TECHNICAL FIELD

The present invention relates to a method to improve robustness and salmon welfare, and reduce unspecific mortality.

BACKGROUND OF THE INVENTION

In intensive salmon aquaculture production in Norway, the average mortality in the seawater phase is currently at 15-20 percent. A large proportion of this loss is due to unspecified mortality that causes large economic losses and poor fish welfare. The agents and causes to unspecific mortality are complex. However, some of the problems may have originated from the fresh water phase and the transition from small to large industrial scale production of farmed fish and its challenges to harmonize with the biological complexity of different fish species. In recent years, the negative effects caused by stress due to sorting, delousing and restocking have increased significantly. High numbers of dead fish normally follow such necessary handling procedures, which may indicate insufficient robust fish for intensive modern aquaculture farming.

It is generally acknowledged that outbreaks of many disease conditions in aquaculture occur as a result of intricate interactions between the host, agents and environmental conditions, combined with effects of site management and stress due to handling.

Cardiovascular diseases and circulatory failure are common in aquaculture and are often characterized as production-related diseases. Frequently cardiovascular diseases and circulatory failure coincide with outbreaks of the disease cardiomyopathy syndrome (CMS). CMS is an increasing problem in salmon farming and since it is usually the largest and seemingly well-fit salmons that die, it is a great economic burden for the farming industry. In addition, cardiovascular and circulatory failure have been observed in diseases like heart and skeletal muscle inflammation (HSMI) and pancreas disease (PD), but mainly as secondary effects. These are widespread infectious diseases of viral character in fish farming. In many cases, however, cardiovascular and circulatory failure are also observed without these viruses being present and thus represent a significant proportion of the non-specific mortality in fish farms. Seasonal variation in fat accumulation and reproduction strategy.

A number of factors govern the fat content of salmon and trout. Generally, fat and fatty acid levels in salmon and trout increase with increasing fish size, ration/feed intake and increased dietary fat. Several studies have also reported seasonal changes in

accumulation, metabolism and burning of fat lipids. Salmon deposition and retention of dietary fat has proven to be dynamic and more season dependent than for example the retention of dietary protein. In particular, the autumn has proven to be a period of high retention and accumulation of fat.

There is a marked increase in fat accumulation in the muscle in the autumn and stagnation/decline in winter, before it increases slightly again the following spring. This pattern of fat deposition is not random but governed by the salmon reproduction strategy to become sexually mature. The sexual maturation process in salmon requires, in addition to light stimuli (rising and falling day length), also sufficient fat/energy reserves. Low energy stores after the sea winter may challenge the reproductive strategy. After initiation of the maturation process in the late winter season, appropriate energy or fat reserves during the spring period seem to be a major factor controlling the progression of the maturation process. Too low energy or fat levels may arrest further progress. Especially female salmon need to invest more energy in gonads compared to their male

counterparts. Therefore, to secure necessary levels of energy/fat in male and female salmon at increasing day lengths during the initiation of the maturation process in the spring, falling day lengths in the autumn before is the key factor for accumulation of sufficient fat reserves for the acquire energy to reproduce.

An overview of the salmon reproduction strategy with regard to fat is shown in figure 1 revealing that spawning takes place in late autumn. The maturation process starts and continues only if the salmon has sufficient energy stores in the spring before (black arrow). The overall reproduction process starts with the crucial accumulation of fat and build-up of energy stores during late summer and early autumn prior the initiation of the maturation process the following spring, more than one year ahead of spawning (gray arrow). Fat storage.

A detailed understanding of factors controlling the body distribution of fat and fat cell recruitment in Atlantic salmon is scarce. The salmon normally stores the depot lipids in visceral white adipose tissue in addition to myosepta surrounding the muscle fibres. Very little of the fat from the diet is stored in the liver and heart under normal conditions. Little is known for fish, but overloaded fat cells may produce adipokines with a wide variety of physiological or pathological functions, and thereby play a role in development of inflammatory responses.

There is a need in the aquaculture production to find a way to reduce and/or prevent mortality of salmons by increasing the robustness of the fish to better withstand production related mortality, inflammatory responses as well as stress due to handling. In addition it is the goal of every fish farmer to continuously increase the weight of each fish during the whole production phase in order to improve the economic profit.

SUMMARY OF THE INVENTION Thus, an object of the present invention is to provide a method for rearing fish of the Salmon salar species wherein

a pre-dietary feed comprising a fat content of less than 27% (wt/wt) is administered to the fish during a pre-dietary phase throughout which the period of daytime increases for each subsequent day. After the end of said pre-dietary phase, a main-dietary Salmon sa/arfeed having a fat content of more than 30% (wt/wt) is administered to the fish throughout a main-dietary phase during which the period of daytime decreases for each subsequent day.

The Salmon salar species comprises smolts and post-smolts.

The pre-dietary feed has a fat content of 15-27% (wt/wt) fat, more preferably a fat content of 18-26% fat, more preferably a fat content of 20-24% fat (wt/wt).

Advantageously the pre-dietary feed contains 24% fat or less,≥ 43% protein, more preferably≥ 47% protein, and more preferably≥ 50% protein (wt/wt) when administered to fish weighing < 400g. Advantageously the pre-dietary feed contains 27% fat or less,≥ 40% protein, more preferably≥ 43% protein, and more preferably≥ 47% protein (wt/wt) when administered to fish weighing≥ 400g.

