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Háskólinn á Akureyri

Viðskipta- og raunvísindasvið

Námskeið: Lokaverkefni – LOK1126 og LOK1226

Heiti verkefnis: Effects of enhancing black soldier fly larvae (Hermetia illucens) meal for Atlantic salmon (Salmo salar) - Probiotic application and chitin removal: A feasibility study

Verktími: Desember 2017 – apríl 2018

Nemandi: Kristófer Leó Ómarsson

Leiðbeinandi: Alexandra Leeper Rannveig Björnsdóttir

Upplag: 5

Blaðsíðufjöldi: 39

Fjöldi viðauka: 5

Útgáfu og notkunarréttur: Lokað í eitt ár. Verkefnið má ekki fjölfalda, hvorki að hluta til né heild, nema með skriflegu leyfi höfundar.

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„Ég lýsi því yfir að ég einn er höfundur þessa verkefnis og að það er afrakstur eigin rannsókna“.

____________________

Kristófer Leó Ómarsson

„Það staðfestist að verkefni þetta fullnægir að mínum dómi kröfum til prófs í námskeiðunum LOK1126 og LOK1226“.

_____________________

Alexandra Leeper

____________________

Rannveig Björnsdóttir

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Summary  (English  Language)  As the human population continues to grow at a rapid rate, combined with a rising pressure on

already over-exploited global wild fisheries, the need for a dependable and risk-free food source

is of great importance. These concerns have fuelled expansion of the aquaculture industry

which today produces over half of the global fish production for food and non-food uses.

However, modern aquaculture practises rely greatly on wild capture fisheries to produce

aquafeed, hence, as the industry has expanded the demand for wild capture fish has too. After

years of research, soybean protein has found its way into the global market as a partial

replacement for fish meal. Soybeans do have their limitations, one in particular being

deforestation. This conundrum has caused researchers to look to alternative feed ingredients.

After widespread research and despite its limitations, insect protein has been considered a

promising replacement.

This trial aimed to bridge some of the knowledge gaps associated with replacing fish meal and

soy protein with alternative sources, including insect protein. The aim of this study was to assess

black soldier fly larvae (Hermetia illucens) (BSF) meal and investigate possible techniques to

enhance it to make it more competitive in the modern market.

A 28-day trial was conducted on juvenile Atlantic salmon (Salmo salar). Fish were fed 6

different formulated, baked test diets in addition to a commercially available extruded diet that

was used for comparison. Two of the test diets were control diets; fish meal control and soy

bean control, and four test diets were insect-based, formulated using 10% treated and untreated

BSF meal.

The current study found that enhancing BSF meal is a feasible option to improve the

performance of this alternative protein source. Treating the BSF for removal of chitin from

insect proteins resulted in improved growth performance compared with the other experimental

diets, and comparable growth with the commercial feed. Likewise, enhancing the untreated

BSF meal diet with probiotic bacteria resulted in comparable growth with the commercial feed.

Two diets, BSF meal treated with the probiotic and untreated BSF meal, resulted in significantly

lower final weight (g) of the fish than the commercial diet. No negative effects of the BSF meal

were reported in this project. This project aims to support future development and application

of enhanced BSF meal.

(Keywords: Salmo salar, alternative feed, insect meal, chitin, probiotics)

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Acknowledgement  I would like to thank everyone that in one way or another helped me during the preparation and

execution of this project. First and foremost, I want to thank my lead instructor Alexandra

Leeper. I could not be more thankful for her positive attitude and willingness to help me during

my writing. Her knowledge, support and guidance was invaluable. Secondly, I thank my

instructor Rannveig Björnsdóttir for incredible support and guidance. Her knowledge on the

subject was extremely helpful when preparing and executing my project. I want to thank Matís

ohf. for the co-operation and for the use of their facility and equipment, with special thanks to

Birgir Örn Smárason and Jón Árnason for their hospitality and support. Finally, I would like to

thank my family and friends for their support during my writing. For her patience and

encouragement, I owe my fiancé Halldóra Pálsdóttir sincere thanks.

April 20th 2018, Akureyri

Kristófer Leó Ómarsson

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Secondary  Summary  (Icelandic  Language)  Hafið hefur verið uppspretta próteinríkrar fæðu fyrir manninn allt frá því að menn hófu að neyta

sjávarfangs. Á undanförnum áratugum hefur mannkyninu fjölgað með ógnvekjandi hraða og

augu beinast nú að því hvernig eigi að fæða allan þann fjölda sem nú hýsir Jörðina. Aukinn

þrýstingur á veiðar á villtum stofnum og þörfin fyrir sjálfbæra og áreiðanlega próteingjafa hefur

drifið áfram stækkun fiskeldisiðnaðarins. Hraður vöxtur fiskeldis á heimsvísu hefur sett

þrýsting á vísindamenn að finna nýja og umhverfisvænni próteingjafa til fóðurgerðar fyrir

fiskeldi. Skordýramjöl ýmiskonar hefur skilað góðum árangri en hefur þó einnig sínar

takmarkanir.

Þetta verkefni miðaði að því að brúa þekkingarbil sem myndast hefur í tengslum við útskiptingu

á fiski- og sojamjöli fyrir aðra próteingjafa, þ.á.m. skordýramjöl. Meginmarkmið verkefnisins

var að rannsaka möguleika þess að auka virkni lirfumjöls úr black soldier fly (Hermetia

illucens) (BSF), annars vegar með notkun Pediococcus acidilactici bætibaktería, og hins vegar

með því að fjarlægja kítín úr mjölinu. Skoðuð voru áhrif þessarar meðhöndlunar á vöxt og lifun

smáseiða Atlantshafslax (Salmo salar) í samanburði við viðmiðunarfóður.

Framkvæmd var 28 daga löng rannsókn þar sem smáseiði Atlantshafslax voru alin á sjö

mismunandi fóðurblöndum. Fjórar þeirra voru útbúnar með lirfumjölinu sem aðalpróteingjafa.

Allar innihéldu þær sama magn BSF mjöls en voru mismunandi að því leyti að BSF blandan

innihélt ýmist kítín og/eða P. acidilactici bætibakteríur eða hvorugt. Til samanburðar var notast

við tvær blöndur sem innihéldu annars vegar fiskimjöl og hinsvegar sojamjöl sem

aðalpróteingjafa. Markaðsselt eldisfóður frá BioMar var auk þess notað til samanburðar.

