The Effect of Dietary Nucleotide Supplementation on Growth ...

The Effect of Dietary Nucleotide Supplementation on Growth and Feed Efficiency of Rainbow Trout (Oncorhynchus mykiss) Fed Fish Meal-Free

and Animal Protein-Free Diets

By

Bing Liu

A Thesis

presented to The University of Guelph

In partial fulfilment of requirements

for the degree of Master of Science

in Animal Biosciences

Guelph, Ontario, Canada

© Bing Liu, May, 2016

i

ABSTRACT THE EFFECT OF DIETARY NUCLEOTIDE SUPPLEMENTATION ON GROWTH AND

FEED EFFICIENCY OF RAINBOW TROUT (ONCORHYNCHUS MYKISS) FED FISH MEAL-FREE AND ANIMAL PROTEIN-FREE DIETS

Bing Liu Advisor: University of Guelph, 2016 D. P. Bureau Effects of dietary nucleotide supplementation on growth and feed efficiency in rainbow

trout (initial weight = 25 g/fish) were conducted in a 70-day growth trial. One control diet and

two basal diets (25%, 5% and 0% fish meal, respectively) were formulated using high levels of

highly digestible plant protein ingredients to meet all the known nutrient requirements of

rainbow trout. Two basal diets were supplemented with four increasing levels (0, 8, 16 and 24

ppm, respectively) of nucleotides using a commercial supplement (Laltide®, Lallemand Inc.

Qc, Canada). Nucleotide supplementation levels significantly (p<0.05) affected final body

weight and feed efficiency in rainbow trout fed with the 5% fish meal diet. The results of this

study suggest that dietary nucleotide supplementation may be beneficial to young rainbow

trout fed with low fish meal and high levels of plant protein ingredients. The essentiality of

nucleotides to young rainbow trout deserves to be further examined.

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Acknowledgement

I would first like to express my sincere heartfelt thanks to my advisor Dr. Dominique

P. Bureau, thank you for providing me this valuable opportunity to study in your lab which

has been my most precious memory in my life. Thank you for your insightful suggestion

and constant encouragement on my work during my time in your lab which guided me

towards making improvements not only as a researcher but also as a person. You have

inspired me with your brilliant ideas, passion and wealth of knowledge for your research.

Thank you to my advisory committee, Dr. Niel Karrow and Dr. Ming Fan for your

encouragement and great ideas that you have provided me at the beginning of my master

study, and thank you to Dr. Fan for providing me a great opportunity to attend to your

class.

I would like to thank Owen Skipper-Horton and Guillaume Pfeuti for their help and

advice during my experiment. I couldn’t deal with those physical work and system

problems without your help. Thank you to Christopher David Powell, for his help in data

analysis. Your code and explanation really helped me understand the practical application

of statistics. I would also like to thank Chunfang Wang and Mingfei Zhao for their tireless

help during my experiment. I would like to thank all of my lab mates, the lab is a warm and

family-like place for me. I would especially like to thank a former lab mate of mine,

Patricio Saez Albornoz, for his kind help and explanation throughout the preparation of my

experiment. Also, I would like to thank Laura Schnablegger, from learning and curriculum

support team at the university McLaughlin Library, for her commons and suggestion on my

writing.

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I would like to thank the contributor for the funding of this project, Lallemand Inc.

Thank you to Dr. Mathieu Castex for his valuable discussion with Dr. Bureau at the

beginning of my experiment.

Finally, I would like to thank my family, friends and all the people I love, thank you

for your supporting, understanding and encouraging in the last two years, thank you for

taking accompany with me online during my homesick and confused periods, and thank

you for all the wishes that you sent to me. I would especially thank my mom for giving me

her endless love and teaching me how to be a brave and optimistic person. All of you are

always the main props of mine.

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Table of Contents

CHAPTER 1- GENERAL INTRODUCTION ................................................................... 1

Objective ..................................................................................................................................... 3

CHAPTER 2 - LITERATURE REVIEW ........................................................................... 4

2.1 Nucleotides: Structure ........................................................................................................... 4 2.2 The Functions of NT ............................................................................................................... 6 2.3 Biosynthesis of purine NT ...................................................................................................... 8 2.4 Pathways of pyrimidine NT biosynthesis .............................................................................. 12 2.5 Nucleotides in Nutrition ....................................................................................................... 14 2.6 Effects of dietary NT ............................................................................................................ 18 2.6.1 The effect of dietary NT on gastrointestinal tract ................................................................... 19 2.6.2 The effect of dietary NT on growth performance ................................................................... 20 2.6.3 The effect of dietary NT on immune function ......................................................................... 21 2.6.4 The Effect of Dietary NT on Liver Function .............................................................................. 24 2.7 Evolution of Aquaculture and Aquaculture Feed Formulation .............................................. 24 2.8 Conclusion and perspectives ................................................................................................ 27

CHAPTER 3- THE EFFECT OF DIETARY NUCLEOTIDES SUPPLEMENTATION ON GROWTH AND FEED EFFICIENCY OF RAINBOW TROUT (ONCORHYNCHUS MYKISS) FED FISH MEAL-FREE AND ANIMAL PROTEIN-FREE DIETS ................................................................................................... 29 Abstract .................................................................................................................................... 29 3.2 Methods .............................................................................................................................. 32 3.2.1 Experimental Diet Formulation and Preparation .................................................................... 32 3.2.2 Fish, Feeding and Husbandry .................................................................................................. 36 3.2.3 Chemical Analysis .................................................................................................................... 37 3.2.4 Calculations and Statistical Analysis ........................................................................................ 38 3.3 Results ................................................................................................................................. 40 3.3.1 Growth Performance ............................................................................................................... 40 3.3.2 Carcass Composition ............................................................................................................... 43 3.3.3 Retained Nutrients and Retention Efficiencies ....................................................................... 44 3.4 Discussion ............................................................................................................................ 48 3.5 Conclusion ........................................................................................................................... 50

CHAPTER 4 GENERNAL DISCUSSION ....................................................................... 52

References ............................................................................................................................ 58

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List of Tables

Table 2.1. Types of reactions in the purine biosynthesis de novo pathway ((Henderson &

Paterson, 1973) ..................................................................................................................... 10

Table 2.2 Purine and pyrimidine base content of some aquafeed ingredients (%) (Devresse,

2000) ..................................................................................................................................... 15

Table 2.3 Summary of studies examining the effects of dietary NT in fish ......................... 17

Table 3.1 Ingredient composition of the experimental diets ................................................. 33

Table 3.2 Nucleotide supplementation added level .............................................................. 34

Table 3.3 Total potentially available nucleotides (TPAN) in the Laltide® .......................... 34

Table 3.4 Proximate composition of experimental diets. ..................................................... 35

Table 3.5 Performance of rainbow trout (initial weight = 25 g/fish) in response to being fed

increasing NT content with different fish meal inclusion levels diets for 10 weeks. ........... 45

Table 3.6 Comparison of some parameters of rainbow trout in response to being fed diet 4

with Control diet ................................................................................................................... 45

Table 3.7 Proximate composition of whole carcass of rainbow trout in response to being fed

increasing NT content with different fish meal inclusion levels diets for 10 weeks,

expressed on a wet weight basis. .......................................................................................... 46

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 NT content with different fish meal inclusion levels diets for 10 weeks. ........... 47

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List of Figure

Figure 2.1. Nucleotide structure. .......................................................................................... 5

Figure 2.2. The de novo synthesis of purines (Henderson & Paterson,1973; King, 2016) ..... 9

Figure 2.3. Catabolism and the salvage pathway of purine NT (Henderson &

Paterson,1973; King, 2016) ................................................................................................. 10

Figure 2.4. The de novo pathway of pyrimidine biosynthesis (Henderson & Paterson,1973;

King, 2016) .......................................................................................................................... 13

Figure 2.4 Digestion and absorption of dietary nucleotides (Hess & Greenberg, 2012). .... 15

Figure 3.1 Live body weight (g/fish) of rainbow trout in response to being fed

experimental diets containing increasing NT levels with different fish meal inclusion levels.

............................................................................................................................................. 41

Figure 3.2 Final body weight (g/fish) of rainbow trout in response to being fed


............................................................................................................................................. 41

Figure 3.3 Thermal-‐unit growth coefficient (TGC) of rainbow trout in response to being fed


............................................................................................................................................. 42

1

CHAPTER 1- GENERAL INTRODUCTION

Nucleotides are important metabolites that are involved in almost all cellular processes and

play major roles in structural, metabolic, energetic and regulatory functions (Rudolph 1994). The

physiological functions of these compounds include encoding and deciphering genetic

information, mediating energy metabolism and cell signaling as well as serving as components of

co-enzymes, allosteric effectors and cellular agonists (Carver and Walker, 1995).

Studies have indicated that dietary nucleotide supplementation had positive effect on

various physiological functions in many species, especially during early life stages (Gil, 2002). It

has been hypothesized that during periods of rapid growth or high metabolism, some tissues such

as immune cells and gastrointestinal cells may have high demands for nucleotides. There are

three ways that an animal can obtain the NT: de novo synthesis, the salvage pathway, and

through the diet (Quan et al., 1990). The de novo synthesis and salvage pathways provide

insufficient amounts of nucleotides for the needs of these tissues, which are only partially able to

produce nucleotides or unable to produce them at all (Holen & Jonssona, 2004; Peng et al.,

2013).

Research with various animals has shown that dietary nucleotides can enhance the

proliferation of epithelial cells of the gastrointestinal tract (Sanderson & He, 1994), improve

immune responses (Carver 1994), reduce hepatic lipid accumulation (Carver 1994) and

beneficially modify intestinal microflora (Uauy 1990). Evidence also suggests that the dietary

supply of nucleotides may optimize the function of rapidly dividing tissues (Carver 1994).

Experimental evidence suggests substantial advantages of dietary nucleotides in different

aquatic species, including turbot (Low et al., 2003; Peng et al., 2013), red drum (Li et al 2007a;

Cheng et al., 2011), Coho salmon (Burrells et al., 2001a), Atlantic salmon (Burrells et al.,

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2001b), rainbow trout (Adamek et al., 1996, Burrells et al., 2001a; Tahmasebi-Kohyani et al.,

2011; Mohebbi et al., 2013), common carp (Sakai et al., 2001), grouper (Lin et al., 2009), tilapia

(Ramadan et al., 1991; Ramadan et al., 1994), hybrid striped bass (Li et al, 2004), puffer fish

(Kiyohara et al., 1975), American lobster (Mackie, 1973), Pacific white shrimp (Li et al., 2007b)

and black tiger shrimp (Huu et al., 2012).

Feed for most aquaculture species were in the past formulated with high levels of fish

meal. However, growth of the demand for aquaculture feeds and the limited supply and high

price of fish meal has led aquaculture feed manufacturers to progressively decrease the level of

fish meal in feeds and increase their reliance on more economical plant protein ingredients.

Studies have shown that good growth and feed efficiency can be achieved with feeds based

primarily on plant ingredients. However, performance of fish generally drops significantly with

complete replacement of fish meal by plant ingredients, especially in young, fast-growing,

animals, even if all known nutrient requirements appear to have been met. Fish meal is an

excellent source of digestible essential amino acids, vitamins, minerals as well as of a wide

variety of compounds, including nucleotides (Zinn et al., 2009; Watanabe et al., 1997). In some

markets, processed animal proteins (PAPs) have filled in void left by decreasing fish meal levels

in aquaculture feeds. However, feed safety regulations and consumer concerns and demands are

preventing the use of PAPs in some markets. Most PAPs are also excellent sources of amino

acids, minerals and various compounds, including nucleotides. Conversely, plant protein

ingredients are significantly poorer sources of nucleotides. In this context, the level of

nucleotides in aquaculture feeds has likely decreased significantly and may, in part, explain the

lower performance of fish fed diets with very high levels of plant protein ingredients. It can be

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thus hypothesized that nucleotides could potentially be beneficial essential nutrients for young

fast growing fish.

This thesis examines the effect of dietary nucleotide supplementation on growth

performance of young rainbow trout fed plant-based diets with no or only very low levels of fish

meal.

Objective

The objective of this thesis was to investigate the effects of dietary nucleotides

(nucleosides) on growth and the efficiency of feed, protein and energy utilization in rainbow

trout fed plant-based diets containing very low levels of fish meal or no fish meal.

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CHAPTER 2 - LITERATURE REVIEW

2.1 Nucleotides: Structure

Nucleotides (NT) are low molecular weight molecules consisting of a nitrogenous base, a

pentose sugar, and one to three phosphate groups (Cosgrove, 1998) (Figure 2.1). The

nucleobases are classified as either purines including adenine (A), and guanine (G), or

pyrimidines including cytosine (C), thymine (T), and uracil (U). Pentose sugars include ribose

and deoxyribose. A purine or pyrimidine base attaches a pentose sugar to constitute a nucleoside

(NS), while a phosphate ester of NS is a NT (Carver &Walker, 1995; Li & Gatlin, 2006). NT are

the building blocks for DNA and RNA synthesis.

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Figure 2.1. Nucleotide structure.

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2.2 The Functions of NT

NT can either participate in reactions or synthesize other molecules. Purine and pyrimidine

NT generally have five types of functions. These functions of NT are as: 1. structure units; 2.

energy storage; 3. parts of coenzymes; 4. intermediates in some biosynthetic pathways; 5. part of

intracellular signalling molecules (Henderson & Paterson, 1973).

From the genetic perspective, the key roles of NT are the activated precursors of nucleic

acids. These precursors are connected to each other with a phosphodiester bond between the 3’

carbon of one and the 5’ carbon of the next. The pentose sugars contain five atoms of carbon,

which can be numbered from 1’ through 5’, and are attached to a nitrogenous base at the 1’

carbon position. A phosphate group is attached with the 3’ carbon of the one and 5’carbon of the

next, where the phosphodiester bond is formed. Thus, nucleic acids, are formed when NT are

joined as phosphodiester bonds. As a result, an RNA or DNA strand will have a 3’-hydroxyl end

and a 5’-phosphate or triphosphate end (Guttman, 2013).

NT, nucleoside diphosphates and triphosphates, are the main energy carriers in cells

(Guttman, 2013). Adenosine triphosphate (ATP) takes part in many endergonic metabolic

reactions of animals and plants as an energy source, and experiences change in formation,

consumption and recycling (Wischke et al, 2014). In most reactions, ATP transfers one or two

terminal phosphate group(s) or phosphoryl group(s) to some other molecule, leaving behind a

nucleoside diphosphate (ADP) or a nucleoside monophosphate (AMP) (Guttman, 2013). Some

reactions are shown below:

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NT can also be components of co-enzymes. For example, adenosine along with cysteine

and pantothenate are the precursors of coenzyme A (CoA). Two other important coenzymes,

nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD), are also

synthesized by adenine NT (Cosgrove, 1998; Pesnot et al, 2011; Deluca & Kaplan, 1958).

