The concept of functional aquafeeds represents an emerging new paradigm to develop diets for fish and crustaceans. Functional feeds extend beyond the satisfying basic nutritional requirements of the cultured organism to improve growth and feed utilisation also the support of general health and stress resistance of the animals. The increasing concerns about residues of antibiotic or anabolic agents in food production lead to a restrictive use of these substances to stimulate growth and treat bacterial diseases in most animal industries. The alterations in drug-use policies and consumer attitudes have already impacted aquaculture and thus have prompted interests in developing alternative strategies for growth and health management. Disease prevention and treatment strategies such as vaccinations and drugs are currently limited in large-scale aquaculture due to regulatory constrains or inconvenient administration protocols. Without any doubt it would be beneficial for the aquaculture industry if cultured organisms were conferred with enhanced growth performance, feed efficiency and disease resistance. This would reduce production costs and simultaneously improve consumers’ perception.
One goal in all types of aquaculture is to maximise the efficiency of production to optimize profitability. Intensification of production is one means of increasing production efficiency but also may lead to increased susceptibility of the cultured organism due to deterioration of water quality and elevation of stress. Bacterial, viral or parasite infections often affect the cultured organisms whose immunity may be compromised by the stressful conditions. In case of the occurrence of bacterial infections in aquaculture the most common means of treating was the administration of antibiotics. However, these drugs have been criticized for potential development of antibiotic resistant bacteria and destruction of environmental microbial flora as well as being cost intensive and in some cases with marginal effects only. Certain antibiotics have also been shown to suppress the immune system, making organisms in aquaculture more susceptible to viral and parasite infections. This has led to the ban of sub- therapeutic use of antibiotics in Europe and more stringent regulations on application of antibiotics in lots of other countries. The development of alternative strategies for disease control turned out to be more and more interesting for livestock and aquaculture. In recent years there had been heightened research in developing dietary supplementation strategies in which various health-promoting compounds have been evaluated.
It has been documented in a number of terrestrial animals and humans that the microbiota of the gastrointestinal tract plays important roles in affecting nutrition and health of the host. This similarly should apply to aquaculture too. In the following paragraphs some possibilities to increase general health of aquatic organisms will be compiled.
Probiotics
Probiotics are dietary supplements containing potentially beneficial live microorganisms. According to the currently adopted definition by FAO/WHO, probiotics are “Live microorganisms which when administered in adequate amounts confer a health benefit on the host”.
This health benefit is established by affecting the intestinal microbial balance of the host organism. The addition to or altering of the intestinal microbiota has been done to achieve such positive effects as enhanced growth, digestion, immunity and disease resistance. Examples of probiotics include gram-positive bacteria such as Bacillus sp, Carnobacterium inhibens K1 and Lactobacillus sp as well as gram-negative bacteria such as Aeromonas hydrophila, Pseudomonas fluerescens and Vibrio fluvialis. Other probiotics are bacteriophages and yeast (Saccharomyces cerevisiae, Phaffia Rhodozoma, Debaryomyces hansenii) as well as microalgae (Tetraselmis suecica). Probiotics are usually selected to control specific pathogens through competitive exclusion or enhancement of fishes’ immune system. This means that the list of probiotics is steadily increasing.
In early work on probiotics the microbial preparations were either applied directly to the water or added to the feed. It turned out that the normal gut microbiota needs to be identified and characterized in order to provide the adequate cocktail of probiotics for a given organism or species. Moreover questions on the ability of the probiotics to adhere to the mucosal surface of the gut were raised by some scientists. How long will the microorganisms survive in the intestinal tract and what really happens in the intestinal tract and in the environment? These questions as well as issues on optimal dosage or survival of the probiotics in or on pelleted feeds were addressed. It turned out that there is really some recent success by the application of probiotics. But still there is additional work needed to answer questions concerning optimal dosage, the organisms used as probiotics and their presentation.