Niðurstöður verkefnisins leiddu í ljós að virkni BSF mjöls má bæta með ofangreindum

aðferðum þannig að vöxtur fiska verði sambærilegur við vöxt á því markaðsfóðri sem notað er

í dag. Engin neikvæð áhrif á lifun af völdum BSF mjölsins komu í ljós við framkvæmd

verkefnisins. Markmið þessa verkefnis er að styðja við og hvetja til áframhaldandi rannsókna á

lirfumjöli og notkun þess í fiskeldisiðnaði.

(Lykilorð: Salmo salar, fóðurhráefni, skordýraprótein, kítín, bætibakteríur)

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Table  of  Contents  1 Introduction ............................................................................................................................. 1  

1.1 Aquaculture production ................................................................................................... 1  

1.2 Unsustainable feeding methods in aquaculture ................................................................ 2  

1.3 Knowledge gaps in aquafeed production ......................................................................... 3  

1.4 Alternatives to fish and plant meal in salmonid aquaculture ........................................... 4  

1.5 Insect meal ....................................................................................................................... 5  

1.6 Black soldier fly larvae .................................................................................................... 6  

1.7 Chitin ............................................................................................................................... 7  

1.8 Probiotic application ........................................................................................................ 7  

1.9 Pediococcus acidilactici .................................................................................................. 8  

1.10 Study outline and aims ................................................................................................... 8  

2 Materials and Methods .......................................................................................................... 10  

2.1 Study location and conditions ........................................................................................ 10  

2.2 Test organisms and experimental diets .......................................................................... 11  

2.3 Feeding methods ............................................................................................................ 12  

2.4 Ethical approval ............................................................................................................. 13  

2.5 Experimental setup ......................................................................................................... 13  

2.5.1 Mortality ................................................................................................................. 13  

2.5.2 Growth .................................................................................................................... 13  

2.6 Statistical analysis .......................................................................................................... 14  

3 Results ................................................................................................................................... 15  

3.1 Mortality and growth ..................................................................................................... 15  

4 Discussion ............................................................................................................................. 19  

4.1 Mortality and growth ..................................................................................................... 19  

4.2 Future perspectives ........................................................................................................ 21  

5 Final Conclusion ................................................................................................................... 23  

6 References ............................................................................................................................. 24  

Appendices ............................................................................................................................... 29  

   

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List  of  Figures   Figure 1: Black soldier fly larvae at the ideal size and development stage for harvesting ....... 6  

Figure 2: Configuration of tank rows ..................................................................................... 10  

Figure 3: Distribution of test tanks ......................................................................................... 11  

Figure 4: Automatic belt feeder used in the trial .................................................................... 12  

Figure 5: Growth performance data comparing BSF diets and control diet treatments ......... 16  

Figure 6: Growth performance data comparing BSF diets and the COM diet treatment ....... 16  

Figure 7: Specific growth rate data comparing BSF diets and control diet treatments .......... 17  

Figure 8: Specific growth rate data comparing BSF diet and COM diet treatments .............. 18  

List  of  Tables  

Table 1: Proximate feed composition of the experimental diets ............................................. 11  

Table 2: Macronutrient composition of the commercial BioMar Inicio Plus diet .................. 12  

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List  of  Appendices   Appendix 1: FAO feed table for Atlantic salmon (Salmo salar) ............................................ 29  

Appendix 2: European Directive 2010/63 EU on ethical standards ....................................... 29  

Appendix 3: Results of Tukey post hoc test for final weight (g) of BSF and control diets .... 30  

Appendix 4: Results of Tukey post hoc test for final weight (g) of BSF and COM diets ..... 30  

Appendix 5: Results of Tukey post hoc test for SGR% of BSF and COM diets ................... 31  

List  of  Equations  

Equation 1: Specific Growth Rate (%) ................................................................................... 14  

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1  Introduction  Rising global demand for food is unequivocally linked to the expanding population. With a

predicted global population of 9,8 billion people by the year 2050 (United Nations, 2017), food

production must not only increase, but be environmentally and socially sustainable.

Sustainability is often described as ‘the rate of which a resource is used as to not exceed the

Earth's ability to replace it’ (United Nations, 2017). That was certainly the case hundreds of

years ago when the world population was only a fraction of what it is today. The current state

of technology and population requires the concept of sustainability to be re-examined (Godfray

et al., 2010). There are other factors that need to be considered, including food and consumer

safety, production methods and their impact on the environment. New methods to produce food

with emphasis on minimising the impact on the environment, and relieving pressure on already

over-exploited ecosystems is imperative to maintain sustainability in food production (Behrens

et al., 2017). This has caused an increased reliance on aquaculture to provide for our expanding

society (Watts et al., 2017).

1.1  Aquaculture  production  In recent decades, aquaculture’s rapid growth has outpaced its main competitors, terrestrial

livestock and capture fisheries (Schmidt et al., 2016). With a total world aquaculture production

of 66,6 million tonnes of food fish in 2012, totalling a first-sale price of approximately

US$137,7 billion, the aquaculture industry had grown almost thirty times since 1970 (FAO,

2014). Almost all fish produced by the aquaculture industry is utilized for human consumption,

with by-products being used for non-food purposes. Aquaculture accounted for approximately

44,1 percent of the global fish production (non-food fish included) from capture fisheries and

aquaculture in 2014 (FAO, 2016) and close to 50 percent in 2016 (Schmidt et al., 2016). In the

present day, production of farmed Atlantic salmon exceeds one million tonnes, constituting

over 90 percent the of the farmed salmon market and over 50 percent of the global market

(FAO, 2018).

Latest findings show a general trend in increase of aquaculture’s share in total fish production

worldwide. As aquaculture’s share in the global food-fish production continues to expand

(FAO, 2016), researchers predict aquaculture will produce an estimated 62 percent of all

seafood to be consumed in 2020 (Schmidt et al., 2016). Regardless of its sharp expansion,

aquaculture on a global scale is a reasonably undeveloped industry and therefore has its

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limitations. This study will focus on some of those limitations, in particular fish feed, and

directly link them to salmonid aquaculture.

