increase soybean meal (SBM) inclusion levels in trout feeds by elucidating the resistance to the development of distal intestinal enteritis observed in a selective-bred strain of rainbow trout. The proposed objectives will determine the effects of high dietary soy on the expression of immunogenic genes during the progression or resistance to the development of distal enteritis in selected and non-selected strains of rainbow trout. Understanding the ability of selected strain fish to consume higher amounts of SBM without developing significant distal intestinal enteritis will assist in efforts to increase FM replacement with SBM in salmonid diets. Examining the underlying differences in immune response between selected and non-selected fish will increase our understanding of the relatively unclear mechanisms by which SBM inclusion in feed induces intestinal enteritis in susceptible species.This goal leverages the collaborative, USDA-funded, long-term selection program on rainbow trout at the Hagerman Fish Culture Experiment Station. It will advance current knowledge in rainbow trout nutrition using rainbow trout selected for resistance to SBM-derived distal enteritis and selected for faster growth on an all plant diet.GOAL 2: Optimize Fishmeal and Fish oil in diets of selected line rainbow troutOur long-term goal is to characterize strain selection and dietary strategies in rainbow trout that improve their production efficiency in the United States and minimize the use of FM and FO in aquafeeds. Toward that end, our three objectives are to (1) Assess differences in limiting amino acid metabolism between selected vs. non-selected lines of rainbow trout; (2) Assess fatty acid differences in selected vs. non-selected lines of rainbow trout; (3) Disseminate project results broadly to growers, feed manufacturers, and aquaculture research and extension specialists to provide for informed decisions regarding supplementation of diets with rainbow trout selected to utilize plant proteins and plant oils. This goal also leverages the collaborative, USDA-funded, long-term selection program on rainbow trout at the Hagerman Fish Culture Experiment Station. This study will provide mechanistic, practical information for fish nutritionists, feed manufacturers and culturists regarding catabolic and anabolic metabolism of long-chain polyunsaturated fatty acids (lcPUFAs) and two essential amino acids which are often limiting in plant proteins (lysine and methionine). This knowledge will enhance our understanding of how selected rainbow trout use fatty acids and amino acids differently compared to current commercial lines and help move the industry forward by optimizing FO and FM use in aquaculture diets.
Project Methods
Objective 1: The experimental design compares expression of immunogenic genes in the distal intestine of a selected-strain of rainbow trout showing resistance to enteritis to a susceptible strain when fed a high SBM (40%) diet. A two-treatment design using a triplicate experimental units (tanks) of selected- and wild-type strain rainbow trout will be utilized (i.e. a 2 x 2 factorial design). An expreimental diet will be formulated and fed to rainbow trout replacing fishmeal protein with protein from SBM as in Blaufuss et al. (2019). All diets will be formulated to meet or exceed other known nutrient requirements (NRC 2011). Yttrium oxide will be included as an indigestible inert marker to quantify nutrient digestibility/absorption. Rainbow trout will be stocked into a recirculating system consisting of 320-L poly tanks and water temperature will be maintained at 15 °C. Fish will be fed twice daily 6 days per week for 12 weeks to apparent satiation; consumed feed will be recorded weekly. All fish in a tank will be counted and weighed every 4 weeks. A subset of fish from each tank will be sampled at 1, 2, 4, 8, and 12 weeks for mid-intestine and distal intestine; mid-intestine as described in Blaufuss et al. 2019. Histological examinations will be conducted to determine if any significant pathological changes are present by following established criteria (as per Penn et al. 2011; Sealey et al. 2013; Krogdahl et al. 2015). Proximate composition of diets, feces and whole-body samples will be performed according to standard methods (AOAC 1995). Amino acids in diets and whole fish samples will be quantified on a Biochrom 30+ amino acid analyzer. Physiological indicators of stress/inflammation in serum will