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Assessing the Bioavailability of Synthetic Methionine and Lysine from Different Sources in Rainbow Trout (Oncorhynchus mykiss). By Christopher David Powell A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Animal and Poultry Science Guelph, Ontario, Canada © Christopher D. Powell, August, 2014 i ABSTRACT ASSESSING THE BIOAVAILABILITY OF SYNTHETIC METHIONINE AND LYSINE FROM DIFFERENT SOURCES IN RAINBOW TROUT (ONCORHYNCHUS MYKISS). Christopher David Powell University of Guelph, 2014 Advisor: Professor D.P Bureau Relative bioavailability of L-methionine and a hydroxy methionine analogue (MHA-Ca) were compared to the commercially prevalent DL-methionine in a 12-week growth trial. A separate 12-week trial investigated relative bioavailability between L-lysine HCL and L-lysine sulphate. Basal diets were formulated to be deficient in methionine or lysine and were supplemented with increasing equimolar levels of methionine or lysine from three sources of methionine or two sources of lysine. Using a linear slope-ratio assay, bioavailability of L-methionine and DL-methionine were determined to be similar (p>0.10). Differences in bioavailability between DL-methionine and MHA-Ca were observed (p<0.05), with MHA-Ca being 69, 60 and 73% as bioavailable as DL-methionine based upon weight gain, growth rate (thermal-unit growth coefficient) and retained nitrogen response parameters respectively. L-lysine HCL and L-lysine sulphate were effective sources of lysine with no significant differences in bioavailability (p>0.10). In conclusion, differences in bioavailability exist between sources of synthetic methionine, but not lysine. ii Acknowledgements I would first like to thank my advisor Dr. Dominique P. Bureau for his guidance and the wealth of knowledge that he has provided for me during my time in his lab. His ability to keep in communication even while on the opposite side of the world is actually quite astounding. Dr. Bureau’s ability to provide feedback yet encourage independent work along with forcing me outside my comfort zone I feel has not only improved my ability as a researcher but also as a person. Thank you, Dom. Thank you to my advisory committee, Dr. John Cant and Dr. Cornelis F. M. de Lange for the help and ideas that you have provided me throughout the process. Thank you John in particular for your help in regards to my statistical analysis and coding. Kees I appreciate all your questions and ideas that you put forward during our meetings. I would also like to thank Dr. Andreas Lemme and Dr. Claudia Silva from Evonik Industries AG for spearheading this effort. I would also like to thank Dr. Margaret Quinton for her help in developing the initial statistical models used in my research. Our meetings and my analysis of your code helped me understand the practical application of statistics. I would also like to thank Jamie Hooft and Owen Skipper-Horton for all their help in teaching me the finer points of the statistical software SAS. I would like to thank all of my lab mates, there are too many to mention, but the lab really has developed into a second family for me. I would especially like to thank a former lab mate of mine Dr. M.A. Kabir Chowdhury for all his help through my project; your help was pivotal to my success. All the undergraduate volunteers who helped me run my trials, your work was much appreciated and much needed thank you. I would like to thank Evonik Industries AG (Hanau, Germany) for funding this research along with OMAFRA and the University of Guelph for provided me with a very generous scholarship. iii Finally I would like to thank those who are the most important in my life, my family and friends and all those I love. In particular I would like to thank my parents David and Judith for their support, both emotionally and financially throughout my Master’s and academic career as a whole. iv Table of Contents CHAPTER 1 – GENERAL INTRODUCTION ..............................................................................................1 1.1– Objectives.......................................................................................................................................... 3 CHAPTER 2 – LITERATURE REVIEW ......................................................................................................4 2.1 Amino Acids: Chemistry and Structure ............................................................................................... 4 2.2 Methionine Biochemistry & Metabolism........................................................................................ 133 2.3 Methionine Requirements .............................................................................................................. 177 2.4 Lysine Biochemistry & Metabolism................................................................................................. 211 2.5 Lysine Requirements ....................................................................................................................... 244 2.6 Strategies for Meeting Essential Amino Acid Requirements .......................................................... 288 2.7 Introduction to Synthetic Amino Acids ........................................................................................... 331 2.7 Estimating Bioavailability of Essential Amino Acids ........................................................................ 366 2.8 Conclusions and perspectives ......................................................................................................... 444 CHAPTER 3 – ASSESSING THE BIOAVAILABILITY OF L-METHIONINE AND A HYDROXY METHIONINE ANALOGUE (MHA-Ca) COMPARED TO DL-METHIONINE IN RAINBOW TROUT (ONCORHYNCHUS MYKISS) ......................................................................................................................................... 466 Abstract ................................................................................................................................................. 466 3.1 Introduction .................................................................................................................................... 477 3.2 Methods ............................................................................................................................................ 49 3.3 Results ............................................................................................................................................. 588 3.4 Discussion........................................................................................................................................ 700 v 3.5 Conclusion ....................................................................................................................................... 744 CHAPTER 4 - ASSESSING THE BIOAVAILABILITY OF L-LYSINE SULPHATE COMPARED TO L-LYSINE HCL IN RAINBOW TROUT (ONCORHYNCHUS MYKISS) ................................................................................. 766 Abstract ................................................................................................................................................. 766 4.1 Introduction .................................................................................................................................... 777 4.2 Methods ............................................................................................................................................ 79 4.3 Results ............................................................................................................................................. 844 4.4 Discussion........................................................................................................................................ 955 4.5 Conclusion ....................................................................................................................................... 988 CHAPTER 5 GENERAL DISCUSSION .................................................................................................... 99 BIBLIOGRAPHY............................................................................................................................. 1033 vi List of tables Table 2.1 Essential and non-essential amino acids ....................................................................................... 6 Table 2.2 Biological compounds and amino acid precursors in animals. ..................................................... 9 Table 2.3 Essential amino acid requirements of rainbow trout (dry matter basis). ................................. 122 Table 2.4 Amino acid profiles of common animal feed ingredients. ........................................................ 122 Table 2.5 Methionine + cysteine and methionine requirements of various finfish species. ...................... 19 Table 2.6 Estimates of methionine requirement of rainbow trout. ........................................................... 19 Table 2.7 Estimated and recommended lysine requirement for various fish species. ............................... 26 Table 3.1 Ingredient composition of experimental diets.......................................................................... 511 Table 3.2 Experimental design – methionine inclusion from various synthetic sources. ......................... 522 Table 3.3 Analyzed essential amino acid content of experiential diets.................................................... 522 Table 3.4 Proximate composition of experimental diets. ......................................................................... 533 Table 3.5 Formulated and analyzed levels of methionine and MHA in experimental diets. .................... 533 Table 3.6 Performance of rainbow trout in response to being fed increasing equimolar levels of methionine from different sources over a 12 week experimental period. .............................................. 644 Table 3.7 Proximate composition of whole carcass of rainbow trout in response to being fed increasing equimolar levels of methionine from various sources for 12 weeks, expressed on a wet weight basis. 655 Table 3.8 Retained nitrogen (RN), recovered energy (RE), nitrogen retention efficiency (NRE), and energy retention efficiency (ERE) of rainbow trout in response to being fed increasing equimolar levels of methionine from various sources for 12 weeks. ...................................................................................... 666 Table 3.9 Relative bioavailability of L-Met and MHA-Ca compared to the standard DL-Met based on weight gain, growth rate and retained nitrogen values. ............................................................................ 69 Table 4.1 Ingredient composition of experimental diets.......................................................................... 800 Table 4.2 Experimental design- addition of L-lysine from supplemental L-lysine sources. ...................... 811 vii Table 4.3 Analysed essential amino acid composition of experimental diets (% dry matter).................. 811 Table 4.4 Proximate composition of experimental diets (dry matter basis). ........................................... 822 Table 4.5 Formulated and analyzed total lysine and free (supplemental) lysine of experimental diets (dry matter basis). ............................................................................................................................................ 822 Table 4.6 Performance of rainbow trout in response to being fed diets containing increasing equimolar levels of L-lysine from two supplemental sources. ................................................................................... 900 Table 4.7 Proximate composition of whole carcasses of rainbow trout in response to being fed diets containing increasing equimolar levels of L-lysine from two supplemental sources, on a wet weight basis. .................................................................................................................................................................. 911 Table 4.8 Retained nitrogen (RN), recovered energy (RE), nitrogen retention efficiency (NRE), and energy retention efficiency (ERE) of rainbow trout in response to being fed diets containing increasing equimolar levels of L-lysine from two supplemental sources. ................................................................. 922 Table 4.9 Relative bioavailability of L-lysine sulphate compared to the standard L-lysine HCL based on weight gain, TGC and retained nitrogen values. ....................................................................................... 944 viii List of figures Figure 2.1 Metabolic pathways of sulphur amino acids. ............................................................................ 15 Figure 2.2 Degradation of lysine to acetyl CoA with enzymes: 1 L-amino acid oxidase; 2, specific aminotransferase. ....................................................................................................................................... 23 Figure 2.3 Increasing crude protein content of a diet as a strategy for meeting essential amino acid requirements. ........................................................................................................................................... 311 Figure 2.4 Use of multiple protein sources as a strategy for meeting essential amino acid requirements. .................................................................................................................................................................. 311 Figure 2.5 Use of synthetic amino acids to supplement a deficient diet.................................................. 322 Figure 2.6 Four commonly used assays for determining bioavailability of a nutrient. .............................. 40 Figure 2.7 Determining nutrient bioavailability using a slope-ratio assay RBV=XS/XT ……………………….…….42 Figure 3.1 Growth curves of rainbow trout in response to being fed experimental diets containing graded equimolar levels of methionine from three synthetic sources. ................................................... 611 Figure 3.2 Weight gain of rainbow trout in response to being fed diets containing increasing equimolar levels of methionine from DL-Met (■), L-Met (▲), and MHA-Ca (▼)...................................................... 622 Figure 3.3 Thermal-unit growth coefficient (TGC) of rainbow trout in response to being fed diets containing increasing equimolar levels of methionine from DL-Met (■), L-Met (▲), and MHA-Ca (▼). 622 Figure 3.4 Retained Nitrogen (g/fish) content of rainbow trout in response to being fed diets containing increasing equimolar levels methionine from DL-Met (■), L-Met (▲), and MHA-Ca (▼). ...................... 633 Figure 3.5 Weight gain of rainbow trout in response to being fed increasing equimolar levels of methionine from DL-Met (■), L-Met (▲), and MHA-Ca (▼). ................................................................... 688 Figure 3.6 Thermal-unit growth coefficient (TGC) of rainbow trout in response to being fed diets containing increasing equimolar levels of methionine from DL-Met (■), L-Met (▲), and MHA-Ca (▼). 688 ix Figure 3.7 Retained nitrogen (g/fish) of rainbow trout in response to being fed diets containing increasing equimolar levels of DL-Met (■), L-Met (▲), and MHA-Ca (▼). ................................................ 69 Figure 4.1 Growth curves of rainbow trout in response to being fed experimental diets containing increasing equimolar levels of L-lysine from two supplemental sources. ................................................ 877 Figure 4.2 Weight gain of rainbow trout in response to being fed diets containing increasing equimolar levels of lysine from L-lys sulphate (■) and L-lys HCL (▲). ....................................................................... 888 Figure 4.3 Thermal-unit growth coefficient of rainbow trout in response to being fed diets containing increasing equimolar levels lysine from L-lys sulphate (■) and L-lys HCL (▲). ........................................ 888 Figure 4.4 Retained nitrogen content (g/fish) of rainbow trout in response to being fed diets containing increasing equimolar levels of lysine from L-lys sulphate (■) and L-Lys HCL (▲). ..................................... 89 Figure 4.5 Weight gain of rainbow trout in response to being fed diets containing increasing equimolar levels of L-lysine from L-lys sulphate (■) and L-lys HCL (▲). .................................................................... 933 Figure 4.6 Thermal-unit growth coefficient (TGC) of rainbow trout in response to being fed diets containing increasing levels of L-lysine from L-lys Sulphate (■) and L-lys HCL (▲). ................................ 933 Figure 4.7 Retained nitrogen (g/fish) of rainbow trout in response to being fed diets containing increasing equimolar levels of L-lysine from L-lys sulphate (■) and L-lys HCL (▲). ................................. 944 x CHAPTER 1 – GENERAL INTRODUCTION The ability to successfully feed diets containing high levels of plant protein ingredients to carnivorous salmonids is due to the improved understanding of their nutritional requirements and the greater characterization of available nutrients in feed ingredients. Digestibility and amino acid profiles of cost-effective protein sources are of particular interest, as well as, the essential amino acid requirements of the animal. In comparison to good quality fish meal, most plant proteins have a poorer amino acid profile and are often deficient in one or several essential amino acids. In particular methionine and lysine are often found in low levels in common plant protein ingredients including soybean meal, corn gluten meal, wheat gluten and most cereal grains (NRC, 2011). Therefore, diets containing high levels of these plant proteins can frequently contain levels of methionine and lysine that are below the requirement of the animal. Besides their importance in protein synthesis, lysine and methionine play key biochemical and structural roles in animals, both methionine and lysine are precursors to important molecules including carnitine which is responsible for the transportation of fatty acids to the mitochondria in order to generate metabolic energy. Methionine is a methyl donor and a precursor to many important biological molecules used in a diverse array of metabolic reactions. Animals fed diets that are deficient in methionine result in decreased animal growth. High incidence of cataracts and spinal deformities may arise in fish fed methionine deficient diets (Poston et al., 1977; Keembiyehetty and Gatlin, 1993; Walton et al., 1982, Rumsey et al., 1983; Cowey et al., 1992). Lysine plays an important structural role in all types of proteins including transmembrane proteins and various connective tissues, with depressed growth and health issues, such as fin erosion, arising when fish are fed lysine deficient diets (Di Lullo et al., 2002, Ketola, 1983, Walton et al., 1984, 1986). Estimates of methionine and lysine requirements for 1 rainbow trout on a dry matter basis are 0.7% and 2.4% of the diet, respectively, in order to achieve maximum growth (NRC, 2011). Crystalline amino acids have been used in the animal feed industry for over 50 years allowing for the wider use of economical feed ingredients and greater flexibility in feed formulations while ensuring amino acid requirements are met. Unlike protein-bound amino acids, crystalline amino acids are not part of a polypeptide chain and are commonly referred to as free amino acids. Crystalline amino acids may be referred to as synthetic or supplemental amino acids in reference to their production technique. As methionine and lysine are most commonly the first and second most limiting essential amino acids a variety of commercially produced synthetic sources of these amino acids exist. Commercial synthetic methionine sources include the widely used DL-methionine along with the hydroxy analogue of methionine (MHA), both products of chemical synthesis. Other sources of synthetic methionine exist, such as L-methionine which is used primarily for laboratory purposes as commercial production techniques are inefficient. Instead of being products of chemical synthesis, supplemental sources of lysine including L-lysine HCL and L-lysine sulphate are produced through bacterial fermentation. Various sources of both methionine and lysine are commercially available, as differences between sources exist, their relative ability to provide a metabolically active, utilizable form of methionine or lysine is of interest. Bioavailability is defined as the extent to which an ingested nutrient is digested and absorbed in a form that can be utilized by the animal (Batterham, 1992; Lewis and Bayley, 1995). Bioavailability of synthetic amino acids between sources can be compared in order to understand the relative ability of these sources to supply a particular amino acid in an utilizable form. One method that allows for the direct and practical assessment of bioavailability of individual amino acids is the slope-ratio assay which has been used extensively in farmed animals (Lewis and Bailey, 1995). 2 In a slope-ratio assay, a basal diet is formulated to be deficient in the nutrient under investigation yet meet all other known nutritional requirements of the animal. The basal diet is then supplemented with increasing levels of the test nutrient from different test sources. Animal response, weight gain, growth rate etc. are then regressed against nutrient supplementation level and slopes of the lines are compared against a reference nutrient source in order to determine relative bioavailability. Slope-ratio assays have successfully been applied to determine relative bioavailability of amino acids in protein-bound ingredients as well as between crystalline or supplemental sources of essential amino acids (Boebel and Baker, 1982; Wang et al., 2007, Yi et al., 2006; Smiricky-Tjardes et al., 2004; Batterham et al., 1979, Li et al., 2009; El-Haroun and Bureau, 2007; Rodehutscord et al., 2000). 