1.2  Unsustainable  feeding  methods  in  aquaculture  With constant expansion and increasing production of the aquaculture industry, fish feed has

become a seriously limiting factor, both restraining production volume and compromising the

sustainability of the industry (Gachango et al., 2017). A key ingredient in commercial aquafeed

is fish meal and fish oil sourced from the ocean, thus has aquaculture put a further strain on the

marine ecosystem (Schmidt et al., 2016). In 2011, aquaculture accounted for the consumption

of 60 to 70 percent of the annual fish meal production in the world and 80 to 90 percent of all

fish oil production. Poultry, pork and pet feed accounted for most of the rest (Marine Harvest,

2017). The main source of fish meal and fish oil is from various small pelagic fish including

herring, capelin, pilchard and anchovies. These species are at risk due to high demand in fish

meal and fish oil, not to mention decades of unsustainable fisheries and direct consumption.

(Tacon and Metian, 2008). Studies have recommended that these species be used minimally for

reduction into fishmeal and fish oil and have instead encouraged the use of these species for

human consumption in food scarce regions (Tacon and Metian, 2013).

Salmonids are highly carnivorous fish that need protein rich feed. For that reason, studying a

hugely commercially important species, with a very well documented biology, like Atlantic

salmon is especially important. This is crucial on the topic of dietary requirements. As will be

later mentioned in this paper, dietary requirements differ not only between species of fish, but

also across different developmental stages within the same species. At early stages of

development, fry and juvenile fish are more sensitive to proteins and other nutrients. Therefore,

examining effects of feed enhancements on juvenile Atlantic salmon will give a good idea about

effects in salmon in later stages of development.

Not only is nutritious feed important, but well-balanced feed at a cost-effective price is very

important to fish farmers and a prerequisite for a profitable production. On-farm feed in

salmonid production can cut costs and encourage productivity with fish farmers (FAO, 2016).

Rising fish meal and fish oil costs have driven companies to look to other protein sources

including soy protein and other plant-based proteins (Gatlin et al., 2007).

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1.3  Knowledge  gaps  in  aquafeed  production  Extensive studies have been performed in search of a feasible replacement for fish meal in the

aquafeed industry. Numerous types of plant-based meals have been tested, including meals

from soybeans, barley, canola, corn, cottonseed, wheat, peas and lupines (Gatlin et al., 2007).

Although all are readily available, soybeans have been the main focus-point for plant protein,

largely due to its high digestible protein content as well as a suitable nutritional composition

and a good amino acid profile (Yaghoubi et al., 2016). Aforementioned nutritional benefits and

a reasonable price has made soybean meal a widely popular alternative to fish meal in the

industry. After years of research, soy protein has now become commercially accepted and has

gained considerable popularity over its traditional market competitor, fish meal (Refstie et al.,

2001). Multiple trials have been conducted on adult Atlantic salmon where a 25-35 percent

inclusion of soy protein in their diet did not have negative effects on growth and productivity

(Gatlin et al., 2007; Carter et al., 2000). However, with increased inclusion, salmonids begin

showing negative effects (Gatlin et al., 2007). Although previous trials have reported positive

effects of using high-inclusion soy-based meals in fish feed, trials involving salmon being fed

this type of feed have largely been negative. Not all species of fish have the same tolerance

against replacing traditional protein sources for a plant-based protein, that being most visible

with carnivorous fish (Yaghoubi et al., 2016). Inclusion of soy protein may not be a direct cause

of mortality, but could have inhibiting effects due to the presence of anti-nutritional factors

(Francis et al., 2001).

Anti-nutritional factors are substances that reduce the availability of one or more nutrient when

introduced through either animal feed or water (Yacout, 2016). When it comes to feeding any

type of fish, nutritional composition and protein content plays a vital role. Without adequate

nutrition, fish are more likely to grow slower and often exhibit various negative traits such as

muscle wasting, weight loss and an increased risk of infections and diseases. It is important to

note that these traits can also be derived from poor environmental conditions and bad work-

ethics by fish keepers (Poli, 2009). A trial conducted on juvenile Atlantic salmon (Salmo salar)

in sea cages showed that juvenile salmon exhibited distal enteritis which lead to diarrhoea,

digestive disturbances, a slight reduction in the concentration of muscles and limited nutrient

uptake (Refstie et al., 2001). This goes to show that juvenile salmon are likely to react

differently to feed trials than adult salmon since they have a higher protein requirement and are

more sensitive to anti-nutritional factors. Furthermore, limiting nutrient uptake is especially bad

for the producer as it increases feeding costs. Earlier studies report that high content of

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indigestible carbohydrates in soybean meal can have an effect on digestibility of fat in soybean

meal, consequently inducing an inflammatory condition in the fish distal intestine (Krogdahl et

al., 2003; Refstie et al., 2001). It is important to note that even though fish feeds with high

volume of soybean meal show insignificant effect on weight gain, fish welfare and the effect

that plant-protein has on their digestive system and health must be considered. An unhealthy

fish may put other fish in its environment in jeopardy, therefor endangering an entire production

stock for a fish farmer.

It is worth mentioning, although being popular with fish farmers due to cost efficiency and

adequate growth results, soy is somewhat economically unattractive. This is especially relevant

in the European salmon farming industry. Reliance on a single ingredient as a replacement for

fish meal is risky and because soybeans cannot be grown in Europe, in part due to improper

agro-climatic conditions, soy must be imported from other countries. That means relying on

outside-markets and fluctuating prices. Additionally, soybean production requires large open

fields which in return requires deforestation. By increasing the demand for soybeans, so does

the demand for deforestation. For a sustainable future, a different approach must be taken. An

emphasis must be put on growing other protein sources in Europe (FEFAC, 2018).

1.4  Alternatives  to  fish  and  plant  meal  in  salmonid  aquaculture  The need for research into alternative protein sources for aquafeeds is of great importance if we

are to continue to produce sustainable and protein rich food for human consumption (United

Nations, 2017). Numerous studies have shown that several species of carnivorous fish,

including Atlantic salmon, are well equipped to consume and digest various types of feed. That

includes poultry meals, pork by-product meals, soy, grains and nuts. These findings present an

opportunity to produce aquafeeds which include little to no fishmeal. A number of studies have

investigated the effect of fishmeal-free feeds on farm raised fish, including juvenile Nile tilapia

(Oreochromis niloticus) fed soybean meal and juvenile rainbow trout (Oncorhyncus mykiss)

fed a mixture of soybean- and cornmeal (Furuya et al., 2004; Adelizi et al., 1998). Largely,

results have shown that these feeds have little to no effect on various performance metrics,

including growth, palatability, nutrition, fatty acid composition of fillets and water quality

(Schmidt et al., 2016). This indicates that aquafeeds without fishmeal as a main raw material

are certainly viable (Schmidt et al., 2016).

