1

Investigating the effects of a dietary inclusion of Actigen and

Aquagard on the health and overall performance of

yellowtail kingfish.

Shayla Stefanetti

Supervisors: Gavin Partridge and Alan Lymbery

Fish Health Unit, School of Veterinary and Life Sciences. Murdoch University, Murdoch WA

6150.

Australian Centre for Applied Aquaculture Research (ACAAR), Challenger Institute of

Technology, Fremantle, Western Australia, 6160.

A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Animal

Science, Murdoch University, 2016.

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Contents

Declaration……………………………………………………………………………………… 3

Acknowledgements…………………………………………………………………………….. 4

Literature Review……………………………………………………………………………… 5

Research Article……………………………………………………………………………….. 21

Abstract………………………………………………………………………………………... 22

1 .Introduction………………………………………………………………………………… 22

2. Materials and Methods…………………………………………………………………….. 26

2.1 Experimental design………………………………………………………………………... 26

2.2 Data analysis……………………………………………………………………………….. 28

3. Results………………………………………………………………………………………. 28

3.1 Growth……………………………………………………………………………………... 28

3.2 Survival…………………………………………………………………………………….. 29

3.3 Food conversion ratio……………………………………………………………………… 30

3.4 Blood tests…………………………………………………………………………………. 31

3.5 Lysozyme activity…………………………………………………………………………. 32

3.6 Gut villus and mucous cell count………………………………………………………….. 33

4. Discussion…………………………………………………………………………………... 34

5. Conclusions………………………………………………………………………………… 39

Literature Review References……………………………………………………………….. 41

Scientific Paper References………………………………………………………………….. 46

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Declaration:

This thesis has been composed by myself and has not been accepted in any previous application

for a degree. The work, of which this is a record, has been done by myself and all sources of

information have been cited.

Signed:

Shayla Stefanetti

4/11/2016

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Acknowledgements:

I would like to thank my supervisors Gavin Partridge and Alan Lymbery for their overall

guidance, assistance, patience and advice.

I would also like to thank the team at the Australian Centre for Applied Aquaculture Research

(ACAAR) for their knowledge and assisting me in completing this project.

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Literature Review

1. Introduction

Aquaculture can be defined as the production and culture of animals and plants in either fresh

or marine water. The global market demand for fish is continuously increasing, and currently

provides 20% of animal protein for three billion people (Bowyer, 2008). Fish farming in

enclosed and monitored conditions is an effective way to increase production with the extra

benefits of being able to control variables such as diet, nutrition and improve in the

development of dietary additives and formulations (Murray et al., 2005). By providing fish

with better and more effective diets, aquaculture can assist in producing a higher quality

product for consumers.

The capture of fish has been declining in recent years, due to factors such as extreme

exploitation and pollution of the environment, as well as an increase in demand for fish

products. This is why aquaculture production must expand in order to contribute and provide

fish for the global demand, and has been seen to be growing at 7.7% per year for the last

decade (Stone, 2013). However, the industry is still plagued with having to contend with

severe under-investment, the continuing rising of costs and a huge amount of inconsistences

in the approaches to achieving a quality product, due to the lack of research in aquaculture,

especially in Australia. There is also the large issue of disease which currently negatively

impacts the aquaculture industry and causes major production loss (Murray et al., 2005).

Australian aquaculture is one of the fastest growing primary food industries, and is the 4th

most valuable food industry in the country, behind only beef, wheat and milk, and had a

gross production value of $868 million in 2008 (ABARE, 2009). The majority of production

is from the coastal zone, although Australian aquaculture does use both marine and

freshwater resources. The industry has been growing at 4% per year over the last decade, and

produces 50 species commercially. The most important species being produced are southern

bluefin tuna, the pearl oyster, Australian prawns, Atlantic salmon and yellowtail kingfish, as

these species make up 90% of the total production value in Australia. The culture of finfish is

based on four major species, Atlantic salmon, southern bluefin tuna, barramundi and

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yellowtail kingfish, with these four industry sectors producing approximately $477.7 million

in 2007 (Stone, 2013).

2. Yellowtail kingfish (Seriola lalandi)

Yellowtail kingfish are a temperate, carnivorous finfish species of marine fish that are found

widely throughout the Pacific and Atlantic oceans (Bowyer et al., 2012). Yellowtail kingfish

are a fast growing species that are well suited to sea cage conditions, making them an

increasingly popular fish to be used in aquaculture. The entire yellowtail kingfish or Seriola

industry is at a production of 200,000 tonnes, with approximately 90% of this production

coming from Japan. It is important to state that there are several different species of

yellowtail kingfish, with the Japanese industry comprising of three different species of

yellowtail. This review will focus on the specific species Seriola lalandi which makes up

only a small proportion of this figure (Bowyer, 2008).

In Australia, the yellowtail kingfish industry is at a production of approximately 4,000 tonnes

per year and is worth $29.2 million (Econsearch, 2010). South Australia is responsible for the

majority of production in Australia where juvenile yellowtail kingfish are produced in

hatcheries, which is in contrast to Japan where juveniles are sourced from the wild. In Japan,

yellowtail kingfish are the second most popular species produced, with Bluefin Tuna being

the most valuable product. However, with increased pressures to reduce catching quotas for

Bluefin Tuna, the demand for Yellowtail kingfish is likely to increase in both Japan and

around the world (Ma, 2009).

Yellowtail kingfish are considered to be a premium quality product and are a circumglobal

species, supporting both commercial and recreational industries worldwide. The fish can be

marketed as a whole fish, as fresh or frozen fillets, or as cutlets and loins. In Japan, the

species is popular as sashimi, along with bluefin tuna. Therefore, due to the large popularity

and increasing importance of yellowtail kingfish in the aquaculture industry, new research

into effective diets and supplements is necessary in order to produce a better quality product

and provide efficiently for the increasing global demand (D’Antignana, 2008).

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3. Disease in Aquaculture

Infectious diseases in fish are caused by either bacteria, viruses or parasites and are one of

the most primary concerns in aquaculture, and need to be effectively controlled in order to

achieve success in the industry. The maintenance of fish at high densities, allows for the easy

spread of pathogens, so although there are many measures put in place to control and

minimise disease, infections will still occur on monitored fish farms (Ma, 2014).

Factors such as the occurrence, severity and spread of infectious diseases between fish in

aquaculture are similar to factors associated with diseases found in humans and other species

of animal. However, there is one important factor which is unique to other terrestrial animals

which is the water environment in which fish live. Water facilitates in the spread of disease

in two ways: vertical transmission, where pathogens can be spread from parents to the

offspring; or horizontal transmission, where the pathogens are spread from one fish to

another directly through the water (Murray et al., 2005).

How a disease then develops following exposure involves a variety of variables including:

virulence, the immune strength of the fish, previous exposure to pathogens, the genetic and

physiological condition of the fish, their nutrition status, the amount of stress the fish is

under, and the population density. The population density is an important variable as the

denser a population is, the easier it will be for disease to spread in a population as there is

increased opportunity for infected fish to associate with uninfected fish (Murray et al., 2005).

It is also common for one pathogen to have many different strains which can all vary in how

severely they can cause disease, and all fish will differ in how susceptible they are to these

different pathogens. These variables, along with the pathogen, host and environment will all

affect the severity of disease and its development (Meyer, 1991).

The control of pathogens can be achieved through the attempt of effective management

practices and using approved drugs, antibiotics or vaccines. However, a severe problem for

the aquaculture industry is the extensive use of vaccines and antibiotics and the possibility of

this leading to antibiotic resistant strains of bacteria in the environment (Defoirdt et al.,

2011). While vaccines and antibiotics have been seen to be effective against the treatment

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and prevention of some diseases, additional methods are often required to control the spread

of disease. Also, unlike the treatment of humans or other animals, there are few drugs and

vaccines available for treating disease in fish (Fielder, 2011). These issues with antibiotics,

vaccines and other chemical treatments, means that much more emphasis is put into other

ways of prevention such as having good management practices and the possible use of

immunostimulants which will be discussed further in this review.

