Reviews in Aquaculture (2017) 0, 1–25 doi: 10.1111/raq.

12209

Beneficial roles of feed additives as immunostimulants in

aquaculture: a review
Mahmoud A.O. Dawood1,2

, Shunsuke Koshio2 and Maria Angeles Esteban3
1 Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University, Kagoshima, Japan
2 Department of Animal Production, Faculty of Agriculture, Kafrelsheikh University, Kafrelsheikh, Egypt
3 Fish Innate Immune System Group, Department of Cell Biology and Histology, Faculty of Biology, Regional Campus of International Excellence
“Campus Mare Nostrum”, University of Murcia, Murcia, Spain

Correspondence
Mahmoud A.O. Dawood, Department of
Animal Production, Faculty of Agriculture,
Kafrelsheikh University, 33516 Kafrelsheikh,
Egypt.
Email: mahmouddawood55@gmail.com
Received 29 March 2017; accepted 12 July
2017.
Abstract
The use of antibiotics in the aquafeeds to mitigate infectious diseases or to boost
growth performance is commonly practiced. Recently, the prophylactic use of
antibiotics and chemotherapies have been criticized which eventually led to their
ban in aquaculture by law on many countries. In view of the current restrictions
on antibiotic use, there is a critical need to evaluate other possible alternatives.
One potential substitute for antibiotics in aquafeeds is the use of functional feed
additives. The present review is a comprehensive and an updated compilation of
the available works on different feed additives, their examples, modes of action
and useful applications for improving aquafeeds. It highlights several functional
feed additives including probiotics, prebiotics, synbiotics, immunostimulants,
organic acids, nucleotides and medicinal herbs. Apart from boosting aquafeeds
and safeguarding general health of aquatic animals, some of these substances have
been found to possess beneficial immunostimulant and anti-stress relieving prop-
erties. Furthermore, the use of these natural substances increases the consumer
confidence of farmed fish. Due care has been taken to cover the on-going trends
and recent advances with a perspective vision and their holistic usages and benefi-
cial applications in aquatic animals’ systems. The current study also shed light
regarding the management and production aspects of aquatic animals which will
enlighten farmers and producers for better economic growth.
Key words: aquafeeds, disease resistance, feed additive, immune response, immunostimulants,
sustainable aquaculture.

in aquaculture (Dawood et al. 2015a). A worldwide effort

Introduction
The rapid growth of modern aquaculture is driven by a
variety of factors, which include the increasing use of for-
mulated aquafeeds and intensification of the culture sys-
tems (Tal et al. 2009; Dawood et al. 2014). Efforts to
intensify aquaculture of valuable aquatic animal species can
lead to increased stress, limited growth performance and
poor welfare in farmed specimens (Dawood & Koshio
2016a). For long time, antibiotics have often been used in
the aquaculture industry not only to prevent and/or control
infectious diseases caused by bacterial pathogens, but also
as growth promoters (Hern
andez-Serrano 2005). Public
awareness regarding the prophylactic use of antibiotics in
aquafeeds, which may lead to the transfer of immunity of
bacterial species pathogenic to humans, has led to their ban
to minimize and eventually eliminate the use of antibiotics
for growth-promoting purposes in the aquaculture industry
started with the ban on the use of sub-therapeutic antibi-
otics in the European Union on January 2006 (European
Parliament and Council Regulation (EC) No 1831/2003)
(Ng & Koh 2016). The potential substitute for antibiotic
additives is to use additives as immunostimulants that
improve resistance to diseases in aquatic animals by
enhancing innate defence mechanisms for the continued
expansion of the global aquaculture industry (Fuchs et al.
2015).
Feed additives have been defined as non-nutritive
ingredients or non-nutritive components of ingredients
that are included in formulations to either influence
physical or chemical properties of the diet or affect

© 2017 Wiley Publishing Asia Pty Ltd 1

M. A. O. Dawood et al.
aquatic animals’ performance or quality of resulting
products (Barrows 2000). The chemical nature of these
feed additives is quite diverse, and their use in commer-
cial diet formulations for aquatic species varies consider-
ably. Additives that influence feed quality in commercial
formulations include pellet binders, preservatives (such
as antimicrobial compounds and antioxidants) and feed-
ing stimulants (NRC 2011). Such additives are com-
monly included to achieve and maintain optimal
physical and chemical characteristics. Other additives
that may directly affect fish performance or product
quality including probiotics, prebiotics, acidifiers and
plant- or animal-derived extracts, are also commercially
available for aquatic animals (Dawood et al. 2015b,c; Ng
& Koh 2016). Previous studies have evaluated several of
these additives on their effect on growth performance,
immune response and disease resistance, as well as intesti-
nal microbial communities for various aquatic animal spe-
cies (Balc
azar et al. 2006; Kesarcodi-Watson et al. 2008;
Merrifield et al. 2010; Ringø et al. 2010a,b). Among the
many other substances and extracts investigated as diet
additives, macroalgae and macroalgal extracts are rich in
polysaccharides (e.g. alginic acid, laminarin, fucoidan) and
contain numerous bioactive substances (e.g. vitamins or
polyphenols) that are known to improve growth perfor-
mance and health condition in a variety of fish species
(Fleurence 1999; Ahmadifar et al. 2009; Gupta & Abu-
Ghannam 2011; Holdt & Kraan 2011; Heidarieh et al.
2012; Sheikhzadeh et al. 2012). In addition, probiotics or
beneficial bacteria are known to control pathogens
through a variety of mechanisms and affect intestinal
microbial communities (Kesarcodi-Watson et al. 2008;
Navarrete & Tovar-Ram
ırez 2014; Newaj-Fyzul et al.
2014). Prebiotics also exhibited various effects on the
aquatic animal growth, immune response and tolerance
against different stressors (Burr et al. 2005; Daniels &
Hoseinifar 2014; Akhter et al. 2015; Dawood & Koshio
2016a). Dietary uptake of nucleotides, isolated from yeast,
may optimize cell proliferation to promote rapid growth,
as the synthesis of nucleotides is a metabolically costly
process (Sanderson & He 1994). Particularly under stress-
ful conditions, additional nucleotides can be needed, for
instance, for further signal transduction or immune cell
proliferation (Carver & Walker 1995; Li & Gatlin 2006).
The application of nucleotides has demonstrated a positive
influence on growth performance when added to formu-
lated fish diets (Burrells et al. 2001a,b; Lin et al. 2009;
Tahmasebi-Kohyani et al. 2012). Furthermore, acidifiers
consisting of organic acids and their salts, used for
instance as preservatives, are considered as promising
growth promoters in aquatic animals (L€ uckst€adt 2008).
For example, dietary supplementation of citric acid and
potassium diformate showed improved growth and feed
2 © 2017 Wiley Publishing Asia Pty Ltd
utilization in some fish species (Baruah et al. 2007; Hos-
sain et al. 2007).
Supplementation of the diet with small fragments of
carbohydrates also manipulates the gut ecosystem (NRC
2011). The organic acids have got bacteriostatic properties
as well as anti-mycotoxic effects (Bai et al. 2015). Various
herbal products have got enhanced digestive activity,
antimicrobial, antioxidant and immunomodulatory prop-
erties and can be used as alternative to chemotherapeutic
agents (Van Hai 2015).
Despite the progress made with various fish species, the
effect of the above-mentioned feed additives on growth
performance and feed utilization of commercially impor-
tant aquatic animal species not yet well documented (Li
et al. 2008; Yun et al. 2011; Peng et al. 2013; Abdelkhalek
et al. 2017). The current study is needed to fill the knowl-
edge gaps regarding feed additives’ potential to support the
increased demand for aquatic animal species. This review
article will discuss about the main diverse groups of feed
additives and their functions in aquatic animal nutrition.
Reviews on the use of different feed additives in aquacul-
ture have recently been published as proceedings or book
chapters to present the beneficial roles of feed additives
individually (Bai et al. 2015; Lee 2015). However, the pre-
sent review aims to gather, evaluate and analyse the current
knowledge on the use of most of the feed additives and
includes recent research results with different aquatic ani-
mal species. Our aim is to benefit researchers, feed manu-
facturers, feed additives companies, farmers, seafood
processors and policymakers related to the aquaculture
industry. It includes peer-reviewed research published in
mainstream journals as well as selected research from con-
ference proceedings, magazine articles and other grey litera-
ture. This review seeks to provide scientific evidence for a
viable alternative to the use of antibiotics, thereby con-
tributing to the sustainability of aquaculture and safety of
the resultant seafood products.
The current use of antibiotics in aquaculture
Antibiotics can be defined as substances that have the
capacity to kill or inhibit the growth of micro-organisms
which can be derived from natural sources or have syn-
thetic origins (Romero et al. 2012). Antibiotics should be
safe to the host, allowing their use as chemotherapeutic
agents for the treatment of bacterial infectious diseases, and
their use can be categorized as therapeutic, prophylactic or
metaphylactic (Cabello 2006). In aquaculture, antibiotics at
therapeutic levels are frequently administered for short
periods of time via oral route to groups of aquatic animals
that share tanks or cages (Defoirdt et al. 2011). All drugs
legally used in aquaculture must be approved by the gov-
ernment agency responsible for veterinary medicine (e.g.
Reviews in Aquaculture (2017) 0, 1–25