Although studies have shown promising results, many questions are still unanswered. Several

studies have found that feeding diets based on materials that are not readily available in the

fishes’ natural environment can have negative effect on their metabolism and health. Such

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effects include high excretion of ammonia and nitrate, and differences in gut morphology and

microbiota. These findings clearly show that further research is needed concerning alternative

feeds, both its effect on the fish digestive systems and its impact on the microbial environment

which is very important for the growth and welfare of the fish (Schmidt et al., 2016).

1.5  Insect  meal  Insects in fish diets have been widely discussed in the recent, and not so recent, literature. Not

only are they thought to have beneficial properties for animal feed but also for human

consumption. Insects have multiple positive properties including easy growth and reproduction

and a high feed conversion ratio (Tran et al., 2015; Makkar et al., 2014). A promising benefit

of insects is the ability to rear them on organic waste. They can break down a wide range of

organic materials which gives an additional benefit for waste disposal. Due to their high feed

conversion rate, one kilogram of insect biomass can be produced from an average of two

kilograms of organic waste (Makkar et al., 2014). This, coupled with their fast growth and

reproduction, makes them almost an ideal candidate to replace other raw protein sources like

fish meal. Also, it is a known fact that many fish in the wild readily eat insects as a part of their

natural diet. (Tran et al., 2015). It is also worth mentioning that only recently was it made legal

in Europe to feed insects, reared on vegetable based food waste, to fish. This change in law has

prompted researchers to start looking into incorporating these insect into fish feeds.

Various insects have been researched for their ability to replace fish meal and plant-based meals

in feed for fish. The most promising candidates include; maggots, mealworm larvae, silk worm

pupae, adult Orthoptera (grasshoppers and locusts) and black soldier fly larvae (Hermetica

illucens) (BSF) (Tran et al., 2015). A research conducted in 2012 concluded that the use of

insects on an industrial scale is technically feasible. The most promising insect species included

BSF and mealworms (Veldkamp et al., 2012). It must be stated that despite having the suitable

nutritional value, insect-based feed may not be suitable for all species of fish whereas different

species of fish have different nutritional needs. For example, highly carnivorous fish like

Atlantic salmon and rainbow trout need much more protein and lipids than the common tilapia

(Tilapia sparrmanii). It goes without saying that a fish that demands high volumes of protein

and lipids must be reared on nutrient rich feed. Consequently, a protein rich insect must be an

ideal candidate for such fish. These are qualities that evidence suggests BSF will have and so

they have been considered in this study for further investigation.

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1.6  Black  soldier  fly  larvae  Past studies have shown a potential for inclusion of up to 50 percent of BSF meal for

carnivorous fish. A recent study by Stamer et al. (2014) on juvenile rainbow trout concluded

that fish meal could be substituted with up to 50 percent BSF meal without risking losses in

body weight gain, feed conversion ratio and protein retention ratio. Furthermore, even though

an inclusion of 75 percent BSF meal did result in poorer growth performance, no signs of

nutrient deficiency nor higher mortality rates were reported. As already stated, the BSF has

shown great promise in attacking the feed protein deficit.

The BSF occurs in both warm and temperate climates and its larvae can therefore be reared

almost anywhere in the world (Figure 1). As applies with many other fly larvae, the BSF can

be reared on organic materials, including organic waste. By reducing the volume of organic

materials by 50%, the BSF will produce a biomass with a protein content of up to 42% and a

fat content of up to 35% (Bondary and Sheppard, 1981; Sheppard et al., 1994). In a world where

circular economy1 is key, the importance of recovering waste materials and re-using them as

insect feed to produce animal feed cannot be understated. In a perfect world we would be able

to substitute 100 percent of fish/soy-derived protein with BSF meal. To reach such an ambitious

objective we must find a way to optimize the use of black soldier fly – and determine what

factors in insect proteins are limiting its market competitiveness.

1 An economy where resources are in use for as long as possible. While in use, these resources are extracted for their maximum value and at the end of their service life, are recovered and used to regenerate products and materials (WRAP, 2018).

Figure 1: Black soldier fly larvae at the ideal size and development stage for harvesting. Reference: Royal Society of Chemistry (2018)

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1.7  Chitin    One way the BSF meal could be enhanced is with the removal of chitin. Chitin is a highly

insoluble material found not only in insect exoskeleton, but also in their internal structures

including the inner surface of their gut (Merzendorfer, 2006). Chitin functions naturally as a

structural polysaccharide, much like cellulose, and has low chemical reactivity (Kumar, 2000).

It is an essential element in insect growth and development as insects regularly shed and replace

their cuticular exoskeleton, which is mostly formed by chitin (Merzendorfer, 2006). In a study

on Atlantic salmon, Karlsen et al. (2017) reported that an inclusion of up to 1% chitin had no

effect on length, weight, condition, liver size or specific growth rate of Atlantic salmon. On the

other hand, an inclusion of >1% of dietary chitin had negative effect on growth and nutrient

utilization. Faecal protein increased significantly with increased dietary chitin, however faecal

dry matter and lipid did not increase. The conclusion was that Atlantic salmon is likely to have

low tolerance to dietary chitin inclusions. Recent studies have reported that the presence of

chitin may influence nutrient uptake of juvenile turbot by limiting availability and digestibility

of important nutrients. An observation was made that decrease in growth performance could be

linked to low palatability in the feed due to the presence of chitin. Incorporation of BSF meal

is possible but further research on BSF meal and effects of chitin on nutrient uptake to increase

utilization in fish needs further research (Kroeckel et al., 2012). Removing chitin may only be

a part of the problem. Methods of enhancing BSF meal without having to remove chitin may

prove beneficial to both fish and producers. Such methods include probiotic application.

1.8  Probiotic  application  In recent years, use of probiotics as a mean of improving the intestinal balance of test animals

has shown promise (Watts et al., 2017). Additionally, increasing digestibility of chitin by

enzyme application has been done but preliminary studies have not reported promising results

(Gasco et al., 2016). In 2001, Food and Agriculture (FAO) and World Health Organization

(WHO) described probiotics as “live microorganisms which, when administered in adequate

amounts, confer health benefit on the host” (Watts et al., 2017). This study investigated the

possibility of a probiotic treatment, where applying a microbial organism to fish feed diets, in

hopes of improving growth performance. In order to do so, we must fully understand the

concept of probiotics. There are many different types of microbial applications available with

a range of potential aquaculture applications. Therefore, proposing a joint definition for all

those treatments is required. Following Merrifield’s example, we assume that a probiotic is any

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microbial cell, introduced via the diet, that benefits the host fish regarding improved fish

appetite, growth performance and feed utilization (Merrifield et al., 2010). Several types of

probiotics have been tested in fish with the aforementioned benefits, one of them being

Pediococcus acidilactici.