Prebiotics
The restrictions on application of probiotics in aquaculture include the costs as well as the insufficient evaluation of the biological consequences and the potential influence on natural microbial diversity. In addition, there are some potential constrains to dietary application of probiotics including the susceptibility of these live organisms to inactivation by heat during extrusion. It is well established that some microbial products such as lipopolysaccharides and β-Glucans can stimulate the cell-mediated immune system. Some of these products can be delivered orally without complete degradation and thus may be used as potential immunostimulants for aquaculture.
The various potential constrains to probiotic application as well as some of the positive influences of live microbes such as increased growth and disease resistance have resulted in the evaluation of the so-called prebiotics. These are non-digestible feed ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the intestinal tract, and thus improve host health. The main advantage of prebiotics over probiotics is that they are natural feed ingredients and thus regulatory control over dietary supplementation should be limited. The classic definition of prebiotics today only fits on fructooligosaccharide (FOS), galactooligosaccharides (GOS) and transgalactoologosaccharides (TOS). Other feed supplements often referred to as being prebiotics like for example mannanoligosaccharides (MOS) have effects on the composition of the intestinal microflora but their mode of action is different as they are not directly utilized or metabolized by the bacteria.
The inclusion of prebiotics in the diet has been reported to increase the uptake of glucose and the bioavailability of trace elements most likely by decreasing the pH of the intestinal tract. Moreover effects on volatile fatty acids (VFAs) have been observed.
Synbiotics
Synbiotics refer to nutritional supplements combining probiotics and prebiotics to form a symbiotic relationship. This is a new concept for aquaculture which needs further evaluation and more in-deep research to more fully characterize the effects in aquatic organisms.
Improving general health and performance in aquaculture by stimulating the growth of specific microbes in the intestinal tract or directly stimulating the immune system basically is a good and rational strategy. The development of functional feed including nutraceuticals will be the objective of feed millers worldwide for the next years. The perspective to simultaneously improve the performance and the general health of the animals by addition of specific feed supplements is appealing not only for farmers but also for feed producers in times of increasing competitions on the markets. The success of probiotics and prebiotics in livestock industry might spill over to aquaculture. Nevertheless, the basic requirements of a diet to supply adequate quantities of essential and non-essential nutrients for the various organisms must not be neglected. The stimulation of growth of intestinal bacteria by pro- or prebiotics involves the supply of sufficient amounts of other nutrients to enable the multiplication of these microbes. If the supply of basic nutrients can not be guaranteed by the diet or the animal itself, the beneficial effects of pro- and/or prebiotics will be reduced or even unverifiable. It is still questionable if there are not other possibilities to increase performance and at the same time the immunity of fish or crustaceans without the risk of extensive biological and environmental consequences. Although prebiotics are widely regarded as being safe, their usability in aquaculture is still in its infancy and it will take several years to establish cheap, profitable and feasible protocols for their usage.
Nowadays there are other already scientifically approved feed supplements available which must be regarded as nutraceuticals achieving the dual advantage of improving performance and general health of fish and crustaceans at the same time.
Nucleotides
For years, nucleic acids and nucleotides were not considered essential nutrients for use in any dietary programmes. It was thought that all organisms can supply sufficient amounts of nucleotides to meet their physiological demands. However, under certain conditions, including rapid growth, limited food supply, stress, immunological challenges and some others, dietary nucleotides turn to conditionally essential nutrients. Balanced formulations of purified dietary nucleotides modulate innate and adaptive immune response as reported in numerous scientific publications.
Fish fed nucleotide-supplemented diets generally have shown enhanced resistance to viral, bacterial and parasitic infections. The reasons for the immunofacilitating properties of RNA/nucleotides include dietary provision of physiologically required levels of nucleotides due to limited synthetic capacity of certain tissues, inadequate energetic expenditure for de novo synthesis or salvage, immunoendocrine interactions and modulation of gene expression patterns.
