ANDREW R. COURSEY, for the Master of Science degree in Zoology, presented 24 June 2011 at Southern Illinois University Carbondale TITLE: ALTERNATIVE FEEDING STRATEGIES TO MAXIMIZE MARINE FEEDSTUFF REPLACEMENT IN LARGEMOUTH BASS Micropterus salmoides CULTURE WHILE MAINTAINING PRODUCTION PERFORMANCE Advisors: Chris Kohler and Jesse Trushenski Harvest of marine resources for feedstuff production is at its maximum sustainable yield and alternative feedstuffs are needed to supply livestock production for an expanding human population having a growing desire for seafood. Aquaculture is currently the fastest growing sector of agriculture, but the high cost and limited supplies of marine feedstuffs for aquafeeds could ultimately limit production of foodfish. Replacement of marine feedstuffs poses a dilemma, however, as replacement of fish meal can affect animal growth, and replacement of fish oil in aquafeeds leads to altered fillet fatty acid profiles without the maximum benefit of high omega-3 (n-3) and long-chain polyunsaturated fatty acids (LC-PUFA) content. Two feeding trials were conducted to determine if poultry byproducts can replace fish meal and fish oil in diets for largemouth bass Micropterus salmoides without affecting production performance or fillet fatty acid composition. Poultry byproducts were able to replace fish meal and fish oil while maintaining production performance of largemouth bass, however, the altered fatty acid profiles of the feeds were reflected in the fillets. Finishing diets containing high levels of fish oil were implemented to restore n-3 and LC PUFA concentrations in the fillet. Twelve weeks of finishing proved suitable to partially restore n-3 and LC-PUFA concentrations while limiting the overall use of marine resources in the largemouth bass production cycle.

Cobia (Rachycentron canadum) aquaculture is poised for expansion, due in part to rapid growth rate, tolerance of culture conditions, and high market value of this species. Similar to other carnivorous marine fishes, the high monetary cost and long-range unsustainability of reliance on fish oil as the principle lipid source for cobia feeds necessitates evaluation of alternative lipid sources to spare or replace fish oil. Unfortunately, alternative lipid-based feeds may affect production performance, and typically yield fillets with reduced levels of long-chain polyunsaturated fatty acids (LC-PUFAs) such as docosahexaenoic acid (DHA, 22:6n-3). Recent research has indicated that diets rich in saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs), coupled with the application of so-called "finishing feeds" (feeds containing elevated levels of fish oil relative to grow-out feeds) at the end of the production cycle, may limit or attenuate these effects of fish oil sparing. Accordingly, I conducted two trials to evaluate the usefulness of alternative lipids and finishing in cobia culture. In the first trial, I assessed growth performance and tissue fatty acid composition of juvenile cobia fed diets (~11% lipid, ~48% protein) containing fish oil (control), or 50/50 blends of fish oil and alternative lipids (beef tallow, pork lard, partially and fully hydrogenated soy oils, and traditional soy oil) supplemented with an algal meal (to meet the 22:6n-3 requirement of cobia) for 8 weeks. Although minor differences were observed in feed intake, growth performance was otherwise equivalent among the dietary groups. Tissue fatty acid composition varied significantly among treatments, however, with alternative lipids containing higher levels of saturated (SFAs) and monounsaturated fatty acids (MUFAs) yielding tissue profiles that were most similar to those associated with the fish oil-based control feed. Although beef tallow and hydrogenated soybean oil were largely equivalent in terms of growth performance and fatty acid profile change, beef tallow was selected for further evaluation because of its low cost. In the second trial, beef tallow replaced fish oil in feeds at one of four substitution levels: 0% (100% fish oil), 33%, 67%, or 100% tallow (with algal 22:6n-3 meal included in all feeds as before). Juvenile cobia were raised on these feeds for 8 weeks, then switched to the 100% fish oil-based control feed for an 8-week finishing period. During finishing, subsamples of fish were collected every 2 weeks to quantify changes in tissue fatty acid profile over time as a result of finishing. In the second trial, the overall similarity of tissue fatty profiles to those in the 100% fish oil control treatment increased over the course of finishing, mostly as a result of declines in SFAs and MUFAs. These results suggest that SFA and MUFA-rich lipids, such as fully hydrogenated soy oil and beef tallow, are effective and strategically valuable as partial substitutes for fish oil in cobia feeds, and that finishing is at least partially effective in restoring cobia tissue fatty acid composition to a state approximating that of cobia fed only fish oil as dietary lipid.