1.9  Pediococcus  acidilactici  P. acidilactici is a species of lactic acid bacteria (LAB) that can grow in a wide range of pH,

temperature and osmotic pressure, and therefore has the ability to colonize the intestinal tract

(Merrifield et al., 2010). Merrifield et al. (2010) reported that probiotics, including P.

acidilactici, can provide nutritional benefits and protection against pathogens in the host. A

probiotic application by Ferguson et al. (2010), where red tilapia (Oreochromis niloticus) was

given feed containing P. acidilactici, concluded that the bacteria did in fact colonize the

intestinal tract and remained in the intestinal tract for several weeks after being removed from

the diet. Also, the presence of probiotic bacteria showed a general trend towards elevated

growth, survival and immune stimulation of the fish (Ferguson et al., 2010). Additional studies

concur with these results (for review see Gatesoupe, 2002; Zhou et al., 2010). Nonetheless, not

all studies have been as positive and Shelby et al. (2007) reported a lack of growth promoting

or immune stimulating effects in channel catfish. Despite reporting positive results, studies on

salmonids using P. acidilactici need further attention (Standen et al., 2013; Ferguson et al.,

2010).

1.10  Study  outline  and  aims  The aim of this study was to address knowledge gaps associated with replacing traditional

protein sources in commercial aquaculture feeds. The existing literature supports the use of

BSF in aquafeeds as a promising replacement for traditional protein sources. This study will

investigate two potential methods of optimising BSF meal inclusion to improve its use in

salmonid feeds. In this study, fish meal was replaced with BSF meal. BSF-based diets, either

untreated or treated by removing chitin, were supplemented with the Pediococcus acidilactici

probiotic to determine potential effects on the mortality and growth of the test organisms.

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After reviewing past studies and determining the most important knowledge gaps, two key

research question were formed:

1. Is there a growth benefit of either enhancement to BSF meal compared with a) untreated

BSF and b) with traditional diet ingredients?

2. Will combining these two enhancements with BSF meal result in a growth benefit

compared with a) untreated BSF and b) with traditional diet ingredients?

For this trial, a test organism with a commercial importance and a carefully documented biology

had to be selected. With those elements at hand, and being readily available, juvenile Atlantic

salmon (Salmo salar) were chosen as the ideal candidate. There are a number of key reasons

why juvenile salmon were selected:

-­‐   During this life stage a crucial development period takes place that impacts growth and

survivability in adult fish.

-­‐   Over the course of this life stage, protein requirements are much greater than at later stages

of development. As a result, results in this trial can aid in preparing diets for adult fish too.

-­‐   During juvenility, growth is very rapid so a short term trial (like this one) can provide

reliable results (Leeper, unpublished data).

 

10

2  Materials  and  Methods  

2.1  Study  location  and  conditions  This trial was a part of a collaborative project with Matís ohf., an independent research company

in Iceland. The trial was performed at the Matís Aquaculture Research Station (MARS) in

Reykjavík, Iceland where Atlantic salmon (Salmo salar) were fed 6 experimental diets and 1

commercial comparison diet, used for comparison. The diets were provided for 28 days, starting

on March 12th, 2018. During the trial, test organisms were kept in 28 separate cylindrical tanks

with 15 L of fresh water (Figure 2). For filtration, a flow-through system was used where flow

rates were kept consistent to maintain an oxygen saturation higher than 90% in the runoff water.

By keeping a flow rate of 10 mL per 2,3 seconds (average), total tank water was replaced 3

times per hour. Each tank was stocked with 30 fish during the trial. For the duration of the trial,

environmental conditions were monitored daily. Temperature was maintained at 9,0+-5°C.

Light conditions were kept stable 24 hours a day using 40 Lux +-10.

To avoid any order effect on feeding, replicate tanks with different feed formulations offered

were distributed along the rows in a random order (Figure 3).

Figure 2: Configuration of tank rows.

11

2.2  Test  organisms  and  experimental  diets  Atlantic salmon (Salmo salar) fry were obtained from Stofnfiskur, a large scale commercial

supplier in south Iceland, and transported to MARS. Before the trial started the fish were

divided in two groups in duplicate plastic tubs, with identical water parameters, and acclimated

for a period of one week. The fish were fed ad libitum on commercial fish meal-based (FM)

feed, provided by BioMar, before the initial weighing and switching to the experimental diets.

The main ingredients of the experimental diets were soy bean-, fish- and BSF meals, wheat,

cornmeal, fish oil and a mineral/vitamin pre-mix. Six experimental diets were designed for this

trial (Table 1). All prepared feeds were baked and then crumbled into 0,5-1,0 mm sized pellets.

Table 1: Proximate feed composition of the experimental diets.

Ingredient (%) FM1 SPC2 BSFC+3 BSFC-4 BSFC+P+5 BSFC-P+6

Fish Meal 63,0 46,8 40,6 41,7 40,7 42,0 Fish Oil 10,1 12,0 9,7 9,5 10,1 10,0 PreMix 1,0 1,0 1,0 1,0 1,0 1,0 Wheat 18,1 13,3 11,7 10,9 11,3 10,1 Cornmeal 7,8 7,8 7,8 7,8 7,8 7,8 Soy Protein Concentrate 0,0 19,2 19,2 19,2 19,2 19,2 BSF Meal 0,0 0,0 10,0 10,0 10,0 10,0

1 Fish meal control 2 Soy protein control 3 BSF meal with chitin

4 BSF meal without chitin

5 BSF meal with chitin and with the P. acidilactici probiotic

6 BSF meal without chitin and with the P. acidilactici probiotic

Figure 3: Distribution of test tanks. Colors represent each diet group where an R-number corresponds to the tank replicate within each group. The different diets are; Commercial BioMar feed (COM), fish meal control (FM), soy protein control (SPC), untreated BSF meal (BSFC+), treated BSF meal (BSFC-), untreated BSF meal with the probiotic (BSFC+P+) and treated BSF meal with the probiotic (BSFC-P+).

