EP2293688A2 - Balanced ara/epa ratio in salmon gill and kidney tissues to improve sea water performance - Google Patents

Abstract

The present invention describes a salmon smolt comprising an arachidonic acid (ARA) to eicosapentaenoic acid (EPA) ratio of one or greater, specifically in gill and kidney tissues. The invention discloses a feed additive containing ARA and its use in a sufficient amount to balance the excess amount of EPA contributed from the marine oils used in the standard feed for salmon. The invention also discloses a method to modulate the gill and kidney ARA/EPA ratio to the favor of ARA. Smolt of the present invention demonstrates superior growth recovery and stress resistance after their sea water transfer.

Description

BALANCED ARA/EPA RATIO IN SALMON GILL AND KIDNEY TISSUES TO IMPROVE SEA WATER PERFORMANCE

CROSS REFERENCE TO RELATED APPLICATION

[001] This application claims priority to U.S. Provisional Application No: 61/052,802 filed in the United States Patent and Trademark Office on May 13, 2008, the contents of which are hereby incorporated by reference herein for all purposes.

BACKGROUND OF THE INVENTION

Field of the Invention

[002] The present invention relates to salmon smolt feeding, and more specifically, to the enhancement of growth performance of the salmon smolt when transferring from fresh water hatcheries to salt water environment by introducing a feeding regime with increased levels of arachidonic acid (ARA) prior to such sea water transfer to increase the ratio of ARA to eicosapentanoic acid (EPA) to at least 0.9 in gill and/or kidney tissue of the salmon smolt at the stage of sea water transfer.

Background of Related Art

[003] A critical stage of current salmon farming practice is the transfer of smolts from freshwater hatcheries to ocean net pens when they have attained a critical size of approximately 70-110 grams body weight (for reviews see: (Eddy 1981; Bley and Moring 1988; Bakke, Bjerknes et al. 1991; Jonsson, Hansen et al. 1993). The newly placed smolt in the ocean net pens do not grow optimally during their first 40-60 day interval in seawater because of the presence of osmotic stress that delays their feeding (Salminen, Erkamo et al. 2001). This also results in a higher susceptibility to pathogens and a reduction in fish immunocompetence (Barton and Iwama 1991), leading to an increase in mortality from disease. The reduced growth performance demonstrated by smolts, following their saltwater entry, also affects final harvest weight and timing as well as their resistance to disease. It is, therefore, valuable for the smolt to have a high level or resistance to a chronic exposure of osmotic stress in order to survive the first two months of life in seawater.

[004] In smolt-rearing farms, it is vital that smoltification is accurately monitored so corrective action can rapidly be taken as stress-induced needs are identified. Current methods used in the industry simply assure that all fish have undergone the process and have made all the necessary changes to successfully transfer to the sea, after achieving a critical size. This management method is time consuming and expensive in that: 1) it requires frequent sampling and testing of the smolts for their 24-48 h survival in seawater; 2) a significant number of smolts do not attain a critical size for a timely seawater transfer and are left in freshwater for transfer the following year; and 3) the presence of a narrow transition window, together with delays in achieving optimal smolt growth, prolongs the grow out interval needed to obtain market size.

[005] The use of timing and size cut-offs as exclusive methods for smolt transfer is simple, but cannot guarantee high quality smolts, since the fish still have a limited ability to maintain homeostasis after the abrupt transfer to seawater without an adaptation period. Other salmonids, such as trout, are much less tolerant to abrupt transfers from freshwater to seawater as compared to juvenile Atlantic salmon. As a result, many commercial seawater trout producers transfer their fish to brackish water sites located in estuaries or downstream river openings instead of the full strength seawater present in standard ocean net pens. Trout are then transferred to more standard ocean net pen sites to complete their grow-out cycle at a later date after an adaptation period.