1.1– Objectives The objective of this study was to investigate the relative bioavailability between three sources of synthetic methionine (DL-methionine, L-methionine and MHA-Ca) and two sources of synthetic lysine (L-lysine sulphate and L-lysine HCL) in two separate growth trials using a slope-ratio assay methodology. 3 CHAPTER 2 – LITERATURE REVIEW 2.1 Amino Acids: Chemistry and Structure Proteins are organic compounds found in all living organisms and serve crucial structural and metabolic roles. At the molecular level proteins are simply chains of bound amino acids folded into a particular structure. These chains consist of amino acids bound to one another through amide bounds which bind the α-amino group of one amino acid to the carboxyl group of another (Brody, 1999). Chains of amino acid monomers are classified by the number of amino acids bound in the chain; dipeptides consist of two bound amino acids, while polypeptides contain a continuous linear non-branching chain of amino acid monomers (Brody, 1999). The general structure of amino acid follows the H2NCHRCOOH template whereby “R” represents a side chain which distinguishes amino acids from one another. Individual amino acid molecules contain both an amine and carboxyl group along with the “R” side chain whose properties influence the size, shape and electrical charge of a specific amino acid. With the exception of proline and hydroxyproline all amino acids found in living organisms are considered α-amino acids meaning the primary amino group and the carboxyl group are attached to the same (α) carbon (Lloyd et al., 1978). With the exception of glycine, the α-carbon of an amino acid is bound to four different groups; a carboxyl group, an amino group, an R side chain and a hydrogen atom. The four different groups that are bound to the α-carbon are able to exist in two different spatial arrangements that are non-superimposable mirror images of each other. The two asymmetric spatial arrangements are commonly referred to as L or D isomers of an α-amino acid, with L isomers arranged with the amino group to the left of the α-carbon and hydrogen atom to the right while the opposite is true for D isomers. With few exceptions, amino acids are naturally found in the L-configuration which is commonly referred to as an L-isomer. The amino acids 4 that make up proteins are solely L-isomers as cells specifically synthesize the L isomer of an amino acid due to the asymmetry of enzyme activity sites which causes the reactions that they catalyze to be stereospecific (Nelson & Cox, 2000). Twenty primary amino acids are used by cells during protein biosynthesis, and are commonly referred to the twenty standard amino acids of proteins. Each of these twenty amino acids contain a unique R group, however properties of R groups may be similar between individual amino acids allowing their classification into five categories. The five categories are: nonpolar aliphatic R groups, aromatic R groups, polar uncharged R groups, positively charge basic R groups and negatively charged acidic R groups. The properties of these R groups influence the structural role that these amino acids have in proteins, with the nonpolar aliphatic and aromatic R groups being hydrophobic and stabilizing proteins through hydrophobic interactions, while polar uncharged R groups are hydrophilic. The charged R groups are also highly hydrophilic and due to their charges influence protein folding and structure. The standard twenty amino acids can be separated into two categories: Essential amino acids (EAAs) and non-essential amino acids (NEAAs) (Table 2.1). Both essential and non-essential amino acids are equally important for protein synthesis, the differences between the two is based on the ability of the body to synthesis, or synthesize in sufficient amounts, a particular amino acid. Non-essential amino acids can be synthetized from precursors in the body of the organism through transamination reactions whereby amino groups from either an amino acid or a simple amine are transferred to a suitable carbon source creating a non-essential amino acid. Essential amino acids, although capable of transamination reactions, either cannot be synthetized in the body or cannot be synthetized in the body at a sufficient rate as to meet the physiological needs for this amino acid by the organism (Nelson & Cox, 2000). Cysteine and tyrosine are considered semi-essential or conditionally essential as they can be synthesized from the essential amino acids methionine and phenylalanine respectively. 5 Table 2.1 Essential and non-essential amino acids Essential amino acid Non-essential amino acid Tryptophan Cysteine1 Valine Tyrosine1 Phenylalanine Proline Arginine Glutamate Lysine Glutamine Leucine Glycine Methionine Serine Isoleucine Alanine Threonine Aspartate Histidine Aspartic Acid 1 Conditionally essential Adapted from D’Mello (2003a) 6 2.1.1 Amino Acid Metabolism and Catabolism Both dietary amino acids and amino acids which result from the endogenous breakdown of body proteins enter the metabolic pool and depending on the current metabolic demands on the animal, may be used for protein synthesis and/or as precursors for other biological molecules (Kaushik & Seiliez, 2010). Protein synthesis is the formation of polypeptides and finally proteins through successive attachment of amino acids according to highly specific template. The ability to bio-synthesis proteins depends on the configuration of the amino acid whereby D-isomers must undergo a metabolic racemization to the L-isomer form before they are able to be incorporated into a protein (Friedman & Gumbmann 1984). Protein deposition in an animal occurs when the rate of protein synthesis is greater than the rate protein degradation, in the majority of fish and shrimp species protein synthesis and associated body protein deposition accounts for 25-55% of total amino acids consumed (NRC 2011). Protein deposition has been recognised to be the driving force behind weight gain as a strong correlation between protein deposition, water deposition and live weight gain exist (Dumas et al., 2007). Along with their role in protein synthesis amino acids may serve as precursors for many biological molecules such as enzymes, hormones, neurotransmitters and many other metabolic intermediates. Table 2.2 displays various important biological compounds their amino acid precursors and the physiological function of these compounds. Besides their role in anabolism, amino acids may also follow catabolic pathways which are dependent on the current metabolic demands of the animal. When dietary energy intake is limiting protein deposition, EAA may be partitioned away from protein synthesis and towards catabolism to meet a certain metabolic need, commonly referred to as preferential catabolism (Moughan, 2003). However, when the energy supply of a diet is not limiting protein synthesis or amino acid intake is below an animal’s requirement for maximum protein deposition, inevitable amino acids catabolism will still 7 occur through active catabolic pathways (Moughan, 1995). Finally, amino acids that are in excess of the cumulative amino acid demands of protein synthesis, maintenance requirements, and losses due to inevitable and preferential catabolism, will result in the catabolism of these excess amino acids (NRC, 2011). 8 Table 2.2 Biological compounds and amino acid precursors in animals. Biological compound Amino acid precursor Physiological function Purines and pyrimidines Glycine and aspartic acid Constituents of nucleotides and nucleic acids Creatine Glycine and arginine Energy storage as creatine phosphate in muscle Glcoholic and taurocholic acids Glycine and cysteine Bile acids, aid in fat digestion and absorption Thyroxine, epinephrine, and Tyrosine Hormones norepinephrine Ethanolamine and choline Serine Constituents of phospholipids Histamine Histidine A vasodepressor Serotonin Tryptophan Transmission of nerve impulses Porphyrins Glycine Constituent of hemoglobin and cytochromes Niacin Tryptophan Vitamin Melanin Tyrosine Pigment of hair, skin, and eyes Methyl groups Methionine DNA methylation Glutathione Cysteine Important antioxidant Carnitine Lysine and methionine Transportation of fatty acids Adapted from Lloyd (1978). 9 2.1.2 Essential Amino Acid Requirements In animal-nutrition, two techniques were traditionally used to determine if a specific amino acid is considered an essential amino acid; isotopic labelling studies or feeding trials, with feeding trials being the more popular technique (Wilson, 1989). Through the systematic deletion of certain amino acids in the diet and analysing the corresponding performance of the fish, essential amino acids can be differentiated from non-essential amino acids for a particular species of fish (Wilson, 1989; Cowey 1994). Using this technique, Nose et al. (1974) tested the essentially of 18 amino acids for common carp and found that by excluding any 10 out of the 18 amino acids resulted in significant growth reduction. These amino acids were arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine which are the same 10 essential amino acids (Table 2.3) required by most animals (Ketola, 1982; Wilson, 1989; NRC, 1993). Along with the 10 essential amino acids, both tyrosine and cysteine are considered semi/conditional essential as they can be synthesized from the essential amino acids, phenylalanine and methionine respectively. Although the 10 essential amino acid do not differ between animals, the specific requirements of these 10 essential amino acids vary between both species and life stage. Initial studies on specific essential amino acid requirements for fish species starting in the 1950’s whereby essential amino acid requirements for Chinook salmon were first experimentally determined using a feeding trial (Halver, 1957). Currently well over 200 papers have been published estimating the dietary requirement of essential amino acids for fish and shrimp species, including channel catfish, common carp, Nile tilapia, Pacific salmon and rainbow trout along with many others (NRC, 2011 ). Table 2.3 contains minimal essential amino acid requirement in order to achieve maximum performance as percentage of diet on a dry-matter basis for rainbow trout. It is important to consider that these requirements are based off experimental data under optimal conditions using highly digestible purified feed ingredients. Modern animal feeds contain various protein sources, with each ingredient having a different amino acid profile 10 as seen in table 2.4. Modern feeds are being formulated to include higher levels of plant protein ingredients in partial substitution of animal proteins. Most cereal grains and plant proteins contain low levels of lysine or methionine. Consequently, these two amino acids are commonly the first and second most limiting essential amino acids in diets containing high levels of plant proteins. 11 Table 2.3 Essential amino acid requirements of rainbow trout (dry matter basis). Amino acid Amino acid requirement % of diet Arginine 1.5 Histidine 0.8 Isoleucine 1.1 Leucine 1.5 Lysine 2.4 Methionine 0.7 Methionine + cysteine 1.1 Phenylalanine 0.9 Phenylalanine + tyrosine 1.8 Threonine 1.1 Tryptophan 0.3 Valine 1.2 NRC (2011) Table 2.4 Amino acid profiles of common animal feed ingredients (% as-fed basis). DM Ingredient Arg His Ile Leu Lys Met Cys Phe Tyr (%) Fish meal 92 3.73 1.53 3.64 4.69 7.30 2.20 1.60 2.68 2.10 Blood meal, 93 2.35 5.00 0.80 10.3 7.10 1.00 1.40 5.10 2.30 sprdry Meat and 94 3.60 0.96 1.70 3.20 2.60 0.67 0.33 1.70 1.30 Bone meal Poultry byproduct 89 4.32 1.05 2.30 4.27 3.32 1.29 0.92 1.66 1.21 meal Gelatin 88 6.62 0.76 1.38 2.91 3.55 0.73 0.13 1.79 0.52 Soybean 89 3.23 1.17 1.99 3.42 2.83 0.61 0.70 2.18 1.69 meal Canola 93 2.32 1.10 1.51 2.60 2.02 0.77 0.97 1.50 0.99 meal Corn gluten 91 1.90 1.20 2.30 9.40 1.07 1.90 1.10 3.80 0.87 meal Wheat 89 0.97 0.44 0.70 1.10 0.70 0.12 0.19 0.50 0.29 middlings Adapted from NRC (2011) 12 Thr Trp Val 2.49 0.67 3.26 3.80 1.00 5.20 1.70 0.26 2.25 2.14 - 3.65 1.76 0.05 2.09 1.73 0.61 2.40 1.50 0.46 1.94 2.00 0.30 2.70 0.51 0.20 0.75 2.2 Methionine Biochemistry & Metabolism Of the 20 amino acids that make up the primary structure of proteins, two amino acids, methionine and cysteine, contain a sulphur atom. Methionine is an essential α-amino acid with the chemical formula HO2CCH(NH2)CH2CH2SCH3 and is non-polar. While cysteine is considered a semiessential amino acid as it can be synthesized from serine requiring a trans-sulphuration reaction with methionine. Cysteine contains a highly reactive thiol side chain resulting in it having an important role in the structure and therefore function of many proteins and enzymes (NRC 2011). Besides its role in protein synthesis, L-methionine, can also be converted into S-adenosylmethionine and then Sadenosylhomocysteine releasing a methyl group in the process (Lewis, 2003). This methyl group can then be used in several metabolic processes, including DNA methylation and synthesis of carnitine from lysine, adrenaline from noradrenaline, and creatine from guanidine acetate (Simon, 1999). The conversion of methionine into cysteine, and the associated intermediate compounds including S-adenosylmethionine (SAM), is well understood and major intermediate compounds in this metabolic conversion (figure 2.1). Methionine can be activated by ATP to S-adenosylmethionine (SAM), facilitated by co-enzyme methionine adenosyltransferase. S-adenosylmethionine is an important methyl donor, and is second only to ATP in number of enzymes that require it (Brosnan and Brosnan, 2006). Once S-adenosylmethionine donates its methyl group to an appropriate acceptor it forms Sadenosylhomocysteine (SAH). SAH undergoes hydrolysis by SAH hydrolase forming homocysteine. The culmination of these reactions are referred to as transmethylation. Homocysteine is an important intermediate as it can be converted back to methionine following one of two remethylation pathways or homocysteine may follow the transsulfuration pathway resulting in the formation of the semi-essential amino acid cysteine. To regenerate methionine from homocysteine, a methyl group must be transferred back to homocysteine via a remethylation pathway (Brosnan and Brosnan, 2006). In these pathways, a 13 methyl group from either betaine or 5-methly-THF is donated to homocysteine resulting in the formation of methionine. The culmination of the transmethylation and remethylation pathways is referred to the methionine cycle. The methionine cycle does not involve the catabolism of methionine, this occurs in the transsulfuration pathway. During the transsulfuration pathway homocysteine is converted first to cystathionine then to cysteine by cystathionine b-synthase (CBS) and cystathionine g-lyase (CGL) respectively (Brosnan and Brosnan, 2006). The transsulfuration pathway, unlike the methionine cycle, is an irreversible reaction resulting in the ability of cysteine to be synthesized from methionine but the inability of methionine to be synthesized from cysteine. 14 Figure 2.1 Metabolic pathways of sulphur amino acids. Brosnan and Brosnan (2006) 15 Since methionine is an α-amino acid two isometric forms, L-methionine and D-Methionine, exist. L-methionine is the naturally occurring isomer of methionine, while D isomers may be formed through acid, base or heat damage to L-isomers (Friedman and Gumbmann, 1984). Additionally, certain invertebrates and bacteria have the ability to biosynthesize D-methionine, while chemical synthesis of methionine results in an isometric mixture of D and L isomers (Gomes and Kumar, 2005). L-methionine is the biologically active form of methionine, therefore D-methionine must undergo a metabolic transformation to L-methionine before being used for protein synthesis or other metabolic processes. The conversion of D-methionine to L-methionine is a two-step conversion; D-methionine is first oxidized by D-amino oxidase resulting in an α-keto organic acid (2-keto-4-(methylthiol)butyric acid). The resulting α-keto organic acid is then aminated by the transfer of an amino group from glutamate resulting in Lmethionine (Lewis, 2003). Just as D-methionine is required to be converted to the L-isomer before becoming biologically active, other synthetic methionine products such as the hydroxy analog of methionine, 2-hydroxy-4-(methylthio)butanoic acid (HMB or OH-Met), must undergo metabolic conversions. The chemical synthesis of HMB results in racemic mix of D and L isomers. D-HMB and LHMB require different enzymes, a dehydrogenase and oxidase respectively, in order to convert the isometric analogues of methionine to an α-keto acid (2-keto-4-(methylthiol)butyric acid) (Baker 2006). Once converted the α-keto acid follows the same metabolic pathway as the conversion of D-methionine to L-methionine. With the exception of primates, most animals including fish and crustacea species are able to synthesise D-amino oxidase allowing for the conversion of D-methionine to L-methionine (Lewis, 2003; NRC 2011). 16 2.3 Methionine Requirements Methionine and cysteine are both sulphur-containing amino acids and are commonly referred collectively as total sulphur amino acid (TSAA) in respect to the requirement of an animal for these two amino acids. Cysteine is considered a non-essential amino acid as it can be synthetized from the essential amino acid methionine. Therefore, when a diet does not meet the requirement of an organism for cysteine, dietary methionine can be converted to cysteine. Similarly if level of dietary cysteine is sufficient to meet the cysteine requirement of an animal, lower levels of methionine may be included in the diet in comparison to a cysteine free diet as methionine will not be converted to cysteine (Baker, 2006). The ability of cysteine to spare methionine is supported by research conducted by Walton et al. (1982) whereby the methionine requirement of rainbow trout decreased when dietary cysteine content increased from 0% to 2%. In agreement with Walton et al. (1982), the sparing effect of cysteine methionine in rainbow trout has also been reported by Rumsey et al. (1983) and Cowey et al. (1992). Unlike the ability of methionine to fulfil or partially fulfil the requirement of cysteine, cysteine is not able to fulfill the methionine requirement of an organism. This is a result of the cysathionine synthase reaction being one directional preventing cysteine being converted back to homocysteine which through the methionine cycle can be converted to methionine (Brosnan and Brosnan, 2006). The ability of methionine to be converted to cysteine and help meet the cysteine requirement of an animal causes difficulties in estimating methionine requirements. Therefore, it common to see requirements for methionine and cysteine expressed as either “Met+Cys” or simply TSAA. Depending on the study and experimental conditions estimates of TSAA requirements of rainbow trout range from 0.8% to 1.1% of the diet on a dry matter basis, with TSAA requirement of 1.1% recommended by the NRC (Rumsey et al., 1983; Cowey et al., 1992; NRC, 2011). TSAA of common fresh and marine fish according to the NRC (2011) can be found in table 2.5. As methionine can be readily converted to 17 cysteine the inclusion of cysteine in a diet has a sparing effect for methionine. It has been estimated that cysteine can replace methionine at an efficiency of 40-60% in order meet the TSAA of many fish species (Wilson, 2002). Cysteine replacement value of methionine vary between species with 60% replacement for channel catfish (Harding et al. 1977), 44% for blue tilapia (Liou, 1989), 42% for rainbow trout (Kim et al., 1992) and 40% for both red drum and hybrid striped bass (Moon and Gatlin, 1991; Griffin et al., 1992). However, there is a limit to the extent in which cysteine can spare methionine, studies by Kim et al. (1992) found a breaking point at 0.3% dietary cysteine, whereby any additional amounts of cysteine supplementation did not result in further sparing of methionine. Common feed ingredients contain relatively high levels of cysteine resulting in most practical fish feeds meeting cysteine requirements, subsequently it is often more practical to focus on meeting the methionine requirement of an animal. In order to accurately estimate methionine requirement, diets must contain excess cysteine in order to prevent the conversion of methionine to cysteine and the resulting overestimation of methionine requirement. Estimates of methionine requirement for rainbow trout can be found in table 2.6 with estimates of methionine requirement range from 0.5% to 0.9% of the diet on a dry matter basis (Rodehutscord et al., 1995). Differences in estimates can be attributed to many factors including the model used to determine requirement, life stage the fish etc. Based on the available literature, the NRC (2011) recommends a dietary methionine requirement of 0.7% on a drymatter basis for rainbow trout. Although it may be easier to estimate and meet the total sulphur amino acid requirements of a fish it is recommended to formulate diets to meet the methionine and cysteine requirements of a fish separately, or alternatively meet the methionine and methionine + cysteine requirements in order to properly meet their nutritional requirements (Rodehutscord et al., 1995). 