DEPARTMENT OF BIOLOGICAL AND ENVIRONMENTAL SCIENCES

THE PROTEIN REQUIREMENT OF THE ATLANTIC WOLFFISH (ANARHICHAS LUPUS) AND THE EFFECTS ON GROWTH AND INTESTINAL FUNCTION A study for understanding the impact of protein inclusion and the intestinal functions of the Atlantic wolffish

Josefin Roos Degree project for Master of Science (120 hec) with a major in Biology BIO727, Physiology and cell biology 60 hec Second cycle Semester/year: Autumn 2017 – Spring 2018 Supervisor: Henrik Sundh, Department of Biological & Environmental Sciences Examiner: Michael Axelsson, Department of Biological & Environmental Sciences

Photograph by Author

Table of content Abstract ...... 3 Sammanfattning ...... 3 1. Introduction ...... 4 1.1. Aquaculture ...... 4 1.1.1. The Atlantic wolffish ...... 4 1.1.2. Fish feed & protein requirement ...... 4 1.2. Intestinal morphology & physiology ...... 5 1.3. Stress, health & welfare ...... 6 1.3.1. Stress ...... 6 1.3.2. Health ...... 6 1.3.3. Welfare ...... 6 1.3.4. Measurements of the intestinal health ...... 6 2. Aims ...... 7 3. Materials and Methods ...... 7 3.1. Protein requirement trial (Study 1) ...... 7 3.1.1. Research animal and holding conditions ...... 7 3.1.2. Experimental Feed composition ...... 7 3.1.3. Experimental design ...... 8 3.2. Characterisation of wolfish intestine (Study 2) ...... 9 3.2.1. Research animal and holding conditions ...... 9 3.2.2. Experimental design ...... 9 3.3. Barrier function and transport ...... 9 3.4. Histology ...... 11 3.4.1. Modified Pas stain ...... 11 3.4.2. Immunohistochemistry ...... 12 3.5. Growth ...... 12 3.6. Statistical analysis ...... 12 3.7. Ethical permit ...... 13 4. Result ...... 13 4.1. Protein requirement (Study 1) ...... 13 4.1.1. Growth ...... 13 4.1.2. Barrier function and transport...... 14 4.1.3. Histology ...... 15 4.2. Intestinal characterisation (Study 2): ...... 16 4.2.1. Barrier function and transport...... 16 4.2.2. Histology ...... 17 5. Discussion ...... 18 5.1. Protein requirement (Study 1) ...... 18 5.1.1. Protein requirement of the Atlantic wolffish ...... 18 5.1.2. Protein effects on intestinal barrier function ...... 19 1

5.2. Intestinal characterisation (Study 2) ...... 21 5.2.1. Functional morphology of the intestine ...... 21 Conclusion ...... 22 Acknowledgements ...... 23 References ...... 24 Should the wolffish eat less protein and how does its intestine function? ...... 28

2

Abstract Aquaculture is a growing industry and Sweden aims to increase the production of farmed fish. A potential species for cold-water aquaculture is the Atlantic wolffish (Anarhichas lupus). In farms, fish do not have access to their natural food source and therefore the farmer need to provide a feed that meets their requirement. The protein in the feed is the main energy source, however, manufactures tend to over-exceed the protein content in the feed compared to the species requirement, which generates an economical loss. On the other hand, low protein content can have negative effects on the intestinal functions. The protein requirement of the Atlantic wolffish is not yet known, therefore the aim was to investigate the effects of different dietary protein levels on the intestinal functions and the welfare on the wolffish. Six experimental diets were produced with varied protein content (35%, 40%, 45%, 50%, 54%, 59%). Both intestinal function and welfare were investigated by analysing the barrier functions and nutrient transport of the intestine in the extreme diets with Ussing chamber system together with intestinal morphological evaluation using histology. The intestinal barrier and transport function were not affected in 54% and 59%, while in the 35% diet caused an increased permeability which suggests a decreased barrier function. The growth tended to be greater in the two diets with the highest protein. The caudal part of the proximal intestine, had a higher transport, while the cranial part had a higher resistance. Low protein diet (35%) reduced the intestinal barrier function which indicated impaired welfare. From an intestinal function point of view these results suggests that higher protein content should be used. In support, there was an indication of higher growth at 59% but there was no difference between 50-59%. This indicates the protein content could be 50-59% without affecting the welfare and growth.

Sammanfattning Vattenbruk är en växande industri där Sverige strävar att öka sin produktion av odlad fisk. En ny potentiell art för det svenska vattenbruket är havskatten (Anarhichas lupus). På odlingar har fiskarna inte tillgång till sin naturliga födokälla och behöver därför tillhandahållas ett foder som uppfyller deras behov. Proteinet i fodret är den huvudsakliga energikällan för fisken, men tillverkarna tenderar att överskrida proteinhalten i fodret, vilket bidrar till ekonomisk förlust. Dock kan låga proteinhalt medföra negativa hälsoeffekter. Proteinbehovet för havskatten är ännu inte känt, därför var målet att undersöka proteinhalterna i fodret påverkar tarmfunktionen och havskattens välfärd. Sex experimentella dieter framställdes med varierat proteininnehåll (35%, 40%, 45%, 50%, 54%, 59%). Både funktion och välfärd undersöktes genom analys av barriärfunktioner och näringstransporter hos tarmen i de extrema dieterna med Ussing-kammare tillsammans med analysering av tarmens morfologi via histologi. Tarmfunktionen och transporten påverkades inte i dieterna med 54% och 59%, medan i den 35% dieten orsakades en ökning i permeabilitet vilket tyder på en minskad barriärfunktion. Tillväxten tenderade att vara större i de två dieterna med de hösta proteinnivåerna. Dock skilde sig inte 59% från 50% eller 54%. Den caudaladelen hade högre transport, medan kranialdelen hade en högre motståndskraft. Låg proteindieten (35%) minskade tarmfunktionen vilket indikerar en lägre välfärd. Ur ett tarmfunktions perspektiv bör därför en högre proteinhalt användas. Detta stöds av att det var en indikation på högre tillväxt vid 59% men det var ingen skillnad mellan 50-59%. Detta tyder på att proteinhalten kan vara 50-59% utan att påverka välfärd och tillväxt.

3

1. Introduction 1.1. Aquaculture Aquaculture is the fastest growing sector of animal food production and in 2015 it contributed with 53% of the world’s food fish consumption (FAO 2017). Aquaculture provides food security for a growing population compared to fisheries (FAO 2016). China is the world’s largest aquaculture producer with a production of 45.5 million tons which corresponds to approximately 60 % of the global production, while in Europe Norway is the largest producer with 1.3 million tons (FAO 2016). Sweden, compared to its neighbour Norway, has a relatively low aquaculture production with 13.4 thousand tons (Jordbruksverket 2017), approximately 99 % lower than Norway (Jordbruksverket 2012, Jordbruksverket 2017). However, Sweden has a great potential for aquacultural growth due to the long coastlines (Jordbruksverket 2012). The Swedish government has, in the new goals for 2014-2020, an ambition to expand the production of aquaculture and increase the Swedish aquaculture competition on the global market (Jordbruksverket 2014). 1.1.1. The Atlantic wolffish The Atlantic wolffish (Anarhichas lupus) is a carnivorous fish that can be found in deep waters in North Atlantic and North Pacific oceans. The Atlantic wolffish together with five other species, make up the small family of Anarhicadidae (Rountree 2002). The Atlantic wolffish, together with Spotted wolffish, were selected as species with high potential in marine cold-water aquaculture for the Swedish west coast (Albertsson et al 2012). The Atlantic wolffish has multiple traits that are of interest in aquaculture. It can start feeding on formulated diets after hatching with low mortality rates which reduces costs (Moksness et al 1989). They also have low level of aggression and cannibalism (Moksness et al 1989). Besides the physiological and behavioural traits, it’s also commercially sought after in Sweden with a high market price (Albertsson et al 2012). However, there are few papers related to aquaculture of the Atlantic wolffish compared to the Spotted wolffish. From a farming perspective, the focus has been directed on the spotted wolffish as Moksness (1994) found the spotted wolffish to reach 2 kg in 2 years which was twice the size of the Atlantic wolffish during the same time span. However, the Atlantic wolffish is the native species to Sweden and can be farmed in the higher temperatures range that can be found in Swedish waters which reduces the cost of cooling. Even though, the Atlantic wolffish has been selected as a potential species, there is still a need for more research in e.g. farming environment and feed development to develop a sustainable wolffish aquaculture (Albertsson et al 2012). 1.1.2. Fish feed & protein requirement In most farms, the farmed fish do not have access to their natural food sources (Houlihan et al 2008). Therefore, the feed provided by the farmer should contain the right amount of protein, fat, carbohydrates, vitamins, and minerals to archive optimal growth of the fish (Craig & Helfrich 2009). Protein is an important nutrient as it is used for maintenance, repair, energy and muscle growth (Werner 1981). Other components that are also utilized for energy is carbohydrates and lipids (Jobling 2011). Fish meal, often originating from wild-caught fish (Glencross et al 2007, Naylor et al 2000) is a common protein source in fish feeds for both omnivorous and carnivorous species (Glencross et al 2007). The fishmeal is an expensive product (Naylor et al 2000), and is also contributing to the reduction in the wild-fish stocks. Hence, fishmeal is an unsustainable source of protein (Naylor et al 2009). Another problem is that manufacturers risk to over-exceed the protein levels due to insufficient information of the protein requirement of different species (Naylor et al 2000). Therefore, to limit the waste of recourses, a feed should contain the optimal levels of protein, as wells as the optimal amounts of fats, carbohydrates and minerals (Glencross et al 2007). A few studies have investigated the Atlantic wolffish basic requirements of protein in the feed. Stefanussen et al (1993) investigated the effects of feed with 49.9 - 63.1 % protein in