NT derivatives are integral participants in many biochemical processes in addition to

intercellular base units (Cosgrove, 1998). For example, UDP-glucose participates in the

biosynthesis of glucose (Cosgrove, 1998). During glycogenesis, UDP-glucose is formed from

glucose-1-phosphate with the help of the enzyme UDP-glucose phosphorylase; and the UTP is

converted into PPi. Phosphoglucomutase and UDP-glucose pyrophosphorylase drive the

biosynthesis of nucleotide sugar precursors. These precursors are involved in the biosynthesis of

polysaccharides (Ji et al., 2015).

The last key role of NT is that they can act as signaling molecules. For example, in the

autocrine purinergic signaling systems, pannexin 1 channels can release ATP from cells to drive

the autocrine activation of P2 receptors (Chekeni et al., 2010; Junger, 2011; Idzko et al., 2014).

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Also, the extracellular release of NT is associated with inflammatory disease conditions

(Eltzching et al., 2006; Faigle et al., 2008; Lazarowski, 2012).

2.3 Biosynthesis of purine NT

There are two major pathways for synthesis of purine NT: the de novo pathway and the

salvage pathway. These two pathways for NT synthesis are mostly controlled by the liver

(Carver & Walker, 1995; Cosgrove, 1998; Grimble & Westwood, 2000; Fustin et al, 2012).

Biosynthesis of purine NT by the de novo pathway occurs within the cytosol of mammalian cells

from some precursors and small molecules, such as 5-phosphoribosyl-1-pyrophosphate (PRPP),

THF derivatives, CO2, glutamine, glycine, aspartate and formate (Cosgrove, 1998; Zrenner et al.,

2006; Li & Gatlin, 2006). Purine bases and purine nucleosides participate in the salvage

pathways for the synthesis of NT. The de novo pathway is more complicated and consumes more

energy than the salvage pathway (Cosgrove, 1998; Li & Gatlin, 2006).

Synthesis of inosine monophosphate (IMP) de novo starts with PRPP. Enzymes, ATP,

glutamine, glycine, THF derivatives and aspartate are utilized during the biosynthesis of IMP.

IMP can be converted into either AMP or GMP; the conversion of IMP to AMP needs to be

catalyzed by GTP, while ATP catalyzes the conversion of IMP to GMP. Thus, IMP represents a

branch point for purine biosynthesis (Henderson & Paterson, 1973; Zrenner et al., 2006). The 10

enzymatic reactions of the purine biosynthesis de novo pathway are summarized in Figure 2.2

and Table 2.1.

In the purine biosynthesis de novo pathway, the major rate-limiting steps happen at the

activation of ribose-5-phosphate and the first step of the pathway. Ribose-5-phosphate, along

with the PRPP synthetase and ATP are required to generate the activated sugar, PRPP

(Henderson & Paterson, 1973). This activation is feedback-inhibited by purine-5’-nucleotides,

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especially AMP and GMP (King, 2016). Also, glutamine, glycine and aspartate can be rate-

limiting factors for purine synthesis in some cells (Henderson & Paterson, 1973). Additionally,

the biosynthesis of purine de novo is controlled by the conversion of IMP to AMP and GMP;

ATP and GTP inhibit some enzymes during the biosynthesis of purines de novo (Zrenner et al.,

2006).

Figure 2.2. The de novo synthesis of purines (Henderson & Paterson,1973; King, 2016) Abbreviations in figure 2.2: PRPP: 5-phosphoribosyl-1-pyrophosphate; PRA: 5-phosphoribosylamine; PPi: pyrophosphate; GAR: glycinamide ribonucleotide; 10F-THF:10-formyl tetrahydrofolate; FGAR: formylglycinamide ribonucleotide; FGAM: formylglycinamidine ribonucleotide; AIR: 5-aminoimidazoleribonucleotide; CAIR: 4-carboxy aminoimidazole ribonucleotide; SAICAR: N-succinyl-5-aminoimidazole-4-carboxamide ribonucleotide; AICAR: 5-aminoimidazole-4-carboxamide ribonucleotide; FAICAR: 5-formaminoimidazole-4-carboxamide ribonucleotide; IMP: inosine monophosphate.

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The catabolism and the salvage pathway of purine NT is summarized in Figure 2.3. Free

bases and nucleosides are from the degradation/catabolism of NT, and then are recycled by the

salvage reaction (Zrenner et al., 2006). Catabolism of the purine nucleotides in animal and other

higher primates leads to the final production of uric acid, which is insoluble and is excreted as

the main source of nitrogen waste (Zrenner et al., 2006).

Figure 2.3. Catabolism and the salvage pathway of purine NT (Henderson & Paterson,1973; King, 2016) Abbrevations in figure 2.3: APRT: adenine phosphoribosyl transferase; HGPRT: hypoxanthineguanine phosphoribosyl transferase; ADA: adenosine deaminase; PNP: purine nucleotide phosphorylases Table 2.1. Types of reactions in the purine biosynthesis de novo pathway ((Henderson & Paterson, 1973) Number Reaction Bond formed or

broken

Reversible ATP

required

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1 Phosphoribosyl pyrophosphate

amidotransferase

Carbon-nitrogen No No

2 Phosphoribosyl glycineamide

synthetase

Carbon-nitrogen Yes Yes

3 Phosphoribosyl glycineamide

formyltransferase

Carbon-nitrogen No No

4 Phosphoribosyl

formylglycineamidine synthetase

Carbon-nitrogen No Yea

5 Phosphoribosyl aminoimidazole

synthetase

Carbon-nitrogen No Yes


carboxylase

Carbon-nitrogen Yes No


succinocarboxamide synthetase

Carbon-nitrogen Yes Yes

8 Adenylosuccinate lyase Carbon-nitrogen Yes No


carboxamide formyltransferase

Carbon-nitrogen Yes No

10 IMP cyclohydrolase Carbon-nitrogen Yes No

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2.4 Pathways of pyrimidine NT biosynthesis

The biosynthesis of pyrimidine NT can also occur either by the de novo pathway or by the

salvage pathway (Carver & Walker, 1995; Cosgrove, 1998; Grimble & Westwood, 2000; Fustin

et al, 2012). However, the synthesis of pyrimidine NT is easier than that of purine NT because

the pyrimidine base is simpler. Pyrimidine NT synthesis differs in two significant aspects from

that of purine NT. Firstly, the ring structure is not assembled on PRPP, but built as a free base.

Secondly, there is no branch in the pyrimidine synthesis pathway. The salvage pathway of

pyrimidine NT has not been included as an illustration because it is less significant than that of

purine bases due to the solubility of the byproducts of pyrimidine catabolism.

Within the de novo pathway, pyrimidine NT are synthesized from glutamine, aspartate, and

CO2 to form UMP in the cytosol and the mitochondria of mammalian cells (Li & Gatlin, 2006).

There are six steps in this pathway as diagrammed in the Figure 2.4.

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Figure 2.4. The de novo pathway of pyrimidine biosynthesis (Henderson & Paterson,1973; King, 2016) Abbreviations in figure 2.4: CAD: CPS-II activity; CA: carbamoylaspartate; DHO: dihydroorotate; OMP: orotidine 5 -monophosphate; UMP: uridine-5-monophosphate

Carbamoyl phosphate (CP) joins the pyrimidine biosynthetic pathway with the help of

aspartate transcarbamoylase (ATCase) and aspartate to form CA; CP is a precursor for arginine

biosynthesis as well (Zrenner et al., 2006). Then, the enzyme dihydroorotase (DHOase) catalyzes

the conversion of CA to produce DHO, which is oxidized by dihydroorotate dehydrogenase

(DHODH) to form orotate. Orotate is condensed with PRPP to form OMP (Zrenner et al., 2006),

which is decarboxylated by orotidylate decarboxylase (ODCase) to generate UMP.

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2.5 Nucleotides in Nutrition

NT are present as free NT and nucleic acids in various natural foods, for example, organ

meats, seafood and dried legumes contain purines and RNA (Clifford & Story, 1976).

Unfortunately, there are few publications describing the NT content of different foods, but any

biomass of plant, microbial or animal is generally made up of cellular material and should thus

contain nucleotides. High concentrations of NT are found in high cell density foods and

metabolically active tissues. As a result, animal based foods contain more NT than plant based

foods.

In aquaculture feed ingredients, certain animal by-products contain high NT

concentrations, such as poultry by-product (360-950 ppm) (Nates, 2013). In contrast, oils,

oilseeds and grains contain low levels of nitrogenous bases. Levels have been observed to be

well below 5 mg/g (Devresse, 2000). Purine and pyrimidine base content of some aquaculture

feed ingredients is shown in Table 2.2.

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Table 2.2 Purine and pyrimidine base content of some aquafeed ingredients (%) (Devresse, 2000) Ingredient Adenine Guanine Cytosine Thymine Uracil Total

bases Complete fish meal 0.2 0.9 0.1 0.1 0.1 1.4 Fish solubles 0.2 2.3 0.1 0.1 0.1 2.8 Yeast 0.3 0.3 0.2 0.0 0.2 0.9 Yeast extract 0.7 0.6 0.1 0.0 0.7 2.3 Single cell protein 0.2 0.9 0.1 0.4 0.6 2.1

Figure 2.4 Digestion and absorption of dietary nucleotides (Hess & Greenberg, 2012).

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In the diet, nucleotides are mostly consumed along with protein bound as nucleoproteins

(Carver, 1995). Digestion takes place in the small intestine as protease and nuclease enzymes

break the nucleoproteins and then nucleotides are degraded into nucleosides; Furthermore,

nucleosides can be degraded by nucleosidases to purines and pyrimidines (Figure 2.4),

nucleoside if the preferred form being absorbed into the cells of the gut (Bronk & Hastewell,

1987; Sanderson & He, 1994; Li & Gatlin, 2006; Hess & Greenberg, 2012). Rumsey et al.

(1992) reported that rainbow trout fed increasing levels of yeast nucleic acid extracts presented

significant increasing in their growth and nitrogen retention, but research on digestibility and

bioavailability of nucleic acids in natural aquaculture feed sources is limited.

In aquatic animals, dietary NT have long been implicated as feed attractants in both

vertebrate and invertebrate species (Carr and Thompson, 1983; Carr et al., 1984). For example,

early research showed that the puffer fish have chemoreceptors on their lips that can respond to

AMP, IMP, UMP and ADP through electrophysiological changes (Kiyohara et al.,1975). Kubitza

et al. (1997) reported that dietary IMP (2.8 g/kg) inclusion enhanced feed intake of largemouth

bass by 46%, compared to non-supplemented soybean meal-based diet. Also, inosine and IMP

have been found to be potent gustatory feeding stimulants and chemo-attractive substance for

turbot and American lobster (Mackie, 1973; Mackie and Adron, 1978).

Research into potential growth and health benefits of dietary NT in aquaculture species did

not begin until Burells et al. (2011a, b) reported that dietary supplementation of NT enhanced

resistance of salmonids to viral, bacterial and parasitic infections as well as improved efficacy of

vaccination and osmoregulation capacity of the animals. A summary of studies that focused on

the effect of dietary NT in a variety of fish species is provided in Table 2.3.

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Table 2.3 Summary of studies examining the effects of dietary NT in fish Species Outcomes References

Atlantic salmon Growth+ Antibody titer+ mortality-

plasma chloride- intestinal fold+

Burrells et al., 2001b;

Rainbow trout Growth+ survival after challenge

with specific bacterium and virus+

lipid peroxidation-

Burrells et al., 2001a;

Tahmasebi-Kohyani et al.,

2011;

Mohebbi et al., 2013;

Common carp Phagocytosis+ respiratory

burst+complement+ lysozyme+

A.hydrophila infection-

Sakai et al., 2001;

Red drum Intestinal fold+ Weight gain+ feed

efficiency+ during first week

Li et al., 2007;

Cheng et al., 2011

Turbot Altered immunogene expression in

many tissues; enterocyte height+

Low et al., 2003;

Peng et al., 2013;

Tilapia Feed intake + (at 0.1% NT

supplementation) H2O2 + (0.05% NT

supplementation)

Barros et al., 2015

Sole Plasma cortisol+ glucose levels+ Palermo et al., 2013

Grouper Weight gain+ Lin et al., 2009

Symbols + and - represent increase and decrease, respectively, in the specified response

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Traditionally, NT have been deemed to be non-essential nutrients because the synthesis of

NT through the salvage or the de novo pathways is adequate to meet the metabolic needs of

animals (Carver & Walker, 1995; Li and Gatlin III, 2006). The absence of NT from human and

animal diet does not cause a classic clinical deficiency syndrome. However, evidence is

mounting that endogenous supply, either de novo or salvage synthesis, may be inadequate for

normal function of certain tissues under certain conditions (Carver & Walker, 1995; Van Buren &

Rudolph, 1997). Dietary NT may be highly beneficial and may be considered semi- and/or

conditionally essential nutrients, particularly during periods of rapid cell replication (Carver &

Walker, 1995; Van Buren & Rudolph, 1997; Li and Gatlin 2006; Welker et al. 2011).

Dietary NT may help in provision of physiologically required levels of NT owing to

limited synthetic capacity of certain tissues (e.g. intestinal mucosa, haematopoietic cells,

lymphocytes and the brain), the high cost for de novo synthesis, immune-endocrine interactions

and modulation of gene expression patterns (Saikai et al. 2001; Low et al. 2003). Various studies

have suggested that dietary NT deficiency may lead damage into important tissues such as liver,

heart and intestine, and may also affect the functionality of immune cells (Grimble and

Westwood, 2000).

2.6 Effects of dietary NT

Studies indicated that growth and feed utilization of juvenile Atlantic salmon (initial body

weight: 35 g and 43 g), juvenile red drum (10 g), juvenile grouper (6 g and 10 g), post-larvae

Pacific white shrimp (1 g) and juvenile black tiger shrimp (6 g and 6 g) benefitted from dietary

NT supplementation (Burell et al. 2001b; Li et al. 2007a, b; Lin et al. 2009; Huu et al., 2012).

Experimental evidence from several studies suggest that dietary NT can exert positive effects on

19

the gastrointestinal tract, growth performance, immune response and liver function of different

species (Li & Gatlin, 2006; Sauer et al, 2011; Peng et al., 2013).

In the majority of studies, commercial NT products have been supplemented in fish

diets. These products are derived from yeast and contain other components such as

polysaccharides and trace elements (Lin et al., 2009), which are also known to provide

immunostimulation in fish (Sakai, 1999). Only a handful of studies have evaluated the effects of

purified nucleotide mixtures added to diets, such as those on red drum (Li et al., 2007a) and

Pacific white shrimp (Li et al., 2007b).

