Nucleotides

Despite the accelerating knowledge in understanding metabolism and nutrition and the considerable improvement in farm management in aquaculture, financial losses due to diseases still strike producers severely everywhere in the world. Often the reasons for the outbreak of diseases are not obvious but increased mortalities or reduced meat quality are at least in part triggered by stressful husbandry, overstocking, poorly adjusted feed levels or inferior feed, as well as unsatisfactory water conditions. In land animals it has been shown, that stress has a direct impact on molecular and cellular processes. This affects the overall constitution as well as the immunity of animals. Stress suppresses the primary defence against pathogens and increases susceptibility to bacterial, viral or parasitic infections. The animal’s natural drive to cope effectively with health challenges for its survival reduces performance and productivity.

These observations in land animals apply equally to aquaculture. The immunity of fish is closely related to that of other vertebrates although, in fish, innate mechanisms contribute significantly more to the immune response than the specific or acquired responses. Innate immune response includes the stimulation of phagocytic cells as well as different defence proteins. The acquired immune response is a memory-based reaction of specific cells triggered during or after an initial contact with a bacterial, viral or parasitic antigen. In crustaceans a very basic, innate immune response is established comprising different agglutinating proteins and other, molecular and cellular, responses without a recognisable memory component.

Within the last few years the progress that has been achieved in good husbandry practices in aquaculture has, at least to some degree, decreased the risk of outbreak of epidemics. Nutritionists, veterinarians and last, but not least, farmers strive to maintain the highest standards of feed quality and farm management alongside targeted health management procedures to minimise the risk of epidemics. The use of antibiotics or other chemicals in aquaculture is minimised due to the expense to the producers, the potential reactions of consumers and the consequences for the environment. Moreover, these compounds may lead to impaired growth of the animals as well as encourage the spread of drug resistance.

This has led to discussions on ways of maximising the immunocompetence of stocks while simultaneously minimising the use of therapeutic chemicals. An increasing number of feed additives claiming to enhance health in a general way by supporting and enhancing immunity has appeared on the market. Probiotics, prebiotics and the combination of both, the so-called “synbiotics”, have been extensively studied in terrestrial animals. Although being fairly effective in livestock, the effects at the moment are still questionable for fish and crustaceans because of the lack of scientifically approved studies.

All living organisms are semi-closed chemical systems only; they are not closed to the surroundings and are therefore exposed to changes in the environment as well as to the challenges of various pathogens. To operate properly they need to produce energy and different kinds of bioactive molecules. These are assembled or synthesised in specialised cells or organelles within these cells. This so-called cellular metabolism includes the uptake of raw materials or nutrients, the building of cellular components, converting molecules and releasing by-products. All vital functions of an organism occur within cells and all cells contain the hereditary information necessary for regulating cell function and for transmitting information to the next generation of cells. It would be expected that organisms can produce all essential, smaller molecules and macromolecules themselves. Food or feed would be required merely to supply sufficient energy to cover all the biochemical reactions executed in every single cell thereby maintaining the functionality of the organism. However it has been shown that this is only partially true. Although more or less self-sustaining, there are certain specific nutrients that must be acquired from the diet in order to maintain functionality and optimise performance. This class of nutrients, required for normal systemic functioning, but which cannot be synthesised by the organism, are classified as “essential” nutrients. Different species have different essential nutrients. Nutritionists recognise the relevance of certain feed ingredients and the importance of, for example, trace elements, vitamins, fatty acids or amino acids in a finished feed in livestock or aquaculture, is not questioned. Diets lacking amino acids for instance, will never lead to satisfying performance or stabilised health of animals in agriculture or aquaculture. Additionally, some compounds that are synthesised under normal conditions may cease to be manufactured in adequate amounts or at an appropriate rate under other conditions such as stress and therefore need to be acquired from the diet, partially (semi-essential) or wholly (essential). Only feeds containing a balance of essential as well as semi-essential nutrients facilitate systemic functions to assure high performance and allow an appropriate handling of health challenges with reduced or no medication.