4. The Immune System

The most important physiological mechanism for animals for protection against pathogens is

their immune system. The immune system can be separated into two different types: innate

immunity which uses germline-encoded molecules to detect invading microbes, and acquired

immunity where detection is dependent on molecules being produced by somatic

mechanisms during the ontogeny period of an animal (Tort et al., 2003). The innate system

however is still important in vertebrates as it plays an important role in homeostasis and the

acquired immune response, and is the fundamental defence mechanism used by fish

(Magnadottir, 2006).

4.1 The acquired system

The acquired system plays an important role in providing protection against recurrent

infections by producing memory cells and specific receptors such as T cell receptors and

immunoglobulins, which allow for specific pathogens to be efficiently removed. The

production of vaccines relies heavily on the principle of the adaptive immune system;

however, this does not mean that it is more important than the innate system.

4.2 The innate system

The innate system acts as the first line of defence to prevent the attack of pathogens and so

this is why immunostimulants should act through the enhancement of the innate immune

response (Tort et al, 2003). The innate immune system is able to recognise non-self and

signal alarms through the use of germ-line encoded receptors which are able to detect certain

pathogens through their molecular pattern (Gómez & Balcázar, 2008). There are factors

which can affect the activity of innate parameters, both internal and external, and include

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temperature changes in the water, and stress such as handling. Other factors however such as

immunostimulants have the ability to enhance the innate immunity parameters. The main

cells primarily associated with the innate immune system are phagocytes including

macrophages, neutrophils, as well as other granulocytes, cytotoxic and epithelial cells

(Sweetman, 2010). The innate system is divided into three parts: physical barriers, cellular

components and humoral components. The initial physical barriers include scales, mucous

surfaces, the gills, and the epidermis which act as the first line of defence against infection

(Magnadottir, 2006). Mucous plays an important defence role and is widely documented in

literature and will be discussed further in this review. Cellular components include the

various macrophages and cytotoxic cells which are key cells of the innate immune system

and will also be discussed further in this review. The humoral parameters include enzymes,

growth inhibitors, antibodies, cytokines and precipitins (Magnadottir, 2006).

Unfortunately, literature states that there are limited studies and research on both the

ontogenic development of the innate and adaptive immune system of fish. What also makes

research difficult is that the immune parameters and acquired defence differs vastly between

fish species, but what is known is that they generally develop late. It is also known that

because of this late development, active phagocytes and enzyme activity occur in the early

development of a fish, either before or immediately after hatching, because the innate

defence is the only form of protection during this period (Infante et al., 2001).

4.3 Mucous

It has been well established in literature that the mucosal layer of the gut acts as the first line

of defence for fish, using both a physical barrier as well as chemical and cellular barriers in

order to prevent an infection. Mucous is secreted by epithelial tissue and lines all the external

surfaces of fish, is present in the gills, and lines all the internal surfaces of the gut, creating

barriers to stop the potential invasion of pathogens. Mucous in the gut however, will exhibit

different roles than external mucous such as the production of factors to help digestion,

enhancing healthy microflora in the gut, and also the regulation of the defence of the immune

system (Zhao, 2015). Mucous contains a number of substances to aide in chemical defence

including glycoproteins, cytokines, lectins, proteases, lysosomes and antibodies, which can

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work either directly or indirectly to stop infection causing bacteria. Research also states that

mucous has natural anti-microbial activity, and studies show that it is capable of neutralising

infectious viruses in fish (Bansemer, 2009).

4.4 Mucous cells

Intestinal epithelium contains a variety of cells including enterocytes, goblet mucous cells

and enteroendocrine cells to aide in absorption. Enterocytes are narrow in structure, with a

long nuclei and lamellar structures which increase basolateral surface area, and literature

notes that fish lack certain lateral characteristics of enterocytes of mammals. Surface area is

essential for ion regulation and nutrient uptake. Enterocytes have a membrane which consists

of microvilli which forms a brush border, providing 90% of the total epithelial surface area

which creates an absorption interface where enzymes are found, and where absorption and

transport will happen (Infante et al., 2001). The microvilli are very tightly packed and so

creates a sieving effect, preventing large particles from entering the space of the microvilli.

Morphologically, enterocytes are designed primarily for an absorptive function, in particular

for lipids and proteins (Campbell, 1988).

The majority of mucous cells in the intestines are goblet cells, and are named as their

structure resembles a goblet shape. The cells have a tapered stem which can both widen and

constrict, allowing the secretion of mucous through a pore. The cells contain mucin granules,

and literature states that the majority will also contain sialomucin which is an acidic

mucosubstance (Campbell, 1988). Enteroendocrine cells are seen throughout the epithelium

in the gastrointestinal tract in all species of fish. They are easily identified by their secretory

vesicles which is found in their cytoplasm. The main role of enteroendocrine cells is to work

together with the pancreas to constitute the gastroenteropancreatic endocrine system (Infante

et al., 2001).

4.5 Lysosome Activity

When fish are exposed to pathogens, their first line of defence in fighting off an infection is

their innate immune system. Lysozyme activity is an important component of innate

immunity in fish and is an effective indicator in assessing a fish’s health and response to

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pathogens. Lysosomes can be defined as membrane bounded organelles which differ in their

shape and size. These organelles are found in both animal and plant cells, and hydrolyse

linkages in the cell wall component of bacteria, producing lysozymes (Saurabh & Sahoo,

2008). Lysosomes main functions are to assist with material that is degrading which has been

taken in from outside the cell and to deal with degraded components inside the cell. Recent

studies on lysosomes indicate that the organelles contain hydrolytic enzymes or lysozymes

which are inactivated at the time (Saurabh & Sahoo, 2008). These lysozymes are then

activated when a lysosome combines with another organelle, and then digestive reactions

will take place. These enzymes act as a defence molecule and are well documented in

literature concerning many different species of animal (Le, 2014).

Seen in both invertebrates and vertebrates, lysosomes use multiple pathways to ultimately

kill bacteria within the body. The first mechanism in which lysosomes kill bacteria is through

enzymatic and non-enzymatic mechanisms. The second, is that lysosomes can regulate the

overall response the body will have to bacteria. It is well documented in literature that fish

have the ability to exhibit lysozyme activity against both Gram-positive and Gram-negative

bacteria. Lysozyme activity is opsonic and are also seen to activate phagocytes to assist in the

killing of bacteria, and can be seen in mucous, lymphoid tissue and the plasma of the blood

(Saurabh & Sahoo). Several studies on several fish species including sea bass and salmonid

species have detected the presence of lysosomes in both fertilised eggs and in the larval

stages, and research shows that the detection of lysosomes in the eggs and embryos has the

ability to prevent the vertical transfer from mother to progeny of pathogens (Magnadottir,

2006). Lysozyme activity has also been seen to differ depending of various factors such as

the size, sex, age, water temperature, season, pH, and the severity of the infectious stressors

(Le, 2014).

5. Role of Immunostimulants in Aquaculture

In Western Australia, kingfish are the subject of industry development, but they have been

seen in the past to suffer multiple health issues, some of which have been associated with a

poor diet. This can lead to severe losses for producers and results in large amounts of money

being spent on various treatments. The use of feed additives or immunostimulants has

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become the preferred way to stimulate the immune system of the fish, and improve their gut

health and resistance to diseases (Galindo-Villegas & Hosokawa, 2004).