18 Table 2.5 Methionine + cysteine and methionine requirements of various finfish species. Species Methionine + cysteine (%) Methionine (%) Atlantic Salmon 1.1 0.7 Common carp 1.0 0.7 Tilapia 1.0 0.7 Channel catfish 0.9 0.6 Hybrid striped bass 1.1 0.7 Rainbow trout 1.1 0.7 Asian sea bass 1.2 0.8 Cobia 1.1 0.8 Red drum 1.2 0.8 Yellowtail 1.2 0.8 NRC (2011) Table 2.6 Estimates of methionine requirement of rainbow trout. Requirement (% diet) Reference 0.8 Cowey et al., 1992 0.5 Kim et al., 1992 0.4-0.9 Rodehutscord et al., 1995 0.55-0.75 Rumsey et al., 1983 19 Like all amino acids required for proteins synthesis, if methionine is deficient the rate of protein synthesis will be negatively affected resulting in poor growth and a reduced feed efficiency rate. Diets that are deficient in an essential amino acids, including methionine, result in decreased feed intake which is a concern in any animal production system. Although the mechanism behind this depression in feed intake is not completely understood several studies have tried to describe the underlying biochemical mechanisms. Harper and Rogers (1965) hypothesised that drastic difference in amino acid patterns between muscle and blood plasma invoke an appetite-regulating response from the animal as a result of being fed an unbalanced diet. Besides its effect on growth and feed intake methionine deficiency can also cause physiological changes to the organism. This includes the formation of disulfide bonds in the lens of the eye resulting in the opacity of the lens and referred to at a cataract, this condition is evident in many fish species including; rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), lake trout (Salvelinus namaycush) and hybrid sea bass (Morone chrysops x M. saxatilis) (Poston et al., 1977; Keembiyehetty and Gatlin, 1993; Walton et al., 1982, Rumsey et al., 1983; Cowey et al., 1992). Rate of formation and severity of the cataract depends on the extent in which a diet is deficient in methionine, with highly deficient diets resulting in cataract formation within 3 weeks and mass mortalities occurring after 4 weeks in hybrid striped sea bass (Keembiyehetty and Gatlin, 1993). Cataracts are detrimental to fish vision and result in reduced feed intake which further reduces growth. In terms of the formation of cataracts, cysteine is a precursor of glutathione, which plays a role in preventing oxidative damage to the protein bound thiol groups in the lens of the eye (Ferrer et al., 1990; Cowey et al., 1992). Therefore, fish fed diets that do not meet the methionine or cysteine requirement of an animal may not be able protect against oxidation of methionine in the lens or be able to replace the oxidized methionine molecules resulting in the formation of disulfide bonds between molecules. The formation of these disulfide bonds leads to the insolubility of leans proteins resulting in a cloudy or opaque lens which is referred to as a cataract (Simmons et al., 1999). 20 Like diets deficient in methionine, diets that contain methionine in great excess of requirement of that animal may also result in depressed growth. Methionine is the most toxic of the amino acids that are required for protein synthesis, with reduced growth being seen in mammals as a result of being fed diets containing methionine in excess of two to three times the requirement level of the animal (Edmonds and Baker, 1987; Baker 2006). Similar results have been found in rainbow trout whereby fish fed diets containing 1.5-2.0% total methionine resulted in decreased growth (Poppi et al., 2011). Methionine toxicity is thought to be caused by the accumulation of S-adenosylmethionine (SAM), a metabolite of methionine, in the liver (Regina et al., 1993). Methionine toxicity can be alleviated through the addition of either supplemental glycine or serine. The addition of serine encourages the removal of SAM through the transulfuration pathway (Figure 2.1), while glycine facilities the catabolism of SAM (Baker, 2006). The accumulation of SAM in the liver causes visible histological damage resulting in hepatic dysfunction (Regina et al., 1993). Although high levels of methionine can be toxic to an animal it is not a common concern when formulating feeds. This is due to the relatively low methionine content of feed ingredients and the fact that in order to see adverse toxic effects on the animal the level of methionine in the diet would have to be higher than any practical feed formulation would contain. 2.4 Lysine Biochemistry & Metabolism Lysine is an essential α-amino acid with the chemical formula HO2CCH(NH3)(CH2)4NH2. Lysine contains a positively charged ε-amino group, a primary amine, resulting in lysine commonly forming hydrogen bonds and having important structural roles in proteins. Lysine plays an important tertiary structural role in transmembrane proteins, the positively charged hydrophilic R group of lysine encourages contact with water, while the hydrophobic nature of the rest of the molecule encourages contact with lipid molecules in the transmembrane layer (Buxbaum, 2007). Along with its structural role in transmembrane proteins lysine can also be found abundantly in body proteins of animals. In 21 particular lysine and lysine metabolites are found in collagen, the main structural protein in various connective tissues, and the most abundant protein in mammals (Di Lullo et al., 2002). The stability of the collagen tissue is due to the cross-linking of aldehydes, either lysine or hydroxylysine derived aldehydes, between collagen fibres. Lysyl oxidase is the enzyme that is responsible for converting the amine side chain of lysine and hydroxylysine, a post-translational hydroxyl modification of lysine, into these crosslinking stabilizing aldehydes (Eyre et al., 1984). Beside the role that lysine plays in protein structure and protein synthesis, lysine also follows various important catabolic pathways. Lysine is ketogenic, meaning during catabolism of the carbon skeleton of the amino acid a ketone is produced in the form of Acetyl CoA. The degradation of lysine into Acetly CoA is a multi-step process facilitated by L-amino acid oxidase and specific aminotransferase enzymes (D’Mello, 2003a). The main intermediates and associated enzymes of the degradation of lysine to Acetyl CoA can be found in figure 2.2. Acetly CoA can then enter the citric acid cycle which is the final common energy producing pathway for carbohydrates and lipids along with the carbon skeleton of amino acids. 22 Figure 2.2 Degradation of lysine to acetyl CoA with enzymes: 1 L-amino acid oxidase; 2, specific aminotransferase. D’Mello (2003a) 23 As lysine contains an α-carbon two isomers, L-lysine and D-lysine, occur. As all amino acids must be in the biologically active L-configuration to be utilized by an organism D-isomers must undergo oxidative deamination to an α-keto acid analogue then undergo L-specific re-amination by an amino acid specific aminotransferase. However, no aminotransferases exist for lysine rendering D-lysine nutritionally inactive (D’Mello, 2003a). Additionally, the ε-amino group of lysine is reactive in nature which causes lysine to be susceptible to both heat damage and non-enzymatic glycolyzation, resulting in the formation of Maillard reaction products (Moughan and Rutherfurd, 1996). During Maillard reactions, the carbonyl group of glucose and the ε-amino group of lysine react to form fructosyllysine (εDFL) (Moughan et al., 1996). Fructosyllysine undergoes acid hydrolysis to form pyridosine, regenerated lysine and furosine. However, once a feedstuff undergoes a Maillard reaction irreversible damage occurs to lysine resulting in decreased availability (Moughan and Rutherfurd, 1996; Moughan et al., 1996; Carpenter, 1960). The reactive ε-amino group of lysine and the inability of the body to transform Dlysine to L-lysine results in lysine commonly being the first limiting essential amino acid in feeds especially those formulated with high level of cereal grains or ingredients exposed to high temperature conditions. Additionally, conventional methods of amino acid analysis are unable to distinguish between reactive lysine and mallard products, leading to the overestimation of biologically available lysine in feeds that have being processed in high temperature and pressure conditions. 2.5 Lysine Requirements Lysine is an essential amino acid and therefore the requirement for this amino acid must be fulfilled through dietary intake. As lysine is bound as part of proteins the crude protein level of an ingredient will have an effect on the lysine content of that ingredient. For example, high quality fish meal containing 72% crude protein contains higher levels of lysine then wheat middlings which only contains 17% crude protein, 7.3% vs. 0.7% on a DM basis (NRC, 2011). However, lysine content also 24 varies between protein sources, even when comparing ingredients with similar levels of crude protein. Anchovy fish meal and corn gluten meal contain comparable levels of crude protein, 65% and 64% respectively, while the lysine content of fish meal (5.1%) is substantially higher than in corn gluten meal (1.1%). Additionally since lysine is susceptible to heat damage, due to the reactive nature of the ε-amino group, ingredients processed under high temperatures will also contain low levels of lysine. Since lysine is commonly the first limiting essential amino acid in diets containing high levels of plant proteins or processed under harsh conditions, extensive research has been on conducted on the estimated lysine requirement of most farmed animals, including many commercially important fish species (table 2.6). Based on these estimates, the NRC estimated the lysine requirement of rainbow trout to be about 2.4% (DM basis) (NRC, 2011). Estimates of lysine requirements of other commonly cultured fish species can also be found in table 2.7. Estimates of amino acid requirements are conducted under laboratory conditions, using semi-purified or purified feed ingredients with close to 100% digestibility. 25 Table 2.7 Estimates of lysine requirement for various fish species. Species Estimated lys Reference requirement (% of diet) Atlantic salmon 2.0% Anderson et al. (1993) Nile tilapia Rainbow trout 2.4% NRC (2011) 1.4 % Santiago and Lovell (1988) 1.3-1.4% Furuya et al. (2004) 1.44% Furuya et al. (2006) 1.6% NRC (2011) 1.9% Walton et al. (1984) 2.1-2.7% Wang et al. (2010) 1.9% Walton et al. (1986) 1.3% Kim et al. (1992) 1.4-2.5% Rodehutscord et al. (1997) 1.8-2.3% Cheng et al. (2003) 1.8-2.3% Encarnacao et al. (2004) 2.4% NRC (2011) Besides the negative impact that lysine deficiency has on growth performances of the fish (growth, feed efficiency), lysine deficiencies also result in damages to fish tissues. Along with reduced 26 performance, rainbow trout fed diets highly deficient in lysine resulted in a high degree of caudal fin erosion along with mortality rates of roughly 50% (Ketola, 1983). Similarly, Walton et al. (1984, 1986) reported fin erosion in rainbow trout fed lysine deficient diets but mortality rates of these fish were not significantly impacted. Assuming equal sanitary conditions and thus equal level of waterborne pathogens between studies, the differences in mortality rates may be attributed to degree of lysine deficiency between studies. Ketola (1983) fed highly deficient diets containing 0.7% lysine (DM basis) while Walton et al. (1984,1986) fed diets containing either 1.0% and 1.9% lysine (DM basis). Regardless, it is apparent that lysine deficiency has a large impact on both fish growth and health and highly deficient diets can result in high mortality rates. Amino acid antagonisms are structurally similar amino acids that have deleterious interactions with one another (D’Mello, 2003b). An antagonism relationship exists between lysine and arginine and this relationship is credited for depressed growth in various animals when fed diets containing excess lysine or arginine (Jones, 1961; D’Mello and Lewis 1970). Since lysine and arginine are structurally similar they are transported by the same amino acid carrier and competitive inhibition between these two amino acids exist (Kaushik and Fauconneau, 1984). Therefore, when an animal is fed a diet containing high levels of lysine while still meeting the arginine requirement, the animal may display symptoms of arginine deficiency. This is due to competition for shared amino acid carriers, rate of arginine absorption and transportation could be affected. The lysine-arginine antagonism is found in some mammals but is prevalent in avian species as they lack the ability to synthesize arginine (D’Mello, 2003b). Studies conducted on fish species including channel catfish, European sea bass, and Japanese flounder did not display an effect of feeding excess lysine on growth or plasma arginine levels in these species (Robinson et al., 1981; Tibaldi et al., 1994; Alam et al., 2002). 27 2.6 Strategies for Meeting Essential Amino Acid Requirements Like in other animal production industries feed is a leading cost of production and in aquaculture accounts for between 50-60% of total production costs (Sinha et al., 2011). Therefore meeting the nutritional requirements of the fish in a cost-efficient manner is a primary concern in enabling aquaculture to be an economically sustainable industry. Common ingredients in fish feed formulations include plant and animal proteins along with by-products such as fish meal, poultry byproduct meal, meat and bone meal, soybean meal, soy protein concentrates and corn gluten meal. Byproduct meals along with plant proteins are commonly included in fish feed formulations as they are economical and shown to have relatively high nutritive value to fish (Tacon et al., 2006). However, many of these ingredients, especially certain plant proteins, contain low levels of certain essential amino acids. Feeds which include high levels of these ingredients may result in diets that are deficient in certain essential amino acids. Therefore, in cases whereby a diet is deficient in essential amino acids there are three general feed formulation strategies used to help meet the essential amino acid requirement of an animal: 1) Increase total protein content of the diet, 2) combine various protein sources with complementary amino acid profiles, 3) supplementation with crystalline (synthetic) amino acids (NRC, 2011). The simplest approach to help meet the essential amino acid requirement of an animal when a formulated diet is deficient in one or more essential amino acids is to further increase the protein level of that diet. As amino acids are bound in proteins by further increasing the protein level of the diet the quantity of amino acids in the diet also increases. In cases whereby a diet is slightly deficient in certain essential amino acids increasing the amount of protein can result in amino acid levels meeting the requirement of the animal as seen in figure 2.3. However, there are limitations to this approach as increasing the inclusion of a protein source that is highly deficient in a particular essential amino acid 28 may result in a diet with a very high crude protein content, yet still deficient in that particular essential amino acid. This is common in swine nutrition whereby corn and soybean based diets with high inclusion rates can still result in a lysine deficient diet (Smiricky-Tjardes et al., 2004). Along with the limitations associated with this technique, proteins often represent the most expensive ingredient in feeds. Therefore, the strategy of increasing crude protein level of a diet in order to meet the essential amino acid requirement of an animal may result in a feed that is overly expensive, in addition to the possibility of depressing feed intake of the animal which is associated with being fed a diet with an imbalanced amino acid profile. Furthermore, water quality issues arise when increasing crude protein content of the diet as greater proportions of nitrogen may be expelled as waste into the water. Subsequently other formulation strategies may be more economically feasible and promote better growth. Each source of protein has unique amino acid profiles with some protein sources containing low levels of some essential amino acids while another protein source may contain high levels of that particular essential amino acid. For example, soybean meal contains 2.2% lysine while blood meal contains 8.2% lysine. The basis of the second formulation strategy is to use multiple sources of protein with complementary amino acid profiles in order to meet all essential amino acid requirement of an animal. Figure 2.4 provides a visual representation of how a single protein source “A” is not able to provide sufficient lysine or arginine in order to meet the requirement of these amino acids for an animal but contains an excess of methionine. Protein source “B” contains low levels of methionine but contains high amounts of lysine and arginine, both these source of protein contain low levels of at least one essential amino acid, but when combined all essential amino acid requirements are met. The use of multiple protein sources is a common practise in aquaculture as decreasing reliance on fish meal had led to the use of multiple economical plant and by-product protein sources in feed formulations (Tacon and Metain, 2008). However limitations in this feed formulation technique exist. Concerns regarding high 29 levels of plant protein inclusion exist due to their high fibre and indigestible carbohydrate content and presence of anti-nutritional factors (Dabrowski et al., 1989; Refstie et al., 2000). Furthermore an increasingly wide variety of ingredients are being included in the formulation of compound fish feeds but many aren’t properly characterized (Gatlin et al., 2007; Hardy, 2010; Sinha et al., 2011; Tacon and Metian, 2008). Ongoing research is being conducted to properly understand the nutritive value of these ingredients in the context of fish nutrition (Bureau, 2008; Gatlin et al., 2007). A final formulation strategy is the use of crystalline amino acids to supplement the amino acid profile of a diet. As seen in figure 2.5 if a diet is deficient in certain amino acids, for example lysine and arginine, synthetic lysine and arginine can be added in precise calculated amounts to the deficient diet in order to meet the requirement of the animal for these amino acid. Crystalline amino acids have been in use in the animal feed industry for over 50 years with the benefit of allowing greater flexibility in diet formulation. Economical ingredients can be used at higher inclusion rates as deficiencies in the amino acid profile of the resulting diet can be balanced by the addition of corresponding crystalline amino acids. Therefore, crystalline amino acids are becoming a key component in cost-effective diets. 30 Figure 2.3 Increasing crude protein content of a diet as a strategy for meeting essential amino acid requirements. NRC (2011) Figure 2.4 Use of multiple protein sources as a strategy for meeting essential amino acid requirements. NRC (2011) 31 Figure 2.5 Use of synthetic amino acids to supplement a deficient diet. NRC (2011) 32 2.7 Introduction to Synthetic Amino Acids Over the past 50 years crystalline amino acids have been shown to be utilized effectively by farm animals including many fish species. However, some debate surrounds the effectiveness of crystalline amino acids in comparison to traditional protein-bound amino acids. Several studies on a variety of fish species, including rainbow trout, Atlantic salmon and Asian seabass, have shown that crystalline amino acids are utilized as effectively as protein-bound amino acids in meeting the requirement for essential amino acids (Kim et al., 1991; Espe and Lied., 1994; Rodehutscord et al., 1995; Williams et al., 2001; Rollin et al., 2003; Espe et al., 2006). Contrarily studies conducted on the same fish species including, rainbow trout and Atlantic salmon along with studies on channel catfish, common carp, turbot concluded that protein-bound amino acids are utilised better than crystalline amino acids (Schuhmacher et al., 1997; Zarate and Lovell., 1997; de la Higuera et al., 1998; Refstie et al., 2001; Sveier et al., 2001; Peres and Oliva-Teles, 2005; Dabrowski et al., 2010). It is important to consider that in the majority of these studies crystalline amino acids were used in replacement of protein sources, however when formulating practical diets crystalline amino acids are used to supplement protein sources. Differences in absorption rate and site of absorption may be a possible explanation of the differences in utilization between protein-bound and free (crystalline) amino acids. By being absorbed more rapidly than proteinbound amino acids, the inclusion of crystalline amino acids may result in higher amino acid levels in plasma and tissues resulting in a greater proportion of these amino acids being catabolized rather than being utilized for protein synthesis (Batterham and Murison, 1981; Cowey and Walton, 1988; Tantikittii and March, 1995; Schuhmacher et al., 1997; Zarate and Lovell, 1999). Although discrepancy in utilization efficiency occurs between protein-bound and crystalline amino acids, crystalline amino acids are still widely included in diets with a greater “safety margin” being advised by the NRC (2011) during feed 33 formulations when crystalline amino acids are used to meet a significant portion of an amino acid requirement. As methionine and lysine are commonly the first and second most limiting essential amino acids in animal feeds they were the first synthetic amino acids to be commercially produced for use in animal feeds. The production process differs between lysine and methionine with lysine being a product of bacterial fermentation while methionine is produced through chemical synthesis. Chemical synthesis of amino acid results in the production of a racemic mixture of D and L isomers, as L-lysine is the only isomer of nutritional value all L-lysine must be produced through a fermentation process (Leuchtenberger, 1996). In 1956 in an effort to discover a glutamate excreting organism Corynebacterium glutamicum was discovered, from this bacteria lysine excreting mutations were produced (Kinoshita et al., 1957; Udaka, 1960). Common substrates for the industrial production of lysine via bacterial fermentation