For years, nucleic acids and nucleotides were not considered essential nutrients for use in any dietary programmes. It was thought that all organisms were able to supply sufficient amounts of nucleotides to meet their physiological demands via de novo synthesis or the so called “Salvage Pathway”, a recycling of nucleotides from dead cells (Fig 1). However scientific research has discovered remarkable and measurable benefits from supplementing RNA/nucleotides in a diet. Because of the central and key role played by nucleotides in cell metabolism, changes in their concentrations and availability to cells, particularly where the levels are sub-optimal, can have very far reaching, multifaceted effects on metabolism. For example it has been shown in various trials that formulations of RNA/nucleotide-based feed additives may ameliorate disease, accelerate immune responses, reduce mortality during stressful periods, reduce parasite infestations, improve reproductive output, improve feed conversion and increase growth in livestock and aquaculture.   

Fig 1: The de novo synthesis of nucleotides takes numerous biochemical steps. Precious raw materials are used to synthesize nucleotides. The synthesis taxes the organism’s energy resources and takes time. Although faster, the efficiency of the salvage pathway is a weak point. At least 25% of the deficit in nucleotides needs to be covered by de novo synthesis. When suitably purified, dietary RNA/nucleotides are immediately available for the organism and are transported via the blood stream to organs or cells with high demand for nucleotides.  

For a single cell to divide, it is necessary first to duplicate its DNA which contains the genetic construction plan and the operating instructions for the diverse functions. The gene pool in a cell contains approximately three billion nucleotides. However animals must produce millions of new cells every second in order to simply maintain the status quo.

Most of the cells of the body are capable of producing sufficient nucleotides to maintain a satisfactory supply to the organism for normal metabolic activities and life. For a healthy animal or human, this constant re-supply of nucleotides is very well balanced and is appropriately adjusted in response to occasional stress by overproduction of these molecules by the liver. However, the increased production of nucleotides takes time and energy and stresses the body’s supply of basic raw materials to produce more nucleotides. The internal production of nucleotides is determined by evolution and is based on average requirements with allowances for occasional short-term increase. However, during times of extraordinary stress, such as rapid growth, reproduction, environmental change, combating disease and recovery from injury, trillions of additional nucleotides must be readily available for cell proliferation. However, since the organism must first produce these nucleotides, this continual process is slow, metabolically taxing and reduces performance..

Under intensive farming conditions a degree of stress is always present in the farmed populations; it is a permanent condition and continually poses a threat to health. Stress decreases the replication of special and crucial white blood cells and therefore negatively interferes with the body’s natural immune defence. Under times of high demand, the accelerated need for nucleotides has to be met either by internal synthesis or salvage, which are insufficient in most cases, or from external sources such as the diet. The external supply of nucleotides is of utmost importance for, for example, cells of the immune system, gastrointestinal cells or blood cells since these cells are only partially capable of producing nucleotides or lack the potential to synthesize them at all.

Nucleotides have universally valid, essential physiological and biochemical functions including encoding and deciphering genetic information, mediating energy metabolism and cell signalling as well as serving as components of coenzymes, allosteric effectors and cellular agonists. Dietary nucleotides need to be absorbed through the intestinal wall before they become available for the organism. Some parts are enzymatically metabolised to facilitate the passage from the intestinal lumen into the blood stream. Nevertheless, the reassembly of nucleotides in the intestinal tract requires less time and energy compared with de novo synthesis. Interestingly, nucleic acids found in many feed and food ingredients are protected by specific binding proteins which allow only about 30% of them to be nutritionally available for the organism. By purifying RNA and single nucleotides, the protective proteins are eliminated, which allows the formulation of uniquely balanced feed additives with a nucleotide availability of over 95%. This can provide well proportioned dietary requirements for developing animals in terms of purified nucleotide and nucleic acid content.

Fig 2: The general uptake of nutrients occurs in the small intestine. The chemical and enzymatic decomposition and metabolism of nutrients starts in the stomach. This is suitable for most essential and semi-essential as well as all other nutrients. The degradation of cellular material to release nucleotides from the cell nucleus on the other hand is inefficient. Dietary purified RNA/nucleotides can easily be absorbed in the small intestine to serve cellular processes.