[006] The smoltification process in salmonids has received a lot of attention as an important factor in the quality of hatchery-reared salmonids, since their survival and growth in the marine environment depends on successful smoltification (Iwama 1992). Although several environmental factors (e.g., water temperature, photoperiod, etc.) and biological factors (e.g., size, health, etc.) affect smoltification and subsequent marine survival (Zaugg and Beckman 1990; Shrimpton, Bernier et al. 1994), very little is known about the effects of dietary essential fatty acids on smoltification in salmonids (Sheridan, Woo et al. 1985). [007] During the natural process of a salmon's migration from freshwater to seawater, the fish slowly develop a heightened osmoregulatory capability that increases salinity tolerance and preference. An influx of salts from seawater causes an increase in sodium levels in the salmon, which triggers the secretion of the steroid hormones, thyroxine and growth hormone (Olsen, Reitan et al. 1993). These cause an increase in the number of chloride cells available to transport salts across the gills to the exterior (Uchida, Kaneko et al. 1997). These changes, which take place over a prolonged interval of osmotic adaptation in wild smolts, initiate increased activity of the NaVK⁺- ATPase (a key enzyme of ion transport), as well as increased salt secretion necessary to counter-balance the large intake of sodium from drinking and eating in the seawater environment. In fact, gill Na⁺/K⁺-ATPase is often analyzed to assess the level of smoltifϊcation in juveniles (Zaugg 1982). Fish that are not transferred from freshwater to seawater at the proper size or after a proper acclimation period are sluggish, potentially increasing their susceptibility to disease and predation.

[008] Heat shock proteins (HSPs) have been implicated in adaptation to hyperosmotic stress in Atlantic salmon (Smith, Tremblay et al. 1999). HSPs are ubiquitous and highly conserved across the plant, animal, and microbial kingdoms. Their induction is not limited to heat shock, but includes other cellular insults such as oxidative stresses, nutritional deficiencies, UV irradiation, chemical insults, and viral infections (Pockley 2001). There are several families of inducible HSPs (generally categorized on the basis of molecular weight), as well as constitutive Iy expressed HSPs (molecular chaperones), involved in the normal assembly and folding of oligomeric proteins (Pockley 2001). By implication, there is a general consensus that HSPs are involved in either protection of certain fundamental cellular processes or the repair of cellular damage.

[009] Although the induction of HSPs may involve multiple pathways and regulatory factors, there is increasing evidence that products of arachidonate metabolism play a significant role in the regulation of gene transcription (Jurivich, Sistonen et al. 1994; Jurivich, Pangas et al. 1996). Polyunsaturated fatty acids, particularly ARA and docosahexaenoic acid (DHA), have also been found to be potent modulators of HSPs response in rainbow trout leukocytes (Samples, Pool et al. 1999). Nevertheless, relatively little attention has been paid to aspects of lipid and essential fatty acid nutrition in relation to smoltification and their effect on growth and survival after the saltwater transfer.

[0010] Thus, it would be advantageous to develop an enhanced salmon smolt and a method of feeding such salmon smolt and/or parr prior to and during smoltification to increase ability to survive such transition from fresh water environment to the salt water environment.

SUMMARY OF THE INVENTION

[0011] The present invention provides for farmed raised salmon smolt using standard aquaculture practices and further defined by a specific fatty acid composition of the gills of the raised salmon smolt that comprise an ARA/EPA ratio of greater than 0.9 and that subsequently grow faster and are more resistant to stress associated with the transfer from fresh to sea water.

[0012] One aspect of the present invention provides for a method of feeding farm raised salmon smolt and/or parr to prepare same for the transition from a fresh to salt water habitat, the method comprising: providing a smolt/parr feed composition comprising ARA and EPA wherein the ratio of ARA to EPA is at least 0.2; and administering the feed composition to the smolt/parr for at least 7 weeks prior to the transfer from fresh to sea water habitat, and more preferably from about 10 to 15 weeks prior to such transfer thereby providing for smolt salmon having an ARA/EPA ratio of greater than 0.9 in gill and/or kidney tissue at the time of transfer.

