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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