The Effect of Diet on Intestinal Structure and Function in the Abalone (Haliotis laevegata). Robert Kemp B.Ag Sc, (Ilons), Adelaide University A TIIESIS SI]BMITTED TO THE T]NIVERSITY OF ADELAIDE FOR THE DEGREE OF'DOCTOR OF PHILOSOPHY November 2001. r,= t ( -ø' \r. "-'.,n- .,' ¡. I s ltt, t', t Nf., " 20u!; I Table of Contents: Table of Contents. ....11 List of Figures ...... ...ix List of Tables........ xiii List of Plates,........ .xiv Abbreviations ....... .xvi Abstract .xix Declaration .xxi 1. Review .........1 1.1. GenerallntroductiontoAbaloneAquaculture........... .......................1 1.2 The Farmed Abalone Industry in Australia. Production Factors other than Nutrition.. ..................2 1.2.1 Tank Design ..........2 t.2.2. Water Quality .......3 1.2.3 Stocking Rates .5 1.2.4. Translocation of Abalone ..... 6 I.2.5. Parasite Infestation ................6 t.3. Feeding Behaviour ,.''.'.'........7 1.3.1 Natural Diet. ................. ................7 1.3.2 Growth Rate.................. ................8 1.3.3 Protein Requirements. .. '.'.,..''...'...8 1.3.4 Energy Requirements. .. .............. l0 1.3.5 Mineral Requirements. .......,...... 11 1,4. Diet Development .',',.,,.',,,'12 1.5. AustralianNutritionalResearch. ..............13 1.5.1. Base Diet. ..........,...13 1.5.2. Diet Trials With Variable Nutritional Inclusion levels.. ,.........,.14 1.5.3. Nutrient Digestibility Studies. ................14 1.5.4. Protein to Energy Ratios. t6 1. 5.6. Current Nutritional lnvestigations. ...... l6 1.5.1. Growth Trials........ ................17 1.6. The Structure and Function of the Gastrointestinal Tract of Abalone. ............. 18 1.7. Intestine Development. ............. ..................20 1.7.1. Age related ..20 1.7.2. Horizontal axis...... 2t 1 .7 .3 . Vertical axls. .. , . .. .. L7,4. Effects of Diets on the Intestine. ... 1.7.5. Anti-Nutrient Factors in Legumes. 1.8. Digestion in the Intestine. 1.8.1. Erzymes in Abalone 1.8.2. Frzyme Diet and Species Interaction Effects. 1.8.3. Diet Related Enzyme Changes. .. 1.8.4. Enzymes of Microbial origin...... 1.9. Nutrient Absorption in the Intestine 1.10 Adaptation to Dietary Effects on the Intestine. 1. 1 1 Summary. LI2 Potential Benefits of the Research. 1.13 Objectives of the Research. 2. General Materials and Methods and Initial Experiments. 2.1 Aquarium System. 2.2 Abalone Tissue Sampling: ...... JJ 2.3 Histology: ......3s 2.3.1 Morphology and Mucin Assessment... ......35 2.3.2 Image Analysis. ......35 2.4 Electron Microscopy:.....,,.,......, ......35 2,4,1 ScanningElectron Microscopy ......35 2.4.2 Transmission Electron Microscopy. .... ......36 2.5 Apical Membrane Preparation: ............,... ......36 2.6 Apical Membrane Enzyme Assays: ......36 2.6.1 Alkaline Phosphatase..............., ......37 2.6.2 Maltase, Sucrase and b-galactosidase.. ......37 2.6.3 Leucine Aminopeptidase. ......37 2.7 . Intracellular Membranes: .............. ....,...............38 2.7.1 Succinate dehydrogenase. .................... 38 2.7.2 Acid Phosphatase........ .................... 38 2.8 BasolateralMembrane SodiumÆotassiumATPase: .............,......38 2.9 Protein: ....................39 ill 2.10 Initial Experiments: ............... 