the Food and Drug Administration in the USA). For
instance, in the USA, the following antimicrobials are
authorized for use in aquaculture: oxytetracycline, florfeni-
col and sulfadimethoxine/ormetoprim. These regulatory
agencies may set rules for antibiotic use, including permis-
sible routes of delivery, dose forms, withdrawal times, toler-
ances and use by species, including dose rates and
limitations. The most common route for the delivery of
antibiotics to fish occurs through mixing the antibiotic
with specially formulated feed. However, fish do not effec-
tively metabolize antibiotics and will pass them largely
unused back into the environment in faeces. It has been
estimated that 75% of the antibiotics fed to fish are
excreted into the water (Burridge et al. 2010).
Intensive fish farming has promoted the growth of sev-
eral bacterial diseases, which has led to an increase in the
use of antimicrobials (Defoirdt et al. 2011). Current levels
of antimicrobial use in aquaculture worldwide are not easy
to determine because different countries have different dis-
tribution and registration systems. Nevertheless, Burridge
et al. (2010) reported that the amount of antibiotics and
other compounds used in aquaculture differed significantly
between countries. Defoirdt et al. (2011) estimated that
approximately 500–600 metric tons of antibiotics were used
in shrimp farm production in Thailand in 1994; he also
emphasized the large variation between different countries,
with antibiotic use ranging from 1 g per metric ton of pro-
duction in Norway to 700 g per metric ton in Vietnam.
Furthermore, many antibiotics are used in aquafeeds as
growth promoters for improving health of aquatic animal
species. Other growth-promoting agents and feed additives
comprise of probiotics, prebiotics, synbiotics, organic acids,
medicinal herbs, antioxidants and nucleotides which have
been used widely to promote aquatic animal species health
and production (NRC 2011). Antibiotics are given at sub-
therapeutic dosage for stabilization of the microflora of
intestine and for improving the performance in general
along with prevention of certain specific pathological con-
ditions of the intestine (Romero et al. 2012).
Risks of antibiotics application in aquaculture
Antibiotics are used in aquaculture to prevent diseases by
treating the water or host before infectious disease occurs
(Alderman & Hastings 1998; Christensen et al. 2006).
While this prophylactic method of preventing, disease is
profitable because it prevents loss and allows the host to
grow more quickly, there are several downsides. The over-
use of antibiotics can create antibiotic-resistant bacteria.
Antibiotic-resistant bacteria can spontaneously arise when
selective pressure to survive results in changes to the DNA
sequence of a bacteria allowing that bacteria to survive
antibiotic treatments (Anderson et al. 2003). Because some
Reviews in Aquaculture (2017) 0, 1–25
© 2017 Wiley Publishing Asia Pty Ltd
Feed additives in aquafeeds
of the same antibiotics are used to treat fish that are used to
treat human disease, pathogenic bacteria causing human
disease can also become resistant to antibiotics because of
treatment of fish with antibiotics (Angulo 2000). For this
reason, the overuse of antibiotics in treatment of aquatic
animal species could create public health issues. As a result,
many countries have banned or are banning the inclusion
of antibiotics in aquafeeds as a routine means of growth
promotion.
Additionally, the use of a wide variety of antibiotics in
large amounts, including non-biodegradable antibiotics
used in human medicine, ensures that they remain in the
aquatic environment, exerting their selective pressure for
long periods of time (Iwashita et al. 2015). Also, the
exchange of genes associated with antibiotic resistance
between bacteria in the aquaculture environment and
pathogenic bacteria associated with other animals and
humans in the terrestrial environment, and between animal
micro-organisms and human pathogens, has also been
remarked (Cabello 2006). Briefly, the accumulation of
antibiotics can be a potential risk to consumers and the
environment (Yousefian & Amiri 2009). The excessive use
of antibiotics in commercial aquaculture can produce
residual, non-metabolized molecules within the environ-
ment. This may result in undetected consumption of thera-
peutic contaminants in fish, with the added potential
alteration of the normal fish/host bacterial flora, potentially
affecting susceptibility to infections, antibiotic-resistant
micro-organisms, allergies and toxicity (Cabello 2006).
Recently, the application of antibiotics and chemothera-
peutics to control these diseases caused many other prob-
lems such as the spread of drug-resistant pathogens,
suppression of aquatic animal’s immune system and envi-
ronmental hazards (Allameh et al. 2016). Therefore, an
alternate approach to enhance disease resistance, immune
responses and other health benefits is the administration of
probiotics, prebiotics and other feed additives which have
various health-promoting properties for aquatic animal
species are encouraged.
Feed additives as antibiotics alternatives
A promising alternative to promote growth performance
and to increase immune resistance in fish is the use of func-
tional feed additives (Dawood et al. 2015b). They can be
used in addition to chemotherapeutic agents and vaccines
(Iwashita et al. 2015). Moreover, immunostimulants
increase infectious disease resistance over stimulation of
immunity, which is considered a promising alternative to
chemotherapy and vaccines (Anderson 1992). Several feed
additives with direct and indirect modes of action can
replace the effects of in-feed antibiotics used for growth
promotion in aquatic animals. Recently, an intensive
3
M. A. O. Dawood et al.
amount of research has been focused on the development
of alternatives to antibiotics to maintain aquatic animal
health and performance. The most widely researched alter-
natives include probiotics, prebiotics, synbiotics, acidifiers,
plant extracts, nucleotides and immunostimulants such as
b-glucan and lactoferrin (LF) as mentioned before (Misra
et al. 2006; Yokoyama et al. 2006; Dawood et al. 2015a,b,
2016c, 2017; Dawood & Koshio 2016a; Hossain et al.
2016). Information on the development of alternatives to
antibiotics in aquaculture were discussed deeply in recent
published articles, reviews and textbooks (Not exclusive,
Table 1).
Since these additives have been more than adequately
covered in previous reviews, the focus of this review will be
on less traditional alternatives. Based on a thorough review
of the literature, it is evident that a long and growing list of
compounds exist which have been tested for their ability to
replace antibiotics as functional feed additives in diets fed
to aquatic animals. Unfortunately, most of these com-
pounds produce inconsistent results and rarely equal
antibiotics in their effectiveness. Therefore, research is still
needed in this area and that the perfect alternative to
antibiotics does not yet exist.
Feed additives and immunological defences of fish
Several studies hypothesized that functional feed additives
could activate the innate immune system of aquatic animals
in two ways: (i) by directly stimulating the innate immune
system, or (ii) by enhancing the growth of commensal
microbiota (K€ uhlwein et al. 2013, 2014). However, addi-
tional work is needed to characterize the ligand–receptor
interactions, signal transduction pathways involved and the
types of cytokines secreted.
The cellular and humoral immune responses of fish are
phagocytosis, respiratory burst and lysozyme activities,
complement system, and cytokines. Fish depend completely
on their innate immunity to contrast pathogens due to lim-
itations in the adaptive immune system (Esteban et al.
2005; Little et al. 2005) by the production of different cells
and soluble components involved in immune responses.
Leucocytes are the main force in the immune system; they
include lymphocytes (T cells, B cells, large granular lym-
phocytes), phagocytes (mononuclear phagocytes, neu-
trophils and eosinophils) and auxiliary cells (basophils,
mast cells, platelets). The head kidney is considered the
principal immune organ responsible for phagocytosis, anti-
gen processing, and the formation of IgM and immune
memory through melanomacrophacic centres (Tsujii &
Seno 1990; Meseguer et al. 1995). The spleen represents an
important secondary lymphoid organ in fish. The popula-
tions of lymphocytes and macrophages are actively
involved in phagocytosis and they are located close to the
4 © 2017 Wiley Publishing Asia Pty Ltd
antigen trapping sites. Accordingly, antigens may be
detained for long periods of time and for this reason the
spleen plays an important role in immunological memory
(Uribe et al. 2011; Secombes & Wang 2012). In the follow-
ing sections, detailed information will be discussed to reveal
the specific information on the applications of functional
feed additives in aquaculture and the problems facing aqua-
culture in terms of feed supplementation, mode of action
and disease outbreaks.
Probiotics as feed additives
There are a range of approaches available to protect farmed
aquatic animals against the effect of infectious diseases. Of
these approaches, probiotics have become widely used for
the control of diseases (Hai 2015; Adel et al. 2017). Probi-
otics are defined as cultured products or live microbial feed
supplements, which beneficially affect the host by improv-
ing the intestinal (microbial) balance (Fuller 1989). A pro-
biotic is a mono or mixed culture of live micro-organisms
to improve the properties of the indigenous microflora
(Havenaar et al. 1992). Probiotics are defined as live
intestinal bacteria that promote the viability of the host
(Skjermo & Vadstein 1999). Probiotics can also be defined
as microbial cells administered through the gastrointestinal
(GI) tract to improve the health of the hosts (Gatesoupe
1999). As the intestinal microbiota in aquatic animals con-
stantly interacts with the environment and the host func-
tions, a probiotic is defined as a live microbial adjunct
which provides beneficial effects including (i) modifying
the host-associated or ambient microbial community, (ii)
improving the use of feed or enhancing its nutritional
value, enhancing the response of the host towards diseases
or (iii) improving the quality of its ambient environment
(Verschuere et al. 2000). The definition of probiotics was
as ‘live micro-organisms which when administered in ade-
quate amounts confer a health benefit to the host’. A probi-
otic is a live, dead or component of a microbial cell, which
is administered via the feed or to the rearing water, benefit-
ing the host by improving disease resistance, health status,
growth performance, feed utilization, stress response or
general vigour, which is achieved via improving the hosts
microbial balance or the microbial balance of the ambient
environment (Merrifield et al. 2010; Dawood et al. 2016a).
Inactivated probiotic preparations appeared as an alterna-
tive to live probiotics, which could potentially cause safety
problems in open aquatic environments (Salinas et al.
2006; Dawood et al. 2015a,b, 2016b,c).
The use of probiotics in aquaculture includes bacteria
and non-bacteria, with application via water routine and
feed supplement (Fuller 1989). Probiotics provide benefits
to the hosts such as improving the host growth (Kumar
et al. 2006; Boonthai et al. 2011; Silva et al. 2013), reducing
Reviews in Aquaculture (2017) 0, 1–25
 Feed additives in aquafeeds