include starch and molasses. These substrates provide C. glutamicum with a sources of glucose, fructose and sucrose which are converted into pyruvate which enters the citric acid cycle producing oxaloacetate which through the addition of glutamate produces LAspartate (Wittmann and Becker, 2007). C. glutamicum has a split biosynthesis pathway for the production of lysine, through a series of metabolic steps facilitated by a wide range of enzymes, LAspartate is converted into L-Piperidine-2,6 dicarboxylate which enters either of the two biosythethis pathways resulting in the formation of L-lysine (Schrumpf et al., 1991; Sonntag et al., 1993). The process encompassing bacterial fermentation and the production of L-lysine is referred to the upstream process, depending on the downstream process, different L-lysine products can be created. The product of bacterial fermentation is a fermentation broth containing L-lysine, the bacteria biomass, and by-products of the fermentation process. Traditionally lysine was purified from the broth via ion exchange, the addition of chloride ion, and resulting evaporation and the release of ammonia 34 (Hermann, 2003). The resulting product undergoes crystallization resulting in a hydrochloric salt which when dried forms L-lys HCL containing 78.8% free L-lysine (Schutte and Pack, 1994). Changes to downstream processing techniques have resulted in many different forms of lysine preparations such as liquid lysine, liquid lysine sulphate or granulated lysine sulphate which contain various levels of L-lysine, 50%, 20-30% and 40-50% respectively (Kelle et al., 2005). L-lysine sulphate is a another common lysine product that varies in downstream processing from L-lysine HCL, in the formation of L-lysine sulphate the fermentation broth is left to undergo evaporation and granulation along with the free lysine. This results in a product that contains 50.7% free lysine along with 15% sulphate and small quantities of other nutrients such as phosphate and different amino acids (Smiricky-Tjardes et al., 2004). Crystalline methionine is produced predominantly though a chemical process based on the principle of Strecker synthesis. Strecker synthesis is a technique developed by Adolph Strecker in 1850 whereby α-amino acids were first chemically produced de novo through a series of chemical reactions. In Strecker synthesis, a condensation reaction between ammonium cyanide and either an aldehyde or a ketone compound results in the formation of α-amino nitriles. These nitriles are then hydrolysed to release α-amino acids (Lyer et al., 1996). Barger and Coyne (1928) first used Strecker synthesis to produce methionine using β-methylthiolpropaldehyde and ammonium cyanide to produce a racemic mixture of methionine at a yield of 6% (Barger and Weichselbaum, 1931). Although the Strecker technique has been continuously improved in order to meet commercial demand for synthetic methionine, due to the nature of chemical synthesis the process continues to produce a racemic mixture of D and L isomers of methionine. Although the D-isomer of methionine have been shown to be well utilized by swine, poultry and various fish species, some mammals including humans are not able to convert the D-isomer of methionine into the biologically active L-isomer (Friedman and Gumbmann, 1984). Therefore L-methionine can also be produced through bacterial fermentation much like L-lysine. However, due to limitations in the ability of microorgansims to produce high concentrations of L35 methionine commercial application of this technique has not yet been established (Gomes and Kumar, 2005). In addition to DL-methionine a hydroxy methionine analogue, 2-hydroxy-4-(methylthio)butanoic acid, commonly referred to DL-methionine-hydroxyanalog (MHA) is a also a product of chemically synthesis and available in a variety of forms including the granular calcium salt (MHA-Ca) and liquid free acid (MHA-FA). As MHA is a product of chemical synthesis commercial MHA contains a racemic mixture of D and L isomers of MHA (Baker, 2006). MHA differs structurally from methionine as a hydroxyl group is bound to the alpha carbon of MHA instead of the amino group found in methionine. This results in MHA being classified as an organic acid rather than an amino acid (Dibner, 2003). The aqueous solution of MHA, MHA-FA, contains 88% MHA while MHA-Ca contains 84% MHA with the reminder of the product consisting of the calcium salt (Baker, 2006). 2.7 Estimating Bioavailability of Essential Amino Acids Protein synthesis drives growth of an animal through the deposition of protein which requires amino acids at the site of synthesis. Therefore, not only is the dietary level of essential amino acid important but their ability to be absorbed, transported and utilized for protein synthesis. Instead of assuming that the all dietary amino acids are utilized equally well the “biological availability” of essential amino acids has become increasingly an area of interest. Bioavailability is commonly defined as the extent to which an ingested nutrient is digested and absorbed in a form that can be utilized by the animal (Batterham, 1992; Lewis and Bayley, 1995). For example lysine containing heat-treated feed ingredients often result in the formation Maillard reaction products. These Maillard reaction products are readily absorbed by the animal but cannot be utilized for protein synthesis (Carpenter, 1960; Moughan and Rutherfurd, 1996). Therefore, in a given feed the formulated level of lysine may 36 theoretically meet the requirement of the animal but due to a portion of lysine being converted into Maillard reaction products, the biological availability of lysine is lower in this feed. Bioavailability studies have focused primarily on the availability of nutrients in novel feed ingredients, however with the emergence of multiple synthetic amino acid products the bioavailability of these products is also of interest. In order to determine bioavailability of amino acids two major invivo methods are used; amino acid digestibility and growth assays. Apparent digestibility refers to the disappearance of a nutrient from the digestive tract and is calculated by comparing the nutrient content of the feed and fecal matter (Mosenthin and Rademacher, 2003). While determining apparent digestibility of amino acids in feed stuffs plays an important role in feed formulation, apparent digestibility only measures one aspect of bioavailability. Apparent digestibility measures the disappearance of the nutrient from the digestive tract but does not measure how well the nutrient is utilized once it leaves the digestive tract. Furthermore, microbial fermentation in the hindgut causes amino acid disappearance from the digestive tract resulting in overestimation of bioavailability, however this is not a major concern in fish nutrition due to limited microbial fermentation in the fish gut (Lewis and Bayley, 1995). Feed ingredients that are processed under high heat and pressure conditions may have a high apparent digestibility value but once absorbed may not utilized well by the animal and, therefore, have a lower bioavailability value. In addition fish feed and excrete waste in water which poses a unique challenge when measuring digestibility compared to land based agriculture. Therefore, a method that is able to account for both digestibility and utilization of a nutrient would better measure bioavailability. Growth assays for determining bioavailability of essential amino acid involve measuring the growth of an animal in response to being fed a test diet and are often considered the gold standard to which other methods are compared (Lewis and Bayley, 1995). While digestibility studies cannot fully 37 measure bioavailability as nutrient utilization post absorption is not accounted for, well designed and executed growth assays are a good representation of bioavailability as a growth response of an animal is the cumulative function of digestion, absorption and utilization of nutrients. The principle of growth assays for determining bioavailability is based off the formulation of a basal diet that is highly deficient in the test essential amino acid but meets all other known nutritional requirements of the animal. From the basal diet, test diets containing increasing level of the test essential amino acid can be created. Certain dietary limitations exist when using a dose-response slope-ratio assay to calculate relative bioavailability and a well-designed study must take these limitations into consideration. Firstly differences in the response of an animal to being fed test diets must be due solely to the test nutrient and not influenced by any other nutrients in the diet, including the presence of growth depressing antinutrients and growth enhancing nutrients such as probiotics (Batterham, 1992). To accomplish this test diets are formulated to meet all known nutritional requirements of the animals with the exception of the test nutrient. Secondly nutrient deficient diets affect feed intake, with increasing deficiency of an essential nutrient causing an increasing depression of feed intake (Harper and Rogers, 1965). In order to distinguish if the animals’ response is due to the effect of the test nutrient or due to differences in test nutrient intake, pair or equal feeding strategies can be implemented (Baker, 1986). Therefore meaningful comparison of relative bioavailability between sources of nutrients must be due solely to the effect that these nutrients have on the animal and not influenced by other exogenous factors. In terms of a response variable, growth of the animal is most commonly used in this type of study, however studies have also successful measured bioavailability using protein deposition and feed efficiency as response variables (Lewis and Bayley, 1995). The independent variable for growth assays measuring amino acid availability is either the dietary inclusion rate of the test amino acid or the total intake of that amino acid. When feed intake is not controlled, studies expressing test amino acid intake as the independent variable are believed to result in more valid assays (Gupta et al., 1958; de 38 Muelenaere et al., 1967; Oh et al., 1972; Cave and Williams, 1980). In contrast when feed intake is equalized between treatments either test amino acid intake or dietary concentration may be used as the independent variable (Carpenter et al, 1963). In order to calculate bioavailability of a nutrient slope-ratio assay can be applied to these growth assays. Slope-ratio assay allow for the comparison of nutrient from a test source to a standard source. Using regression analysis the slope of the growth curve of the animal being fed the test diets can be calculated and compared to the slope of the growth curve of animals being fed a standard source of the nutrient. The standard used is assumed to be 100% bioavailable, and relative bioavailability of the test ingredient can be calculated by comparing the slope of the test source to the standard. Figure 2.6 displays graphically the concept of slope-ratio assay along with other commonly used assay used to determine bioavailability. With the exception of parallel line assays, whereby data is subjected to a logarithmic transformation prior to regression and intersects are compared to determine bioavailability, bioavailability is determined through the comparison of slopes. Slope ratio assays were first applied by Batterham et al. (1979) to estimate lysine availability in plant protein ingredients in swine and rats and by Major and Batterham (1981) to determine availability of amino acids in protein concentrates in poultry. Slope-ratio assays have also successfully been applied in fish nutrition to estimate relative bioavailability of amino acids between various protein sources, and to compare relative bioavailability between various sources of synthetic amino acids (El-Haroun and Bureau, 2007; Poppi et al., 2011; Rodehutscord et al., 2000). Depending on the nature of the response both linear and non-linear regression can be applied to the data in order to calculate relative bioavailability. 39 Figure 2.6 Four commonly used assays for determining bioavailability of a nutrient. 40 Bioavailability of a test nutrient is calculated according to the ratio xs/xt whereby xt and xs are the amounts of the test nutrient and standard nutrient in the diets that when fed to the animal result in an equal response of the dependent variable. However, before any form of regression can be applied to the data set, assumptions for validity of slope-ratio assay must be tested. In order for slope-ratio to be valid the following criteria must be met: 1) common intercepts of all regression lines, including standard and test sources, at the Y-axis; and 2) the common intercepts of all regression lines intersect at the basal level, whereby xs and xt=0 (Finney et al., 1971; Littell et al., 1997). Figure 2.7 illustrates a linear slope-ratio assay that meets both the requirement for common intercept between standard and test nutrient regression lines and have equal intercept when xs and xt equal zero. 41 Figure 2.7 Determining nutrient bioavailability using a slope-ratio assay RBV=XS/XT 42 The most commonly used model for slope-ratio assay is the linear model whereby linear regression in the form of y=a+bxXx is applied to both the standard and the test nutrient. In this equation y= the performance parameter being measured, bx is the slope of the line, and Xx is the level of the nutrient. The resulting linear equations can be represented as f(xs)=a+bSXs for a standard nutrient and g(xt)=a=btXt for the test nutrient. One of the validity assumptions of slope-ratio assay is that both lines must share an equal intercept, therefore if f(xs)=g(xT) relative bioavailability =xs/xT=bT/bs which is the ratio between the slopes of the lines. A multiple regression equation can then be used to combine the linear equations of the two nutrients into Y= a + bsXs + bTxT. Linear models have been used to determine bioavailability of both protein bound and synthetic amino acids in a wide variety of terrestrial animals including poultry (Boebel and Baker, 1982; Wang et al., 2007) and swine (Yi et al., 2006; Smiricky-Tjardes et al., 2004; Batterham et al., 1979), along with fish species including rainbow trout and hybrid striped bass (Li et al., 2009; El-Haroun and Bureau, 2007; Rodehutscord et al., 2000). When measuring growth responses of an animal being fed a diet containing an essential amino acid between 0 and 100% of the requirement for maximum growth of an animal, a curvilinear response is expected. Examining the growth curve of an animal when being fed a diet containing an essential amino acid at a level that corresponds with meeting between about 30-70% of the requirement for the animal in order to achieve maximal growth, constant utilization of the essential amino acid occurs resulting in a linear response of the growth curve. At levels above about 70% of requirement of an essential amino acid, slope of the growth curve begins to decrease as requirement begun to be met for individual animals in the sample that have the lowest requirement (Baker, 1986). Therefore variability between animals, and the effect they have on calculated bioavailability, can be reduced by applying linear models to slope-ratio assays whereby essential nutrient levels are between 30-70% of the requirement of an animal for maximal growth. 43 In addition to the use of linear regression for slope-ratio assays, exponential regression models are commonly applied to non-linear responses. The non-linear equation follows the form: Y =b1 + b2* (1-e^(bsxs + btxt)) whereby b1 represents the intercept, b2 is the difference between lowest and highest response, bs and bt represent the steepness co-efficient of the curves for the standard and test nutrient respectively, and xs and xt represent the level of the standard and test nutrient respectively. In order to calculate relative bioavailability of a test amino acid compared to the standard source using a non-linear model ratios between steepness coefficients are calculated whereby relative bioavailability=bt/bs (Noll et al., 1984; Littell et al., 1997). A potential source of variability when determining bioavailability using a non-linear model is aberrant results tend to increase as the growth curve transitions from a linear response, representing constant utilization of the nutrient, to the curvilinear portion of the growth curve where variability in nutrient utilization between animals is often greatest (Baker,1986). 2.8 Conclusions and perspectives Modern fish feed formulation strategies increasingly involve the use of alternative plant proteins in substitution of traditional animal based protein sources in an effort to increase both environmental economic sustainability of the aquaculture industry. Although diets containing these plant protein sources can be utilized well by many fish species the amino acid profiles of these ingredients often result in diets that are limiting in the essential amino acids lysine and methionine. As both methionine and lysine are essential amino acids they cannot be synthetized by the fish and, therefore, all requirements for these amino acids must be fulfilled through dietary sources. In addition to their roles in protein synthesis, lysine and methionine play other important roles in an animal. Methionine is a pre-cursor to SAM which is second only to ATP in terms of molecules required by enzymes, while lysine plays an important role in protein structure. Diets that fail to meet an organisms’ requirement for either lysine or methionine result in poor growth as a result of an impaired rate of 44 protein synthesis and death in the case of severe deficiency. In addition to the impact that lysine and methionine deficiencies have on the rate of protein synthesis, cataract formation and fin erosion are common clinical signs of methionine and lysine deficiency seen in fish. Maximizing growth and feed efficiency is crucial in terms of economic sustainability of the aquaculture industry. Therefore, formulation strategies such as the supplementation of synthetic amino acids, have been implemented to ensure diets are not deficient in an essential amino acid. Over the past 50 years the inclusion of synthetic amino acids in diets of agriculture and aquaculture species has increasingly become a common practise. However, the production of synthetic amino acids is much older with chemical synthesis of methionine first produced in 1928 while lysine production via bacterial fermentation occurring in the early 1960s. With the widespread acceptance of synthetic amino acids in the agri-food industry commercial production of synthetic methionine and lysine has increased resulting in the production of various forms of these two synthetic amino acids including; L-lysine HCL, L-Lysine-sulphate, DL-methionine, L-methionine, and the hydroxyl analogue of methionine MHA-Ca. As production techniques and chemical properties and structure differ between the various forms of these synthetic amino acids the relative bioavailability between forms is important in terms of their ability to properly fulfill either the lysine or methionine requirement of an animal. Dose response slope-ratio assays have commonly been used to determine the nutrient requirement of an animal, such as an essential amino acid. However, a slope-ratio assay can be applied to a carefully planned dose response experiment in order to determine relative bioavailability of different sources or forms of a synthetic amino acids. By determining relative bioavailability of these various synthetic forms of methionine and lysine feed manufactures will be better able to formulate diets in a cost-effective manner that contain these synthetic amino acids ensuring that the lysine or methionine requirement of an animal is properly met. 45 CHAPTER 3 – ASSESSING THE BIOAVAILABILITY OF L-METHIONINE AND A HYDROXY METHIONINE ANALOGUE (MHA-Ca) COMPARED TO DLMETHIONINE IN RAINBOW TROUT (ONCORHYNCHUS MYKISS) Abstract Supplementing synthetic methionine (Met) to diets of farm animals in order to meet their nutritional requirements for this essential amino acid is a common practice. Different synthetic forms of supplemental met are produced commercially by various manufacturers, limited information exists on the bioavailability of these different forms to fish species particularly in regards to commercially relevant fish species. The relative bioavailability of L-methionine and a hydroxy methionine analogue (MHA-Ca) was compared to DL-methionine in a commercially relevant species, rainbow trout, and assessed using a linear slope-ratio assay method. The ability of rainbow trout to effectively convert the D-isomer of methionine into the biologically active L-isomer was also studied by comparing the relative bioavailability of L-methionine to DL-methionine. Nine (9) diets were created from a basal diet which was supplemented with graded equimolar levels of methionine (0.1, 0.2, 0.3% of diet by weight) from DL-methionine, L-methionine and methionine hydroxyl analogue calcium (MHA-Ca) and fed to triplicate groups of rainbow trout (IBW=24.0±0.6g) for 12 weeks using a pair-feeding protocol. The basal diet was formulated to be deficient in both methionine (0.5%) and cysteine (0.3%) while meeting all other known nutritional requirements of rainbow trout. MHA-Ca was found to be less available (p<0.05) than DLmethionine with relative bioavailability values of 69, 60 and 73% based upon weight gain, growth rate (TGC) and retained nitrogen values, respectively. Although numerical differences in relative bioavailability exited between L-methionine and DL-methionine, differences were not significant (P>0.05) leading to the conclusion that rainbow trout can effectively convert D-isomer of methionine into the L-isomer without affecting fish performance. 