Aquaculture is currently the fastest growing sector of protein production, and is expected to overtake the harvest of wild fisheries. Limitations in nutrition, specifically fatty acid nutrition, are preventing even more dramatic growth of many species of commercial importance. Currently, much of the research involving fatty acids examines requirements as being correlated to thermal guilds (warm vs. cool water) or salinity tolerance (marine vs. freshwater). However, recent studies have revealed the potential for trophic level to be as much, if not more, influential in determining fatty acid requirements of a species. As such, two feeding trials were conducted to determine the requirements of two species of different trophic levels (Oreochromis niloticus and Trachinotus carolinus) based on C18 PUFA vs. LC-PUFA. Nile Tilapia, O. niloticus, exhibited similar growth regardless of the inclusion of C18 PUFA or LC-PUFA, however, tissue fatty acid profiles were influenced per the diet provided. As such, it was concluded that Nile Tilapia exhibit the capacity to effectively synthesize LC-PUFA from C18 PUFA as is seen in many species that occupy low trophic levels. Florida Pompano, T. carolinus, did not exhibit any significant differences in growth regardless of the diet provided, but numerical differences indicated benefits towards inclusion of dietary LC-PUFAs. Similar to O. niloticus, tissue fatty acid profiles were significantly affected by dietary treatment. Based on numerical differences in growth performance and significant differences in tissue fatty acids, it was concluded that Florida Pompano show a typical carnivorous requirement for LC-PUFA.

Aquatic habitats are closely linked to surrounding terrestrial environments via reciprocal subsidies. Much of the research on these subsidies has been focused on streams, while subsidies across aquatic-terrestrial boundaries of other aquatic systems, like temporary ponds, have received little attention. Temporary ponds are seasonally inundated aquatic habitats and patterns of energy transport across transient aquatic-terrestrial boundaries are not well understood. To address the lack of information regarding these cross-habitat subsidies, I quantified: 1) leaf litter inputs, 2) amphibian egg inputs, 3) terrestrial insect inputs, 4) amphibian metamorph emergence, and 5) aquatic insect emergence for eight temporary ponds. Terrestrial insect inputs to ponds represented substantial high-quality subsidies to ponds that are generally unaccounted for in similar studies. While larger ponds produced greater total fluxes to terrestrial habitats, smaller ponds were often more productive per unit area. Therefore, a mosaic of small ponds may produce greater or equivalent subsidies and may be of greater value to terrestrial food webs than a single large pond. Resource subsidies of energy and nutrients can be transported via physical forces or biotic processes, such as animal migration or emigration. I quantified net nutrient fluxes associated with amphibian migrations across aquatic-terrestrial boundaries of eight temporary ponds in Illinois. I measured carbon (C), nitrogen (N), and phosphorus (P) subsidies from forests to ponds, in the form of eggs, and the reciprocal subsidies from ponds to forests via juvenile emigration. Juvenile emergence biomass did not resemble egg biomass, as not all species bred successfully in each pond, resulting in variability in the magnitude of nutrient fluxes both across ponds and species. The forest was not always the recipient system of net nutrient fluxes. Hydroperiod, trophic interactions, and species composition explained some dynamics of N and P subsidies. Most studies have focused on the magnitude of cross-boundary fluxes, but the impact of a subsidy is mediated by the size of the flux as well as the quality and relative abundance of similar resources in the recipient habitat. Long-chain polyunsaturated fatty acids (LC-PUFAs) are necessary for proper physiological function, are unevenly distributed across the landscape, and animals differ greatly in their ability to synthesize them de novo. This creates the potential for limitation and increases their possible importance as a subsidy. I examined LC-PUFA tissue concentration and export in eight species of emerging amphibian metamorphs across eight temporary ponds in a wetland complex. I found that tissue concentrations and export of LC-PUFAs varied across species, but were generally within the ranges of several freshwater fish and aquatic insects. Anurans exported higher amounts of LC-PUFAs than salamanders, largely due to the higher emergence biomass of anurans. Further, I explored fatty acid profiles and immune function of wolf spiders in wetland and upland habitats. Wetland spiders had higher tissue levels of aquatic-derived LC-PUFAs and elevated immune function compared to upland spiders. These patterns suggest aquatically derived nutrients, such as LC-PUFAs, may benefit organisms exploiting freshwater habitats.