[0013] Such feed may comprise conventional ingredients well known to those with experience in salmon aquaculture and preferably contain ARA at levels of from 2% to 5% of total fatty acids while maintaining EPA levels from 5% to 10% of total fatty acids. The feed composition may comprises, fish meal, fish oil lipid components, and/or microalgal oil or microalgal biomass containing long chain fatty acids such as ARA, EPA and DHA. Further, the feed comprises a DHA/EPA ratio of greater than 0.5. [0014] Another aspect of the present invention provides for a feed wherein the proportion between ARA and EPA in the lipids is sufficient to increase the ARA proportion in gill and kidney tissues of smolt to at least a level that matches the levels of EPA.

[0015] Yet another aspect of the present invention provides for a method to increase levels of heat shock proteins for enhanced adaptation of hyperosmotic stress in smolt salmon after transfer from fresh to salt water habitat, the method comprising: providing a smolt/parr feed composition wherein the feed composition comprises ARA and EPA and wherein the ratio of ARA to EPA is at least 0.2, and more preferably a ratio of at least 0.5 to 2; and administering the feed composition to the smolt/parr for at least 7 weeks prior to the transfer from fresh to sea water habitat, and more preferably from about 10 to 15 weeks prior to such transfer thereby providing for smolt having at least twice the levels of heat shock proteins relative to smolt consuming a feed lacking a ratio of ARA to EPA of at least 0.2.

[0016] A still further aspect of the present invention relates to a method of feeding smolt salmon before transfer of the smolt salmon from fresh to salt water habitat, the method comprising: providing smolt salmon having a fish weight of about 30 g; feeding the smolt salmon a feed containing a balanced proportion of ARA/EPA of at least 0.2 for at least 4 weeks prior to the transfer of from fresh to salt water habitat or until the smolt salmon reaches the weight of 100 g.

[0017] In an alternative method, the transferred smolt salmon are fed the feed containing a balanced proportion of ARA/EPA of at least 0.2 for additional 30 days after the sea water transfer.

[0018] Another aspect of the present invention relates to a smolt salmon having a weight of approximately 80 to 110 grams and having an ARA/EPA ratio of greater than 0.9 in gill and/or kidney tissue. Preferably, the gill and/or kidney tissue is tested for an ARA/EPA ratio level greater than 0.9 in the 10 day period before transfer from the fresh to salt water environment.

[0019] Yet another aspect relates to a feed comprising fish meal or fish oil substantially balanced by the addition of ARA.

[0020] Other aspects and advantages of the invention will be more fully apparent from the ensuing disclosure and appended claims

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Definitions

[0022] The term "smolt" is used to describe a salmon fingerling that is cultured in fresh water and is about to transfer to seawater.

[0023] The term "parr" is used to describe a salmon fingerling that is undergoing physiological changes that result in visible morphological and behavior changes known as smoltification.

[0024] Salmon aquaculture typically involves the raising of salmon fry in freshwater followed by their transfer to sea water where they are grown to a commercial size on diets containing predominantly fish meal and fish oil. During the freshwater to sea water transition (i.e., smoltification), the fish are under a high degree of stress and grow poorly and are susceptible to disease.

[0025] Tissues of the salmon have an osmolality of about 5-7 parts per thousand (ppt). In conventional aquaculture practice (and in the wild) salmon eggs are hatched in fresh water (ca. 2-4 ppt) and the salmon fry matures into a parr that then must go through the smoltification process of gradual increases in salinity up to full strength seawater (ca. 25-28 ppt) prior to stocking seawater pens for the grow-out phase of the commercial process. This transition period is very stressful on the fish and results in reduced growth rates and increases in disease susceptibility. Conventional salmon aquaculture feeds are rich in fish oil (ca. up to 25% by weight) which itself is rich in EPA. The present invention has shown that surprisingly the addition of a certain long chain omega-6 lipid, that being ARA, to the diet of pre-smolt fish allows these animals to pass through this process with a minimal impact on growth and disease sensitivity. The net result is a significant improvement of growth and shortening the time to reach market size. Such an invention can significantly improve the economics of commercial salmon production.