Table 1 Influences, dose and administration methods of feed additives in aquatic animal species

Feed additive Growth Immunological and Disease Administration References

performance physiological resistance
and feed responses
utilization

Antibiotics (+)† (+++) (+++) Medicated feed, Alderman and Hastings (1998), Sakai (1999),
injection Defoirdt et al. (2011), Ku €mmerer (2009), Romero
and baths and dips et al. (2012), Rico et al. (2013), Ringø et al. (2014a,
b) and Sommerset et al. (2014)
Probiotics (+++) (+++) (+++) Oral/water routine Merrifield et al. (2010), Ringø et al. (2010a,b), Van
and feed additives Hai and Fotedar (2010), Aguirre-Guzm
an et al.
(2012), Mart
ınez Cruz et al. (2012), Zhou and
Wang (2012), Lakshmi et al. (2013), Mohapatra
et al. (2013), Rico et al. (2013), De et al. (2014),
Merrifield and Ringo (2014), Navarrete and Tovar-
Ram
ırez (2014), P
erez-S
anchez et al. (2014), Akhter
et al. (2015), Iwashita et al. (2015), Dawood and
Koshio (2016a), A. M. Haygood and R. Jha (under
review) and Ringø and Song (2016)
Prebiotics (++) (+++) (++) Oral/water routine Yousefian and Amiri (2009), Merrifield et al. (2010),
and feed additives Ringø et al. (2010b, 2014a,b), Merrifield and Zhou
(2011), Patel and Goyal (2012), Ganguly et al.
(2013), Daniels and Hoseinifar (2014), Merrifield
and Ringo (2014), Song et al. (2014), Akhter et al.
(2015), Iwashita et al. (2015) and Dawood and
Koshio (2016a)
Synbiotics (+++) (+++) (+++) Oral/water routine Hosseinifar and Mahious (2007), Cerezuela et al.
and feed additives (2011), Cerezuela et al. (2011), Hoseinifar et al.
(2014), Dawood and Koshio (2016a) and Ringø and
Song (2016)
Immunostimulants (++) (+++) (++) Feed additives Deachamag et al. (2006), Lin et al. (2011), Ringø
et al. (2011), Alishahi and A€ıder (2012), Immanuel
et al. (2012), Ringø et al. (2012), Ganguly et al.
(2013), Kitikiew et al. (2013), Meena et al. (2013)
and Ringø and Song (2016)
Nucleotides (++) (+++) (+) Feed additives Carver and Walker (1995), Cosgrove (1998), Fegan
et al. (2006), Li and Gatlin (2006), Ringø et al.
(2011, 2012), Ringø et al. (2012) and Hossain et al.
(2016)
Medicinal plants (+) (++) (++) Feed additives Shangliang et al. (1990), Citarasu (2010),
Chakraborty and Hancz (2011), Direkbusarakom
(2011), Harikrishnan et al. (2011), Lee and Gao
(2012), Pandey et al. (2012), Hwang et al. (2013),
Ramudu and Dash (2013), Reverter et al. (2014),
Vaseeharan and Thaya (2014), Dikel (2015),
Syahidah et al. (2015) and Van Hai (2015)
Organic acids (+) (+) (+) Feed additives Gislason et al. (1994, 1996), Vielma et al. (1999),
or acidifiers €ckst€adt (2007, 2008), Weimin (2010), Mine and
Lu
Boopathy (2011), Tabrizi et al. (2012), da Silva
et al. (2013), Bai et al. (2015), Elala and Ragaa
(2015) and Ng and Koh (2016)
Antioxidants ( ) (++) (++) Feed additives Murata and Yamauchi (1989), Frigg et al. (1990),
Gatlin et al. (1992), Lygren et al. (1999), Wilhelm
(2006) and Bai et al. (2015)

†Where (+++) means highly significant; (++) means significant; (+) means possible and ( ) means doubtful.