The pre-dietary feed is administered to the fish during a pre-dietary phase. The term "pre- dietary phase" as used herein is intended to mean a time of the year during which the period of daytime increases for each subsequent day. This means that for each subsequent day during this phase the period of daytime becomes longer. In the Northern Hemisphere the pre-dietary phase starts around mid-January and ends in late June. In the Southern Hemisphere the pre-dietary phase starts mid-July and ends in late December. The pre-dietary feed comprising a fat content of less than 27% (wt/wt) is administered to the fish throughout a pre-dietary phase during which the period of daytime increases for each subsequent day until the pre-dietary phase has ended and the period of daytime starts decreasing for each subsequent day. The advantage of administering the fish a low- fat diet during this phase is an increased robustness of the fish, reduced mortality with as much as 50% compared to fish administered a fish feed containing > 30% fat (see Example 1 and 3).

After the pre-dietary phase has ended and the period of daytime starts decreasing for each subsequent day (i.e. at the end of June in the northern hemisphere and at the end of December in the southern hemisphere, the administration of pre-dietary feed stops and instead a main-dietary Salmon sa/arfeed is administered to the fish.

The main-dietary feed, as opposed to the pre-dietary feed, has a fat content of 30-40% fat, more preferably a fat content of 33-39% fat, more preferably a fat content of 35-38% fat (wt/wt).

The main-dietary Salmon sa/arfeed is administered to the fish during a main-dietary phase when the period of daytime decreases for each subsequent day. Advantageously, the main-dietary phase starts about one month after the change in period of daytime from increasing to decreasing daylight (summer solstice in late June in the Northern

hemisphere, whereas in late December in the Southern hemisphere). The main-dietary phase stops about the time of vernal equinox (about 7 months after the start), when day and night are of about equal lengths (i.e. in late March in the Northern hemisphere, and in late September in the Southern hemisphere, 12h nighttime:12h daylight). The main-dietary Salmon sa/arfeed when administered to fish weighing < 400g contains 30% fat or more and≥ 35% protein, more preferably≥ 38% protein, more preferably≥ 42% protein (wt/wt).

The main-dietary Salmon sa/arfeed when administered to fish weighing≥ 400g contains 33% fat or more and≥ 30% protein, more preferably≥ 33% protein, more preferably≥ 35% protein (wt/wt).

The advantage of first administering the salmon a pre-dietary feed comprising a fat content of less than 27% during a pre-dietary phase, and thereafter administering a main- dietary Salmon sa/arfeed having a fat content of more than 30% throughout a main- dietary phase is that the growth is significantly improved compared to salmon not given the pre-dietary feed during the pre-dietary phase. In addition, the robustness of the salmon is increased regarding handling of stress, which leads to reduced unspecific mortality and improved fish welfare. Salmon administered a pre-dietary feed with a fat content of less than 27% experience a reduced mortality by 50%. If the salmon is not already slaughtered (at normal slaughter weight >4 kg), after the main-dietary phase with decreasing periods of daylight for each subsequent day has ended and a new second phase wherein the period of daylight increases above 12 hours (vernal equinox), a post-dietary feed comprising a fat content of less than 32% and more than 35% protein (wt/wt) is administered to the salmon to maintain robustness and improve the slaughter yield.

The method as described herein is also applicable to salmon reared in closed

environments using artificial lights. The advantage of rearing salmon in closed

environments is that the periods of slaughter may be distributed throughout the year to provide a constant supply of salmon. With the method described herein the salmon farmer may control the artificial light to mimic the natural fluctuation of daylight throughout the calendar year to initiate the pre-dietary and main-dietary feeding phases for salmon as described herein at any time of the year. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In the following detailed description, reference is made to the accompanying set of drawings that form a part of the description hereof and in which are shown by way of illustration several specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 displays a visualization of the salmon reproduction strategy hypothesis relative to timing of fat accumulation and requirement of energy for spawning.

Figure 2A discloses how the period of daytime changes for the northern hemisphere during the course of one year, and Figure 2B illustrates the rate of change expressed as percentage change.

Figure 3 reveals the significant decrease in mortality as a result from feeding the pre-test diet P(T) compared to the pre-control diet P(C).

Figures 4 A and B document the increased unspecific mortality in the late seawater phase at two different research locations. Stippled line shows the time of dietary change from low to high fat content in the test diet.

Figures 5 A and B illustrate the sudden increase in unspecific mortality (5A) and reduced growth (5B) observed in the late seawater phase (from mid-January 2016) at the R&D location in the North of Norway. Vertically stippled line shows the time of dietary change from low to high fat content in the test diet. Figure 6 exemplifies the significantly higher total level of fat observed in the liver of dead compared to in live salmon in the two net pens administered test diet at the R&D location in the North of Norway.

Figure 7 demonstrates that starvation reduces daily unspecific mortality (7A) in the test pens and the problem of yellowish fatty liver (7B). Figure 8 discloses the presence of higher levels of the liver enzyme alanine

aminotransferase (ALT) in the blood of dead/moribund salmon compared to living salmon.

Figure 9 discloses the significant reduction in daily unspecific mortality among salmon (>1 kg) administrated , pre-dietary test feed, and ½ ration pre-dietary test feed compared to the pre-dietary control feed during the pre-dietary spring and summer period (May to August).

Figures 10A and B disclose the effects on muscle fat content (1 OA) and bodyweight (1 OB) in salmon from a change in administration of the pre-dietary feeds during a the pre-dietary phase to administration of a main Salmon sa/arfeed during the main-dietary phase.

Figures 1 1 A and B disclose that administration of both pre-dietary feeds had a highly significant positive effect on growth (1 1 A) and weight gain (1 1 B) throughout the 7-month main-dietary feeding phase (Aug-Mar).

Figure 12 discloses the dispersion in weight of the different pre-dietary, ½ ration and control groups during the pre-dietary phase and the main-dietary phase.

