1a. Objectives (from AD-416):
The objective of this research is to improve the competitiveness of rainbow trout production by reducing feed costs through the development of improved ingredients, feeds, and strains of rainbow trout.
1b. Approach (from AD-416):
Ingredients will be selected based upon nutrient and anti-nutrient profiles, and cost on a protein unit basis. Special emphasis will be placed upon barley and oat cultivars, but any ingredient that has favorable profiles will be considered. Some marine oils and proteins maybe evaluated at low inclusion levels to enhance feed palatability and the final product fatty acid profile. Practical feed formulations, will be produced using cooking extrusion (commercial conditions) for rapidly growing juvenile fish. Traditional evaluation procedures, including growth performance and nutrient retention, and cost per unit gain, will be used to evaluate the improved diet formulations. Additional evaluation procedures will be developed and applied to fish feeding trials as follows: (1) clinical assays of blood plasma for electrolyte levels, cholesterol, triglycerides, ASAT, ALAT, and routine plasma parameters; (2) whole blood analysis for hematocrit percentages; (3) gene expression activities for genes associated with protein synthesis and degredation, fatty acid elongation and desaturation, bone and/or cartilage metabolism and energy metabolism plus cellular redox status. Reproductive performance of rainbow trout fed diets containing plant-derived ingredients in place of fishmeal and fish oil (partial replacement or total replacement for selected growth periods) will be assessed by measuring fecundity, egg fertility, and egg hatchability, plus chemical analysis of eggs to determine effects of diet on nutritional content. Bone density and imaging may be explored for fish fed diets lacking fishmeal to determine if replacement mineral supplements in plant-based feeds support normal bone development and absence of skeletal deformities.
3. Progress Report:
The collaborative research between ARS and University of Idaho focused on three key tasks: 1) Identify rainbow trout families with improved phenotypes for growth and utilization of a grain-based fish feed through a genetic selection program, begin to assess performance of selected families as triploid fish and begin to establish sex-reversed broodstock to produce all-female populations, the primary types of rainbow trout used in commercial production; 2) Conduct feeding trials to evaluate nutritional value of grain-based products; 3) Conduct feeding trials in support of the goal of developing a commercial trout feed based on oilseed and grain products without inclusion of marine protein and/or oil. Each of these tasks apply to the Parent Project Objective 3: Determine nutritional value of alternative ingredients (protein, lipid, energy) and develop practical feed formulations for improved strains of fish, Objective 4: Determine optimal nutrient supplementation levels for specific life stages of improved strains of trout, Objective 5: Use gene expression analyses to advance the understanding of gene targets for improving nutrition, growth, and development processes under production conditions, or Objective 6: Identify phenotypic differences in rainbow trout for growth and utilization of plant-based sustainable diets and determine the genetic variation for the identified traits. For Task 1, 176 single family crosses were made using rainbow trout families in fall 2012 and evaluated for performance when fed an all plant protein diet. Eggs, fry and fingerlings from the crosses were incubated and reared separately over a period of 7-8 months to maintain cross identity. Performance of family cross groups fed the plant protein diet was compared to that of cohorts fed a conventional fishmeal-based diet. After evaluation of performance and upon reaching approximately 200g in average weight, the top 40 families were identified and the top 15 fish from each of the top families were marked with a PIT tag and pooled in several outdoor raceways for communal rearing to maturation in fall of 2014 at two years of age. Meanwhile, tagged fish from the previous year class (2011) were communally reared and will spawn in fall 2013. In 2012, a pressure chamber was purchased to induce triploidy in rainbow trout eggs soon after fertilization. However, the chamber did not arrive until late in the spawning period. Attempts to induce triploidy by high pressure in 2012 were unsuccessful due to failure of the steel frame upon which the pressure chamber was mounted. A replacement frame of higher quality was