No description available.

Investigating the biosynthesis of long-chain (C20–24) polyunsaturated fatty acids (LC-PUFA), physiologically important compounds including arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in fish is crucial to identify dietary requirements for essential fatty acids (EFA). Moreover, knowledge of the C20–24 LC-PUFA biosynthetic capability of farmed fish species enables us to understand their ability to utilise commonly used raw materials such as vegetable oils, which naturally lack LC-PUFA but include C18 PUFA that are metabolic precursors of LC-PUFA. Studies have shown that the potential of a species for LC-PUFA biosynthesis is associated with the complement and function of fatty acyl desaturase (fads) and elongase of very long chain fatty acid (elovl) genes existing in that species. The present study therefore aimed to investigate these genes in the African catfish (Clarias gariepinus), the most commercially important farmed fish in sub-Saharan Africa. A fads2, a fads6 and four elovl (elovl2, elovl4a, elovl4b, elovl8) cDNAs were cloned and functionally characterised by heterologous expression in yeast. The Fads2 was a bifunctional desaturase enzyme with ∆6∆5 and ∆8 activities, and thus catalysing all the desaturation reactions required for ARA and EPA biosynthesis from C18 precursor fatty acids. Moreover, the C. gariepinus Fads2 enzymes also desaturated 24:5n-3 to 24:6n-3, a ∆6 desaturation required for the biosynthesis of DHA through the so-called “Sprecher pathway”. Functional characterisation of Fads6 by heterologous expression in yeast did not reveal its function. With regards to elongases, the C. gariepinus Elovl2 demonstrated the ability to elongate C20 and C22 PUFA and thus complements the Elovl5 with elongase capability towards C18 and C20 PUFA. The Elovl8 was capable of only limited elongation of C18 and C20 PUFA. Elovl4a and Elovl4b, enable the biosynthesis of very long-chain (>C24) fatty acids, compounds with major roles in vision and fertility of vertebrates. The present study confirmed that C. gariepinus possess all the enzymatic capabilities required for the biosynthesis of ARA, EPA and DHA and, therefore, its physiological EFA requirements could be satisfied with dietary provision of C18 PUFA.