Figure 13 discloses the relation between muscle fat content for salmon in the pre-dietary, ½ ration and control groups at the end of the pre-dietary phase (x-axis), and the increase in fat percentage in non-edible (visceral) to edible (muscle) part of the salmon during the first month of the main-dietary phase (Aug-Oct). Figure 14 displays seasonal accumulation of muscle fat relative to changes in day length in Salmon sa/arfarmed at 62°N in Norway.

Definitions

Certain terms are used throughout the description and the claims that, while for the most part are well known. However, the following terms may require some explanation: It should be understood that the mentioned percentages are per weight (weight/weight or wt/wt) percentages unless otherwise stated. For example a fish feed that has a protein content of 40% means that a pelleted fish feed that weighs 100 g (as is), 40 g constitutes proteins of some type as explained below.

In the context of the present invention the term "fish feed" relates to a feed suitable for being administrated to fish. The fish feed comprises ingredients normally used for salmon such as proteins, lipids, carbohydrates and minerals.

The term "protein" when used herein is intended to mean both proteins derived from a source of protein and to free amino acids in general, therefore the term "protein" is meant the total content of proteinaceous material including free amino acids (if present). The detailed make-up of the protein source is not believed to be critical to the present invention, and all protein sources suitable for fish feed can be used. Thus, the protein may be any of dairy proteins, animal proteins, vegetable proteins, cereal proteins or a combination thereof. However, in the study described in the examples, the protein used in the control diet and test diet comprises the same source of protein (within each R&D location) in order to have the same amino acid profile in the test diet and the control diet. Hereby, the results obtained in the study are comparable.

The amount of protein in relation to the total fish feed is present in the fish feed in relation to the total fish feed is in the ranges as indicated herein. The protein(s) in the protein source may be intact (non-hydrolysed) protein or partially hydrolysed or a combination of non-hydrolysed and hydrolysed proteins. In a preferred embodiment the protein is non-hydrolysed protein. In another embodiment of the invention, the protein is intact protein.

It is preferred to use non-hydrolysed protein, since high levels of hydrolysed protein may result in a negative effect on feed intake, i.e. if too high levels of hydrolysed proteins are used, the appetite may be reduced.

The term "fat" when used herein is intended to mean one or more lipid compounds and may be in the form of monoglycerides, diglycerides, triglycerides and fatty acids, preferably the lipid or fat is an oil. The lipid may be derived from any vegetable or animal source.

The lipid is in the context of the present invention edible. By edible is meant that the lipid composition is suitable for being consumed. Thus, the lipid of the fish feed is suitable for being eaten by an animal, e.g., fish without being sick and/or violating internal organs. In an embodiment of the invention, the lipid is a mixture of marine and vegetable oil. The lipid source may be any lipid or fat which is suitable for use in fish feed.

Preferred fat sources include a mixture of marine and vegetable oil. However, in the study described in the examples, the lipid used in the control diet and test diet comprises the same source of lipid (within each R&D location) in order to have the same fatty acid profile in the test diet and the control diet. Hereby, the results obtained in the study are comparable. The amount of lipid present in the fish feed in relation to the total fish feed is in the range as indicated herein.

The term "carbohydrate" when used herein is intended to mean any carbohydrate suitable for use in fish feed. The specific source of carbohydrate is not believed to be critical for the invention and therefore all sources of carbohydrate suitable for fish feed may be used. Preferably, the carbohydrate content of the fish feed is 5-20% by weight, such as 5-15% by weight, more preferably form 7-12% by weight.

The salmon dietary feed may also comprise other ingredients such as minerals, vitamins, ash and pigments. The method described herein is advantageously used for Salmon salar. All salmonids spawn in fresh water but in many cases the fish spend most of their lives at sea returning to the rivers only to reproduce. They are predators feeding on small crustaceans, aquatic insects and smaller fish.

When used herein the term "smolt" is intended to mean a juvenile salmon, which undergoes a process of physiological changes that allows them to survive a shift from freshwater to saltwater (smoltification), regardless of what time of year the smoltifi cation takes place. The term smolt as used herein also includes in-season smolt, S1 , and out-of- season smolt, SO.

The term "post smolt" is an all year around salmon adapted to seawater, where the early grow-out phase, and later the adult grow out phase, is in a water environment where the salinity is sufficiently high to avoid de-smoltification. The water environment could be either on land or in sea, irrespective of type of containment systems (open, semi-closed or closed).

The term "closed environments" as used herein is intended to mean environments that is not normally exposed to natural photoperiods. To apply the present method described herein, the salmon farmer may use artificial light to mimic the natural fluctuation of daylight described in the present invention to initiate the pre-dietary and main-dietary feeding phases for salmon as described herein at any time of the year.

As used herein the term "period of daytime" is roughly defined as the period between sunrise, i.e. when the earth's rotation towards the east first causes the sun's disc to appear above the horizon, to sunset, i.e. when the continuing rotation of the earth causes the sun's disc to disappear below the horizon to the west.

As the earth moves around the sun, the period of daytime (defined as the time between sunrise and sunset) changes. The extent to which the period of daytime changes (i.e. increases or decreases) depends on latitude, as shown in the graph in figure 2A. Figure 2A discloses how the period of daytime changes for the northern hemisphere during the course of one year. Here it is seen that the period of daytime changes much more during the year the further away you get from the equator.

Generally, around the equinoxes in March and September, the days are getting longer/shorter at their fastest rate (i.e. the steepest slopes). The rate of change is illustrated as percentage change in Fig. 2B. In this graph, it is seen that at higher latitudes the length of day changes quite noticeable in early January and from mid-November. At the latitude 60°N the rate of change around February and October is around + and -5 min or more per day (see figure 2B). The change in period of daytime from one day to the next at the time of the winter solstice in late December is around zero (see Figs. 2A and 2B). Similarly, there is little change in period of daytime from day-to-day at the time of the summer solstice in June. The reverse will be true for the Southern Hemisphere where the period of daytime will increase for each day after the winter solstice in late June and decrease after the summer solstice in late December.