4

juvenile Atlantic wolffish and found no significant effect on growth rate. Stefanussen et al (1993) suggested that the Atlantic wolffish should have a feed with more than 50 % protein. Similar Moksness (1990) found no differences in growth rate in feed with 48 - 55 % and neither when comparing 42.6 % in moist pellets. Other studies on the spotted wolffish have investigated the protein requirements and found good growth rates using 45-50 % protein in feed, as well as 55-62 % protein (Foss et al 2004). Potentially the minimum requirement of the wolffish could be within a lower range than those from previous studies. However, low protein levels in the feed have been shown to reduced growth, feed conversion and appetite (Ketola 1982). 1.2. Intestinal morphology & physiology Many studies on feed development focus on growth, and sometimes body composition, as the increased growth give economical gain in farming. However, Babaei et al (2017) argue that growth and body composition it is not enough to fully understand the digestion and absorption of a diet. The main organ for nutrient uptake is the intestine, still few studies have included the intestine as a focal tissue in regard to diet in wolffish. The fish intestine is in general categorised into four regions; the proximal intestine with or without pyloric caecas, the middle intestine, the distal intestine and the rectum (Olsson 2011). The Atlantic wolffish’s intestine, however, has a more diffuse regionalization. According to Hellberg and Bjerkas (2000), the Atlantic wolffish have a long, narrow proximal intestine that lack pyloric caecas and a short, wide distal intestine with a rectum (Figure 1). The proximal intestine can be further divided into three parts; the cranial part, the middle part and the caudal part. Hellberg and Bjerkas (2000) also investigated the morphology and histology of the gastrointestinal tract in the Atlantic wolffish. They found that the Atlantic wolffish have a short intestine (Hellberg & Bjerkas 2000) like many carnivore species (Olsson 2011). The intestinal wall consists of different layers. The layer closest to the cavity of the intestine, i.e. the lumen, is the intestinal epithelium. The intestinal epithelium consists mainly of epithelial cells and the mucus producing goblet cells. The cells in the epithelium are held together via a network of proteins at the apical membrane called the tight junctions (Anderson & Van Itallie 2009, Günzel & Fromm 2012). Together, they separate the luminal and interstitial fluids (Jutfelt 2011) and limiting uncontrolled diffusion of ions and molecules between these fluid compartments (Günzel & Fromm 2012, Loretz 1995). Active uptake of nutrients, ions and fluid is mainly driven by the sodium–potassium ATPase + + (Na /K ATPase), which build up a sodium gradient across the epithelium that drives other transport of other ions and nutrients into the enterocytes (Sundell & Rønnestad 2011). The Figure 1 Overview of the gastro-Intestinal tract by Hellberg & Bjerkås (2000). (1) oesophagus, (2) stomach, epithelium is also an essential physical barrier (3-5) Proximal intestine, (3) Cranial part of the proximal towards pathogens and harmful particles from intestine, (4) middle part of the proximal intestine, (5) the gaining access to the circulatory system caudal part of the proximal intestine, (6) distal intestine, (P) pyloric sphincter, (I) intestinal valve. (Schneeberger & Lynch 2004, Sundh & Sundell 2015). The goblet cells produce and secrete mucus which contain antimicrobial properties (Olsson 2011, Shephard 1994). The mucus functions as a physical barrier by limiting the access to the epithelium and at the same time causing particles to adhere to the mucus causing the particles to follow the mucus out through the intestinal tract (Segner et al 2012). Thus, the mucus acts as a protective barrier against pathogens or other harmful materials. Beneath the intestinal epithelium is a layer of connective tissue called the lamina propria. Underneath the lamina propria is the submucosa layer that is followed by the layers of circular and longitudinal muscles (Olsson 2011). The changes in these intestinal structures and functions can give a good indication of the fish’s wellbeing (Sundh et al 2010).

5

1.3. Stress, health & welfare 1.3.1. Stress When an animal cannot maintain the body’s stable condition, i.e. homeostasis, it is experiences stress. Stress causes the animal to express a range of behavioural and physiological responses that in turn can lead to whole-body responses, e.g. reduced growth, and/or behavioural responses (Broom 1986), e.g. reduced appetite (Wendelaar Bonga 2011). One physiological response to stress is the activation of the neuro-endocrine pathway, i.e. the hypothalamic-pituitary-interrenal- axis (Wendelaar Bonga 1997) where stress is sensed by the brain and elicits the primary stress response that result in the release of the stress hormones, adrenaline and cortisol (Barton & Iwama 1991, Wendelaar Bonga 1997). The stress hormones bind to target tissues and elicit secondary responses (Barton & Iwama 1991, Wendelaar Bonga 2011), which includes e.g. increased ventilation, heart rate (Wendelaar Bonga 1997) and a decreaed in the blood flow to the gastrointestinal tract (Axelsson & Seth 2011, Sundh et al 2018), changes in osmoregulation and impaired primary barrier function (Segner et al 2012). These secondary responses use energy from other functions to counteract the negative effects of the stressor in an attempt to regain homeostasis (Broom 1988, Segner et al 2012). Depending on the duration of the stress (Wendelaar Bonga 2011), it can cause tertiary stress responses such as decreased growth, reduced reproduction and increased risk of disease or even death (Broom 1988, Wendelaar Bonga 2011). 1.3.2. Health In aquaculture, the fish health is normally defined as the absence of disease aquaculture. However, the definition also includes pathology, i.e. the detrimental rearrangements of molecules, cells and tissue (Segner et al 2012). The pathology causes a change in the animal’s ability to cope with a stressor and can result in chronic stress but can also be a result of stress (Broom 1988, Segner et al 2012). 1.3.3. Welfare There is a growing concern from the public about the welfare of animals (Duncan 2005) and that they might experience pain, hunger, etc., i.e. emotional suffering, similar to humans (Dawkins 1990). Welfare is complex issue (Duncan 2005, Segner et al 2012) and is defined in various ways. Often the definitions include three things; the ability to express natural behaviour, absence of psychological suffering and the ability to maintain homeostasis (Chandroo et al 2004, Segner et al 2012). Stress and poor health are direct threats to the welfare of the fish because they reduce ability of the fish to cope with additional changes in the environment (Broom 1988). Therefore, farmed fish should, for both ethical and economic reasons, have good welfare (Broom 1998). 1.3.4. Measurements of the intestinal health There are various ways of measuring health and welfare. However, species as well as individuals show variations in which physiological responses they tend to utilize (Broom 1988), therefore, welfare should be estimated by combining different measurements, e.g. growth, physiology, disease and/or behaviour parameters (Broom 1988, Smidt 1983). As stated above, intestinal health can be assessed by the presence of pathology or impaired function. Histology is a common tool to investigate pathological changes to tissue morphology. The appearance of the mucosal folds is known to be affected by feeds composition, where food antigens induce intestinal inflammation which manifests as a widening of the lamina propria and shortening of the mucosa folds (Baeverfjord & Krogdahl 1996, Knudsen et al 2008, Krogdahl et al 2003). Other factors like acute and chronic environmental stress or the presence of pathogens can also induce inflammation and cellular damage (Niklasson et al 2011, Soderholm & Perdue 2001). During stress the mucus layer generally increases (Shephard 1994) due to increased secretion (Szakolczai 1997). During prolonged stress Szakolczai (1997) found the goblet cells numbers to decrease and the mucus layer decreases. Therefore, the change in the amount of goblet cells can be used as an indicator for stress.

6

Besides the histological parameters, permeability of the intestine can also give an indication of health and stressors. Cortisol is known to increase the permeability in trout (Sundell & Sundh 2012). The change in permeability can lead to disturbances in ion transport and reduced nutrient uptake both of which in turn can cause a reduced growth (Sundh et al 2010). Even though the intestine is the organ for absorption of nutrients (Sundell & Rønnestad 2011) and the first barrier pathogens encounter (Segner et al 2012), the wolffish intestine has not been subjected to investigations of protein requirement.

2. Aims Different protein levels have been tested in the Atlantic wolffish, however the results showed no significant difference in growth rate between 45-62 %. The minimum required levels of protein could hence be outside of the tested ranges. This project aimed to investigate the protein requirement of the Atlantic wolffish and the health and welfare effects with a focus on the intestine. Another aim was to provide more descriptive information about the functions of the intestine of the wolffish in regard to barrier properties and active transport functions.

3. Materials and Methods 3.1. Protein requirement trial (Study 1) 3.1.1. Research animal and holding conditions 360 juveniles of 40 g (± 0.2 SEM) Atlantic wolffish (Anarhichas lupus) reared from wild-caught eggs obtained from Island were kept in an indoor recirculation system with 10oC aerated artificial seawater (31 psu) in pre-experimental holding tanks (70 L). The photoperiod was set to 12 hours of light and 11 hours of darkness (12L:11D) with 30 minutes of dimming between each shift in light and dark period to lessen the disturbance caused by the shift. The wolfish were fed with commercial dry pellets (Amber Neptune St 2, Skretting AS, Norway) until satiation twice a day on weekdays and once a day on weekends. During the protein requirement trail (see 3.1.3) the wolffish were kept in experimental tanks (17 L) with the same water, temperature and photoperiod as the pre-experimental holding conditions. The tank contained a stand pipe in the middle of the tank that allowed water to flow trough. The pipe was removed once a day to clean the tank from faeces. The fish were fed with experimental diets (Table 1). The expected final growth was taken into consideration to not exceed the recommended density of fish during the experiment, and to ensure that there would be no need to transport fish to bigger tanks during experiment. 3.1.2. Experimental Feed composition Six different diets with similar energetic content but varied protein content (35 %, 40 %, 45.1 %, 50.2 %, 54.4 % and 59.2 %) were produced. Where the 55 % diet were used as a reference diet as 55 % protein is normally used in commercial marine feeds. The ingredients for each diet (Table 1) were mixed with industrial mixer (Livsmedel teknik AB, Sweden) for 15 to 17 minutes dependent on the gluten content. The gluten tended to form clumps and required more mixing. For each diet, 250 ml of heated water with 60 g gelatine were added to into 940 g of diet mix to bind the ingredients. Diet were then extruded (die 2 mm, Nima AB, Sweden) and heated to 120 oC with steam for 1 minute to gelatinise the starch. The diets were then dried in 40-45 oC for 17 hours and manually broken down before being minced in a cut-o-mat (Livsmedel Teknik AB, Sweden) into adequate size (approximately 0.5 cm).

