Nucleotide nutrition in fish: Current knowledge and future applications

Aquaculture 251 (2006) 141 – 152 www.elsevier.com/locate/aqua-online

Review article

Nucleotide nutrition in fish: Current knowledge and future applications Peng Li, Delbert M. Gatlin IIIT Department of Wildlife and Fisheries Sciences and Faculty of Nutrition, Texas A&M University System, College Station, TX 77843-2258, USA Received 7 December 2004; received in revised form 13 January 2005; accepted 14 January 2005

Abstract The roles of nucleotides and metabolites in fish diets have been sparingly studied for over 25 years. Beside possible involvement in diet palatability, fish feeding behavior and biosynthesis of non-essential amino acids, exogenous nucleotides have shown promise most recently as dietary supplements to enhance immunity and disease resistance of fish produced in aquaculture. Research on dietary nucleotides in fishes has shown they may improve growth in early stages of development, enhance larval quality via broodstock fortification, alter intestinal structure, increase stress tolerance as well as modulate innate and adaptive immune responses. Fishes fed nucleotide-supplemented diets generally have shown enhanced resistance to viral, bacterial and parasitic infection. Despite occasional inconsistency in physiological responses, dietary supplementation of nucleotides has shown rather consistent beneficial influences on various fish species. Although nucleotide nutrition research in fishes is in its infancy and many fundamental questions remain unanswered, observations thus far support the contention that nucleotides are conditionally or semi-essential nutrients for fishes, and further exploration of dietary supplementation of nucleotides for application in fish culture is warranted. Hypothesized reason(s) associated with these beneficial effects include dietary provision of physiologically required levels of nucleotides due to limited synthetic capacity of certain tissues (e.g. lymphoid), inadequate energetic expenditure for de novo synthesis, immunoendocrine interactions and modulation of gene expression patterns. However, currently there are numerous gaps in existing knowledge about exogenous nucleotide application to fish including various aspects of digestion, absorption, metabolism, and influences on various physiological responses especially expression of immunogenes and modulation of immunoglobulin production. Additional information is also needed in regard to age/size-related responses and appropriate doses and timing of administration. Thus further research in these areas should be pursued. D 2005 Elsevier B.V. All rights reserved. Keywords: Nucleotide; Immunonutrition; Immunostimulant

T Corresponding author. Tel.: +1 979 847 9333; fax: +1 979 845 4096. E-mail address: [email protected] (D.M. Gatlin). 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.01.009

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Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Biochemistry of nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Digestion and absorption of nucleotides and related metabolites . . . . . . . 4. Chemo-attractive and feeding stimulatory effect of dietary nucleotides . . . . 5. Gastrointestinal influences of dietary nucleotides . . . . . . . . . . . . . . . 6. Nucleotide effects on fish growth . . . . . . . . . . . . . . . . . . . . . . . 7. Nucleotides and reproduction . . . . . . . . . . . . . . . . . . . . . . . . . 8. Effects of nucleotides on innate immunity . . . . . . . . . . . . . . . . . . 9. Effect of nucleotides on adaptive immunity . . . . . . . . . . . . . . . . . . 10. Effects of nucleotides on stress responses . . . . . . . . . . . . . . . . . . . 11. Nucleotides and resistance to infectious diseases . . . . . . . . . . . . . . . 12. Concerns about dose, administration timing and types of dietary nucleotides. 13. Future research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Nucleotides have essential physiological and biochemical functions including encoding and deciphering genetic information, mediating energy metabolism and cell signaling as well as serving as components of coenzymes, allosteric effectors and cellular agonists (Carver and Walker, 1995; Cosgrove, 1998). However, the roles of nucleotides administered exogenously have been debated for many years. Because neither overriding biochemical malfunctions nor classical signs of deficiency are developed in human or animal models, nucleotides have traditionally been considered to be non-essential nutrients. However, this opinion has been challenged by successive research publications which suggest that dietary nucleotide deficiency may impair liver, heart, intestine and immune functions (reviewed by Grimble and Westwood, 2000a). The modulatory effects of dietary nucleotides on lymphocyte maturation, activation and proliferation, macrophage phagocytosis, immunoglobulin responses as well as genetic expression of certain cytokines have been reported in humans and animals (reviewed by Gil, 2002). Nucleotide supplementation has been one important aspect of research on clinical nutrition and functional food development for humans. Besides certain application of exogenous nucleotides in clinical situations such as partial hepatectomy (Grimble, 1996), nucleotide fortification of breast milk substitutes has been recommended to