2.6.1 The effect of dietary NT on gastrointestinal tract

Dietary NT play an important role in the development of proper functions in the

gastrointestinal (GI) tract of different species. For example, the intestine of Atlantic salmon fed a

diet with a commercial NT supplement (“Optimun” 0.03% of the total feed) had a significantly

greater mean fold height of the proximal, mid and distal intestine than that of fish fed the control

diet, which may have led to a greater gut surface area (Burrells et al., 2001b). Similar results

were found in turbot (Peng et al., 2013) and red drum (Cheng et al., 2011) where researchers

observed that the intestinal morphology, including distal intestine fold height, enterocyte height

and microvillus height of fish fed a NT-supplemented diet (0.1% nucleotide premix (Sigma

Aldrich) in turbot; 0.5% nucleotide product (Canadian Bio-Systems Inc.) in red drum) was

significantly improved compared to those of fish fed control diets. In studies with rats and

piglets, dietary NT can also influence intestinal morphology including higher mitosis and lower

apoptosis rates (Sauer et al., 2011).

20

Dietary NT also has a potential role in influencing the gastro-intestinal flora. Sauer et al.,

(2010) observed that dietary NT had the ability to modulate the composition of the

gastrointestinal microbiota, after weaning in piglets exposed to stressors (Andrés-Elias et al.,

2007; Moore et al., 2011). The authors suggested that dietary NT could have the potential to be

used as alternatives to antimicrobial growth promoters in piglets.

2.6.2 The effect of dietary NT on growth performance

Dietary NT have effects on growth performance for some species; notably in the early

growth period, such as in Atlantic salmon (Burrells et al., 2001a,b), tilapia (Ramadan et al.,

1994), rainbow trout (Tahmasebi-Kohyani et al., 2011; Mohebbi et al., 2013), grouper (Lin et al.,

2009), Pacific white shrimp (Li et al., 2007b), weanling pigs (Li et al., 2015) and rats (Xu et al.,

2013).

During early growth stages, de novo synthesis might be insufficient to support rapid cell

replication, so dietary NT could reduce the high demand for NT synthesis (Li & Gatlin, 2006).

This positive effect on growth performance might also be related to the amount of feed

consumed by animals since NT may have feed attractant properties. For example, IMP

supplementation (2.8%) has increased feed intake of largemouth bass (Micropterus salmoides)

significantly (Kubitza et al., 1997). However, some experiments showed that the positive effects

of dietary NT were not related to the feed attractant properties of these compounds since similar

quantities of the diet consumed across treatments (Carver, 1994).

21

2.6.3 The effect of dietary NT on immune function

The maintenance of normal immune function may require dietary NT (Carver et al., 1990).

Studies with terrestrial animals have indicated that NT can influence phagocyte activity (Gil,

2002), interleukin-2 production (Van et al., 1985) and natural killer cell activity (Carver et al.,

1990). Also, dietary NT can also affect lymphocyte activity and immunoglobulin production

(Leonardi et al., 2003; Li & Gatlin, 2006).

Dietary NT have a positive effect on resisting infection, and are conducive to the

immunoglobulin response in early life (Gil, 2002). Enhancing resistance to infection is the

evidence that NT can affect immune function. For example, groups of mice fed NT-

supplemented diets showed less mortality after being challenged with the infection of

Staphylococcus aureus and Candida albicans compared to the groups of mice fed non-NT diets

(Kulkarni et al., 1986a,b; Carver, 1994). Also, the number of Aeromonas hydrophila was

reported to be significantly lower in liver, kidney and blood after an intraperitoneal injection

challenge in the fish fed a NT supplemented diet (Sakai et al., 2001). This may be because of the

increased phagocytic activity of murine peritoneal macrophages (Kulkarni et al., 1986a), the

enhanced T-cell dependent antibody production (Jyonouchi, 1994), the increased interleukin-2

(IL-2) production (Carver, 1994) and the intensive bone marrow cell and peripheral neutrophil

number (Matsumoto et al., 1995). The exogenous supply of a nucleoside-nucleotide mixture

might be necessary to enhance the proliferation of some host defense cells, such as bone marrow

cells and peripheral blood neutrophils, after induction of sepsis (Matsumoto et al., 1995).

Li et al. (2005) reported that dietary inclusion of 0.2% NT (Optimun) did not exert any

significant impact on growth, stress tolerance (assessed by plasma cortisol following

confinement) and in situ challenge by co-habitation with A. ocellatum in juvenile red drum. Li et

22

al. (2007a) also investigated the possible effect of purified mixtures of NT in juvenile red drum,

and a transient growth enhancing effect was found in the first week, and then it disappeared.

However, dietary supplementation of 0.5 or 1% NT (Ascogen) was effective in enhancing

immune function as assessed by superoxide anion production of head kidney macrophages and

intestine fold and microvillus height in the same fish species (Cheng et al. 2011).

Burrells et al. (2001a) observed that dietary NT (0.03%, Optimun) inclusion improved

survival rates of rainbow trout challenged with V. anguillarum and with the infectious salmon

anaemia virus. Dietary NT (Optimun) reduced plasma cortisol release, and increased number of

B lymphocytes and resistance to challenge with infectious pancreatic necrosis virus (Leonardi et

al. 2003). Recently, Tahmasebi-Kohyani et al. (2011, 2012) found that dietary 0.15-0.2% NT

supplementation promoted growth, immune function (ACH50 level, lysozyme activity and IgM

level), handling and crowding stress tolerance (plasma cortisol, glucose and ion concentration)

and resistance (survival) of fingerling rainbow trout to S. iniae.

Although dietary supplement of 0.2% NT (Aquagen) didn’t improve the growth of red-tail

black shark (Epalzeorhynchos bicolor), its inclusion enhanced the resistance (survival) to S. iniae

infection in vaccinated and unvaccinated fish (Russo et al., 2006). Similarly, 0.4% yeast RNA

inclusion in purified diets improved blood innate immune capacity (leucocyte count, respiratory

burst activity, total protein, globulin and A/G ratio) and the relative percent survival after

challenge with Aeromonas hydrophila in rohu (Labeo rohita) juveniles (Choudhury et al. 2005).

Haemato-immunological response and resistance of Catla catla juveniles to A. hydrophila also

increased when 0.8% yeast RNA was added to the purified diets (Jha et al. 2007).

Dietary NT inclusion of (0.2 or 0.5%) exerted positive impact on the innate immunity of

Pacific White Shrimp, Litopenaeus vannamei (Murthy et al. 2009). Huu et al. (2012)

23

investigated the NT supplementation (0.44% and 0.56%) of a semi-purified diet could obtain

optimal growth rate of juvenile black tiger shrimp (Penaeus monodon) at different days. They

also indicated declining requirement for NT with the increase in prawn size in this dose-titration

stud. Lin et al. (2009) also studied the NT requirement of grouper employing a purified diet

supplemented with a purified mixture of NT or single NT, and found that dietary 0.15% of

purified mixture or AMP was effective in promoting growth and immune function of the fish.

Dietary single supplementation of inosine monophosphate was also studies in olive flounder

(Paralichthys olivaceus), and it was effective in improving growth at levels of about 0.1-0.2%.

IMP inclusion can enhance innate immunity (myeloperoxidase and lysozyme activities) and

disease resistance against S. iniae at levels ranging from 0.1-0.4%.

Dietary 0.25% NT (Optimun) inclusion was reported to improve growth performance,

blood innate immune response (blood protein, albumin, albumin/globulin ratio, red blood cells,

white blood cells, lymphocyte content, alkaline phosphatase and lysozyme activity and cortisol

in serum) and stress tolerance of confinement and salinity of Caspian brown trout (Salmo trutta

caspius) (Kenari et al. 2013). In another study, 0.25-0.35% NT supplement increased the

tolerance of low water level stress in Beluga sturgeon Huso huso (Yousefi et al. 2012). However,

Welker et al. (2011) fed channel catfish with graded levels (0, 0.1%, 0.3%, 0.9% and 2.7%) of

purified nucleotide mixture for 8 weeks and observed that addition of nucleotides produced a

dose-dependent reduction in survival of to Edwardsiella ictaluri. It is hypothesized that the high

levels of NT in that study may have led to the decrease in disease resistance, which was not

related to innate immune and adaptive antibody responses.

24

2.6.4 The Effect of Dietary NT on Liver Function

A study examining the effects of dietary NT on hepatic composition and morphology

indicated that the liver weight as a percentage of the body weight was significantly higher in the

animals fed NT-supplemented diets compared to that of the animals fed basal diets (Carver,

1994). Moreover, the total serum bilirubin level and total lipid, lipid phosphorous and cholesterol

levels in the livers were lower, while the glycogen level in the livers was higher in the animals

fed NT-supplemented diets than the animals fed basal diets (Carver, 1994). A nucleotide-

nucleoside mixture diet is recommended to provide a better nutritional supplement to the

cirrhotic rats following partial hepatectomy, because the diet stimulated a hepatic RNA level and

a hepatic fraction protein synthetic rate (Usami et al., 1996).

Research on the role of dietary NT has shown in consistent results in a variety of species;

therefore, further study on these components is warranted.

2.7 Evolution of Aquaculture and Aquaculture Feed Formulation

Fish, crustacean and mollusk production increased from 3.9 percent of total by weight in

1970 to 33 percent in 2005, and about one third of fish consumed in the world is farm raised

(Gatlin III et al., 2007). The growth and the intensification of aquaculture production has

contributed to the higher aquaculture feed production. In 2015, the production of aquaculture

feed is 4% share of the global livestock feed market at 41 million tonnes, where 1.8 million

tonnes of aquaculture feed was produced in North America (Alltech Global Feed Survey, 2015).

Aquaculture feed formulations have traditionally relied on fish meal as a major protein

source. Fish meal is rich in highly digestible amino acids, essential fatty acids, vitamins, minerals

25

and nucleotides (Zinn et al., 2009; Watanabe et al., 1997). The increasing demand for this

ingredient combined with limited supply has led to a significant increase in the price of fish

meal. Aquaculture feed manufacturers have had to increasingly rely on more economical protein

sources of animal and plant origins (Wang et al., 2010; Hu et al., 2013; Zinn et al., 2009).

Studies have shown that good growth and feed efficiency can be achieved with feeds

based primarily on plant ingredients. However, performance of fish generally drops significantly

with complete replacement of fish meal by plant ingredients, especially in young, fast-growing,

animals, even if all known nutrient requirements appear to have been met. Fish meal is an

excellent source of digestible essential amino acids, vitamins, minerals as well as of a wide

variety of compounds, including nucleotides (Zinn et al., 2009; Watanabe et al., 1997). In some

markets, processed animal proteins (PAPs) have filled in the role traditionally played by fish

meal in aquaculture feeds. However, feed safety regulations and consumer concerns and

demands are preventing the use of PAPs in some markets. Most PAPs are also excellent sources

of amino acids, minerals and various compounds, including nucleotides.

Conversely, plant protein ingredients are significantly poorer sources of nucleotides

because they have low cell density compare to animal protein sources, although some plant

ingredients are high in protein content and well balanced amino acids, and have a reasonable

price and stable supply (Storebakken et al., 2000; Mente et al., 2003; Burel et al., 2000a). In this

context, the level of nucleotides in aquaculture feeds has likely decreased significantly and may,

in part, explain the lower performance of fish fed diets with very high levels of plant protein

ingredients. Thus, it is hypothesized that nucleotides are beneficial essential nutrients for young

fast growing fish.

26

From nutritional and environmental perspectives, partially or totally replacing fish meal

with plant protein is reliable and cost effective, even though the presence of some anti-nutritional

factors, such as protease inhibitors, phytates, lectins, saponins, and high fiber content in plant

protein ingredients may limit how much of these ingredients are used in feeds (Spinelli et al.,

1983; Davies et al., 1990; Krogdahl et al., 1994; Storebakken et al., 1998; Hendricks, 2002).

Several studies have examined replacing fish meal with plant protein in a multitude of species,

such as Atlantic salmon (Mente et al., 2003), rainbow trout (Oliva-Teles et al., 1994; Kaushik et

al., 1995; Refstie et al., 2000; Yurkowski et al., 1978; Burel et al., 2000b), Chinook salmon

(Higgs et al., 1982), tilapia (Davies et al., 1990), common carp (Viola et al., 1982), grass carp

(Dabrowski & Kosak, 1979; Tan et al., 2013), turbot (Regost et al., 1999; Fournier et al., 2004),

Japanese flounder (Pham et al., 2007), cobia (Chou et al., 2004), puffer fish (Zhong et al., 2011),

tiger puffer (Lim et al., 2011), African catfish (Toko et al., 2008), red sea bream (Biswas et al.,

2007), gilthead seabream (Robaina et al., 1997; Pereira & Oliva-Teles, 2003), sunshine bass

(Lewis & Kohler, 2008) and Japanese seabass (Men et al., 2014).

Most studies have led to the conclusion that it is possible to produce successful feeds

containing high levels of plant protein ingredients and minimal fish meal levels (Hua and

Bureau, 2012). However, complete replacement of fish meal and other animal ingredients has

proved very difficult, notably in diets for salmonids and other carnivorous fish species, even

when formulating to digestible essential nutrient levels in excess of all known requirements of

these animals. Growth and feed efficiency of rainbow trout fed fish meal and animal protein free

diets are generally significantly less good than that of fish fed diets with some levels of fish meal

and/or animal protein ingredients (Cho et al., 1974; Gomes et al., 1995; Adelizi et al., 1998;

Glencross et al., 2011; Burr et al., 2012). Review of evidence suggest that the lower performance

27

is unlikely due to differences in digestible amino acids, energy or essential nutrient contents of

the diets or due to the effect of anti-nutritional factors. The results of studies appear to indicate

that fish meal may have contributed some dietary components that may be beneficial to these

fish. Growth performance and feed efficiency of the fish remain nonetheless relative good and

the fish did not present any overt signs of deficiency or nutritional pathologies. This may indicate

that the dietary components contributed by fish meal may be nutrients or metabolites that the

animal is able to synthesize but unable to synthesize sufficient quantities of in order to enable

higher growth and higher efficiency of feed and nutrient utilization.

Since studies have indicated that dietary nucleotide supplementation had positive effect on

various physiological functions in many species, especially during early life stages (Gil, 2002).