46 3.1 Introduction Methionine is commonly one of the first-limiting essential amino acids in diets of farmed animals. These diets are often supplemented with synthetic methionine (Met) from a variety of sources in order to meet the animals Met or total sulphur amino acid (TSAA) requirements. Various synthetic methionine products are commercially available including; DL-Methionine, liquid methionine-hydroxy analogue, and methionine hydroxy analogue calcium salt (MHA-Ca). Other Met products, such as Lmethionine (L-Met), are used primarily for laboratory purposes. Both DL-methionine and MHA-Ca are products of chemical synthesis resulting in the formation of a racemic mixture of D and L isomers. MHA differs structurally from methionine as it contains a hydroxyl group in place of the amino group found in methionine, therefore MHA is classified as an organic acid instead of an amino acid (Dibner, 2003). As MHA is an organic acid it is not a utilizable source of methionine, therefore, it must undergo a series of transformation, first to α-keto analogue of methionine, and then to L-methionine through a dehydrogenase and transaminase reactions, respectively (Lloyd et al., 1978). As only L-amino acids can be utilized by the body D-isomers must first undergo a metabolic conversion to the L-isomer form before becoming metabolically available to the animal (Friedman and Gumbmann 1984). The metabolic conversion from the D-isomer to the L-isomer of an amino acid requires the enzyme D-amino oxidase, which most fish species are able to synthesis (Lewis, 2003). There is evidence that supplemental amino acids are well utilized by fish and crustacea. However, synthetic amino acids appear to be absorbed faster by the intestinal epithelium than dietary protein-bound amino acids (Batterham and Murison, 1981; Cowey and Walton, 1988; Tantikittii and March, 1995; Schumacher et al., 1997; Zarate and Lovell, 1999). The faster absorption rate may cause higher blood plasma concentration of these synthetic amino acids, leading to potentially higher levels of catabolism in the liver and other organs which results in lesser amounts of these amino acids being 47 available for metabolic functions. The imbalance of amino acids in the tissue caused by the rapid absorption of synthetic amino acids compared to protein-bound amino acids have been shown to be mitigated through increased feeding frequency (Batterham & Bayley, 1989). By increasing feeding frequency both synthetic and protein-bound amino acids are supplied on a more regular basis which reduces the disparity between absorption rates and ultimately reduces the proportion of synthetic amino acids which are catabolized and, therefore, improve utilization of the synthetic amino acid (Batterham, 1974). Recent studies have shown that supplemental amino acids may be 20-30% less efficiently utilized than amino acids supplied as intact proteins (El Haroun and Bureau, 2007). Due to synthetic amino acids appearing to be utilized slightly less well than protein-bound amino acids, the NRC recommends the use of safety margins when formulating synthetic amino acid-containing diets. Regardless synthetic amino acids allow for greater flexibility in feed formulations and the use of more economical feed ingredients, resulting in their widespread use in animal feeds. In salmonid nutrition, modern feeding concepts of reducing fish meal and animal protein inclusion levels while balancing dietary amino acid profiles with synthetic amino acids are established. As multiple methionine products, including DL-Met and MHA-Ca, are commercially available the relative ability of these products to meet the methionine requirements of an animal is of interest. Bioavailability, defined as the extent to which an ingested nutrient is digested and absorbed in a form that can be utilized by the animal, can be compared between sources in order to understand the relative ability of these products in meeting the methionine requirements of an animal (Batterham, 1992; Lewis and Bayley, 1995). Research in poultry (Lemme et al., 2002, Sauer et al., 2008) and swine (Kim et al., 2006; Baker, 2006; Sauer et al., 2008) suggest that the bioavailabilities of MHA products are considerably lower compared to DL-Met. Research in fish suggests similar conclusions (Keembiyehetty and Gatlin, 1995, 1997; Goff and Gatlin, 2004; Kelly et al., 2006). Alternatively, limited studies in poultry 48 nutrition suggest similar bioavailability between MHA and DL-methionine (Richards et al., 2005; Dibner and Knight, 1984; Romer & Abel, 1999). However, additional conclusive studies on commercially relevant species are needed. Therefore, in this study, MHA-Ca was tested against DL-Met in a commercially important salmonid species, rainbow trout. In addition, the ability of rainbow trout to utilize the D-isomer in DL-methionine was explored by comparing relative bioavailability of L-Met to DLMet. The objective of this study was to determine the relative bioavailability of two methionine sources, L-methionine (L-Met) and the calcium salt of the hydroxyl analogue of methionine (MHA-Ca) compared to DL-methionine (DL-Met) in rainbow trout. Bioavailabilities of the two methionine sources, L-Met and MHA-Ca, were determined through the analysis of growth performance and nutrient retention criteria of a dose-response trial using multi-linear regression in comparison to a standard (DL-Met) following the method proposed by Littell et al. (1997). 3.2 Methods 3.2.1 Experimental Diet Formulation and Preparation In total, 10 diets were included in this experiment; one basal (control) diet and nine (9) diets consisting of three graded levels of each of the three test products (3 test products at 3 inclusion levels). A master batch of the basal diet (control) was produced which was formulated to be deficient in both methionine (0.45% digestible Met) and marginally deficient in cysteine (0.26% digestible Cys). From the master batch, 10 isonitrogenous (39% CP, 36% DP) and isoenergetic (20 kJ/g DE) test diets were created. Nine diets were created by adding one of the three synthetic sources to equal aliquots of the master batch at three graded levels (Table 3.1). The diets were formulated to contain equimolar amounts of methionine and methionine equivalent (MHA) per level across all three sources (Table 3.2). The analyzed essential amino acid composition of the test diets are presented in Table 3.3. The basal diet was formulated to meet all known nutritional requirements for rainbow trout with the exception of 49 methionine according to recommendations of the NRC (2011). Proximate composition of all experimental diets is presented in table 3.4. Formulated and analyzed levels of total methionine, methionine equivalent (MHA) and total methionine of experimental diets are found in table 3.5. Prior to mixing, all ingredients were weighed, individually bagged and sorted according to diet. All dry ingredients, excluding test ingredients, were first mixed as one master batch in a Marion Mixer for 30 minutes (Marion Mixers Inc., Marion, Iowa, USA). The master batch was separated into 10 equal portions, with each portion representing a separate test diet, and individual diets were placed separately into a Hobart mixer (Hobart Ltd, Don Mills, ON, Canada). Respective test ingredients were then added and hand mixed to ensure no loss of test ingredients during mechanical mixing. Using the Hobart mixer dry ingredients plus test ingredient were mixed for five minutes. Wet ingredients, i.e. fish and canola oil, were added slowly during mixing and the resulting mash was mixed for an additional 10 minutes. The resulting diets were stored at 4°C until pelleted. Diets were steam-pelleted using a laboratory-scale team pellet mill using a 2mm diameter dye and a pellet length of 5mm (California Pellet Mill, San Francisco, CA, USA), and dried overnight under forced air at room temperature using a drying oven (Precision & Scientific Co., USA), sieved to remove fines and broken pellets, and stored at 4°C until used. 50 Table 3.1 Ingredient composition of experimental diets. Ingredient Fish meal Blood meal, spray dried Gelatin Soybean meal Starch, gel. Lentil, green Wheat gluten Wheat middlings Fish oil Canola oil Vitamin premix1 Ascorbic acid Amino acid premix2 Lysine (BioLys) DL-Met (MetAmino) L-Met MHA-Ca Choline chloride Mineral premix4 Ca(H2PO4)2 Arbocel (cellulose) Total Diet (% as is ) 5 6 10 10 1 10 2 10 3 10 4 10 7 10 8 10 9 10 10 10 3 3 3 3 3 3 3 3 3 3 8 8.9 10 11 4.6 7 11 8 1 0.03 8 8.9 10 11 4.6 7 11 8 1 0.03 8 8.9 10 11 4.6 7 11 8 1 0.03 8 8.9 10 11 4.6 7 11 8 1 0.03 8 8.9 10 11 4.6 7 11 8 1 0.03 8 8.9 10 11 4.6 7 11 8 1 0.03 8 8.9 10 11 4.6 7 11 8 1 0.03 8 8.9 10 11 4.6 7 11 8 1 0.03 8 8.9 10 11 4.6 7 11 8 1 0.03 8 8.9 10 11 4.6 7 11 8 1 0.03 5 5 5 5 5 5 5 5 5 5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 - 0.1 0.2 0.3 - - - - - - 0.1 0.2 0.3 0.1183 0.2363 0.3533 0.345 0.345 0.345 0.345 0.345 0.345 0.345 0.345 0.345 0.345 1 1 1 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3 3 3 5 5 5 5 5 5 5 5 5 5 100 100 100 100 100 100 100 100 100 100 1Provides per kg of diet: Retinyl acetate (vit. A), 75mg; Cholescalciferol (vit. D), 60mg; dl-a-tocopherol-acetate (vit. E), 300mg; Menadione Na-bisulfate (vit. K), 1.5mg; Cyanocobalamine (vit. B12), 30mg; Ascorbic acid monophosphate, 300mg; Biotin, 210mg; choline chloride (chloride, 50%), 15mg; D-calcium pantothenate, 32.6mg; pyrodpxone-HCL (vit. B6), 7.5mg; Riboflavin (vit. B2), 9mg; Thiamin-HCL (vit. B1), 1.5mg; Caro-Pink (Astaxanthin), 500mg. 2Provides per kg of diet: Tryptophan,3500mg; Threonine, 8000mg; Isoleucine, 8000gm; Histidine, 7000mg; Valine, 6000mg; Leucine, 6000mg; Arginine, 2500mg; Phenylalanine, 9000mg. 3Test products were added in equimolar As the commercial product MHA-CA contains 84% MHA and 14-16% Ca. 4Provides per kg: sodium chloride (NaCl, 39% Na, 61% Cl), 3077mg; potassium iodide (KI, 24% K, 76%I), 10.5mg; ferrous sulphate (FeSO4-H20, 20% Fe),65mg; manganese sulphate (MnSO4, 36% Mn), 88.9mg; zinc sulphate (ZnSO4-H2O, 40% Zn), 150mg; copper sulphate (CuSO4-H20, 25% Cu), 28mg; yttrium oxide, 100mg. 51 Table 3.2 Experimental design – methionine inclusion from various synthetic sources. Treatment 1 2 3 4 5 6 7 8 9 10 Met source Addition of test source (% of product) Basal1 DL-Met DL-Met DL-Met L-Met L-Met L-Met MHA-Ca MHA-Ca MHA-Ca 0.1 0.2 0.3 0.1 0.2 0.3 0.1182 0.2362 0.3532 Addition Of met and met equivalent (%) 0.1 0.2 0.3 0.1 0.2 0.3 0.13 0.23 0.33 1 Met + cys deficient diet: 0.45% digestible Met and 0.26% digestible Cys. products were added in equimolar amounts, with molar weight of 149 g/mol for DL-Met /L-Met, and 150 g/mol for MHA. The commercial product MHA-Ca contains 84% MHA and 14-16% Ca. 3 MHA is considered an equivalent of methionine on a molar basis. 2 Test Table 3.3 Analyzed essential amino acid content of experimental diets (dry matter basis). Diet1 MET MHA Cys LYS THR TRP ARG ILE LEU VAL HIS PHE % 1 Basal 0.50 0.31 3.49 1.79 0.58 2.43 1.76 2.77 2.03 1.51 2.26 2 DL-Met-1 0.61 0.30 3.92 1.77 0.60 2.37 1.77 2.74 1.98 1.49 2.25 3 DL-Met-2 0.70 0.30 3.43 1.76 0.59 2.36 1.73 2.81 2.08 1.53 2.24 4 DL-Met-3 0.83 0.30 3.61 1.74 0.60 2.40 1.76 2.75 2.02 1.48 2.20 5 L-Met -1 0.60 0.30 3.79 1.73 0.58 2.35 1.70 2.77 2.07 1.51 2.22 6 L-Met-2 0.69 0.30 3.54 1.74 0.59 2.41 1.73 2.76 2.08 1.51 2.22 7 L-Met-3 0.81 0.30 3.99 1.73 0.56 2.42 1.74 2.83 2.13 1.54 2.24 8 MHA-Ca-1 0.50 0.10 0.30 3.41 1.73 0.59 2.37 1.74 2.73 2.02 1.51 2.24 9 MHA-Ca-2 0.47 0.19 0.28 4.16 1.68 0.59 2.32 1.66 2.66 1.97 1.46 2.17 10 MHA-Ca-3 0.49 0.30 0.31 4.05 1.73 0.57 2.41 1.75 2.85 2.07 1.52 2.25 Requirements: 0.7 0.4 2.4 1.1 0.3 1.5 1.1 1.5 1.2 0.8 0.9 NRC (2011)2 1 Dietary treatments= DL-Met graded levels 1-3, L-Met graded levels 1-3, MHA-CA graded levels 1-3 based upon equilmolar levels of methionine for each level. 52 Table 3.4 Proximate composition of experimental diets. Diet 1 2 3 4 5 6 Crude 40.6 40.6 40.9 40.4 40.5 39.5 protein, % Lipid, % 21.5 21.6 21.5 21.6 21.8 21.6 Ash 6.4 6.6 6.3 6.4 6.6 6.6 GE kJ/g 23.8 23.8 23.6 23.7 23.8 23.6 7 8 9 10 40.8 39.8 40.6 40.4 21.9 6.4 23.7 21.5 6.5 23.1 21.1 6.8 23.6 21.6 6.4 23.7 Table 3.5 Formulated and analyzed levels of methionine and MHA in experimental diets. Formulated levels Analyzed levels Diet 1 Basal 2 DL-Met-1 3 DL-Met-2 4 DL-Met-3 5 L-Met -1 6 L-Met-2 7 L-Met-3 8 MHA-Ca-1 9 MHA-Ca-2 10 MHA-Ca-3 Met % MHA % 0.50 0.60 0.70 0.80 0.60 0.70 0.80 0.50 0.50 0.50 0.101 0.201 0.301 Total met + met equivalent2 % 0.50 0.60 0.70 0.80 0.60 0.70 0.80 0.603 0.703 0.803 1 Met % MHA % 0.50 0.61 0.70 0.83 0.60 0.69 0.81 0.50 0.47 0.49 - 0.101 0.191 0.301 Total met + met equivalent2 % 0.50 0.61 0.70 0.83 0.60 0.69 0.81 0.593 0.693 0.803 % of MHA from MHA-Ca source. As the commercial product MHA-Ca contains 84% MHA and 14-16% Ca, supplementation levels of product will be 0.118, 0.236, and 0.353% MHA-Ca, resulting in 0.10, 0.20 and 0.30% MHA supplementation levels 2 Includes MHA as an equivalent to methionine 3 Total methionine from basal diet plus methionine equivalent from MHA-Ca 53 3.2.2 Fish, Feeding and Husbandry Rainbow trout fingerlings, initial average body weight of 10.7g, were obtained from the Alma Aquaculture Research Station (Elora, Ontario, Canada) and transferred to the Fish Nutrition Research Lab at the University of Guelph prior to the start of the trial. The juvenile fish, average initial weight of 24.0 ± 0.6g, were stocked into 30 (60L) fiberglass tanks with a stocking density of 15 fish per tank. Water was supplied through a partial recirculation system (+/- 30% make up water per pass) at a flow rate of ~3L/min per tank. Prior to the start of the trial, biomass in each tank was equalized to be within 3% of the mean. Each tank was individually aerated using an air stone and water temperature was controlled thermostatically to 15°C. Photoperiod in the room housing the experimental tanks was controlled using a timer to ensure a 12h light: 12h dark schedule. The 10 experimental diets were allocated in three replicates to tanks using a complete randomized design. Prior to the experiment the fingerling were transferred into the experimental system and fed a high quality commercial diet (ProfishentTM High Energy Feed, Martin Mills, Elmira, Ontario, Canada), in order to acclimate the fish to the experimental housing. Fish were hand-fed using a pair-feeding protocol whereby each tank was pair fed to the lowest intake replicate of the basal diet. During weekdays fish were fed three times per day, 1000h, 1300h and 1600h and once daily on weekends for a total of 12 weeks (84 days). Feed intake were recorded weekly and fish were weighed in bulk every 28 days, with feed being withheld prior to weighing. All animals were treated in compliance with the guidelines of the Canadian Council on Animal Care and the University of Guelph Animal Care Committee. At the conclusion of the 84-day trial, fish were weighed in bulk and 6 fish per tank were randomly selected and sacrificed by an overdose of an anesthetic (MS-222). These 6 fish were dissected immediately and whole body, liver and viscera weights were recorded. 54 3.2.3 Chemical Analysis At the start of the trial an initial pooled sample of 36 fish was obtained, while final samples consisting of an additional 6 fish per tank. Fish were sacrificed, using an overdose of MS-222, for proximate composition analysis. Live weights were recorded and 6 fish per tank were pooled into plastic autoclave bags and stored at -20°C until processing. Samples were thawed overnight prior to being cooked in an autoclave (LabTech LAC-5100S Autoclave). After autoclaving samples were homogenized into a slurry using a food processor (Cuisinart DLC-X PLUS), freeze dried (FTS Systems Dura Dry MP freeze drier), and ground to a fine consistency. Ground samples were stored at -20°C until further analysis was conducted. Diets, major ingredients (>10% inclusion in diets; fishmeal, gelatinized starch, lentils and soy protein concentrate), and initial and final carcass samples were analyzed in duplicate. Crude protein content of experimental diets and major test ingredients was analyzed by EVONIK Inc. (Hanau, Germany). Crude protein content of carcass samples was determined by a contracting lab using the LECO combustion method (Lab Services, University of Guelph, ON, Canada). Crude lipid content was determined using a high pressure lipid extractor (Ankon XT-20 Lipid extractor – ANKOM Technology, Macedon, USA). Dry matter was determined by placing samples in a drying oven (Fisher Scientific Isotemp oven, Markham, ON, Canada), at 100°C for 12 hour. Ash was determined by placing dried samples in a muffle furnace (Fisher Scientific Isotemp muffle furnace, Markham, ON, Canada) and ashing for 4 hours at 550°C. Gross energy (GE, Mj/kg) was determined using a Parr 1271 bomb calorimeter (Parr Instruments, Moline, IL, USA). Amino acid analysis of diets was performed by an outside laboratory (EVONIK, Hanau, Germany). 55 3.2.4 Calculations and Statistical Analysis All data was submitted to the Brown & Forsythe test, which is a modified Levene test, to compare variance between treatments. The effect of supplemental methionine and methionine equivalent on weight gain, thermal-unit growth coefficients (TGC), feed efficiency, retained nitrogen (RN) , recovered energy (RE), nitrogen retention efficiency (NRE), energy retention efficiency (ERE) and proximate carcass composition, and any possible interactions between source and level were investigated using SAS general linear model in addition to the use of orthogonal polynomial contrasts (PROC GLM, SAS Version 9.2, SAS Institute, Cary, NC, USA). Performance and proximate composition data were analyzed using a complete randomized design with ANOVA with source and level of synthetic methionine as sources of variation. In all these analyses the tank was the experimental unit. Weight gain, TGC and retained nitrogen data were regressed against supplemental methionine and methionine equivalent concentrations using a linear model (PROC GLM). After confirming that the model satisfied the assumptions for slope-ratio assay according to Littell et al. (1997) a common intercept linear model was used to compare the slopes of L-met and MHA-Ca to DL-met to determine the relative bioavailability of L-met and MHA-Ca compared to DL-Met. The following linear model was used (Littell et al. 1997). Y = a + b1x1 + b2x2 + b3x3 Where: Y = performance criterion a = intercept b1 = regression coefficient for product x1(DL-Methionine) b2 = regression coefficient of x2 (L-Methionine) b3 = regression coefficient of x3 (MHA-Ca) 56 x1, x2, x3 = formulated dietary methionine/methionine equivalent inclusion level of DL-Met, L-Met, and MHA-Ca Relative bioavailability for products x2 and x3 can be calculated by b2/b1 and b3/b1. Growth performance parameters were calculated as follows: i) Thermal-Unit Growth Coefficient (TGC): TGC = 100 x (FBW1/3 – IBW 1/3) / ∑ (Temp(°C) x Number of days) Where: FBW = final body weight (g); IBW = initial body weight (g); Temp = average water temperature (°C). ii) Feed Efficiency (FE): FE = weight gain (as-is basis) / feed intake (DM basis) Where: weight gain (g/fish) = (FBW/ final number of fish)-(IBW/initial number of fish); feed intake = total feed (DM basis) / ∑(number of fish x days) iii) Retained Nitrogen (RN, g/fish): Retained Nitrogen = (FBW x % CP final) – (IBW x % CP initial) Where %CP final = final carcass crude protein content (as-is basis); %CP initial = initial carcass crude protein content (as-is basis) iv) Recovered Energy (RE, Kj/fish): Recovered Energy = (FBW x GE final) – (IBW x GE initial) Where GE final = final carcass gross energy content (kJ/g BW); GE initial = initial carcass gross energy content (kJ/g BW) v) Nitrogen and Energy Retention Efficiencies (NRE and ERE): NRE (%) = ([FBW x % N final) – (IBW x %N initial)]/IN x 100 ERE (%) = ([FBW x GE final) – (IBW x GE initial)]/IE x 100 Where: NRE = nitrogen retention efficiency; ERE = energy recovery efficiency; 57 %N final = nitrogen content (%) in final carcass sample; %N initial = nitrogen content (%) in initial carcass sample; IN = nitrogen intake (g); IE=GE intake (kJ) vi) Viscerosomatic index (VSI): VSI = (Viscera Weight/FBW) x 100 Where: Viscera Weight = dissected weight of all viscera (g) at end of experimental period vii) Hepatosomatic index (HSI): HSI = (Liver Weight/FBW) x 100 Where: Liver Weight = dissected liver weight (g) at end of experimental period 3.3 Results 3.3.1 Mortalities Over the course of the 84-day trial one mortality was due to fish illness. A mechanical failure in one of the systems housing the experimental tanks resulted in the loss of water in two adjoining tanks. These two tanks represented replicates of two different treatments. Due to potential adverse effects on fish growth due to stress and low levels of oxygen associated with this event, these two tanks were excluded from data analysis. 3.3.2 Growth Performance Figure 3.1 presents the growth curves of the fish fed the 10 experimental diets over the 84-day trial. Using non-linear regression on all four inclusion levels of methionine figures 3.2, 3.3 and 3.4 illustrate the response of live weight gain, TGC and retained nitrogen respectively, of fish fed diets containing increasing levels of methionine and a methionine equivalent from different sources. Growth performance parameters from the 84-day feeding trial are presented in table 3.6. Fish that received supplemental methionine exhibited higher weight gain (p<0.001), growth rate (p<0.001) 58 and feed efficiency (p<0.001), while displaying lower VSI (p<0.001), and HSI (p<0.01) scores when compared to fish fed the basal diet. Increases in weight gain (p<0.001), TGC (p<0.001) and feed efficiency (p<0.001) occurred in response to increasing levels of methionine supplementation while VSI (p<0.001) and HSI (p<0.05) decreased in response to increasing methionine supplementation. When comparing means of growth criteria between sources of methionine, weight gain (p<0.05) and growth rate (p<0.05) exhibited significant differences between DL-Met and MHA-Ca, while no significant differences were observed between these two products when comparing feed efficiency (p>0.10), VSI (p>0.10) or HSI (p>0.10). Growth rate of fish exhibited a significant interaction effect between product and level of supplementation (p<0.05) while interaction effects were not significant (p>0.05) for the other performance parameters measured. No significant differences were observed between DL-Met vs L-Met and L-Met vs MHA-Ca in any of the performance criteria measured (p>0.10). 