12

In order for the trial to be directly relevant to the aquaculture industry, a standard industry

commercial diet (COM) BioMar Inicio Plus, with a pellet size of 1,1 mm, was used as further

comparative (Table 2).

Table 2: Macronutrient composition of the commercial BioMar Inicio Plus diet

*Since this is a commercial industry feed the full ingredient list is not available

2.3  Feeding  methods  Daily feed allowance of diets was calculated using a FAO table which provides

recommendations specific for Atlantic salmon (Appendix 1). In view of the fact that the

objective of this trial was purely to examine the effect of the ingredients in the diets, not the

availability of the feed, an overestimate of figures in the FAO table was made to ensure that the

fish were getting adequate feed (Leeper, unpublished data). All tanks were fed 15 times per day

in 3 minute intervals between hours 09:00 and 01:00 (16 hours) using an automatic belt feeder,

(Figure 4).

Macronutrients in COM* Inclusion (%) Crude protein 56 Crude fat 18 Ash 10,2 Crude fibre 0,4 Phosphorous 1,65 Calcium 2,9 Sodium 1,01

Figure 4: Left: Automatic belt feeder used in the trial. An electric motor (1) connected to a timer (2) that activates the motor every hour, pulling the white strips that carry the feed. The feed then hits a barrier (3) causing it to fall into the tank.

13

All tanks received the same weight of feed across all treatments during the trial. Feed was

placed on the automatic belt feeder and in approximately one hour intervals the belt would

move the feed into a plastic barrier causing it to fall into the tank.

2.4  Ethical  approval  Over the course of this trial, various procedures were performed where fish either had to be

handled or moved with a fish net. All procedures involving live fish were done with fish welfare

on the forefront and in accordance with the ethical standards of Matís ohf which follow the

European Directive 2010/63 EU2. Fish behaviour and welfare was monitored daily throughout

the experiment. In case fish showed signs of severe injury, illness or discomfort humane

endpoints (euthanasia by an excess of anaesthetic dose) were planned and performed.

2.5  Experimental  setup  For this project, focus was primarily on assessing any differences on fish growth and survival

between the experimental diets. Mortality data was collected daily over the course of this trial

and changes in total wet biomass (g) was recorded in the beginning and the end of the trial.

2.5.1  Mortality  

Throughout the trial, survival was monitored daily with weight and size of any dead fish being

recorded. At the end of the trial, accumulated mortalities were tallied for each feed to judge for

any differences between the different feed treatments.

2.5.2  Growth  

Total wet biomass (g) data was collected during the initial measurement, and during final

measurements at the end of the 28-day trial. During initial measurement, a randomly selected

number of individuals were weighed to give data on wet weight (g) and collected in the

experimental tanks where number of fish in each tank was 30 and the total biomass for each

replicate was 40 g.

After 28 days of feeding the experimental diets, a final weight assessment was carried out where

data on total wet biomass (g) per tank was collected. Additionally, accumulated mortalities

were calculated for each feed.

2 A further read and a link to the European Directive is provided in Appendix 2

14

The data collected was used to calculate the Specific Growth Rate (SGR%) according to the

following equation:

where (t) represents the number of trial days.

All fish were starved for 24 hours prior to initial and final biomass weighing. For weighing, a

digital scale with an accuracy range of 0,01 grams was used.

2.6  Statistical  analysis   In order to assess growth and mortality variables for equality of variance, a Levene’s test was

used. As variables were parametric, a two-level nested ANOVA test was applied to statistically

assess for differences between the diet treatments. For this test, treatments were assigned to the

standard (fixed) factor with the tank replicate as a random factor nested within the diet

treatments. Subsequently, to assess where differences existed, a Tukey post hoc test was used.

 

Equation 1:

15

3  Results  

3.1  Mortality  and  growth   Initial measurements of the Atlantic salmon total wet biomass (g) showed no significant

difference between the 28 groups of fish prior to offering the experimental diets. For the

remainder of this chapter, total wet biomass (g) will be referred to as final weight (g).

After being fed the different diet treatments; Commercial BioMar feed (COM), fish meal

control (FM), soy protein control (SPC), untreated BSF meal (BSFC+), treated BSF meal

(BSFC-), untreated BSF meal with the probiotic (BSFC+P+) and treated BSF meal with the

probiotic (BSFC-P+) for 28 days, total wet biomass (g) and Specific Growth Rate (%) was

calculated for the trial period.

In total, five mortalities were observed, all associated with fish jumping out of their tanks. Since

no fish were found dead inside their tanks, no mortality was associated with the diets.

When comparing the experimental insect feed treatments to the control diets (SPC and FM),

revealed a significant difference across the diet treatments regarding final weight

(F(5,15)=2,9133 p-value=0,0494). The Tukey post hoc test revealed the source of this

significant difference (Figure 5). The BSFC- treatment revealed the highest final weight for

this comparison. No significant difference was found across the FM, BSFC-, BSFC+,

BSFC+P+ and BSFC-P+ diet treatments. The SPC diet produced a significantly lower final

weight than the BSFC- treatment, however it was not significantly different from the other

treatments. A table containing the Tukey post hoc results can be found in Appendix 3.

When comparing the experimental insect feeds to the COM diet treatment, a significant

difference was found across the diet treatments concerning final weight (F(4,12)=4,7077 p-

value=0,0162). The Tukey post hoc test revealed the source of this significant difference

(Figure 6). The COM treatment revealed the highest final weight across all diets for this project.

No significant difference was found between the COM, BSFC- and BSFC+P+ treatments,

however the BSFC+ and BSFC-P+ treatments revealed a significantly lower final weight when

compared to the COM diet. A table containing the Tukey post hoc results can be found in

Appendix 4.

16

Figure 5: Growth performance data comparing BSF diets and control diet treatments. Final measurements of total wet weight (g). Letters a and b indicate differences between treatments. Treatments not sharing a common letter are significantly different.

Figure 6: Growth performance data comparing BSF diets and the COM diet treatment. Final measurements of total wet weight (g). Letters a and b indicate differences between treatments. Treatments not sharing a common letter are significantly different.

