Improved Quantification of Geosmin and 2-Methylisoborneol in Farmed Fish Using Stable Isotope Dilution Gas Chromatography-Mass Spectrometry.

A requisite to improving the taste and odor attributes of farmed fish is the availability of accurate and practical analytical methods to quantify 2-methylisoborneol (MIB) and geosmin (GSM). Solid-phase microextraction (SPME) enables reliable measurement of nanogram per liter quantities of MIB and GSM in water. In contrast, direct headspace (HS)-SPME of biological matrices with variable proximate compositions can increase bias and uncertainty in off-flavor determinations. Analytical recovery plays a crucial role in the accurate determination of MIB and GSM in fish, and this study investigates strategies to maximize and account for this recovery factor. MIB and GSM values in off-flavor catfish and trout were measured using direct HS-SPME and distillation as sample preparation techniques. Trout samples prepared by distillation yielded 10-fold higher GSM recoveries than those from direct HS-SPME (31% versus 3%). A stable isotope dilution method (SIDM) was implemented by routinely spiking samples with known quantities of deuterium-labeled MIB and GSM, allowing for the correction of sample-to-sample recovery deviations. SIDM-determined GSM values generated recoveries of 106 and 95% for direct HS-SPME and distilled trout, respectively. Aspects of the strategies and techniques presented can be incorporated into existing analytical methods to improve the accuracy and sample throughput. Particularly, routine inclusion of SIDM in the evaluation of MIB and GSM can facilitate identification of reliable practices to control off-flavors in aquaculture.

Characterization of maternal immunity following vaccination of broodstock against IHNV or Flavobacterium psychrophilum in rainbow trout (Oncorhynchusmykiss).

Infectious hematopoietic necrosis (IHN) is a significant viral disease affecting salmonids, whereas Flavobacterium psychrophilum (Fp), the causative agent of bacterial coldwater disease (BCWD), remains one of the most significant bacterial pathogens of salmonids. We explored maternal immunity in the context of IHN and BCWD management in rainbow trout (Oncorhynchus mykiss) aquaculture. Two experimental trials were conducted where different groups of female broodstock were immunized prior to spawning with an IHNV DNA vaccine or a live attenuated F. psychrophilum (Fp B.17-ILM) vaccine alone, or in combination. Progeny were challenged with either a low or high dose of IHNV at 13 days post hatch (dph) and 32 dph or challenged with F. psychrophilum at 13 dph. Mortality following a low-dose IHNV challenge at 13 dph was significantly lower in progeny from vaccinated broodstock vs. unvaccinated broodstock, but no significant differences were observed at 32 dph. Mortality due to BCWD was also significantly reduced in 13 dph fry that originated from broodstock immunized with the Fp B.17-ILM vaccine. After vaccination broodstock developed specific or neutralizing antibodies respectively to F. psychrophilum and IHNV; however, antibody titers in eggs and fry were undetectable. In the eggs and fry mRNA transcripts of the complement components C3 and C5 were detected at much higher levels in progeny from vaccinated broodstock and showed a significantly increased and rapid response post-challenge compared with unvaccinated broodstock. After challenges pro-inflammatory cytokine expression was immediately and considerably elevated in the fry from vaccinated broodstock vs. unvaccinated broodstock, whereas adaptive immune genes were elevated to a lesser degree. Results suggest that maternal transfer of innate and adaptive factors at the transcript level occurred because development of lymphomyeloid organs is not complete in such young fry. In addition to documenting maternally derived immunity in teleosts, this study demonstrates that broodstock vaccination can confer some degree of protection to progeny against viral and bacterial pathogens.

Oxytetracycline does not cause growth promotion in finfish.

Until recently, use of antibiotics to enhance terrestrial animal growth performance was a common, U.S. Food and Drug Administration (FDA)-approved, but controversial practice. There are no FDA-approved production claims for antibiotic drug use in fish, but it is a common misconception that antibiotics are widely used for this purpose in U.S. aquaculture. Antibiotics are not thought to be effective growth promoters in fish, but there is little quantitative data available to address whether there are growth-promoting effects that might incentivize the use of antibiotics in this way, despite legal prohibitions. Therefore, this study was conducted to determine if oral administration of oxytetracycline, an antibiotic with known growth-promoting effects in terrestrial livestock, has a similar effect when applied to channel catfish Ictalurus punctatus, hybrid striped bass Morone chrysops × M. saxatilis, Nile tilapia Oreochromis niloticus, or rainbow trout Oncorhynchus mykiss. Oxytetracycline products with production claims are typically applied at doses substantially lower than the approved therapeutic doses for the same products. Medication (0, 0.24, or 1.2 g oxytetracycline dihydrate kg-1 feed) and feeding rates (3% BW d-1) were selected to achieve target daily doses of 0, 16, or 80 mg kg-1 fish representing control, subtherapeutic, and therapeutic treatments. Replicate groups of fish (N = 4) were fed accordingly for 8 wk. Overall, oral administration of oxytetracycline did not affect survival or promote growth of the selected taxa, with no significant differences observed for weight gain, feed conversion ratio, or specific growth rate (P > 0.05 in all cases). Few differences were observed in organosomatic indices and in the frequency of tissue abnormalities; where present, these differences tended to suggest a negative effect of long-term dietary exposure to oxytetracycline. These data demonstrate that there is no benefit to dietary supplementation with oxytetracycline for nontherapeutic purposes in a range of economically important finfish species. As such, our results indicate there is little incentive to misuse oxytetracycline products for purposes of growth promotion in U.S. aquaculture.