The main source of omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA) in our diet is supplied by fish, and an ever-increasing proportion of these are being produced by aquaculture. The drive for the growing market demand and production from sustainable sources has led to the use of high-energy (fat) diets and, recently, to the replacement of fishmeal and fish oil with non-marine components, such as plant meals and vegetable oils that are devoid of n-3 LC-PUFA. Both changes impact greatly on lipid and fatty acid metabolism in fish, with health implications for the fish and the human consumer. This impact highlights the need to investigate the basic molecular mechanisms underlying the regulation of lipid and fatty acid metabolism in fish, specifically focussing on the pathways of lipid homeostasis and LC-PUFA synthesis. The aim of this study was to develop and utilise Chinook salmon embryo (CHSE-214) cell line as a model for Atlantic salmon, Salmo salar L., to enable an integrated approach to study the biochemical and molecular regulation of lipid metabolism in fish. In particular, α-linolenic acid (LNA, 18:3n-3) and linoleic acid (LOA, 18:2n-6), which are essential fatty acids abundantly found in vegetable oils, and are precursors of LC-PUFA, were supplemented in combination with other fatty acids, to explore the effect of these on total lipid content, lipid class, FA composition and gene expression of CHSE-214 cell line. Total lipid content was extracted, followed by determination of lipid class and fatty acid analyses. Gene expression analyses of transcription/nuclear factors and various target genes in Atlantic salmon, including those involved in pathways of LC-PUFA synthesis and fatty acid oxidation, were carried out. The results demonstrated that CHSE-214 cell line, under experimental conditions, is able to convert LNA to eicosapentaenoic acid (EPA, 20:5n-3), and LOA to arachidonic acid (ARA, 20:4n-6), but not LNA and/or EPA to docosahexaenoic acid (DHA, 22:6n-3), highlighting the activity of elongase and desaturase enzymes during the conversion process. Changes occurring on the fatty acid profile and also at molecular level were observed. Understanding the role that transcription factors play in the regulation of lipid biosynthesis in fish will allow endogenous LC-PUFA synthesis to be optimised. The results from this study could be used to improve the efficiency of alternative, sustainable diets in aquaculture, while maintaining the nutritional quality of farmed fish for the final consumer. CHSE-214 cell line can therefore be used as a model to study the molecular mechanisms involved in the LC-PUFA biosynthesis, particularly in the conversion of LNA to EPA, which can then be reproduced in vivo, saving time and money.

Au cours des dernières années, l'augmentation de la production aquacole a nécessité un changement dans la composition des aliments pour poissons, pour remplacer la farine (FP) et l’huile de poisson (HP) par des sources végétales plus disponibles. Dans ce contexte, le travail de ma thèse avait pour but d'analyser les effets d’une substitution totale et concomitante de la FP et HP par des ingrédients végétaux dans les aliments distribués pendant tout le cycle de vie de la truite arc-en-ciel, de la première alimentation jusqu’à la reproduction. Ce travail a montré pour la première fois que la truite peut survivre, se développer et avoir une descendance viable, lorsqu’elle est nourrie tout au long de son cycle de vie avec un régime 100% végétal. Un tel remplacement alimentaire a entraîné des changements radicaux dans le profil en acides gras des tissus des femelles. Cependant, nous avons constaté que la truite était capable de synthétiser des acides gras polyinsaturés à longue chaîne n-3 (AGPI-LC n-3) à partir de précurseurs présents dans l'aliment, et de stocker dans les ovules une partie de ces acides gras néosynthétisés dans le foie , permettant ainsi une reproduction efficace. Nous avons également étudié les possibles effets trans-générationnels d’une alimentation 100% végétale des géniteurs, sur la survie et la croissance de la descendance et sur sa réponse à différents régimes aux stades précoces, en nous appuyant sur des analyses du transcriptome des alevins entiers. Aucun effet significatif de l’alimentation maternelle avec des régimes à base de végétaux n’a été observé sur les descendants avant leur première alimentation, à l'exception d'un poids corporel légèrement inférieur (-13%) à celui des descendants de femelles nourries avec un aliment contenant des FP et HP. En revanche, des effets significatifs de l'alimentation maternelle sur le transcriptome des alevins sont apparus après 3 semaines d'alimentation. L'histoire nutritionnelle de la mère a affecté principalement des gènes impliqués dans la croissance/contraction musculaire et dans les métabolismes énergétique et