Immunostimulants can be defined as “a naturally occurring compound that modulates the

immune system by increasing the host’s resistance against diseases that in most

circumstances are caused by pathogens (Montet & Ray, 2009).” Immunostimulants can be

divided into various groups based on their sources, not their mode of action: bacterial, algae-

based, animal-based, nutritional factors, and hormones (Bricknell & Dalmo, 2005). Many

studies have demonstrated that immunostimulants are beneficial to fish in that they protect

the animal from bacteria and infectious diseases and overall assist in increasing survival

rates, especially in larval fish where it is most beneficial to aid the innate immunity response

(Gannam, 1999).

Immunostimulants enhance both the humoral and cellular response in both specific and non-

specific ways. The specific immunostimulation relates to the host’s ability to react to a

specific antigen, while non-specific immunostimulation relates to the ability of the immune

system to respond when a host is exposed to pathogens and may be immune-compromised

(Galindo-Villegas & Hosokawa, 2004).

An ideal immunostimulant will have certain characteristics such as being non-toxic, even

when fed at a high rate, and be non-carcinogenic with no long term side effects. The

stimulant should be capable of stimulating both the innate and adaptive immune responses

and the breakdown of the product be inactive or easily degradable in the environment. It also

important that the stimulant is activated by the oral route as ingestion is vital in order to

achieve an immune response. Finally, the stimulant should be able to function with a

synergist relationship with antibiotics, as well as be relatively cheap and palatable (Montet &

Ray, 2009).

Several studies have shown that particular immunostimulants have increased lysozyme

activity and also provided protection against Photobacterium damsela, are bacteria which

frequently cause mortalities in the aquaculture industry (Zhao et al., 2015). Literature states

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though that there is still only a small amount of immunostimulants available on the market as

they are relatively new to the industry, with further research being needed. It is to be said

also that if immunostimulants are fed in too high a dose this could cause immunosuppression

and be detrimental to the health of the fish (Montet & Ray, 2009).

There are particular types of immuostimulants which are popularly used in aquaculture

today, the first being Muramyl dipeptide (Anderson, 1996). This is a simple glycoprotein and

a purified form of mycobacteria. Another immunostimulant commonly used is Levamisole

which is an anthelmintics chemical shown to assist in suppressor cell function (Li et al.,

2006). The third is Glucans which are the most popular immuostimulants used in aquaculture

as it is shown to have excellent immunostimulatory properties, and will be discussed further

in this review.

5.1 Advantages of an Effective Oral Treatment of Immunostimulants

The inclusion of an immunostimulant as a feed additive has obvious advantages over other

forms of treatment for aquaculture, such as bathing and injection (Clarke, 2008). The

inclusion of a feed additive means that there is less labour and time involved, and also less

stress that the fish has to undergo. Repeated handling of fish, the time lost for feeding and the

loss of dissolved oxygen that occurs during bath treatments has the likely potential to cause

mortalities and loss of growth due to stress (Conte, 2004).

By including immunostimulants into feed, there are wider safety margins and fish are not

handled and exposed to stressful situations. There is also the potential to increase treatment

efficiency as stimulants can be added to feed in mass amounts and fed to all fish, allowing all

fish to be treated quickly and efficiently, and also means that the fish can maintain their

regular feeding regime. Immunostimulants as a feed additive also will have a smaller

environmental impact than other treatment methods, such as bathing where chemicals are

released directly into the water (Grant, 2002).

Unfortunately, there are some disadvantages to being included as a feed additive. As the

immunostimulant is added to feed this means that it is impossible to uniformly spread the

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concentration evenly between fish, something that can be more accurately achieved in a

method such as injection. Smaller, slower fish may not ingest the same amount as larger

more voracious fish might, resulting in fish receiving different amounts of the

immunostimulant (Shet & Vaidya, 2013). There is also the possibility that certain

immunostimulants will decrease the palatability of feed which can result in fish eating less

and will slow their growth (Williams et al., 2007).

6. Role of Yeast in Aquaculture

It has been identified that yeast is part of the normal microbiota present in both the gut and

gastrointestinal tract of wild and farmed fish and plays an important role in fish health and

nutrition. Yeast has been shown to have extensive metabolic potential which is seen by the

production of various enzymes as well as containing components such as β-glucans and

mannoproteins which act by stimulating the immune system of fish. Through understanding

the role of yeast microbiota in fish health, an increase in production performance is likely to

be seen (Navarrete et al, 2014).

When compared to bacterial cells, yeast cells can be a hundred times larger, which is why

feeding fish a diet which includes just a low population of yeast can have major health

benefits. Recent studies have shown that diets supplemented with yeast stimulate better

growth, feed efficiency and conversion, bloody chemistry, reduced mortality rates, gut mucus

lysozyme activity and non-specific immune responses in species including catfish and

rainbow trout (Zhao et al., 2015). Studies have shown that yeast stimulates the activity and

expression of digestive enzymes (trypsin, lipase, and amylase) which are secreted from the

pancreas, and indicate gut maturation (Navarrete et al., 2014). Various literature indicates

that yeast can be introduced to fish in different ways, including adding yeast directly into

water, fed in live feed using rotifers, or administered as an additive into a formulated diet

(Navarrete et al, 2014).

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6.1 Yeast β-glucans: Structure, Role and Mechanisms of Action

β-glucan is a glucose polymer and is a major structural component of the cell wall of yeast,

as well as other sources such as plants, bacteria and fungi. These molecules have various

immune modulating properties, with studies showing that when included in an oral diet can

stimulate the immune system of the animal (Meena, 2013). β-glucan molecules are part of a

group of modifiers called biological response modifiers and are physiological active

compounds, capable of interacting with cell surface receptors in order to induce a response

(Meena, 2013).

β-glucan molecules differ in terms of structure, their size and overall physiological function,

with the literature describing the structure and activity of β-glucan molecules proving to be

controversial. Most evidence appears to show that the larger the molecular weight of a β-

glucan molecule, the more active that molecule is likely to be, when compared to a smaller

molecule (Meena, 2013). The solubility also has been seen to affect the activity of a

molecule, with soluble β-glucan molecules showing more activity than non-soluble

molecules (Kumari & Sahoo, 2006). β-glucan molecules have shown to be effective in many

species of animals, including humans, where they have seen to provide health benefits

including anticancer mechanisms, the prevention of metabolic syndromes, lower cholesterol

and promote skin health (Kumari & Sahoo, 2006).

In aquaculture, yeast β-glucans have been used to stimulate the innate immune system of the

fish in order to reduce mortality rates. Until fish have developed adaptive immune responses

against pathogens they are easily vulnerable to infections, which is why the inclusion of β-

glucans as a feed additive has been seen to be successful in improving survival rates in fish,

as well as other health benefits. β-glucan can be sourced from a variety of ways, and most

often comes from Saccharomyces cerevisiae or known as Baker’s yeast (Kumari & Sahoo,

2006). Now as aquaculture continues to grow, there is also the production of commercial

glucan feed additives currently on the market. Some of these include MacroGard ®, Betagard

A ®, and Aquagard ®, and are increasing in popularity (Navarrete et al., 2014).

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Studies have shown that the inclusion of β-glucans in fish diets has resulted in specific

immune effects on the production of antibodies, gene expression, increased survival rates and

stress resistance, resistance to infectious diseases, and growth and weight enhancement

(Kumari & Sahoo, 2006).

6.2 Mannan-Oligosaccharides: Structure, Role and Mechanisms of Action

In recent years, more has been discovered about the complex carbohydrate structures of the

yeast cell wall. Mannan-oligosaccharides are a glucomannanprotein complex which are

produced when enzymes hydrolyse the inner cell wall of fungi or yeast. The outer wall of the

fungi or yeast contains a carbohydrate glucomannoprotein which acts to bind pathogens,

providing various methods of toxin binding which act to have a synergistic effect with one

another (Dimitroglou, 2010).