Reviews in Aquaculture (2017) 0, 1–25

© 2017 Wiley Publishing Asia Pty Ltd 5
M. A. O. Dawood et al.
the incidence of diseases (Irianto & Austin 2002;
Newaj-Fyzul et al. 2007; Silva et al. 2013) and requiring less
chemotherapy (Azad & Al-Mazouk 2008; Hai & Fotedar
2009). Moreover, probiotics can be used in various aquatic
environments: freshwater (Rahiman et al. 2010), brackish
water and sea water (Vijayan et al. 2006). Generally, probi-
otics are live and/or dead microbial feed supplements or
water additives in the form of mono, multiple strains or in
combination with prebiotics or other immunostimulants,
which are administered to improve the rearing water qual-
ity, to enhance the physiological and immune responses of
aquatic animals and to reduce the use of chemicals and
antibiotics in aquaculture.
In aquatic animals, there are two main modes of actions
of probiotics viz., competitive exclusion and immunomod-
ulation. Probiotics occupy and colonize in digestive tracts,
particularly the mucosal epithelium (Macey & Coyne 2006;
Merrifield et al. 2010; Lazado et al. 2011; Korkea-Aho et al.
2012), they adhere to (Mahdhi et al. 2012) and growth in
the intestinal mucus (Sorroza et al. 2012). Competition for
adhesion receptors with pathogens may be the first probi-
otic effect (Montes & Pugh 1993). Thus, they reduce the
ability of pathogens (Chabrillon et al. 2005), and/or antag-
onize their effects (Luis-Villasenor et al. 2011). However,
although Microbacterium ID3-10, Ruegeria RA4-1, Pseu-
doalteromonas RA7-14 and Vibrio RD5-30 originated from
Atlantic cod (Gadus morhua) larvae intestines, their colo-
nization was just a transient presence in the larvae (Skjermo
et al. 2015). In summary, probiotics can be used as a suit-
able alternative to the prophylactic use of antibiotics and
chemicals. They can compete for chemicals, nutrition/en-
ergy or even oxygen, enhance health and immune systems,
elevate growth and survival rates as well as feed utility, and
improve water quality.
Prebiotics as feed additives
Prebiotics are non-digestible fibres that increase beneficial
gut bacteria in the host (Roberfroid 2005). It has been well
established that the by-products originated when beneficial
commensal bacteria ferment prebiotics play a major role in
improving host health (Delgado et al. 2011). In this
respect, prebiotics are used as energy sources for the gut
bacteria and can be referred to as functional saccharides
(Roberfroid 1993). The immunomodulatory activity of pre-
biotics is mediated through direct interactions with the
innate immune system, or by enhancing the growth of
commensal microbiota (Song et al. 2014; Dawood et al.
2015b, 2017). Several review articles have investigated the
different aspects of prebiotic application (e.g. inulin,
oligofructose, xylooligosaccharide, fructooligosaccharide,
mannanoligosaccharide, galactooligosaccharide, b-glucan)
in aquatic animal species, and the results revealed that
6 © 2017 Wiley Publishing Asia Pty Ltd
prebiotics are promising and have beneficial effects on
growth performance, gut microbiota, immunity and disease
resistance (Cerezuela et al. 2008, 2013a,b; Ringø et al.
2010a,b, 2014a,b; Ganguly et al. 2013; Daniels & Hoseinifar
2014; Song et al. 2014; Torrecillas et al. 2014; Dawood
et al. 2015b; Dawood & Koshio 2016a).
Prebiotics have the potential to enhance many aquatic
animal biological responses and reduce the mortality
caused by invasion of pathogens (Dawood & Koshio
2016a). They also modify the microbial community within
the GI tract to boost non-specific immune responses (Song
et al. 2014). However, anaerobic, intestinal tract microbiota
need to be investigated to determine whether there are bac-
terial species to be enhanced using prebiotics (Burr et al.
2008). Studies indicate that microflora in the colon ferment
the prebiotic compounds and cause significant modifica-
tion of the gut environment owing to the oligosaccharides
serving as a substrate for the growth of anaerobic bacteria
that inhibit the growth of putrefactive and pathogenic bac-
teria (Roberfroid 1993, 2005).
Prebiotic substances can also stimulate the immune sys-
tem, thus enhancing the host’s protection against infections
(Dawood & Koshio 2016a). The host benefits from an
increase in the production of volatile fatty acids in the GI
tract by recovering some of the lost energy from indi-
gestible dietary constituents and from the inhibition of
potentially pathogenic bacteria (Ganguly et al. 2013). The
manipulation of the intestinal microbiota to achieve
favourable effects, such as enhancing growth, digestion,
immunity and disease resistance of the host organism, has
been investigated in various aquatic animal species
(Mahious & Ollevier 2005; Merrifield et al. 2010; Ringø
et al. 2010a,b; Merrifield & Zhou 2011; Patel & Goyal 2012;
Merrifield & Ringo 2014; Iwashita et al. 2015).
Synbiotics in aquafeeds
Synbiotics contain both probiotics and prebiotics (Akhter
et al. 2015; Dawood & Koshio 2016a). The synbiotic con-
cept may give the benefit of both prebiotics and probiotics
on growth of aquatic animals mainly due to the synergistic
effect (Dawood et al. 2015b). The rationale behind their
combined use is that it may improve the survival of the
probiotic organism because fermentation can be imple-
mented more effectively as its required specific substrate is
readily available; hence, simultaneous presence of live
micro-organisms (i.e. probiotic) and prebiotics rewards the
host in a proper manner (Dawood & Koshio 2016a).
Recent attempts in fish include the use of fructooligosac-
charide, mannanoligosaccharide and inulin as a prebiotic,
in combination with probiotics to achieve long-term health
benefits via the gastro-intestinal immune system (Hoseini-
far et al. 2014). The results showed that synbiotic could
Reviews in Aquaculture (2017) 0, 1–25