be measured using colormetirc assays for L-Lactate, Glucose, Superoxide Dismutase, total antioxidants, TBARS, Catalase, Glutathione peroxidase and HSP70. Quantification of RNA transcripts will be carried out as described in (Blaufuss et al.2019). Genes to be examined include: proliferating cell nuclear antigen, GATA3, forkhead box p3, RAR-like orphan receptor-γ, T-Bet, tumor necrosis factor α, transforming growth factor β, interferon γ, and interleukins 1β, 4/13, 6, 10, 17, and 22. Samples of distal intestine will be cryo-sectioned and cells dissected using laser microdissection with a PALM MicroBeam (Carl Zeiss AG, Oberkochen, Germany). Dissected cells will be pooled for expression analysis of immunogenic and master regulatory genes associated with the inflammatory response and the recognized subtypes of T helper cells. Data will be analyzed using PROC MIXED in SAS version 9.3 (SAS Institute, Inc., Cary SC) with post hoc tests (Tukeys, SNK). Tanks are considered the operational unit.Objectives 2 and 3: Ongoing selection studies at the Hagerman Fish Culture Experiment Station between the USDA-ARS and University of Idaho, spawn approximately 130 families of CX line rainbow trout yearly. In even years, approximately 40 additional families from an additional line selected for enhanced ability to biosynthesize and store lcPUFAs when fed feeds containing only plant oils (CLX line). Following the spawning and early rearing of CX and CLX fish, we will differentially PIT tag 150 of each strain along with 150 commercial line cohorts that are cultured on the Hagerman Station. PIT-tagged fish will be returned to the general population and will be treated the same regarding sampling, feeding of the selection diet (Overturf et al. 2013b) and ponding in outside raceways. PIT-tagged fish will be separated from the general populations following monthly weighing at approximately 100, 200, 400 g. A random sample of fish from each group (CX, CLX and commercial line) will be placed separately by group into 140 L tanks and held off feed for 36 hours and then be gavaged with a slurry of 1% BW of feed containing 1% of [13C] or 15N labeled lysine or methionine or [13C] labeled LNA, EPA, or DHA. At 0, 6, 12 and 24 h post-gavage, fish will be sampled for blood, liver muscle, and intestine. The metabolic fate of isotopic lysine and methionine in rainbow trout tissues will be determined using GCMS detection (Thermo Trace 1310) with modifications used by Mackay et al. (2015) specifically for amino acids. Figure 2 show recovery of isotpic phenylalanine in our lab using this technique. Similar percent recoveries of stable isotopes of ALA, DHA and EPA are also examined using the same instrument with modifications. Overall profiles of amino acids in diets and whole fish samples will be quantified on a Biochrom 30+ amino acid analyzer. Overall fatty acid profiles in tissue samples will be determined according to a method modified from AOAC method 991.39. Fish will be sampled for whole-body proximate composition, relative liver size (hepatosomatic index), and viscerosomatic index. Proximate composition will be determined according to standard methods (AOAC 1995). Amino acid retention efficiencies will be determined according to methods in Gaylord and Barrows (2009). Amino acid digestibility from each diet will be quantified according to the methods of Gaylord et al. (2010) utilizing yttrium oxide. All qPCR primers are currently used our laboratory. Quantification of transcripts will be carried out with a QuantStudio 6 Flex Sequence Detection System (Life Technologies). Genes to be examined include: Fatty acid Binding protein 2 (FABP2), Delta-6 desaturase, Delta-5 elongase, Delta-9 elongase, Carnitine Palmitoyl Transferase (CPT1), peroxisome proliferator activated receptor alpha (PPARa), peroxisome proliferator activated receptor gamma (PPARg), apolipoprotein (ApoE), and Fatty acid synthase (FAT). Factorial ANOVA will be utilized to test for effects between size class and efficiencies and selected (CX and CLX) vs. control fish for fatty acid conversion and/or utilization. Growth and physiological measures along with qPCR data will be analyzed using PROC MIXED in SAS version 9.3 (SAS Institute, Inc., Cary SC) with post hoc tests (Tukeys, SNK). Tanks are considered the operational unit for growth performance data. Canonical analyses will be used to assess correlates between gene expression, fatty acid utilization and growth indices.