Studies have shown that mannan-oligosaccharides can bind to pathogen, such as frequently

seen bacteria Staphylococcus aureus, Escherichia coli and Pseudomonas spp. This means

that the bacteria cannot colonize in the gastrointestinal tract and infections will be less

frequent. Studies also show that mannan-oligosaccharides will especially benefit young

animals that have an intestinal tract which is still in its maturing phase and have yet to

establish a mature population of microflora (Salze, 2008). Literature states that they are

popularly used in conjunction with antibiotics or other immunostimulants such as β-glucans

in order to achieve the most effective result and can then exhibit the ability to assist in times

of stress and have many other benefits to the productivity of the animal (Torrecillas et al.,

2015).

Mannan-oligosaccharides work in one method by providing a source for attachment of

pathogens, which is done using villi which protrude from the lining of the gastrointestinal

tract and contain large amounts of lectins, which are important in the attachment process.

Normally, the pathogens would bind to the villi and then infect the host, however the

mannan-oligosaccharides will adsorb the bacteria once it attaches, and will also travel

through the gastrointestinal tract in order to prevent any further colonization of the bacteria

and keep the animal free from disease (Torrecillas et al., 2015).

17

Another method used by mannan-oligosaccharides is to assist in the growth of healthy

bacteria which will then out compete the disease causing bacteria. Studies show that being a

class of carbohydrate that is indigestible by intestinal enzymes, it allows mannan-

oligosaccharides to stimulate the growth of bacteria such as Lactobacillus spp which has

shown to stop the proliferation of the infectious bacteria. Pathogens are then unable to

multiply and colonize (Dimitroglou, 2010).

The final role used by mannan-oligosaccharides is their use of mannose binding protein.

Mannose stimulates the liver to produce the protein which can then bind to bacteria and

triggers what is called the complement cascade. This results in mannan-oligosaccharides

acting as an immune-activator, enhancing the effect of a healthy immune system (Staykov et

al., 2007).

7. Commercial Immunostimulants from Yeast

7.1 Actigen

The product Actigen is a yeast cell wall Mannan oligosaccharide which was produced by

Alltech Inc. and was developed upon the improvement of a previous product called Bio-

MOS. The Actigen product has shown to be more effective than Bio-MOS in numerous trials

on different species of fish. Actigen contains a Mannan oligosaccharide which has an effect

of an immunostimulant with evidence of improved growth performance and utilization of

feed (Hung, 2015).

According to many studies it was found that a supplementation of 0.08%- 0.12% of Actigen

was an ideal amount to achieve optimal results. Specific studies conducted on Catfish and

Rainbow trout resulted in improved weight gains of 13.7%, reduced mortality, increased feed

intake and improved indicators of immune status including increased lysosome activity and

leukocyte count (Hung, 2015).

Many studies also challenged fish with Edwardsiella ictaluri bacteria while being treated

with Actigen and results showed an increase in survival rates when compared to those

18

untreated with Actigen (Zhao et al., 2015). This increased survival rate was a result of an

improved immune response from the fish which was shown by an increase in lysosome

activity and leukocyte count. It has been concluded in studies that fish that are treated with

Actigen are overall healthier than those not treated with an immunostimulant (Bentea et al.,

2014).

7.2 Aquagard as an Immunostimulant

The product Aquagard is developed from food grade Bakers’ yeast through the use of

proprietary procedures such as autolysis. This allows for the activation of the hydrolytic

enzymes in the yeast, aiding in self-digestion. The cell walls of yeast can also be further

digested through the use of gluconases to produce mannan oligosaccharides and 1-3 and 1-6

beta glucans. Aquagard contains a polysaccharide where glucan is hydrolysed to produce

glucose units which are linked with 1-3 and 1-6 bonds (Duffus et al., 1982). Beta glucans

have the ability to prevent the attachment of harmful toxins and therefore decrease the

incidence of disease, which is achieved through the adsorption of the bacteria to an inert

material which then passes through the gastrointestinal tract and leaves the body (Kumari &

Sahoo, 2008).

Aquagard can be added to the regular diet of fish, and can be done either after the

manufacture or during the manufacture. Aquagard is recommended to be added at 0.1% of

the total weight of feed, and should be fed on a continuous basis, preferably 6 weeks on and

then a period of 2 weeks off (Hudson, 2015). It is recommended to be fed prior to fish during

periods of high risk of disease or high risk of stress, such as handling times, transport, or the

administering of vaccinations or other treatments (Hudson, 2015). Best results are seen when

the immune system of a fish has been stimulated before being challenged with a pathogen

(Sommerville et al., 2009).

Several studies of the oral treatment of Aquagard on various fish species including Atlantic

salmon, rainbow trout, Catfish, sand whiting and turbot have resulted in many benefits in the

prevention of disease. Reduced mortality rates and an increase in lysosome activity were seen

in fish tested against bacterium such as Vibrio anguillarum, Edwardsiella ictaluri and Vibrio

19

vulnificus, indicating an enhanced immunity in response to the addition of Aquagard in the

diet (Hudson, 2015). Aquagard has also been seen to have many benefits over other

treatments. The product is heat stable to temperatures which are used in the manufacture of

feed and so can be added to feed during the manufacturing process. It has also been found

that the product is not associated with being hazardous to the environment and so can be

disposed of safely with no residue concerns (Hudson, 2015).

8. Conclusions and Aims of Project

As aquaculture continues to grow and increase in production and profit, the industry is still

severely negatively impacted with disease and causes major losses on fish farms. An

effective way to combat disease in aquaculture is the use of immunostimulants as a feed

additive. Immunostimulants enhance the innate immunity system of fish, allowing them to

have higher growth rates, increased feed intake, higher survival rates and show an increase in

immunity in the form of lysosome activity and leukocyte counts. Immunostimulants can

contain mannan oligosaccharides, a glucomannanprotein complex of a yeast cell wall capable

of toxin binding and killing pathogens. β-glucans are also a major cell component of yeast

which are capable of stimulating cell surface receptors in order to induce a response from

toxins.

The gastrointestinal tract is the prime way fish become infected with pathogens, and so by

feeding immunostimulants orally this allows the protection of the gut from bacteria and an

increased immune system with the ability to fight off disease. Treating fish with an

immunostimulant orally as a feed additive also has other benefits such as less stress through

minimal handling, and the fact that fish are kept to their regular feeding regime so their feed

intake will not be negatively impacted. Immunostimulants are still new to the aquaculture

industry with not a large amount being available commercially yet. This project aims to

determine whether inclusion of the immunostimulants Actigen and Aquagard in the diet of

cultured yellowtail kingfish will have health and performance benefits when compared to a

standard diet. Specifically, I hypothesise that the immunostimulants will increase survival

rates, growth rates, feed conversion rates, and enhance the immune system by showing

20

evidence in blood parameters as well as increased lysosome activity, villi length and mucous

cell counts.

There are two products, Actigen and Aquagard which have shown promising results on

various species of fish, with increased survival rates, increased growth and feed intake, and

enhanced immune systems providing protection against disease. Although these products

have not yet been tested on Seriola lalandi, the previous studies on various species of fish

give evidence that the dietary inclusion of Actigen and Aqugard will show benefits to both

the gut health and overall performance of the fish.

21

Research Article

Investigating the effects of a dietary inclusion of Actigen and Aquagard on the health and overall performance of

Yellowtail Kingfish.