One of the reasons for the success of RNA/nucleotide-supplementation of diets is that the response of the immune system is accelerated because the proliferation of the cells involved is facilitated. The immune response simply is the ability to establish an effective defence against pathogens by either producing specific antibodies or stimulating phagocytic cells as well as the expression of different kinds of defence proteins. Acquired immune response comprises memory-based reactions of specific cells in the organism which are assembled during or after the initial contact of the animal with an antigen (bacterial, viral or parasitic). In crustaceans, a very basic non-specific immunity consisting of various agglutinating proteins involved in inflammatory-type of reactions is found. In general, the immune response is activated through the production of millions of specialized cells. This process as well as the activation of specific protein expression is accelerated, when more nucleotides are made available. A strong immune response is moreover equally important to respond to stress factors, such as injuries, environmental changes, physical exertion and growth. Every pressure taxes the immune system and the ability to survive or adequately react to changes during lifetime. 

The benefits of RNA/nucleotide supplemented feed on performance and general health as well as development of young animals was tested in numerous trials in agriculture and aquaculture. In the following, some remarkable trials in aquaculture are described showing the positive effects of RNA/nucleotide supplemented feed aligned with the effects observed.  

The nature of the mode of action attributed to RNA/nucleotides in the immune function of animals would be expected to be manifested in greater resistance and therefore better survival rates in the face of disease under experimental and field conditions when diets containing nucleotide supplements are used. A number of workers have reported similar findings in several fish species.

The performance of RNA/nucleotides in fish artificially infected with IPNV was tested by Leonardi et al although the number of fish in the trial, primarily designed to study stress parameters, was small. Eight rainbow trout, fed RNA/nucleotides for 60 days and 8 fish on standard diet (Controls) were challenged by IP injection with virulent IPNV. All of the Control fish died within 8 days; all fish on the RNA/nucleotide diet survived. Such a trial would need to be repeated on a larger scale and allowed to proceed for a longer time to quantify the level of protection that might be expected but, even so, this is an indication that the immune system was substantially affected by the diet used.

Sakai et al (2001) used clearance of bacteria from the blood, liver and kidney as an indicator of resistance to infection. Common carp were intubated with nucleotide suspensions and challenged, intraperitoneally, with Aeromonas hydrophila. In the RNA/nucleotide treated fish no bacteria were detected after 12 hours whereas in the non-treated fish the bacteria reached a titre of 10³ cfu*ml^-1 in the same time.

Protection against viable challenge with Streptococcus iniae was reported by Li et al (2004) in juvenile, hybrid striped bass:

 

The most detailed work to date in fish has been carried out by Burrells et al (2001).The data from a series of viable challenge trials reported in their work, are summarised below:

 

None of the fish used in these trials was vaccinated; the effects seen are solely from the incorporation of RNA/nucleotides in the diet. It is therefore clear that in a number of commercially important species, over a wide range of diseases (bacterial, viral and parasitic) and under different trial conditions, the addition of nucleotides to the diet of fish significantly improves their ability to resist disease.

2. Immune Responses.

Sakai et al (2001) described a number of effects of nucleotides on the innate (non-specific) immune system. Components of the alternative complement pathway, lysozyme activity, phagocytosis and anion production in the head kidney of carp were all increased although Burrells (2001) failed to show any effect on the oxidative burst in the head kidney of Atlantic salmon. Detailed work by Low et al (2003) in turbot recorded a decrease in lysozyme activity in some organs, including the kidney, but a significant increase in the amount of Interleukin1-β produced. The picture therefore varies between species of fish and probably under different experimental conditions. However it is clear that RNA/nucleotides do have a significant effect on these important components of immunity. 

Lymphocyte activity in general has been found to be higher with a RNA/nucleotide supplement but the most pronounced effect is on immunoglobulin production. The work of Ramadan and of Burrells, reported in sections below, indicates enhancement of antibody production and this is reflected in enhanced protection. The precise mechanism of this enhancement has not been clarified in fish but the inability of immune cells to produce purines efficiently, essential when disease challenge requires rapid multiplication and differentiation, is one strong possibility in explanation. 