Comparison of Sampling and Detection Methods for Chinook Salmon and Steelhead Naturally Infected with Myxobolus cerebralis.

Myxobolus cerebralis (Mc) is a myxozoan parasite causing whirling disease in hatchery- and natural-origin salmonids. To minimize spread of this parasite and the incidence of its associated disease, fish health professionals routinely screen fish for Mc before stocking or moving the fish to Mc-free waters. Sample collection for Mc traditionally entails lethal sampling of cranial tissue followed by pepsin-trypsin digest (PTD) and screening of the sample for mature myxobolid myxospores (PTD method); however, nonlethal sampling methods would be advantageous in some circumstances, such as when dealing with rare or otherwise valuable fish. Accordingly, we compared Mc detections in cranial cartilage by using the PTD method with PCR assays of fin biopsies collected from juvenile Chinook Salmon Oncorhynchus tshawytscha and adult steelhead O. mykiss. Cranial samples were also analyzed using PCR methods for comparative purposes. Results indicated that Mc could be detected by PCR in fin clips, but the results generated by this approach differed significantly from those associated with PTD- and/or PCR-based analysis of cranial cartilage samples. Polymerase chain reaction-based analysis-of individual head samples and head digest pools in both species as well as fins in steelhead-yielded more positive detections than PTD analysis alone. The PCR-based analysis of head and fin tissues yielded different Mc detection rates in both species, but the nature of the detection disparity varied depending on the species and/or life stage of the fish. We conclude that for lethal cranial samples, neither PTD nor PCR should be used alone, but using these techniques in concert may provide the most complete and accurate estimation of Mc presence in a group of salmonids. If imperiled or highly valuable fish are in question, nonlethal fin samples may be used to generate some information regarding Mc status, with the understanding that parasite DNA detections do not necessarily signify mature infections or disease.

Uncoupling EPA and DHA in Fish Nutrition: Dietary Demand is Limited in Atlantic Salmon and Effectively Met by DHA Alone.

Due to the scarcity of marine fish oil resources, the aquaculture industry is developing more efficient strategies for the utilization of dietary omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA). A better understanding of how fish utilize EPA and DHA, typically provided by fish oil, is needed. However, EPA and DHA have different physiological functions, may be metabolized and incorporated into tissues differently, and may vary in terms of their importance in meeting the fatty acid requirements of fish. To address these questions, Atlantic salmon were fed experimental diets containing, as the sole added dietary lipid source, fish oil (positive control), tallow (negative control), or tallow supplemented with EPA, DHA, or both fatty acids to ~50 or 100% of their respective levels in the positive control diet. Following 14 weeks of feeding, the negative control diet yielded optimum growth performance. Though surprising, these results support the notion that Atlantic salmon requirements for n-3 LC-PUFA are quite low. EPA was largely ß-oxidized and inefficiently deposited in tissues, and increasing dietary levels were associated with potential negative effects on growth. Conversely, DHA was completely spared from catabolism and very efficiently deposited into flesh. EPA bioconversion to DHA was largely influenced by substrate availability, with the presence of preformed DHA having little inhibitory effect. These results clearly indicate EPA and DHA are metabolized differently by Atlantic salmon, and suggest that the n-3 LC-PUFA dietary requirements of Atlantic salmon may be lower than reported and different, if originating primarily from EPA or DHA.

Analysis of persistent halogenated hydrocarbons in fish feeds containing fish oil and other alternative lipid sources.