Shayla Stefanetti

Murdoch University

22

Abstract

Bacterial disease can have significant impacts in the culture of yellowtail kingfish, Seriola

lalandi. Immunostimulants induce an immune response to better effectively fight off disease and

have many advantages over the use of antibiotics. There are few commercially available

immunostimulants for fish, with Actigen and Aquagard being two products which have shown

potential. Recent studies on these two products have suggested they have the ability to increase

survival and growth rates, and improve feed conversion ratios, as well as stimulate the immune

system-shown by increased lysozyme activity, blood parameters and villus and mucous cell

counts, which is what this study hypothesised. These immunostimulants, however, have never

been tested in yellowtail kingfish. In this 16-week study, these parameters were measured in

yellowtail kingfish fed a commercial diet coated with Actigen or Aquaguard and compared

against the same diet without any immunostimulants. Survival of fish in the Aquagard treated

group was significantly higher than those fed the control diet and the Actigen diet following a

natural infection of Photobacterium damselae and Vibrio harveryi. The growth rates, feed

conversion ratios, blood parameters, lysozyme activity, villus height and mucous cell count did

not significantly differ among the treatment groups. This study indicates that the

immunostimulant Aquagard has the potential to enhance the immune system, however further

investigation is required to optimise dose and frequency of administration and gain a better

understanding of the long-term effects of immunostimulant treatment.

Key words: Immunostimulant, Actigen, Aquagard, Pelagica, yellowtail kingfish.

1. Introduction

Infectious disease in fish caused by bacteria, viruses and parasites are of major concern in

aquaculture. The farming of fish at high densities allows for the easy spread of pathogens, so

although there are many measures put in place to control and minimise disease, disease

outbreaks still occur commonly on fish farms (Ma, 2014). Vaccines, antibiotics and paraciticides

have been effective in the treatment and prevention of some diseases, however there are few

chemical compounds available and/or registered for treating disease in fish, particularly in

Australia, and additional methods are often required to control the spread of disease (Fielder,

2011).

23

In many countries, the regulation of antibiotics and paraciticides is very strict, with only a few

antibiotics being licensed for use in aquaculture. However, a large proportion of the global

aquaculture production occurs in countries with more permissive regulations, leading to risks to

public health. There is the potential for antibiotic-resistant bacteria from fish products to pose a

threat to consumers as resistance can be transferred among bacteria and lead to human pathogens

which cannot effectively be treated by antibiotics (Romero et al., 2012). Duran & Marshall

(2005), for example, tested various brands of ready-to-eat prawns from grocery stores and found

that 42% of 1,564 bacterial isolates from these prawns had resistance to antibiotics, as well as

finding several human pathogens including Escherichia coli, Salmonella and Vibrio spp. It is

very possible that the high proportions of antibiotic-resistant bacteria that are found in

aquaculture environments are a threat to the outside environment and to human consumers

(Romero et al., 2012). This means that increasing emphasis is being placed on preventative

disease management, such as the use of immunostimulants. In many species of cultured fish,

immunostimulants have become the preferred way to stimulate the immune system of the fish

and improve their gut health and resistance to diseases (Galindo-Villegas & Hosokawa, 2004).

Immunostimulants can be defined as “naturally occurring compounds that modulate the immune

system by increasing the host’s resistance against diseases that in most circumstances are caused

by pathogens (Montet & Ray, 2009).” Immunostimulants work by enhancing both the humoral

and cellular immune system and an ideal treatment will have certain characteristics such as being

non-toxic and non-carcinogenic (Galindo-Villegas & Hosokawa, 2004). It is also important that

the immunostimulant is capable of stimulating both innate and adaptive immune responses, and

is easily activated by the oral route as this is the preferred method of administration by producers

(Montet & Ray, 2009). There are particular types of immunostimulants used today in

aquaculture, with mannan-oligosaccharides and glucans being some of the most popular (Li et

al., 2006). Various studies have shown that mannan-oligosaccharides and glucan

immunostimulants can provide protection against bacteria, and show other health benefits such

as increased growth rate and feed conversion efficiency (Zhao et al., 2015).

Mannan-oligosaccharides are produced when enzymes hydrolyse the cell wall of yeast

containing a carbohydrate glucomannoprotein which binds to pathogens, using various methods

24

of toxin binding which act synergistically (Dimitroglou, 2010). They work principally by

adsorbing pathogens after they attach to the villi (Torrecillas et al., 2015), promoting the growth

of healthy bacteria to out compete disease causing bacteria, and can also release a mannose

binding protein, which can bind to bacteria to degrade it (Staykov et al., 2007).

Actigen is a product produced by Alltech Inc. and is a yeast cell wall mannan oligosaccharide.

This product has been demonstrated to improve growth performance and utilisation of feed in

fish with a supplementation rate of 0.08%- 0.12% (Hung, 2015). A study conducted on both

catfish (Ictalurus punctatus) and rainbow trout (Oncorhynchus mykiss) showed improved weight

gains, increased survival rates, improvement in feed intake and various improvements in immune

status such as increased lysosome activity and leukocyte count (Hung, 2015). A study conducted

where fish were challenged with the bacteria Edwardsiella ictaluri after being treated with

appropriate levels of Actigen, showed an increase in survival rates when compared to those

untreated with the immunostimulant (Zhao et al., 2015). It has been concluded from these studies

that fish treated with Actigen have an improved immune response and are overall healthier than

fish left untreated (Bentea et al., 2014).

β-glucan molecules are a type of biological response modifiers which are capable of interacting

with cell surface receptors to induce a response (Klis, 1994). They are a major structural

component of the cell wall of yeast and contain various immune modulating properties (Brown

& Gordon, 2001). These beta glucans have a demonstrated ability to prevent harmful toxins from

attaching to a host and can therefore decrease the incidence of disease, by absorbing the bacteria

to an inert material which can then be excreted by the fish through their gastrointestinal tract

(Kumari & Sahoo, 2008). β-glucans have been shown to be effective in many species of animals,

providing health benefits such as anticancer mechanisms, preventing metabolic syndromes,

lowering cholesterol and increasing survival (Kumari & Sahoo, 2006). Beta glucans differ from

mannan oligosaccharides by how they are absorbed in the body. Enterocytes facilitate the

transfer of beta glucans across the intestinal cell wall to the lymph where they then interact with

macrophages to activate an immune response (Frey et al., 1996). The structure of the two

different polysaccharides are similar as they are both structural components of the yeast cell wall

(Refstie et al., 2010). Studies conducted on cultured fish have resulted in specific immune

25

responses being seen with fish showing resistance to infectious diseases, growth enhancement

and an overall reduction in mortalities (Kumari & Sahoo, 2006).

Aquagard (Aquatic Diagnostic Services International Pty Ltd), is manufactured from food grade

bakers’ yeast. Using a process called autolysis, the hydrolytic enzymes in the yeast are activated

and 1-3 and 1-6 beta glucans are produced (Duffus et al., 1982). Studies on a number of species

of fish including Atlantic salmon (Salmo salar), rainbow trout (Ocorhynchus mykiss), catfish

(Ictalurus punctatus), sand whiting (Sillago ciliate) and turbot (Scophthalmus maximus) have

found several health benefits of Aquagard, including increased survival rates and an increase in

lysosome activity (Engstad et al., 1992; Jorgensen et al., 1993; Baulney et al., 1996). It was also

found that when these fish were challenged with the bacterium Vibrio anguillarum, Edwardsiella

ictaluri and Vibrio vulnificus, there was reduced mortality for those fish treated with Aquagard,

suggesting an enhanced immune response (Chen and Ainsworth, 1992).

Yellowtail kingfish (Seriola lalandi) are a marine finfish species found throughout most of the

world’s temperate oceans, widely supporting both commercial and recreational fisheries, as they

are a highly regarded eating fish (Bowyer et al., 2012). The entire Seriola industry produces

200,000 tonnes annually, with a significant portion of this production coming from Japan, which

has the largest marine fish aquaculture industry in the world (Bowyer, 2008). In Australia, the

culture of yellowtail kingfish is a developing industry and has suffered in the past due to multiple

health issues, some of which have been associated with a poor diet (Diggles & Hutson, 2005).