The release of cortisol under conditions of stress has marked inhibitory effects on a number of important physiological pathways related to disease response. It may result in immunosuppression, reduced feed intake, reduction in growth rates and increased susceptibility to disease (Anderson, 1996). The work of Burrells showed that exogenous RNA/nucleotides were very effective in maintaining the health of young salmon (smolts) during the stressful period of transfer to seawater and deduced that a reduction in cortisol levels was the likely cause of this reduction in stress. Growth and survival in smolts maintained on a supplemented diet around the period of transfer was significantly better than controls. The proof of this was established by Leonardi et al (2003) who showed that salmon, stressed by infection with IPNV, had lower cortisol levels than control fish that were not fed exogenous RNA/nucleotides. Although the mechanism through which the nucleotides function in this way is not clear, it is clear that supplementary feeding with RNA/nucleotide enriched diets is an important means of reducing stress both at sea-transfer and in other stressful on-farm processes such as ectopic treatments and vaccination. 

Crustacea, such as shrimp, have an immune system that is primarily, if not entirely, dependent on those mechanisms which fall into the innate immunity category. Although words such as ‘vaccine’ are occasionally used for simplicity, the two basic requirements of the immune system that allow that terminology to be used validly, the presence of antibodies and the induction of  immune memory, are absent.

For this reason compounds that are used widely in shrimp to stimulate the immune mechanisms, including those referred to as ‘vaccines’, function either as immunopotentiators or nucleotides supplements.

Experiments and trials with RNA/nucleotides have established both protection against disease as well as enhanced growth rate and they are used increasingly in shrimp husbandry. However the collection of data in large-scale trials with shrimp is notoriously difficult and many data do not stand up to rigorous statistical analysis. Also not all nucleotide products perform equally well in these functions as shown in trials where a direct comparison has been made.

Early, unpublished data from laboratory trials by Appelbaum indicated that growth in Litopenaeus vannamei was markedly increased by regularly feeding with nucleotides and also survival in the face of infection with Whitespot virus.

Under field conditions in Thailand both weight and survival benefits were reported in shrimp trials that were conducted over the complete shrimp cycle of 110 days in one case and 87 days in a second (Promchaiwong, 1995 unpublished).

Achupalas, in a large scale field trial in Ecuador, where 5 ponds were treated with a RNA/nucleotide supplemented diet and 6 ponds maintained on standard diet as controls, showed that survival rate was 5.9% improved and feed conversion 9.8% However, in this trial, the differences observed in growth rates were not statistically significant.

Perhaps the most extensive and detailed data have been collected by Hertrampf, Mishra and Krishna from trials inIndia.

In the first of these trials, using only 30 shrimp per replicate, both survival and growth rate was significantly better in the treated groups under conditions where regular changes in salinity were used as an additional stress factor. 

The second trial was carried out  on ‘pond-scale’.  One pond containing 75000 post larval (pl) shrimp was allocated as a control and another, containing 100,000 pl, as the treatment group in which the diet was supplemented with 0.2% RNA/nucleotide. Throughout the trial there were again wide variations in salinity which would have created stress conditions in the two ponds. The trial was continued for 98 days.

Nucleotide products vary in the extent of the processing used in their manufacture and the degree of purity achieved. Although a degree of performance enhancement can be accomplished with many of the crude yeast extracts marketed, when these are directly compared with highly purified nucleotides in the same experiment, a significant difference is seen. This difference in performance has been demonstrated in both shrimp and salmon in commercial, farm-scale trials. 

3000 Litopenaeus vannamei at PL-30 were each distributed at random into one of three commercial ponds designated Control, RNA/nucleotide (1) and RNA/nucleotide (2)  and grown on for 84 days. At the end of the trial period 5% (50) of the individuals in each cage were removed for measurement; the rest were harvested for market. RNA/nucleotide (1) comprises >95% purified nucleosides, a form which promotes easier adsorption in the gut and the balance of the purified components is adjusted according to the ratio ‘preferred’ by the target species. RNA/nucleotide (2) is a less pure product where no adjustment of the ratio of the components has been made.

The RNA/nucleotide (1) showed a 6.6% growth advantage (p ≤ 0.05) over the RNA/nucleotide (2) product. In both weight and length shrimp fed the RNA/nucleotide (2) product were smaller than the Control, standard diet shrimp although this difference was not significant at the 5% level.

A similar picture was seen in survival which was assessed at the end of the trial period from the number of individuals harvested.