39 2.10.1 Initial Isolation Procedure of Abalone Intestinal Apical Membranes. ..... 39 2.10.2 Comparison of lntestinal Enzyme Activities in Wild-reared Abalone of Sizes 55-70mm and 113-l45mm 3. Impact of Rearing Regime on Intestinal Structure and Function in the Abalone Haliotis laevegata.. 3.1 Abstract: 42 3.2 Introduction:............ 42 3.3 Methods:....... 3.3.1 Animals,... .....45 3.3.2 Diets and Feeding. .....45 3.3.3 General Methods. .....45 3.3.4 Chemicals. .....46 3.3.5 Statistical Analysis .....46 3.4 Results: .....46 3.4.1 Morphological Studies: ....... .....46 3.4.2 Functional Characteristics: .. ..........,.55 3.5 Discussion:................ ....,.......58 3.5. I Morphological Studies: ...,... ............ s8 3.5.2 Functional Analysis: ............61 3.6 Conclusion,...... 64 4, Effect of Diet Constituents on the Abalone Halìotìs laevegøta Intestinal Structure and Function. ....,...... 66 4.1 Abstract. 66 4.2 Introduction: .............. 67 4.3 Materials and Methods: .............. 69 4.3.1 Animals. 69 4.3.2 Diets and Feeding.... 69 4.3.3 General Methods. .... 69 4.3.4 Chemicals. 70 4.3.5 Statistical Analysis ....... 70 4.4 Results 70 lv 4.4.1 Morphological Studies: .............. 4.4.2 Functional Capabilities: ............... 4.4.2.1 Raw and Raw + Phytase Treatments. 4.4.2.2 Legumes with Heat Treatment. 4.5 Discussion:.. 4.5.1 Raw Protein Source;.. 4.5.1.1 Morphological Studies 4.5.2.7 Functional Analysis.... 4.5.2 Raw Protein Source Plus Phytase:.. 4.5.2.1 Morphological Studies 4.5.2.2 Functional Analysis.... 4.5.3 Heat Treated Protein Source. 4.5.3.1 Morphological Studies 4.5.3.2 Functional Analysis.... 4.5.4 Mucus Cover: 4.6 Conclusion: ................ 5. Carbohydrate Digestion in the Abalone Halíotis laevegata Intestine: Effects of Processed Maize Starch, Endogenous Legume Starch and Inclusion Level. 5.1 Abstract: 5.2 Introduction: ...................... 5.3 Methods:.. 5.3 1 Animals................ s.3 2 Diets and Feeding.,....... 5.3 3 General Methods. 5.3 4 Chemicals. 5.3 5 Statistical Analysis ....... 5.4 Results: 5.4.1 Reduced Pregelled St¿rch Inclusion: 5.4.2 Reduced Legume Inclusion and Altered Starch: 5.5 Discussion:.,.............. 5.5.1 Reduced Pregelled Starch Inclusion: 5.5.2 Reduced Legume Inclusion and Altered Starch: v 5.6 Conclusion 111 6. A Preliminary Study on the Effect of Inert Fillers on the Abalone Halíotis laevegata Intestinal Digestive Enzyme Function. 6.1 Abstract: 6.2 Introduction:.................. 6.3 Methods:.. 6.3.1 Animals: ... 6.3.2 Diets and Feeding.... 6.3.3 General Methods. .... 6.3.4 Chemicals. 6.3,5 Statistical Analysis,....... 6.4 Results: 6.4.1 Inert Fillers 5Vo Inclusion: 6.4.2 Increasing Levels of the Inert Filler Kaolin: .,. 6.5 Discussion:................ 6.5.1 Inert Filler 5% Inclusion.............. 6.5.2 Increasing Levels of the Inert Filler Kaolin: ... 6.6 Conclusion: ................ 7. Carbohydrate Binding and the Agglutinating Activity of Antinutrient Factors of Various Legumes Sources on the Intestine of the Abalone Halíotís laevegata.. ......125 7.1 Abstract: ................125 7.2 Introduction:.... ...............125 7.3 Methods: ...............127 7.3.1 Agglutination Experiment. ........... ...............127 7.3.1.1 Heat Treaftnent of Legumes......,.... ...............127 7.3.1.2 Animals ............... 