significantly enhance growth performance, digestive
enzyme activity which leads to digestive system efficiency
and the immune-haematological response in fish (Hos-
seinifar & Mahious 2007; Cerezuela et al. 2011; Hoseinifar
et al. 2014; Dawood et al. 2015b; Dawood & Koshio 2016a;
Ringø & Song 2016). However, further studies regarding
the implications and the effects of different probiotics, pre-
biotics and synbiotics on gut morphology, permanent
adherence of the probiont, gut immunology and challenge
tests of aquatic animals are of high importance to investi-
gate, as less information is available on these topics.
Products act as immunostimulants
Scientific reports indicate that dietary nutrients and addi-
tives could stimulate the immune response of aquatic ani-
mal species (Lin et al. 2011; Alishahi & A€ıder 2012; Kiron
2012; Ringø et al. 2012; Ganguly et al. 2013; Meena et al.
2013). The application of chemotherapeutics is no more
appreciated in aquaculture and leads to undesirable effects
that can be a threat to human health and a host of environ-
mental problems (Harikrishnan et al. 2011). In this con-
text, the application of natural, safe and cost-effective
additives that can stimulate the immunological response of
aquatic animal species has received heightened attention
among the feed manufacturers. Consequently, research has
aimed to identify promising safe, natural and cost-effective
immunostimulating agents.
Several definitions have been proposed for immunostim-
ulants. An appropriate definition was provided by Bricknell
and Dalmo (2005): ‘An immunostimulants is a naturally
occurring compound that modulates the immune system
by increasing the hosts’ resistance against diseases that in
most circumstances are caused by pathogens’. Furthermore,
immunostimulants have been classified in different ways,
including by their origin, mode of action and the way they
are administrated. Inactivated natural microbes or micro-
bial products such as b-glucans, lipopolysaccharides, LF,
chitin, fucoidan and peptidoglycans can stimulate the
immune system (NRC 2011). Some of these products can
be delivered orally without complete degradation in the
digestive system and taken up by used as potential
immunostimulants for aquaculture.
Lactoferrin is a non-hem iron binding glycoprotein that
is a part of the transferrin protein family and is found in
high concentration in mammal’s milk and other body
secretions (Gonz
alez-Ch
avez et al. 2009). Iron binding is
one of the most important LF specifications, which causes
LF to bind with iron and other metals. Many biological
specifications of LF relate to this feature (L€
onnerdal & Iyer
1995; Van der Strate et al. 2001). LF can affect the immune
system either directly or indirectly; for instance, LF could
increase the generation of granulocytes and neutrophils,
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© 2017 Wiley Publishing Asia Pty Ltd
Feed additives in aquafeeds
phagocytic activity and respiratory burst activity (Sakai
et al. 1993; Miyauchi et al. 1998); however, some contra-
dictory effects of LF on the immune system have also been
proven (Caccavo et al. 2002). Other biological functions of
LF include the bactericidal, antifungal, antiviral, antipara-
sitic, enzymatic, anti-inflammatory and anticarcinogenic
activities as well as the growth promotion of some animal
cells (Brock 2002; Gonz
alez-Ch
avez et al. 2009; Jenssen &
Hancock 2009; Tomita et al. 2009). It has been reported
that the dietary supplementation of LF enhanced mucus
secretion in orange spotted grouper (Epinephelus coioides)
(Yokoyama et al. 2006), growth and nutritional status in
goldfish (Carassius auratus) (Kakuta 1996a), phagocytosis
in rainbow trout (Oncorhynchus mykiss) (Sakai et al. 1995)
and gilthead seabream (Sparus aurata) (Esteban et al.
2005) and lysozyme activity in Asian catfish (Clarias batra-
chus) (Kumari et al. 2003). The resistance of some fish spe-
cies to parasites, bacterial infections and lethal stress has
improved by the oral administration of LF (Kakuta 1996b;
Yokoyama et al. 2005; Welker et al. 2007). As a feed addi-
tive, dietary LF adjusted iron absorption in Nile tilapia
(Oreochromis niloticus) and altered stress response in fish
by intensifying and or suppressing and the fish responsive-
ness to stress (Kakuta 1996a, 1998; Kakuta et al. 1998;
Yokoyama et al. 2005; Welker et al. 2007). In contrast, in
some experiments on fish, growth performance, physiologi-
cal and immunological parameters were not affected by
dietary LF (Lygren et al. 1999; Yokoyama et al. 2006;
Welker et al. 2007). It seems that the LF effects on fish can
be different depending on experimental conditions and
species specificity.
Recently, b-glucans have been extensively as immunos-
timulants for aquatic animal species (Dawood et al. 2015b,
c; Dawood & Koshio 2016a). They are ubiquitous in nature
and can be found in the cell walls of yeasts, cereal grains,
algae, bacteria and fungi (Zekovi
c et al. 2005). These com-
pounds generally have been shown to influence the
immune response including blood neutrophil oxidative
radical production and superoxide anion production in
activated macrophages (Gatlin 2002). These changes may
lead to significant reductions in mortality of fish exposed
to different pathogenic organisms. Discoveries of b-glucan
receptors such as Dectin-1 and Toll-like receptors on
leukocyte surfaces of vertebrates have provided some
understanding about mechanisms of immune responses
induced by b-glucan (Brown & Gordon 2003). b-glucans
exhibited beneficial roles in different fish species such as
red sea bream (Pagrus major) (Dawood et al. 2015b),
Atlantic Salmon (Salmo salar L.) (Paulsen et al. 2001),
Rainbow trout (O. mykiss) (Walbaum 1792) (Lauridsen &
Buchmann 2010), rohu (Labeo rohita) (Misra et al. 2006),
koi (Cyprinus carpio koi) (Lin et al. 2011) and mirror carp
(C. carpio L.) (K€uhlwein et al. 2014).
7
M. A. O. Dawood et al.
Chitin is a polymer of glucosamine and is found in shells
or cell walls of invertebrates, fungi and yeasts and this com-
pound is the main component of crustacean exoskeletons
and consisting of calcium oxide and protein units. Chi-
tosan, an amino polysaccharide, is prepared from shellfish
chitin by treatment with alkali. Both chitin and chitosan
exerted immunostimulatory effects on fish (Esteban et al.
2000, 2001; Gopalakannan & Arul 2006; Yan et al. 2017a).
Using of natural immunostimulants in aquafeeds for the
activation of the immune response is promising to increase
disease resistance (Traifalgar et al. 2013). The seaweed algae
are wealthy with different minerals, vitamins, amino acids
(AA), alginic acid and fucoidan (El-Boshy et al. 2014).
Fucoidan is seaweed algal sulphated polysaccharide with a
wide variety of biological activities including detoxification
of heavy metals, antiviral, antibacterial and antiparasitic
action, antioxidant effect (Davis et al. 2003; Chotigeat et al.
2004; Immanuel et al. 2012). The importance of fucoidan
in disease resistance and immunomodulatory has been
highlighted in several farmed aquatic species (Deachamag
et al. 2006; Immanuel et al. 2012; Kitikiew et al. 2013; El-
Boshy et al. 2014; Isnansetyo et al. 2016; Prabu et al.
2016).
Nucleotides
Nucleotides traditionally have not been considered to be
essential nutrients because they are metabolically synthe-
sized, and no signs of efficiency are observed when aquatic
animals fed nucleotide-free diets (Ringø et al. 2012).
Nucleotides have essential physiological and biochemical
functions including encoding and deciphering genetic
information, mediating energy metabolism and cell sig-
nalling as well as serving as components of coenzymes,
allosteric effectors and cellular agonists (Carver & Walker
1995; Cosgrove 1998; Li & Gatlin 2006). The modulatory
effects of dietary nucleotides on lymphocyte maturation,
activation and proliferation, macrophage phagocytosis,
immunoglobulin responses, gut microbiota as well as
genetic expression of certain cytokines have been reported
in aquatic animals (Gil 2002; Singhal et al. 2008). It has
been described that dietary supplementation of nucleotides
enhanced resistance of salmonids to viral, bacterial and par-
asitic infections as well as improved efficacy of vaccination
and osmoregulation capacity (Burrells et al. 2001a,b; Tah-
masebi-Kohyani et al. 2011).
Research on nucleotide nutrition in fish and shrimp is
needed to provide insights concerning interactions between
nutrition and physiological responses as well as provide
practical solutions to reduce basic risks from infectious dis-
eases for the aquaculture industry (Hossain et al. 2016).
Diverse hypothesized that nucleotides are a key nutrient for
the shrimp immune system and supplementation of
8 © 2017 Wiley Publishing Asia Pty Ltd
nucleotides or other nucleic acid-rich ingredients such as
yeast or yeast extract may enhance disease resistance and
growth of shrimp (Devresse 2000). Li and Gatlin (2006)
summarized and evaluated knowledge of nucleotide nutri-
tion in fish as compared with that of terrestrial animals.
Beside this, possible involvement in diet palatability, fish
feeding behaviour and biosynthesis of non-essential AA,
exogenous nucleotides have shown promise most recently
as dietary supplements to enhance immunity and disease
resistance of fish produced in aquaculture (Ringø et al.
2012). Research on dietary nucleotides in fish has shown
that they may improve growth in early stages of develop-
ment, enhance larval quality via brood stock fortification,
alter intestinal structure, increase stress tolerance as well as
modulate innate and adaptive immune responses (Li &
Gatlin 2006). Dietary nucleotide supplementation has been
shown to enhance growth of certain fish species disease
resistance of all male hybrid tilapia (Saratheradon niloticus
♀ 9 Saratheradon aureus ♂) (Ramadan et al. 1994), Atlan-
tic salmon (S. salar L.) (Burrells et al. 2001a), common
carp (C. carpio L.) (Sakai et al. 2001), hybrid striped bass
(Morone chrysops 9 Morone saxatilis) (Li et al. 2004),
grouper (Epinephelus malabaricus) (Lin et al. 2009), red
drums (Sciaenops ocellatus) (Cheng et al. 2011a), rainbow
trout (O. mykiss) (Tahmasebi-Kohyani et al. 2012) red sea
bream (P. major) (Hossain et al. 2016) and Japanese floun-
der (Paralichthys olivaceus) (Song et al. 2012). Supplemen-
tation of nucleotides was also reported to increase stress
tolerance in Atlantic salmon (Burrells et al. 2001b) and GI
physiology and morphology of tilapia (Ramadan et al.
1994), Atlantic salmon (Burrells et al. 2001b) and red drum
(Cheng et al. 2011a,b).
Medicinal plants
The alternative herbal bio-medicinal products in the aqua
cultural operations, which have the characteristics of
growth-promoting ability and tonic to improve the
immune system, act as appetite stimulators (Van Hai 2015;
Adel et al. 2016). They increase consumption, induce mat-
uration and have antimicrobial capability and also anti-
stress characteristics that will be of immense use in the cul-
ture of shrimps and other fin fishes without any environ-
mental and hazardous problems (Citarasu 2010; Ramudu
& Dash 2013; Van Hai 2015). Several studies have proved
that herbal plants can be used as promising antibiotics that
after challenging with pathogens, the survival rates of
infected fish priority fed various immunostimulants, vacci-