The term "pre-dietary phase" as used herein is intended to mean a time of the year during which the period of daytime increases for each subsequent day. This means that for every day the period of daytime becomes longer. Theoretically this increase in day length starts at the winter solstice in around 21 December and ends at the summer solstice around 21 June in the Northern Hemisphere. In the Southern Hemisphere, the pre-dietary phase will start around the 21 June and end around 21 of December. However, from the graphs 2A and B it is seen that the change in period of daytime is not very significant around the solstices and it is not until about a month after the winter solstice (mid-January in the Northern Hemisphere and mid-July for the Southern Hemisphere) that the period of daytime starts increasing noticeably. Thus, for the purpose of the method to be used in natural light conditions as described herein, the term "pre-dietary phase" starts about four weeks after the winter solstice, and ends about four weeks after the summer solstice, preferably it ends about five weeks after the summer solstice, more preferably it ends about six weeks after the summer solstice.

The term "main-dietary phase" as used herein is intended to mean a time of the year during which the period of daytime decreases for each subsequent day. This means that for every day the period of daytime becomes shorter. Theoretically this decrease in daytime starts at the summer solstice in around 21 June and ends at the winter solstice around 21 December in the northern hemisphere. In the southern hemisphere, the main- dietary phase will start around the 21 December and end around 21 of June. However, as seen in the graphs 2A and 2B it is seen that the change in period of daytime is not very noticeable around the solstices, and it is not until about a month after the summer solstice (mid-July in the northern hemisphere and mid-December for the southern hemisphere) that the period of daytime starts increasing significantly. Thus, for the purpose of the method as described herein the term "main-dietary phase" starts about four weeks after the summer solstice, preferably it starts about five weeks after the summer solstice, more preferably it starts about six weeks after the summer solstice.

The described method of light regimes for pre-, main- and post-dietary phases as defined above also include artificial light used in close environmental systems. In a closed environment system, artificial light may be used to mimic the natural fluctuation in periods of daytime throughout the calendar year. The present invention is directed to a method for rearing fish wherein during a pre-dietary phase, throughout which the period of daytime increases for each subsequent day, a pre- dietary feed comprising a fat content of less than 27% (wt/wt) is administered to the fish. After the end of said pre-dietary phase, a main-dietary Salmon sa/arfeed having a fat content of more than 30% (wt/wt) is administered to the fish throughout a main-dietary phase during which the period of daytime decreases for each subsequent day.

The inventors have surprisingly found that the above described feeding regime, increased the robustness of fish compared to fish being administered a fish feed having a fat content of more than 30% throughout both the pre-dietary and main-dietary phases. Fish administered the pre-dietary feed reached a higher body weight and demonstrated an improved biological feed conversion ratio (FCRb) i.e. the efficiency with which the nutrients in the feed are convert to fish body weight, compared to fish administered a feed with higher fat content. Therefore, the method for rearing fish as described herein can advantageously also be used for increasing weight in fish of the Salmon salar species. In the Examples below it is seen that when Salmon salar is being fed pre-dietary feed comprising a fat content of less than 27% (wt/wt) during a pre-dietary phase throughout which the period of daytime increases for each subsequent day, and after the end of said pre-dietary phase, a main-dietary Salmon sa/arfeed having a fat content of more than 30% (wt/wt) is fed to the fish throughout a main-dietary phase during which the period of daytime decreases for each subsequent day, the Salmon salar showed a significantly improved weight gain resulting in a higher bodyweight compared to fish having been fed a regular Salmon sa/arfish feed throughout the year. Surprisingly, the pre-dietary feed also had a higher positive influence on the mortality for fish of a smaller size compared to the mortality for fish of a larger size. This positive influence started in late pre-dietary phase and continued until the first period of the main- dietary phase. The dietary regime of administering a pre-dietary feed during the pre- dietary phase and thereafter switching to a main-dietary feed during the main-dietary phase will both improve the robustness of the fish (i.e. higher bodyweight, reduced unspecific mortality, improved biological feed conversion ratio and reduced disease related mortality) during the pre-dietary phase, as well as improve the growth rate and bodyweight increase during the main-dietary phase, thereby providing an improved economic profit to the fish farmer.

The following studies were conducted at Nofima commercial scale research &

development (R&D) facilities in the south (Rad0y in Hjeltefjorden, Hordaland), in the middle (Halsa in Halsafjorden, M0re og Romsdal) and the North of Norway (Bjark0y in Harstad, Nordland). The invention will now be explained in more detail in the following:

Rearing facilities and fish R&D facility in the Middle of Norway:

The site consists of four net pens (circumference of 120 m) each with approximately 100 000 salmon. At the facility, Atlantic salmon (Salmon salar) transferred to sea in April 2014 as in-season (S1 ) smolt with an average weight of 106 grams were reared in the four net pens during the period April 2014 - July 2015. The dietary treatments (test or control) were randomly allocated among the four net-pens (2 pens per diet).

R&D facility in the South of Norway: The site consists of six net pens (circumference of 120 m), each with approximately 100 000 Salmon salar. The salmon were transferred to sea in September and October 2014 inducing two different weight classes at sea transfer (88 gram in September and 75 gram in October) as out-of-season smolt (SO). The dietary treatments (test or control) were randomly allocated among the six net-pens (3 pens per diet regarding mortality and 2 pens per diet regarding repeated sampling for production and quality data).