3.3.3 Proximate Carcass Composition The results from proximate analysis of pooled carcass samples are presented in Table 3.7. Fish receiving synthetic methionine supplementation showed significantly higher levels of carcass crude protein content (p<0.001) while having significantly lower levels of lipid (p<0.001) and gross energy content (p<0.05) when compared to unsupplemented fish. Ash content of carcasses was not influenced by treatment (p>0.05). Moisture (p<0.001) and crude protein (p<0.001) content of carcasses increased in response to increasing levels of methionine supplementation while lipid (p<0.001) and gross energy (p<0.001) content of carcasses decreased. When comparing between sources of methionine, significant differences in carcass lipid contents were found when comparing treatments that received DL-Met vs. MHA-Ca (p<0.001) and L-MET vs. MHA-Ca (p<0.001), with fish receiving diets containing MHA-Ca having higher carcass lipid content. When analyzing for a source x level interaction, lipid content (p<0.01) of 59 carcasses responded significantly while all other proximate parameters, with the exception of GE (p=0.0587), were not significantly affected (p>0.10). 3.3.4 Retained Nutrients and Retention Efficiencies Results for retained nitrogen (RN), recovered energy (RE), nitrogen retention efficiency (NRE), and energy retention efficiency (ERE) are presented in table 3.8. Comparing methionine supplemented to unsupplemented fish, carcasses of fish that received supplemental methionine showed higher rates of retained nitrogen (p<0.001), recovered energy (p<0.01), and higher rates of nitrogen retention efficiency (p<0.001) and energy retention efficiency (p<0.01). Comparing between levels of methionine supplementation, retained nitrogen (p<0.001) and nitrogen retention efficiency (p<0.001) increased in response to increasing levels of methionine supplementation while energy retention efficiency (p<0.05) decreased, and retained energy was not affected (p>0.05). Methionine source effected retained energy (p<0.05), nitrogen retention efficiency (p<0.05) and energy retention efficiency (p<0.05). Differences in recovered energy (p<0.01) and energy retention efficiency (p<0.05) were found when comparing DLMet vs. MHA-Ca. Differences in nitrogen retention efficiency (p<0.05) were found when comparing between the sources L-Met and MHA-Ca and significant differences in recovered energy (p<0.05) were also found when comparing DL-Met to L-Met. 60 Figure 3.1 Growth curves of rainbow trout in response to being fed experimental diets containing graded equimolar levels of methionine from three synthetic sources. Live Weight (g/fish) 120 100 Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6 Diet 7 Diet 8 Diet 9 Diet 10 80 60 40 20 0 56 28 84 Time (days) Diet 1 represents the basal diet, 2-4 contain graded levels of methionine from DL-methionine, diets 5-7 are supplemented with L-methionine and 8-10 with MHA-Ca. Supplemental levels are based on equimolar amounts methionine from each synthetic supplemental source. Refer to table 3.2 for further diet detail. 61 Figure 3.2 Weight gain of rainbow trout in response to being fed diets containing increasing equimolar levels of methionine from DL-Met (■), L-Met (▲), and MHA-Ca (▼). 105 DL-Met L-Met MHA-CA Gain (g/fish) 100 95 90 85 80 75 70 0.5 0.6 0.7 0.8 Total Methionine1 (%) 1Total dietary methionine, basal diet contains 0.5% methionine with increasing inclusion levels of 0.1% supplemental methionine or methionine equivalent (MHA). Lines regressed using a 2nd order polynomial function. Figure 3.3 Thermal-unit growth coefficient (TGC) of rainbow trout in response to being fed diets containing increasing equimolar levels of methionine from DL-Met (■), L-Met (▲), and MHA-Ca (▼). 0.175 DL-Met L-Met MHA-CA 0.170 TGC % 0.165 0.160 0.155 0.150 0.145 0.140 0.135 0.5 0.6 0.7 0.8 Total Methionine1 (%) 1Total dietary methionine, basal diet contains 0.5% methionine with increasing inclusion levels of 0.1% supplemental methionine or methionine equivalent (MHA). Lines regressed using a 2nd order polynomial function. 62 Retained Nitrogen (g/fish) Figure 3.4 Retained Nitrogen (g/fish) content of rainbow trout in response to being fed diets containing increasing equimolar levels methionine from DL-Met (■), L-Met (▲), and MHA-Ca (▼). 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 0.5 DL-Met L-Met MHA-Ca 0.6 0.7 0.8 Total Methionine1 (%) 1Total dietary methionine, basal diet contains 0.5% methionine with increasing inclusion levels of 0.1% supplemental methionine or methionine equivalent (MHA). Lines regressed using a 2nd order polynomial function. 63 Table 3.6 Performance of rainbow trout in response to being fed increasing equimolar levels of methionine from different sources over a 12-week experimental period. Feed Weight Feed Inclusion TGC1 Intake gain efficiency2 VSI3 level % Met % Diet g/fish g/fish gain:FI 1 0.0 91 78.5 0.145 0.86 15.4 2 0.1 91 87.0 0.154 0.96 14.6 3 0.2 91 94.9 0.164 1.04 14.3 4 0.3 93 94.5 0.162 1.02 14.0 5 0.1 91 86.3 0.152 0.95 14.6 6 0.2 89 91.4 0.160 1.03 13.7 7 0.3 90 94.4 0.161 1.04 13.5 8 0.15 91 83.2 0.148 0.92 14.8 5 9 0.2 91 90.6 0.158 1.00 14.3 10 0.35 89 93.8 0.163 1.05 13.7 S.E.M <2.0 <0.003 <0.02 <0.3 6 Significance DL-MET vs. MHA-CA * * N.S N.S L-MET vs. MHA-CA N.S N.S N.S N.S DL-MET vs. L-MET N.S N.S N.S N.S Among Levels *** *** *** ** Among Sources N.S N.S N.S N.S Source*level N.S * N.S N.S Basal vs. avg. (added) *** *** *** *** 1 TGC=thermal unit growth coefficient, %TGC= 100 x (FBW1/3-IBW1/3)/∑(Temp (◦C) x Number of days). efficiency= gain: dry matter feed intake. 3 Viscerosmatic index = 100 x viscera weight (g)/ whole body weight (g). 4 Hepatosomatic index=100 x liver weight (g)/ whole body weight (g). 5 Inclusion of MHA as a methionine equivalent 6Significance indicated by *, ** and *** at the p<0.05, 0.01 and 0.001 level; N.S = not statistically significant (p≥0.05). 2 Feed 64 HSI4 1.20 1.08 1.00 1.05 1.08 1.01 1.01 1.10 1.06 0.97 <0.04 N.S N.S N.S N.S N.S N.S ** Table 3.7 Proximate composition of whole carcass of rainbow trout in response to being fed increasing equimolar levels of methionine from various sources for 12 weeks, expressed on a wet weight basis. Diet Dietary Moisture Crude Lipid Ash GE 1 treatment % protein % % % kJ/g 1 Basal 69.4 14.7 13.8 2.3 9.0 2 DL-Met-1 68.1 15.8 13.6 2.6 9.2 3 DL-Met-2 70.2 15.7 11.9 2.3 8.4 4 DL-Met-3 70.3 16.1 11.6 2.6 8.4 5 L-Met -1 68.8 15.7 13.5 2.4 9.0 6 L-Met-2 70.2 16.0 11.6 2.6 8.3 7 L-Met-3 70.1 16.5 11.7 2.5 8.3 8 MHA-Ca-1 68.8 15.4 13.4 2.6 8.8 9 MHA-Ca-2 69.0 15.9 12.9 2.4 8.8 10 MHA-Ca-3 69.8 16.2 12.1 2.4 8.7 S.E.M <0.5 <0.12 <0.4 <0.2 <0.2 Significance2 DL-MET vs. MHA-CA N.S N.S * N.S N.S L-MET vs. MHA-CA N.S N.S * N.S N.S DL-MET vs. L-MET N.S N.S N.S N.S N.S Among Levels *** *** *** N.S *** Among Sources N.S N.S *** N.S N.S Product*level N.S N.S *** N.S N.S Basal vs. avg. (added) N.S *** *** N.S * 1 Dietary treatment=DL-Met, L-Met and MHA-Ca based on equimolar amount of methionine/methionine equivalent (MHA) for each level. 2 Significance indicated by *, ** and *** at the p<0.05, 0.01 and 0.001 level; N.S = not statistically significant (p≥0.05). 65 Table 3.8 Retained nitrogen (RN), recovered energy (RE), nitrogen retention efficiency (NRE), and energy retention efficiency (ERE) of rainbow trout in response to being fed increasing equimolar levels of methionine from various sources for 12 weeks. Diet Dietary RN RE NRE ERE treatment1 g/fish kJ/fish %IN %IE 1 Basal 1.8 765.1 30.5 35.3 2 DL-Met-1 2.2 868.2 37.2 40.1 3 DL-Met-2 2.4 850.3 40.1 39.6 4 DL-Met-3 2.4 839.3 40.9 38.1 5 L-Met -1 2.2 844.4 36.9 40.3 6 L-Met-2 2.4 799.8 41.9 38.2 7 L-Met-3 2.5 824.4 42.6 38.4 8 MHA-Ca-1 2.1 791.4 35.2 37.4 9 MHA-Ca-2 2.3 849.2 39.1 38.7 10 MHA-Ca-3 2.5 804.1 41.6 37.2 S.E.M <0.06 <19.9 <0.8 <0.9 Significance3 DL-MET vs. MHA-CA N.S ** N.S * L-MET vs. MHA-CA N.S N.S * * DL-MET vs. L-MET N.S * N.S N.S Among Levels *** N.S *** * Among Products N.S * * * Product*level N.S N.S N.S N.S Basal vs. avg. (added) *** ** *** ** 1 Dietary treatment=DL-Met, L-Met and MHA-Ca based on equimolar amount of methionine/methionine equivalent (MHA) for each level. 2 Significance indicated by *, ** and *** at the p<0.05, 0.01 and 0.001 level; N.S = not statistically significant (p≥0.05). 66 3.3.5 Relative Bioavailability Using polynomial contrasts a linear response of weight gain (p<0.001), TGC (p<0.01), and retained nitrogen content (p<0.001) were observed regardless of methionine source when including three levels of methionine supplementation. In contrast both linear (p<0.001) and quadratic responses (p<0.05) were observed when including four levels of methionine supplementation. The inability to compare steepness-coefficients between linear and quadratic responses requires the three methionine level linear model to be used over the four methionine level model, which contains both significant linear and quadratic responses. Figures 3.5, 3.6 and 3.7 illustrate the linear responses of live weight gain, TGC and retained nitrogen content of experimental fish in response to being fed diets containing graded levels of three sources of supplemental methionine. Table 3.9 displays the calculated relative bioavailability values of L-methionine and MHA-Ca compared to DL-methionine. Weight gain, growth rate, and retained nitrogen were regressed against source inclusion level and slope was calculated for each of the synthetic sources of methionine to calculate relative bioavailability according to the method of Littell et al. (1997). Significant differences in slopes, and therefore bioavailability, were tested by comparing the interaction effect of level and source of supplemental methionine on the weight gain, growth rate and retained nitrogen. MHA-Ca was found to be less bioavailable (p<0.05) than DL-Met based upon weight gain, growth rate and retained nitrogen, with calculated relative bioavailability values of 69, 60 and 73% respectively. Comparing L-methionine to DL-methionine, relative bioavailability values of L-Met were 82, 78 and 95% respectively. However bioavailability of L-Methionine was not significantly different from DL-methionine. 67 Figure 3.5 Weight gain of rainbow trout in response to being fed increasing equimolar levels of methionine from DL-Met (■), L-Met (▲), and MHA-Ca (▼). 105 DL-Met L-Met MHA-CA Gain (g/fish) 100 95 90 85 80 75 70 0.5 0.6 0.7 Total Methionine 1 (%) 1 Percent methionine supplemented to basal diet from varying synthetic sources, MHA is considered a methionine equivalent and added in equimolar amounts to that of DL and L methionine. Linear equation for determining bioavailability: Y = 78.6 + 81.7xDL-met +66.6xL-Met+56.2xMHA-Ca. Figure 3.6 Thermal-unit growth coefficient (TGC) of rainbow trout in response to being fed diets containing increasing equimolar levels of methionine from DL-Met (■), L-Met (▲), and MHA-Ca (▼). 0.175 DL-Met L-Met MHA-CA 0.170 TGC % 0.165 0.160 0.155 0.150 0.145 0.140 0.135 0.5 0.6 0.7 1 Total Methionine (%) 1 Percent methionine supplemented to basal diet from varying synthetic sources, MHA is considered a methionine equivalent and added in equimolar amounts to that of DL and L methionine. Linear equation for determining bioavailability: Y = 0.144 + 0.103xDL-met +0.080xL-Met+0.062xMHA-Ca. 68 Retained Nitrogen (g/fish) Figure 3.7 Retained nitrogen (g/fish) of rainbow trout in response to being fed diets containing increasing equimolar levels of DL-Met (■), L-Met (▲), and MHA-Ca (▼). 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 0.5 DL-Met L-Met MHA-Ca 0.6 0.7 Total Methionine 1 (%) 1 Percent methionine supplemented to basal diet from varying synthetic sources, MHA is considered a methionine equivalent and added in equimolar amounts to that of DL and L methionine. Linear equation for determining bioavailability: Y = 1.8 + 2.2xDL-met +2.1xL-Met+1.6xMHA-Ca. Table 3.9 Relative bioavailability of L-Met and MHA-Ca compared to the standard DL-Met based on weight gain, growth rate and retained nitrogen values. Parameters DL-Met L-Met MHA-Ca Weight gain (g/fish) 100a 82ab 69b a ab Thermal growth coefficient (%) 100 78 60b Retained N (g/fish) 100a 95ab 73b a,b Differences of letters between a row letters significant differences (p<0.05) 69 3.4 Discussion Relative bioavailability of two sources of synthetic methionine, L-methionine and MHA-CA, compared to DL-methionine in rainbow trout was assessed in this study. Increasing supplementation level resulted in increases in weight gain, growth rate (TGC), feed efficiency, retained nitrogen and retained nitrogen efficiency (p<0.05) regardless of source of methionine. The increased performance of supplemented fish and the associated increase in performance associated with increasing levels of synthetic methionine suggests that all three methionine products were able to be utilized by the fish in order to at least partially fulfill their requirements for methionine. The ability of DL-methionine, Lmethionine and MHA-Ca to help fulfill the dietary methionine requirement of an animal is established (Keembiyehetty and Gatlin, 1995, 1997, Cheng et al., 2003, Goff and Gatlin, 2004, and Kelly et al., 2006), supporting the notion that both synthetic methionine and MHA can be used as a source of methionine for fish. Body composition, specifically crude protein, moisture and lipid content of carcasses, was significantly affected by methionine inclusion level, regardless of source. Fish fed the basal diet, containing the lowest level of methionine, had the lowest crude protein content, with increasing levels of supplementation resulting in progressively higher carcass crude protein content. As the basal diet was formulated to be limiting in methionine, the increase in carcass crude protein which resulted from increasing levels of dietary methionine was expected (Simmons et al., 1999; Goff & Gatlin, 2004; Kim et al., 1992). The opposite was true for carcass lipid content, which was highest in fish which received the basal diet, and decreased in response to being fed diets containing increasing levels of dietary methionine. These findings are supported by those of Dumas et al., (2007) which describes the positive relationship between moisture and crude protein carcass content, and the inverse relationship between lipid and moisture carcass content. Significant differences in lipid content of carcasses were observed 70 between sources of methionine. Fish fed MHA-Ca-containing diets displayed higher carcass lipid content than those fed diets containing DL-methionine or L-methionine. The apparent lower bioavailability of MHA-Ca compared to DL-methionine may explain the higher lipid content of fish fed MHA-Ca as these fish may not have been able to utilize MHA-Ca as effectively for protein deposition as those fed DLmethionine. However, the crude protein and moisture contents of carcasses were not significantly different between DL-methionine and MHA-Ca in this study. The basal diet used in this study contained 0.5% methionine with three equimolar product inclusion levels of 0.1, 0.2 and 0.3% of the diet, resulting in total methionine content of the diets being 0.6, 0.7 and 0.8% for each source of methionine. Suggested total methionine requirement of rainbow trout is 0.7% of the diet (NRC, 2011), which corresponds with the second (0.2%) product inclusion level used in this study. In figures 3.2, 3.3 and 3.4 weight gain, growth rate, and retained nitrogen were regressed against total dietary inclusion level of methionine which ranged from 0.5% to 0.8%. In these figures a departure from linearity occurs for two methionine sources, DL-methionine and L-methionine, above the second inclusion level (0.2%). Additional supplementation of DL-methionine or L-methionine above the second inclusion level did not result in increased weight gain, growth rate or carcass retained nitrogen. When weight gain, growth rate and retained nitrogen were regressed against total methionine levels up to 0.7%, a linear response was evident. Similarly, a deviation from linearity did not occur in the response of MHA-Ca, even at inclusion levels that are above the methionine requirement of rainbow trout as seen in figures 3.2, 3.3, and 3.4. An explanation is the lower bioavailability of this product, and therefore, even at an inclusion level that corresponds with 0.8% total methionine MHA-Ca is not able to meet the 0.7% methionine requirement of the animal. One could predict a possible deviation from linearity, and eventual plateau, at inclusion levels slightly higher than those used in this study (>0.3% MHA or 0.8% total methionine). 71 Conditions for a valid slope-ratio assay including, linear response of each nutrient source, common intercepts between standards and tests sources, and response to the blank, the basal diet, is equal to common intercept of standard and test lines were tested and confirmed according to statistical methods proposed by Littell et al. 1997. Once conditions were satisfied linear regression lines with a common intercept were fitted to the data. When calculating relative bioavailability the current study excluded the highest inclusion level of methionine for all sources as these levels corresponded with values exceeding the methionine requirement for rainbow trout and thus caused a curvilinear response. Using linear regression and comparing slopes of regression lines between sources, MHA-Ca was found to be significantly (p<0.05) less available than DL-methionine on an equimolar level based upon weight gain, growth rate (TGC) and retained nitrogen values with relative bioavailability values of 69, 60 and 73% respectively . Major controversy surrounds the relative bioavailability of MHA products compared to DL-methionine. A large meta-analysis of data from poultry suggests a relative bioavailability of MHA to DL-methionine ranging from 75-80% based upon equimolar inclusion levels (Jansman et al., 2003; Baker, 2006; Sauer et al., 2008). However, a number of studies in poultry (Richards et al., 2005; Dibner and Knight, 1984; Romer & Abel, 1999) suggest equal bioavailability between MHA and DL-methionine. Differences in experimental design, statistical model, inclusion levels of product, animals used and type of comparison (equimolar or weight-to-weight) vary between studies. The differences in these factors result in difficulties when comparing bioavailability results between studies. However, the majority of studies, including this study and those conducted on other fish species conclude that MHA is significantly less available then DL-Methionine (Keembiyehetty and Gatlin, 1995, 1997; Cheng et al., 2003; Goff and Gatlin, 2004; Kelly et al., 2006). Differences in bioavailability between these two products may also be due to differences in absorption across the brush border membrane (Drew et al., 2003). Between 10-20% of dietary MHA remained in the distal section of the small intestine of broiler chickens compared to 4-5% for DL-methionine (Esteve-Garcia and Austic, 1993; Lingens and Molnar, 72 1996; Maenz and Engele-Schaan, 1996a). These findings suggest that MHA is absorbed slower, and thus remains in the lumen longer, than methionine. This slower absorption may be due to differences in absorption mechanisms. D and L-methionine are actively transported across the cell membrane by system B amino acid transporter, while MHA is transported passively by an H+ dependent transporter (Maenz and Engele-Schaan, 1996b). In addition system b amino acid transporter was found to have a higher affinity for L-methionine and higher maximal velocity of transport compared to affinity of the H+ dependent transporter for L-MHA (Maenz and Engele-Schaan, 1996b). In order for an animal to utilize dietary MHA for protein synthesis, MHA must be converted into L-methionine. Therefore, an additional explanation of the apparent lower bioavailability of MHA compared to DL-methionine is the possible inability to completely convert all of the ingested dietary MHA into the biologically active L-methionine isomer. The conversion of MHA to L-methionine occurs in two steps. During the first reaction, dehydrogenation of the alpha-hydroxyl group of MHA forms an alpha-keto analogue of methionine. The alpha-keto analogue of methionine then undergoes a transamination reaction whereby an amino group is transferred to the alpha-carbon resulting in the formation of L-Methionine (Lloyd et al., 1978; Dibner, 2003). It is important to note that the conversion of DL-methionine to L-methionine shares the intermediate, alpha-keto acid of methionine, as the second step in the conversion of MHA to Lmethionine. Therefore, differences in relative bioavailability between DL-methionine and MHA may be due to the slow rate of the first conversion step. Calculated relative bioavailability of L-methionine compared to DL-methionine were 82, 78 and 95% based upon weight gain, growth rate (TGC) and retained nitrogen values, but differences between these values were not statistically significant. As bioavailabilities of these two sources were found to be similar, the D-isomers in DL-methionine appear to be readily converted to the biologically active Lisomers in rainbow trout. Studies have reported that the two isomerases, D-amino acid oxidase and D- 73 aspartate oxidase, the enzymes responsible for converting D-isomers to L-isomers, have been found in significant quantities in fish (Kera et al. 1998; Sarower et al. 2003). 