This has led to severe losses for producers and resulted in large amounts of money spent on

various treatments (Defoirdt et al., 2011). Despite immunostimulants, such as Actigen and

Aquagard being increasingly used in fish culture, their efficacy has not been tested in yellowtail

kingfish. The aim of this study was to test the hypothesis that the dietary inclusion of Actigen

and Aquagard will have health and performance benefits to yellowtail kingfish, when compared

to a standard diet. This was tested through surface coating feeds with Actigen and Aquagard and

comparing these to an untreated control.

26

2. Materials and Methods

2.1 Experimental design

The trial ran for 16 weeks and was conducted at the Australian Centre for Applied Aquaculture

Research (ACAAR) in Fremantle, Western Australia. The trial was undertaken in nine

experimental tanks of 10,000 litres each, containing 80 fish in each tank. The average starting

weight of the fish was 939g. Each tank was supplied with seawater sourced from a marine bore

with a flow rate of 100 litres per minute. The flow created a circular flow path which forced

uneaten feed and other wastes into the drain pipe located in the centre of the tank. Each tank

contained an air stone located in the centre of the tank which was used to maintain circulation

and provide oxygen.

During the trial, fish were fed to satiety twice daily (0900 and 1400 hours) on Ridley ‘Pelagica

Sink’ 6mm and 9mm pellets (changed after 6 weeks). The two immunostimulant treatments,

Actigen (Alltech Inc.) and Aquagard (Aquatic Diagnostic Services International Pty Ltd.) were

adhered to this diet with gelatin. Uncoated pellets of the same diet acted as the control treatment.

Three replicate tanks of fish received each diet treatment. To produce the gelatin coating, 200g

of powdered gelatin was added to 1000ml of water and dissolved at 55⁰C on a magnetic mixer.

Supplementation inclusion levels were calculated based on a 20kg batch of feed, and active

inclusion level recommendations from the product companies (0.1% for each product). Actigen

and Aquagard were added directly in powder form to the batches of pellets and mixed

thoroughly. Gelatin was then poured over the batch of pellets and mixed in a clean cement mixer

to ensure homogenous coverage across the whole batch of pellets. The pellets were then moved

into a cold room overnight for the gelatin to set. Daily food intake was recorded by weighing out

the pellets before feeding, and then weighing out the remaining pellets after feeding. The food

conversion ratio (FCR) was then calculated by dividing the amount of food consumed (g) by the

weight gain of the fish (g). Any mortalities during the trial were removed from the tank and sent

to the Fish Health Laboratories, Western Australian Department of Fisheries for a post mortem

analysis. At the end of each trial month all fish in each tank were lightly anaesthetised with

AQUI-S® (20mg/L), individually weighed, and one randomly selected fish per tank was

euthanised using a high dose (40mg/L) of AQUI-S® for a health assessment, including

measurement of the blood-parameters outlined in Table 1:

27

Table 1. Blood metabolites tested

At the end of the trial, histology was conducted on three sections of the gut (fore, mid and

hindsection). In these sections, mucous cells were stained with Hematoxylin and eosin, and a

combination of Alcian Blue and Periodic acid–Schiff. The quantification of mucous cells and

measurements of villi and their abundance was conducted at the end of the 4-month trial on these

three gut sections. These measurements were performed using the programme ImageJ. Multiple

images of each gut section were taken using a microscope and the image containing the most

intact gut villi was chosen. Every intact villus was then measured from the base of the villi to the

tip, and every mucous cell per villi was counted using the ImageJ programme.

Lysozyme was measured in blood serum at the end of the 4-month trial using an EnzChek

Lysozyme Assay Kit (E22013) (ThermoFisherScientific) using a fluorescence microplate reader.

Blood Metabolites

Packed cell volume Albumin

Creatine kinase Globulin

Alanine aminotransferase Calcium

Glutamate dehydrogenase Phosphorus

Urea Magnesium

Creatine Glutathione peroxidase

Cholesterol Hemoglobin

Sodium High-density lipoprotein

Potassium Low-density lipoprotein

Chloride

28

2.2 Data analysis

The number of replicate tanks per treatment was determined prior to the trial using a power

analyses for a one-way ANOVA design, assuming that response variables were measured on a

per tank basis. A sample size of three replicates provided 80% power to detect a significant

difference among treatments at alpha level of 0.05 and an effect size of ∆ = 2.5.

Differences among treatments in weight over time, feed conversion ratio and blood parameters

were measured using a one-way ANOVA, while differences in villi height and mucous cell count

among treatments were analysed using a two-way ANOVA, treatment diet and gut section as the

two factors.

Mortality rates were compared among treatments using a generalized linear mixed model

(GLMM), assuming a binomial distribution with a logit link function and tank nested within

treatment as a random effect. As treatment had a significant effect on the risk of mortality,

differences in mortality percentages between each pair of treatments were tested by Chi-square,

using a Bonferroni correction for multiple comparisons. In addition, survival times were

compared among treatments by the Kaplan-Meier method, with a Chi-square approximation to

the log-rank test. As treatment had a significant effect on survival time, differences between each

pair of treatments were tested by Chi-square, using a Bonferroni correction for multiple

comparisons.

All analyses were conducted using the programs SPSSv19 and JMPv10.

3. Results

3.1 Growth

Figure 1 shows the growth rate of fish in each treatment diet over the time of 4 months. Fish fed

the control Pelagica diet increased in size from 1012g to 2258g. Fish treated with Actigen

increased in size from 1003g to 2265g. Fish treated with Aquagard increased in size from 1014g

to 2351g which was the largest increase of the treatment groups, however there was no

significant difference in final growth weights among the diets at the end of the 4-month period

(one-way ANOVA; p = 0.45).

29

Figure 1. The average growth of fish fed each treatment diet over the 4-month period.

3.2 Survival

Over the 4 months of the trial, mortality rates were 19.5% for fish on the Pelagica diet, 13.5% for

fish on the Actigen diet and 5.6% for fish on the Aquaguard diet. Mortality rates differed

significantly among treatments (GLMM; χ22 = 0.003), with pairwise comparisons finding a

significant difference only between the Aquaguard and Pelagica diets (χ22 < 0.001), with the

Bonferonni.

Survival times are shown in Figure 2. Most mortalities occurred in the first 20 days of the trial

due to the naturally occurring bacteria Photobacterium damselae and Vibrio harveyi. Times to

death, analysed using the Kaplan-Meier method, differed significantly. There was found to be a

significant difference among treatments (χ22 = 0.002), with pairwise comparisons again finding a

significant difference only between the Aquaguard and Pelagica diets (χ22 < 0.01), with the

Bonferonni.

0

500

1000

1500

2000

2500

3000

0 1 2 3 4

Wei

ght (

g)

MonthActigen Aquaguard Pelagica

30

Figure 2. Survival probabilities over time for yellowtail kingfish fed Pelagica (control), Actigen and Aquagard diets.

3.3 Food Conversion Ratio

Figure 3 shows the food conversion ratio for each treatment diet at the end of the 4 month period.

Fish fed the Pelagica control diet had an average food conversion ratio of 1.83, fish treated with

Actigen had an average food conversion ratio of 1.87, and fish treated with Aquagard had a food

conversion ratio of 1.68. There was no significant difference in food conversion ratio among the

treatment groups (F₂, ₆ =2.02; p=0.21).

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

0 20 40 60 80 100 120

Surv

ival

Pro

bbal

ity

DayActigen Aquagard Pelagica

31

Figure3. Feed conversion ratios for yellowtail kingfish fed each treatment diet over the 4-month trial period.

