Download Assessing the Bioavailability of Synthetic Methionine and Lysine

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
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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.
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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.
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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.
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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
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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
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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
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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
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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
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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
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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
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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.
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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.
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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)
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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).
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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. If the relative bioavailability of these
synthetic sources were not investigated and assumed to be 100% available, resulting feeds containing
these supplemental sources of synthetic amino acids may still be deficient in the amino acid being
supplemented for. This is especially relevant to aquaculture as modern feed formulation strategies
include the substitution of fishmeal with economic plant proteins which are characterized by poor
amino acid profiles in particular low levels of both methionine and lysine.
102
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