nes and probiotics, increased (Gudmundsd
ottir & Mag-
nad
ottir 1997; Sakai 1999; Brunt et al. 2007).
Recently, a growing interest has emerged in using medic-
inal plants that have attracted a lot of attention globally,
and has become the subject of active scientific
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investigations (Sivaram et al. 2004; Takaoka et al. 2011;
Abdel-Tawwab 2012; Caruso et al. 2013; Mehrim & Salem
2013; Adel et al. 2015a,b; Awad et al. 2015; Bahi et al.
2016) because they are cheap and easy to prepare, and are
effective with fewer side effects during the treatment of dis-
eases and without any environmental and hazardous prob-
lems (Jian & Wu 2004; Citarasu 2010). Medicinal plants
include herbs, spices, seaweeds, herbal extracted com-
pounds, traditional Chinese medicines and commercial
plant-derived products.
Although medicinal plants have been used as
immunostimulants for thousands of years (Tan &
Vanitha 2004), the immunostimulating activity of herbal
components has been most widely studied in mice,
chickens or human cell lines (Cao & Lin 2003; Lin &
Zhang 2004; Lin et al. 2006). In aquaculture, medicinal
plants are also used as chemotherapeutics and feed addi-
tives (Chang 2000). They have the properties of growth-
promoting ability, a tonic to improve the immune sys-
tem, antimicrobial capability, and stimulating appetite
and anti-stress characteristics (Citarasu 2010). Several
plants or their by-products contain phenolic, polypheno-
lic, alkaloid, quinone, terpenoid, lectine and polypeptide
compounds, many of which are effective alternatives to
antibiotics, chemicals, vaccines and other synthetic com-
pounds (Harikrishnan et al. 2011). In addition, medici-
nal plants are rich in a wide variety of nutrients (Chang
2000). They can be administered as a whole plant or
parts (leaf, root or seed) or extract compounds, via
water routine or feed additives, either singly or as a
combination of extract compounds, or even as a mixture
with prebiotics or other immunostimulants. A better
understanding of modes of action may lead to effective
and appropriate applications of medicinal plants in aqua-
culture because effects of administration of herbal adju-
vant may be species specific (Zakez
s et al. 2008).
Herbs contain many types of active components, such as
polysaccharides, alkaloids or flavonoids (Ard
o et al. 2008).
The herbal-compound extracts act as immunostimulants to
enhance the immune response of fish (Siwicki 1990; Hardie
et al. 1991; Thompson et al. 1993) viz. lysozyme, comple-
ment, antiprotease, meloperoxidase, reactive oxygen species
(ROS), reactive nitrogen species, phagocytosis, respiratory
burst activity, nitric oxide, total haemocytes, glutathione
peroxidase (GPx) and phenoloxidase, against bacterial, fun-
gal, viral and parasitic diseases (Harikrishnan et al. 2011).
Numerous studies have investigated the efficacy of various
plant products in aquafeeds, such as herbs (Ard
o et al.
2008; Yin et al. 2009), roots (Sharma et al. 2010), seed meal
(Ahmad & Abdel-Tawwab 2011), onions (Cho & Lee
2012), green tea (Hwang et al. 2013), microalgae (Supa-
mattaya et al. 2005) and bamboo charcoal (Thu et al.
2010).
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Feed additives in aquafeeds
Organic acids
Organic acids are short-chain fatty acids, volatile fatty acids
or weak carboxylic acids, such as formic, citric, benzoic and
lactic acid (Bai et al. 2015; Elala & Ragaa 2015; Ng & Koh
2016). Organic acids have been used for decades as food
preservatives in livestock feeds due to their antimicrobial
properties, as well as their ability to enhance growth, nutri-
ent utilization and disease resistance of aquatic animals (Ng
& Koh 2016). In the intestinal tract of aquatic animals,
organic acids inhibit the growth of Gram-negative bacteria
by penetrating through the cell wall and releasing protons
in the cytoplasm (Bai et al. 2015). Thus, the bacteria con-
sume a large amount of adenosine triphosphate (ATP) to
excrete protons in trying to keep the balance of intracellular
pH, thus depleting cellular energy and subsequently leading
to death (Defoirdt et al. 2009). Organic acids and their salts
can also contribute in nutritional ways; they are compo-
nents in several metabolic pathways for energy generation,
such as for ATP generation in the citric acid cycle or car-
boxylic acid cycle (da Silva et al. 2013).
To date, limited comprehensive research has been con-
ducted to evaluate the efficacy of organic acids or their salts
in aquatic animals. Few studies had been published on the
use of organic acids in aquafeeds (As reviewed by: Ng &
Koh 2016). More recently, many studies have been con-
ducted to determine the effects of dietary organic acids and
their salts on growth performance, nutrient utilization and
disease resistance in several commercially important
farmed fish species, such as rainbow trout, salmon, carp
and tilapia (Ringø 1991; Gislason et al. 1994; Vielma & Lall
1997; Vielma et al. 1999). The major impacts of these diet-
ary organic acids in fish and shrimp are summarized by Ng
and Koh (2016). Despite the discrepancies among the pub-
lished data, it appears that organic acids and/or their salts
have good potential as dietary supplements to improve
growth performance, feed utilization, nutrient digestibility,
disease resistance as well as the alteration of the gut micro-
biota populations in several aquatic animal species (Lim
et al. 2010).
Functional amino acids as feed additives
Dietary AA are crucial for fish as energy substrates, for
endogenous protein synthesis and to regulate metabolic
pathways (Andersen et al. 2016; Yan et al. 2017b). More
than half of the AA consumed by fish may be deposited
into body protein, and the requirement of essential AA cor-
responds to the AA tissue content (Kaushik & Seiliez 2010).
Protein is a significant component of fish diets, and is gen-
erally higher in carnivorous than herbivorous (NRC 2011).
Most of the AA are protein bound but can also be supplied
in the form of crystalline AA to fulfil the AA requirement,
9
M. A. O. Dawood et al.
as regulated by national legislation of feed additives, espe-
cially when using alternative protein sources (Espe et al.
2006). Recently, increased interest in using AA to increase
disease resistance, immune response and reproduction as
well as aquatic animal welfare, including arginine, glu-
tamine, sulphur AA (methionine, cysteine and taurine),
histidine and branched chain AA (leucine, isoleucine and
valine) (Table 2). This has led to a boost of commercially
available functional fish feeds that aim to optimize fish per-
formance and quality of the product.
Arginine effects on both the innate and adaptive immune
response have been demonstrated in fish, where arginine
may act through nitric oxide to combat pathogens, through
polyamines, directly by affecting gene expression or by reg-
ulating nutrient availability for immune cells by endocrine
control (Andersen et al. 2016). Increased inclusion of argi-
nine in channel catfish diets has shown to correlate with
survival when exposed to the bacteria Edwardsiella ictaluria
(Buentello & Gatlin 2001). This was linked to increase
nitric oxide production in activated macrophages after
increased plasma arginine (Buentello & Gatlin 2001). Fur-
ther in vivo and in vitro experiments in channel catfish con-
firmed this positive effect on the immune system, as
arginine supplementation improved macrophage killing
and phagocytosis abilities (Buentello et al. 2007; Pohlenz
et al. 2012). Moreover, arginine increased haematocrit,
haemoglobin, erythrocyte count and lysozyme activity, as
well as enhanced native T-cell and B-lymphocyte prolifera-
tion after mitogenic exposure (Buentello et al. 2007; Poh-
lenz et al. 2012). Costas et al. (2011) found that dietary
arginine supplementation to Senegalese sole (Solea sene-
galensis) increased the respiratory burst after mitogenic
exposure, and this correlated with increased nitric oxide
production in head kidney leucocytes. In a later paper, they
found that plasma cortisol levels were reduced in stressed
turbot (Scophthalmus maximus) after arginine supplemen-
tation (Costas et al. 2013). Both arginine and lipopolysac-
charide exposure induce nitric oxide production in head
kidney macrophages (Buentello & Gatlin 1999; Tafalla &
Table 2 Effects of amino acids supplementation in aquafeeds
Species Amino acids
Channel catfish (Ictalurus punctatus) Arginine
Senegalese sole (Solea senegalensis Kaup, 1858) Arginine
Gilthead seabream (Sparus aurata) Trypthophan
Red drum (Sciaenops ocellatus) Glutamine
Channel catfish (I. punctatus) Glutamine
Yellow catfish (Pelteobagrus fulvidraco) Methionine
10
Novoa 2000). Substrate availability of arginine may thus be
of importance to produce a sufficient immune response
when required. This highlights the importance of sufficient
dietary supply of arginine during exposure to pathogens as
plasma arginine is known to decrease in fish during stress
(Aragao et al. 2008). Arginine increased abundance of
phosphorylated p38MAPK in Atlantic salmon head kidney
and liver cells in vitro, suggesting anti-inflammatory effects
of arginine (Berge et al. 2002). Notably, small concentra-
tions of nitric oxide can protect the cell from apoptosis and
pathogens by activating heat shock proteins and inducing
macrophage activity (Mori 2007).
Glutamate and glutamine play a crucial role in intestinal
health of fish, by modulating intestinal structure, protecting
against oxidative damage and acting as energy substrate for
the enterocytes. Glutamine supplementation increased
growth and intestinal structure in red drum (S. ocellatus)
(Cheng et al. 2011a,b), Jian carp (C. carpio var. Jian) (Yan
& Qiu-Zhou 2006) and hybrid striped bass
(M. chrysops 9 M. saxatilis) (Cheng et al. 2012), as well as
increasing growth and differentiation of carp enterocytes
in vitro (Jiang et al. 2009). The role of glutamine in
immune response appears to be species specific. Glutamine
has been proposed to aid the immune response by modu-
lating the nitric oxide response of macrophages and act as
energy substrate for leucocytes. Glutamine further
increased superoxide anion and neutrophil oxidative radi-
cal production in kidney macrophages from red drum
(Cheng et al. 2011a,b), and improved macrophage super-
oxide anion production and lysozyme activity in hybrid
striped bass (Cheng et al. 2012). In channel catfish macro-
phages on the other hand, no effect was observed from glu-
tamine supplementation on phagocytosis or bactericidal
activity after pathogen exposure (Pohlenz et al. 2012). Glu-
tamine is demonstrated to regulate cytokine production,
expression of immune-related genes and inhibit apoptosis
in mammals (Wu 2010), while these interactions are yet to
be investigated in fish. Synergistic effects of glutamine and
arginine on the immune response have been examined in
Effects References
Increased disease resistance Buentello and Gatlin (2001)
Increased disease resistance Costas et al. (2011)
Enhanced innate immune response Cuesta et al. (2008)
Improve immune response Cheng et al. (2011a,b) and
Protect against oxidative damage Hu et al. (2014)
Improve immune response Pohlenz et al. (2012)
Improve antioxidant capacity, Elmada et al. (2016)
innate immune response and
disease resistance
Reviews in Aquaculture (2017) 0, 1–25
© 2017 Wiley Publishing Asia Pty Ltd