R&D facility in the North of Norway:

The site consists of four net pens (circumference of 120 m), each net pen with

approximately 100 000 Salmon salar. The salmon were transferred to sea during May 2015 as post-smolt. The salmon was originally an out-of-season smolt (SO) kept at a land- based facility in fresh water until January 2015. In January 2015, 400 000 salmon post- smolt were divided into two tanks (200 000 in each tank) supplied with brackish water (14%o salt). Each of the two tanks received different dietary treatments (pre-test or p re- control) from Mid-January until sea transfer in May. The pre-test feed contained less than 24% fat. In May (after four months in brackish water), the fish given the pre-test diet had an average weight of 135 grams (initial weight 80 gram), whereas the fish given the pre-control diet (24-28% fat) had an average weight of 165 grams (initial weight 85 gram).

At sea transfer, the fish from the tanks were allocated among four net pens with 100 000 fish in each pen (two pens with an average fish weight of 135 grams and two pens with an average fish weight of 165 gram). One net pen of each weight class/pre-dietary group were assigned to a new dietary treatment of control and test diet during the seawater phase.

Sampling and growth estimations All net pens incorporated in Nofima R&D licenses at the different R&D facilities of Nofima (two net pens per diet at each site) were regularly sampled during the grow-out period in sea. At each sampling, the length and weight of approximately 100-150 fish per pen were individually measured. The data obtained from the samplings, together with daily feeding amount and estimated feed conversion rate of different fish sizes, were used to obtain models for weight development, estimated growth and feed utilization. At the R&D sites in the Middle and North of Norway, fish weights and biomass were also estimated with BM Storvik infrared light frames (biomass estimation system; Storvik AS, Sunndals0ra, Norway), with 400-1500 fish being measured in each cages over 1 -3 days with approximately monthly intervals.

At all R&D sites, the fish were fed to satiation using underwater camera by feeding several meals throughout the day (With the help of central feeding systems and modern feed barges). The feeding volume (kg day ^"1 ) and dead fish were recorded daily.

Diets

All experimental feed used in the R&D facilities were made from the same feed manufacture. The control diet contained protein/fat ratios that are commonly used in commercial feed according to fish size (Control diets) and the test diet had a higher protein/fat ratio according to fish size (Test diets). Table 1 shows the dietary range of fat and protein content (%) used from 75 until 200 gram, 200 until 500 gram, 500 until 1000 gram, 1000 until 2500 and 2500 until≥ 5000 gram of bodyweight.

The diets comprised conventional feed ingredients that commonly are used in fish feed such as protein, lipids, carbohydrates, vitamins and minerals.

Table 1 : Range of dietary content of protein and fat in the test and control diets used for different fish size intervals at the R&D facilities.

Table 1

Fish size (g) Test diet Control diet

Protein and fat content (%)

75 - 200 g

Protein (%) 47-50 44-46

Fat (%) 17-24 27-28

200 - 500 g

Protein (%) 46-47 43-44

Fat (%) 24-27 29-30

500 - 1000 g

Protein (%) 46-47 41 -42

Fat (%) 24-27 30-32 1000 - 2500 g

Protein (%) 45-47 35-36

Fat (%) 24-27 35-36

2500 -≥5000 g

Protein (%) 40-47 33-34

Fat (%) 24-35 36-39

Example 1 - Positive effects of the test diet in all locations first period in sea R&D facility in the Middle of Norway:

The estimated growth and feed utilization during the period from the sea transfer in April 5 2014 and until January 18 ^th 2015, revealed an overall positive effect of the test diet. Fish given the test diet had a higher body weight and improved biological feed conversion ratio (FCRb) compared with the fish given the control diet (see table 2).

R&D facility in the South of Norway:

At the R&D facility in the South of Norway, similar positive effects as observed in the0 Middle of Norway were detected for the fish given the test diet. The fish given the test diet had a higher bodyweight and better biological feed conversion ratio (in the period from sea transfer until April 2015) compared to the fish given the control diet (table 2).

In June 2015, a coinfection of heart and skeletal muscle inflammation (HSMI) and Pancreas Disease (PD) were detected at the site and disease related mortality started in5 the beginning of July. The accumulated total mortality (during the acute and sub-acute phase July - September) revealed an overall significant positive dietary effect (control = 8.0% vs test = 4.0%), showing that the test diet reduced mortality by 50%. In addition, reduced mortality for fish fed the test diet was observed within both weight classes. The reduced total mortality was significantly higher for the smaller fish than for the bigger0 ones. In addition, the test diet improved growth until late August (table 2).

R&D facility in the North of Norway:

In the R&D facility in the North, the accumulated mortality during the phase May 2015 - January 2016, revealed a positive pre-dietary effect on mortality (Figure 3). The pre-test diet (P(T)) used during the time at the land-based facility (January - May) significantly reduced mortality by almost 40% during the initial seawater phase compared to the pre- control diet (P(C)) (pre-control = 2.24% vs pre-test = 1 .40%) irrespective of diet given in sea phase (control or test diet). Taken together, initial overall positive effects of the test diet with a high protein/fat ratio were detected at all R&D facilities of Nofima. The positive effects are summarized in table 2.

Table 2

Test location Test diet Control Differences Positive diet _/-r . _ . increase in

(Test vs. Control) , v ¹ percentage

(Test vs. Control)

R&D Mid-Norway

Bodyweight in Jan 15 31 19 g 2935 g + 184 g 6.3%

FCRb (Apr 14 - Jan 15) 0.94 0.98 -0.04 4.1 %

R&D South-Norway

Bodyweight in Apr 15 small fish 728 g 706 g +22 g 3.1 %

FCRb (Sept - Apr 15) small fish 0.86 0.89 -0.03 3.4%

Bodyweight in Apr 15 large fish 993 g 921 g +72 g 7.8%

FCRb (Sept - Apr 15) large fish 0.95 0.97 -0.02 2.1 %

Bodyweight in Aug 15 small fish 1663 g 1314 g +349 g 27%

Bodyweight in Aug 15 large fish 2686 g 2182 g +504 g 23%

Total mortality Sept 15 Small fish 5.3% 1 1.3% -6% 53%

Total mortality Sept 15 Large fish 2.7% 4.6% -1 .9% 41 %

R&D North-Norway

Mortality, regards to pre-dietary

treatment (May 15 - Jan 16) 1 .4% 2.24% -0.84 37.5% Example 2 - Surprisingly increased unspecific mortality in all groups fed the main test diet during their last period in sea.

