UUnniivveerrssiittyy ooff RRhhooddee IIssllaanndd DDiiggiittaallCCoommmmoonnss@@UURRII Open Access Dissertations 2014 NNUUTTRRIITTIIOONNAALL AANNDD IIMMMMUUNNOOLLOOGGIICCAALL EEVVAALLUUAATTIIOONN OOFF SSOOYY-- BBAASSEEDD DDIIEETTSS FFOORR SSUUMMMMEERR FFLLOOUUNNDDEERR ((Paralichthys dentatus)) AAQQUUAACCUULLTTUURREE Daniel Ward University of Rhode Island, [email protected] Follow this and additional works at: https://digitalcommons.uri.edu/oa_diss RReeccoommmmeennddeedd CCiittaattiioonn Ward, Daniel, "NUTRITIONAL AND IMMUNOLOGICAL EVALUATION OF SOY-BASED DIETS FOR SUMMER FLOUNDER (Paralichthys dentatus) AQUACULTURE" (2014). Open Access Dissertations. Paper 199. https://digitalcommons.uri.edu/oa_diss/199 This Dissertation is brought to you for free and open access by DigitalCommons@URI. It has been accepted for inclusion in Open Access Dissertations by an authorized administrator of DigitalCommons@URI. For more information, please contact [email protected]. NUTRITIONAL AND IMMUNOLOGICAL EVALUATION OF SOY- BASED DIETS FOR SUMMER FLOUNDER (Paralichthys dentatus) AQUACULTURE BY DANIEL WARD A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIRMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOLOGICAL AND ENVIRONMENTAL SCIENCE UNIVERSITY OF RHODE ISLAND 2014 DOCTOR OF PHILOSOPHY DISSERTATION OF Daniel Ward APPROVED: Dissertation Committee: Marta Gomez-Chiarri David Bengtson Chong Lee Roxanna Smolowitz Gavino Puggioni Dean of the Graduate School: Nasser H. Zawia UNIVERSITY OF RHODE ISLAND 2014 ABSTRACT Summer flounder aquaculture has progressed significantly in the last twenty years, and diets have been optimized to provide optimal nutrition, ensuring a high quality final product. Recently, the focus of research has evolved toward finding a replacement for fish meal in diets for many species, as the demand for fish meal worldwide has caused the primary protein product to become increasingly economically volatile. Therefore, a series of experiments were designed to systematically evaluate feeds for summer flounder (Paralichthys dentatus) including soy-based products as the primary protein source. The first feed trial involved the comparison of three diets; a fish meal control, a 60% fish meal replacement diet with soybean meal (SBM), and a 60% fish meal replacement diet with soybean meal and soy protein concentrate (1:1 ratio, SBM/SPC), in order to investigate the impact of including soy products in diets for summer flounder on growth and survival following challenge (post-feeding trial) with a bacterial pathogen of summer flounder (Vibrio harveyi). The diet with the majority of the FM replaced with SBM/SPC produced significantly greater growth, and survival following bacterial challenge, suggesting that a combination of soybean meal and soy protein concentrate could provide a good alternative to fish meal-based replacement diets by providing equal growth to fish meal diets and enhanced resistance to bacterial challenge. A second trial was designed to systematically evaluate which combination of soybean meal and soy protein concentrate would result in the best growth and survival to bacterial challenge. A diet comprised of 40% fish meal, 48% soy protein concentrate, and 12% soybean meal resulted in significantly better growth and survival following bacterial challenge compared to a fish meal control diet, confirming the results from the first trial. Additional feed trials were designed to investigate the compounds responsible for both the reduced growth and the increased survival following bacterial challenge, in order to determine which compounds of soybean meal, but absent in soy protein concentrate, may be responsible for the effects on growth and survival to bacterial challenge. In the first feed trial in this series, ,in addition to a fish meal-based control, four additional diets were formulated to contain 60% soy protein concentrate and 40% fish meal as protein sources, with 0.53%, 1.23% or 1.94% soy molasses (w/v) added to the diet. Growth was reduced with soy molasses added to the diet, with the most decreased growth in the group fed the 1.94% soy molasses-supplemented diet, suggesting that antinutritional factors present within soy molasses are responsible for decreased growth observed in summer flounder fed soybean meal-based replacement diets. Therefore, a second feed trial was designed to further investigate the role of compounds present in soybean molasses on growth and survival. Soy molasses was fractionated using n-butanol to phase-separate the different compounds within the soy molasses into either a water (primarily enriched with oligosaccharides), interphase (containing a mix of saponins, oligosaccharides, ash and proteins) or butanol (including saponins) phase and 3 diets were prepared by adding each sub-fraction to a soy protein concentrate replacement (60%) diet at a level corresponding to a 12% soybean meal replacement level (best results from trial 2). Growth was significantly lower for all diets with soy molasses fractions added compared to groups fed either the fish meal or soy protein concentrate control diets. Survival following challenge was highest for the groups fed the diets containing either the water phase or butanol phase of soybean molasses, suggesting that oligosaccharides, alone or in combination with low levels or other antinutritional factors, may be responsible for increased survival to bacterial challenge compared to the fish meal diets. Finally, a feed trial was designed to test the effect of three different levels of oligosaccharide supplementation (0.2%, 0.4% or 0.6% supplementation with stachyose hydrate and raffinose pentahydrate combined at a 3.16:1 ratio (w/w)) to a 60% soy protein concentrate replacement on growth and survival to bacterial challenge. The diet which