several fish species (Cheng et al. 2011a,b, 2012; Pohlenz
et al. 2012), with various results, showing both synergistic
and inhibitory effects. Glutamine is a precursor for synthe-
sis of purine and pyrimidine nucleotides, and could thus
regulate DNA and RNA synthesis and affect lymphocyte
proliferation. Nucleotides are commercially used as an
immune-stimulant in Atlantic salmon feed, as dietary sup-
plementation is known to increase antibody titres in Atlan-
tic salmon after pathogen exposure (Burrells et al. 2001a).
Whether dietary glutamine may stimulate the immune
response in fish through endogenous nucleotide synthesis
is still unknown.
More research is needed to understand the underlining
mechanisms of functional AA supplementation and sig-
nalling between body compartments to fully take advantage
of this information when designing fish diets and optimiz-
ing fish health and welfare.
Vitamins
Using of natural vitamin sources, which are required for
normal cell function, growth and development of aquatic
organisms, leads to the belief that the absence of vitamins
leads to characteristic deficiency diseases in aquatic species
(Fracalossi et al. 2001; Ai et al. 2004; Chen et al. 2015).
They are unable to be synthesized by the host in sufficient
amounts to meet the physiological needs of the animal and
therefore must be obtained from the diet. In aquatic animal
nutrition, vitamins are among the most expensive ingredi-
ents used in complete diet formulation (Gaylord et al.
1998). Vitamins play essential roles in the organism, acting
principally as cofactors for enzymes; thus, inadequate sup-
ply leads to reduced enzyme activities and in turn to both
non-specific responses such as poor growth, low survival
and increased susceptibility to infections and diseases
(NRC 1993, 2011; Halver 2002) as well as more specific
deficiency signs and symptoms (Dawood & Koshio 2016b).
Through their coenzyme functions and other properties,
vitamins maintain normal metabolic functions and optimal
health (Bender 2003; Sauberlich & Machlin 1992).
Vitamin C is one of the important nutrients because it is
a powerful antioxidant and immunomodulator for fish/
shrimps. The fish and shrimp body need vitamin C to
remain proper health condition (Dawood & Koshio
2016b). Vitamin C has been known to be an important
micronutrient correlating with enhanced aquatic animal
performances (Gao et al. 2014; Shahkar et al. 2015). Many
fish and crustacean species have a limited ability to synthe-
size vitamin C (Lightner et al. 1979), due to the absence of
L-gulonolactone oxidase that is responsible for vitamin C
biosynthesis (Fracalossi et al. 2001; Ai et al. 2004) so that
signs of deficiency are observed when vitamin C excluded
from the diet. Vitamin C is a strong antioxidant that can
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Feed additives in aquafeeds
scavenge ROS (Bae et al. 2012). It participates in synthesis
of steroid hormones and collagen (Lightner et al. 1979;
Cavalli et al. 2003; Mustafa et al. 2013), tolerance to toxi-
cants and environmental stressors (Merchie et al. 1995). It
has also been proposed to be potentially beneficial in
increasing immune response (Roosta et al. 2014; Kim &
Kang 2015; Shahkar et al. 2015) and reducing oxidative
damage to tissues (Huang et al. 2017; Liang et al. 2017)
although its exact mechanism has not been demonstrated.
Vitamin C also promotes beneficial effects on serum bacte-
ricidal activity, phagocytic activity, antibody levels and
lysozyme activity (Ren et al. 2005). Phagocytic activity of
cells of the immune system in fish produce reactive oxygen
radicals that are potent microbicidal factors (Secombes
et al. 1988). However, there is still limited information on
the relationship between dietary vitamin C and its antioxi-
dant effects with different degrees of lipid oxidation for fish
species. Vitamin C also facilitates the absorption of iron
(Hsu & Shiau 1999), thus preventing the anaemia often
observed in case of vitamin C deficiency in fish. Moreover,
vitamin C may play a role in reproduction of cultured fish
species. Reduced reproductive performance has been
reported in female tilapia (Oreochromis mossambicus) and
rainbow trout (Salmo gairdneri) fed vitamin C deficient
diets (Sandnes et al. 1984). Furthermore, supplementation
of vitamin C in egg, larval development and broodstock
showed enhanced immune function and overall health
(Hamre et al. 2013; Shahkar et al. 2015).
Vitamins C and E are major antioxidant additives used
in the food industry and have been shown to reduce the
oxidative stress in animals (Chien & Hwang 2001; Tocher
et al. 2003; Gao et al. 2013). Vitamin C acts primary as an
electron donor and therefore a reducing agent that plays an
important role as a water-soluble antioxidant able to scav-
enge free radicals including ROS and reactive nitrogen spe-
cies and avoid cellular components from radical damages.
This antioxidant function is also strengthened by its ability
to regenerate vitamin E from a-tocopheryl radical in mem-
branes (Jim
enez-Fern
andez et al. 2015).
Interactive effects between vitamins C and E in prevent-
ing lipid peroxidation have been investigated (Montero
et al. 1999; Lewis-McCrea & Lall 2007). Vitamin C could
regenerate and/or spare vitamin E in fish, such as in Atlan-
tic salmon (Hamre et al. 1997), gilthead sea bream (S. au-
rata) (Montero et al. 1999), juvenile lake sturgeon
(Acipenser fulvescens) (Moreau et al. 1999), channel catfish
(Lim et al. 2000), yellow perch (Perca flavescens) (Lee &
Dabrowski 2003) and red sea bream (P. major) (Gao et al.
2012b). In addition to the protective effects of both vita-
mins against lipid peroxidation, high doses of vitamin E
may even exert pro-oxidative effects in human low-density
lipoproteins (Bowry & Stocker 1993). Vitamin C may also
act as both pro- and antioxidant in biological systems
11
M. A. O. Dawood et al.
depending on the physiological conditions (Lee et al.
2001). A higher dose of antioxidants needs to be provided
in diets when oxidized lipid is used (Gao et al. 2012a).
The presence of a vitamin C/E interaction mechanism
has been suggested as an explanation for the large variabil-
ity observed in sensitivity to vitamin E deficiency in cul-
tured aquatic species (Lovell et al. 1984). Gatlin et al.
(1986) examined the hypothesis that marginal vitamin C
status would increase sensitivity of channel catfish to vita-
min E deficiency. Channel catfish fed diets without vita-
min C had reduced weight gain and feed efficiency
regardless of vitamin E supplementation. Although ele-
vated lipid peroxidation was observed, signs of vitamin E
deficiency were not observed in fish fed the vitamin E–de-
ficient diet with supplemental vitamin C. Wilson et al.
(1984) and Gatlin et al. (1986) suggested that vitamin E–
deficient diets supplemented with high or adequate levels
of vitamin C still caused elevated lipid peroxidation, as the
lack of a vitamin C sparing effect on vitamin E nutrition
of channel catfish. Frischknecht et al. (1994) and Hamre
et al. (1997) demonstrated the ability of dietary vitamin C
to protect rainbow trout (O. mykiss) and Atlantic salmon
from anaemia and mortality due to dietary deficiency of
vitamin E. However, more studies are required to deter-
mine the effects of various levels of vitamins C and E, and
their interactive effects on oxidative stress condition of
culture species.
Trace minerals
General function of minerals includes constituents of the
exoskeleton, balance of osmotic pressure, structural con-
stituents of tissues and transmission of nerve impulse and
muscle contractions (NRC 2011). Minerals serve as essen-
tial components for enzymes, vitamins, hormones, pig-
ments, and co-factor in metabolism, catalysts, and enzyme
activators (Antony Jesu Prabhu et al. 2017). Shrimp can
absorb or excrete minerals directly from the aquatic envi-
ronment via gill and body surfaces. So, the dietary require-
ment of minerals is largely dependent on the minerals
concentration of the aquatic environment in which the
shrimp is being cultured.
The micro-elements or trace minerals, such as chro-
mium, cobalt, copper, iodine, iron, manganese, molybde-
num, selenium and zinc, are required in small quantities
and participate in a wide variety of biochemical processes
(Antony Jesu Prabhu et al. 2017). They are involved in cel-
lular metabolism, formation of skeletal structures, mainte-
nance of colloidal systems, regulation of acid–base
equilibrium, immunity enhancer, stress releaser, disease
resistance and other physiological functions. They are
important components of hormones and enzymes, and
serve as cofactors and/or activators of a variety of enzymes.
12
Dietary bioactive food components that interact with the
immune response have considerable potential to reduce
susceptibility to infectious diseases. A functional immune
system is essential for the survival and performance of
shrimp/fish in aquaculture. Every trace mineral compo-
nents are having their specific role in immunity of cultured
animals, but the crucial trace metals that have been associ-
ated with an improvement in immunity or function that
support immunity are Zn, Mn, Cu and Se (El Basuini et al.
2016, 2017). The immune system uses several methods to
detoxify these foreign agents or antigens. The trace ele-
ments that have been combined with an improvement in
immunity, or function that support immunity. The micro-
elements have especially been strengthened by the impor-
tance of their roles in immune defence and antioxidative
protection.
Copper (Cu) is an essential trace element for animals
including fish (Tan et al. 2011). Copper plays an important
role in large number of biological processes including syn-
thesis of haemoglobin, formation of bone, maintenance of
myelin within the nervous system and as a primary compo-
nent of key enzymes (such as cytochrome oxidase, dopa-
mine hydroxylase ferroxidase and tyrosinase) (O’Dell 1976;
Tan et al. 2011). Copper is also important to reduce the
activities of liver superoxide dismutase and heart cyto-
chrome oxidase in fish (Lall 2002). Administration of diet-
ary Cu has been reported to enhance growth performance
and immunological function in various fish species (Lin
et al. 2008; Sabatini et al.2009; Mohseni et al. 2014).
Selenium (Se) is an essential trace element for the normal
growth of several cultured fish species, and has been con-
sidered as an essential micro-nutrient for sustainable aqua-
culture (Pacitti et al. 2016). Selenium is a component of
the enzyme GPx, which protects cell membrane from
oxidative stress (Pacitti et al. 2016). Administration of diet-
ary Se has been reported to enhance growth performance,
immune and antioxidative responses in several fish species
(Zhu et al. 2012, 2017; Ashouri et al. 2015). Furthermore,
Se is involved in gene transcription and cell signalling cas-
cades, thyroid hormone metabolism and reproduction
(Pacitti et al. 2016).
Zinc is the key essential element which acts as a growth
promoter and plays significant part in several other cellular
functions including cell proliferation, co-factor reproduc-
tion, immune function and defence against free radicals
(Bray & Bettger 1990; Powell 2000). It is considered as the
intracellular trace element which is vital for both genetic
stability and function (Dreosti 2001). Zinc acts as cofactors
for several metabolic pathways in many enzymatic systems,
and is also a major component of many metalloenzymes
such as carbonic anhydrase, carboxypeptidase, alcohol
dehydrogenase, glutamic dehydrogenase, D-superoxide dis-
mutase (Salgueiro et al. 2000). It is well-established fact
Reviews in Aquaculture (2017) 0, 1–25
© 2017 Wiley Publishing Asia Pty Ltd