R&D facility in the Middle of Norway:

5 A change from 27 to 31 % dietary fat content in the test diet group and from 35 to about 38% dietary fat content in the control diet group, took place in early January 2015. From the midst of January and until June 2015, the monthly unspecific mortality in fish group given test diet was consistently higher than in the fish group given control diet (figure 4A). Increased mortality in the test group was particularly observed during months when

10 handling procedures were conducted as delousing and/or net change in the cages

(January, March and April).

R&D facility in the South of Norway:

Similar observations regarding unspecific mortality as in the R&D facility in the Middle of Norway were detected in the R&D facility in the south. At this R&D site, the change from

15 24 to 35% dietary fat content in the test diet group and from 35 to about 38% dietary fat content in the control diet group took place in October/November 2015. From October 2015 and until March 2016, the test group had a higher monthly unspecific mortality than the control group (figure 4B). The mortality in the test group was also here markedly increased during months with handling (routine delousing during October, January,

20 February and March).

R&D facility in the North of Norway:

At the R&D facility in the North of Norway, a change from 27 to 35% dietary fat content in the test diet group and from 35 to 38% dietary fat content in the control diet group was made in early December 2015. A sudden increased mortality in the two net pens given the

25 test diet i.e. groups P(C)-D(T) and P(T)-D(T)) were observed in the late seawater phase (from the mid-January 2016) at the R&D facility in North of Norway (figure 5A). At the same time, a significant drop in growth was documented (figure 5B). No increase in mortality or drop in growth was registered in the two net pens given the control diet. P(T) and P(C) are pre-test diet and pre-control diet used in the land base, respectively,

30 whereas D(T) and D(C) is the respective feeds used in the sea production. Due to this, a disease outbreak was suspected and the local veterinary service

(Vesteralen Fiskehelsetjeneste ASA) was contacted. The veterinary services arrived at the location on the 25 ^th of January and conducted a simple autopsy of moribund fish in the test group. In addition, organs for histology and heart tissue were sampled for real time RT-PCR analysis. The autopsy revealed symptoms of circulation disorder by blood clot in the heart cavity and bloody fluid in the intestinal cavity. Yellowish livers and swollen spleens were also observed. The National Veterinary Institute of Norway (NVI, Harstad) analyzed the heart tissue samples by real time RT-PCR analysis for Piscine myocarditis virus (PMCV - causing CMS) and the infectious salmon anemia virus (ISAV - causing ISA). The analysis results showed that the analyzed fish were not infected with PMCV nor ISAV virus.

Sampling of the fish was conducted at the R&D facility in the North on February 3 due to high mortality rates during this period. Moribund fish and normal healthy fish from the two pens given the test diet and likewise from the control pens were sampled at this occasion. The autopsy of the moribund fish from the test group revealed the same symptoms as observed by the veterinary service, with signs of circulation disorder and yellowish livers. The sampled moribund fish were seemingly well fit (normal to high condition factor) and almost 500 grams larger than the estimated mean weight of the net pens.

During this sampling, livers from all fish were collected and later analyzed for total fat content. Figure 6 exemplifies the significantly higher total level of fat observed in the livers of dead compared to in live salmons in the two net pens fed test diet D(T) at the R&D location in the North of Norway. No difference in liver fat was observed between live salmon from all four net pens. The low liver fat among dead fish in dietary group P(T)- D(C) (in figure 6) was probably due to the fact that only small fish were dead the day of sampling (February 18 ^th 2016). D(T) and D(C) represent test- and control diet used in sea, respectively.

On 18 of February, a second sampling was conducted during a period with increased mortality at the R&D site. Since the fish had clear symptoms of circulation disorder (similar symptoms as CMS) in combination with increased levels of fat in the liver, a so far unknown nutrition-related cause of mortality was suspected. Starvation was therefore chosen as a method to reduce the level of fat in the liver and also to potentially lower the mortality in the test group. The test group was starved (i.e. not fed at all) from 16 of February and until 1 1 of March. The starvation significantly decreased the mortality rates which were back to normal within 3-4 weeks (figure 7A). A comparison of visual colour score before and after starvation revealed significant change of liver colour from a yellowish fatty liver to a light brown normal colour (figure 7B). D(T) represents the test-diet used in sea. Feeding of the test groups was recommenced on 1 1 of March, and fish were given a diet with a lower fat content (30% fat). No increase in mortality was observed after the recommencement of feeding.

During the sampling on February 18 ^th , blood were collected from 10 normal and 10 moribund fish in each of the two net pens of the test group (40 samples), with highly increased mortality rates. The blood samples were analyzed by the Central Laboratory at Norwegian University of Life Sciences for alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP) and other parameters that are commonly used as indicators of dysfunctional liver. The analysis revealed up-regulated levels of ALT in the blood of moribund (dead) fish compared to normal (alive) fish in the two net pens of the test group (figure 8). Taken together, surprisingly overall negative effects of the test diet after changing to increased dietary fat content were detected at all R&D facilities during the last period in sea. This may indicate a reduced robustness of the fish given test-diet. In addition, observation in the R&D facility in the North during the winter indicated a so far unknown nutrition-related cause of mortality related to circulation failure. Especially, observation of fatty liver in salmon fed only the low fat test diet was very surprising. The negative effects are visualized in figures 4, 5 and 6.