A trade-off exists between beneficial n-3 long-chain polyunsaturated acids and toxic persistent halogenated hydrocarbons (PHHs), both of which primarily originate from fish oil commonly used in fish feeds. Alternative lipid sources are being investigated for use in fish feeds to reduce harmful contaminant accumulation, hence, research is needed to evaluate PHHs in fish feeds with various lipid compositions. An analytical method was developed for PHHs including nine organochlorine insecticides (OCPs), 26 polychlorinated biphenyls (PCBs) and seven polybrominated diphenyl ethers (PBDEs) in fish feeds with differing proportions of fish oils and alternative lipid sources by GC-ECD after accelerated solvent extraction, gel permeation chromatography (GPC), and sulfuric acid cleanup. The GPC removed the majority of the neutral lipids and sulfuric acid treatment effectively destroyed the polar lipids. Thus, the combination of the two methods removed approximately 99.7% of the lipids in the extracts. The method detection limits were less than 5 ng/g dry weight (dw) for most PHHs, while recoveries were 75-118%, 67-105%, 69-92%, 63-100% and 94-144% with relative standard deviations of 0.2-39%, 0.3-20%, 0.5-12%, 1.5-18% and 1.5-15% for PHHs in five types of fish feeds made from different lipid sources. Although the source of lipid showed no impact on cleanup efficiency and the developed method worked well for all feeds, fish feeds with 100% fish oil contained background PHHs and more interference than feeds containing alternative lipids.

Converting crude glycerol derived from yellow grease to lipids through yeast fermentation.

Cryptococcus curvatus, an oleaginous yeast was observed to grow on crude glycerol derived from yellow grease. When cultured in a one-stage fed-batch process wherein crude glycerol and nitrogen source were fed intermittently for 12 days, the final biomass density and lipid content were 31.2 g/l and 44.2%, respectively. When cultured in a two-stage fed-batch operation wherein crude glycerol was supplemented at different time points while nitrogen source addition was discontinued at the middle of the experiment, the biomass density was 32.9 g/l and the lipid content was 52% at the end of 12 days. Compared with other oil feedstocks for biodiesel production, lipid accumulated by C. curvatus grown on glycerol has high concentration of monounsaturated fatty acid, which makes it an excellent source for biodiesel use.

Use of sweet sorghum juice for lipid production by Schizochytrium limacinum SR21.

Stalk juice from sweet sorghum grown in Southern Illinois, USA, was examined for lipid production through microalgal fermentation. Juice concentrations at 100%, 75%, 50%, and 25% led to different biomass, lipid, and docosahexaenoic acid (DHA) production by Schizochytrium limacinum SR21. Biomass dry weight as 9.4g/l at 50% juice concentration was similar to that from pure glucose (10.9g/l). But with a 73.4% lipid content, this dose resulted in higher lipid and DHA production than those from pure glucose. Major fatty acids in cells grown on juice were identical to those fed by other substrates. Among the three sugars - glucose, fructose, and sucrose in sorghum juice, only glucose was utilized for growth. Spent medium after algal removal may be further processed for white sugar production in a traditional way since sucrose content remained the same throughout the algal fermentation process. Algal cells or lipids harvested can be utilized as fish meal, human nutrition supplements, or for biodiesel purpose.

New developments in fish amino acid nutrition: towards functional and environmentally oriented aquafeeds.

Recent evidence shows that some amino acids and their metabolites are important regulators of key metabolic pathways that are necessary for maintenance, growth, feed intake, nutrient utilization, immunity, behavior, larval metamorphosis, reproduction, as well as resistance to environmental stressors and pathogenic organisms in various fishes. Therefore, conventional definitions on essential and nonessential amino acids for fish are challenged by numerous discoveries that taurine, glutamine, glycine, proline and hydroxyproline promote growth, development, and health of aquatic animals. On the basis of their crucial roles in cell metabolism and physiology, we anticipate that dietary supplementation with specific amino acids may be beneficial for: (1) increasing the chemo-attractive property and nutritional value of aquafeeds with low fishmeal inclusion; (2) optimizing efficiency of metabolic transformation in juvenile and sub-adult fishes; (3) surpressing aggressive behaviors and cannibalism; (4) increasing larval performance and survival; (5) mediating timing and efficiency of spawning; (6) improving fillet taste and texture; and (7) enhancing immunity and tolerance to environmental stresses. Functional amino acids hold great promise for development of balanced aquafeeds to enhance the efficiency and profitability of global aquaculture production.

Fatty acid profile of sunshine bass: I. Profile change is affected by initial composition and differs among tissues.

