United Soybean Board Domestic Programs Report Form

Transcription

1 United Soybean Board Domestic Programs Report Form Project # and Title Reporting Period , Genomic and Physiological Assessment to Identify Changes Allowing High-soy Use in Genetically-improved Line of Rainbow Trout: Breaking the 20% Soy Protein Barrier in Feeds for Marine Fish Final Report The University of Idaho was notified of this award on September 27, Post-award negotiations between the Office of Sponsored Programs (OSP), University of Idaho, and SAA began on that date. The contract was fully executed on February 26, 2013 and an internal budget was established for the investigators on March 27, Although there was a six month delay between notification of the award and establishment of a budget for the project, the PIs began the study during the first quarter of the period. Investigators must deal with biological and logistical factors that dictate when various elements of studies can be conducted during the year. Rainbow trout from selected and non-selected broodstock at the Hagerman Station spawn during the fall months, producing offspring that are used in feeding trials associated with the USDA-ARS rainbow trout selection program during winter and early spring months. Although these fish grow rapidly and reach post-juvenile stages (>100g) by spring, they occupy most of the fish rearing tank in the UI facility during this period. Thereafter, the investigators began the research work by addressing Objective 2 to utilize available tank space and also because fish for this study had been retained for this purpose and raised to be the correct size for the study in fall, Objective 2: Determine if differences in digestion and absorption of amino acids are the determining factor lowering protein retention of fish fed soy protein-based feeds. Objective 2, designed to sample plasma amino acids levels in selected or non-selected post-juvenile rainbow trout following a single meal, was initiated and completed during the October to December period. In this study, relatively large fish, average weight 450g, were obtained from selected and non-selected strains and, over a period of two months, used as subjects to assess amino acid levels in hepatic portal vein blood and circulating blood obtained from the caudal vein. Prior to this work, no researchers had sampled blood in this fashion from the hepatic portal vein of rainbow trout, so considerable time was spent developing an appropriate and efficient protocol for sampling blood from the hepatic portal vein. Hepatic portal vein blood samples contain amino acids from intestinal absorption before they reach the liver and are distributed to tissues for protein synthesis. Caudal vein -1-

2 blood samples represent the amino acid supply to cells throughout the body after hepatic metabolism. Six feed ingredients and the two feed samples (plant protein-based with or without supplemental amino acids) were force-fed to rainbow trout and samples of plasma from two locations were obtained at intervals after feeding up to 24 hours. The six feed ingredients were soybean meal, soy protein concentrate, soy protein isolate, corn gluten meal, wheat gluten meal and fishmeal. The plant protein blends contained soybean meal, soy protein concentrate, corn gluten meal and wheat gluten meal in proportions similar to those found to be suitable as a fishmeal replacement blend in previous studies with rainbow trout (Burr et al., 2013). Three amino acids were supplemented to the protein blend, lysine, methionine and threonine. These were chosen based upon feed formulation practices for trout and salmon, and the levels of supplementation were calculated to be very similar to levels that would be added to commercial feeds containing mainly plant protein ingredients to meet the dietary requirements of rainbow trout based upon the NRC (2011) bulletin, the standard used by the fish feed industry. Each feed ingredient and the blends were prepared into slurries to force-feed the fish. Prior to this, preliminary testing was conducted to assess the optimum amount of material to intubate into the stomach using a large syringe and tube in a single feeding. We found that feeding 0.75% of body weight or more resulted in regurgitation after fish were returned to tanks, whereas feeding 0.5% of body weight did not result in regurgitation. Therefore, fish were force-fed at 0.5% body weight using six single feed ingredients and two plant-protein mixtures, with or without supplemental amino acids. The six single feed ingredients were soybean meal, soy protein concentrate, soy protein isolate, corn gluten meal, wheat gluten meal and fishmeal. Each ingredient was fed and all sampling occurred over a five-day period. Fish were fed at 20 minute intervals and placed in tanks for each time point. In this manner, fish were sampled for each time point by sampling all fish in an individual tank without disturbing the remaining fish in other tanks destined for later sampling. Two tanks of fish were sampled at each time point, one for each trout strain (selected strain and non-selected strain). At 3, 6, 12, 18, and 24 hrs after force feeding, fish were anesthetized and blood samples taken from four fish from each stock (selected and non-selected) in heparinized syringes. Blood sampling for plasma amino acid analysis (plasma deproteinization followed Frank and Powers (2007). The steps in blood sampling are outlined below. 1. Anaesthetize fish in 100 ppm MS-222. Remove fish from water when opercula movements have almost stopped. 2. Open the abdominal wall of fish and collect about 0.2 to 0.3 ml of blood from the hepatic portal vein with a heparinized winged infusion set (butterfly needle; 12 inch tubing, 23 G and 3/4 inch ultra-thin wall -2-