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the U.S. Food and Drug Administration for approval (Aggett et al., 2003). Although initial efforts in evaluation of dietary supplementation of nucleotides for fishes could be traced to the early 1970s, research at that time mainly focused on the possible chemo-attractive effects of these compounds (Mackie, 1973; Kiyohara et al., 1975; Mackie and Adron, 1978). World-wide heightened attention on nucleotide supplementation for fishes was aroused by the reports of Burrells et al. (2001a,b), indicating that dietary supplementation of nucleotides enhanced resistance of salmonids to viral, bacterial and parasitic infections as well as improved efficacy of vaccination and osmoregulation capacity. To date, research pertaining to nucleotide nutrition in fishes has shown rather consistent and encouraging beneficial results in fish health management (Table 1), although most of the suggested explanations remain hypothetical and systematic research on fishes is far from complete. Because increasing concerns of antibiotic use have resulted in a ban on subtherapeutic antibiotic usage in Europe and the potential for a ban in the US and other countries (Patterson and Burkholder, 2003), research on immunonutrition for aquatic animals is becoming increasingly important (Gatlin, 2002). Research on nucleotide nutrition in fish is needed to provide insights concerning interactions between nutrition and physiological responses as well as provide practical solutions to reduce basic risks from

Table 1 Research on dietary supplementation of nucleotides (NT) with fishes Nucleotide form

Dose and/or feeding regime

Ramadan and Atef (1991) Ramadan et al. (1994) Adamek et al. (1996) Burrells et al. (2001a)

Ascogen S, (Chemoforma, Augst, Switzerland) Ascogen (Chemoforma, Augst, Switzerland) ASCOGEN (Chemoforma, Augst, Switzerland) Optimuˆn, (Chemoforma, Augst, Switzerland)

2 and 5 g kg

Burrells et al. (2001b)

Optimuˆn, (Chemoforma, Augst, Switzerland)

Sakai et al. (2001) ribonuclease-digested yeast RNA (Amano Seiyaku Co-op, Tokyo, Japan) Leonardi et al. Optimuˆn, (Chemoforma, (2003) Augst, Switzerland) Low et al. (2003) Optimuˆn, (Chemoforma, Augst, Switzerland) Li et al. (2004a,b) Ascogen P (Canadian Biosystem Inc. Calgary, Canada) a

5 g kg

1

1

diet

diet

0.62, 2.5 and 5 g kg 1 diet at 1% bw day 1 2 g kg 1 diet, containing 0.03% NT, 2% bw day 1 2 g kg 1 diet, containing 0.03% NT, 1% bw day 1 2 g kg 1 diet, containing 0.03% NT, 2% bw day 1 2 g kg 1 diet, containing 0.03% NT, 2% bw day 1 2 g kg 1 diet, containing 0.03% NT at 1.5% bw day 1

Length of administration

Species

Initial size

Effecta

16 weeks

Hybrid tilapia

21 days old

Growthz, Survivalz

120 days

Hybrid tilapia

30 days old

37 days

Rainbow trout

163.4 – 169.7 g fish

Antibody titer after vaccinationz, Mitogenic response of lymphocytez Growthz

3 weeks

Rainbow trout

217F62 g

2 weeks

Rainbow trout

53 – 55 g

3 weeks

coho salmon

100 g

3 weeks

Atlantic salmon

60 g

Survival after challenge with V. anguillarum z Survival after challenge with infectious salmon anaemia virusz Survival after challenge with Piscirickettsia salmonisz Sea lice infectionA