17

There was no significant difference found across the control diets and BSF treatments for

SGR% (F(5,15)=2,6881 p-value=0,0629) (Figure 7). The result of comparing the COM diet

treatment to the experimental insect feeds revealed a significant difference across the treatments

for SGR% (F(4,12)=4,5655 p-value=0,018). A post hoc test revealed the source of this

significant difference (Figure 8). Identical to the post hoc test performed on the final weight

when comparing the BSF and COM treatments, the COM diet revealed the highest value for

SGR%. The BSFC- and BSFC+P+ revealed no significant difference compared to the COM

diet. However, the BSFC+ and BSFC-P+ revealed a significantly lower SGR% compared to

the COM diet but were not significantly different from the other BSF-based diets. A table

containing the post hoc results can be found in Appendix 5.

Figure 7: Specific growth rate (SGR%) data comparing BSF diets and control diet treatments. No significant difference was found across treatments.

18

Figure 8: Specific growth rate (SGR%) data comparing BSF diet and COM diet treatments. Letters a and b indicate differences between treatments. Treatments not sharing a common letter are significantly different.

19

4  Discussion  Facing a future protein deficit in Europe, the development of a sustainable aquafeed may be

key to solving that problem. Already, black soldier fly larvae (BSF) meal has been studied and

tested on fish with promising results at moderate inclusion levels. However, these studies share

the common denominator that a high inclusion of BSF meal has a limiting effect on growth

performance (Stamer et al., 2014; Kroeckel et al., 2012; Karlsen et al., 2017). The aim of this

project was to bridge a knowledge gap by assessing the impact on growth of enhancing insect-

based diets for juvenile Atlantic salmon. BSF-based diets, untreated or treated by removing

chitin, and with or without the probiotic, Pediococcus acidilactici, were compared with

traditional protein-based feeds The objective was to investigate if enhancing BSF meal could

make it comparable to commercial fish feed. This aim has been achieved and key findings

suggest that either removing chitin from, or adding probiotic to the untreated BSF meal shows

potential to increase the competitiveness with commercial fish feed. In the following section,

these findings will be discussed in detail along with recommendations for further research.

4.1  Mortality  and  growth  In this project, we assessed statistical differences in total wet biomass (g) and specific growth

rate (SGR%) in juvenile Atlantic salmon after feeding 7 different diet treatments over a 28-day

period. By gathering tank biomass data from all the diet treatments; COM3, FM4, SPC5,

BSFC+6, BSFC-7, BSFC+P+8 and BSFC-P+9, it was determined that the BSFC- and BSFC+P+

treatments were statistically comparable with the COM diet.

The only few mortalities observed were associated with fish jumping out of their tanks. Since

no fish were found dead inside their tanks, no mortality was associated with the diets and so it

is considered that all diets have the potential to be used.

The COM presented the highest final weight and SGR% values across all diets. Following the

COM diet were the BSFC- and BSFC+P+ treatments, but interestingly they were not

significantly different from the COM. This suggests that treating BSF meal and supplementing

3 Commercial BioMar Inicio Plus 4 Fish meal control 5 Soy protein control 6 BSF meal with chitin 7 BSF meal without chitin 8 BSF meal with chitin and with the P. acidilactici probiotic 9 BSF meal without chitin and with the P. acidilactici probiotic

20

untreated BSF meal with a probiotic will make both diets statistically comparable to the

commercial feed. Growth results for the BSFC+ diet revealed a significantly lower final weight

and SGR% compared to the COM diet. This suggests that the probiotic in the BSFC+P+ diet

may have counteracted the negative effects of the chitin.

Earlier studies, where an inclusion of chitin is thought to be limiting feed and nutrient uptake

in fish, do support the poor results of the BSFC+ treatment (Kroeckel et al., 2012; Olsen et al.,

2006). The BSFC-P+ treatment also showed significantly lower final weight than the COM

diet. The promising results for the BSFC- treatment does raise a question if a combination of

the probiotic and the treated BSF meal was in any way counteracting beneficial effects of the

meal. The success of the BSFC+P+ diet may be linked to the inclusion of P. acidilactici,

supporting earlier studies where tilapia fed diets enriched with P. acidilactici revealed no

negative effects on growth performance (Ferguson et al., 2010). Furthermore, Zare et al. (2017)

reported increased growth performance in newly hatched sturgeon (Huso huso) larvae when

fed Artemia-based feed enriched with P. acidilactici. Results from the current study therefore

imply that either removing chitin or counteracting the negative effect of chitin by using a

probiotic may improve the feed. The results are in accordance with earlier studies that report

positive effects of probiotic application on growth performance in newly hatched and juvenile

fish (Zare et al., 2017; Zhou et al., 2010).

Comparing the insect-based experimental diets to the control diets, FM and SPC, revealed that

the BSFC- delivered the highest final weight across all diets. All diets apart from the SPC were

not significantly different from the BSFC-. Therefore, the SPC diet performed significantly

worse than the BSFC- diet over the course of this project regarding both final weight and

SGR%. This may have been caused by lower inclusion of fish meal in the SPC diet and the

effect of replacing fish meal with soy protein in a juvenile salmon diet. Juveniles, being more

sensitive to protein replacement, may have experienced a limiting effect on growth performance

by receiving a soy-based diet. A study on juvenile Atlantic salmon (initial weight 5,5 g) and

rainbow trout (initial weight 19,5 g) supports these findings by reporting decrease in growth

performance with increased SPC inclusion (Burr et al., 2012). It is important to mention that in

tank number 10, containing fish fed the SPC diet, were three mortalities linked to fish jumping

out of the tank. Additionally, one fish in in the same tank was euthanized due to being bullied

by other fish. This may have brought down the average final weight for the SPC treatment,

thereby obscuring the results to some extent.

The FM diet performed well and was not significantly different from the BSFC- diet, as already

stated. Although not being significantly different, the FM diet did result in lower final weight

21

and SGR% than the aforementioned BSFC- and BSFC+P+ diets. That may have been a result

of using baked feeds instead of extruded, as will be discussed further below.

Previous studies have shown that BSF meal can successfully partially replace fish meal in some

salmonid diets (Stamer et al., 2014). A study by Olsen et al. (2006) stated that substituting fish

meal for Antarctic krill by up to 100 percent is possible in Atlantic salmon. Krill shell, much

like the insect used in this study, is rich in chitin. That may be the reason why authors report

that at higher inclusion the salmon needed to eat more of the krill than the fish meal diet to

maintain growth. A later study supports these results, reporting limiting effects on growth

performance when feeding BSF-based diets to juvenile turbot. When fed a 33 percent inclusion

BSF-based diet, turbot exhibited a decrease in feed intake which was likely caused by low

palatability, and decreased growth rate (Kroeckel et al., 2012). Limiting nutrient uptake and

decreasing palatability in the feed may be equally responsible for reduced feed intake in

salmonids. This may cause fish to have to eat more feed compared to fish meal-based diets, or

increased left-over feed when feeding fish at farming facilities. This is a negative point because

the idea is to make BSF meal a sensible replacement for traditional protein sources; fish meal

and soy protein. Increasing the amount of feed needed to reach the commercial slaughtering

weight of the fish, thereby increasing feed costs and further risk to fish welfare, is unlikely to

be appealing to producers in the industry. In conclusion, numerous studies all agree that that

increased chitin inclusion will produce a negative effect on growth performance.