3 needle) connected to a 1 ml syringe. 3. After gently inverting the syringe 3-4 times for proper mixing, transfer the blood to a 0.6 ml conical Eppendorf tube on ice. 4. Draw ml blood from the caudal vein in a 3-ml heparinized syringe with 22 G 1.5 inch needle. 5. After gently inverting the syringe 3-4 times for proper mixing, transfer the blood to a 2 ml round bottom Eppendorf tube on ice. 6. Centrifuge blood at 2000 x g for 5 min at 4 C, collect only the upper plasma layer taking care not to touch the red blood cells or buffy coat (white blood cells). 7. Transfer the plasma to a labeled tube. 8. Add 20 ul plasma and 180 ul milliq water into a separately labeled tube. 9. Precipitate the plasma proteins by adding 200 ul 0.5 M perchloric acid, vortex, and centrifuge at x g for 5 min at room temperature. 10. Collect ~350 ul supernatant, filter it in a Spin-X 0.2 um microcentrifuge filter by centrifugation at x g for 2 min at room temperature. 11. Store the filtrate (deproteinized plasma sample) at -80 C until further analysis. A total of 480 samples were obtained as follows: Five sampling times x two samples per fish (hepatic portal vein and caudal vein) x 3 fish per sampling x 2 strains of fish x 8 dietary treatments (6 ingredients and 2 diets). We sampled 4 fish at each point with the intention of analyzing 3 fish and having a reserve sample in case we need it. Tissue samples were also taken from the intestine of the fish for gene expression analysis by RT-QPCR to determine if the expression levels of genes coding for intestinal peptide and amino acid transporters was affected by amino acid supplementation, and to measure expression levels over time following a single feeding. Liver and muscle tissue were also sampled from fish after plasma sampling for gene expression. All tissues for gene expression analysis were placed in RNA-later to preserve them until analysis. Methods and Results are described below. Plasma amino acid analysis: Samples were transferred on dry ice to the Department of Animal and Veterinary Sciences, University of Idaho, Moscow, Idaho. Analysis was conducted using a Waters HPLC system and AccQ-Tag derivatization system following protocols described in the Waters manual. Analysis of plasma samples proved more time consuming than anticipated. In contrast to hydrolyzed samples, the run time of plasma samples was greatly extended. Further, the analytical -3-

4 parameters had to be modified to adequately separate amino acids on the chromatograph. A decision was made to analyze the sample from fish fed the protein blends first. This involved analyzing 120 samples in duplicate. This analysis was completed in late fall, Because we were analyzing samples using shared equipment, we did not have exclusive use of the Waters HPLC over the entire period. In late fall, 2013, other faculty needed to use the Waters HPLC, so we retrieved our samples and continue to store them at the Hagerman Station at - 80C pending analysis. Our intention is to complete the analysis of these samples in In the meantime, all data from feeding the plant protein blends was statistically analyzed and prepared into figures and tables. This portion of the study was presented at the Aquaculture America meeting in Seattle in February, 2014 and also at the annual Soy Aquaculture Alliance meeting held at the same time. Presentations were made by Dr. Ron Hardy, Dr. Ken Overturf and Andreas Brezas, a PhD student working on the SAA project. Mr. Brezas applied for and was awarded a travel grant from SAA to attend the meeting in Seattle. Results from analysis of plasma samples from the protein-blend mixes show clearly that plasma levels differ in selected compared to nonselected fish. In selected fish, plasma levels of essential amino acids follow a more-or-less constant pattern, increasing slightly after feeding and declining gradually to near baseline levels after 24 hours. In contrast, plasma levels of essential amino acids in non-selected fish follow a more complicated pattern, generally showing either a decline three hours after feeding, followed by a more distinct increase after 6-12 hours. More significantly, supplementing the protein-blend mixes with amino acids altered the pattern of amino acids in the plasma compared to levels measured in plasma of fish fed the diet without amino acid supplementation, not just for the amino acids being supplemented but also for other essential amino acids. This data shows for the first time that supplementing deficient amino acids to a plant protein-based diet alters the pattern of plasma levels of other essential amino acids that are protein-bound (intact proteins). This information suggests a possible explanation for the observation that protein retention in fish fed plant-based diets is lower than that of fish fed fishmeal-based diets even when all deficient amino acids are present in the diet at required levels as a result of supplementation. First, supplemented amino acids are absorbed in the intestine and appear in the plasma within a few hours, whereas amino acids present in the plant-based ingredients take hours longer to appear in the plasma. This indicates a problem with synchronization of plasma amino acid levels that has implications for their use in protein synthesis in peripheral tissues. All essential amino acids must be present in correct ratios at the same time for protein synthesis to operate efficiently in cells. Second, the results show that selected strains of rainbow trout differ from non-selected fish in that the pattern of plasma amino acids following a single feeding is more modulated and balanced. This is more favorable for efficient protein synthesis and is likely a factor that contributes to the observation that -4-