Example 3 - A method to increase robustness and reduce unspecific mortality

Materials and methods

Rearing facilities, fish and dietary treatments The following study was conducted at a seawater research station at Ekkils0y on the west coast of Norway during the period May 201 1 to March 2012. The fish material used in the study were sampled from groups of sexually immature Atlantic salmon originating from the same population of post-smolts transferred to sea in July 2010 as S1 smolts with an average weight of 62 grams. The study embraced two periods: May to August 201 1 (referred to as the pre-dietary phase) and August 201 1 to March 2012 (main-dietary phase). In May 201 1 , the post-smolt were stocked into three pens (7 x 7 x 7 m) with 650 fish per pen. Prior to this, length and weight were recorded and Passive Integrated Transponders (PIT) tags were implanted into the abdominal cavity of each individual fish. Average bodyweight per pen was 1085 grams. Each pen received different dietary treatments: a normal high-fat diet (control-diet: 35% protein and 35% fat), a low-fat high protein diet (test-diet: 50% protein and 18% fat). In addition, one extra group received only half the daily ration of the low-fat high protein diet. The net-pens were fed the respective diets until August (during the pre-dietary phase).

In August 201 1 , the fish from the three net pens were redistributed, 50 fish from each net pen into 4 new net pens (5 x 5 x 5m), resulting in four net pens with 150 fish in each pen. In this way, all three pre-dietary groups were stocked together in each pen. From August 201 1 until March 2012, all four pens received the same dietary treatment (35% protein and 35% fat - referred to as the main-dietary phase).

Sampling The fish were sampled in May and August (30 fish in May, and 30 fish for each pre-dietary treatment in August) for general status of muscle fat level. During the main-dietary phase (August 201 1 - March 2012), the fish were sampled every second month (October, December and March). The PIT-tag was scanned and the weight and length of all fish in every pen were individually recorded at each sampling point. Thereafter ten fish from each pre-dietary group (control and test), representing the average weight of the groups were collected. Individual muscle samples (Norwegian Quality Cut, NQC) for each collected fish were then analysed for fat by digital image analysis according to Folkestad et al. (Folkestad, A., Wold, J. P., Rervik, K. A., Tschudi, J., Haugholt, K. H., Kolstad, K., & Morkore, T. (2008). Rapid and non-invasive measurements of fat and pigment

concentrations in live and slaughtered Atlantic salmon (Salmo salar L). Aquaculture, 280 (1 ), 129-135). All collected fish were anesthetized (MS 222 metacaine, 0.1 g L ^"1 ) and killed by a blow to the head.

Results

In line with the initial overall positive effects of the test diets with a high protein/low fat ratio detected at all R&D facilities of Nofima (Example 2 and summarized in table 2), a significant higher mortality (unspecific) among salmon administrated pre-diet with 35% fat compared to both groups fed 18% dietary fat were observed. It is seen from figure 9 that, irrespective of whether the salmon were fed full or half the daily ration during the pre- dietary phase at increasing daylight, grow out salmon fed low fat diets had significantly less unspecific mortality than the control group given normal high fat diets for salmon larger than 1 kilo during the pre-dietary phase. The pre-dietary feeding (May - Aug 201 1 ) significantly influenced the muscle fat content of the dietary groups, in an expected manner. The fish fed control diet had a significantly higher muscle fat content (16.4%) than the fish fed the test diet (13.2%) and the half- ration diet (1 1 .3%) at the end of the pre-dietary phase (see figure 10A).

After restocking these pre-dietary groups in the same net pens, marked with pit tag, they were fed the same commercial diet with 35% fat for 7 months from August until October. As clearly seen in figure 10A, already after two months feeding in early autumn the significant differences in fat were equilibrated. This indicates a total replenishing of fat stores in salmon given both test diets during the pre-dietary phase.

The muscle fat content did not increase further after October, and the muscle fat did not differ significantly between the fish given different pre-dietary treatments during the samplings in October, December and March (Figure 10A). This indicates a total replenishing of fat stores/reserves in fish given the test diet and the half-ration during the pre-dietary phase.

Although the salmon was exposed to some stress during sampling throughout the main- dietary period, total accumulated unspecific mortality was very low (24 of 650 salmon died), and no significant differences between the three pre-dietary groups were detected. This result, in contrast to the observed increased mortality for the pre-dietary groups during the main-dietary phase described in Example 2, strongly indicates that by changing the fat content of the feed in the early period of falling day length ensures a robust salmon throughout the whole grow out phase.

The significant reduction in bodyweight due to that only half ration was fed to the half ration pre-dietary group (Figure 10B) was surprisingly more than compensated for, relative to the control, after changing to a high fat test diet in early autumn (indicated by the vertical stippled line). The pre-dietary treatments did not have any negative effects on growth or bodyweight increase during the later main-dietary phase. On the contrary, a highly significant improved growth in the test fed salmon (TGC test=3.4, TGC ½ ration = 3.9 and TGC Control = 2.9) was observed throughout the whole main-dietary phase with a duration of 7 months (figure 1 1 A). The test fed fish had about 0.65 kg higher bodyweight gain compared to the control fed salmon (4306 vs. 3641 gram) during the 7 months. The ½ ration fed salmon had an improved bodyweight gain (4623 gram), i.e. an increase of almost 1 .0 kilo relative to the control (figures 1 1 A and B). Taken together, results from the half ration pre-dietary group surprisingly reveal that growth in the almost 7 months main- dietary grow out period was highly related to the fat content in the muscle of the salmon at termination of the pre-dietary period. From this it can be concluded that a robust fish is expected to have improved appetite, growth and survival.