obtained and the system is now operational. Triploidy will be induced in eggs during the 2013 spawning in October and November. Approval was obtained to begin sex-reversed broodstock development from selected trout families and the process to sex-reverse trout was begun. Sex-reversed fish will be spawned in fall of 2014, leading to the production of all-female rainbow trout. The aim of this work is to assess the growth performance of all-female trout from selected family lines when fed all plant protein diets. A new study was initiated to evaluate differences in trout family lines in the ability to convert linolenic acid, the only omega-3 fatty acid in plant oils, to EPA and DHA, long-chain, polyunsaturated omega-3 fatty acids found in marine oils and some insects that are prey for trout in freshwater. A batch of the plant protein-based trout selection diet, which is nearly devoid of omega-3 fatty acids except for added lipid, was prepared without the added lipid by Dr. Rick Barrows and shipped to the Hagerman Station, University of Idaho. Linseed oil, the only commercial source of linolenic acid, was added to a portion of the feed, and salmon oil was added to another portion. The linolenic-supplemented feed was fed to trout fingerlings from 25 family crosses, each in separate tanks. The salmon oil-supplemented feed was fed to a commercial strain of trout. Fish muscle tissue was sampled by biopsy from fish in each tank and analyzed for fatty acid composition using GC-MS. Total lipid was also measured in the samples. The sampling did not harm the fish but revealed differences in EPA+DHA content among families, suggesting that families differed in metabolic ability to elongate and desaturate linolenic acid to long-chain omega-3 fatty acids. The fish will be raised to maturity and spawned, and offspring will be evaluated in a similar manner to estimate heritability of this metabolic ability, and to begin to develop genetic markers to allow selection for this trait. Fish will spawn in fall of 2014. For Task 2, a 12-week feeding trial was completed with post-juvenile rainbow trout fed either a fishmeal-based or all plant-product diets supplemented various levels of zinc, supplied as zinc sulfate. Plant-based trout feeds contain lower baseline levels of zinc than do fishmeal-based feed and further contain phytic acid, a potential zinc antagonist that reduces zinc digestibility. Fish weight gain increased with zinc supplementation up to 30 ppm but no significant differences associated with dietary zinc supplementation were observed in weight gain of fish fed the fishmeal-based diet. Other response variables, such as gene expression and tissue histology, are being assessed. For Task 3, a second trial was conducted for 24 weeks using juvenile rainbow trout (initial average weight 3 g) fed experimental diets lacking fish meal (and therefore residual fish oil containing omega-3 fatty acids) and containing varying amounts of Alaska fish oil and canola oil to achieve 0.15, 0.45, 0.75, 1.05, 1.35, 1.65 and 1.95% long-chain, polyunsaturated omega-3 fatty acids at constant lipid levels among diets. In a previous feeding trial, dietary levels of omega-3 fatty acids ranged from 0,20 to 0.92%, and at the end of an 18-week feeding trial, final fish weights did not reach a plateau with increasing long-chain, polyunsaturated omega-3 fatty acid levels. In the second trial, fish reached over 400g final weight (initial weight was 2.7g). A plateau was reached at 1.5% omega-3 fatty acid level in the diet, demonstrating that at dietary levels beyond 1.5 %, no additional growth response was evident. Depending on the regression model used on the fish weight data, the requirements varied: 0.73% in the second-order polynomial regression (omega-3 level corresponding to 95% of the maximum growth response) and 0.83% in one-slope broken line regression. No clinical signs of omega-3 fatty acid deficiency were observed. Additional feeding trials were conducted with supplemental funding from the Soy Aquaculture Alliance to determine if differences in the rate of protein digestion and amino acid absorption between fishmeal protein and plant proteins (soy, corn and wheat protein) were partially responsible for differences in growth performance and/or feed efficiency between fishmeal-based and plant protein-based feeds. Trout were force-fed various feed ingredients and complete feeds with and without supplemental amino acids and both hepatic portal vein and caudal vein blood samples were obtained at intervals for 24 hours following feeding. Samples are being analyzed and results are expected in fall, 2013.