The survival rate in RNA/nucleotide (1) treated shrimp was significantly different from the RNA/Nucleotide (2) although not significantly different from the Control. The Control and RNA/nucleotide (2) were insignificantly different from each other.

Increasing the performance of a vaccine may occur at different parts of the immune on-set process and is normally achieved by the use of an adjuvant that is part of the vaccine formulation. Good adjuvanting may improve recognition of the antigen, the level of protection eventually achieved and the length of time for which this protection lasts. Similar effects may be achieved by the application, during the vaccination process, of external treatments and it has been shown that RNA/nucleotides administered in feed can act in this way.

Ramadan et al (1994) fed RNA/nucleotides to Tilapia before and after vaccination against Aeromonas hydrophila and compared their survival with that of unvaccinated fish and fish vaccinated, but not fed RNA/nucleotides. Significantly higher antibody titres were seen in nucleotide treated fish (humoral response) and in macrophage, migration inhibition (cell mediated response). These measures translated into superior protection of the treated fish, when challenged with live Aeromonas hydrophila, 2352 degree days after vaccination.  

The data show, as expected, that the injection method of vaccination confers the better protection. In the injection data both treatments (standard vaccinates and RNA/nucleotide fed vaccinates) give a significant level of protection. The nucleotide-fed fish outperform those on a standard diet (2 v 3), although the difference is not significant (P ≤ 10.1%) probably due to the small numbers used.

The difference however becomes marked and significant in the same comparison made by immersion administration. Here the vaccinates that do not receive nucleotides are no better protected than the non-vaccinates (1 v 2); the RNA/nucleotide fed fish are protected significantly better than those on standard diet (2 v 3). This indicates that the use of nucleotides may have a significant adjuvanting effect when used in conjunction with commercial, immersion vaccines such as are widely used in salmonid fry, bass, bream and other fish too small to be injected.

Burrells et al (2001) evaluated the performance of commercial, furunculosis (Aeromonas salmonicida) vaccines administered to Atlantic Salmon that had been fed RNA/nucleotides in the diet for 3 weeks prior and 5 weeks after, vaccination. Serum antibody titres and survival after viable challenge were used as performance indicators. Compared with salmon maintained on standard, non-RNA/nucleotide diet the treated salmon had a significantly higher specific antibody titre (conditions). Five weeks after vaccination, the mean group titre of specific antibody in fish fed for 8 weeks with the nucleotide diet (1/144) was significantly greater P≤0.05 than that of the control fish (1/60).

Survival after challenge was significantly higher in vaccinated, compared with non-vaccinated fish whether on RNA/nucleotide or standard diet indicating the high performance of the vaccine used. The nucleotides enhanced the survival rate of the vaccinates but, due to the high performance of the injectable vaccine and the size of the experiment, this difference was not significant.

In the non-vaccinated group of fish fed the control diet, mortalities first occurred at 11 days post-challenge and rose steadily to a cumulative total of 38% at 36 days post-challenge. At this time, mortalities in the vaccinated group fed the control diet had been significantly reduced to 6% (P≤0.05), a relative percent survival RPS of 84%, as a consequence of the vaccination. In the group of fish fed the RNA/nucleotide diet for 3 weeks before and for 5 weeks following vaccination, no further mortalities occurred after day 11 and these remained significantly lower P≤0.05 than the non-vaccinated group at just 2% throughout the period (RPS 95%). However, because of individual tank variation, this further reduction in mortalities was not shown to be significant. 

When weights were recorded 3 weeks after feeding, the RNA/nucleotide diet fish had a mean weight 5.4% greater than fish fed the control diet, although this was not shown to be significant. By the time of the challenge, 5 weeks after vaccination, the same fish were 14.9% heavier (82.5 g) compared with the controls (71.8 g). This was significant (P≤0.001). At the end of the study, the mean weight of the fish previously fed RNA/nucleotide diet (121.4 g) was still 8.7% in excess of those given control diet, 111.7 g,  throughout (P≤0.05).

As in the trials of Ramadan, it is clear that there is an adjuvanting effect of RNA/ nucleotides when used in conjunction with vaccines. However, where an efficient injection vaccine is used, especially one such as the powerful oil adjuvanted furunculosis vaccine used here, statistical significance of the added effect of the RNA/nucleotides can be seen only in a large scale trials where the numbers allow the treatment effect to be separated from the background error.