7

Table 1: Ingredients of the six experimental diets. Each ingredient is expressed in grams per 100 grams of the total diet.

Ingredients (g/100 g) D35 D40 D45 D50 D55 D60 Wheat meal 10,0 10,0 11,0 10,0 10,0 3,0 Fish oil 14,5 14,0 13,0 11,0 10,0 8,5 Gelatine 6,0 6,0 6,0 6,0 6,0 6,0 Mineral mix 2,0 2,0 2,0 2,0 2,0 2,0 CMC binder 1,0 1,0 1,0 1,0 0,8 0,8 Fish meal 28,5 32,5 37,0 41,0 44,0 49,5 Wheat gluten 3,0 5,0 9,0 12,0 16,0 18,0 Lecithin 1,0 1,0 1,0 1,0 1,0 1,0 Corn starch 17,0 10,0 5,0 2,0 0,0 0,0 Casein 5,0 4,5 2,0 2,0 1,0 1,0 Soybean 2,0 4,0 6,0 6,0 6,0 6,0 Cellulose 9,1 9,3 6,5 5,6 2,8 4,0 Methionine 0,8 0,7 0,5 0,4 0,2 0,0 Total content 99,9 100,1 100,1 100,0 99,8 99,8 Nutritional composition (% DM) Crude protein 35,0 40,0 45,1 50,2 54,4 59,2 Lipid % 16,2 15,9 15,3 13,5 12,7 11,4 GE (MJ/kg) 18,0 18,1 18,4 18,3 18,6 18,4

3.1.3. Experimental design The fish were individually tagged with pit-tags (Hdx 12 mm, Biomark). The fish were randomly netted, weighed and, if within the weight range of 25-60 g, transferred to experimental tanks. The weight range was set between 25-60 g to avoid cofounding factors of individuals with below or above the average growth rate. The 360 fish were split between 18 experimental tanks to correspond to an individual average weight of 40 g (± 0.2 SEM) and simultaneously contain 20 individuals per tank. Three replicate tanks were randomised and used for each of the six diets. The wolffish were acclimatised to the new feed for three weeks, where the inclusion of the experimental diet increased over time. During the experiment the fish were feed twice a day during week days and once during weekends. Each feeding was performed in two rounds where half the feed was distributed to the fish and they were then left for 20 minutes before the rest were distributed. After an additionally 20 minutes the left-over feed was siphoned out of the tanks. The amount of experimental feed fed per tank corresponded to 0.25 % of the total weight of the fish. The fish weight and length were recorded approximately every three weeks (13th of December, 11th of January, 5th of February, 5th of March and 15th of March) to not disturb the fish more than necessary and the feed rations were then updated according to the new average weight of the tank. After 91 days the fish were starved for 24 hours before sampling. Twelve growing fish per diet were netted and euthanized in an over-dose of Finquel MS-222 (0.4 g l-1, Tricaine mesylate, Scanvacc, Årnes, Norway) and sampled for histology, visceral fat, body length, body and liver weight. After sampling the remaining fish were fed the experimental diet for an additionally week before sampling twelve fish from the extreme levels (D35, D60) and the reference diet (D55) were measured with Ussing chamber system for barrier functions and transport. The cranial part of the proximal intestine and the intermediate area, containing both the middle and caudal part of the proximal intestine (Figure 2), were sampled for the measurements of both histology and Ussing chambers.

8

3.2. Characterisation of wolfish intestine (Study 2) 3.2.1. Research animal and holding conditions 12 juvenile Atlantic wolffish, with a mean weight of 41 g (± 2.0 SEM) were kept in the same holding conditions as the pre-experimental holding condition in study 1. 3.2.2. Experimental design The fish were randomly selected for intestinal characterisation of the middle part and the caudal part of the proximal intestine (Figure 2). The samples were analysed for morphological changes with histology together with analysis of the barrier functions and transport with Ussing chambers.

S 1 3

Figure 2: Atlantic wolffish (Anarhichas lupus) intestinal tract. Stomach (S), cranial part of the proximal intestine (1), middle part of the proximal intestine (2), caudal part of the proximal intestine (3) and distal intestine (4). White arrows mark where the areas begins and ends are defined. The beginning of the caudal part (white arrow between 2 and 3) was defined as where the circumference of the intestine notably increased compared to the prior part.

3.3. Barrier function and transport 12 fish from the extreme diets (D35 and D60) and 10 from control (D55) for study 1 and 12 fish for study 2 were randomly netted. Fish with abnormities were excluded to avoid including effects of abnormal individuals. The fish were euthanised with Finquel MS-222 (0.4 g l-1, Tricaine mesylate, Scanvacc, Årnes, Norway) and the proximal intestine were dissected out. The tissue samples were then stored on ice in a beaker with wolffish ringer (160 mM NaCl, 2 mM KCl,1 mM MgCl2 x 6H2O, 1.6 mM CaCl2 x 2H2O, 0.5 mM L-Lysine, 0.7 Na2HPO4 x 2H2O, 7 mM NaHCO3, 5 mM Na HEPES, 2.9 g/l Glutamine and 1,8 g/l Glucose) to keep the tissue viable until the tissue could be mounted in modified Ussing chambers. The tissue was mounted to in-between two half-chambers exposing the mucosal side of the tissue in one half of the chamber and the serosa side in the other half chamber. Four ml ringer was added to each side of the. The system was connected to a cooler to keep the chambers at 10oC to resemble the fish body temperature by the use of cooling-mantles. The Ringer solution was bubbled with air (99.7 % air and 0.3 % CO2) to maintain the viability of the tissue and enable mixing of the Ringer. The Ussing chamber was set-up according to Sundell et al (2003), including the further modifications from Sundell and Sundh (2012). The set-up consists of chambers with two pairs of electrodes per chamber (Figure 3). One pair of KCL-electrode that was used to detect the trans-epithelial potential difference across the membrane (TEP) and one pair of platinum electrodes to detect the current. Voltage were applied via the ringer solution in an alternating manner resulting in currents between -30 to 30 µA cm-2. These U/I pairs were fitted to a line using the least square method, and the slope of the line represents the trans-epithelial resistance (TER). TEP was measured in undisturbed tissue and the short circuit current (SCC), was calculated as:

9

= 𝑇𝑇𝑇𝑇𝑇𝑇 The tissue was allowed 𝑆𝑆𝑆𝑆𝑆𝑆 − to stabilise for 1 hour in 𝑇𝑇𝑇𝑇𝑇𝑇 the chambers before the measuring the electrical parameters (TER, TEP and SCC). The paracellular permeability of the intestinal tissue were assessed with mannitol, known to pass through tight junctions (Anderson & Van Itallie 2009). While the nutrient uptake was assessed with the amino acid lysine’s transport across the epithelium. In order to measure the permeability Figure 3: Ussing chamber set-up with four chambers (red) with one pair of KCL-electrodes and nutrient uptake After (yellow arrows) and one pair of platinum electrodes (white arrows) per chamber. the acclimation period, the Ringer in the serosal side was exchanged with plain Ringer containing 4.41 µl/ml 14C labelled mannitol (58 mCi mmol-1, Moravek Inc., Brea, USA) and 0.44 µl/ml 3H labelled L-Lysine (32 Ci mmol-1, Moravek Inc., Brea, USA) In order to get the initial concentration of the label in the mucosal chamber and monitor the accumulation of radioisotopes in the serosa chamber 100 µl ringer were extracted from each chamber at T0 and put into vials. Additional 100 µl were sampled from the serosa side of the chamber at T20, 25, 30, 60, 80, 85, 90 for study 1 and at T20, 30, 60, 80, 90 for study 2 to assess the transport across the epithelium over time. For each 100 µl that were extracted a 100 µl of ringer was added into the chamber side. The radioactivity content of the samples was measured with a liquid scintillation counter (Wallac 1409, Turku, Finland) with a dual label protocol (C14 & H3) after filling the vials with 5 ml Ultima Gold TM (PerkinElmer). The permeability of C14 labelled mannitol (Papp) and H3 L-lysine transport were calculated accordingly to Sundell et al (2003) and Vidakovic et al (2016): 1 = ×

𝑎𝑎𝑎𝑎𝑎𝑎 𝑑𝑑𝑑𝑑 𝑃𝑃 0 1 𝑑𝑑𝑑𝑑 𝐴𝐴=𝐴𝐴 × 𝑑𝑑𝑑𝑑 Where dQ/dT is the rate of appearance𝐿𝐿 − 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 of mannitol𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 or lysine in the serosa chamber, A is the area 2 𝑑𝑑𝑑𝑑 𝐴𝐴 -1 (cm ) and C0 is the concentration at T0 on the mucosal side (mole mL ). In study 1 samples from T30-60 in was removed due to pipetting error in a few samples and as a precaution all samples were treated the same.