Dietary NT supplementation have been observed to have positive effect on fish growth

performance, immune responses and GI tract of different fish species (Burrells et al., 2001b;

Sakai et al., 2001; Li & Gatlin, 2006; Cheng et al., 2011; Sauer et al, 2011; Tahmasebi-Kohyani

et al., 2011; Mohebbi et al., 2013; Peng et al., 2013). It has been hypothesized to be due to high

metabolic demands for nucleotides. Fish meal is rich in nucleotides while plant protein

ingredients are poor sources of these compounds. It has therefore been suggested that NT

supplementation of diet containing high levels of plant protein ingredients and low levels of fish

meal or animal protein ingredients may be an effective strategy to improve performance achieved

with such diets (Watanabe et al., 1997; Zhang et al., 2012).

2.8 Conclusion and perspectives

NT are ubiquitous intracellular compounds with important roles in cellular function and

metabolism (Cosgrove, 1998). They have been traditionally thought of as non-essential nutrients,

28

but recent research has suggested that they are semi-essential nutrients for different animal

species. An abundance of data from nutritional studies with fish species indicates that dietary NT

supplementation can influence aquatic animals’ biology, mainly in terms of growth performance,

GI tract morphology and functionality, resistance to diseases, and efficiency of nutrient

utilization.

Feeds containing high levels of fish meal probably contain sufficient quantity of NT to

meet the requirement of the animals (Huu et al., 2012). The increase in the demand and cost of

fish meal is leading feed manufacturers to reduce fish meal levels in feeds to a minimal level and

to increasingly rely on plant protein ingredients. However, feed formulated with very high levels

of plant protein ingredients and no fish meal generally perform relatively poorly. Plant protein

ingredients are less good sources of certain nutrients than fish meal and contain certain anti-

nutritional factors. Plant protein ingredients are also much less good sources of NT than fish

meal. Dietary NT supplementation has also been shown to improve growth, intestinal

morphology and functionality and immune function of fish and other animals. It would be of

interest to examine the effect of dietary NT supplementation of diets formulated to contain high

levels of plant protein ingredients and low fish meal and other animal protein ingredients.

29

CHAPTER 3- THE EFFECT OF DIETARY NUCLEOTIDES SUPPLEMENTATION ON GROWTH AND FEED EFFICIENCY OF RAINBOW TROUT (ONCORHYNCHUS MYKISS) FED FISH MEAL-FREE AND ANIMAL PROTEIN-FREE DIETS

Abstract

The effect of dietary nucleotide supplementation on growth and feed efficiency of rainbow

trout (initial weight = 25.3±0.7 g/fish) was examined in a 70-day growth trial. One control diet

was formulated to contain 25% fish meal and no supplemental nucleotides. Two basal diets were

formulated using high levels of highly digestible plant protein ingredients and to meet all the

known nutrient requirements of rainbow trout. One of the basal diet contained 5% fish meal

while the other contained 0% fish meal. These two diets were supplemented with four increasing

levels (0, 8, 16 and 24 ppm, respectively) of nucleotides (measured as total potentially available

nucleotides, TPAN) using a commercial supplement (Laltide®, Lallemand Inc. Qc, Canada).

Differences (p<0.05) in final body weight, thermal-unit growth coefficient (TGC), feed

efficiency, retained nitrogen (RN) and recovered energy (RE) were observed among diets with

different fish meal inclusion levels. Trout fed the fish meal-free diet performed less well

(p<0.05) than trout fed the fish meal-containing diets. Nucleotide supplementation levels

significantly (p<0.05) affected final body weight and feed efficiency in rainbow trout fed with

the 5% fish meal diet. The growth rate of fish fed the 5% fish meal diet with 16 ppm of dietary

supplementation of nucleotides was not different from that of the fish fed the control diet with

25% fish meal. The feed efficiency of fish fed the control diet with 25% fish meal was better

than of fish fed all the other diets. The results of this study suggest that dietary nucleotide

supplementation may be beneficial to young rainbow trout fed with low fish meal and high levels

of plant protein ingredients. The essentiality of nucleotides to young rainbow trout deserves to be

further examined.

30

3.1 Introduction

Nucleotides (NT), the phosphate esters of nucleosides (NSs), are the building blocks for

DNA and RNA, which provide energy for normal cellular bioprocesses, thus, are essential to

growth performance and development of humans and animals (Van Buren et al., 1994). An

animal can obtain NT in three ways, the de novo pathway, the salvage pathway and through the

diet. Dietary NT have been characterized as non-essential nutrients, but this position has been

over-turned by recent research, indicating that dietary NT are semi-essential nutrients. Since the

de novo synthesis pathway may be limiting under conditions like infection, rapid growth and

development, dietary NT is essential when the de novo pathway and the salvage pathway are not

able to synthesize sufficient amount of NT to meet the needs of all tissues. The results from a

number of studies suggest that dietary NT supplementation has the effects on growth, intestinal

morphology and functionality and immune function of different fish species. (Ramadan et al.,

1994; Adamek et al., 1996; Sakai et al., 2001; Burrells et al., 2001a, b; Li & Gatlin, 2006; Sauer

et al, 2011; Cheng et al., 2011; Tahmasebi-Kohyani et al., 2011; Mohebbi et al., 2013; Peng et

al., 2013).

Fish can obtain NT from their diets, notably diets that contain significant levels of fish

meal. Fish meal is a good source of nutrients, such as amino acids, minerals and NT. An

increasing demand of fish meal due to the growth of the aquaculture feed industry in recent years

has led to it becoming a very expensive commodity. The replacement of fish meal by other more

cost-effective ingredients has been the focus of much research in aquaculture nutrition. Studies

have indicated that feeds can be formulated with high levels of plant protein sources provided

attention is paid to meeting the nutritional requirements of the animals and that diets do not

contain excessive levels of anti-nutritional factors (Watanabe et al., 1997; Hua and Bureau,

31

2012).

However, feed formulated with very high levels of plant protein ingredients and no fish

meal generally perform relatively poorly. Plant protein ingredients are less good sources of

certain nutrients and other less well characterized components than are found in abundant

quantity in fish meal. Plant protein ingredients are notably much less good sources of NT than

fish meal. The performance of fish generally drops significantly with complete replacement of

fish meal by plant ingredients, especially in young, fast-growing animals, even if all known

nutrient requirements appear to have been met. In this situation, the level of NT in aquaculture

feeds has likely decreased significantly and may partly explain the lower performance of fish fed

diets with high levels of plant protein ingredients. Thus, it is hypothesized that nucleotides are

beneficial essential nutrients for young fast growing fish.

Currently, there are gaps in existing knowledge about supplementation of nucleotides in

diets of fish and the optimal dose of NT supplementation in rainbow trout. Therefore, it has been

suggested that NT supplementation of diet containing high levels of plant protein ingredients and

low levels of fish meal or animal protein ingredients may be an effective strategy to improve

performance, achieved with such diets (Watanabe et al., 1997; Zhang et al., 2012).Thus, it would

be of interest to examine the effect of dietary NT supplementation of diets formulated to contain

high levels of plant protein ingredients and low fish meal and other animal protein ingredients on

growth and efficiency of feed and nutrient utilization in rainbow trout.

32

3.2 Methods

3.2.1 Experimental Diet Formulation and Preparation

In total, nine (9) diets were prepared in this experiment (Table 3.1). All the experimental

diets were formulated to contain 38% digestible protein and 18 MJ/kg digestible energy and

exceed all the known nutritional requirements for rainbow trout recommended by NRC (2011). A

diet containing 25% fish meal, with no NT supplementation was included in the study as positive

control. Two basal diets were formulated to contain 0% and 5% of fish meal and using a

combination of several highly digestible plant protein ingredients. Eight (8) experimental diets

were produced by supplementing two basal diets with four (4) graded levels of a commercial NT

supplement (Laltide®, Lallemand Inc. Quebec, Canada) (Table 3.2) to produce four levels of NT,

(0, 8, 16 and 24 ppm of total potentially available nucleotides (TPAN) as presented as Table 3.3).

All the dry ingredients of each diet were first mixed as one batch in a Hobart mixer for 10

min (Hobart Ltd, Don Mills, ON, Canada). Fish oil and canola oil were subsequently added

slowly after mixing of dry ingredients and the resulting mash was mixed for an additional 10

min. The nine (9) resulting mashes were stored at 4°C until pelleted. Diets were steam-pelleted

using a laboratory-scale team pellet mill using a 3 mm diameter dye and a pellet length of 3 mm

(California Pellet Mill, San Francisco, CA, USA), and dried under forced air at 36°C for 24 h

using a drying oven (Precision & Scientific Co., USA). Pelleted diets were sieved to remove fine

and broken pellets, and stored at 4°C until used. The fines and broken pellets were collected and

stored at 4°C as well. Samples of the dried diets were analyzed as described in Section 3.2.3 and

the proximate composition of the diets is reported in Table 3.4.

33

Table 3.1 Ingredient composition of the experimental diets Diets Ingredients (g/kg) 1 2 3 4 5 6 7 8 9 Fish meal, herring, 68% CP 250 50 50 50 50 0 0 0 0 Rapeseed protein conc, 62% CP 20 108 108 108 108 150 150 150 150 Rapeseed meal, 44% CP 140 140 140 140 140 145 145 145 145 Wheat gluten, 80% CP 50 50 50 50 50 50 50 50 50 High oil soy protein conc, 60% 20 150 150 150 150 160 160 160 160 Corn gluten meal, 60% CP 60 60 60 60 60 60 60 60 60 Hipro sunflower meal, 46% CP 150 150 150 150 150 150 150 150 150 Wheat middlings, 17% CP 113 68.5 68.5 68.5 68.5 56.5 56.5 56.5 56.5 Fish oil 80 90 90 90 90 90 90 90 90 Canola oil 80 80 80 80 80 80 80 80 80 Vitamin premixa 5 5 5 5 5 5 5 5 5 Vitamin E premixb 1 1 1 1 1 1 1 1 1 Bio-Lys®(51% lys) 10 20 20 20 20 20 20 20 20 DL-Met (99%) 1 1.5 1.5 1.5 1.5 2 2 2 2 L-Histidine 0 1 1 1 1 1.5 1.5 1.5 1.5 Choline chloride 60% 3 3 3 3 3 3 3 3 3 Mineral premixc 2 2 2 2 2 2 2 2 2 Dicalcium phosphate 10 15 15 15 15 15 15 15 15 Sodium chloride 2 2 2 2 2 2 2 2 2 Rovimix Stay-C 1 1 1 1 1 1 1 1 1 Carophyll® pink 1 1 1 1 1 1 1 1 1 Laltide (g/kg) 0 0 0.05 0.1 0.15 0 0.05 0.1 0.15 aProvides 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. bProvides per Kg of diet: Vitamin E, 0.375g; Wheat middling, 1g. cProvides 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.

34

Table 3.2 Nucleotide supplementation added level

Diet Diet Type + Nucleotide Supplementation

(expressed as ppm TPAN)

1 Control (25% fish meal)

2 Basal diet (5% fish meal) + 0 ppm








Table 3.3 Total potentially available nucleotides (TPAN) in the Laltide®

LALTIDE® inclusion Actual TPAN added

g/kg ppm

0 0

0.05 8

0.1 16

0.15 24

35

Table 3.4 Proximate composition of experimental diets. Diets

1 2 3 4 5 6 7 8 9

Dry Matter % 98.2 98.0 97.7 98.0 97.8 97.8 97.9 97.9 98.0

Crude Protein % 42.6 43.3 43.5 43.3 43.4 42.9 42.3 42.9 43.2

Lipid, % 21.0 20.5 21.0 20.7 20.3 20.9 20.8 20.8 20.6

Ash, % 7.4 7.3 7.0 7.2 7.1 6.9 7.0 6.9 6.8

36

3.2.2 Fish, Feeding and Husbandry

Rainbow trout fingerlings were obtained from Alma Aquaculture Research Station (Elora,

Ontario, Canada) and transferred to the Fish Nutrition Research Laboratory at the University of

Guelph before the start of the experiment. The juvenile rainbow trout, average initial weight of

25.3±0.7g, were stocked into 27 fiberglass tanks (60L) with a stocking density of 12 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. Before the start of the experiment, biomass in each tank

was equalized to be within 3% of the mean. Water temperature was maintained at 15.0 °C. Fish

was kept under a 12 h light: 12 h dark photoperiod regime. The nine (9) experimental diets were

each allocated to three replicates groups (tanks) using a complete randomized design.

Fish were acclimated to the experimental conditions for two weeks prior to the start of

the experiment. During the acclimation period, fish was fed a commercial trout feed (Profishent,

Martin Mills Inc., Elmira, ON, Canada) once daily. During the 10-week-trial (70 days), fish was

hand-fed to near satiation three times daily on weekdays, 9:00, 13:00 and 16:00 and once daily

on weekends. Feed intake was recorded weekly and bulk weight of the fish in each tank were

recorded every 28 days. Experimental tanks were cleaned twice a week on each Monday and

Thursday according to the Standard Operating Procedures of the Fish Nutrition Research

Laboratory. All animals were treated in compliance with the guidelines of the Canadian Council

on Animal Care and the University of Guelph Animal Care Committee.

37

3.2.3 Chemical Analysis

At the beginning of the trial, an initial pooled sample of ten fish was taken for the

determination of the initial carcass composition. At the end of the trial, a sample of five fish per

tank was taken for the determination of the final carcass composition. Fish was humanly killed

by an overdose of tricaine methane sulfonate (MS-222) followed by a sharp blow to the head.

Live weights were recorded and 10 (initial) and/or 5 (final) fish per tank were pooled into plastic

autoclave bags and stored at -20°C until processing. Samples for carcass composition

determination were thawed overnight prior to being cooked in an autoclave (LabTech LAC-

5100S Autoclave). After autoclaving, samples were thoroughly ground into homogeneous slurry

using a food processor (Cuisinart DLC-X PLUS), freeze-dried (FTS Systems Dura Dry MP

freeze drier), and ground into a fine powder using a food processor and kept at -20 °C until

further analysis.

Another sample of three fish per tank was taken for determination of individual body

weight and length. Viscera and liver from each fish were dissected, weighed, and snap frozen in

liquid nitrogen and stored at -80°C.

Diets, ingredients, initial and final carcass samples were analysed in duplicate. Dry

matter was determined with a drying oven at 100°C for 12 h (Fisher Scientific Isotemp oven,

Markhan, ON, Canada). Ash was determined using a muffle furnace at 550°C for 4 hours (Fisher

Scientific Isotemp muffle furnace, Markham, ON, Canada), crude protein (% N × 6.25) was

determined by LECO (LECO Corp., St. Joseph, MI, USA) and lipid analysis was performed with

an Ankom XT20 fat analyser (Ankom Technology, Macedon, NY, USA) using petroleum ether.