34.7F9.6 g

Antibody titerz, MortalityA

43F3.0 g

Plasma chlorideA, Growthz

Atlantic salmon

205 g

Intestinal foldz

Common carp

100 g

Phagocytosisz, Respiratory burstz, Complementz lysozymez, A. hydrophila infectionA

3 weeks before Atlantic salmon vaccination and 5 weeks post-vaccination 8 weeks Atlantic salmon

2 g kg 1 diet, containing 0.03% NT at 1.5% bw day 1 2 g kg 1 diet, containing 10 weeks 0.03% NT 15 mg fish 1, by intubation 3 days

NA

120 days

2 g kg 1 diet, containing 15 weeks 0.03% NT to hand satiation daily 5 g kg 1 diet, fixed ration 7 weeks approaching satiation daily

1

ball-femaleQ rainbow 80 –100 g trout Turbot Scophthalmus 120.9F5.1 g maximus

B lymphocytesz, Resistance to IPN virusz, Plasma cortisolA Altered immunogene expression in various tissues

Hybrid striped bass

Neutrophil oxidative radical productionz, Survival after challenge with Streptoccus iniaez

7.1, 9.1 g

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Authors

Symbols represent an increase (z) or decrease (A) in the specified response. 143

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infectious diseases for the aquaculture industry. This review summarizes and evaluates current knowledge of nucleotide nutrition in fishes as compared with that of terrestrial animals.

2. Biochemistry of nucleotides Nucleotides consist of a purine or a pyrimidine base, a ribose or 2V-deoxyribose sugar and one or more phosphate groups. The term nucleotide in this context refers not only to a specific form of the compounds but also to all forms that contain purine or pyriminidine bases. Major purine bases include: adenine, guanine, hypoxanthine and xanthine and form nucleotides through a glycosidic bond between the N-8 nitrogen and C-1V carbon of the pentose, while the C-5V hydroxyl is esterified with the phosphoryl group(s) (Rudolph, 1994). Major pyrimidine bases include: uracil, thymine and cytosine and form nucleotides in a similar way. Purines and pyrimidines are synthesized from de novo pathways or obtained from salvage pathways (reviewed by Rudolph, 1994; Carver and Walker, 1995; Grimble and Westwood, 2000a). Purine rings are synthesized in the cytosol of mammalian cells from glycine, aspartate, glutamine, tetrahydrofolate derivatives and CO2 with considerable energy input, while pyrimidines are synthesized from aspartate, glutamine and CO2 in cytosol and mitochondria of mammalian cells. Presumably these pathways are also operative in fish. The salvage pathway conserves energy and maintains nucleotide homeostasis. It is known from mammalian research that salvage and de novo pathways vary markedly among various tissues and may be significantly influenced by metabolic needs or physiological functions. Although this information needs to be confirmed in fish, nucleotide turnover in erythrocytes, lymphocytes, heart and brain of mammals primarily depends on supply from the salvage pathway. It has been noted that dietary nucleotides modulate liver nucleotide metabolism (Lo´pez-Navarro et al., 1995). The liver is uniquely adapted to rapid induction of nucleotide supply (Grimble and Westwood, 2000a), and it is the most important organ for nucleotide storage and interorgan transport to meet physiological needs.