[0026] One conclusion from these studies could be that the larval Cortisol level is more sensitive to tissue ARA than DHA during salinity changes. The accumulating findings suggest that the array of eicosanoids produced from dietary ARA plays a major role in modulating HSPs induction, Cortisol synthesis and Na⁺/K⁺-ATPase activity during the stress response, and therefore could be a central factor influencing post-stress growth and survival. However, the positive or negative modulation of dietary and tissue ARA during smoltification, and in particular during smolt transfer to seawater, has never been tested.

[0027] ARA can be produced by the elongation and desaturation of omega-6 fatty acid precursors such as linoleic acid. EPA can be produced by the elongation and desaturation of omega-3 precursors such as linolenic acid. However, the enzymatic conversion of omega-6 precursors to ARA is antagonized by long chain omega-3 fatty acids, such as EPA and its eicosanoid derivatives (Bell, Castell et al. 1995). Similarly, the enzymatic conversion of omega-3 precursors to EPA is antagonized by ARA and its eicosanoid derivatives. Thus, complex interactions exist between fatty acids and the metabolic pathways that determine eicosanoid biosynthesis in regulatory tissues (Bergstrom 1989; Bessonart, Izquierdo et al. 1999; Sargent, Bell et al. 1999; Tocher, Bell et al. 2000).

[0028] One of the long chain omega-3 fatty acids, DHA, plays a key structural role in biomembranes of nerve and muscle cells and is critical for the optimal functioning of the animal. A number of papers have reported on the effect of long chain omega-3 fatty acids, particularly DHA, on stress resistance (Kanazawa 1997; Tago, Yamamoto et al. 1999; Harel, Gavasso et al. 2001). However, as no eicosanoids are derived from DHA (i.e., eicosanoids are 20-carbon molecules and DHA is a 22-carbon molecule), its specific role in Cortisol synthesis remains unclear. On the other hand, several studies have demonstrated the clear role of ARA in modulating fish responses to stress. Studies have reported that ARA was preferentially retained in various species together with DHA during starvation, suggesting an important metabolic priority for conserving these fatty acids (Rainuzzo, Reitan et al. 1994; Izquierdo 1996). In turbot, dietary deficiencies in ARA resulted in high mortality and obvious pathology (Bell, Henderson et al. 1985), while Castell and co-workers (Castell, Bell et al. 1994) reported a positive effect of ARA on survival. Bessonart and co-workers (Bessonart, Izquierdo et al. 1999) found that ARA was more effective in improving survival of gilthead seabream larvae if provided in the presence of a high dietary DHA/EPA ratio. Koven and coworkers (Koven, Van Anholt et al. 2003) showed that dietary ARA fed to gilthead seabream larvae from 3-19 days after hatching markedly improved survival following the acute handling stress during transfer from rearing tanks to aquaria. Moreover, feeding ARA prior to handling was much more effective in improving post-stress survival compared to feeding this fatty acid following this stress event. This suggests that the larvae previously fed ARA had an advantage derived from a pre-existing store of tissue ARA. Finally, it has been reported that wild salmon smolts contain much higher proportions of ARA in their total lipids compared to aquacultural-produced smolts (Ackman and Takeuchi 1986; Bergstrom 1989).

[0029] The present invention with the results shown herein provide evidence that when the ratio of the essential fatty acids ARA and EPA in gill and kidney tissues are greater than 0.9, the fish can more efficiently pass through the smolting process. Further, the present invention shows that this fatty acid balance can be modulated only by the addition of dietary ARA and in a preferred embodiment this is in conjunction with the provision of dietary long-chain omega-3 fatty acids in a condition where the maximal amount of DHA can be provided with the minimum of EPA (i.e. DHA/EPA ratio greater than one).

[0030] Sources of ARA and its esters may include ARA-containing microbial oils or biomass (Barclay 2008), ARA containing algal oils or biomass (Kyle 2005). ARA extracts from animal byproducts such as brain or liver (Abril and Wills 2008), ARA from recombinant plants or microbes (Chen 2008) or ARA from egg yolk. Sources or DHA and its esters that have minimal levels of EPA and its esters include micoralgal oils and their biomass (Bailey, Dimasi et al. 2008), recombinant microbial products (Chen 2008; Metz, Weaver et al. 2008), recombinant plant products (Mukerji and Pereira 2008) and certain natural fish oils (e.g., tuna eye socket oil) (Bakkene, Nordvi et al. 2007) or fish oils that have been processed or modified to increase the DHA/EPA ratio (Fabritius, Reimann et al. 2008).