10

3.4. Histology Tissues were dissected out from euthanized fish (0.4 g l-1 Finquel MS-222 Tricaine Methanesulfonate, Scanvacc, Årnes, Norway). A section of approximately 1-1.5 cm of each part of the proximal intestine were used for histology. In study 1 the cranial and intermediate area was stretched to increase the visibility of the mucosal folds by inverting the swish rolling technique as desribed by Bialkowska et al (2016). The technique is prominent to get longitudinal histological sections where the length of the whole intestinal region can be studied. In brief, the inverted swish rolling technique was performed by cutting the dissected intestinal tissue longitudinally and flatting the section with the mucosal side facing downward. Then by pinching the anterior side of the tissue, and by rotating the tweezers, coiling the tissue into a small role and thereby causing the mucosal side facing outwards, instead of inwards. In study 2, due to the lower number of individuals, the middle and caudal part was stretched by pining the sections to wax slides. The tissue sections were fixated, for 24 hours in study 1 and for 24-27 hours in study 2, in 4% paraformaldehyde in 0.1 M Sorenson’s buffer (pH 7.4). The intestinal tissues were then dehydrated in ethanol (30 % and 50 %) and then stored in 70 % ethanol. The tissue was further processed with a tissue processor (Leica TP1020, Leica Biosystems). The processors first dehydrated the tissue using an ethanol gradient (70 %, 80 %, 95 %, 100 %) where the tissue was dipped in three sequential baths of the 100 % ethanol. The tissue was then submerged in three separate baths of histo-clear (Histolab products AB, Askim, Sweden) and finally dipped in three separate baths of wax (Paraplast, Lecia Biosystems, Nussloch, Germany). The processor submersed the tissue for one hour in each bath. Tissue was then embedment in wax by hand with Paraffin embedding station (Leice EG1150 H, Leice Biosystems, Nussloch, Germany). The wax block from six fish from each of the extreme diet (D35, D60) together with the control diet (D55) in study 1 and six fish from study 2 were selected for further processing. These samples were sectioned (5 µm) with microtome (Finesse Me, Shandon, Cambridge, UK) and mounted on 2 % 3-Aminopropyltriethoxysilne (Acros, New Jersey, USA) coated glass slides. Tissue were then stained with Modified PAS stain (3.4.1) or immunohistochemistry (3.4.2). 3.4.1. Modified Pas stain The paraffin wax was removed from the sections by submersing it twice for three minutes in separate baths of histo-clear (Histolab products AB, Askim, Sweden). The sections were then rehydrated in an ethanol gradient (100 %, 90 %, 80 %, 70 %) and washed in milliQ before the sections were then transferred into Alcian blue 8GX (Histolab products AB, Askim, Sweden), that stains the goblet cells, for 25 minutes, and washed in milliQ for three minutes before staining in Schiff reagent (Histolab products AB, Askim, Sweden) for 10 minutes. The sections were then washed in milliQ for 3 minutes before staining the structures with Harries haematoxylin (Histolab products AB, Askim, Sweden) for 5 minutes. The excess of the haematoxylin was washed off by submersing the sections in milliQ for 3 minutes. The sections were then dipped in acid alcohol (1 % acetic acid, 70 % ethanol) three times before rinsing them in milliQ for 10 minutes and staining for 1 minute in the complementary stain eosin (Histolab products AB, Askim, Sweden). The sections were then dehydrated in an ethanol gradient (70 %, 80 %, 90 %, 100 %) before being submerged in two sequential baths of histo-clear for 3 minutes. The sections were mounted with Pertex glue (Histolab products AB, Askim, Sweden) and then left to dry before being analysed with Microscope (Eclipse E1000, Nikon), and photographed with mounted digital camera (DXM1200, Nikon). The sections were analysed for mucosal fold length and amount of goblet cells per µm epithelium. The mucosal fold length was measured from the base to the top of the of the fold, following lamina propria. Complex folds and branches of the fold were not measured, due to risk of overestimation on length of folds. Goblet cells per µm were counted and the amounts of goblet

11

cells where divided by the double mucosal fold length (µm) as goblet cells are on both sides of the fold. Goblet cells on fold branches were not included. 3.4.2. Immunohistochemistry The wax was removed from the slides with two sequential baths in Histo-clear for ten minutes then rehydration with ethanol gradient with two baths of 100 % ethanol for ten minutes each, followed by two baths of 90 %. The rehydration continued with single baths of 80 % and 70 %. The sections were then washed in two baths of milliQ before they were submerged in three baths of TBST (0.05 M Tris, 0.9 % NaCl, 0.05 % Tween, pH 7.6) before incubated blocking solution for one hour. The blocking solution hinder the primary antibody from attaching to unspecific structures. All sections except one, the negative control, were incubated with a dilution 1:1000 of the primary antibody (α5), which binds to the Na+/K+ ATPase, in TBST overnight at 5 oC. The negative control prevents an over estimation of the distribution of the Na+/K+ ATPase by visualising unspecific binging. The primary antibody was then rinsed off in three sequential baths of TBST for 3 minutes each. The secondary antibody (cy-3-antimouse) with a dilution of 1:800 in TBST were then added. The sections were then incubated for one hour in darkness in room temperature. The incubation of the secondary antibody in darkness were kept in darkness to maintain the fluorescent properties. The excess of second antibody were then rinsed off in three baths of PBST (0.01 M PBS, 0.05 % Tween, pH 7.2) three minutes each. The sections were left in the baths for five minutes if nothing else was stated. The sections were mounted with Prolong Gold Antifade reagent with DAPI glue (Thermo fisher scientific, Waltham, USA).

3.5. Growth The weight of the each individual wolffish were measured during the protein requirement trial. The fish were weighed approximately every three weeks (13th of December, 11th of January, 5th of February, 5th of March and 15th of March).

3.6. Statistical analysis All statistical analyses were made in SPSS (IBM SPSS statistics software version 25; IBM Corp., Armonk, NY, USA). All data were tested for normal distribution and heterogeneity of variances (Levene’s test of Equality of Variance) if nothing else is stated. Data that was not normally distributed were transformed before running the tests. A significance level of 0.05 was used in all tests. All data are presented as mean ± SEM. In study 1 the Ussing and histology data were analysed with a Mixed Linear Model analysis. The analysis included diet and intestinal region as fixed factors and their interaction. The random effects of tank were nested within diet. Significant main effects were subjected to Bonferroni- corrected Post hoc analysis. In study 2 all data was analysed with Independent-sample t-test, except for mannitol as it could not be transformed to a normal distribution. Mann-Whitney U test was used for the non-normally distributed data. Growth data was tested with Nested ANOVA with the random effects of tank nested within diet. Significant effects were further investigated with Tukey´s Post hoc test.

12

3.7. Ethical permit The studies were conducted at a certificated animal facility at the department of Biological & Environmental Sciences at the university of Gothenburg Sweden. The studies were conducted under the license number 208-2014 which were granted by the ethical committee for Animal research in Gothenburg.

4. Result 4.1. Protein requirement (Study 1) 4.1.1. Growth The mean weight in all the diets (Figure 4) increased over time. Where the weight in general increased with the increased content of protein in the diet. However, D45 had the lowest overall growth and had a significant difference from the higher diets.

58.00 a a

53.00 a,b D35

D40 D45 48.00 D50 Growth (g) a,b D55 a,b D60 43.00 b

38.00 21-nov 11-dec 31-dec 20-jan 09-feb 01-mar 21-mar

Figure 4: The values presented in the graph are the average growth of the six experimental diets with SEM error bars. Diet D35 contained 35% protein, D40 contained 40 % protein, D45 contained 45 % protein, D50 contained 50 % protein, D55 contained 54% protein and D60 contained 59% protein. The growth was measured in grams at five different occasions (13th of December 11th of January 5th of February 5th of March and 15th of March). In all the diets there were individuals that did not increase in growth (Table 2). The number of individuals that did not grow decreased with the increased protein content. D35 and D45 had the highest number of non-growing individuals, where 30 individuals out of initial 60 did not grow in these diets. Table 2: The amount of fish that did not grow (Non-growing individuals) in diets with different protein content. The protein content of the diets varied from 35 – 60 % protein. Each diet had a total of 60 individuals.

Diet D35 D40 D45 D50 D55 D60 Non-growing individuals 30 23 30 14 15 6

13

4.1.2. Barrier function and transport The analysis of Papp showed an overall effect of intestinal region (p < 0.001) and diet (p < 0.001), while no interaction between diet and region could be found (p > 0.05). The regional effect showed that Papp was higher in the intermediate region compared to the cranial part in all diet groups. Post hoc test of diet effects revealed higher Papp in the D35 group compared to D55 (p < 0.05) and D60 (p < 0.01) (Figure 6A). Regional average of the cranial region 4.05 e7 ± 0.58 e7 cm s-1, and the intermediate part 8.38 e7 ± 0.89 e7 cm s-1. The barrier function assessed as TER was affected by intestinal region (p < 0.001). However, there was no overall effect of neither diet (p > 0.05) nor any interactions (p > 0.05). The regional effect showed a higher TER in the cranial region compared to the intermediate in all diet groups (p < 0.001; Figure 6B). Regional average of the cranial region was 84.19 ± 7.2 Ω x cm2, while the intermediate part had an average of 34.27 ± 2.07 Ω x cm2.

0.0000016 a b b a a a A B 120

0.0000012 100

) ) 1 2 - - 80 x cm 0.0000008 C C Ω 60 I I TER ( Papp (cm (cm x Papp s 40 0.0000004 20

0 0 D35 D55 D60 D35 D55 D60

Figure 5: The barrier function of the extreme diets (D35, D60) and the control diet (D55). A) The permeability of mannitol (Papp) differed significantly both between diets and regions. D35 had a significant increase in the cranial part of the proximal intestine (C). B) The trans-epithelial resistance (TER) differed stigmatically between the intestinal regions but not between diets. The Cranial part (C) differed significantly from the intermediate area (I).