Total carbohydrate content was determined by the following equation, total carbohydrate

content= 100 – (crude protein + lipids + ash), and gross energy (GE) was determined by the

38

following equation, GE=[(CP*23.4 kJ/g)+(Lipid*39.2 kJ/g)+(DM-Ash-CP-Lipid

kJ/g)*17.2]/100 (Cho, 1982).

3.2.4 Calculations and Statistical Analysis

The effect of dietary nucleotide level in the diet, level of fish meal and possible

interaction between the two, on final body weight, thermal-unit growth coefficients (TGC), feed

intake (FI), feed efficiency (FE), retained nitrogen (RN), recovered energy (RE), nitrogen

retention efficiency (NRE), energy retention efficiency (ERE) and proximate carcass

composition were investigated using SAS general linear model in addition to use linear and

quadratic contrasts (PROC GLM, SAS Version 9.2, SAS Institute, Cary, NC, USA). Growth

performance and proximate composition data were analyzed using a complete randomized

design with ANOVA with fish meal level and NT level as sources of variation. In all these

analyses the tank was the experimental unit and the significance level was P<0.05.

Growth performance parameters were calculated as follows:

a) Weight gain (WG, g/fish)

WG= FBW – IBW

Where: FBW= final body weight (g/fish); IBW= initial body weight (g/fish).

b) Thermal unit growth coefficient (TGC)

TGC= 100×[(FBW1/3−IBW1/3)×(sum T×D)-1]

where: FBW= final body weight (g/fish); IBW= initial body weight (g/fish); sum T×D= sum

39

Celsius degrees×days.

c) Feed efficiency (FE, gain:feed)

FE= live body weight gain/ feed intake (DM basis)

Where: live body weight gain= (FBW/final number of fish)−(IBW/initial number of fish);

feed intake= total dry feed/number of fish.

d) Retained nitrogen (RN, g/fish)

RN= (FBW×N contentfinal)−(IBW×N contentinitial)

Where: N contentfinal= nitrogen content (%) of the final carcass sample;

N contentinitial= nitrogen content (%) of the initial carcass sample

e) Recovered energy (RE, kJ/fish)

RE= (FBW×GE contentfinal)−(IBW×GE contentinitial)

Where: GEfinal= gross energy (kJ/g) content of the final carcass sample;

GEinitial= gross energy (kJ/g) content of the initial carcass sample

f) Nitrogen retention efficiency (NRE) and energy retention efficiency (ERE)

NRE (% IN)= [[(FBW×N contentfinal)−(IBW×N contentinitial)]/IN]×100

ERE (% IE)= [[(FBW×GE contentfinal)−(IBW×GE contentinitial)]/IE]×100

where: N contentfinal= nitrogen content (%) of the final carcass sample; N contentinitial= nitrogen

content (%) of the initial carcass sample; GEfinal= gross energy (kJ/g) content of the final carcass

sample; GEinitial= gross energy (kJ/g) content of the initial carcass sample; IN=ingested nitrogen

(g/fish); IE= ingested energy (kJ/fish).

40

g) Viscerosomatic index (VSI, %)

VSI =100×Individual viscera weight/Individual FBW

Where: Individual viscera weight = weight of viscera (g) at the end of experimental period

h) Hepatosomatic index (HIS, %)

HIS =100×Individual liver weight /Individual FBW

Where: Individual liver weight = weight of liver at the end of experimental period.

3.3 Results

3.3.1 Growth Performance

During the 70-day trial, no mortality was observed. Figure 3.1 presents the live body

weight of the fish fed the nine experimental diets over the 70-day trial. The results of final body

weight, TGC and feed efficiency, of fish fed diets containing increasing levels of NT with

different levels of fish meal inclusion, are presented in Figure 3.2, 3.3 and 3.4, respectively.

41

Figure 3.1 Live body weight (g/fish) of rainbow trout in response to being fed experimental diets containing increasing NT levels with different fish meal inclusion levels.

Figure 3.2 Final body weight (g/fish) of rainbow trout in response to being fed experimental diets containing increasing NT levels with different fish meal inclusion levels.

1 Dietary NT supplementation level.

0.0

20.0

40.0

60.0

80.0

100.0

120.0

140.0

160.0

180.0

200.0

Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6 Diet 7 Diet 8 Diet 9Day 0 Day 28 Day 56 Day 70

Live

body weight (g/fish)

1

42

Figure 3.3 Thermal-unit growth coefficient (TGC) of rainbow trout in response to being fed experimental diets containing increasing NT levels with different fish meal inclusion levels.

1 Dietary NT supplementation level. Figure 3.4 Feed efficiency of rainbow trout in response to being fed experimental diets containing increasing NT levels with different fish meal inclusion levels.

1 Dietary NT supplementation level. Lines regressed using a 2nd order polynomial function.

1

1

43

The growth performance parameters of rainbow trout fed different diets are presented in

Table 3.5. Increases in final body weight (p<0.001), TGC (p<0.001) and feed efficiency (p<0.01)

of rainbow trout were found in response to increasing fish meal level in diets, but no significant

effect on VSI and HIS score occurred when comparing different fish meal levels. However,

dietary NT supplementation level affected feed intake (p<0.05) and final body weight (p<0.05)

significantly.

NT supplementation has shown effects on feed intake (p<0.05) and final body weight

(p<0.05) of rainbow trout were fed with 5% fish meal diets in significant linear responses, no

such difference was found in fish fed with 0% fish meal diets (Figure 3.2; Figure 3.3). In Table

3.6, feed intake, final body weight and TGC of rainbow trout showed no statistical difference

between fish fed with Diet 4 and fish with control diet. However, feed efficiency of fish fed Diet

4 was significantly lower (p<0.01) than that of fish fed control diet.

Only a significant quadratic increase in response to increasing dietary NT levels in VSI

(p<0.05) was observed in rainbow trout fed with 0% fish meal diets. Nonetheless, no statistical

interaction effect between fish meal levels and NT levels on growth performance was detected in

this 70-day trial.

3.3.2 Carcass Composition

The results from proximate analysis of pooled carcass samples are presented in Table 3.7.

Fish meal and NT supplementation did not significantly influence whole body composition with

the exception of ash content of fish fed with 0% fish meal which increased in a significantly

decreased (p<0.05) in a linear fashion. However, significant interactions were observed between

44

fish meal level and NT level on moisture (p<0.05), lipid (p<0.01), ash (p<0.05) and gross energy

(p<0.05) which indicates influence of NT supplementation on whole body composition as a

function of fish meal level.

3.3.3 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. Fish meal level had a

significant positive effect on RN (p<0.05) and RE (p<0.05), but no effect on NRE and ERE. NT

supplementation did not significantly affect RN, RE, NRE and ERE. However, NRE of fish fed

0% fish meal inclusion diets responded to NT supplementation significantly (p<0.05) in a

quadratic fashion. A significant interaction response on ERE (p<0.05) was noted between fish

meal levels and NT supplementation.

45

Table 3.5 Performance of rainbow trout (initial weight = 25 g/fish) in response to being fed increasing NT content with different fish meal inclusion levels diets for 10 weeks.

Inclusion level of

NT

Feed Intake

Final Weight

TGC1 Feed efficiency2

gain:FI

VSI3 HIS4

Ppm g/fish g/fish % gain:feed % %

Control5 - 147 194 0.274 1.17 13.6 1.11 5 % FM Diet 2 - 133 163 0.247 1.04 15.4 1.20 Diet 3 8 137 171 0.257 1.09 15.0 1.15 Diet 4 16 152 187 0.270 1.09 16.0 1.18 Diet 5 24 142 176 0.259 1.06 15.6 1.16 S.E.M 2.8 3.4 0.003 0.01 0.2 0.03 Significance6 Linear * * N.S N.S N.S N.S Quadratic N.S N.S N.S * N.S N.S 0 % FM Diet 6 - 133 162 0.246 1.03 16.5 1.22 Diet 7 8 122 151 0.233 1.04 15.0 1.09 Diet 8 16 134 165 0.249 1.04 15.5 1.24 Diet 9 24 127 156 0.236 1.02 16.7 1.26 S.E.M 2.4 2.6 0.003 0.01 0.371 0.03 Significance Linear N.S N.S N.S N.S N.S N.S Quadratic N.S N.S N.S N.S * N.S Among FM Levels *** ** *** ** N.S N.S Among NT Levels * * N.S N.S N.S N.S FM Levels*NT Levels

N.S N.S N.S N.S N.S N.S

Control vs. Average7

* *** *** *** ** N.S

1TGC=thermal unit growth coefficient, %TGC= 100 x (FBW1/3-IBW1/3)/Σ(Temp (◦C) x Number of days). 2Feed efficiency= (final body weight - initial body weight) : feed intake as dry matter basis. 3Viscerosmatic index = 100 x viscera weight (g)/ whole body weight (g). 4Hepatosomatic index=100 x liver weight (g)/ whole body weight (g). 5Containing 25% fish meal 6Significance indicated by *, **, *** at the p<0.05, 0.01 and 0.001 level; N.S = not statistically significant (p≤0.05). 7Average of Diet 2, 3, 4, 5, 6, 7, 8 and 9. Table 3.6 Comparison of some parameters1 of rainbow trout in response to being fed diet 4 with Control diet

Feed intake g/fish

Final body weight g/fish

TGC %

Feed efficiency %

Diet 4 vs. Control diet N.S N.S N.S 0.007 1parameters with significant effects after comparison, on the contrary, other parameters are not significantly affected by the NT supplementation. Significance indicated by *at the p<0.05level; N.S = not statistically significant (p≤0.05).

46

Table 3.7 Proximate composition of whole carcass of rainbow trout in response to being fed increasing NT content with different fish meal inclusion levels diets for 10 weeks, expressed on a wet weight basis.

Inclusion level of NT (ppm)

Moisture %

Crude Protein

%

Lipid %

Ash %

GE1 kJ/g

Control2 - 66.8 15.2 14.9 2.4 9.4 5 % FM Diet 2 - 66.7 15.3 15.0 2.5 9.3 Diet 3 8 65.3 15.5 16.2 2.7 9.9 Diet 4 16 67.6 14.7 15.2 2.2 9.1 Diet 5 24 66.2 15.8 15.0 2.5 9.6 S.E.M 0.4 0.2 0.2 0.1 0.1 Significance3 Linear N.S N.S N.S N.S N.S Quadratic N.S N.S N.S N.S N.S 0 % FM Diet 6 - 65.4 15.0 16.2 2.6 9.9 Diet 7 8 66.0 16.0 14.7 2.6 9.6 Diet 8 16 65.3 15.6 15.7 2.6 9.9 Diet 9 24 66.9 15.3 14.9 2.2 9.5 S.E.M 0.2 0.2 0.2 0.1 0.1 Significance Linear N.S N.S N.S * N.S Quadratic N.S N.S N.S N.S N.S Among FM Levels N.S N.S N.S N.S N.S Among NT Levels N.S N.S N.S N.S N.S FM Levels*NT Levels

* N.S ** * *

Control vs. Average4

N.S N.S N.S N.S N.S

1 Gross energy (kJ/g)

2 Containing 25% fish meal 3Significance indicated by *, **, *** at the p<0.05, 0.01 and 0.001 level; N.S = not statistically significant (p≤0.05). 4Average of Diet 2, 3, 4, 5, 6, 7, 8 and 9.

47

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 NT content with different fish meal inclusion levels diets for 10 weeks.

Inclusion level of NT

(ppm)

RN g/fish

RE kJ/fish

NRE %IN

ERE %IE

Control1 - 4.0 1645 40.3 49.0 5 % FM Diet 2 - 3.3 1344 36.0 44.2 Diet 3 8 3.6 1512 37.5 48.2 Diet 4 16 3.7 1528 35.3 43.9 Diet 5 24 3.8 1506 38.3 46.8 S.E.M 0.1 39 0.5 0.8 Significance2 Linear N.S N.S N.S N.S Quadratic N.S N.S N.S N.S 0 % FM Diet 6 - 3.2 1431 35.2 46.9 Diet 7 8 3.2 1270 38.9 45.8 Diet 8 16 3.5 1452 37.6 47.4 Diet 9 24 3.1 1286 35.3 44.2 S.E.M 0.1 32 0.7 0.5 Significance Linear N.S N.S N.S N.S Quadratic N.S N.S * N.S Among FM Levels * * N.S N.S Among NT Levels N.S N.S N.S N.S FM Levels*NT Levels N.S N.S N.S * Control vs. Average3 ** ** * *

1 Containing 25% fish meal

2Significance indicated by *, **, *** at the p<0.05, 0.01 and 0.001 level; N.S = not statistically significant (p≤0.05). 3Average of Diet 2, 3, 4, 5, 6, 7, 8 and 9.

48

3.4 Discussion

In this study, a series of experimental diets were formulated to contain essential nutrients

in excess of all the known nutritional requirements for rainbow trout according to NRC (2011)

recommendations. One control diet contained 25% fishmeal was formulated. The other two basal

diets were formulated with two fish meal levels (5% fish meal and 0% fish meal) and each of

them had been supplemented with four increasing levels (0, 8, 16 and 24 ppm, respectively) of

NT using a commercial supplement (Laltide®, Lallemand Inc. Qc, Canada).

In the present study, the level of fish meal in diets decreased from 25% down to 5% and

0%, which negatively affected the growth and feed efficiency of rainbow trout. Plant protein

ingredients in this study have been previously examined and shown to be sources of highly

digestible essential amino acids, contain low levels of anti-nutritional factors and be of high

nutritive value to rainbow trout (unpublished results). Previous results from related studies on the

same ingredients are suggesting that the differences in performance achieved with the diets with

different fish meal and plant protein ingredients levels were not likely due to different in

digestible amino acids, energy or essential nutrient contents of the diets or to the effect of anti-

nutritional factors. Similar results were observed in another fish meal replacement trial, rainbow

trout fed diets containing low (less than 20%) fish meal source exhibited decreased growth

(Gomes et al., 1995; Adelizi et al., 1998; Glencross et al., 2011), although the nutrient contents

in the experimental diets appeared to be similar and meet all known requirements of rainbow

trout (NRC, 2011). However, complete replacement of fish meal and other animal ingredients

has proved to be very difficult in salmonid diets even when formulating to digestible essential

nutrient levels in excess of all known requirements of these animals. For example, Burr et al.

49

(2012) observed that rainbow trout which received 0% fish meal had significantly lower weight

gain, lower TGC and lower feed efficiency than fish which received higher fish meal levels.

In this study, the feed intake in rainbow trout fed with diet 4 was not significantly different

from that of fish fed with control diet. It could be hypothesized that dietary NT may have a

potential effect on palatability of diet since some NTs have been found to have significant

attractant properties for aquatic animals. For example, inosine and AMP were found to be potent

chemo-attractants for lobster (Mackie, 1973) and dietary IMP supplementation (2.8%)

significantly increased feed intake of largemouth bass (Micropterus salmoides) fed soybean-

based diet (Kubitza et al., 1997). However, the fact that NT supplementation was only effective

for feed containing 5% fish meal suggest that enhancement of performance could be related to

different mechanisms.