3.4 Blood Tests

Table 2 shows the mean(+SE) blood parameter results from fish in each treatment at the end of

the 4-month period. There were no significant differences in any of the blood parameters tested.

1.45

1.5

1.55

1.6

1.65

1.7

1.75

1.8

1.85

1.9

1.95

2

Pelagica Actigen Aquagard

Food

con

vers

ion

ratio

Diet

32

Table 2. Blood parameter results for yellowtail kingfish from each treatment diet after the 4-month period.

3.5 Lysozyme activity

Figure 4 shows the average lysozyme activity at the end of the 4-month trial for each diet. Fish

fed the control Pelagica diet had an average of 4088 U/ml, fish fed the Actigen treatment had an

average of 3883 U/ml, and fish fed the Aquagard treatment had an average of 3045 U/ml. No

significant difference (p= 0.082982) was found between the diet treatments.

Blood Parameters Pelagica (control) Actigen Aquagard P value

PCV 49.2±0.9 46.0±0.9 48.0±0.8 0.63 CK 484.8±235.8 866.3±200.9 1301.3±251.6 0.24 ALT 23.8±6.5 17.7±10.2 40.7±8.5 0.19 GLDH 23.2±6.5 14.7±1.5 15.5±3.2 0.14 UREA 3.0±0.5 2.2±0.3 2.2±0.1 0.20 CREAT 22.0±0.2 23.0±0.5 22.3±0.4 0.91 CHOL 6.5±0.1 6.6±0.1 6.8±0.1 0.90 Na 202.3±2.4 206.3±2.2 207.0±1.5 0.74 K 5.4±0.5 6.0±0.3 4.6±0.4 0.67 Cl 174.3±0.8 176.3±1.0 176.3±0.9 0.94 SPROT 42.7±1.5 46.0±2.5 43.3±1.0 0.39 ALB 13.2±3.2 14.7±1.2 13.7±0.5 0.33 GLOB 29.5±2.0 31.3±0.5 29.7±0.9 0.48 Ca 3.5±0.8 3.8±0.9 3.5±0.4 0.19 P 3.2±0.2 3.3±0.5 2.5±0.9 0.35 Mg 1.9±0.1 1.9±0.1 1.6±0.1 0.89 Gpx U/g Hb 60.7±5.6 57.3±4.7 69.7±4.8 0.45 Hb g/L 238.0±22.4 278.7±15.3 228.7±17.3 0.25 Hb g/L WHOLE 139.4±25.6 193.5±19.4 153.0±12.7 0.43 HDL 2.3±0.1 2.4±0.1 2.4±0.1 0.25 LDL 3.3±0.5 2.8±0.4 3.9±0.3 0.21

33

Figure 4. Average lysozyme activity for yellowtail kingfish fed each treatment diet at the end of

the 4-month trial period.

3.6 Gut Villus length and mucous cell count.

Figure 5 shows the mean gut villus length by treatment diet and gut section. Two-way ANOVA

revealed no effect of treatment diet (F₂=0.42; p= 0.05) or gut section (F₂=0.53; p = 0.33) on

villi length, nor their interaction (F₄=0.68; p= 0.18).

Figure 5. Mean villus length (with SE bars) in fore-, mid- and hind-gut sections of yellowtail kingfish fed different treatment diets.

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Pelagica Actigen Aquagard

Lyso

zym

e Ac

tivity

(U/m

l)

Diet

0

200

400

600

800

1000

1200

1400

Actigen Aquagard Pelagica

Villi

Len

gth

(um

)

Diet Treatment

Fore Mid Hind

34

Figure 6 shows the mean mucous cell count by treatment diet and location of the gut section. No

significant effect of diet (F₂=0.42; p = 0.67) or their interaction (F₄=0.62; p = 0.36) was found

on mucous cell count. There was a significance difference between the gut sections (F₂=7.15;

p=0.005), with the hindgut section having significantly lower mucous cell counts when

compared to the foregut (p=0.017) and midgut (p=0.008).

Figure 6. Mucous Cell count for both the treatment diet and gut section location.

4. Discussion

The aim of this study was to determine whether inclusion of the immunostimulants Actigen and

Aquagard in the diet of cultured yellowtail kingfish would have health and performance benefits

when compared to a standard diet. Specifically, I hypothesised that the immunostimulants would

increase survival rates, growth rates, feed conversion ratios, and enhance the immune system by

showing effects on blood parameters as well as increased lysosome activity, villus length and

mucous cell counts. The results did partially support this hypothesis, with significantly decreased

mortality rate and survival time in fish fed the Aquagard diet, compared to the other treatment

groups. However, no significant benefit was evident in mortality rate or survival time for the

Actigen treatment, or between any of the diets for growth rates, feed conversion ratios, blood

metabolites, lysosome activity, villus length or mucous cell counts. It should be noted that there

0

20

40

60

80

100

120

140

160

180

200

Actigen Aquagard Pelagica

Muc

ous c

ells

per v

illi

Diet Treatment

Fore Mid Hind

35

was a significant difference in mucous cell counts found between the gut sections, and so

although this is not an effect of the treatments, it contradicts studies such as Lazado & Caipang

(2004) and Merrifield et al., (2011) which found that immunostimulants increases the count of

mucous cells in the gut.

The beneficial effects of Aquagard on fish survival were expected, because previous studies have

demonstrated the success of immunostimulants such as Aquagard in increasing survival rates

when compared to feeding a standard diet. Onarheim et al., (1992), for example, concluded that

Atlantic salmon pre-smolts that were fed a diet which included beta glucans (Aquagard) resulted

in reduced mortality rates when they were challenged with Aeromonas salmonicida, when

compared to those not fed beta glucans. The current trial did not challenge the fish with bacterial

infections like many of the previous studies, with mortalities instead being caused by

Photobacterium damselae and Vibrio harveyi which are naturally occurring infections (Austin,

2010). These naturally occurring infections are common causes of mortalities in the aquaculture

industry. The results of this trial show that the β-glucan molecules contained in the Aquagard

treatment significantly improved survival. Although the feeding of Aquaguard had a beneficial

effect on survival, there was no significant effect of the Actigen treatment. This is in contrast to

other studies such as Zhao (2015) which found that catfish fingerlings fed a diet supplemented

with Actigen, took significantly longer to die when challenged with Flavobacterium columnare

bacteria when compared to an unsupplemented diet.

Lysosomes contain active proteases, lipases and hydrolytic enzymes called lysozymes which can

generate toxic oxidative compounds that assist in microbial degradation, and high levels of

lysozyme can therefore be considered as an indicator that the fish is immunocompetent and has

produced an immune response against an infection (Mock & Peters, 1990; Roos and

Winterbourn, 2002). This study hypothesised that lysozyme levels would be increased as a direct

result of the addition of the immunostimulants to the diet. Other studies such as Zhao (2015),

which treated channel catfish with Actigen over a period of nine weeks, Engstad (1992) which

treated Atlantic salmon with beta-glucans for 7 weeks, and Chen and Ainsworth (1992) which

treated rainbow trout with beta-glucans for 9 weeks, have found increased lysozyme activity and

an enhanced immune response. Hung (2015) found that channel catfish fingerlings saw a

significant increase in serum lysosome levels in those fish which were treated with the inclusion

36

of Actigen to their diet after 10 weeks. Engstad et al., (1992) found that Atlantic salmon had

significant increases in serum lysozyme activity when the beta-glucans were included in their

diet over a 3 week period.