3.5 Conclusion The objective of this study was to compare the relative bioavailability of two sources of methionine, L-methionine and MHA-Ca, to DL-methionine. Diets were received well, with fish fed diets that were supplemented with synthetic methionine, regardless of source, displaying better growth performance than those fed the unsupplemented basal diet. This leads to the conclusion that all sources of synthetic methionine tested in this study were able to be utilized by rainbow trout and help fulfill their methionine requirement. Growth curves of fish followed a linear response up to the highest inclusion level of supplemental methionine whereby a curvilinear response begin to occur which corresponded with the total methionine requirement of rainbow trout beginning to be met. The linear portion of the response curve was used to calculate relative bioavailability between methionine sources On the basis of weight gain, growth rate (TGC), and retained nitrogen, MHA-Ca was found to be statistically less available then DL-methionine on an equimolar basis with relative bioavailability values of 69, 60 and 73% respectively. The finding of lower bioavailability of MHA-Ca compared to DLmethionine in rainbow trout is in agreement with findings of other fish species along with findings in swine and poultry nutrition. Relative bioavailability of L-methionine was also compared to DLmethionine to study the ability of rainbow trout to effectively convert the D-isomers contained in DLmethionine into the biologically active L-isomer. No statistical differences in relative bioavailability were seen between these two products suggesting that the D-isomers contained in DL-methionine are able to be effectively converted into the L-isomer by rainbow trout. This is supported by D-amino acid oxidase and D-aspartate oxidase, the two enzymes responsible for converted D to L isomers being found in abundance in fish (Kera et al. 1998; Sarower et al. 2003). MHA-Ca was less bioavailable than DL- 74 methionine, but rainbow trout were still able to partially fulfill their requirement for methionine through the use of MHA-Ca. An economic analysis of cost of each products would be interesting to see if greater inclusion of MHA-Ca at a rate as to equal the responses seen with DL-methionine would be economically viable. 75 CHAPTER 4 - ASSESSING THE BIOAVAILABILITY OF L-LYSINE SULPHATE COMPARED TO L-LYSINE HCL IN RAINBOW TROUT (ONCORHYNCHUS MYKISS) Abstract A growth trial was conducted to determine relative bioavailability between two commercially available sources of supplemental lysine in rainbow trout. A basal diet, which was formulated to be deficient in lysine while meeting all other known nutritional requirements for rainbow was supplemented with increasing levels (0.1, 0.2, 0.4 & 0.6%) of lysine on an equimolar basis from one of two supplemental lysine sources; L-lysine sulphate (Bio-lys) or L-lysine HCL. Diets were fed in triplicate to groups of rainbow trout (IBW=25±0.6g) for 12 weeks using a pair-feeding protocol. Fish which received diets containing supplemental lysine from either source exhibited improved weight gain (p<0.05), growth rate (p<0.05), retained nitrogen (p<0.05), retained nitrogen efficiency (p<0.05) and feed efficiency (p<0.01) compared to fish which received the unsupplemented basal diet. Similarly increasing levels of lysine supplementation from either source resulted in improved performance of all aforementioned parameters with performance plateauing as total lysine content of diets approached recommended lysine requirements for rainbow trout. Performance parameters specifically, weigh gain (g/fish), growth rate (TGC %) and carcass retained nitrogen (g/fish), were regressed against dietary supplemental lysine inclusion and relative bioavailability compared between sources of synthetic lysine using a linear slope-ratio assay. Comparing sources of lysine supplementation, L-lysine sulphate was found to be 112, 110 and 92% as available as L-lysine HCL based upon weight gain (g/fish), growth rate (TGC%) and retained nitrogen (g/fish) values respectively. Although numerically L-lysine sulphate appears to be more available then L-lysine HCL, no statistical differences (p>0.05) in bioavailability were 76 observed between these two sources. Therefore, based on equimolar levels of L-lysine supplementation these two sources of supplemental lysine appear to be equally available to rainbow trout. 4.1 Introduction Common feed ingredients such as fish and blood meal contain high levels of the essential amino acid lysine, while plant protein ingredients notably cereal grain by-products contain low levels of lysine. The low levels of lysine in these ingredients often result in lysine being the first limiting amino acid in animal feeds, especially those formulated with high levels of plant proteins (NRC 2011). It has become common practise to supplement these diets with additional lysine (lys) in order to meet the lysine requirements of animals. The D-isomer of lysine is nutritionally inactive as animals lack an aminotransferase for converting the D-isomer into the biologically available L-isomer (D’Mello, 2003a). Therefore, chemical methods of lysine production which would yield a racemic mixture of L and D isomers are not practical. An alternative to chemical synthesis is the production of amino acids through bacterial fermentation, whereby only the biologically active L-isomer of lysine is produced. Conventionally, L-lysine HCL has been added to deficient animal feeds in order to meet their dietary requirements for lysine. Due to changes in post-fermentation processes a new supplemental lysine source, L-lysine sulphate has been developed. During the post-fermentation process the bacterial fermentation broth is separated from the bacterial biomass in L-lysine- HCL while the fermentation broth is not separated and is found as dried biomass containing L-lysine sulphate (Schutte and Pack 1994). In the feed industry, lack of accurate information on both the amino acid content of certain ingredients and the availability of amino acids in feed additives have led to increased production costs as feed formulators are forced to implement amino acid safety margins (Bureau, 2008). As two sources of L-lysine are now commercially available, the ability of L-lysine sulphate to provide utilizable lysine in 77 comparison to L-lysine HCL, or their relative bioavailability, is of interest. Bioavailability is defined as the extent to which an ingested nutrient is digested and absorbed in a form that can be utilized by the animal (Batterham, 1992; Lewis and Bayley, 1995). Bioavailability of ingredients or supplemental products, such as L-lysine sulphate and L-lysine HCL, can be compared in order to understand their relative ability to supply a nutrient. Slope-ratio assays are a direct and practical method which allow researchers to assess the bioavailability of individual amino acids, and have been used extensively with farmed animals (Lewis and Bayley, 1995). In a slope-ratio assay, a basal diet is formulated to be highly deficient in the test nutrient while meeting all other known nutritional requirements of the animal. To this basal diet, graded levels of the test ingredient are added and fed to animals whereby growth and other response variables such as body composition are measured in response to the increasing level of the test ingredient. Depending on the data set, linear or non-linear regression may be applied, with animal performance being regressed against dietary level of the test ingredient. Bioavailability is then calculated by comparing the slope of the test nutrient regression line to the slope of a regression line representing the response to graded levels of a standard source of the test nutrient. Prior to a sloperatio assay being fit to the data three conditions must be satisfied; all regression lines have a common intercept at the Y-axis, linearity of response (linear vs. non-linear) and the common intercept shared between the basal diet and the regression lines (Finney, 1971; Littell et al., 1997). Linear slope-ratio assays have been used to determine bioavailability of amino acids in proteinbound ingredients as well as in sources of free amino acids in a variety of animals including; poultry (Boebel and Baker, 1982; Wang et al., 2007) and swine (Yi et al., 2006; Smiricky-Tjardes et al., 2004; Batterham et al., 1979), along with fish species including rainbow trout and hybrid striped bass (Li et al., 2009; El-Haroun and Bureau, 2007; Rodehutscord et al., 2000). Limited research exists on the relative bioavailability between sources of supplemental lysine, in particular that of L-Lysine sulphate to L-lysine HCL, in commercially relevant fish species. The objective of this study was to determine the relative 78 bioavailability of L-lysine sulphate compared to L-lysine HCL in rainbow trout, a commercially relevant species in Ontario. Bioavailability was compared through the analysis of growth performance and nutrient retention criteria using a linear slope-ratio assay following the method proposed by Littell et al. (1997). 4.2 Methods 4.2.1 Experimental Diet Formulation and Preparation In total, nine (9) diets were included in this experiment; one basal (control) diet and eight diets consisting of four increasing equimolar levels of L-lysine from each of the two test products (L-lysine HCL and L-lysine sulphate). A master batch of the basal diet was produced and formulated to be deficient in lysine (1.6% digestible lys) yet meet all other known nutritional requirements for rainbow trout. From the master batch, nine isonitrogenous (41% CP, 38% DP) and isoenergetic (18 kJ/g DE) test diets were created. Eight diets were created by adding one of the two supplemental sources of lysine at four increasing equimolar levels of L-lysine to equal aliquots of the master batch (Table 4.1). Since L-lysine HCL and L-lysine sulphate contain different concentrations of L-lysine the, diets were formulated to contain equimolar amounts of L-lysine between the two sources (Table 4.2). The analyzed essential amino acid composition of the test diets are presented in Table 4.3. All diets were formulated to meet all known nutrient requirements for rainbow trout except lysine according to recommendations of the NRC (2011). Proximate composition of all nine experimental diets are presented in table 4.4. Formulated and analyzed levels of total lysine and free lysine of experimental diets are found in table 4.5. Diet preparation, including mixing, pelleting, drying and storage followed the same procedure and used the same equipment as outlined in section 3.2.2. 79 Table 4.1 Ingredient composition of experimental diets. Ingredient (%) Fish meal Corn gluten meal Soy protein conc. 300 Soybean meal Starch, gel. Wheat gluten Wheat middlings Fish oil Canola oil Vitamin premix1 Ascorbic acid Amino acid premix2 L-Lys sulphate 3 L-Lys-HCL3 Choline chloride Mineral premix4 Ca(H2PO4)2 Arbocel (cellulose) Total 1 14 3 6 9 10 20 9 12 5.5 1 0.03 0.6 0.345 1 2.7 4.4 100 2 14 3 6 9 10 20 9 12 5.5 1 0.03 0.6 0.197 0.345 1 2.7 4.4 100 Diet (% as is ) 4 5 6 14 14 14 3 3 3 6 6 6 9 9 9 10 10 10 20 20 20 9 9 9 12 12 12 5.5 5.5 5.5 1 1 1 0.03 0.03 0.03 0.6 0.6 0.6 0.789 1.183 0.127 0.345 0.345 0.345 1 1 1 2.7 2.7 2.7 4.4 4.4 4.4 100 100 100 3 14 3 6 9 10 20 9 12 5.5 1 0.03 0.6 0.394 0.345 1 2.7 4.4 100 1Provides 7 14 3 6 9 10 20 9 12 5.5 1 0.03 0.6 0.254 0.345 1 2.7 4.4 100 8 14 3 6 9 10 20 9 12 5.5 1 0.03 0.6 0.508 0.345 1 2.7 4.4 100 9 14 3 6 9 10 20 9 12 5.5 1 0.03 0.6 0.761 0.345 1 2.7 4.4 100 per kg of diet: Retinyl acetate (vit. A), 75mg; Cholescalciferol (vit. D), 60mg; dl-a-tocopherol-acetate (vit. E), 300mg; Menadione Na-bisulfate (vit. K), (1.5mg; Cyanocobalamine (vit. B12), 30mg; Ascorbic acid monophosphate, 300mg; Biotin, 210mg; choline chloride (chloride, 50%), 15mg; D-calcium pantothenate, 32.6mg; pyrodpxone-HCL (vit. B6), 7.5mg; Riboflavin (vit. B2), 9mg; Thiamin-HCL (vit. B1), 1.5mg; Caro-Pink (Astaxanthin), 500mg. 2 Provides per kg: Methionine, 900mg; Tryptophan, 300mg; Threonine, 1200 mg; Isoleucine, 600 mg; Histidine, 3000mg 3 Test products were added in equimolar amounts with an L-Lys content of 50.7% for L-lysine sulphate and 78.8% for L-Lys HCl.,. 4 Provides per kg: sodium chloride (NaCl, 39% Na, 61% Cl), 3077mg; potassium iodide (KI, 24% K, 76%I), 10.5mg; ferrous sulphate (FeSO4-H20, 20% Fe),65mg; manganese sulphate (MnSO4, 36% Mn), 88.9mg; zinc sulphate (ZnSO4-H2O, 40% Zn), 150mg; copper sulphate (CuSO4-H20, 25% Cu), 28mg; yttrium oxide, 100mg. 80 Table 4.2 Experimental design- addition of L-lysine from supplemental L-lysine sources. Addition of L-Lys Addition of test source2 Lys Source from test product (% of product) Diet (%) 1 Basal1 2 L-Lys Sulphate 0.1 0.197 3 L-Lys Sulphate 0.2 0.394 4 L-Lys Sulphate 0.4 0.789 5 L-Lys Sulphate 0.6 1.183 6 L-Lys HCl 0.1 0.127 7 L-Lys HCl 0.2 0.254 8 L-Lys HCl 0.4 0.508 9 L-Lys HCl 0.6 0.761 1 Lys deficient diet: 1.6% lysine on a dry matter basis. Supplemental lysine sources were added in equimolar amounts, with an L-lysine content of 50.7% for L-lysine sulphate and 78.8% for L-Lys HCl. 2 Table 4.3 Analysed essential amino acid composition of experimental diets (% dry matter). Diet1 LYS MET CYS THR TRP ARG ILE LEU VAL 1 Basal 1.70 0.81 0.63 1.40 0.45 2.04 1.60 3.09 1.74 2 L-lys sulphate -1 1.84 0.88 0.64 1.43 0.44 2.07 1.63 3.01 1.77 3 L-lys sulphate -2 1.96 0.83 0.63 1.43 0.43 2.05 1.65 3.03 1.79 4 L-lys sulphate -3 2.14 0.85 0.63 1.41 0.46 2.06 1.65 3.02 1.78 5 L-lys sulphate -4 2.36 0.81 0.62 1.39 0.45 2.06 1.58 2.96 1.72 6 L-lys HCl -1 1.84 0.84 0.62 1.39 0.44 2.04 1.63 2.96 1.75 7 L-lys HCl -2 2.01 0.83 0.65 1.45 0.43 2.09 1.64 3.12 1.79 8 L-lys HCl -3 2.21 0.82 0.64 1.41 0.44 2.09 1.63 3.03 1.78 9 L-lys HCl -4 2.35 0.81 0.64 1.43 0.45 2.14 1.66 3.05 1.80 Requirements: NRC 2.4 0.7 0.4 1.1 0.3 1.5 1.1 1.5 1.2 (2011) 1 Dietary HIS PHE 1.20 1.95 1.24 1.95 1.25 1.97 1.24 1.95 1.21 1.91 1.22 1.91 1.27 1.99 1.23 1.97 1.24 1.97 0.8 0.9 treatments= L-lysine sulphate graded levels 1-4, L-lys HCl graded levels 1-4 based upon equilmolar levels of lysine for each level. Values presented as percentage of diet on a dry-matter basis. 81 Table 4.4 Proximate composition of experimental diets (dry matter basis). Diet 1 2 3 4 5 6 Crude protein, % 42.0 42.1 41.8 42.5 42.1 41.4 Lipid, % 20.8 21.2 21.0 20.9 20.8 20.9 Ash, % 7.0 7.2 7.1 7.0 7.0 7.1 GE kJ/g 23.6 23.9 24.0 23.9 23.8 23.7 7 41.9 21.2 7.1 23.8 8 42.1 20.8 7.1 23.9 9 42.2 20.8 7.0 23.8 Table 4.5 Formulated and analyzed total lysine and free (supplemental) lysine of experimental diets (dry matter basis). Formulated levels Analyzed levels Total lys % Free lys1 % Total lys % Free lys1 % Diet 1 Basal 1.6 1.70 0.03 2 L-Lys sulphate -1 1.7 0.1 1.84 0.14 3 L-Lys sulphate -2 1.8 0.2 1.96 0.24 4 L-Lys sulphate -3 2.0 0.4 2.14 0.43 5 L-Lys sulphate -3 2.2 0.6 2.36 0.63 6 L-Lys HCl -1 1.7 0.1 1.84 0.15 7 L-Lys HCl -2 1.8 0.2 2.01 0.27 8 L-Lys HCl -3 2.0 0.4 2.21 0.47 9 L-Lys HCl -4 2.2 0.6 2.35 0.68 1 Products L-lys sulphate and L-lys HCl contain 50.7% and 78.8% L-lys respectively. 82 4.2.2 Fish, Feeding and Husbandry Rainbow trout fingerlings, at an initial average body weight of 10.7g, were obtained from Alma Aquaculture Research Station (Alma, Ontario). Fish were reared at the Fish Nutrition Research Lab at the University of Guelph until the start of the trial. The juvenile fish (average initial weight = 25.0±0.6g) were stocked into 27 (60L) fiberglass tanks with a stocking density of 15 fish per tank. Fish husbandry conditions along with the pair-feeding protocol followed the same procedures as described in section 3.2.3. 4.2.3 Chemical Analysis Initial and final carcass samples along with analysis of major feed ingredients, diets and carcass samples followed the procedures detailed in section 3.2.4. 4.2.4 Calculations and Statistical Analysis Calculations and statistical analysis of all data followed the procedure outlined in section 3.2.5 with the exception of the linear model for calculating relative bioavailability which incorporated two sources instead of three as described in section 3.2.5. Whereby: y = a + b1x1 +b2x2 Where y = performance criterion a = intercept b1 = regression coefficient for product x1(L-lys HCL) b2 = regression coefficient of x2 (L-lys sulphate) x1, x2, = dietary lysine level from L-lys HCL and L-lys sulphate Biological efficacy for product x2 can be calculated by b2/b1 83 4.3 Results 4.3.1 Mortalities Over the course of the 84-day trial, two fish exhibited signs of bacterial infection, these fish were removed from their respective experimental tanks and euthanized using an overdose of MS-222. No other mortalities or fish illness occurred in the remainder of the trial. 4.3.2 Growth Performance Figure 4.1 displays growth curves of experimental fish in response to being fed experimental diets containing increasing levels of supplemental lysine from either L-lysine HCL or L-lysine sulphate over the course of an 84-day growth trial. Figures 4.2, 4.3, and 4.4 illustrate weight gain, growth rate and retained nitrogen values of these fish in response to being fed four increasing equimolar levels of supplemental lysine from either L-lysine HCL or L-lysine sulphate. Growth performance parameters of rainbow trout, represented by weight gain (g/fish), thermalunit growth coefficient (TGC %), and feed efficiency (gain: feed (DM basis)) are presented in table 4.6. Fish which received diets supplemented with lysine, regardless of source, exhibited higher weight gain (p>0.01), growth rate (p<0.05), and feed efficiency (p<0.01) than fish who received the unsupplemented basal diet. Increased fish weight gain (p<0.01), growth rate (p<0.01), and feed efficiency (p<0.01) resulted from increases of supplemental lysine level, regardless of source. When comparing mean response of performance parameters of rainbow trout fed either L-lysine HCL or L-lysine sulphate, no differences in weight gain (p>0.10), growth rate (p>0.10), or feed efficiency (p>0.10) were observed. Similarly no significant interaction effect was present between lysine supplementation level and source of lysine on weight gain (p>0.10), growth rate (p>0.10) and feed efficiency (p>0.10). 84 4.3.3 Proximate Carcass Composition Table 4.7 presents the proximate analysis of pooled carcass samples in response to increasing equimolar inclusion levels of supplemental lysine from either L-lysine HCL or L-lysine sulphate. Fish which received diets supplemented with either L-lysine HCL or L-lysine sulphate exhibited higher levels of carcass moisture (p<0.01) and crude protein content (p<0.01) than those which received the unsupplmented basal diet. In contrast, fish which received the lysine-supplemented diets exhibited lower lipid (p<0.01) and gross energy (p<0.01) carcass content than those fish which received the unsupplmented diet. Fish carcass moisture (p<0.05) and crude protein (p<0.001) content increased with increasing levels of dietary lysine supplementation while lipid (P<0.001) and gross energy (p<0.01) content decreased. Ash content of carcasses was not affected by supplemental lysine inclusion level (p>0.10). Carcass crude protein (p>0.05) and ash (p>0.05) content were not affected by the source of lysine supplementation, while moisture (p<0.001) content of fish fed L-Lys sulphate was higher than those fed diets containing L-Lys HCL. Similarly, fish fed diets supplemented with L-lysine sulphate exhibited lower lipid (p<0.01) and gross energy (p<0.01) carcass content than those fed diets supplemented with L-Lys HCL. Source of lysine supplementation did not affect carcass ash content. Proximate composition of carcasses were not affected by an interaction between source and level of lysine supplementation (p>0.10). 4.3.4 Retained Nutrients and Retention Efficiencies Retained nitrogen (RN), recovered energy (RE), nitrogen retention efficiency (NRE), and energy retention efficiency (ERE), of rainbow trout fed various equimolar levels of supplemental lysine from either L-lysine HCL or L-lysine sulphate are presented in table 4.8. Fish which received diets supplemented with lysine, regardless of source, exhibited higher retained nitrogen (p<0.01) values and 85 greater NRE (p<0.01) than those fed the unsupplemented basal diet. No differences were observed between fish fed diets supplemented with lysine and those receiving the basal diet on the basis of recovered energy (p>0.05) or ERE (p>0.05). Comparing responses of fish to increasing levels of lysine supplementation, both retained nitrogen (p<0.001) and NRE (p<0.001) increased in response to increasing levels of lysine supplementation while recovered energy (p>0.10) and ERE (p>0.10) were not affected. With the exception of ERE (p<0.05), source of lysine supplementation did not significantly affect any of the retained nutrients or retention efficiencies measured (p>0.10). Although no differences in energy retention efficiencies were observed when comparing between diets, comparing the mean of responses based on source of lysine supplementation showed differences between L-lysine HCL and Llysine sulphate (p<0.05). None of the retained nutrient or retention efficiencies measured displayed a source by supplementation level interaction (p>0.10). 4.3.5 Relative Bioavailability Figures 4.5, 4.6 and 4.7 represent the linear response of live weight gain, growth rate (TGC %) and carcass retained nitrogen to increasing equimolar levels of lysine supplementation from L-lysine HCL and L-lysine sulphate. Relative bioavailabilities of L-lysine sulphate compared to L-lysine HCL, based upon live weight gain, growth rate (TGC %) and retained nitrogen, are presented in table 4.9. L-lysine sulphate was found to be 112, 110, and 92% as available as L-lysine HCL based upon weight gain, growth rate and retained nitrogen values, respectively. These values were not significantly different from one another and therefore no differences in bioavailability between sources exist (p>0.10). 