Performing Department
(N/A)
Non Technical Summary
For the past several decades, aquaculture has been the fastest growing sector of animal agriculture, with worldwide production of fish and shellfish tripling in volume produced since 1995. Currently, aquaculture and fisheries each contribute approximately half to the demand for aquatic protein. A recent estimate indicates that aquaculture production must increase by an estimated 157% to meet the protein demand of an ever-growing global population and avoid increased reliance upon capture fisheries. Both expanding aquaculture production capacity and improving production efficiency are essential to meet the future demand for aquaculture-derived aquatic protein. Selective breeding has been applied in aquaculture as a strategy to increased production efficiency. Fish breeders have primarily focused on increasing growth, fillet yield, and disease resistance since these traits have high heritability and directly benefit production efficiency and animal welfare. However, it is recognized that breeding for trait improvement can have unintended negative consequences on other production traits, such as tolerance to low dissolved oxygen (hypoxia). Salmonid fish species, such as rainbow trout, require high concentrations of dissolved oxygen for optimal growth. However, adding supplemental oxygen into raceways of large rainbow trout operations is not economically feasible so farmers use alternative strategies such as reducing fish density and increasing water flow to keep dissolved oxygen within acceptable limits; unfortunately, these strategies also reduce production capacity. This project addresses this by characterizing genetic variation in hypoxia tolerance among lines of rainbow trout with improved production traits and investigating nutritional strategies that increase hypoxia tolerance.The first objective of this project is to characterize how genetically improved lines of rainbow trout with high fillet yield andimproved disease resistance respond to hypoxia stress. Although these select lines are valued by producers because of their high product yield and improved health, it is unknown whether their hypoxia tolerance differs from standard lines. For this study, standard and genetically improved lines of rainbow trout will be exposed to grow-out conditions characterized by chronic hypoxia. Growth performance will be analyzed for how well fish perform under these conditions; global gene expression will also be evaluated to identify genetic and physiological mechanisms contributing to performance differences. Findings from this study will indicate if selection for increased fillet yield or disease resistance has a negative consequence on hypoxia tolerance and determine whether line-specific husbandry strategies are beneficial when these genetically improved lines are used for production.A second objective focuses on the potential for dietary choline to alleviate negative effects of hypoxia stress on reproductive performance in rainbow trout. In this study, female rainbow trout will consume choline-supplemented diets and be exposed to chronic hypoxia as they progress through sexual maturation. Indices of reproductive performance such as egg size, egg yield, and hatch rate will be evaluated to determine whether excess dietary choline provided broodstock with a performance advantage. In addition, growth and hypoxia tolerance of offspring will be evaluated to determine if maternal treatments have transgenerational effects. These findings can identify a nutritional approach to improving reproductive success when broodstock are exposed to hypoxia stress. Additionally, identification of transgenerational effects in offspring may introduce broodstock conditioning as a husbandry strategy for improving hypoxia tolerance.Together, these two objectives will improve our understanding of genetic selection effects on non-selectedphenotypes and maternal effects on offspring performance. This information will aid in stakeholder's understanding of a few basic husbandrytechniques that can be altered slightly to affect performance.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
The long-term goal of this research is to combine genetic selection strategies with feeding andhusbandry strategies in rainbow trout production in order to optimize key epigenetic mechanismsregulating growth and stress tolerance to enhance growth performance while selecting genetics fordisease resistance or fillet yield and relying less on marine resources in feeds.This project will clearly identify metabolic regulatory mechanisms and hypoxia-relatedpathways affected by key nutrients, methyl donors, to increase production and economic efficiency. Atwo-tier approach will be used to build on the existing genetic selection program at the National Centerfor Cool and Cold Water Aquaculture (NCCCWA, ARS, USDA, Leetown, WV) that has been selectingfor two traits independently, disease resistance and fillet yield. The disease resistant selected line wasrecently released to commercial fish operations (e.g., TroutLodge). Our specific objectives are to 1)determine how specific trait selection affects stress tolerance, 2) identify how maternal dietary cholineintake affects offspring growth potential, fillet yield, and stress tolerance, and 3) demonstrate howmaternal dietary choline intake interacts with maternal stress exposure to imprint offspring. To satisfythese objectives, we will test the following hypotheses: 1) genetic selection for performance-relatedtraits, like disease resistance or fillet yield, reduces hypoxic stress tolerance, 2) dietary choline intakeattenuates negative hypoxic stress effects on reproductive performance and offspring stress tolerance,and 3) maternal exposure to acute hypoxic events alters offspring hypoxia tolerance through maternalimprinting (epigenetics).