5 selected lines of rainbow trout more efficiently convert dietary protein into tissue protein when fed plant protein-based feeds. Gene expression: Tissue samples were also taken from the fish that were force-fed the protein-blend mixes to examine gene expression levels at intervals after feeding. Samples from the proximal intestinal region, liver and muscle were processed to extract RNA and expression levels of genes were measured for amino acid transporters known to be expressed at the apical membrane of the proximal intestine and representing the capacity to uptake all the classes of amino acids (neutral, cationic, anionic and tri-dipeptides). From the results obtained so far (intestinal peptide and amino acid transporter expressions) the expression of Pept1 (SLC15A1) was statistically significant higher in the protein blend supplemented with amino acids regarding the 3h and 6h time points. This is a surprising and novel finding. The amino acid transporters responsible for the neutral, basic and anionic amino acid uptake at the apical membrane revealed diet (amino acid supplementation vs. no supplementation) differences and strain (selected vs. commercial) differences. In more detail, the neutral amino acid transporter B 0 AT1 (SLC6A19), which is considered to be a potential marker regarding protein utilization, showed higher expression when the protein blend was supplemented with amino acids at 3, 6, 12 and 18 hours after feeding. Further, there was a strain effect with the selected trout strain showing different pattern between 3 and 12 hours (peak at 6 hours). The second transporter that we analyzed, responsible for the uptake of neutral amino acids ASCT2 (SLC1A5), did not show any strain differences but confirmed again the diet differences. The amino acid supplemented diet showed a more homogeneous pattern of plasma amino acids through time compared to the un-supplemented diet suggesting that this transporter is not a good indicator of diet quality. Regarding the cationic amino acid transporter b 0,+ AT (SLC7A9) responsible for the uptake of arginine and lysine, there was a diet effect as expected (when lysine was supplemented), but also a strain effect was evident between 3 and 12 hours. The selected strain receiving the amino acid-supplemented diet had a more leveled regression gene expression response rather than an up-and-down pattern observed in the non-selected (commercial) trout strain. Finally, regarding the anionic amino acid transporter EAAT3 (SLC1A1) there were both diet and trout strain effects. The selected strain receiving the supplemented diet responded differently between 3 and 12 hours showing the same pattern as in the case of the cationic amino acid transporter (B 0 AT1). This supports the observation that there are genetic differences between the selected and non-selected trout strains that have important implications concerning soy protein utilization to support muscle tissue growth. The results obtained so far from gene expression levels of peptide and -5-

