K. Hoffmann (CHEMOFORMA Ltd.)
The global aim of aquaculture is to optimize overall performance and growth of high quality fish. As in livestock, the outbreak of diseases can be a major concern. The high susceptibility to stress and the rapid spread of diseases in water have forced fish farmers to concentrate on maintaining their fish in good health in order to achieve economic performance.
Farming of healthy fish requires them to be able to develop defence mechanisms against different kinds of bacterial, viral or parasite infections. The non specific and the specific immune response both contribute to immune reactions in vertebrates. In fish the non-specific mechanism is more important whereas the specific immune response is far more important in mammals or higher vertebrates. Ameliorating the immune response improves the vaccination efficiency. Vaccines induce a specific immune response and increase the capacity to kill pathogens by non-specific defence mechanisms.
During the early development of the salmon industry, antibiotics were commonly used in the treatment of diseases. However, the consumption of drugs has progressively been reduced due to environmental as well as regulatory concerns and to increased resistance of pathogens. Furthermore, the medicative effect of orally administered drugs is diminished by the fact that diseased fish often do not feed. The extirpation of major diseases by improved husbandry and vaccination programmes has reduced mortality levels considerably. Although some efficient vaccines against major diseases of finfish are available today, others need to be improved in terms of efficiency and duration of protection.
Intensively raised fish are exposed to stressful situations which often result in a depressed immune status. Good management practices reduce stress and therefore help to maintain healthy animals. However, since not all stressing situations can be avoided, fish with enhanced defence mechanisms will be better primed to combat the negative effects of stress.
Fish are the most primitive vertebrates. They possess the non-specific defence mechanisms of invertebrates such as phagocytic mechanisms developed by macrophages and granular leukocytes, but develop both cellular and humoral immune responses mediated by lymphocytes. The main lymphoid organs of fish are the anterior kidney, the thymus and the spleen. In contrast to mammals, the nonspecific immunity is considered as the first line of defence in fish and represents a considerable part of the immune response.
When a pathogen infiltrates the body, the non-specific immune mechanisms may be sufficient to stop the infection. If not, the disease will develop and the specific immune mechanisms must be involved. If the animal survives, it will be protected against a re-infection with the same pathogen, due to the development of a specific immunological memory. This memory is less developed in fish than in mammals.
Non-specific immune mechanisms are host-defence mechanisms which do not require a specific recognition of the antigen. However, some functions of the non-specific immune response are also involved in the specific response.
Many factors can affect the immune response of fish. Among them are stressors and environmental factors of natural origin. Nutrients, micronutrients and substances of no nutritional values can also modulate the immune response. Depending on their type, the amount and the duration of exposure, their effect can be either negative or positive. Substances with immunostimulating properties can compensate the immunodepression caused by other factors, e.g. the immunodepression caused by a stressor can be compensated by an increased intake of immunomodulators or specific nutrients supporting or facilitating immune response before a predictable stress event such as grading (Figure 1).
Temperature: Fish are poikilotherm organisms. Their physiological processes are influenced by the temperature of the surrounding water. Major defence mechanisms are temperature-dependent and develop faster at the optimal temperature of the fish species concerned. Low temperatures are known to decelerate all metabolic processes including the immune response. However, high temperatures can also depress the immune functions. It becomes obvious that antigen processing and cellular co-operation between macrophages and lymphocytes are temperature-sensitive. The normal function of fish lymphocytes is highly dependent on homoviscous adaptation of membrane lipids. Fatty acid composition and environmental temperature are factors establishing the fluidity and permeability of membranes as well as the activity of membrane-associated receptors and enzymes.
Stress conditions influence the health status of fish. Immunodepression is known to be a major secondary effect in the response of an organism to stress. Many situations such as transport, stocking density, handling and bad water quality can cause stress responses. The fish will react by secreting high levels of stress hormones (corticosteroids) which are known to be immunosuppressive. The stress response is accompanied by lymphocyte depletion in blood and in lymphoid organs.
Pollutants and heavy metals are also known to have detrimental effects on the fish immune system causing various effects depending on the nature of the substance. Drugs such as antibiotics can also be immunosuppressive.
A well-balanced diet is essential for adequate host defence mechanisms as well as to optimise growth and the quality of fish for human consumption.
Micronutrients: Antioxidant vitamins such as vitamins C and E have been demonstrated to have immunomodulatory properties when fed at elevated doses. The presence of carotenoids in the diet has also been demonstrated to improve the health status of pigmented fish.
Immunomodulators other than micronutrients: Adjuvants are substances that enhance specific immune response in addition to non-specific defence response when combined with antigens. Generally adjuvants reduce the rate of antigen elimination thereby prolonging antigen contact with macrophages and lymphocytes and augmenting the specific immune response. This is the principle of adjuvanted vaccines.
Figure 1: Factors influencing proper functionality of the immune system in fish
The quality of the feed is a major factor in sustaining healthy fish. Over the last 50 years the developments in nutritional research was significantly improved, particularly with emphasis on protein, amino acid, mineral and energy requirements. This became necessary to understand and meet the needs of fish. Nowadays the knowledge on metabolism and nutrition of fish entails to highly sophisticated feeds. It has been shown that the immune system can be enhanced by the use of immunomodulators such as antioxidant vitamins, carotenoids and other feed additives. The combination of good management, vaccination and nutritional prophylaxis will insure higher survival rates and improve growth in intensive farming systems.