Great variation in individual bodyweight at slaughter is a problem in commercial salmon farming. To reduce the variation salmon farmers tends to sort the salmon at least once during the grow-out period. In this experiment the group fed half the daily ration of test diet (0.5 Test), had as expected the highest dispersion in bodyweight at the end of the pre- dietary phase. However, at termination of the main-dietary phase in March, salmon fed the control diet during the pre-dietary phase ended up with a significantly higher dispersion in bodyweight compared to in both groups fed the test diet (Figure 12).

Surprisingly, this was observed despite of the fact that the test groups at even higher bodyweight compared to the control group (mean bodyweight >6 kilo). This shows that both test-groups had more homogeneous growth among individual salmon compared to the control group during the main-dietary phase, but only when they had been fed low fat test diets in the pre-dietary period.

Normally, when evaluating dietary effects on growth performance, analyses of nutrient and energy in the whole body of the salmon is examined. However, in the present study as the importance of the salmon reproduction strategy for growth and growth pattern was investigated, the fat stores in muscle and visceral tissues were analyzed separately.

Hence, the fat accumulation was analyzed separately in edible and non-edible parts of the salmon. The relation between, on one hand the fat content in the muscle in the three pre- dietary groups after increasing day length in spring, versus, on the other hand, the relative increase in fat percentage in non-edible (visceral) to edible (muscle) part of the salmon during the first month of falling daylight in the main-dietary autumn period (Aug-Oct) were investigated.

Surprisingly, it was clearly shown that the percentage of fat accumulation in the non- edible part, relative to the edible part, was strongly dependent on the fat content in the muscle at termination of the pre-dietary phase (Figure 13). Thus, e.g. to achieve higher levels of healthy (for human) omega-3 fatty acids in the edible part of the salmon and simultaneously maintain sustainability by having these important marine fatty acid profiles in the feed, is better attained by feeding low fat pre-dietary test diets in the spring.

Furthermore, increased fat deposition in internal organs, combined with overloaded fat cells, will evidently increase the risk of fatty liver, inflammatory diseases, increase risks for circulatory heart problems and, therefore, significantly decrease the robustness of the fish.

According to the present disclosure, the fat/protein ratio in the feed for Atlantic salmon influences the recruitment of fat cells. Fat-storage in salmon is strongly influenced by day length as visualized in figure 14 which shows changes in muscle fat of salmon during the autumn and winter period (M0rk0re, T., & Rorvik, K. A. (2001 ). Seasonal variations in growth, feed utilisation and product quality of farmed Atlantic salmon (Salmo salar) transferred to seawater as 0+ smolts or 1+ smolts. Aquaculture, 199(1 ), 145-157; Alne, H., Oehme, M., Thomassen, M., Terjesen, B., & R0rvik, K. A. (201 1 ). Reduced growth, condition factor and body energy levels in Atlantic salmon Salmo salar L during their first spring in the sea. Aquaculture Research, 42(2), 248-259). Salmon administered a lean diet in earlier stages of life develop fewer fat cells than salmon receiving a high-fat diet. If salmon fed the lean diet suddenly are administered a high-fat diet at a time of the year when they normally store less fat (late autumn), or are not storing fat at all (e.g. during winter, see figure 14), they will not be able to produce a sufficient amount of new fat cells. Hence, they will not have the capacity to store the sudden excess amount of fat in normal depots. Instead the excess fat will deposit in the liver.

Therefore, such dietary-change may lead to reduced growth, less robust salmon, and higher unspecific mortality during handling (Figure 4A and B). In extreme situations as in the R&D facility in Northern Norway during the wintertime, high mortality due to circulatory failure (similar to CMS) may also occur (Figures 5A and 6). However, here a test for CMS- virus was negative, indicating a so far unknown dietary related cause of mortality.

This new information strongly indicates that salmons fed a low fat diet, in a period in life where they normally deposit high amounts of lipids in white adipose tissue, will recruit less new fat cells than a fish fed a high fat diet. The fat cells available will be overloaded if the fish are fed high fat diets later in life. This phenomenon will lead to increased fat deposition in the internal organs, liver and heart. Increased fat deposition in the internal organs, combined with overloaded fat cells, will evidently increase the risk of inflammatory diseases and significantly decrease the robustness of the fish. If instead dietary-fat content is increased in periods when salmon normally deposits high levels of fat (e.g. early autumn, Figure 14), no increase in unspecific mortality in late autumn and during the winter will take place. This is most probably due to a substantial recruitment of fat cells during the early autumn. The present invention reveals that the timing when the different feeds (high fat or low fat diets) are administrated is of crucial importance. A low-fat diet is beneficial during the pre- dietary phase (spring in the Northern Hemisphere) to increase the robustness (increased survival during handling). However, if the salmon is administrated low-fat diet throughout the period of falling day length (early autumn in the Northern Hemisphere), the initial robustness gained during the pre-dietary phase is lost and the salmon is worse off than salmon fed a high-fat control diet later on in the grow-out-period. It is therefore crucial that the administration of a high-fat diet starts at the beginning of the main-dietary phase when the period of day length decreases at a steady rate (Example 3). If the timing of this phase fails, the salmon may end up less prepared to withstand later challenges, such as handling of stress and circulation failure etc. (see Example 2). A correctly timed switch to a high-fat diet will surprisingly increase the weight-gain (Figure 10B and 1 1 B) compared to fish having been fed a normal high-fat diet throughout the year. Taken together, the present inventions reveals the importance of dynamic feeding methods related to season to improve appetite, growth, robustness and welfare of the salmon during the whole production phase in sea.