Project Methods
Objective 1 - Determine is genetic selection for enhanced performance traits (disease resistance or fillet yield) affects chronic hypoxic tolerance. For this objective, fish from each genetically selecte lline will be subjected to chronic or acute hypoxia.To induce chronic hypoxic conditions, aerationwill be removed and water flow will be reduced sothat dissolved oxygen concentrations remainbetween 4.5-5.5 ppm. Normoxic conditions will be achieved by introducing supplemental oxygenthrough an air stone. Oxygen probes will be submerged in each tank and will record dissolvedoxygen levels every 5 minutes. If oxygen concentrations fall outside the experimental range, analarm will sound and a water technician will respond. Tanks assigned to the control and hypoxiatreatments will be fed on an identical schedule as follows: feed will be provided using automaticfeeders set to dispense feed between 8 am-2:30 pm at a fixed percent of tank biomass thatapproaches satiation. Tanks will then be hand-fed to satiation at 3:00 pm. Body weights and lengthswill be recorded for all fish at the beginning and end of the 4-week study, after which 5 fish fromeach line will be sampled; blood will be removed from caudal vasculature and tissues (liver, gill,head kidney, muscle) will be immediately frozen in liquid nitrogen. Water samples will be collectedat each stage (normoxic, hypoxic) for tank cortisol analysis. A head-off guttted carcass will beweighed as an indicator of fillet yield. Samples will be harvested from the control tanks in anidentical manner.The surviving fish from the 4-week normoxic conditions will be used in an acute hypoxiachallenge approximately two weeks later. Acute hypoxia stress will be induced by stopping the flowof water and oxygen into each tank. Dissolved oxygen levels are expected to drop within an hourand lead to loss of equilibrium in fish. Fish will be euthanized after the first observation of loss ofequilibrium past 45 degrees. The fish will not be allowed to reach the point of asphyxiation.Objective 2 - Determine is maternal dietarycholine intake and/or exposure to acute hypoxia stress imprint stress tolerance benefits in offspring.Approximately 100 all-female rainbow trout families from the FY-H line willbe hatched in the spring of 2024 as part of the NCCCWA selective breeding program. Thesefamilies will serve as broodstock for the study described herein. Hatch dates are coordinated bymodifying egg incubation temperatures so that all families hatch within a five-day window inMarch. Families will be reared in separate tanks from hatch to approximately 5 months post hatch(~15 g) when a pool of ~300 FY-H fish will be created by PIT tagging three fish from each FY-Hfamily. These fish will be commingled in multiple tanks and reared according to standard husbandryprotocols. From the time of first feeding through June 2025 (~16 months post-hatch), fish willconsume a commercially available diet that meets or exceeds dietary requirements for rainbow trout(Zeigler Finfish G).Inrainbow trout, eggmaturation begins to rapidlyprogress at ~18 months posthatch with spawning at ~22months post hatch, therefore the broodstock study willbegin right before thiswindow, when fish are 16months post hatch (July2024). Fish will be separatedinto one of twelve experimental tanks, with 20 fish per tank. The experimental design will be a 2 x 2factorial with the following independent variables: 1) +/- dietary choline supplementation and 2) +/-hypoxia stress. The +choline dietary treatment will be created by top dressing acommercially available broodstock diet (Zeigler Finfish Broodstock) with supplemental choline. The Finfish Broodstock diet (-choline) contains 3600 ppm choline; the +choline diet will doublethose levels to 7200 ppm. The +/-choline diets will be fed for two weeks prior to introducing the +/-hypoxia treatment to allow for adaptation to higher choline intake. Feed will be provided at a fixedpercent of tank biomass. Feeding percentage will be adjusted according to visual observation ofover- or under-feeding and percentages will be consistent across all treatments. Hypoxia stress willbe applied to 6 tanks (3 +choline, 3 -choline) as previously described. Briefly, dissolvedoxygen levels will be low by removing aeration, with normoxic conditions achieved bysupplementing tank water with oxygen purged through an air stone. The normoxia/hypoxia and +/-choline treatments will be applied at 12 months post-hatch through December 2025 and stop whenfemales begin to ovulate. Body weight and length will be recorded bi-monthly through September2025 and at spawning; handling after September will be avoided to minimize handling stress duringrapid gonadal development.Beginning in January 2026, fish will be anesthetized (MS-222, 100 mg/L) and spawn-checked by abdominal palpation to identify females that have ovulated (released eggs into body cavity). The period of ovulation tends to occur over an 8-wk period and the number of females that ovulate each week exhibits a bell-curve shape, with most females ovulating during the middle four weeks. However, the few females that ovulate during the first (1-2 wk) and last (7-8 wk) 2-week periodsoften exhibit poor egg quality. For this reason, only females that ovulate in the middle 4-weekperiod (3-6 wk) will be used to produce offspring, although all collected eggs will be analyzed forindices of egg quality. A total of 96 offspring crosses will be produced (8 females per tank x 3 tanksFigure 10. Experimental setup to test impacts of maternal dietary choline intakeand hypoxia exposure on offspring.16per treatment, n = 24 families per treatment combination). Eggs (1,000) from each female will befertilized with milt from one of two males from the same broodstock family so that effects of siregenetics are equally distributed across all treatment groups. Due to variation in spawning datesspread over the 4-week period, fertilized egg incubation temperatures will be manipulated tocoordinate hatch dates to within a 1-wk period which is a routine approach used by breedingprograms and egg production facilities.Body weight and length will be recorded for all ovulatedfemales during egg collections. The following indices of egg quality will be recorded: egg yield(total weight and number), egg size (diameter), fertilization rate, and hatch rate. Sacfry weight (1-wk post hatch) and fry mortality rate will also be recorded.Each offspring family will be hatched and reared separately in the sacfry and fingerling stage. Prior to first feeding (~14 days post hatch) a subsample of fry from eachoffspring family will be pooled and harvested for transcriptome (RNAseq) and methylome (RRBS)analysis. At first feeding, the number of fish per offspring family will be reduced to 50 fish per tank.Families will be housed in separate tanks until they reach ~15 g (5 months of age). At this time 12fish per family will be anesthetized and PIT tagged and tagged fish will be commingled in six tanks(n=2 fish per family per tank, N=192 fish per tank) and reared according to standard husbandryprocedures. When average weights reach 50 g, 3 tanks will be assigned to a 3-day per week hypoxiachallenge for a period of 8 weekswhile the remaining 3 tanks will remain in consistentnormoxic conditions. Growth performance will be analyzed by measuring body weight and lengthat the beginning, middle (4 wk), and end (8 wk) of the challenge period. Cortisol levels will be measured to assess stress responsiveness. After the final challenge, tissueswill be collected fromchallenged and control fish to analyze patterns of maternal imprinting.