In contrast to these studies however, I found no significant differences in lysozyme activity

between fish fed the immunostimulants and fish fed their normal diet. Indeed, fish fed

Aquaguard had slightly (but not significantly) lower lysozyme levels at the end of the trial. It is

important to note, that although no significant differences were found in lysozyme in this trial,

blood of the same fish were analysed under another trial using flow cytometry and significant

differences were found in lysosome activity. Furthermore, the differences in lysosome activity

found from flow cytometry displayed the same pattern as seen within the lysozyme activity

within this trial. That is, fish on the control Pelagica diet showed the highest lysosome activity

and Aquagard treated fish had the lowest activity. These results are surprising as they indicate

that the fish treated with the immunostimulant Aquagard experienced the opposite effect from

was hypothesised, and instead had their lysosome levels negatively affected by the treatment.

These results can be seen in figure 7.

Figure 7. Average lysozyme activity for yellowtail kingfish fed each treatment diet at the end of

the 4-month trial period and average lysosome activity for yellowtail kingfish fed each treatment

diet in a separate trial.

a

ab

b

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Pelagica Actigen Aquagard

Lyso

zym

e/Ly

soso

me

activ

ity

(U/m

l)

Diet

Lysozyme (this trial) Lysosome (separate trial)

37

Despite the reduction in lysosome and lysozyme activity seen in this trial when fish were fed a

diet containing Aquaguard, mortality rate was decreased (and survival time increased). There are

several possible reasons for these apparently contradictory results. First, my findings may have

been influenced by the length of treatment. The studies on lysosome activity mentioned

previously had been conducted on fish over a 3 to 10 week interval, while the current trial ran for

16 weeks. Due to the majority of trials running for only up to a period of 10 weeks, the long term

effects of immunostimulants are unknown. However, studies have shown that high doses of an

immunostimulant can result in a lack of immune enhancement. Robertsen (1994) reported that

immune response in rainbow trout was increased at glucan concentrations of 0.1-1 mg/ml while

at 10 mg/ml the glucan had no effect. Although there appears to be no previous literature to

indicate that long term feeding of immunostimulants causes negative impacts, the lysozyme and

lysosome results from this trial suggests that prolonged feeding may lead to overstimulation of

the immune system. In this study, mortalities from infection occurred in the early part of the trial

and those fed Aquagard had significantly lower mortality. It is therefore possible that lysozyme

activity in these fish increased up until a certain point in time, but then declined as a result of

overstimulation by the end of the 4-month trial period. There is evidence for this occurring which

is seen in the survival analysis results, as the natural mortality event occurred early in the trial,

supporting the suggestion that the Aquagard treatment provided an immune benefit in the short

term. It is possible, therefore, that initial feeding of the Aquagard improved immunocompetence,

as shown by the reduced mortality rates early in the trial, but prolonged feeding led to

overstimulation of the immune system and a decrease in immunocompetence. More studies are

required, examining lysosome and lysozyme activity regularly over a prolonged period of time,

to test this hypothesis.

Immunostimulant treatments had no significant effects on growth rates, feed conversion rates,

blood parameters, villi length and mucous cell counts, and (in the Actigen treatment) survival

rates. There are multiple reasons as to why these results may have occurred. First, all previous

studies have been conducted on freshwater fish such as catfish, Atlantic salmon and rainbow

trout, not marine fish such as yellowtail kingfish. Although marine finfish such as yellowtail

kingfish are similar to salmonid and catfish species, yellowtail kingfish are unique in their

schooling and feeding habits (Ward et al., 1994). It is also possible that the different species

38

immune systems will vary and so while salmonids and catfish may respond in one way to a

disease, yellowtail kingfish may respond differently.

Whilst there are no major anatomical differences between freshwater and a saltwater fish, there

are major differences in how they control the flow of water across their body and osmoregulate

(McCormick, 2001). The body tissues of a saltwater fish will contain less salt than the outside

environment, causing the saltier outside water to draw water from the body tissues, resulting in

the fish continuously losing water through its skin and gills (Evans et al., 2005). In order to

compensate for this, a saltwater fish must drink large amounts of seawater and produce only a

small amount of urine and secrete salt through the gills (Manzon, 2002). Freshwater fish, by

contrast, contain more salt within their body tissues than the outside environment and so water is

able to flow continually through the skin and gills (Manzon, 2002). Freshwater fish, therefore do

not drink additional water and produce larger amounts of urine (McCormick, 2001). It is possible

that this difference in osmoregulation could have an effect on how immunostimulants are

ingested and passed through the body. Extra drinking may have flushed the immunostimulant

from the digestive tract faster than would occur in a freshwater fish leading to a reduced impact

compared to freshwater fish. Although this trial used the dosage recommended by the

manufacturer in the treatment on yellowtail kingfish, it is possible that these dosage levels are

best suited for different species of fish such as freshwater fish. As these products have never

before been tested on yellowtail kingfish, it is uncertain whether these dosage amounts were

appropriate for this species of fish and is an aspect of the trial that requires further research.

Another possible reason for my results could be the method of administration. In this trial, it was

decided that the method of administration would be oral ingestion and the immunostimulants

were included in the diet of the fish. Oral administration was used because it was less invasive to

the fish; injection requires anesthetising the fish, causing stress, as well as physical damage

during the handling process. In addition, injection involves considerable time and costs, and is

only possible for fish that are more than 10-15 g in weight (Barman et al., 2013). However, it is

important to state that the most effective method of administration of immunostimulants to fish

has been found to be by injection. Sakai (1999), reported that catfish injected with yeast glucan

showed increased resistance to certain bacteria, while those treated with by oral administration

showed no effect. Similarly, Galindo – Villegas and Hosokawa (2004) stated that although oral

39

administration of an immunostimulant showed potential and was most suitable for the

aquaculture industry, the injection method has the biggest effect in enhancing the immune

system and is the most potent immunisation route. A major disadvantage of the injection method

for commercial aquaculture is its short duration of action as it is not possible to administer

regular injections to commercial quantities of cultured fish (Anderson, 1992). In a study

conducted on Atlantic salmon which were injected with a high dose (100 mg/kg) of beta glucans,

maximum benefits were seen at 4 weeks, and at a low dose of injection (2-10 mg/kg) protection

was only shown for one week (Barman et al., 2013). In this trial, it is possible that an injection

route instead of an oral treatment route would have been more effective but the costs involved,

particularly if protection only lasts a limited time, make injection unviable in a commercial

aquaculture operation.

An important limitation to this trial was the lack of routine measurements of immune function

during the trial which may have elucidated. One reason for this trial was that the flow cytometry

method used to test lysosome levels was still being developed and so there is no way of knowing

if the lysosome levels fluctuated throughout the trial period. It should also be noted that the use

of gelatin is unlikely to have interfered with the immunostimulants due to the common use of it

in other trials such as Biller-Takahashi et al., (2012), Saurabh & Sahoo (2008) and Duncan &

Klesius (1996), which still demonstrated improvements in immune function. As of yet, there are

no commercial immunostimulants available to treat specific strains of disease such as the

common bacterium strains mentioned previously (Tafalla et al., 2013). The research into the

design and production of such a product is a future prospect in the aquaculture industry as it

would allow fish farms which know the specific type of disease that is affecting their fish to

directly target this disease, whether it be for prevention or treatment (Tafalla et al., 2013).

Conclusions

The development of immunostimulants is only recently starting to receive more attention in

Australia, especially with marine finfish, with the most successful developments and studies

being conducted within the past few years (Barman et al., 2013). The specific commercial

products Aquagard and Actigen are still considered new products, and, although there was only

limited evidence from this trial that they provide health benefits, and given the promising effects

40

which have been seen in other trials it would be unjust to rule that the products are ineffective

against yellowtail kingfish. It can be concluded that further studies specifically on yellowtail

kingfish using the two products would need to be conducted in order to gain a better

understanding of the true effects.

41

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