Nucleotides are chemical compounds that consist of three portions: a heterocyclic base, a sugar, and one or more phosphate groups. In the most common nucleotides the base is a derivative of purine or pyrimidine, and the sugar is a five-carbon sugar (pentose). 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. RNA and nucleotides provide basic building blocks for cell proliferation in fauna and flora. Unhindered cell proliferation is a prerequisite for growth, repair, development and functionality of organs and regulatory systems (e.g. the immune system).
To date, with an increasing number of publications, rather consistent and encouraging trial results are reported favouring balanced formulations of free and isolated nucleotides for health and performance management in aquaculture. Nucleic acids found in many feed and food ingredients are protected by specific binding proteins only allowing just around 30% to be nutritionally available for the organism. By isolating and purifying RNA/nucleotides, thereby eliminating the protective proteins, it is possible to formulate feed additives with a nucleotide availability of over 95%.
Dietary nucleotides have been shown to have multiple effects on the gastrointestinal tract in animal models, including physiological, morphological and microbiological influences. Proximal, mid and distal intestinal villi are significantly elongated, thereby simultaneously increasing the total gut surface area, compared to control animals. In higher vertebrates the positive effect of RNA/nucleotides on the composition of the intestinal microflora was reported. Although the microbial ecology of fish and crustaceans still needs further research, possible positive effects of dietary nucleotides may be predictable as RNA/nucleotides are ubiquitously used in every animal species and microbes.
It was reported that RNA/nucleotides have direct influence on both humoral and cellular components of the innate immune system in fish. An increase of serum complement and lysozyme activity as well as phagocytosis and superoxide anion production was observed upon feeding nucleotide-enriched diets. An increase in blood neutrophil oxidative radical production was found upon RNA/nuclotides compared to fish fed a basal diet.
An immunopotentiating effect was observed upon direct injection or immersion of fish with killed Aeromonas bacteria after feeding nucleotide-supplemented diets on both humoral and cell mediated response. Antibody titres after vaccination were significantly higher in fish fed nucleotide diets. Identical results were found in mitogenic responses of lymphocytes.
Survival after a challenge is a widespread measure of disease resistance in fish and crustaceans. A reduction in cumulative total mortality was observed upon infection with infectious salmon anaemia, infectious pancreatic necrosis and V.anguillarum, P.salmonis, A.hydrophila and S.iniae. Moreover, dietary nucleotides affected the development of sea lice, thereby reducing the cross-infestation of other fish.
In crustaceans the positive effects of dietary nucleotides on the immune system was also reported. Under conditions of stress, the susceptibility to infections with pathogens increases dramatically resulting in elevated mortalities. In several reports from experiments in tanks as well as in ponds, the positive effect of nucleotide-enriched diets was outlined. In cases of outbreak of white spot virus, the use of nucleotide-supplemented feed not only lead to a reduction in mortalities of shrimp but also to elimination of viruses from the ponds as determined by PCR analysis.
Although the supportive mechanism of balanced formulations of free nucleotides on crustacean immunity is not yet clear, most of the results suggest their impact on stabilization and enhancement of innate immune response by triggering gene expression and/or maturation of defense components.
Conclusion
In contrast to pro-, pre, or synbiotics purified dietary RNA/nucleotides are not restricted to species or applications. Their universally valid use and diverse functionality in every living organism as well as their ecological safety allows them to be regarded as a management tool to control stress, performance and diseases in aquaculture. Prolonged administration of medication or immunostimulants often leads to undesirable side effects on growth as well as disease resistance. RNA/nucleotides do not stimulate innate or acquired immunity. By providing the resource for unhindered cell proliferation, gene expression and signalling they facilitate adequate reactivity upon health challenges. Other processes in the organism e.g. growth, development or reproduction need not be suppressed upon an infection as basic materials for cellular and molecular functionality are adequately supplied. All the scientifically approved functions of purified RNA/nucleotide feed supplements highlight their significance for the development of functional feeds in aquaculture.