The analysis of the transport functions, TEP, SCC and the lysine transport, of the epithelium differed between the intestinal regions. The intermediate region had an overall more negative TEP (- 0.95 ± 0.17 mV) compared to the cranial region (- 0.06 ± 0.11 mV) (p < 0.001; Figure 6A). SCC had a regional effect where the cranial region (2.35 ± 2.16 µA x cm-2) had a lower activity compared to the intermediate region (31.08 ± 5.48 µA x cm-2) (p < 0.001; Figure 6B). Likewise, the lysine transport was affected by region (p < 0.001) with a lower activity in the cranial region (6.46 e-10 ± 0.062 e-10 mole x cm-1 x cm-2) compared to the intermediate area (16.25 e-10 ± 0.78 e-10 mole x cm-1 x cm-2) (Figure 6C). Neither parameter showed an effect of diet (p > 0.05) nor any interaction effects (p > 0.05) (Figure 6).

14

A 0.5 a a a B 50 a a a 40 0.0 D35 D55 D60

30 ) 2 - -0.5 C 20 C

I 10 I TEP (mV) -1.0 SCC (µA x cm 0 -1.5 -10 D35 D55 D60

-2.0 -20

Figure 6:Transport functions in cranial part of the proximal 2E-09 a a a intestine (C) and the intermediate area (I). There was no C difference between either of the three diets with varied protein

) 2 - 1.6E-09 content. 35 % protein (D35), reference diet with 54 % protein content (D55), and 59 % protein (D60). The was a regional x cm

difference between the cranial part of the proximal intestine (C) 1 - 1.2E-09 and the intermediate area (I) in A) Trane-epithelial potential C (TEP) B) The short circuit current (SCC) and the C) Lysine transport. 8E-10 I

4E-10 Lysine (mol x cm

0 D35 D55 D60

4.1.3. Histology The analysis of the histological parameters found a difference between the regions in the mucosal folds length (p = 0.001). The cranial region had an average of 1237.31 ± 122.21 µm, which was longer compared to the intermediate area that had an average length of 948.70 ± 38.57 µm (Figure 7A). Likewise, was there a difference found in the amounts of goblet cells (p = 0.002) between the regions, where the cranial part had fewer goblet cells compared to the intermediate area (Figure 7B). The goblet cells had two different colorations, light blue and dark blue. The number of light blue cells were significantly higher in the intermediate region compared to the cranial, while the dark blue did not differ between the regions. The histology analysis showed no effect of diets on the mucosal fold length (p > 0.05) nor on the amount of goblet cell (p > 0.05). Neither was there any interaction (p > 0.05) found between diet and region.

15

A 1600 a a a B 0.045 a a a 0.04

1400

) 1 - 1200 0.035 µm 0.03 1000 0.025 800 C C 0.02 600 I I 0.015 400 0.01 Goblet cells (cells Goblet (cells cells x Moucosal fold legth (µm) legth fold Moucosal 200 0.005 0 0 D35 D55 D60 D35 D55 D60

Figure 7: The histological data of the protein requirement trail had showed no effect of diet. There was a difference between the cranial part of the proximal intestine (C) and the intermediate area (I). A) the mucosal fold length was greater in the cranial part (C) compared to the intermediate area (I.). B) The number of goblet cells per µm varied between the regions, where the cranial part (C) had a lower number of goblet cells per µm compared to the intermediate area (I).

4.2. Intestinal characterisation (Study 2): 4.2.1. Barrier function and transport There was a significant difference in Papp (p = 0.016) between the two regions in study 2 where the middle part of the proximal intestine had an lower Papp (4.85 e-7 ± 1.41 cm s-1) than the caudal part (9.24 e-7 ± 1.66 cm s-1) (p < 0.016; Figure 8A). The middle and the caudal parts of the proximal intestine did not differ in TER (p > 0.05). The overall average TER of the middle and the caudal part of the proximal intestine was 31.25 Ω x cm2 (± 2.22 SEM). The analysis of the L-lysine transport found a regional effect (p = 0.032), with a lower transport in the middle part (1.61 e-9 ± 0.15 e-9 mole min-1 cm-2) compared to the caudal part (1.99 e-9 ± 0.91 mole min-1 cm-2) (Figure 8B). The middle and the caudal parts of the proximal intestine did not differ in TEP nor SCC. The TEP of both regions had an average of -1.33 ± 0.17 mV. The average of the SCC of both regions was 49.62 ± 7.09 µA.

0.0000012 b 2.5E-09

A B ) b 2 0.000001 - 2E-09

cm

) a 1 1 - 0.0000008 - 1.5E-09 a M M 0.0000006 C C 1E-09 0.0000004 Papp (cm (cm x Papp s

0.0000002 5E-10 Lysine (mol x min

0 0

Figure 8: A) Permeability of mannitol (Papp) differed significantly between the middle part (M) and the caudal part of the proximal intestine (C). B) Lysine transport differed significantly between the middle part (M) and the caudal part of the proximal intestine (C)

16

4.2.2. Histology There was no significant difference in mucosal fold length between the middle and caudal part of the proximal intestine, giving the regions an overall average of 628.59 (± 53.53 SEM). However, the middle part of the proximal intestine had significantly (p = 0.013) fewer goblet cells per um (Figure 9) than the caudal part of the proximal intestine. The middle part had an average of 0.04 cells x um-1 compared to 0.05 cells x um-1 in the caudal part.

0.06 b There was no observed difference in distribution of the Na+/K+ ATPase when 0.05 a comparing the middle part and the caudal part ) 1 - 0.04 of the proximal intestine (Figure 10). There

µm M 0.03 were however, unidentified auto-florescent C structures in both regions (white arrows). The

(cells x (cells 0.02 cranial part of the proximal intestine was not 0.01 investigated. Abundece of goblet cells

0

Figure 9: The amount of goblet cells per µm were higher in the middle part of the proximal intestine (M) compared to the caudal part of the proximal intestine (C).

17

Figure 10: Immunohistochemistry of two parts of the proximal intestine at different magnifications. The Caudal part (D-E) were photographed with a different camera, however expressed the same coloration as the middle (A-C) in the microscope. There were unidentified auto-florescent structures (white arrows) present in all samples. A) Negative control of the Middle part of the proximal intestine. Shows the unspecific fluorescent. Magnification 10x B) The distribution of Na+/K+ ATPases in the Middle part of the proximal intestine magnification 10x C) The distribution of Na+/K+ ATPases in the Middle part of the proximal intestine magnification 20x. D) The unspecific florescence of the negative control of the caudal part of the proximal intestine. Magnification 20x. E) The distribution of Na+/K+ ATPases in the caudal part of the proximal intestine. Magnification 20x.

5. Discussion 5.1. Protein requirement (Study 1) 5.1.1. Protein requirement of the Atlantic wolffish The wolffish is a potential species for Swedish aquaculture and few studies have investigated the protein requirement of the Atlantic wolffish. The earlier studies of Moksness (1990) and Stefanussen et al (1993) found no difference in growth in relation to protein content. In this study, the end weight of D45 differed from D50 and D60. The slow growth of the D45 was unexpected as the diets with even lower protein content had a greater end weight. However, this slow weight increase is likely due to 50 % the individuals that did not increase in weight during the

18

experiment and/or the low feed intake of the fish in that diet. There were non-growing individuals in all diets, but D35 and D45 had 30 individuals out of 60 that did not increase in weight. There can be different explanations for the non-growing individuals. The wolffish in this study originated from a wild population and a higher variation in growth rates are expected in farmed fish originating from a wild population compared to a population that has gone through selective breeding (Kai et al 2012). Another plausible explanation is that social hierarchies are formed in the experimental tanks that causes the individuals lowest in the hierarchy to eat less than those ranked above them. This hierarchy system is known to develop in salmonids during times of scares food sources and space (Chapman 1966) but have not been investigated in wolffish. The individual tracking can therefore be useful for taking into account the non-growers’ effect on growth rate. This is the first study, to our knowledge, that have tracked the individual growth in a protein requirement experiment. Other studies on the Atlantic wolffish (Moksness 1990, Stefanussen et al 1993) and other species have used the average weight per tank and, therefore, might include non-growing individuals in the growth data. The design of the experimental diets prioritised a iso-energetic content with a varied amount of protein. Lipids have a high energy content and the diets could therefore not have the same amount of lipids without affecting the energetic content nor the protein content. The difference in lipid levels in the diets might cause an effect on the fish. One known consequence of low protein and high lipid levels is protein sparing. Protein sparing is the effect where higher lipid levels causes an increase in the utilization of protein for growth instead of energy (Beamish & Medland 1986). The protein sparing effect could therefore cause the diets with higher lipid content (D35, D40, D45) to utilize protein for growth in a higher proportion than the diets with lower levels of lipids (D50, D55, D60) and thereby reduced the visible difference between the diets as the lower protein diets growth was within the variation of the higher protein diets. This protein-sparing effect could therefore explain how the analysis could not find a difference in growth between the diets. The protein content might therefore have a greater effect on growth than what this study can indicate. The protein sparing effect has been observed in some species such as the gilthead sea bream (Vergara et al 1996), and rainbow trout (Beamish & Medland 1986). However, in some species the increase of lipids in the feed does not create a protein-sparing effect, e.g. cod (Lie et al 1988), nor in juvenile Asian seabass (Catacutan & Coloso 1995) and might thus not occur in wolffish. This is further supported by Stefanussen et al (1993) who also concluded that increased fat content, 18.1-26.5 % compared to 13.8-14.6 %, in the feed did not contribute to the growth rates in juvenile Atlantic wolffish. The greatest nutrient difference between the diets was the protein content and therefore assumed to cause the main effect on the growth of the wolffish. Furthermore, carnivorous fish are known for using a high amount of the ingested proteins for energy (Cowey et al 1974, Cowey 1994, German 2011). Lysine is an essential amino acid and a deficiency of lysine cause fin rot and increased mortality (Ketola 1982). However, the lysine transport did not differ between the diet groups, therefore the absorption of lysine, and presumably other amino acids, is not the limiting factor for growth. 5.1.2. Protein effects on intestinal barrier function The intestinal barrier function, i.e., permeability towards ions and molecules, has been used as a measurement of intestinal health of the fish as well as mammals, including humans (Segner et al 2012, Welsh et al 1998). In fish, the permeability of mannitol (Papp) is known to increase during acute and chronic stress (Sundh et al 2010, Sund et al 2018). In the current study the Papp for mannitol was higher in the diet D35 in both the cranial and the intermediate area of the proximal intestine. This indicate that low protein levels may be stressful to the fish. Similar regional effect has been seen in the Atlantic salmon where the Papp was most affected in the proximal region compared to the distal during low oxygen stress (Sundh et al 2010). The effect of low protein diet as a stressor and its impact on the intestinal barrier function have not, to my knowledge, been