However, where injection vaccines are used that have a less powerful performance than the modern, oil adjuvanted, injectable furunculosis vaccines, for example IPNV, or where only an immersion vaccine can be administered, for example fry vaccines, the advantage conferred by the use of the RNA/nucleotides may well be sufficient to improve the performance of the vaccine to an economic level and confer a worthwhile level of protection not possible without them. 

The action of RNA/nucleotides in ameliorating disease is fundamentally different from that of immunopotentiators, such as β-glucans, lipopolysaccharide, zymosan and others. Clearly this distinction will depend partly on the definition of immunopotentiation that is used. True immunopotentiators target mechanisms of the innate immune system such as complement activation, phagocytosis and cytokine secretion in a general way not related to any specific disease or antigen. They are therefore frequently used to boost immune reactivity and improve resistance in order to improve survival rates during the culture cycle. This stimulation is artificial and does not depend on the presence of any external source of infection.

The role of RNA/nucleotides is well described. It may consequentially have some of the effects of immunostimulation but its role is not directly in immune stimulation but in ‘supporting’ immune responses once they have been naturally activated by an external stimulus such as disease. This support is by the provision of essential metabolic components (purines and pyrimidines) required in large quantities by the responding cells.

The nucleotides are a natural component of the diet of animals and, when not adsorbed in an immune response, are used elsewhere in metabolism. On the other hand, the external provision of substances that stimulate the immune systems artificially, as do immunopotentiators, has inherent dangers. The principal use of immunopotentiators in aquaculture is in crustacea which do not possess mechanisms that allow them to respond to vaccination in the same way as higher animals. Smith et al (2003, Fish Shellfish Immunology 15(1) 71-90) reviewed the published data of their use in crustacea. Much of the data, after statistical analysis, was seen either to be invalid or not to support the conclusions claimed. On the contrary, the use of immunostimulation as a routine procedure was occasionally found to be damaging. The innate immune system is able to respond rapidly and effectively to infection by switching on gene pathways which enhance the secretion of certain cytokines, complement, agglutinins, precipitins, interferon etc. Constant, artificial stimulation of these parts of the innate immunity, in the absence of infection, may induce feedback mechanisms and promote imbalances in systems that are ‘programmed’ to respond only when required. The release of potent cytotoxic agents such as lysins, reactive oxygen species and others, may damage the cell itself particularly in the open circulation system typical of crustacea.

The provision of genuine immunostimulators throughout the growth cycle is expensive and may therefore be detrimental, nevertheless it has been claimed that their provision at times of stress and when disease is anticipated, may be of benefit. This is a clear distinction between this family of compounds and RNA/nucleotides since nucleotides may be provided as a normal component of the diet which, in the absence of stress or disease, are metabolised in non-disease related pathways, with beneficial rather than detrimental effects. However, in the presence of disease they are readily, incorporated into the immune response pathways with proven, advantageous effects. 

The means of preparation and, in particular the degree of purity of commercial RNA/nucleotide products, differ and this has been shown to be a significant factor in the performance obtained both experimentally and under field conditions. The highly purified products, although generally more expensive, consistently outperform those of a lower grade. The proportions of the nucleotides in the product have to be adjusted to ratios experimentally determined to be the most appropriate for the target species.

A more extensive piece of work in salmon was conducted by a fish diet manufacturer (unpublished, 2004/5). In a direct comparison with three other diets, the diet supplemented with purified RNA/nucleotides was 7% superior to the nearest competitor. In a second trial, a direct one to one comparison with a diet supplemented with unpurified nucleotides, salmon fry over a 5 week period grew 15.3% better on the purified RNA/nucleotide diet.

In other parameters measured, villous height in the intestine was significantly better at all time points and osmoregulation (determined by blood chloride concentration) and non-specific immunity (measured by lysozyme production) were also superior in the diet supplemented with purified RNA/nucleotides compared with the less pure forms.

It seems clear therefore that the extra cost purifying the nucleotide supplements and adjusting the ratio of the bases creates a consistent performance that is significantly better than unpurified forms or simple yeast extracts. 