glucidique. Quelle que soit l'origine maternelle, l’alimentation des alevins avec des aliments contenant des ingrédients végétaux conduit à une régulation positive des gènes impliqués dans le métabolisme des AA/protéines et le métabolisme du cholestérol, ainsi qu’à des changements dans l'expression des gènes impliqués dans le métabolisme glucidique. Enfin, les effets à long-terme de régimes à base de matières premières végétales chez la truite arc-en-ciel ont également été étudiés chez les juvéniles (10g) et les poissons «en croissance» (250-350g), en se focalisant principalement sur l’expression des gènes dans l’intestin et le foie. Cette étude à long-terme a mis en évidence chez les juvéniles des effets subtils sur des gènes intestinaux et hépatiques (transcriptome), avec principalement une augmentation de l’expression des gènes impliqués dans les voies de synthèse des AGPI-LC et du cholestérol chez les poissons nourris avec des régimes à base de plantes. Ces changements d’expression ont été maintenus chez les poissons «en croissance». L'expression de gènes impliqués dans le catabolisme des protéines, le métabolisme des glucides et dans le transport intracellulaire a également été modifiée par les régimes à base de plantes chez les juvéniles, mais ces résultats ne sont pas entièrement confirmés chez les poissons «en croissance». L'ensemble des résultats de cette thèse fournissent des informations originales sur l'utilisation de régimes alimentaires avec des taux de remplacement élevés de FP et d’HP pendant l'ensemble du cycle de vie des poissons, y compris les reproducteurs et les stades précoces, car la majorité des études précédentes avaient concerné la phase de grossissement. Des ajustements de la formule alimentaire sont encore nécessaires pour continuer à optimiser les performances de reproduction et la croissance des poissons d’élevage. / In the last years, the increase in aquaculture production has forced a change in fish feed composition, with increasing substitution of fish meal and fish oil by more available plant sources. In this context, the present PhD work aimed at analyzing the effects of different levels of concomitant dietary replacement of fish meal and fish oil by plant ingredients during the whole life cycle of rainbow trout (from first feeding to reproduction). This work showed for the first time that rainbow trout was able to survive, grow and produce viable offsprings, when fed a totally plant-based diet throughout the whole life cycle. Such dietary replacement resulted in drastic changes in tissues fatty acid profile of broodstock females. However, we found that trout was capable to synthetize n-3 long chain- polyunsaturated fatty acids (LC-PUFA) from dietary precursors and to transfer large amounts of these fatty acids in ova, so that an efficient reproduction can occur. We also studied the potential carry over generation effects of feeding broodstock a totally plant-based diet on progeny and their response to different diets at early stages, using molecular approaches, including the study of the whole body transcriptome. Considering the effects on progeny, the present study confirmed the capability of trout to survive and grow on a plant-based diet, but with slight differences in terms of weight. While no effects of maternal dietary background were observed before first feeding, except slightly lower body weight (-13%), significant effects on the transcriptome of whole body alevins appeared after 3 weeks of feeding. These effects of maternal nutritional history were mainly related to muscle growth/contraction and carbohydrate and energy metabolism. Irrespective of the maternal origin of progeny, first feeding diets containing plant ingredients resulted in up-regulation of genes involved in AA/protein and cholesterol metabolism, as well as in changes in the expression of genes involved in carbohydrate metabolism. Finally, the effects of long-term feeding trout plant-based diets were also studied in juveniles (10g) and ongrowing fish (250-350g), mainly focusing on intestine and liver gene expression. The long term trial in juveniles highlighted subtle effects on both intestinal and hepatic gene expression (transcriptome), mainly related to LC-PUFA and cholesterol biosynthetic pathways, which were enhanced in fish fed the plant based diets. This transcriptional pattern was maintained in ongrowing fish. Genes involved in protein catabolism, carbohydrate metabolism and trafficking were also affected by plant-based diets in juveniles, but these results were not fully confirmed in ongrowing fish. Overall, the results of the present thesis allowed extending the use of diets with high replacement rates of fish meal and fish oil to the whole life cycle of fish, including broodstock and early stages. Adjustments of the feed-formula are still needed to further optimize reproductive and growth performance.