[0031] EXAMPLES

[0032] Example 1. Preparation of salmon smolt feed containing ARA/EPA ratio of at least 0.2.

[0033] A standard commercial diet for smolt (commercially available as starter feed from Ziegler Bros, PA with 2 and 3 mm pellets) was analyzed for its EPA content. Then the same starter feed was enhanced according to the methods of the present invention by top coating the pellets with ARA oil (40% ARA, Martek BioSci. Columbia, MD) to adjust the ARA/EPA ratio in the diet to at least 0.2. Table 1 provides the fatty acid composition (% of total fatty acids) of a standard commercial diet and diet of the present invention.

TABLE 1

Diet of the present

Fatty Acid

Standard diet invention

C16:l 5.2 5.3

C17:0 0.4 0.5

C17:l 0.1 0.1

C18:0 5.1 5.4

C18:ln9 27.1 24.6

C18:2n6 (LNA) 12.0 11.4

C18:3n3 1.7 1.6

C20:0 0.2 0.3

C20:ln9 1.3 1.4

C20:3n6 0.0 0.0

C20:4n6 (ARA) 0.5 2.1

C20:5n3 (EPA) 7.5 7.8

C22:ln9 1.3 1.3 C22:5n6 0.0 0.0

C22:5n3 0.6 0.6

C22:6n3 (DHA) 5.0 4.9

C24:ln9 0.0 0.0

Unknown 7.7 7.8

ARA/EPA 0.07 0.27

[0034] Example 2. Feeding schedule of salmon smolt the feed of the present invention.

[0035] Each diet was assigned to two tanks containing 120 fish in each starting average size of 30 g. Smoltification was induced when the fish reached an average weight of 85g. Fish were then transferred to sea water and continue feeding for additional 4 weeks. Table 2 shows the ARA and EPA levels in salmon smolt at the stage of sea water transfer that fed standard diet or diet of the present invention.

TABLE 2

Fatty Acid Gill Kidney

Diet of the present C20:4n6 (ARA) 6.7±0.6 6.6±0.8 invention

C20:5n3 (EPA) 5.4±0.2 6.6±0.4

Standard diet C20:4n6 (ARA) 4.0±0.5 3.1±0.5

C20:5n3 (EPA) 5.6±0.7 7.8±1.1

[0036] Example 3. Growth of salmon smolt fed the diet of the present invention in sea water.

[0037] Growth of Smolt fed on standard diet or the diet of the present invention were continue to monitor for 3 month after the sea water transfer. Fish were initially started to feed on the diet in fresh water until they reached an average size of 85g then transferred to seawater for additional 90 days observation. Table 3 show the size of the fish when experiment terminated.

TABLE 3 Diet of the present 139.8 g (a) invention

Standard diet 117 g (b)

[0038] Example 4. The effect of the diet of the present invention on expression of heat shock protein 90.

[0039] Analysis of Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) was used to assay the quantity of transcripts for hsp90 in branchial lamellae. PCR primers were designed using sequences in the public domain to Atlantic salmon (Salmo salar) hsp90 Accession # AF135117). Validation of the primers and optimization of amplification conditions were conducted by standard PCR and nucleotide sequencing of the resulting amplicons. Table 4 shows the relative quantities of Hsp90 in branchial lamellae of juvenile Atlantic salmon fed standard diet or diet of the present invention 24 h after transfer to seawater. Values Hsp90 Diet 1 was arbitrarily set to 1; all other values are relative to these values.

TABLE 4

Diet of the present 2.4 invention

Standard diet 1

[0040] References:

[0041] The contents of all references cited herein are hereby incorporated by reference herein for all purposes.