128 7.3.1.3 Diets and Feeding..... ...............128 7.3.1.4 Agglutination Assay., 7.3.2 General Methods. 7.3.4 Chemicals. 7.3.5 SøtisticalAnalysis vl 7.4 Results 7.4.1 Agglutination Experiment.. 7.4.2 Feeding Trial: 7.5 Discussion:................ 7.5.1 Agglutination Experiments. 7.5.2 Feeding Trial: 7.6 Conclusion:................ 8. The Effects of Legume based Diets on the Assimilation of Fatty Acids in the Abalone Halíotís laevegata Foot Tissue............... 8.1 Abstract: 8.2 Introduction: .............. 8.3 Materials and Methods:............. 8 3.1 Animals.... 8 3.2 Diets and Feeding. 8 3.3 Aquarium System. 8 3.4 Abalone Tissue Sampling. 8 3.5 Lipid content and fatty acid analysis. .............. 8.3.6 Chemicals........ 8.4 Results:...... 8.5 Discussion: 8.6 Conclusion: 9 GeneralDiscussion:............... 9. I Abstract:,.................... 9.2 Introduction:.,............. 9.3 Antinutrient factors. ... 9.4 Intestinal Morphology 9.5 Functional Characteristics... 9.5.1 Heat Treafnent...... 9.5.2 Enzyme Treatment. 9.5.3 Starch Type, 9.5.4 Inclusion Level of Legumes. .......... 9.5.5 Inert Filler Inclusion. 9.5.6 Carbohydrate Binding and Cell Agglutination. vil 9.5.7 Fatty Acid Profile, Dietary Effects 1s6 9.5.8 Environmental Effects. 157 9.6 Conclusions and Future Work. 1s7 ...159 vlll List of Figures: Figure 3.1: Effect of Diet on Intestinal Alkaline Phosphatase Specific Activþ and Total Activity in Isolated Intestinal Apical Membrane from the Abalone Hal¡otis \aevegata...............55 Figure 3.2: Effect of Diet on Intestinal Carbohydrases Maltase (A), Sucrase (B) and p-galactosidase (C) Specific Activity and Total Activity in Isolated Intestinal Apical Membrane from the Abalone Haliotis laevegata. ..............56 Figure 3.3: Effect of Diet on the Protease Leucine amino-peptidase Specific Activity and Total Activity in Isolated Intestinal Apical Membrane from the Abalone Haliotis laevegata.ST Figure 3.4: Effect of Diet on the Amount of Protein per gfam Tissue in Isolated Intestinal Apical Membrane from the Abalone Haliotis laevegata. 57 Figure 3.5: Specific and Total Activities of Commercially Reared Abalone Intestinal Enzymes Relative to Wild Abalone (Haliotis laevegata) Intestinal Enzymes. 62 Figure 4.1: Effect of Dietary Protein Source with Treatments Raw and Raw + Phytase on Alkaline Phosphatase Specific Activity and Total Activity in Isolated Intestinal Apical Membrane from Abalone Haliotis laevegata. ......"".83 Figwe 4.2: Effect of Dietary Protein Source with Treatments Raw and Raw + Phytase on Intestinal Carbohydrases Maltase (A), Sucrase (B) and B-galactosidase (C) Specific Activity and Total Activity in Isolated Intestinal Apical Membrane from the Abalone Haliotis laevegata.. '.....'........' 84 Figure 4.3: Effect of Dietary Protein Source with Treatments Raw and Raw + Phytase on Leucine Amino Peptidase Specific Activity and Total Activity in Isolated Intestinal Apical Membrane from the Abalone Haliotis laevegata. 85 Figure 4.4: Effect of Dietary Protein source