that, zinc has antioxidant properties and protects tissue
from oxidative damage (Ho & Ames 2002) necessary for
normal growth, reproduction and other metabolic pro-
cesses (Eisler 1993); however, it may also become toxic to
aquatic organisms at high concentrations (Bielmyer et al.
2012).
Manganese is one of the important elements required in
fish. It is widely distributed in fish and another animal tis-
sue as well. Mainly manganese is act as co-factor for the
enzymes peptidase, arginase, succinic decarboxylase and
also activates specific enzymes such as glycosyltransferase
and non-specific enzymes such as kinases, transferases,
hydrolases and decarboxylases. The manganese also acts as
an integral part of metalloenzymes. This element has a great
implication in performing oxidative phosphorylation.
Manganese content in mitochondria is higher than
cytoplasm.
Future research and trends
A new era has been brought about in aquafeeds by the
above-mentioned feed additives. They are considered as
powerful alternatives to materialize improvement in health
status and production performances of aquatic animals,
thereby increasing the net economic returns. The achieve-
ment and maintenance of optimal balance of beneficial
microflora in the GI tract can be obtained with the use of
probiotics, prebiotics, immunostimulants, medicinal herbs
and organic acids. A lot of success had been achieved in
aquatic animal culture initially using antibiotics, but they
present some negative aspects with development of micro-
bial resistance, due to their indiscriminate use. On the con-
trary, functional feed additives offer the best viable and safe
alternatives to antibiotics. The use of feed additives in a daily
supplementation regimen has become a popular routine
method in the aquafeeds, particularly following an antibiotic
therapy. The use of feed additives has many potential bene-
fits including the modification of host metabolism,
immunostimulation, exclusion and inhibition of pathogens
in the intestinal tract, enhanced nutrient absorption and per-
formance and ultimately decreased human health risk.
Future studies are required to evaluate the antimicrobial
peptides expression in aquatic animals by functional feed
additives are interesting to reveal the additional beneficial
mechanisms of these compounds. Moreover, exploiting
genetic engineering approaches, it is possible to strengthen
or create completely novel feed additives which may have
revolutionary potentials in oral immuno-therapeutic appli-
cations. Combined with good hygiene and management,
these alternatives have shown to be effective in maintaining
growth and production of aquatic animals by controlling
the stress factors and commonly encountered infectious
and non-infectious diseases.
Reviews in Aquaculture (2017) 0, 1–25
© 2017 Wiley Publishing Asia Pty Ltd
Feed additives in aquafeeds
Concluding remarks
A variety of additives have always played a role, either
alone or as a combination, in compounded aquafeed. A
better understanding of the mechanisms whereby feed
additives influence aquatic animal’s growth performance
and health condition will lead to the development of
alternative feed additives while minimizing the use of
antibiotics. Scientific literature related to this topic sug-
gests that the immunostimulatory effects of several feed
additives vary by fish type, route of administration, dose,
duration and association with other immunostimulants.
Therefore, the optimum dietary level could be related to
the aquatic animal species; proper care should be taken
to explore the beneficial effects and avoid immunosup-
pressive effects.
Acknowledgements
This research was funded by MINECO co-funded with
European Regional Development Funds (ERDF/FEDER)
(grant number AGL2014-51839-C5-1-R) and by the Fun-
daci

on S
eneca de la Regi

on de Murcia (grant number
19883/GERM/15, Grupo de Excelencia).
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