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Claims

ClaimsThat which is claimed is:

1. A method of feeding farm raised salmon smolt and/or parr to prepare same for the transition from a fresh to salt water habitat, the method comprising: providing a smolt feed composition comprising ARA and EPA wherein the ratio of ARA to EPA is at least 0.2; and administering the feed composition to the smolt/parr prior to transfer from the fresh to salt water habitat to provide smolt salmon having an ARA/EPA ratio of greater than 0.9 in gill and/or kidney tissue at the time of transfer.

2. The method of claim 1, wherein the feeding of the smolt with the smolt feed composition comprising ARA and EPA wherein the ratio of ARA to EPA is at least 0.2 for at least 7 weeks prior to the transfer from fresh to sea water habitat.

3. The method of claim 1, wherein the feeding of the smolt with the smolt feed composition comprising ARA and EPA wherein the ratio of ARA to EPA is at least 0.2 for at least 10 to 15 weeks prior to such transfer.

4. The method of claim 1, further comprising feeding of the smolt with the smolt feed composition comprising ARA and EPA wherein the ratio of ARA to EPA is at least 0.2 for about 30 days after the transfer.

5. The method of claim 1, wherein the feed further comprises a DHA/EPA ratio of greater than 0.5.

6. The methods of claim 1, wherein the feed contains ARA at levels of from 2% to 5% of total fatty acids while maintaining EPA levels from 5% to 10% of total fatty acids to provide the ratio of ARA to EPA of at least 0.2.

7. The method of claim 1, wherein the feed composition further comprises fish meal, fish oil lipid components, and/or microalgal oil or microalgal biomass.

8. The method of claim 1, wherein the method further comprises increasing levels of heat shock proteins in smolt salmon for enhanced adaptation of hyperosmotic stress after transfer from fresh to salt water habitat, wherein the smolt has at least twice the levels of heat shock proteins relative to smolt not consuming the feed with a ratio of ARA to EPA of at least 0.2.

9. The method of claim 1, wherein the smolt weigh about 30 g and are administered the feed until the smolt salmon reach the weight of 100 g and then transferred from the fresh to salt water habitat.

10. A farmed raised salmon smolt comprising gill tissue and/or kidney tissue comprising an ARA/EPA ratio of greater than 0.9 and that subsequently grow faster and are more resistant to stress associated with the transfer from fresh to sea water habitat.

11. The farmed raised salmon smolt of claim 10, wherein the levels of ARA in the gill and/or kidney tissue is at least equal to the levels of EPA.

12. The farmed raised salmon smolt of claim 10, wherein the gill and/or kidney tissue is tested for an ARA/EPA ratio level greater than 0.9 in the 10 day period before transfer from the fresh to salt water environment.

13. A feed composition wherein fish meal and fish oil lipid components have been substantially balanced by the addition of ARA, wherein the proportion between the ARA and EPA is adjusted to at least 0.2.

14. The feed composition of claim 13, wherein the ARA is at levels of from 0.5% to 5% of total fatty acids while maintaining EPA levels from 5% to 10% of total fatty acids and a DHA/EPA ratio of greater than 0.5.

15. The feed composition of claim 13, wherein the proportion between ARA and EPA in the lipids is sufficient to increase the ARA proportion in gill and kidney tissues of smolt to at least to match that of the EPA.

16. The feed composition of claim 13, wherein the feed comprises a balanced proportion of ARA/EPA of at least 0.2 thereby providing a salmon smolt comprising a fatty acid composition of the gills that comprises an ARA/EPA ratio of greater than 0.9.

17. The feed composition of claim 13, wherein the feed is administered to the smolt beginning at least 7 weeks prior to transfer of the smolt from fresh to sea water habitat.

18. The feed composition of claim 13, wherein the salmon smolt contain an ARA/EPA ratio of at least 1 at the stage of seawater transfer.

19. The feed composition of claim 13, wherein the feed is administered to the smolt salmon beginning at fish weight of about 3O g.

20. The feed composition of claim 17, wherein the feed is administered to the smolt salmon for additional 30 days after the sea water transfer.

21. The feed composition of claim 19, wherein the feed is administered to the smolt until the smolt reaches a weight of about 100 g.