6 amino acid transporters suggest that supplementing amino acids directs endocrine regulation of the digestion process. In brief, we believe that there is amino acid signaling that acts as a feedback control mechanism to modulate the expression of hormones, transporters and digestive enzymes. This control can be of high importance for the understanding of the mechanisms that lead to lower protein efficiency when soy products are used in feeds for fish. To dissect in more detail this avenue we are evaluating candidate gene expression (CKK, trypsin, somatostatin, aminotransferases and amino acid degradation pathways) in muscle and liver tissue samples from the fish to determine how diet and amino acid supplementation affect cellular protein synthesis and degradation and thereby affect protein turnover in tissues. This is very important because soy-based aquafeeds must be supplemented with amino acids to correct deficiencies. Regarding gene expression analysis of the genes listed above from the liver and muscle tissue samples, RNA extraction and cdna synthesis was completed in fall 2013 and all gene expression analysis was completed in the first quarter of Analysis of data is underway. Objective 1: Identify the transcriptomic, physiological and morphological changes in our selected rainbow trout strain associated with rapid growth when fed high-soy, plant-based feeds. A feeding trial was completed in July 2013 to identify mechanisms of enhanced tolerance to high soy diets in selected lines of rainbow trout compared to that of unselected lines. Next generation, high-throughput sequencing was employed to evaluate key genes, proteins and pathways linked to metabolism and protein regulation utilizing a suite of gene probes and antibodies for phosphorylated (active) and unphosphorylated states of key metabolic proteins. Background: The University of Idaho s Aquaculture Research Institute has had a joint research program underway with the USDA Agriculture Research Service since 2000 in selective breeding of rainbow trout for enhanced growth when fed all plant-protein diets. Over the 14 year life of the project, over $12 million has been spent to improve the growth rate of rainbow trout fed all plant, high-soy diets. The results have been very successful. In the selection diet, all dietary protein is supplied by a blend of soy protein concentrate, soybean meal, corn gluten meal and wheat gluten meal. Amino acid and mineral supplements are included in the formulated diet. Rainbow trout in the selection program mature at two years of age, so as of 2014, seven generations of selection have been completed. At the beginning of the selection program, growth performance of families (single parent crosses) fed the selection diet was inferior to that of growth performance of the families when they were fed a conventional fishmeal-based control diet. Over six generations, growth performance of selected lines has improved, such that these -6-

7 fish now exhibit faster growth rates when fed the plant-protein selection diet than when fed the control diet. Recent studies have shown that selected lines are less susceptible than unselected lines to intestinal inflammation (distal enteritis) when fed the plant protein diet. Therefore, over six generations of selection, fish have distinct morphological and/or physiological differences that make them more tolerant of high soy diets. On December 17 th, 2012, offspring of two single-parent crosses (families), average weight of 5 g, of selected fish were sorted and separated into two tanks of 150 fish per tank. For each family one tank was fed the plant protein selection diet (PM) and the other was fed the control fishmeal diet (FM). This was repeated with two mixed groups of non-selected fish. The resulting 8 tanks of fish were reared for 9 weeks on these diets to an approximate weight of 55 grams. Initial samples were then taken of muscle, liver, kidney, spleen and intestine along with measurements of feed conversion ratio (FCR), specific growth rate (SGR) and HSI. These eight tanks were kept on the same diet fed nine weeks. This was done so that chronic dietary effects could be examined in fish with selected tolerance to soy and plant proteins alongside non-selected fish that have already developed intestinal enteritis. All tissue samples have been taken and RNA isolated from liver, muscle and distal intestine tissue for RNA sequence analysis. This is a unique sample set for looking at mechanisms for improved tolerance and utilization of high soy diets and to determine how the selected strain in the long term establishes a tolerance and preferentially utilizes plant-based diets even more effectively than fishmeal-based diets. At the end of the feeding trial, tissue samples were collected for RNA- Seq analysis. Muscle, liver, and intestine samples were collected and snap frozen in liquid nitrogen or placed in fixative for histological analysis. RNA was isolated from all samples (3 tissues from 10 fish from each experimental treatment for 120 samples) and DNAase treated. Samples were then processed using the Ribo-Zero magnetic gold kit for extraction of mrna and purification of rrna from these samples. The isolated RNA from the 120 samples was then further processed and standardized to generate individual cdna libraries. The relative concentration and uniformity for each library was measured and 12 libraries were found to not meet the required specifications for utilization. The original isolates from these 12 samples were then reprocessed until we were able to confirm that all individual sample libraries met the specified criteria for sequencing. Of the 120 individual libraries, 14 failed during the sequencing. These samples were re-prepared and sequenced again using a paired end sequencing run which will generate increased sequence information useful for blasting, aligning, and identifying unknown sequences of interest. All samples have now been sequenced and have passed an approved quality rating (good criteria rating is >8 million reads per sample).. With all 120 samples now completely sequenced the data -7-