3. Digestion and absorption of nucleotides and related metabolites Nucleotides are naturally present in all foods of animal and vegetable origin as free nucleotides and nucleic acids. Concentrations of RNA and DNA in foods depend mainly on their cell density (Gil, 2002). Clifford and Story (1976) reported the contents of purines and RNA in some foodstuffs including various organ meats, seafoods and dried legumes. Devresse (2000) also reported the total contents (after complete hydrolysis) of purine and pyrimidine bases in common aquafeed ingredients such as fish meal (1.4%), press cake fish meal (0.4%), fish solubles (2.8%), yeast (0.9%), yeast extract (2.3%) and singlecell proteins (2.1%). Rumsey et al. (1992) reported that 12–20% of the total nitrogen in brewers yeast Saccharomyces cerevisiae, a single-cell protein readily used for aquafeeds, can be composed of RNA nitrogen, mainly in the purine and pyrimidine bases of the nucleoproteins. Based on mammalian research (reviewed by Quan and Uauy, 1991), nucleoproteins are degraded by proteases to peptides and nucleic acids. The nucleic acids are cleaved by nucleases to nucleotides. The phosphate groups of nucleotides are removed primarily by intestinal alkaline phosphatase to nucleosides. Sugars may be cleaved by nucleosidases to produce free purine and pyrimidine bases. The nucleosides and nitrogenous bases are absorbed by the gut mucosa. Studies with human and mice indicated there might be species-specific differences in the digestion and absorption of exogenous nucleotides: human absorb combinations of various nucleotide intermediates; whereas, mice absorb nucleotides mainly in the form of nucleosides (Sonoda and Tatibana, 1978). Research on digestion and absorption of nucleotides by fish is currently limited. The presence and characterization of proteases and alkaline phosphatases in fish intestine have been well studied. However, nuclease, the most important enzyme for nucleotide digestion in fish is poorly understood, although its presence has been reported for some fish such as rainbow trout (Roald, 1978). Therefore, the capacity of digesting exogenous nucleotides by various fishes remains unknown at this time. Two hypotheses have been raised to address why fish need exogenous supplementation of nucleotides.

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Borda et al. (2003) reported that nucleotides in their non-free form or in the form of nucleic acids tend to be tremendously stable and difficult to digest and thus recommended a well-balanced cocktail of free nucleotides to overcome certain stressful conditions, while Burrells et al. (2001b) and Low et al. (2003) hypothesized that there is an adequate supply of exogenous nucleotides in standard commercial fish feed when fish are geared towards rapid growth under normal, non-stressful conditions. To the best of our knowledge, digestibility and bioavailability of nucleic acids in natural feed ingredients such as marine protein sources or brewers yeast for fishes remain unknown, although it appears that fish such as rainbow trout can utilize yeast nucleic acid extracts for growth, nitrogen retention and possibly nonessential amino acid synthesis (Rumsey et al., 1992). In addition, Roald (1978) reported nucleases as well as proteases of rainbow trout could be inhibited by environmental contaminants such as lignosulphonates. This may indicate that digestion and absorption of nucleotides are influenced by various environmental and/or physiological factors. The nucleotide concentration in feed ingredients and bioavailability to fishes as well as information about the nucleotide pools in fishes are not fully defined, and information on how environmental factors such as stressors and pollutants exert their effects on the digestion of nucleotides is not known. These issues are fundamental to obtaining a greater understanding of nucleotide nutrition of fishes and require further research.

4. Chemo-attractive and feeding stimulatory effect of dietary nucleotides It is known that certain nucleotides act as taste enhancers for mammals. Mackie (1973) first analyzed the low-molecular weight fraction of squid and hypothesized nucleotide (AMP) and nucleoside (inosine) components as the main chemo-attractants for aquatic animals such as the lobster. Kiyohara et al. (1975) also reported the presence of chemoreceptors on the lips of the puffer fish that responded to nucleotides (AMP, IMP, UMP and ADP) by electrophysiological methods. These early experiments resulted in an important discovery of the chemoattractive effect of dietary nucleotides on fish. Sub-