Disease resistance,

Burrells, C., Williams, P.D. & Forno, P.F. (2001) Dietary nucleotides: a novel supplement in fish feeds. 1. Effects on resistance to disease in salmonids. Aquaculture 199, 159-169. 

Burrells, C., Williams, P.D., Southgate, P.J. & Wadsworth, S.L. (2001) Dietary nucleotides: a novel supplement in fish feeds.2. Effects on vaccination, salt water transfer, growth rate and physiology of Atlantic salmon. Aquaculture 199, 171 – 184.

Leonardi, M., Sandino, A.M. & Klampau, A. (2003) Effect of a nucleotide enriched diet on the immune system, plasma cortisol levels and resistance to Infectious Pancreatic Necrosis (IPN) in juvenile rainbow trout (Oncorhynchus mykiss). Bulletin of the European Association of Fish Pathologists 23(2), 51

Salobir, J., Rezar, V.,Pajk, T. & Levart, A. (2005). Effect of nucleotide supplementation on lymphocyte DNA damage induced by dietary stress in pigs. Animal Science 81, 135-140.

Salobir, J., Rezar, V.,Pajk, T. & Levart, A. (2005)The Effect of Nucleotide Supplementation on Oxidative Stress Induced by High Dietary Fat Intake in Pigs. University of Ljubljana, Slovenia. (Non- peer reviewed poster presentation) 

Ramadan, A., Afifi, N. A., Moustafa, M.M. & Samy, A.M. (1994) The effect of Ascogen on the immune response of Tilapia fish to Aeromonas hydrophila vaccine. Fish & Shellfish Immunology 4(3), 159-166

Burrells, C., Williams, P.D.,Southgate, P.J. & Wadsworth, S.L. (2001) Dietary nucleotides: a novel supplement in fish feeds.2. Effects on vaccination, salt water transfer, growth rate and physiology of Atlantic salmon. Aquaculture 199, 171 – 184. 

Smith, V.J., Brown, J.H. & Hauton. (2003) Immunostimulation in crustaceans: does it really protect against infection? Fish & Shellfish Immunology 15 (1), 71 – 90. 

Inability of lymphocytes to manufacture purines efficiently.

Saviano, D.A. and Clifford, A.J. (1978) Absorption, tissue incorporation and excretion of free purine bases in the rat. Nutritional Reports International 17, 551 – 556. 

Li, P. & Gatlin, D.M.(2006) Nucleotide nutrition in fish: Current knowledge and future applications. Aquaculture 251( 2-4), 141-152 

Metabolic cost to animal of purine synthesis:-

Sanderson, I.R. and He, Y.(1994) Nucleotide uptake and metabolism by intestinal epithelial cells. Journal of Nutrition 124, 131S – 137S

Use of salvage or synthesis nucleotide pathways in different tissues:-

(López-Navarro, A.T., Gil, A. and Sánchez-Pozo, A. (1995) Deprivation of dietary nucleotides results in a transient decrease in acid soluble nucleotides and RNA concentration in rat liver. Journal of Nutrition125, 2090 – 2095.

Protection against Staphylococcus aureus

Kulkarni, A.D., Fanslow, W.C., Rudolph, F.B., Van Buren, C.T., 1986. Effect of dietary nucleotides on response to bacterial infections. Journal of Parenteral & Enteral Nutrition 10, 169–171.

Protection against Candida albicans.

Carver, J.D., 1994. Dietary nucleotides: cellular immune, intestinal and hepatic system effects. Journal of  Nutrition 124  Supplement 1S , 144S–148S.

Protection against Streptococcus iniae

Li, P., Lewis, D.H., Gatlin, D.M. (2004) Dietary oligonucleotide from yeast RNA influences immune responses and resistance of hybrid striped bass (Morone chrysops X Morone saxatilis) to Streptococcus iniae infection. Fish & Shellfish Immunology 16, 561 – 569.

Protection against Aeromonas hydrophila (Use of clearance from blood as protection indicator).

Sakai, M., Taniguchi, K., Mamoto, K., Ogawa, H. And Tabata, M. (2001) Immunostimulant effects of nucleotide isolated from yeast RNA on carp, Cyprinus carpio. Journal of Fish Diseases 24, 433 – 438.