8 has been trimmed for analysis. The data is currently being run through our bioinformatics pipeline to evaluate relative quantified expression differences within the tissues between the fish strains, and dietary treatments in several tissues. From this information isolation and association of SNP markers and annotation of known sequences correlating with changes in measured parameters will be determined. This element will be completed in summer, 2014, and manuscripts will be prepared for publication in scientific journals. Tissue samples were also taken from the distal intestine of these fish for histological examination. Tissues were preserved in buffered formalin and sent to the College of Veterinary Medicine (CVM), Washington State University, to be prepared into tissue slides. These samples have been embedded in paraffin and tissues cut, mounted and stained. Evaluation of the degree of distal enteritis in selected and non-selected trout is now underway by Dr. Kevin Snekvik, Professor in the CVM. He is examining tissue sections under the microscope for changes in structure and will score the sections for severity of distal enteritis. We expect to see changes in the non-selected fish because we could see visual differences in these fish when we took the samples. We do not expect similar changes in selected trout. Finally, samples of tissues were taken from the fish and flash frozen in liquid nitrogen for protein level analysis (Western blotting), specifically measuring the levels of proteins coded for by the genes whose expression levels are elevated. Proteins are extracted from tissues, purified and separated using gel electrophoresis. They are then visualized by staining the gel and identified by comparison with standards. The concentration is estimated by density of the staining. This is a standard technique which is important because it connects gene expression (transcriptomics) with protein synthesis in cells. Summary: All tissue analysis associated with the proposed studies was completed by the end of the project period. Analysis of data is underway and will be completed in the summer of The proposed study was ambitious and unprecedented in the application of next generation, high-throughput molecular technology to address the genomic and physiological changes associated with feeding high-soy diets to rainbow trout. The use of the selected strain of rainbow trout that are unaffected by high-soy diets and do not develop distal enteritis provides a unique tool to identify the genetic changes that are responsible for soy tolerance, providing a new approach to apply this knowledge in other species of farmed fish, thereby increasing soy protein levels in farmed fish feeds. -8-

9 Relative Concentration * * HC SEL Figure 1. Demonstrated difference in the absorption and transport of the essential amino acid between selected (SEL) and nonselected (HC) fish h 6h 12h 18h 24h Figure 2. Plasma amino acid levels in the Hepatic Portal Vein of rainbow trout fed the protein blend supplemented with amino acids. The commercial trout strain shows two major peaks whereas the selected trout strain shows a controlled decreasing pattern over time. -9-

10 Figure 3. Plasma amino acid levels in the Caudal Vein of rainbow trout fed the protein blend supplemented with amino acids. Amino acid levels through time reflect tissue protein synthesis and protein turnover at later time points (18 and 24 hrs.). Note that in the commercial strain, amino levels increase at 18 hours, most likely from tissue protein turnover. Relative Expression * * HC SEL Figure 4. Expression difference for the high affinity amino acid transporter SLC1A1. Significant changes in expression are found at 12 and 18 hours after intubation between selected (SEL) and nonselected fish (HC) h 6h 12h 18h 24h -10-

11 LIBRARY FLOW CELL LANE SAMPLE INDEX 29M 30M 31M 32M 5L 6L 7L 8L ATCACG CGATGT TTAGGC TGACCA ACAGTG GCCAAT CAGATC ACTTGA READS 13,826,406 16,055,433 20,142,224 17,943,102 25,863,498 13,522,352 11,447,998 17,895,328 % READS 9.79% 11.36% 14.26% 12.70% 18.31% 9.57% 8.10% 12.67% Table 1. Example of RNA-seq data from a single flow cell. Muscle and liver samples from selected and non-selected fish with index run identification and linked library. Average number of sequence reads per sample was 17.1 million. Figure 5. Histology of distal intestine in selected fish and non-selected fish reared on plant-based diet containing 20% soybean meal. The breakdown of the mucosal membrane in the non-selected fish, which leads to enteritis, is evident in top left photo. FM-NS FM-SE -11-