19

studies in fish. In humans, Welsh et al (1998) found that malnourished patients have increased paracellular permeability. Another study on rats by Worthington and Syrotuck (1976) found that the protein-deficient rats had damaged apical intercellular junctions, which includes tight junctions and adherence junctions (Ivanov et al 2005). The rats also had a higher uptake of viral and bacterial particles to the intercellular space. The particles were suggested to be absorbed paracellularly due to the apical damage (Worthington & Syrotuck 1976). The protein deficiency induced damage of the tight junctions as described in rats could therefore explain the increased permeability in the D35 diet. The wolffish could also have had a potentially higher uptake of pathogens, similar to the rats in the study by Worthington and Syrotuck (1976). This increased risk of pathogen uptake with a decreased barrier function in fish have also been suggested by Sundh et al (2010). The trans-epithelial potential (TEP) measures the net distribution of cations and anions across the intestinal epithelium (Loretz 1995). In D35 a positive TEP was observed which is the opposite to previous reported TEP values from stenohaline saltwater (SW) fish as well as the TEP in the high protein diet groups in the current study. The negative TEP in SW fish is thought to be due to a net uptake of Cl- ions. Even though the fish intestine performs electroneutral uptake of Na+ and Cl-, the tight junctions are more permeable to Na+ ions, which leaks back into the lumen to a greater extent than Cl- (Loretz 1995, Sundell & Sundh 2012).The positive TEP might have been caused by an altered ion selectivity of the tight junctions in a way that that anions are preferred over cations, without affecting the overall trans-epithelial resistance (TER). Other studies have found a change in the types of tight junction changed the permeability of both cations and anions (Günzel et al 2009, Günzel & Fromm 2012) and it has been demonstrating that the alteration in the expression of tight junction is correlated with barrier dysfunction (Amasheh et al 2011). Another plausible explanation for a positive TEP is the alteration of the active transports, such as an increased secretion of anions into the lumen and/or increased uptake of cations, both of which would generate a more serosa positive TEP. In the current study, no histological indications of impaired intestinal health could be observed as there was no difference in mucosal fold length, which is usually observed during impaired intestinal health such as intestinal inflammation (Knudsen et al 2008, Krogdahl et al 2003). Nevertheless, the barrier function was impaired in D35 in the current study. This suggest that histological assessment of intestinal health is not as sensitive as the intestinal barrier function. This is supported by studies in Atlantic salmon where barrier impairment can be observed without morphological changes (Knudsen et al 2008). Similar observations have been reported in humans (Welsh et al 1998). However, other parameters such as differences in mucosal fold complexity, lamina propria width and submucosa length were not measured due to time restraints. These parameters might have indicated a histological difference between the diets as they have been found to differ during inflammation in Atlantic salmon (Knudsen et al 2008). There were however observable histological differences between the diets. Structures resembling vacuoles in the enterocytes in the mucosal folds of D35 and D55 but not in D60. Similar vacuoles have been reported in Atlantic salmon where they are believed to be involved in transcellular transport (Knudsen et al 2008). And a decrease in vacuoles is a common sign of lowered intestinal health (Knudsen et al 2008). The vacuoles in the Atlantic wolffish appear to have the opposite relationship as they occur only in the diets with lower protein content. The significance and or the function of these vacuoles in Atlantic wolfish are unknown but warrants further studies. Another histological observation where small structures that resemble microscopic intestinal parasites that occurred in all diets and is expected from fish originating from a wild population.

20

5.2. Intestinal characterisation (Study 2) 5.2.1. Functional morphology of the intestine Different species vary in their intestinal morphology. In salmonids the intestine is divided in anterior intestine with pyloric caecas, middle intestine and distal intestine with rectum (Olsson 2011). The Atlantic wolffish has a long proximal and a much shorter distal intestine that is majorly constituted by the rectum (Hellberg & Bjerkas 2000). The cranial part of the proximal intestine differs from the middle and caudal parts both morphologically and functionally. TER represents the sum resistance of the paracellular shunts as well as the transcellular pathways (Sundell & Sundh 2012). However, as the intestinal epithelium of fish is considered a leaky epithelium with much lower TER in the paracellular shunt compared to the transcellular shunt pathway. Therefore, in leaky epithelia TER mainly reflects the paracellular shunt, i.e. tight junctions (Loretz 1995, Sundell & Sundh 2012). The cranial part of the Atlantic wolffish proximal intestine had a higher TER compared to the later parts, which is the opposite to salmonids and Goby (Loretz 1995, Sundell et al 2003, Sundell and Sundh 2012, Sundh et al 2008, Sundh et al 2010). In the Atlantic wolfish the higher TER in the cranial region was observed concomitantly with a low Papp for mannitol. Similar connections between TER and Papp have been reported in salmonids (Sundell & Sundh 2012, Sundh et al 2010). A higher Papp could also indicate in a higher water uptake in these regions as tight junctions are known to be selective barriers of ions and water (Schneeberger & Lynch 2004). However, Papp cannot by itself predict the water uptake as water is assumed to be absorbed both paracellularly and transcellularly (Sundell & Rønnestad 2011). TER did not differ between middle and caudal parts even thou the permeability of mannitol is higher in the caudal. This could be due to, as mentioned before, the different types of tight junctions vary in their permeability to ions and uncharged particles (Günzel et al 2009, Günzel & Fromm 2012) and could therefore, possibly, explain the difference between the regions Papp without affecting TER. TER is also connected to TEP due to that the epithelium forms a barrier, i.e. resistance, which separates the luminal and intestinal fluids. The separation is partly achieved by limiting the ions transported to the intestinal fluids from leaking back into the lumen (Günzel & Fromm 2012, Justfelt 2011). The difference in the ion distribution between the luminal and intestinal fluids can then be measured as TEP (Sundell & Sundh 2012). In the current study, this relationship between TER and TEP is apparent as the TEP is more negative in the cranial part where the TER is higher, while in the later parts neither TER nor TEP differs in the regions. The TEP in all regions were serosa negative in the Atlantic wolffish in the current study. As mentioned before this is in agreement with previous findings in stenohaline marine fish species (Loretz 1995). SCC corresponds to the net ion transport across the epithelium as a result of active ion transport driven mainly by the Na+/K+ ATPase (Sundell & Sundh 2012, Sundell & Rønnestad 2011). The Na+/K+ ATPase drives the main transports in the intestine and thereby the SCC corresponds to the general transport in the intestine (Loretz 1995, Sundell & Rønnestad 2011). This fits well the finding in the current work where the high SCC in the intermediate and caudal part of the proximal intestine coincided with the higher transport rate of lysine in these regions. Further, in fish the lysine uptake is coupled to the uptake of Na+, the cation that would be expected to be highly transported in intestinal tissue with a high abundance of Na+/K+-ATPases (Berge et al 1999, Loretz 1995). The caudal part has a higher lysine uptake but does not differ in the SCC. The explanation behind this observation is unknown, but it could be hypothesised that the caudal part has a higher expression of amino acid transporters. Further studies are needed to conclude the reason behind these results. The functional morphology of the Atlantic wolffish intestine could be speculated to be due to their natural food sources. In the wild, the Atlantic wolffish prey upon crustaceans and molluscs, (Fairchild et al 2015, Simpson et al 2013) especially bivalves in Norwegian waters (Fairchild et al

21

2015), which is tougher to digest compared to fish and therefore might require nutrient absorption further back in the gastrointestinal tract to optimize the uptake. Spotted wolffish and Northern wolffish in Canadian differs in feeding habits from the Atlantic wolffish, where the Northern specialises in fish while the spotted wolffish specialises in echinoderm (Simpson et al 2013). If the feeding habit mainly determines the relationship of the intestinal function, this might indicate that the other wolffish species could have similar intestinal functions to salmonids compared to functions of the Atlantic wolffish. Goblet cells were fewer in the cranial part then in the other parts. This observation contradicts the findings of Hellberg and Bjerkas (2000) that stated that the whole proximal regions had the same amount of goblet cells. The higher amount of goblet cells is assumed to correlate to a higher production of mucus (Al-Hussaini 1949). Al-Hussaini (1949) also stated that a higher density of goblet cells facilitates the transport of faeces in the rectum, however no data was provided to the assumption. Shephard (1994) suggested an alternate function of the mucus where it recycle ions by trapping them in the mucus and therefore increase the accessibility of the ions for transport proteins. This recycling might aid to some extent the transport in the middle and caudal parts. However, more studies are needed to confirm if the mucus has a recycling effect.

Conclusion The diets with 54-59% protein did not disturb barrier and transporting functions of the intestine and therefore, there should be a continues usage of 54% protein in the diet, or even increase to 59%. In agreement, there was a tendency for increased growth in the diets with 50-59 % dietary protein which, together with the barrier functions, indicates better welfare compared to the diet with lower protein content. Diets with 35% protein should be avoided due to it causes a disturbed intestinal barrier function in combination with poor growth. The intestine of the wolffish appears to have reversed functional morphology in comparison to salmonids with lower transport and higher TER in the cranial region of the proximal intestine compared to more caudal parts.