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sequently, Mackie and Adron (1978) tested 47 nucleosides and nucleotides and identified inosine and IMP as the most potent gustatory feeding stimulants for turbot based on feeding behavior of fish fed experimental diets. Ishida and Hidaka (1987) tested gustatory sensitivity of various marine teleosts including aigo rabbitfish, isaki grunt, kampachi amberjack, maaji jack mackerel and masaba chub mackerel and found UMP was the most effective for most species, although ADP and IMP also were effective. In addition, Rumsey et al. (1992) also observed that dietary supplementation with 2.5% and 4.1% yeast RNA extract or 1.85% guanine or 2.17% xanthine significantly increased cumulative feed intake of rainbow trout over a 12-week feeding period. The behavioral or gustatory responses of fishes to exogenous nucleotides may be species specific. It was reported that aigo rabbitfish did not respond to any nucleotides as did most other marine teleosts (Ishida and Hidaka, 1987). It also has been noted that publications pertaining to the stimulatory effect of inosine or IMP on various fishes are not consistent. Person-Le Ruyet et al. (1983) reported dietary inosine enhanced growth of turbot larvae. In subsequent research by this group, turbot larvae fed a diet supplemented with both betaine and inosine showed significantly higher growth than larvae fed diets supplemented only with betaine or inosine and a reduced amount of betaine (Me´tailler et al., 1983). However, Ikeda et al. (1991) used jack mackerel as an experimental model and found that IMP, GMP, UMP, UDP, UTP were effective feeding stimulants while nucleosides (including inosine, adenosine, guanosine, uridine) and other nucleotides (AMP, ADP, ATP, IDP, ITP, GDP, GTP, xanthosine 5V-monophosphate, 3VIMP, 3V-UMP, 2-deoxy-IMP, allyltio-IMP) were not. To date, it has been reported that only IMP, but not inosine has stimulatory effects on feeding of fish species including jack mackerel (Ikeda et al., 1991) and largemouth bass (Kubitza et al., 1997). Kubitza et al. (1997) reported that dietary supplementation of IMP (2800 mg kg 1) enhanced feed intake of largemouth bass by 46% compared to the non-supplemented soybean meal-based diet. However, feed intake of largemouth bass fed the soybean meal diet supplemented with either 2800 or 5600 mg kg 1 IMP was inferior to fish fed 10% fish meal. This is possibly

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because marine organisms have relatively high concentrations of IMP; therefore, the beneficial influence of IMP supplementation generally is not noticeable when fish meal is added to aquafeed formulations. IMP may serve as a primary candidate for feed attractant research to further explore complete replacement of fish meal in aquafeeds.

5. Gastrointestinal influences of dietary nucleotides Dietary nucleotides have been shown to have multiple effects on the gastrointestinal (GI) tract in animal models, including physiological, morphological and microbiological influences. It is known that dietary nucleotides or AMP alone can significantly increase growth and differentiation of the developing GI tract (Uauy et al., 1990). These compounds also have been shown to ameliorate intestinal injury and facilitate bifidobacteria predominance in intestine (Carver and Walker, 1995). Morphological responses of human and terrestrial animal GI tracts to dietary nucleotides include increased villus height (Uauy et al., 1990), increased jejunum wall thickness and villus cell number (Bueno et al., 1994). However, research on gastrointestinal responses of fishes to dietary nucleotides is limited at this time except several intestinal morphological studies. Burrells et al. (2001b) first detected morphological responses of Atlantic salmon intestine to dietary nucleotides by histological examination. In that study, the mean fold height of proximal, mid and distal intestine as well as total gut surface area of fish fed a nucleotide-supplemented diet was significantly greater than those of fish fed the control diet. Borda et al. (2003) recently reported similar observations in juvenile sea bream. It appears clear that dietary nucleotides can beneficially influence intestinal health of fish. Because the intestine is a very important immune organ and a significant portion of dietary nucleotides are retained in the GI tract, the possible influences of dietary nucleotides on mucosa associated lymphoid tissue (MALT) has been regarded as a prioritized topic of nucleotide nutrition research, although the knowledge on MALT in fish is very limited. In addition, dietary nucleotides have been reported to promote intestinal microflora such as bifidobacteria in human and various animals. Although bifidobacteria have not been reported as

probiotics in fish, possible effects of dietary nucleotides on microbial ecology of the fish GI tract is an interesting topic deserving further research.