A fish meal-based diet supplemented with the P. acidilactici probiotic, fed to juvenile tilapia

(O. niloticus), did not exhibit negative effects on growth performance when compared to a fish

meal-based control diet (Standen et al., 2013). Interestingly, not only was there no significant

difference in growth performance reported, but survival rate was significantly higher than that

in the control group (Ferguson et al., 2010).

4.2  Future  perspectives  When reviewing the results of this project, limitations must be acknowledged and explained.

Because this was only a 28-day long study, potential long-term effects of feeding the

experimental diets were not revealed. Similarly, only using juvenile salmon implies that the

effects are only relevant to a certain stage in the salmon life cycle. As stated in the aims and

outline chapter of this thesis, juvenile fish grow at a rapid rate during this protein sensitive and

crucial growth period (Yaghoubi et al., 2016). For that reason, a 28-day trial should give reliable

indication of the potential of these new dietary treatments. Nevertheless, this was only a small-

scale project at a small research scale facility and the results can therefore not be linked directly

22

to full scale fish farming facilities at all developmental stages. A major difference in the

commercial feed and other feed treatments in this trial is that the commercial feed was extruded

but all other feeds were baked. A critical characteristic for extruded feeds is that it minimizes

the degradation of nutrients while improving the digestibility of proteins and starches (Azeuz,

n.d.). Baked feeds may not do as well in this sense. The need for further research, where these

limitations are considered and resolved, will undoubtedly aid in further understanding the

concept of enhanced BSF meal application in fish feed. It will also be important to test these

optimized diets at all developmental stages of Atlantic salmon production to make them market

applicable.

Additional studies are also required to fully understand the concept of probiotic application and

treating chitin in aquafeeds. That is especially true for Atlantic salmon, a heavily cultured

species in Europe and a provider of a lot of the human fish protein in the European market. A

closer look into probiotics like P. acidilactici is essential in order to broaden our understanding

of its effect on fish. Future studies should aim to further assess the effects that P. acidilactici

has on fish intestines and gut microbiota and how it can relate to disease prevention and

immunity.

In view of this study’s findings, further research involving probiotic application is needed. By

reporting positive results for an untreated BSF-based diet, supplemented with the P. acidilactici

probiotic, a whole new area of research opens up. Further research regarding its effect on fish

immune system, behaviour, welfare and growth factors will greatly benefit the industry. Chitin

removal is in great need of further research. Its effect on different species of salmonid and

further testing on Atlantic salmon will undoubtedly be beneficial. Future studies should also

aim to assess the potential benefit of coupling chitin removal with various probiotics. If we can

further enhance insect-based meals without having to treat them for chitin removal, then that

will play an important role in advancing future feed production. Furthermore, additional studies

on juvenile salmonids are needed to further understand juveniles’ adaptability and survivability,

when fed new and enhanced feeds.

 

23

5.  Final  Conclusion  This study revealed that enhancing untreated black soldier fly larvae (BSF) meal, by

supplementing the Pediococcus acidilactici probiotic for use in a juvenile Atlantic salmon diets

shows potential. By using fry as the test organism, this study has also demonstrated the benefits

of experimenting on juvenile fish which will be helpful in screening experimental diets for later

stages in salmonid development.

This project revealed that a 10 percent inclusion of treated BSF meal did not significantly affect

growth or mortality of juvenile Atlantic salmon when compared to the commercial diet.

Likewise, by including the P. acidilactici in the untreated BSF meal, the diet did not

significantly differ from the treated insect-based diet and what’s more, produced growth results

that were not significantly different from the commercial diet. These findings suggest that the

P. acidilactici probiotic may counteract the negative effects of chitin, making BSF-based diets

comparable to an already proven BSF-based diet. The untreated BSF treatment performed

worse than the aforementioned diets, as did the treated and probiotic enhanced BSF diet. This

suggests that Atlantic salmon may exhibit lower tolerance to chitin inclusion when not paired

with the P. acidilactici probiotic. Further studies on probiotic application in Atlantic salmon is

needed and additional studies on juvenile fish would undoubtedly benefit ongoing research in

the aquaculture industry.

 

24

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Appendices  

Appendix 2: European Directive 2010/63 EU on ethical standards

Article 14 - Anaesthesia

1.

Member States shall ensure that, unless it is inappropriate,

procedures are carried out under general or local anaesthesia,

and that analgesia or another appropriate method is used to

ensure that pain, suffering and distress are kept to a minimum.

Procedures that involve serious injuries that may cause severe

pain shall not be carried out without anaesthesia.

2. When deciding on the appropriateness of using anaes-

thesia, the following shall be taken into account:

(a) whether anaesthesia is judged to be more traumatic to the

animal than the procedure itself; and

(b) whether anaesthesia is incompatible with the purpose of the

procedure.

For further read, visit

http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2010:276:0033:0079:EN:PDF

Appendix 1: FAO feed table for Atlantic salmon (Salmo salar)

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Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 (Adjusted p values reported -- single-step method)

Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 (Adjusted p values reported -- single-step method)

Appendix 3: Results of Tukey post hoc test for final weight (g) of BSF and control diets. Numbers in first column represent diet treatments (i.e. 2=FM, 3=SPC, 4=BSFC+, 5=BSFC-, 6=BSFC+P+, 7=BSFC-P+)

Appendix 4: Results of Tukey post hoc test for final weight (g) of BSF and COM diets. Numbers in first column represent diet treatments (i.e. 1=COM, 4=BSFC+, 5=BSFC-, 6=BSFC+P+, 7=BSFC-P+)

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Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 (Adjusted p values reported -- single-step method)

Appendix 5: Results of Tukey post hoc test for SGR% of BSF and COM diets. Numbers in first column represent diet treatments (i.e. 1=COM, 4=BSFC+, 5=BSFC-, 6=BSFC+P+, 7=BSFC-P+)