22

Acknowledgements I would like to thank Henrik Sundh for being my supervisor during my thesis project and for his enthusiastic assistance even on short notice. I would also like to thank him for the assistance during sampling and during the Ussing measurements, along with all the feedback along the way. I would also like to thank my practical supervisor James Hinchcliffe for letting me take part in his protein requirement study and for tagging along to Uppsala to learn about the process of feed manufacturing. As well as for teaching me histology and the Na+/K+ ATPase assay, even though it later would not be in the thesis. I would furthermore like to thank, Ida Hedén for the instructions and the assistance with the Ussing chambers systems. Jonathan Roques for showing me around the lab and teaching me fish duty. I am also thankful to the whole gang; James, Ida, Jonathan, Koen de Reus and Felicia Fetscher for all the help with feeding the wolffish and for the good company during the long days of sampling. Furthermore, I would also like to thank Linda Hasselberg Frank for helping me find all the hidden treasures in the lab. I would also like to thank the rest of the FEL group for the opportunity to work with my thesis in the group. Also, I would like to thank my fellow master students Jakob Lundin, Argus Pesqueda and Jennifer Bowman for much needed fika breaks and their support during the writing process!

I wish you all the best of luck in your future endeavours and thank you for all your assistance!

23

References Al-Hussaini AH. 1949. On the Functional Morphology of the Alimentary Tract of Some Fish in Relation to Differences in their Feeding Habits: Anatomy and Histology. Quarterly Journal of Microscopical Science s3-90: 109-39 Albertsson E, Strand Å, Lindegarth S, Sundell KS, Eriksson S, Björnsson BT. 2012. Marin fiskodling på den svenska västkusten: Biologiska förutsättningar, Vattenbrukscentrum Väst, Göteborgs universitet Amasheh S, Fromm M, Günzel D. 2011. Claudins of intestine and nephron–a correlation of molecular tight junction structure and barrier function. Acta physiologica 201: 133-40 Anderson JM, Van Itallie CM. 2009. Physiology and Function of the Tight Junction. Cold Spring Harbor Perspectives in Biology 1: a002584 Ando M, Hara I. 1994. Alteration of sensitivity to various regulators in the intestine of the eel following seawater acclimation. Comparative Biochemistry and Physiology Part A: Physiology 109: 447-53 Ando M, Urita S, Nagahama H. 1975. Active transport of chloride in eel intestine with special reference to sea water adaptation. Comparative Biochemistry and Physiology Part A: Physiology 51: 27-32 Ashley PJ. 2007. Fish welfare: Current issues in aquaculture. Applied Animal Behaviour Science 104: 199-235 Axelsson M, Seth H. 2011. DESIGN AND PHYSIOLOGY OF ARTERIES AND VEINS | The Gastrointestinal Circulation In Encyclopedia of Fish Physiology, ed. AP Farrell, pp. 1132-41. San Diego: Academic Press Babaei S, Abedian-Kenari A, Hedayati M, Yazdani-Sadati MA. 2017. Growth response, body composition, plasma metabolites, digestive and antioxidant enzymes activities of Siberian sturgeon (Acipenser baerii, Brandt, 1869) fed different dietary protein and carbohydrate: lipid ratio. Aquaculture Research 48: 2642-54 Baeverfjord G, Krogdahl A. 1996. Development and regression of soybean meal induced enteritis in Atlantic salmon, Salmo salar L, distal intestine: A comparison with the intestines of fasted fish. Journal of Fish Diseases 19: 375-87 Bakke-McKellep A, Nordrum S, Krogdahl Å, Buddington R. 2000. Absorption of glucose, amino acids, and dipeptides by the intestines of Atlantic salmon (Salmo salar L.). Fish physiology and biochemistry 22: 33-44 Barton BA, Iwama GK. 1991. Physiological changes in fish from stress in aquaculture with emphasis on the response and effects of corticosteroids. Annual Review of Fish Diseases 1: 3-26 Beamish FWH, Medland TE. 1986. Protein sparing effects in large rainbow trout, Salmo gairdneri. Aquaculture 55: 35-42 Berge GE, Bakke-McKellep AM, Lied E. 1999. In vitro uptake and interaction between arginine and lysine in the intestine of Atlantic salmon (Salmo salar). Aquaculture 179: 181-93 Bialkowska AB, Ghaleb AM, Nandan MO, Yang VW. 2016. Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses. Journal of visualized experiments : JoVE Broom D. 1998. Fish welfare and the public perception of farmed fish. report Aquavision 98: 89- 91 Broom DM. 1986. Indicators of poor welfare. British Veterinary Journal 142: 524-26 Broom DM. 1988. The scientific assessment of animal welfare. Applied Animal Behaviour Science 20: 5-19 Buddington RK, Diamond JM. 1987. Pyloric caeca of fish: A 'new' absorptive organ. Am. J. Physiol. 252: G65-G76

24

Bulut M, Yiğit M, Ergün S, Kesbiç OS, Acar Ü, et al. 2014. Evaluation of dietary protein and lipid requirements of two-banded seabream (Diplodus vulgaris) cultured in a recirculating aquaculture system. Aquaculture International 22: 965-73 Catacutan MR, Coloso RM. 1995. Effect of dietary protein to energy ratios on growth, survival, and body composition of juvenile Asian seabass, Lates calcarifer. Aquaculture 131: 125- 33 Chandroo KP, Duncan IJH, Moccia RD. 2004. Can fish suffer?: perspectives on sentience, pain, fear and stress. Applied Animal Behaviour Science 86: 225-50 Chapman DW. 1966. Food and space as regulators of salmonid populations in streams. The American Naturalist 100: 345-57 Collie NL. 1985. Intestinal nutrient transport in coho salmon (Oncorhynchus kisutch) and the effects of development, starvation, and seawater adaptation. Journal of Comparative Physiology B 156: 163-74 Cowey C, Adron J, Blair A, Shanks AM. 1974. Studies on the nutrition of marine flatfish. Utilization of various dietary proteins by plaice (Pleuronectes platessa). British Journal of Nutrition 31: 297-306 Cowey CB. 1994. Amino acid requirements of fish: a critical appraisal of present values. Aquaculture 124: 1-11 Craig S, Helfrich LA. 2009. Understanding fish nutrition, feeds, and feeding. Dawkins MS. 1990. From an animal's point of view: motivation, fitness, and animal welfare. Behavioral and brain sciences 13: 1-9 Duncan I. 2005. Science-based assessment of animal welfare: farm animals. Revue scientifique et technique-Office international des epizooties 24: 483 Duritis I, Mugurevics A. 2016. Distribution and Characterisation of Goblet Cells in the Large Intestine of Ostriches during the Pre‐ and Post‐Hatch Period. Anatomia, Histologia, Embryologia 45: 457-62 Fairchild EA, Tallack S, Elzey SP, Armstrong MP. 2015. Spring feeding of Atlantic wolffish (Anarhichas lupus) on Stellwagen Bank, Massachusetts. Fishery Bulletin 113 FAO. 2016. The State of World Fisheries and Aquaculture 2016. Contributing to food security and nutrition for all., Rome FAO. 2017. World aquaculture 2015: A brief overview, Rome Forster IP. 2011. FOOD ACQUISITION AND DIGESTION | Dietary Requirements of Fish Under Culture Conditions A2 - Farrell, Anthony P In Encyclopedia of Fish Physiology, pp. 1617-22. San Diego: Academic Press Foss A, K. Imsland A, Falk-Petersen I-B, Øiestad V. 2004. A review of the culture potential of spotted wolffish Anarhichas minor Olafsen. Reviews in Fish Biology and Fisheries 14: 277-94 German DP. 2011. FOOD ACQUISITION AND DIGESTION | Digestive Efficiency A2 - Farrell, Anthony P In Encyclopedia of Fish Physiology, pp. 1596-607. San Diego: Academic Press Glencross BD, Booth M, Allan GL. 2007. A feed is only as good as its ingredients – a review of ingredient evaluation strategies for aquaculture feeds. Aquaculture Nutrition 13: 17-34 Grisdale-Helland B, Shearer KD, Gatlin DM, Helland SJ. 2008. Effects of dietary protein and lipid levels on growth, protein digestibility, feed utilization and body composition of Atlantic cod (Gadus morhua). Aquaculture 283: 156-62 Günzel D, Fromm M. 2012. Claudins and other tight junction proteins. Comprehensive Physiology Hellberg H, Bjerkas I. 2000. The anatomy of the oesophagus, stomach and intestine in common wolffish (Anarhichas lupus L.): A basis for diagnostic work and research. Acta Vet. Scand. 41: 283-97 Houlihan D, Boujard T, Jobling M. 2008. Food intake in fish. John Wiley & Sons.