Fatty acid (FA) composition of fillet tissue can be tailored by transitioning fish from alternative lipid-based, low long-chain polyunsaturated fatty acid (LC-PUFA) grow-out feeds to high LC-PUFA "finishing" feeds. To address whether grow-out feed composition influences the responsiveness of fillet tissue to finishing, sunshine bass (SB, Morone chrysops x M. saxatilis) were reared to a submarketable size on grow-out feeds containing fish oil (FO) or a 50:50 blend of FO and coconut (CO), grapeseed (GO), linseed (LO), or poultry (PO) oil. For the final 8 weeks of the trial, fish were either maintained on assigned grow-out feeds or finished with the 100% FO feed. Production performance was unaffected by dietary lipid source, but fillet FA profile generally conformed to nutritional history. Regardless of grow-out regimen, finishing had a significant restorative effect on fillet FA composition; however, complete restoration of control levels of 20:5n-3, 22:6n-3, total LC-PUFA and n-3:n-6 FA ratio was achieved only among fish fed the CO-based grow-out feed. Saturated fatty acids (SFA) appear to be preferential catabolic substrates, whereas medium-chain and long-chain PUFA are selectively deposited in tissues. Provision of SFA in grow-out feeds appears to optimize selective FA metabolism and restoration of beneficial fillet FA profile during finishing.

Fatty Acid Profile of Sunshine Bass: II. Profile Change Differs Among Fillet Lipid Classes.

Fatty acid (FA) profile of fish tissue mirrors dietary FA profile and changes in a time-dependent manner following a change in dietary FA composition. To determine whether FA profile change varies among lipid classes, we evaluated the FA composition of fillet cholesteryl esters (CE), phospholipids (PL), and triacylglycerols (TAG) of sunshine bass (SB, Morone chrysops x M. saxatilis) raised on feeds containing fish oil or 50:50 blend of fish oil and coconut, grapeseed, linseed, or poultry oil, with or without implementation of a finishing period (100% FO feed) prior to harvest. Each lipid class was associated with a generalized FA signature, irrespective of nutritional history: fillet PL was comprised largely of saturated FA (SFA), long-chain polyunsaturated FA (LC-PUFA), and total n-3 FA; fillet TAG was higher in MC-PUFA and total n-6 FA; and fillet CE was highest in monounsaturated FA (MUFA). Neutral lipids reflected dietary composition in a near-direct fashion; conversely, PL showed evidence of selectivity for MC- and LC-PUFA. Shorter-chain SFA were not strongly reflected within any lipid fraction, even when dietary availability was high, suggesting catabolism of these FA. FA metabolism in SB is apparently characterized by a division between saturated and unsaturated FA, whereby LC-PUFA are preferentially incorporated into tissues and SFA are preferentially oxidized for energy production. We demonstrated provision of SFA in grow-out feeds for SB, instead MC-PUFA which compete for tissue deposition, meets energy demands and allows for maximum inclusion of LC-PUFA within fillet lipids.

Modification of fillet composition and evidence of differential fatty acid turnover in sunshine bass Morone chrysops x M. saxatilis following change in dietary lipid source.

Marine oil-based finishing diets have been used to restore fillet FA profile in several "medium-fat" fleshed aquaculture species, and a simple dilution model describing FA turnover has been established to predict and tailor final fillet composition. We evaluated finishing diet efficacy and suitability of the dilution model to describe patterns of FA change in a lean-fleshed model, sunshine bass. Two practical diets (45% crude protein, 15% crude lipid) were formulated, respectively containing corn oil (CO) or menhaden oil (MO) as the primary lipid sources. Sunshine bass (age 1 [approximately 14 mo], 347 +/- 8.6 g, mean individual weight +/- SEM) were stocked in a recirculating system and fed the diets according to different feeding regimens during the final 28 wk of the production cycle. Control groups were fed the CO or the MO feeds exclusively; whereas, the remaining treatment groups were transitioned from the CO diet to the MO diet at 4-, 8-, or 12-wk intervals. Upon completion of the feeding trial, fish were harvested, and production performance and fillet composition were assessed. Replacing MO with CO as the primary lipid source in sunshine bass diets yielded fillets with distinctly different FA profiles; however, finishing with a MO-based diet offered significant compensation for CO-associated reductions in fillet long-chain highly unsaturated FA (LC-HUFA). Although complete restoration was not observed, we achieved significant augmentation of endogenous n-3 FA within 4 wk of feeding the MO diet, and observed a significant increase in LC-HUFA and a beneficial shift in n-3:n-6 FA ratio after 8 weeks. Simple dilution accurately predicted tissue composition for most FA; however, deviations from the model were noted, suggesting selective retention of n-3, PUFA, and LC-HUFA and preferential catabolism of saturates. We conclude marine oil-based finishing diets can rapidly augment beneficial FA levels in sunshine bass fillets; however, simple dilution models do not fully describe selective FA metabolism observed for this lean-fleshed fish.