Lots of discussions have been launched on options of maximising the immunocompetence of animals 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 have 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 because of the lack of scientifically significant studies. Nevertheless, the trend of nutritionists and suppliers of additives to combine specific functionalities with food or feed is comprehensible and reasonable. The target must be achieving most of the feed in terms of nutritional aspects, prevention of diseases and, as a consequence, profitable animal husbandry. These days, although being contraproductive, the generally increasing price of feed ingredients hampers the development of functional feed or the use of nutraceuticals. Nutritionists or feed millers would never disregard the use of specific nutrients in feeds which have been scientifically approved to be essential for the given species but any additional additive is strictly avoided to save cost in production and costs for the customer. It remains questionable if this must be regarded short-sighted as some putative non-essential nutrients may turn essential under specific conditions.
Essential nutrients are nutrients that are required for normal body functioning but can not be synthesized in a given organism. For most classes of essential nutrients like vitamins, minerals, fatty acids or amino acids a ranking in terms of nutritional importance was already compiled for different species. The most important member of each class is called the first limiting followed by the second limiting and so on. All essential nutrients share some specific characteristics:
• They must be available in the feed in a specific form and concentration required by the organism;
• A deficiency of one essential nutrient can not be restored by an excess of another;
• The nutritional bioavailability is vital for the effective utilisation in the organism.
These three characteristics for sure apply to amino acids, some fatty acids, vitamins and minerals. The supply can only be guaranteed when the feed is enriched with adequate amounts of the essential nutrients to fill the corresponding pool.
A second class of nutrients is required from dietary sources under specific circumstances, when the internal supply can not be guaranteed. Under these specific circumstances the same characteristics already listed for essential nutrients likewise apply for these conditionally essential nutrients. Some amino acids as well as vitamins and other micronutrients belong to this group. Moreover, there are other nutrients not yet assigned to be either essential or conditionally essential although being comparably important for the organism.
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. However, scientific research has discovered remarkable and measurable benefits from supplementing RNA/nucleotides in a diet. 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. 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. 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.
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. 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. 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.
The supply of nucleotides in wild animals is controlled and maintained by the use of three autogenous metabolic or catabolic processes (Figure 2). The recycling of nucleotides from dead cells is called “Salvage pathway”. This pathway includes several biochemical and metabolic steps to release the nucleotides from the cells. This pathway is available in adult animals only as there is limited or no salvage in young animals. This process in addition has a limited efficiency in adult animals which means that only up to 75%-80% of the nucleotides can be recycled. The “de-novo synthesis” of nucleotides is a rather complicated biochemical process including 10-14 different biochemical steps. Raw materials for the synthesis of nucleotides are, amongst others, amino acids. The synthesis requires lots of energy which needs to be supplied by other metabolic processes in the organism. Finally, nucleotides may be extracted from the diet as all cellular feed ingredients contain nucleotides in form of DNA present in the nucleus of every cell. This process is by far the most inefficient one as the DNA is protected from degradation by a specific class of proteins, the so-called Histone proteins. These proteins cover and shield the DNA and prevent it from disintegration and degradation. Thus the nutritional availability of nuclear nucleotides may be not more than 5%-10%. Nevertheless these three pathways are sufficient to prevent a depletion of the internal nucleotide pool of the organism under normal conditions.
Figure 2: Under normal conditions the supply of nucleotides is guaranteed through autogenous metabolic and catabolic processes. The de-novo synthesis of nucleotides, or the reuse of nucleotides from dead cells (so-called “salvage pathway”) and the nutritional available dietary nucleotides allow meeting even temporary peaks in the demand for nucleotides without losses in performance.
Under intensive farming conditions a particular 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 (Figure 3).
Figure 3: Under detrimental conditions universal in modern aquaculture, the supply of nucleotides cannot be guaranteed by internal resources. An effective reaction to health challenges or regenerative activities can only be guaranteed at the expense of performance as there are not enough nucleotides available for growth, reproduction or development.
The instinct of self preservation of the animals assesses the disposition of the nucleotides from the internal nucleotide pool. Survival is the preference for the animal regardless of the aim of the farmer to keep up performance and profit. The organism can not be cheated on as the regulatory mechanisms involved are well rehearsed and evolutionary developed. Health challenges or stress typically impair on performance or development as the nucleotide pool is depleted and may not be refilled adequately. The only solution is to add nucleotides to the diet (Figure 4). This external supply of nucleotides is of utmost importance for e.g., 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.
Figure 4: Supplementing feed with adequate amounts of high-quality nucleotides replenishes the internal pool and thereby supports performance even under adverse conditions. Dietary nucleotides are a management tool to maintain general health, enhance performance and manage the harmful effects of stress
The addition of VANNAGEN® to the diet fills the internal nucleotide pool. Every process dependent on unhindered supply with nucleotides is now able to operate unimpeded. One of the reasons for the success of VANNAGEN®-supplementation of diets is that the response of the immune system is accelerated because the proliferation of the cells involved is facilitated. 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 VANNAGEN® supplemented feed on performance and general health as well as development of young animals was tested in numerous trials in agriculture and aquaculture thereby switching nucleotides from non-essential to essential nutrients in modern agriculture or aquaculture. Nucleotides fit into nutraceuticals and support the development of functional feeds combining nutritional as well as sanitary benefits. 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 active ingredients of VANNAGEN® consistently outperform common nucleotide products. The proportions of the nucleotides in VANNAGEN® have been adjusted to ratios experimentally determined to be the most appropriate for the target species. Although not yet defined as essential nutrients, VANNAGEN® must be regarded as a management tool to control stress and disease while maintaining the increasing demand for performance in fish and crustaceans. VANNAGEN® fosters the development, participates in cellular and molecular repair mechanisms and ensures unhindered cell proliferation without affecting growth, development or reproduction upon environmental, physical or pathogenic challenges. Therefore VANNAGEN® must be taken into account when developing functional feed for fish and crustaceans.
Detailed information on VANNAGEN® are available at the booth of CHEMOFORMA (08BV F018) during VIV Europe 2010.
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