NT supplementation level significantly affected feed intake, TGC and feed efficiency of

rainbow trout but only for the basal diet containing 5% fish meal. Positive effect of NT

supplementation of growth and efficiency of feed utilization in several other studies with

different species, such as weanling pigs (Andres-Elias et al., 2007; Li et al., 2015), rats (Xu et

al., 2013), Atlantic salmon (Burrells et al., 2001b), rainbow trout (Tahmasebi-Kohyani et al.,

2011; Zhang et al., 2012), juvenile turbot (Peng et al., 2013), Hybrid tilapia (Ramadan et al.,

1991) and black tiger shrimp (Huu et al., 2012).

In the present study, no positive effect of NT supplementation was observed in fish fed

with 0% fish meal diets. This may suggest that some nutrients may have prevented the fish from

responding to NT supplementation. Theoretically, these different responses of fish should have

resulted in an interaction response between fish meal level and NT supplementation level.

However, no statistical interaction response in growth parameter was detected in this study. This

50

is thought to be the variability of feed intake, final body weight and feed efficiency among tanks

fed the same diet was relatively large. Thus, future studies should be designed to be more

statistically powerful by using larger number of replicates per experimental diet, exploring more

NT inclusion levels to better determine whether NT supplementation is truly beneficial or

whether the results are a simple experimental artifact.

In current study, the basal diets were designed to contain a low percentage of fish meal.

Moreover, the levels of dietary NT supplementation in this study were lower than the other

studies referred to above. More work needs to be done with greater range and types of NT

supplements and more replicates in order to assess whether NT supplementation truly exert a

positive effect on salmonid fish.

3.5 Conclusion

The objective of this study was to evaluate the effect of supplemental dietary NT,

provided through a commercial feed additive, on growth, feed efficiency and efficiency of

protein and energy utilization in rainbow trout fed diets formulated to contain low levels of NT.

The combination of high quality plant protein ingredients used in this study appeared to result in

fairly highly nutritive and palatable diets. The results suggest that dietary NT supplementation

may have a beneficial effect on fish fed low fish meal diets. A significant reduction in

performance with reducing fish meal level in this study also indicated that gaps still exist in our

understanding of nutritional requirements of salmonid fish.

Future studies could put more efforts to on examining the effect of NT and other dietary

components on digestion, cell, tissue or organ integrity, multiplication and functionality, the

51

activity of key enzymes and the expression of genes known to be associated with growth

processes. The effect of these compounds on immune function and disease resistance would also

be highly valuable.

52

CHAPTER 4 GENERAL DISCUSSION

The objective of this study was to evaluate the effect of supplemental dietary NT,

provided through a commercial feed additive, on growth, feed efficiency and efficiency of

protein and energy utilization in rainbow trout fed diets formulated to contain low levels of NT.

Traditionally, aquaculture feeds were formulated to contain high levels of fish meal, which is

characterized by an excellent profile of essential amino acid, minerals, omega-3 fatty acids, fat

soluble vitamins, phospholipids, taurine, and numerous other compounds (Watanabe et al., 1997;

Zinn et al., 2009). Due to high cost and limited global supply of fish meal, modern aquaculture

feeds are increasingly formulated to contain low levels of fish meal and high levels of more cost-

effective ingredients, such as plant protein ingredients. Several limitations, such as the presence

of anti-nutritional factors and poorer levels of essential nutrients, are associated with use of plant

ingredients in feeds. However, it is possible to produce successful rainbow trout feeds containing

high levels of plant protein ingredients at minimal fish meal levels (Hua and Bureau, 2012).

Complete replacement of fish meal and other animal ingredients, however, has proved to be very

difficult in salmonid diets even when formulating to digestible essential nutrient levels in excess

of all known requirements of these animals. Growth and feed efficiency of rainbow trout fed fish

meal and animal protein free diets are generally significantly lower than that of fish fed diets

with some levels of fish meal and/or animal protein ingredients (Cho et al., 1974; Gomes et al.,

1995; Adelizi et al., 1998; Glencross et al., 2011; Burr et al., 2012).

In the present study, the level of fish meal in the diet was from 25% down to 5% and 0%,

and this negatively affected the growth and feed efficiency of rainbow trout, which is consistent

with the findings of earlier studies. In the present study, a series of experimental diets was

formulated with plant protein ingredients that have previously been shown to be a source of

53

highly digestible essential amino acids, contain low levels of anti-nutritional factors and be of

high nutritive value to rainbow trout (unpublished results). This makes it unlikely that

differences in performance were due to different in digestible amino acids, energy or essential

nutrient contents of the diets or to the effect of anti-nutritional factors.

The results appear to indicate that fish meal may have contributed some dietary

components that may be beneficial to these fish. Growth performance and feed efficiency of the

fish remain nonetheless relative good and the fish did not present any overt signs of deficiency or

nutritional pathologies. This may indicate that the dietary components contributed by fish meal

may be nutrients or metabolites that the animal is able to synthesize but unable to synthesize

sufficient quantities of in order to enable higher growth and higher efficiency of feed and

nutrient utilization.

Nucleotides are natural components of all living organisms that are involved in a wide

variety of metabolic and cellular processes. Animals are capable of de novo synthesis of

nucleotides but they can also acquire them through the salvage pathway or through the diet

(Quan et al., 1990). It has been observed that de novo synthesis and the salvage pathways

occasionally provide insufficient amounts of NT for the needs of certain tissues.

Studies have indicated that dietary NT supplementation had positive effect on various

physiological functions in many species, especially during early life stages (Gil, 2002). It has

been hypothesized that during periods of rapid growth or high metabolism, some tissues such as

immune cells and gastrointestinal cells may have high demands for NT. Dietary NT

supplementation can have a positive effect on fish growth performance, immune responses and

GI tract of different fish species (Burrells et al., 2001b; Sakai et al., 2001; Li & Gatlin, 2006;

54

Cheng et al., 2011; Sauer et al, 2011; Tahmasebi-Kohyani et al., 2011; Mohebbi et al., 2013;

Peng et al., 2013).

Fish meal is generally recognized as a good source of NTs, whereas plant protein

ingredients are not (Devresse, 2000). In this thesis, the effect of supplementation of diets

containing with 5% or 0% fish meal with graded levels of NT fed to rainbow trout was

examined. Results suggested that dietary NT supplementation had a beneficial effect on rainbow

trout fed diets with 5% fish meal. The feed intake and final body weight of fish increased

significantly in a linear fashion, and feed efficiency increased significantly in a quadratic fashion

with increasing NT supplementation. However, the fish fed the diet with no fish meal did not

show any significant response to NT supplementation. This may indicate that other nutrients may

have become deficient in this diet and prevented the fish from responding to NT

supplementation. In theory, this difference in response of the fish to NT supplementation should

have resulted in a significant interaction between fish meal level and nucleotide supplementation.

However, no such significant statistical interaction response was detected in this study. The p

value for the interaction response on TGC was 0.17 which is close to the established significance

level (p ≤ 0.05). Overall, variability in terms of feed intake, final body weight and feed

efficiency among tanks fed the same diet was relatively large. Additional feeding trials should be

carried out to determine if the results of this study can be replicated. Future studies should be

designed to be more statistically powerful by using larger number of replicates per experimental

diet, exploring broader and more numerous NT inclusion levels to help better determine whether

NT supplementation is truly beneficial or a simple experimental artifact.

The potential positive effect of NT supplementation in low fish meal diet observed in

this study as well as in other studies (Barros et al., 2015) could be related to different

55

mechanisms. Certain nucleotides have been found to have significant attractant properties for

fish diets. Dietary IMP supplementation (2.8%) significantly enhanced feed intake of largemouth

bass (Micropterus salmoides) fed soybean-based diet (Kubitza et al., 1997). Electrophysiological

studies indicated that, AMP, IMP, UMP and ADP were detected by lips chemoreceptors of

puffer fish (Fugu pardalis) (Kiyohara et al., 1975). Free adenine was found to have potent ability

to inhibit feed intake in rainbow trout (Rumsey et al., 1992). These evidences indicate that NTS

and their metabolites can influence palatability of feeds.

In the present study, the feed intake in rainbow trout fed with Diet 4 (5% fish meal, 16

ppm NT supplementation) was no statistically different (P=0.39) from that of the fish fed with

control diet with 25% fish meal, no NT supplementation. However, the feed efficiency of fish

fed Diet 4 was still significantly (p<0.01) lower than those of fish fed the control diet. These

results suggest a potential effect of NT supplementation on palatability of the diet with 5% fish

meal. However, increase feed intake can be the results of increase palatability or other biological

or metabolic effects. NT could increase metabolic efficiency or growth potential and thus

indirectly influence the feed intake of the animals. The use of a pair-feeding protocol in which all

the groups of fish would have received the same amount of feed would have been an effective

way of determining if the NT supplementation exerted its effect through metabolic effects. Truly

determining the effects of NT on palatability of feeds would require an elaborate protocol. This

could be done through a feed preference assay (Suresh et al., 2011).

As reviewed in some level of details in Chapter 2, NT supplementation has been found to

have significant effect on number of models, notably in young fast growing animals like the

young rainbow trout in this study. Nucleotides are building blocks for rapidly dividing tissues

56

and play many important roles in intermediary metabolisms of all living organisms. As

mentioned above.

The NT supplement used here was a commercial yeast-based feed additive containing

approximately 15% total potentially available nucleotides (TPAN) as well as several other

components, such as amino acids, vitamins, cell wall components, etc. Since NT

supplementation also was done concurrently with these other components, a response to one or

several of these other components cannot be ruled out. Limited information is available on the

actual NT composition of this commercial supplement. Analysis of the NT content of the

experimental diet is still in progress and no results can be reported at this time.

Most other studies examining the effects of dietary NT supplementation have been based

on the use of commercial supplements of poorly characterized composition. Very few studies

have used purified single nucleotides (Kubitza et al., 1997; Lin et al., 2009; Huu et al., 2013).

Different individual NTs or combinations of NTs in different ratios may have different effects.

For example, 0.4% GMP and 0.1% AMP and 0.4% GMP and 0.1% IMP improved growth rate

and weight gain in black tiger prawn (P. monodon) (Huu et al., 2013). Supplementation of the

diet with a nucleotide mixture (IMP:AMP:GMP:UMP:CMP) significantly increased weight gain

in grouper (Epinephelus malabaricus) in a 8-week trial but only increased weight gain

significantly in red drum (Sciaenop ocellatus) during the first week of a 4-week trial (Li et al.,

2007a; Lin et al., 2009). Dietary supplementation of purified AMP in grouper resulted in

significantly higher superoxide anion (O2-) production, a measure of activity of phagocytic cells

(Secombes, 1990), compared to that of fish fed with other purified nucleotides (Lin et al., 2009).

The present study produced valuable results that opens up the door to additional studies.

The reduction in performance in diet with low or no fish meal indicates that our understanding of

57

nutritional requirements for salmonid species is still imperfect. Certain nutrients, compounds or

properties of importance to salmonid fish may be contributed by fish meal and these are poorly

characterized. This also suggests that dietary NT may have a beneficial effect and could

potentially be considered semi or conditionally essential nutrients. These observations deserve to

be examined very carefully in future research efforts.

The experimental diets used in this study represent an interesting model for future studies

on the effect of NT and other dietary components. The combination of high quality plant protein

ingredients used in this study appeared to result in fairly highly nutritive and palatable diets. The

5% fish meal and 0% diets could be supplement with various nutrients and chemical compounds

alone or in combination to determine what is apparently “missing” from fish meal-free diets.

The scope of future studies could be expanded to include assessment of the effect of NT

and other dietary components on digestion, cell, tissue or organ integrity, multiplication and

functionality, assessment of the activity of key enzymes and the expression of genes known to be

associated with growth processes. The effect of these compounds on immune function and

disease resistance would also be highly interesting since dietary NT supplementation have been

observed to have a significant effect on immune function and disease resistance in several animal

models. As such, immune function may represent a highly sensitive indicator of the response of

the animals to dietary NT.

58

References

Adamek, Z., Hamackova, J., Kouril, J., Vachta, R., Stibranyiova, I. (1996). Effect of ascogen

probiotics supplementation on farming success in rainbow trout (Oncorhynchus mykiss)

and wels (Silurus glais) under conditions of intensive culture. Krmiva (Zagreb) 38, 11 –

20.

Adelizi, P., Rosati, R., Warner, K., Wu, Y., Muench, T., White, M., & Brown, P. (1998).

Evaluation of fish-meal free diets for rainbow trout, Oncorhynchus mykiss. Aquac

Nutrition Aquaculture Nutrition, 4(4), 255-262.

Global Feed Survey, 2015. Alltech. Kentucky, USA.

Andrés-Elias, N., Pujols, J., Badiola, I., & Torrallardona, D. (2007). Effect of nucleotides and

carob pulp on gut health and performance of weanling piglets. Livestock Science, 108(1-

3), 280-283.

Barros, M. M., Guimarães, I. G., Pezzato, L. E., Orsi, R. D., Junior, A. C., Teixeira, C. P., Fleuri,

L. F., Padovani, C. R. (2015). The effects of dietary nucleotide mixture on growth

performance, haematological and immunological parameters of Nile tilapia. Aquaculture

Research Aquac Res, 46(4), 987-993.

Biswas, A. K., Kaku, H., Ji, S. C., Seoka, M., & Takii, K. (2007). Use of soybean meal and

phytase for partial replacement of fish meal in the diet of red sea bream, Pagrus major.

Aquaculture, 267(1-4), 284-291.

Bronk, J. R., & Hastewell, J. G. (1987). The transport of pyrimidines into tissue rings cut from

rat small intestine. The Journal of Physiology, 382(1), 475-488.

59

Burel, C., Boujard, T., Tulli, F., & Kaushik, S. J. (2000a). Digestibility of extruded peas,

extruded lupin, and rapeseed meal in rainbow trout (Oncorhynchus mykiss) and turbot

(Psetta maxima). Aquaculture, 188(3-4), 285-298.

Burel, C., Boujard, T., Escaffre, A., Kaushik, S. J., Boeuf, G., Mol, K. A., Van der Geyten, S.,

Kühn, E. R. (2000b). Dietary low-glucosinolate rapeseed meal affects thyroid status and

nutrient utilization in rainbow trout (Oncorhynchus mykiss). BJN British Journal of

Nutrition, 83(06), 653.