produced the greatest growth and significantly better survival than the soy protein concentrate-based control diet included an oligosaccharide supplementation level of 0.4% (w/w). These results demonstrate that soy protein concentrate can replace fish meal in summer flounder diets at a high level (60%) with no negative effects on growth. Furthermore, supplementation of the soy protein replacement diet with crystalline oligosaccharides or an oligosaccharide-rich fraction resulting from the phase-separation of soybean meal may decrease susceptibility to disease. These soy-based diets would provide a sustainable, economically viable alternative to fish meal diets for marine finfish aquaculture. ACKNOWLEDGEMENTS I would like to thank Dr. Marta Gomez-Chiarri for taking a chance on me as a graduate student, and changing the trajectory my life has since taken. I am incredibly grateful for her experience, assistance, and patience in helping to develop me as a scientist. I thoroughly enjoyed working in her lab and learning from her valuable expertise in pathology and all things aquaculture. I have learned many things from Dr. Gomez-Chiarri regarding my graduate research at URI, though I am also thankful for the many things that she was able to teach me regarding research and developing into an independent scientist. Marta has taught me about experimental design, writing grant proposals, how to approach various obstacles that came up throughout the research process, and many other aspects of conducting research that can’t be taught in a class or in a book. For this I am extremely thankful, and she has had an impact on how my scientific knowledge develops throughout the rest of my career. I would also like to thank Dr. David Bengtson for all of his support for the entire time I have been at URI. He helped me design all of my research and was an incredible resource for all of the aquaculture techniques I have learned. I would also like to thank Dr. Chong Lee for all of the assistance he gave as my committee member, as well as everything I learned from him about aquaculture. I would especially like to thank the people I worked with the most during my time at URI: Rachel Bone, Steve Gray, Christy Varga, Alia Chisty, Chris Andrikos and all of the other labmates, URI staff and interns that were especially helpful to my research. Most of all I would like to thank my family, Dad, Mom, Travis, and Jordan, for v supporting me as I went through graduate school, and my wife Jen and my daughters Emily and Sarah for always being there for me. This research was supported by grants and funds from the USDA Sustainable Agriculture, Research and Education (SARE) Program, United Soybean Board, Rhode Island Sea Grant (NOAA), Rhode Island Agricultural Station, URI Graduate Students Association, URI Graduate Assistants Union and URI Graduate School. vi TABLE OF CONTENTS ABSTRACT. ................................................................................................................. ii ACKNOWLEDGMENTS. ........................................................................................... v TABLE OF CONTENTS ........................................................................................... vii LIST OF TABLES .................................................................................................... viii LIST OF FIGURES. ................................................................................................... ix INTRODUCTION. ...................................................................................................... 1 CHAPTER 1 INCORPORATION OF SOYBEAN PRODUCTS IN SUMMER FLOUNDER (Paralichthys dentatus) FEEDS: EFFECTS ON GROWTH AND SURVIVAL TO BACTERIAL CHALLENGE ....................................................................................... 6 CHAPTER 2 IMPACTS OF SOYBEAN MOLASSES AND SUB-FRACTIONATED SOYBEAN MOLASSES ON GROWTH AND SURVIVAL FOLLOWING CHALLENGE IN DIETS FOR SUMMER FLOUNDER (Paralichthys dentatus) ................................. 38 CHAPTER 3 INFLUENCE OF OLIGOSACCHARIDE SUPPLMENTATION OF SOY PROTEIN CONCENTRATE REPLACEMENT ON GROWTH AND SURVIAL TO BACTERIAL CHALLENGE OF SUMMER FLOUNDER (Paralichthys dentatus) .................................................................................................................................... 65 BIBLIOGRAPHY ...................................................................................................... 90 vii LIST OF TABLES TABLE PAGE Table 1. Diet formulations for the 3 experimental diets for Trial 1. ........................... 31 Table 2. Diet formulations for the 7 experimental diets for Trial 2 ............................ 32 Table 3. Performance of summer flounder after a 12 week feeding trial with diets with different protein sources ............................................................................................. 34 Table 4. Effect of diet on survival to bacterial challenge and selected blood parameters. ............................................................................................................... 35 Table 5. Performance of summer flounder after a 9 week feeding trial ..................... 37 Table 6. Diet formulations for the 5 experimental diets for Trial 1 ............................ 56 Table 7. Diet formulations for the 5 experimental diets for Trial 2 ............................ 58 Table 8. Proximate analysis and antinutritional factor content for Trial 1 diets. ...... 59 Table 9. Proximate analysis and antinutritional factor content for Trial 2 diets ...... 62 Table 10. Diet formulations for the 5 experimental diets .......................................... 82 Table 11. Plasma blood chemistry .............................................................................. 88 viii