25

Ivanov AI, Nusrat A, Parkos CA. 2005. Endocytosis of the apical junctional complex: mechanisms and possible roles in regulation of epithelial barriers. pp. 356-65. Hoboken Jobling M. 2011. ENERGETIC MODELS | Bioenergetics in Aquaculture Settings. Elsevier Inc. 1664-74 pp. Jordbruksverket. 2012. Swedish aquaculture – a green industry in blue fields. Strategy 2012– 2020 Jordbruksverket. 2014. Havs- och fiskeriprogrammet för perioden 2014–2020. 2014: 1-163 Jordbruksverket. 2017. Vattenbruk 2016, Statistiska centralbyrån Jutfelt F. 2011. Barrier function of the gut. Encyclopedia of fish physiology: from genome to environment. Academic Press, San Diego: 1322-31 Kai L, C.M. BM, Marc M. 2012. Cultured fish: integrative biology and management of domestication and interactions with wild fish. Biological Reviews 87: 639-60 Ketola HG. 1982. Amino acid nutrition of fishes: requirements and supplementation of diets. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry 73: 17-24 Knudsen D, Jutfelt F, Sundh H, Sundell K, Koppe W, Frøkiær H. 2008. Dietary soya saponins increase gut permeability and play a key role in the onset of soyabean-induced enteritis in Atlantic salmon (Salmo salar L.). British Journal of Nutrition 100: 120-29 Krogdahl Å, Bakke-McKellep AM, Baeverfjord G. 2003. Effects of graded levels of standard soybean meal on intestinal structure, mucosal enzyme activities, and pancreatic response in Atlantic salmon (Salmo salar L.). Aquaculture Nutrition 9: 361-71 Krogdahl Å, Nordrum S, Sorensen M, Brudeseth L, Rosjo C. 1999. Effects of diet composition on apparent nutrient absorption along the intestinal tract and of subsequent fasting on mucosal disaccharidase activities and plasma nutrient concentration in Atlantic salmon Salmo salar L. Aquaculture Nutrition 5: 121-34 Lie Ø, Lied E, Lambertsen G. 1988. Feed optimization in atlantic cod (Gadus morhua): Fat versus protein content in the feed. Aquaculture 69: 333-41 Lignot J-H, Cutler CP, Hazon N, Cramb G. 2002. Immunolocalisation of aquaporin 3 in the gill and the gastrointestinal tract of the European eel <em>Anguilla anguilla</em> (L.). Journal of Experimental Biology 205: 2653 Lionetto M, Maffia M, Vignes F, Storelli C, Schettino T. 1996. Differences in intestinal electrophysiological parameters and nutrient transport rates between eels (Anguilla anguilla) at yellow and silver stages. Journal of Experimental Zoology 275: 399-405 Loretz CA. 1995. 2 Electrophysiology of Ion Transport in Teleost Intestinal Cells In Fish physiology, pp. 25-56: Elsevier Moksness E. 1990. WEANING OF WILD-CAUGHT COMMON WOLFFISH (ANARHICHAS- LUPUS) LARVAE. Aquaculture 91: 77-85 Moksness E. 1994. Growth rates of the common wolffish, Anarhichas lupus L., and spotted wolffish, A. minor Olafsen, in captivity. Aquaculture Research 25: 363-71 Moksness E, Gjosaeter J, Reinert A, Fjallstein IS. 1989. START-FEEDING AND ON- GROWING OF WOLFFISH (ANARHICHAS-LUPUS) IN THE LABORATORY. Aquaculture 77: 221-28 Naylor RL, Goldburg RJ, Primavera JH, Kautsky N, Beveridge MCM, et al. 2000. Effect of aquaculture on world fish supplies. Nature 405: 1017-24 Niklasson L, Sundh H, Fridell F, Taranger GL, Sundell K. 2011. Disturbance of the intestinal mucosal immune system of farmed Atlantic salmon (Salmo salar), in response to long- term hypoxic conditions. Fish & Shellfish Immunology 31: 1072-80 Olsson C. 2011. GUT ANATOMY AND MORPHOLOGY | Gut Anatomy A2 - Farrell, Anthony P In Encyclopedia of Fish Physiology, pp. 1268-75. San Diego: Academic Press Rountree RA. 2002. Wolffishes. Family Anarhichadidae. Bigelow and Schroeder’s Fishes of the Gulf of Maine. 3rd Edition. Smithsonian Institution Press, Washington, DC: 485-96

26

Schneeberger EE, Lynch RD. 2004. The tight junction: a multifunctional complex. American Journal of Physiology-Cell Physiology 286: C1213-C28 Segner H, Sundh H, Buchmann K, Douxfils J, Sundell KS, et al. 2012. Health of farmed fish: its relation to fish welfare and its utility as welfare indicator. Fish physiology and biochemistry 38: 85-105 Shephard KL. 1994. Functions for fish mucus. Reviews in Fish Biology and Fisheries 4: 401-29 Simpson M, Sherwood G, Mello L, Miri C, Kulka DW, Place F. 2013. Feedings Habits and Trophic Niche Differentiation in Three Species of Wolffish (Anarhichas Sp.) Inhabiting Newfoundland and Labrador Waters. Canadian Science Advisory Secretariat. Smidt D. 1983. Advantages and problems of using integrated systems of indicators as compared to single traits In Indicators relevant to farm animal welfare, pp. 201-07: Springer Soderholm JD, Perdue MH. 2001. Stress and gastrointestinal tract. II. Stress and intestinal barrier function. Am J Physiol Gastrointest Liver Physiol 280: G7-g13 Stefanussen D, Lie Ø, Moksness E, Ugland KI. 1993. Growth of juvenile common wolffish ( Anarhichas lupus) fed practical fish feeds. Aquaculture 114: 103-11 Sundell K, Jutfelt F, Ágústsson T, Olsen R-E, Sandblom E, et al. 2003. Intestinal transport mechanisms and plasma cortisol levels during normal and out-of-season parr–smolt transformation of Atlantic salmon, Salmo salar. Aquaculture 222: 265-85 Sundell K, Sundh H. 2012. Intestinal fluid absorption in anadromous salmonids: importance of tight junctions and aquaporins. Frontiers in Physiology 3 Sundell KS, Rønnestad I. 2011. INTEGRATED FUNCTION AND CONTROL OF THE GUT | Intestinal Absorption A2 - Farrell, Anthony P In Encyclopedia of Fish Physiology, pp. 1311-21. San Diego: Academic Press Sundh H, Gräns A, Brijs J, Sandblom E, Axelsson M, et al. 2018. Effects of coeliacomesenteric blood flow reduction on intestinal barrier function in rainbow trout Oncorhynchus mykiss. Journal of Fish Biology Sundh H, Kvamme BO, Fridell F, Olsen RE, Ellis T, et al. 2010. Intestinal barrier function of Atlantic salmon (Salmo salar L.) post smolts is reduced by common sea cage environments and suggested as a possible physiological welfare indicator. BMC Physiology 10: 22 Szakolczai J. 1997. Histopathological changes induced by environmental stress in common carp, Japanese coloured carp, European eel, and African catfish. Acta Veterinaria Hungarica 45: 1-10 Welsh FKS, Farmery SM, MacLennan K, Sheridan MB, Barclay GR, et al. 1998. Gut barrier function in malnourished patients. Gut 42: 396 Wendelaar Bonga S. 1997. The stress response in fish. Physiological reviews 77: 591-625 Wendelaar Bonga SE. 2011. Hormonal Controls | Hormonal Responses to Stress In Encyclopedia of Fish Physiology, ed. AP Farrell, pp. 1515-23. San Diego: Academic Press Vergara JM, Robainà L, Izquierdo M, De La Higuera M. 1996. Protein sparing effect of lipids in diets for fingerlings of gilthead sea bream. Fisheries science 62: 624-28 Werner S. 1981. Protein utilization by rainbow trout (Salmo gairdneri) and carp (Cyprinus carpio): A brief review. Aquaculture 23: 337-45 Vidakovic A, Langeland M, Sundh H, Sundell K, Olstorpe M, et al. 2016. Evaluation of growth performance and intestinal barrier function in Arctic Charr (Salvelinus alpinus) fed yeast (Saccharomyces cerevisiae), fungi (Rhizopus oryzae) and blue mussel (Mytilus edulis). Aquaculture Nutrition 22: 1348-60 Worthington BS, Syrotuck J. 1976. Intestinal Permeability to Large Particles in Normal and Protein-Deficient Adult Rats. The Journal of Nutrition 106: 20-32

27

Should the wolffish eat less protein and how does its intestine function?

During the last decades there has been an increase in farmed aquatic animals and plants, called aquaculture. In 2015 the global aquaculture production of fish for human consumption exceeded the harvest of wild fish. The greatest producer of aquaculture products in Europe is Norway, which produces around 100 times more than Sweden. However, Sweden has a goal to increase its aquaculture products. One potential species for Swedish aquaculture is the Atlantic wolffish. However, not much is known about this species. One challenge to overcome in this effort is the development of an optimal feed. When manufactures produce fish feed they tend to exceed the protein content in the feed compared to what the fish needs. This is due to a limited knowledge about how much protein the species requires. This is a problem due to protein being an expensive product and is often made from wild-caught fish. Therefore, when developing a feed for a new species you should investigate the protein requirements of the species. One study has been made on the Atlantic wolffish where they tested the effect of 45-55% protein content in the feed and found no effect on growth, which might mean that the protein requirement of the Atlantic wolffish is outside this range. However, if the protein levels are too low in the diet it can cause detrimental health effects in the fish. One way of assessing fish health and welfare is to investigate the transportation of nutrients and ions of the intestine as these functions often become disturbed during stress. Another parameter of stress is the barrier function of the intestine, which keep pathogens and other damaging agents from entering the blood stream as well causing an ion gradient on the different sides of the intestine. This gradient causes water to pass in between the cells through selective proteins called tight junctions. Even here there is very little known about these functions in the wolffish intestines. Therefore, the study had two aims, firstly, to investigate the welfare effects of dietary protein levels. The second was to investigate the functions along the intestinal tract. To investigate the protein requirement six experimental diets were produced with 35%, 40%, 45%, 50%, 54% and 59% protein. Both aims were investigated by analysing the barrier functions and the transport of the intestine with a Ussing chamber system. The intestinal function was only affected in the 35% diet. There was an increase in permeability trough the tight junctions, which suggests a decreased barrier function. The weight of the fish was approximately the same in all diets but 50-59 % protein showed an indication towards an increased weight. There was a functional difference dependent on which part of the intestine that were measured. The later part of the intestine, the caudal part, had a greater increase in nutrient and ion transport, while the front part of the intestine had a higher ability to maintain a gradient between the inside of the intestine and the circulation. This relation is the opposite to salmonids which might be due to different feeding habits. In conclusion, a protein content of 35% appears to decrease the barrier function, therefore the feed should contain higher amounts of protein. There was a slightly increased growth with 50- 59% and therefore the protein content is recommended to be at 50-59%. The intestine of the Atlantic wolffish is different compared to more commonly farmed fishes in relation to where the higher uptake of nutrients occurs and in the relationship between uptake and tissue resistance. However, to understand why this difference occur more research is needed.

28