86 Figure 4.1 Growth curves of rainbow trout in response to being fed experimental diets containing increasing equimolar levels of L-lysine from two supplemental sources. Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6 Diet 7 Diet 8 Diet 9 160 Live Weight (g/fish) 140 120 100 80 60 40 20 0 28 56 84 Time (days) Diet 1 represents the basal diet which does not contain any supplemental L-lysine, diets 2-5 containing increasing levels of supplemental L-lysine from L-lysine sulphate while diets 6-9 contain increasing levels of L-lysine from L-lysine HCL. 87 Figure 4.2 Weight gain of rainbow trout in response to being fed diets containing increasing equimolar levels of lysine from L-lys sulphate (■) and L-lys HCL (▲). 140 L-Lys Sulphate L-Lys HCl Gain (g/fish) 135 130 125 120 115 110 1.6 1.7 1.8 1.9 2.0 2.1 2.2 Total Lysine1 (%) 1Total dietary lysine, basal diet contains 1.6% lysine with increasing 0.1, 0.2, 0.4 & 0.6% inclusion levels of supplemental lysine. Lines regressed using a 2nd order polynomial function. Figure 4.3 Thermal-unit growth coefficient of rainbow trout in response to being fed diets containing increasing equimolar levels lysine from L-lys sulphate (■) and L-lys HCL (▲). 0.210 L-Lys Sulphate L-Lys HCl 0.205 TGC (%) 0.200 0.195 0.190 0.185 0.180 0.175 0.170 1.6 1.7 1.8 1.9 2.0 2.1 2.2 Total Lysine1 (%) 1Total dietary lysine, basal diet contains 1.6% lysine with increasing 0.1, 0.2, 0.4 & 0.6% inclusion levels of supplemental lysine. Lines regressed using a 2nd order polynomial function. 88 Retained Nitrogen (g/fish) Figure 4.4 Retained nitrogen content (g/fish) of rainbow trout in response to being fed diets containing increasing equimolar levels of lysine from L-lys sulphate (■) and L-Lys HCL (▲). 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 1.6 L-Lys Sulphate L-Lys HCl 1.7 1.8 1.9 2.0 2.1 2.2 Total Lysine1 (%) 1Total dietary lysine, basal diet contains 1.6% lysine with increasing 0.1, 0.2, 0.4 & 0.6% inclusion levels of supplemental lysine. Lines regressed using a 2nd order polynomial function. 89 Table 4.6 Performance of rainbow trout in response to being fed diets containing increasing equimolar levels of L-lysine from two supplemental sources. Inclusion level Feed intake Weight gain TGC2 % Feed 1 L-lysine % (DM basis) g/fish efficiency3 Diet g/fish gain:FI 1 – Basal 0.0 113 119 0.185 1.05 2 – L-lys sulphate 0.1 112 120 0.186 1.07 3 - L-lys sulphate 0.2 112 125 0.190 1.11 4 - L-lys sulphate 0.4 112 129 0.193 1.14 5 - L-lys sulphate 0.6 112 127 0.193 1.13 6 – L-lys HCL 0.1 112 119 0.184 1.06 7 - L-lys HCL 0.2 113 124 0.190 1.10 8 - L-lys HCL 0.4 112 128 0.193 1.14 9 - L-lys HCL 0.6 112 129 0.195 1.15 S.E.M 1.98 0.002 0.018 Significance4 Among Sources N.S N.S N.S Among Levels ** ** ** Sources*Level N.S N.S N.S Basal vs. avg. ** * ** (added) 1Supplmental L-lysine in equimolar amounts from either L-lysine sulphate or L-lysine HCL unit growth coefficient, %TGC= 100 x (FBW1/3-IBW1/3)/∑(Temp (◦C) x Number of days). 3Feed efficiency= gain: dry matter feed intake. 4 Significance indicated by *, ** and *** at the p<0.05, 0.01 and 0.001 level; N.S = not statistically significant (p≥0.05) 2TGC=thermal 90 Table 4.7 Proximate composition of whole carcasses of rainbow trout in response to being fed diets containing increasing equimolar levels of L-lysine from two supplemental sources, on a wet weight basis. Inclusion level Moisture Crude Lipid Ash Diet L-lysine1 % % protein % % % 1 – Basal 0.0 67.6 15.5 14.6 2.9 2 – L-lys sulphate 0.1 69.1 15.4 13.3 2.5 3 – L-lys sulphate 0.2 68.5 15.6 13.8 2.6 4 – L-lys sulphate 0.4 69.9 16.2 12.2 2.6 5 – L-lys sulphate 0.6 69.3 16.4 12.2 2.6 6 L-lys HCL 0.1 67.9 15.5 14.5 2.7 7 L-lys HCL 0.2 67.8 15.8 14.1 2.7 8 L-lys-HCL 0.4 68.5 16.2 13.5 2.6 9 L-lys-HCL 0.6 68.9 16.7 12.4 2.8 S.E.M 0.35 0.12 0.34 0.10 Significance2 Among Sources *** N.S ** N.S Among Levels * *** *** N.S Source*Level N.S N.S N.S N.S Basal vs. avg. (added) ** ** ** * 1Supplmental 2 Significance L-lysine in equimolar amounts from either L-lysine sulphate or L-lysine HCL indicated by *, ** and *** at the p<0.05, 0.01 and 0.001 level; N.S = not statistically significant (p≥0.05). 91 GE kJ/g 9.4 8.9 9.1 8.6 8.7 9.4 9.4 9.2 8.8 0.13 ** ** N.S ** Table 4.8 Retained nitrogen (RN), recovered energy (RE), nitrogen retention efficiency (NRE), and energy retention efficiency (ERE) of rainbow trout in response to being fed diets containing increasing equimolar levels of L-lysine from two supplemental sources. Inclusion level RN RE RNE ERE 1 Diet L-lysine % g/fish kJ/fish % IN %IE 1 – Basal 0.0 2.9 1193 38.7 44.8 2 – L-lys sulphate 0.1 3.0 1140 39.1 42.5 3 – L-lys sulphate 0.2 3.1 1216 41.5 45.2 4 – L-lys sulphate 0.4 3.3 1167 43.8 43.4 5 – L-lys sulphate 0.6 3.4 1178 44.6 44.1 6 L-lys HCL 0.1 2.9 1205 39.6 45.2 7 L-lys HCL 0.2 3.2 1227 41.6 45.8 8 L-lys-HCL 0.4 3.4 1246 44.3 46.7 9 L-lys-HCL 0.6 3.5 1188 46.0 44.4 S.E.M 0.06 28.0 0.72 1.04 Significance2 Among Sources N.S N.S N.S * Among Levels *** N.S *** N.S Source*Level N.S N.S N.S N.S Basal vs. avg. (added) ** N.S *** N.S 1Supplmental 2 Significance L-lysine in equimolar amounts from either L-lysine sulphate or L-lysine HCL indicated by *, ** and *** at the p<0.05, 0.01 and 0.001 level; N.S = not statistically significant (p≥0.05). 92 Figure 4.5 Weight gain of rainbow trout in response to being fed diets containing increasing equimolar levels of L-lysine from L-lys sulphate (■) and L-lys HCL (▲). 140 L-Lys Sulphate L-Lys HCl Gain (g/fish) 135 130 125 120 115 110 1.6 1.7 1.8 1.9 2.0 Total Lysine1(%) 1Total dietary lysine, basal diet contains 1.6% lysine with increasing 0.1, 0.2 & 0.4% inclusion levels of supplemental lysine. Linear equation for determining bioavailability: Y = 118.1 + 24.6L-Lys HCL +27.6x L-Lys sulphate. Figure 4.6 Thermal-unit growth coefficient (TGC) of rainbow trout in response to being fed diets containing increasing levels of L-lysine from L-lys Sulphate (■) and L-lys HCL (▲). 0.200 L-Lys Sulphate L-Lys HCl TGC (%) 0.195 0.190 0.185 0.180 0.175 0.170 1.6 1.7 1.8 1.9 2.0 Total Lysine1 (%) 1Total dietary lysine, basal diet contains 1.6% lysine with increasing 0.1, 0.2 & 0.4% inclusion levels of supplemental lysine. Linear equation for determining bioavailability: Y = 0.184 + 0.021L-lys HCL +0.023x L-lys sulphate. 93 Retained Nitrogen (g/fish) Figure 4.7 Retained nitrogen (g/fish) of rainbow trout in response to being fed diets containing increasing equimolar levels of L-lysine from L-lys sulphate (■) and L-lys HCL (▲). 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 1.6 L-Lys Sulphate L-Lys HCl 1.7 1.8 1.9 2.0 Total Lysine1 (%) 1Total dietary lysine, basal diet contains 1.6% lysine with increasing 0.1, 0.2 & 0.4% inclusion levels of supplemental lysine. Linear equation for determining bioavailability: Y = 2.9 + 1.2L-lys HCL +1.1x L-lys sulphate. Table 4.9 Relative bioavailability of L-lysine sulphate compared to the standard L-lysine HCL based on weight gain, TGC and retained nitrogen values. Parameters L-lys HCL L-lys Sulphate a Weight gain (g/fish) 100 112a a Thermal growth coefficient (TGC) 100 110a a Retained N (g/fish) 100 92a a,b Differences of letters between a row letters significant differences (p<0.05) 94 4.4 Discussion Relative bioavailability of L-lysine sulphate to L-lysine HCL, two sources of supplemental lysine, were assessed in this study using the commercially relevant rainbow trout as the model species. Increases in weight gain, feed efficiency and growth rate of rainbow trout in response to increasing levels of dietary lysine supplied from either source of supplemental lysine confirmed that the basal diet was deficient in lysine. Similarly, average performance of fish which received experimental diets containing either source of supplemental lysine exhibited greater weight gain, growth rate, feed efficiency and carcass retained nitrogen than fish which received the unsupplmented basal diet. The increased performance of fish which received diets containing supplemental lysine suggest that rainbow trout are able to effectively utilize lysine from both L-lysine sulphate and L-lysine HCL. In agreement with this study, both L-lysine sulphate and L-lysine HCL have been shown to be effective sources of supplemental lysine in many animals including; swine (Smiricky-Tjardes et al., 2004, Schutte and Pack, 1994, Liu et al., 2007), poultry (Ahmad et al., 2007, Wang et al. 2007), and fish (Rodehutscord et al., 2000). Along with increased performance, level of supplemental lysine also affected body composition, specifically moisture, crude protein and lipid content. As diets were formulated to be limiting in lysine, the observed increased carcass crude protein content which resulted from increasing levels of dietary lysine supplementation was expected (El-Haroun & Bureau, 2007; Encarnação et al., 2004; Cheng et al., 2003; Williams et al., 2001). In contrast, carcass lipid content decreased with increasing levels of lysine supplementation regardless of lysine source, with fish being fed diets highly deficient in lysine having the highest carcass lipid content. According to the work conducted by Dumas et al., (2007), an inverse relationship between lipid and moisture carcass content exists, this is in agreement with the findings in 95 this study along with those by Rodehutscord et al., (2000) whereby carcass lipid content of rainbow trout decreased with increasing dietary lysine supplementation. Certain conditions must be satisfied in order to ensure the validity of a slope-ratio assay and enable a confident estimate of relative bioavailability between nutrients. Conditions for a valid sloperatio assay, such as linearity of the responses, a common intercept between test and standard nutrients, as well as the blank or basal diet sharing a common intercept with the test and standard nutrient were ensured according to the statistical approaches of Littell et al. 1997. The experimental design used in this study included five levels of lysine supplementation, including the basal diet, from each source of supplemental lysine. However, only four levels of lysine supplementation, and their associated response variables, were included in the assay in order to calculate bioavailability between sources of lysine. Justification for the exclusion of the highest level of lysine supplementation in the slope-ratio assay is two-fold. Firstly, to satisfy the condition of linearity of responses and secondly to minimize variation in animal responses by ensuring constant utilization of the limiting essential amino acid. The basal diet used in this study contained 1.6% total dietary lysine which, through the supplementation of either Llysine sulphate or L-lysine HCL, increased to 2.2% total lysine in the diet. Recommendations of the NRC (2011) are that rainbow trout diets should contain 2.4% lysine in order to achieve maximal growth. Figures 4.2-4.4 display weight gain, growth rate and carcass retained nitrogen of fish in response to being fed five graded levels of lysine. When total lysine content of the diet exceeded the fourth level, 2.0% total lysine, the performance response of fish to additional lysine supplementation appeared to plateau. In contrast, figures 4.5-4.7, which regress weight gain, growth rate and carcass retained nitrogen against four levels of lysine supplementation, a clear linear trend is observed for all levels of lysine supplementation. The apparent plateau, which occurs after the fourth level of supplementation, coincides with approximately 80% of the lysine requirement of the animal being met. Therefore, when calculating relative bioavailability between L-lysine sulphate and L-lysine HCL, four levels of lysine 96 supplementation were included, ensuring the constant utilization of lysine and the associated linear response. Using linear regression and comparing slopes of regression lines between sources, L-lysine sulphate was found to 112 , 110 and 92% as available as L-lysine HCL on an equimolar basis of lysine supplementation based on weight gain (g/fish), growth rate (TGC %) and carcass retained nitrogen (g/fish), respectively. Relative bioavailability values between L-lysine sulphate and L-lysine HCL were not statistically different from one another and therefore on an equimolar comparison L-lysine sulphate and L-lysine HCL are equally available to rainbow trout. In agreement with the findings of this study, no differences in relative bioavailability between L-lysine sulphate and L-lysine HCL were found in swine (Smiricky-Tjardes et al., 2004, Schutte and Pack, 1994, Liu et al., 2007), poultry (Ahmad et al., 2007, Wang et al. 2007,), and fish (Rodehutscord et al., 2000). Both L-lysine sulphate and L-lysine HCL are products of bacterial fermentation, although final post-fermentation processing differs between products. The difference in post-fermentation processes results in L-lysine HCL containing 78.8% L-lysine on a weight basis and a hydrochloric salt while L-lysine sulphate contains 50.7% L-lysine (Schutte and Pack, 1994; Smiricky-Tjardes et al., 2004). The dried microbial cells which are present in L-lysine sulphate but not L-lysine HCL contain additional essential amino acids, with individual essential amino acids constituting 0.1% – 2.5% of the microbial biomass (Jackson, 2001). The sum of essential and non-essential amino acids contained in the microbial biomass result in the sulphate in L-lysine sulphate containing an additional 12% digestible nitrogen (Wang et al., 2004). As the bacterial biomass in L-lysine sulphate contains additional nutrients, it is possible that differences in performance of animals fed diets containing L-lysine sulphate and L-lysine HCL may be observed. However, in this study all diets were formulated to meet all known nutritional requirements for rainbow trout with the exception of lysine. The additional amino acids provided by the dried bacterial biomass in L-lysine sulphate did not increase the performance of rainbow trout fed these diets 97 and therefore no differences in bioavailability between L-lysine sulphate and L-lysine HCL were found. Therefore, in agreement with studies on a variety of farmed animals, this study has found that L-lysine sulphate can be substituted on an equimolar L-lysine basis as L-lysine HCL without influencing performance of the animal. 4.5 Conclusion The objective of this study was to compare the relative bioavailabilities between two sources of supplemental lysine, L-lysine sulphate and L-lysine HCL. Both sources are products of bacterial fermentation with differing post fermentation techniques resulting in L-lysine sulphate and L-lysine HCL containing 50.7% and 78.8% L-lysine on a weight basis, respectively. Using a linear slope-ratio model and regressing weight gain (g/fish), growth rate (TGC %) and carcass retained nitrogen (g/fish) against dietary equimolar lysine supplementation, L-lysine sulphate was found to be equally available as L-lysine HCL. 98 CHAPTER 5 GENERAL DISCUSSION Historically, aquaculture feeds have contained high levels of fishmeal. However, modern compound aquaculture feeds are being formulated with lower levels of fishmeal and an increasing diversity of animal and plant protein sources. Improvements in processing techniques have resulted in the reduction of anti-nutrients along with increased concentrations of protein and improved digestibility of many plant proteins. These improvements have resulted in diets being formulated to contain higher levels of these cost-effective plant proteins in lieu of traditional fishmeal and animal protein sources. However, the amino acid profiles of these economic plant proteins are often poor compared to fishmeal and certain animal protein sources (NRC, 2011). In particular, lysine and methionine are often the firstmost and second-most limiting essential amino acids in these type of diets. If the requirement for an essential amino acid is not fulfilled through dietary sources, decreased animal growth in addition to health complications may arise. As a result, crystalline amino acids have been used in the animal feed industry for over 50 years to ensure amino acid requirements are and to allow for use of cost-effective feed ingredients and greater flexibility in feed formulation. With lysine and methionine being commonly the first two limiting essential amino acids in animal feeds, many commercial synthetic amino acid products exist, including DL-methionine and Llysine HCL. However, other sources of these synthetic amino acids, including L-methionine, the calcium salt of the hydroxy analogue calcium salt of methionine (MHA-Ca), and L-lysine sulphate are commercially available. Many studies on a variety of animals including poultry (Lemme et al., 2002; Sauer et al., 2008, Ahmad et al., 2007; Wang et al. 2007) and swine (Kim et al., 2006; Baker, 2006; Sauer et al., 2008; Smiricky-Tjardes et al., 2004; Schutte and Pack, 1994; Liu et al., 2007) have compared the ability of these products to fulfill methionine and lysine requirements of an animal. However, very little 99 has been published in regards to fish species in particular those which are relevant to the aquaculture industry (Rodehutscord et al., 1995 & 2000). In this regard the objective of this study was to compare bioavailability between three sources of synthetic methionine and two sources of supplemental lysine using a slope-ratio assay approach in two separate growth trials using the commercially relevant rainbow trout species. Live weight gain, growth rate expressed as thermal-unit growth coefficient and carcass retained nitrogen values were used as response variables for the assays. Findings of this study indicate a significantly lower methionine availability of MHA-Ca compared to DL-methionine and similarly availability between L-methionine and DL-methionine. The study also indicated that lysine availability was very similar between L-lysine HCL and L-lysine sulphate. With increasing levels of product supplementation, regardless of source, an associated increase in performance parameters of rainbow trout was observed. The increased performance resulting from increased product inclusion leads to the conclusion that sources of synthetic amino acids tested in this study are effectively utilized by the animal and able fulfil their dietary requirement for these essential amino acid. The findings of this study are in agreement with findings in swine (Smiricky-Tjardes et al., 2004; Schutte and Pack, 1994; Liu et al., 2007; Kim et al., 2006; Baker, 2006; Sauer et al., 2008), poultry (Ahmad et al., 2007; Wang et al. 2007; Lemme et al., 2002; Sauer et al., 2008) and fish (Rodehutscord et al., 2000; Keembiyehetty and Gatlin, 1995, 1997; Cheng et al., 2003; Goff & Gatlin, 2004; Kelly et al., 2006). When measuring performance of an animal in response to being fed diets containing an essential amino acid between 0 and 100% of the requirement of the animal for maximum growth, a curvilinear performance response is expected (Baker, 1986). With the exception of MHA-Ca, all sources of synthetic methionine or lysine displayed a performance plateau at the highest inclusion levels of 100 these synthetic sources. For lysine sources a plateau occurred beyond the fourth level of lysine supplementation which contained 2.0% total lysine and met just over 80% of the 2.4% dietary lysine requirement for rainbow trout. In contrast performance of fish fed diets containing graded levels of DLmethionine and L-methionine plateaued when fed diets containing 0.7% total methionine, coinciding with 100% of the 0.7 % methionine requirement of rainbow trout. Instead of the anticipated performance plateau occurring at a level whereby roughly 80% of the methionine requirements of the animal is being met it occurred at a level whereby 100% of the methionine requirement was being met for both DL-methionine and L-methionine while no performance plateau was reached for animals fed diets containing MHA-Ca. As fish fed diets containing 0.8% MHA did not exhibit a plateau in performance, it is possible that the 0.7% methionine requirement of rainbow trout was still not being met by MHA. Further supporting the findings of previous studies that MHA-Ca is less available then DL-methionine on an equimolar basis (Keembiyehetty and Gatlin, 1995, 1997; Cheng et al., 2003; Goff and Gatlin, 2004; Kelly et al., 2006). A well designed slope-ratio assay is an effective method for determining bioavailability of nutrients as the response criteria being measured is the sum of the metabolic costs associated with digestion, absorption and utilization of the test nutrient. Other methods such as digestibility trials are only able to measure one facet of bioavailability and are not able to measure the extent to which a particular nutrient is utilized once it leaves the digestive tract. However, growth trials used for sloperatio assays are intensive in terms of labour, time and cost and sound experimental design must be implemented in order to achieve a valid slope-ratio assay. Major differences in experimental design along with differences in statistical models, dietary levels of the test amino acids, deficiency of the basal diet, along with the reporting of equimolar vs. product based comparison of test ingredients must be 101 improved and standardized between institutions and laboratories in order to have meaningful comparisons of the bioavailability of nutrients between studies. In conclusion, the comparison of bioavailability of methionine or lysine between various synthetic sources improves our understanding of the ability of these sources in providing utilizable forms of their respective essential amino acids to an animal. 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