Burr, G. S., Wolters, W. R., Barrows, F. T., & Hardy, R. W. (2012). Replacing fish meal with

blends of alternative proteins on growth performance of rainbow trout (Oncorhynchus

mykiss), and early or late stage juvenile Atlantic salmon (Salmo salar). Aquaculture, 334-

337, 110-116.

Burrells, C., Williams, P., & Forno, P. (2001a). Dietary nucleotides: a novel supplement in fish

feeds 1. effects on resistance to disease in salmonids. Aquaculture, 199(1-2), 159-169.

Burrells, C., Williams, P., Southgate, P., & Wadsworth, S. (2001b). Dietary nucleotides: a novel

supplement in fish feeds 2. effects on vaccination, salt water transfer, growth rates and

physiology of Atlantic salmon (Salmo salar L.). Aquaculture, 199(1-2), 171-184.

Carr, W. E., & Thompson, H. W. (1983). Adenosine 5'-monophosphate, an internal regulatory

agent, is a potent chemoattractant for a marine shrimp. Journal of Comparative

Physiology. A J. Comp. Physiol., 153(1), 47-53.

Carr, W. E., Netherton, J. C., & Milstead, M. L. (1984). Chemoattractants of the shrimp,

Palaemonetes pugio: variability in responsiveness and the stimulatory capacity of

mixtures containing amino acids, quaternary ammonium compounds, purines and other

60

substances. Comparative Biochemistry and Physiology Part A: Physiology, 77(3), 469-

474.

Carver, J.D., Cox, W.I., Barness, L.A. (1990). Dietary nucleotide effects upon murine

naturakiller cell activity and macrophage activation. J Parenter. Enter. Nutr. 14, 18-22.

Carver, J. D. (1994). Dietary nucleotides: cellular immune, intestinal and hepatic system effects.

The Journal of Nutrition, 124, 144S-148S.

Carver, J. D., & Walker, W. A. (1995). The role of nucleotides in human nutrition. The Journal

of Nutritional Biochemistry, 6(2), 58-72.

Chekeni, F., Elliott, M., Sandilos, J., Walk, S., Kinchen, J., Lazarowski, E., Armstrong, A. J.,

Penuela, S., Laird, D. W., Salvesen, G. S., Isakson, B. E., Ravichandran, K. (2010).

Pannexin 1 channels mediate ‘find-me’ signal release and membrane permeability during

apoptosis. Nature, 467(7317), 863.

Cheng, Z., Buentello, A., & Iii, D. M. (2011). Dietary nucleotides influence immune responses

and intestinal morphology of red drum Sciaenops ocellatus. Fish & Shellfish

Immunology, 30(1), 143-147.

Cho, C. Y., Bayley, H. S., & Slinger, S. J. (1974). Partial replacement of herring meal with

soybean meal and other changes in a diet for rainbow trout (Salmo gairdneri). J. Fish.

Res. Bd. Can. Journal of the Fisheries Research Board of Canada, 31(9), 1523-1528.

Chou, R., Her, B., Su, M., Hwang, G., Wu, Y., & Chen, H. (2004). Substituting fish meal with

soybean meal in diets of juvenile cobia Rachycentron canadum. Aquaculture, 229(1-4),

325-333.

Choudhury, A., Pierce, M. E., Nguyen, D., Storace, L., & Confalone, P. N. (2005). Synthesis of

D-D4FC, a biologically active nucleoside via an unprecedented palladium mediated

61

ferrier rearrangement-type glycosidation with an aromatization prone xylo-furanoid

glycal. Tetrahedron Letters, 46(47), 8099-8102.

Cliddord, A., & Story, D. (1976). Levels of purines in foods and their metabolic effects in rats.

Journal of Nutrition, 106(3), 435-442.

Cosgrove, M. (1998). Nucleotides. Nutrition, 14(10), 748-751.

Dabrowski, K., & Kozak, B. (1979). The use of fish meal and soyabean meal as a protein source

in the diet of grass carp fry. Aquaculture, 18(2), 107-114.

Davies, S. J., Mcconnell, S., & Bateson, R. I. (1990). Potential of rapeseed meal as an alternative

protein source in complete diets for tilapia (Oreochromis mossambicus Peters).

Aquaculture, 87(2), 145-154.

Deluca, C., & Kaplan, N. O. (1958). Flavin adenine dinucleotide synthesis in animal tissues.

Biochimica Et Biophysica Acta, 30(1), 6-11.

Devresse, B., 2000. Nucleotides—a key nutrient for shrimp immune system. Feed Mix 8, 20–22.

Eltzschig, H. K., Eckle, T., Mager, A., Kuper, N., Karcher, C., Weissmuller, T., Boengler, K.,

Schulz, R., Robson, S. C., Colgan, S. P. (2006). ATP release from activated neutrophils

occurs via connexin 43 and modulates adenosine-dependent endothelial cell function.

Circulation Research, 99(10), 1100-1108.

Faigle, M., Seessle, J., Zug, S., Kasmi, K. C., & Eltzschig, H. K. (2008). ATP release from

vascular endothelia occurs across Cx43 hemichannels and is attenuated during hypoxia.

PLoS ONE, 3(7).

Fournier, V., Huelvan, C., & Desbruyeres, E. (2004). Incorporation of a mixture of plant

feedstuffs as substitute for fish meal in diets of juvenile turbot (Psetta maxima).

Aquaculture, 236(1-4), 451-465.

62

Fustin, J.M., Masao, D., Yamada, H., Komatsu, R., Shimba, S., Okamura, H. (2012). Rhythmic

nucleotide synthesis in the liver: temporal segregation of metabolites. Cell Reports, 341-

349.

Gatlin III, D. M., Barrows, F. T., Brown, P., Dabrowski, K., Gaylord, T. G., Hardy, R. W.,

Herman, E., Hu, G., Krogdahl, A., Nelson, R., Overturf, K., Rust, M., Sealey, W.,

Skonberg, D., Souza, E. J., Stone, D., Wilson, R., Wurtele, E. (2007). Expanding the

utilization of sustainable plant products in aquafeeds: A review. Aquaculture Research

Aquaculture Res, 38(6), 551-579.

Gil, A. (2002). Modulation of the immune response mediated by dietary nucleotides. European

Journal of Clinical Nutrition, S1-S4.

Glencross, B., Rutherford, N., & Hawkins, W. (2011). A comparison of the growth performance

of rainbow trout (Oncorhynchus mykiss) when fed soybean, narrow-leaf or yellow lupin

meals in extruded diets. Aquaculture Nutrition, 17(2).

Gomes, E. F., Rema, P., & Kaushik, S. J. (1995). Replacement of fish meal by plant proteins in

the diet of rainbow trout (Oncorhynchus mykiss): digestibility and growth performance.

Aquaculture, 130(2-3), 177-186.

Grimble, G.K., Westwood, M.R. (2000). Nucleotides. In: Gershwin, M.E., German, J.B., Keen,

C.L. (Eds.), Nutrition and Immunology: Principles and Practice. Humana Press, Totowa,

NJ, USA, pp. 135-167.

Guttman, B. (2013). Nucleotide. Brenner's Encyclopedia of Genetics, 139.

Henderson, J., & Paterson, A. (1973). Nucleotide metabolism; an introduction. New York:

Academic Press.

63

Hendricks, J. D. (2002). Adventitious toxins. In Halver, J. E., & Hardy, R. W. (Ed.), Fish

Nutrition (pp. 601-649). San Diego: Academic Press.

Hess, J. R., & Greenberg, N. A. (2012). The role of nucleotides in the immune and

gastrointestinal systems: potential clinical applications. Nutrition in Clinical Practice,

27(2), 281-294.

Higgs, D. A., Mcbride, J. R., Markert, J. R., Dosanjh, B. S., Plotnikoff, M., & Clarke, W. (1982).

Evaluation of tower and candle rapeseed (canola) meal and bronowski rapeseed protein

concentrate as protein supplements in practical dry diets for juvenile chinook salmon

(Oncorhynchus tshawytscha). Aquaculture, 29(1-2), 1-31.

Holen, E., & Jonsson, R. (2004). Dietary nucleotides and intestinal cell lines: I. modulation of

growth. Nutrition Research, 24(3), 197-207.

Hu, L., Yun, B., Xue, M., Wang, J., Wu, X., Zheng, Y., & Han, F. (2013). Effects of fish meal

quality and fish meal substitution by animal protein blend on growth performance, flesh

quality and liver histology of Japanese seabass (Lateolabrax japonicus). Aquaculture,

372-375, 52-61.

Hua, K., & Bureau, D. P. (2012). Exploring the possibility of quantifying the effects of plant

protein ingredients in fish feeds using meta-analysis and nutritional model simulation-

based approaches. Aquaculture, 356-357, 284-301.

Huu, H.D., Tabrett, S., Hoffmann, K., Koppel, P., Lucas, J.S., Barnes, A.C. (2012). Dietary

nucleotides are semi-essential nutrients for optimal growth of black tiger shrimp

(Penaeus monodon). Aquaculture 366-367, 115-121.

Idzko, M., Ferrari, D., & Eltzschig, H. K. (2014). Nucleotide signalling during inflammation.

Nature, 509(7500), 310-317.

64

Jha, A. K., Pal, A., Sahu, N., Kumar, S., & Mukherjee, S. (2007). Haemato-immunological

responses to dietary yeast RNA, ω-3 fatty acid and β-carotene in Catla catla juveniles.

Fish & Shellfish Immunology, 23(5), 917-927.

Ji, S., Liu, R., Ren, M., Li, H., & Xu, J. (2015). Enhanced production of polysaccharide through

the overexpression of homologous uridine diphosphate glucose pyrophosphorylase gene

in a submerged culture of lingzhi or reishi medicinal mushroom, ganoderma lucidum

(higher basidiomycetes). International Journal of Medicinal Mushrooms Int J Med

Mushrooms, 17(5), 435-442.

Junger, W. G. (2011). Immune cell regulation by autocrine purinergic signalling. Nat Rev

Immunol Nature Reviews Immunology, 11(3), 201-212.

Jyonouchi, H. (1994). Nucleotide actions on humoral immune response. J. Nutr. 124 ŽSuppl. 1S.,

138S–143S.

Kaushik, S., Cravedi, J., Lalles, J., Sumpter, J., Fauconneau, B., & Laroche, M. (1995). Partial or

total replacement of fish meal by soybean protein on growth, protein utilization, potential

estrogenic or antigenic effects, cholesterolemia and flesh quality in rainbow trout,

Oncorhynchus mykiss. Aquaculture, 133(3-4), 257-274.

Kenari, A. A., Mahmoudi, N., Soltani, M., & Abediankenari, S. (2013). Dietary nucleotide

supplements influence the growth, haemato-immunological parameters and stress

responses in endangered caspian brown trout (Salmo trutta caspius Kessler, 1877).

Aquacult Nutr Aquaculture Nutrition, 19(1), 54-63.

King, M. W., 1996-2016. Nucleotide metabolism: Nucleic acid synthesis.

Kiyohara, S., Hidaka, I., Tamura, T., 1975. Gustatory response in the puffer-II. single fiber

analysis. Bull. Jpn. Soc. Sci. Fish. 41, 383– 391.

65

Krogdahl, Å, Lea, T. B., & Olli, J. J. (1994). Soybean proteinase inhibitors affect intestinal

trypsin activities and amino acid digestibilities in rainbow trout (Oncorhynchus mykiss).

Comparative Biochemistry and Physiology Part A: Physiology, 107(1), 215-219.

Kubitza, F., Lovshin, L.L., Lovell, R.T., 1997. Identification of feed enhancers for largemouth

bass Micropterus salmoides. Aquaculture 148, 191– 200.

Kulkarni, A.D., Fanslow, W.C., Drath, D.B., Rudolph, F.B., Van Buren, C.T. (1986a). Influence

of dietary nucleotide restriction on bacterial sepsis and phagocytic cell function in mice.

Arch. Surg. 121, 169–172.

Kulkarni, A.D., Fanslow, W.C., Rudolph, F.B., Van Buren, C.T. (1986b). Effect of dietary

nucleotides on response to bacterial infections. J. Parenter. Enteral Nutr. 10, 169–171.

Lazarowski, E. R. (2012). Vesicular and conductive mechanisms of nucleotide release.

Purinergic Signalling, 8(3), 359-373.

Leonardi, M., Sandino, A.M., Klempau, A. (2003). Effect of a nucleotide-enriched diet on the

immune system, plasma cortisol levels and resistance to infectious pancreatic necrosis

(IPN) in juvenile rainbow trout (Oncorhynchus mykiss). Bull. Eur. Assoc. Fish Pathol.

23, 52–59.

Lewis, H. A., & Kohler, C. C. (2008). Corn gluten meal partially replaces dietary fish meal

without compromising growth or fatty acid composition of sunshine bass. North

American Journal of Aquaculture, 70(1), 50-60.

Li, P., Lewis, D. H., & Gatlin III, D. M. (2004). Dietary oligonucleotides from yeast RNA

influence immune responses and resistance of hybrid striped bass (Morone chrysops X

Morone saxatilis) to Streptococcus iniae infection. Fish & Shellfish Immunology, 16(5),

561-569.

66

Li, P., Burr, G. S., Goff, J., Whiteman, K. W., Davis, K. B., Vega, R. R., Neill, W. H., Gatlin, D.

M. (2005). A preliminary study on the effects of dietary supplementation of brewers

yeast and nucleotides, singularly or in combination, on juvenile red drum (Sciaenops

ocellatus). Aquaculture Research Aquaculture Res, 36(11), 1120-1127.

Li, P., Gatlin III, D.M. (2006). Nucleotide nutrition in fish: Current knowledge and future

applications. Aquaculture 251:141-152.

Li, P., Gatlin, D. M., & Neill, W. H. (2007a). Dietary supplementation of a purified nucleotide

mixture transiently enhanced growth and feed utilization of juvenile red drum, Sciaenops

ocellatus. J World Aquaculture Soc Journal of the World Aquaculture Society, 38(2),

281-286.

Li, P., Lawrence, A.L., Gastille, F.L., Gatlin, D.M. (2007b). Preliminary evaluation of a purified

nucleotide mixture as a dietary supplement for Pacific white shrimp Litopenaeus

vannamei (Boone). Aquac Res 38, 887-890.

Li, H., Zhao, P., Lei, Y., Li, T., & Kim, I. (2015). Response to an Escherichia coli K88 oral

challenge and productivity of weanling pigs receiving a dietary nucleotides supplement.

Journal of Animal Science and Biotechnology J Animal Sci Biotechnol, 6(1).

Lim, S., Kim, S., Ko, G., Song, J., Oh, D., Kim, J., Kim, J., Lee, K. (2011). Fish meal

replacement by soybean meal in diets for tiger puffer, Takifugu rubripes. Aquaculture,

313(1-4), 165-170.

Lin, Y., Wang, H., & Shiau, S. (2009). Dietary nucleotide supplementation enhances growth and

immune responses of grouper, Epinephelus malabaricus. Aquaculture Nutrition, 15(2),

117-122.

67

Low, C., Wadsworth, S., Burrells, C., & Secombes, C. (2003). Expression of immune genes in

turbot (Scophthalmus maximus) fed a nucleotide-supplemented diet. Aquaculture, 221(1-

4), 23-40.

Mackie, A.M., 1973. The chemical basis of food detection in the lobster Homarus gammarus.

Mar. Biol. 21, 103– 108.

Mackie, A.M., Adron, J.W., 1978. Identification of inosine and inosine 5V-monophosphate as

the gustatory feeding stimulants for the turbot, Scophthalmus maximus. Comp. Biochem.

Physiol. 60A, 79–83.

Matsumoto, Y., Adje, A.A., Yamauchi, K., Kise, M., Nakasone, Y., Shinegawa, Y., Yokoyama,

H., Yamamoto, S. (1995). Mixture of nucleosides and nucleotides increases bone marrow

cell and peripheral neutrophil number in mice infected with methicillin-resistant

Staphylococcus aureus. Biochemical and molecular roles of nutrients. J. Nutr. 125, 815–

822.

Mawson, R., Heaney, R. K., Piskula, M., & Kozlowska, H. (1993). Rapeseed meal-

glucosinolates and their antinutritional effects part 1. rapeseed production and chemistry

of glucosinolates. Food / Nahrung Nahrung, 37(2), 131-140.

Men, K., Ai, Q., Mai, K., Xu, W., Zhang, Y., & Zhou, H. (2014). Effects of dietary corn gluten

meal on growth, digestion and protein metabolism in relation to IGF-I gene expression of

Japanese seabass, Lateolabrax japonicus. Aquaculture, 428-429, 303-309.

Mente, E., Deguara, S., Santos, M. B., & Houlihan, D. (2003). White muscle free amino acid

concentrations following feeding a maize gluten dietary protein in Atlantic salmon

(Salmo salar L.). Aquaculture, 225(1-4), 133-147.

68

Mohebbi, A., Nematollahi, A., Gholamhoseini, A., Tahmasebi-Kohyani, A., & Keyvanshokooh,

S. (2013). Effects of dietary nucleotides on the antioxidant status and serum lipids of

rainbow trout (Oncorhynchus mykiss). Aquacult Nutr Aquaculture Nutrition, 19(4), 506-

514.

Moore, K., Mullan, B., Pluske, J., Kim, J., & D'souza, D. (2011). The use of nucleotides,

vitamins and functional amino acids to enhance the structure of the small intestine and

circulating measures of immune function in the post-weaned piglet. Animal Feed Science

and Technology, 165(3-4), 184-190.

Murthy, H. S., Li, P., Lawrence, A. L., & Gatlin, D. M. (2009). Dietary β-glucan and nucleotide

effects on growth, survival and immune responses of Pacific white shrimp, Litopenaeus

vannamei. Journal of Applied Aquaculture, 21(3), 160-168.

Nates, S. F. (2013). The role of renderers in aquaculture feeds. Latin American Rendering

Association.

NRC (National Research Council). 2011. Nutrient requirements of fish and shrimp. The National

Academy Press, Washington, DC.

Oliva-Teles, A., Gouveia, A., Gomes, E., & Rema, P. (1994). The effect of different processing

treatments on soybean meal utilization by rainbow trout, Oncorhynchus mykiss.

Aquaculture, 124(1-4), 343-349.

Peng, M., Xu, W., Ai, Q., Mai, K., Liufu, Z., Zhang, K. (2013). Effects of nucleotide

supplementation on growth, immune responses and intestinal morphology in juvenile

turbot fed diets with graded levels of soybeam meal (Scophtalmus maximus L.).

Aquaculture 392-395, 51-58.

69

Pereira, T. G., & Oliva-Teles, A. (2003). Evaluation of corn gluten meal as a protein source in

diets for gilthead sea bream (Sparus aurata L.) juveniles. Aquaculture Research Aquac

Research, 34(13), 1111-1117.

Pesnot, T., Kempter, J., Schemies, J., Pergolizzi, G., Uciechowska, U., Rumpf, T., Sippl, W.,

Jung, M., Wagner, G. K. (2011). Two-step synthesis of novel, bioactive derivatives of the

ubiquitous cofactor nicotinamide adenine dinucleotide (NAD). J. Med. Chem. Journal of

Medicinal Chemistry, 54(10), 3492-3499.

Pham, M. A., Lee, K., Lim, S., & Park, K. (2007). Evaluation of cottonseed and soybean meal

as partial replacement for fish meal in diets for juvenile Japanese flounder Paralichthys

olivaceus. Fisheries Science Fisheries Sci, 73(4), 760-769.

Quan, R., Barness, L.A., Uauy, R. (1990). Do infants need nucleotide supplemented formula for

optimal nutrition. Journal of Pediatric Gastroenterology and Nutrition 11, 429-434.

Ramadan, A., Atef, M., & Afifi, N. (1991). Effect of the biogenic performance enhancer

(Ascogen “S”) on growth rate of tilapia fish. Acta Veterinaria Scandinavica, 304-306.

Ramadan, A., Afifi, N. A., Moustafa, M., & Samy, A. (1994). The effect of ascogen on the

immune response of tilapia fish to Aeromonas hydrophila vaccine. Fish & Shellfish

Immunology, 4(3), 159-165.

Refstie, S., Korsøen, Ø J., Storebakken, T., Baeverfjord, G., Lein, I., & Roem, A. J. (2000).

Differing nutritional responses to dietary soybean meal in rainbow trout (Oncorhynchus

mykiss) and Atlantic salmon (Salmo salar). Aquaculture, 190(1-2), 49-63.

Regost, C., Arzel, J., & Kaushik, S. (1999). Partial or total replacement of fish meal by corn

gluten meal in diet for turbot (Psetta maxima). Aquaculture, 180(1-2), 99-117.

70

Robaina, L., Moyano, F., Izquierdo, M., Socorro, J., Vergara, J., & Montero, D. (1997). Corn

gluten and meat and bone meals as protein sources in diets for gilthead seabream (Sparus

aurata): nutritional and histological implications. Aquaculture, 157(3-4), 347-359.

Rudolph, F. (1994). The biochemistry and physiology of nucleotides. J. Nutr. 124(1), 124s-127s.

Rumsey, G.L., Winfree, R.A., Hughes, S.G., 1992. Nutritional value of dietary nucleic acids and

purine bases to rainbow trout (Oncorhynchus mykiss). Aquaculture 108, 97–110.

Russo, R., Mitchell, H., & Yanong, R. P. (2006). Characterization of Streptococcus iniae isolated

from ornamental cyprinid fishes and development of challenge models. Aquaculture,

256(1-4), 105-110.

Sanderson, I., & He, Y. (1994). Nucleotide uptake and metabolism by intestinal epithelial cells.

The Journal of Nutrition, 124(1), 131-137.

Sakai, M., 1999. Current research status of fish immunostimulants. Aquaculture 172, 63– 92.

Sakai, M., Taniguchi, K., Mamoto, K., Ogawa, H., Tabata, M. (2001). Immunostimulant effects

of nucleotide isolated from yeast RNA on carp, Cyprinus carpio L.. Journal of Fish

Diseases, 433-438.

Sauer, N., Bauer, E., Vahjen, W., Zentek, J., & Mosenthin, R. (2010). Nucleotides modify

growth of selected intestinal bacteria in vitro. Livestock Science, 133(1-3), 161-163.

Sauer, N., Mosenthin, R., & Bauer, E. (2011). The role of dietary nucleotides in single-

stomached animals. Nutr. Res. Rev. Nutrition Research Reviews, 24(01), 46-59.

Secombes C.J. (1990) Isolation of salmonid macrophages and analysis of their killing activity.

In: Techniques in Fish Immunology, Vol. 1 (ed. by J.S. Stolen, T.C. Fletcher, D.P.

Anderson, & B.S. Roberson), pp. 137–163. SOS Publications, Fair Haven, NJ, USA.

71

Spinelli, J., Houle, C. R., & Wekell, J. C. (1983). The effect of phytates on the growth of

rainbow trout (Salmo gairdneri) fed purified diets containing varying quantities of

calcium and magnesium. Aquaculture, 30(1-4), 71-83.

Storebakken, T., Shearer, K., & Roem, A. (1998). Availability of protein, phosphorus and other

elements in fish meal, soy-protein concentrate and phytase-treated soy-protein-

concentrate-based diets to Atlantic salmon, Salmo salar. Aquaculture, 161(1-4), 365-379.

Storebakken, T., Refstie, S., & Ruyter, B. (2000). Soy products as fat and protein sources in fish

diets for intensive aquaculture. Soy in Animal Nutrition, 127-170.

Suresh, A. V., Vasagam, K. K., & Nates, S. (2011). Attractability and palatability of protein

ingredients of aquatic and terrestrial animal origin, and their practical value for blue

shrimp, Litopenaeus stylirostris fed diets formulated with high levels of poultry

byproduct meal. Aquaculture, 319(1-2), 132-140.

Tahmasebi-Kohyani, A., Keyvanshokooh, S., Nematollahi, A., Mahmoudi, N., & Pasha-Zanoosi,

H. (2011). Dietary administration of nucleotides to enhance growth, humoral immune

responses, and disease resistance of the rainbow trout (Oncorhynchus mykiss) fingerlings.

Fish & Shellfish Immunology, 30(1), 189-193.

Tahmasebi-Kohyani, A., Keyvanshokooh, S., Nematollahi, A., Mahmoudi, N., & Pasha-Zanoosi,

H. (2011). Effects of dietary nucleotides supplementation on rainbow trout

(Oncorhynchus mykiss) performance and acute stress response. Fish Physiol Biochem

Fish Physiology and Biochemistry, 38(2), 431-440.

Tan, Q., Liu, Q., Chen, X., Wang, M., & Wu, Z. (2013). Growth performance, biochemical

indices and hepatopancreatic function of grass carp, Ctenopharyngodon idellus, would be

impaired by dietary rapeseed meal. Aquaculture, 414-415, 119-126.

72

Toko, I. I., Fiogbe, E. D., & Kestemont, P. (2008). Mineral status of African catfish (Clarias

gariepinus) fed diets containing graded levels of soybean or cottonseed meals.

Aquaculture, 275(1-4), 298-305.

Uauy, R., Stringel, G., Thomas, R., Quan, R., 1990. Effect of dietary nucleotides on growth and

maturation of the developing gut in the rat. J. Pediatr. Gastroenterol Nutr. 10, 497–503.

Usami, M., Iso, A., Kasahara, H., Kotani, G., Haji, S., Kanamaru, T., & Saitoh, Y. (1996). Effect

of a parenteral nucleoside-nucleotide mixture on hepatic metabolism in partially

hepatectomized cirrhotic rats. Nutrition, 12(6), 436-439.

Van Buren, C. T., Kulkarni, A. D., Fanslow, W. C., & Rudolph, F. B. (1985). Dietary

nucleotides, a requirement for helper/inducer T lymphocytes. Transplantation, 40(6),

694-697.

Van Buren, C. T., & Rudolph, F. (1997). Dietary nucleotides: A conditional requirement.

Nutrition, 470-472.

Viola, S., Mokady, S., Rappaport, U., & Arieli, Y. (1982). Partial and complete replacement of

fish meal by soybean meal in feeds for intensive culture of carp. Aquaculture, 26(3-4),

223-236.

Wang, Y., Kong, L., Li, C., & Bureau, D. (2010). The potential of land animal protein

ingredients to replace fish meal in diets for cuneate drum, Nibea miichthioides , is

affected by dietary protein level. Aquaculture Nutrition, 16(1), 37-43.

Watanabe, T., Verakunpiriya, V., Watanabe, K., Kiron, V., & Satoh, S. (1997). Feeding of

rainbow trout with non-fish meal diets. Fisheries Science, 63(2), 258-266.

Windsor, M. (1971). Fish meal. Aberdeen: Torry Research Station.

73

Welker, T. L., Lim, C., Yildirim-Aksoy, M., & Klesius, P. H. (2011). Effects of dietary

supplementation of a purified nucleotide mixture on immune function and disease and

stress resistance in channel catfish, Ictalurus punctatus. Aquaculture Research, 42(12),

1878-1889.

Wischke, C., Weigel, J., Bulavina, L., & Lendlein, A. (2014). Sustained release carrier for

adenosine triphosphate as signaling molecule. Journal of Controlled Release, 195, 86-91.

Xu, M., Liang, R., Guo, Q., Wang, S., Zhao, M., Zhang, Z. (2013). Dietary nucleotides extend

the life span in sprague-dawley rats. The Journal of Nutrition, Health & Aging, 223-229.

Yousefi M, Abtahi B, Kenari A (2011) A hematological, serum biochemical parameters, and

physiological responses to acute stress of beluga sturgeon (Huso huso, Linnaeus 1785)

juveniles fed dietary nucleotide. Comp Clin Pathol.

Yurkowski, M., Bailey, J. K., Evans, R. E., Tabachek, J. L., Ayles, G. B., & Eales, J. (1978).

Acceptability of rapeseed proteins in diets of rainbow trout (Salmo gairdneri). J. Fish.

Res. Bd. Can. Journal of the Fisheries Research Board of Canada, 35(7), 951-962.

Zhang, Y., Øverland, M., Shearer, K. D., Sørensen, M., Mydland, L. T., & Storebakken, T.

(2012). Optimizing plant protein combinations in fish meal-free diets for rainbow trout

(Oncorhynchus mykiss) by a mixture model. Aquaculture, 360-361, 25-36.

Zhong, G., Hua, X., Yuan, K., & Zhou, H. (2011). Effect of CGM on growth performance and

digestibility in puffer (Takifugu fasciatus). Aquaculture International Aquacult Int, 19(3),

395-403.

Zinn, K. E., Hernot, D. C., Fastinger, N. D., Karr-Lilienthal, L. K., Bechtel, P. J., Swanson, K.

S., & Fahey, G. C. (2009). Fish protein substrates can substitute effectively for poultry

by-product meal when incorporated in high-quality senior dog diets. Journal of Animal

74

Physiology and Animal Nutrition, 93(4), 447-455.

Zrenner, R., Stitt, M., Sonnewald, U., & Boldt, R. (2006). Pyrimidine and purine biosynthesis

and degradation in plants. Annual Review of Plant Biology Annu. Rev. Plant Biol., 57(1),

805-836.

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