6. Nucleotide effects on fish growth It is generally recognized that under normal conditions, de novo nucleotide synthesis is sufficient to support growth (Cosgrove, 1998), although supplementation of nucleotides was reported to enhance growth of weaning rats fed low-protein diets (Gyo¨rgy, 1971). Borda et al. (2003) reviewed research pertaining to dietary nucleotide application to sea bream larvae and hypothesized that an exogenous supply of nucleotides may promote growth of fish and crustaceans in early stages to meet their high rate of cell replication. Person-Le Ruyet et al. (1983) also reported that turbot larvae (approximately 100 mg/ fish) fed an inosine-supplemented diet (1.3% of diet for 6 days, 0.13% for 45 days) had significantly enhanced growth and survival after a 55-day feeding trial. Their subsequent study showed that 10 or 20 days of feeding a diet supplemented with 0.77% inosine also significantly increased weight gain of turbot larvae (approximate initial weight of 230 mg/ fish). It was hypothesized that the growth-enhancing effect of inosine resulted from improved feed intake at the beginning of weaning, promoting more rapid food intake that reduced nutrient leaching into the water or possibly playing roles in metabolism (Me´tailler et al., 1983). Beside inosine, growth-enhancing effects of nucleotide metabolite mixtures (such as AscogenR, Chemoforma Co., Basal, Switzerland) occasionally have been observed in fish such as tilapia larva (Ramadan and Atef, 1991) and juvenile rainbow trout (Adamek et al., 1996). However, the growth-enhancing effect on most juvenile or sub-adult fishes appears to be rather marginal (Li et al., 2004a).

7. Nucleotides and reproduction One of the most recognized uses of nucleotide supplementation in human nutrition is related to infants. However, research on dietary supplementation of nucleotides as a maternal strategy is limited in humans as well as in fishes. Gonzalez-Vecino et al.

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(2004) first studied nucleotide nutrition of broodstock haddock and observed that first feeding success of larvae from nucleotide-fortified broodstock was significantly higher than that of larvae from broodstock fed a basal diet without supplemented nucleotides. Survival of larvae from broodstock fed the nucleotidesupplemented diet was over 30% greater than that of larvae from broodstock fed the basal diet. In addition, gut development and size of larvae from broodstock fed the nucleotide-supplemented diet were significantly greater than those from broodstock fed the basal diet.

8. Effects of nucleotides on innate immunity It is recognized that dietary nucleotides can influence macrophage activity such as phagocytosis (Grimble and Westwood, 2000b; Gil, 2002) and activity of natural killer cells (Carver et al., 1990). Research on fish also has shown that exogenous nucleotides can influence both homoral and cellular components of the innate immune system. Sakai et al. (2001) showed that exogenous nucleotides could increase serum complement (alternative pathway) and lysozyme activity as well as phagocytosis and superoxide anion production of head kidney phagocytes of common carp. Li et al. (2004a) also reported that hybrid striped bass fed an oligonucleotidesupplemented (Ascogen PR, Canadian Biosystem, Alberta, Canada) diet had higher blood neutrophil oxidative radical production than fish fed the basal diet. However, an effect of dietary nucleotides on respiratory burst of head kidney cells of salmonids was not demonstrated (Burrells et al., 2001a). Devresse (2000) also suggested that exogenous nucleotides are key nutrients for the shrimp immune system. To the best of our knowledge, however, this hypothesis remains to be tested. Low et al. (2003) first reported the changes in immune gene expression induced by dietary supplementation of nucleotides and discovered that nonspecific immune components such as lysozyme expression were significantly decreased in the spleen and kidney of turbot fed a nucleotide-supplemented diet, but no effect was apparent in the gill. Interleukin1h showed a significant increase in expression in the kidney of nucleotide-supplemented fish; whereas,

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expression of transferrin and transforming growth factor h was unaffected by nucleotide supplementation (Low et al., 2003).

9. Effect of nucleotides on adaptive immunity Nucleotides also influence lymphocyte activity and immunoglobulin production. Jyonouchi et al. (1993, 1994) and Navarro et al. (1996) suggested that nucleotides exert their greatest impact on the immune system by modulating immunoglobulin production. Ramadan et al. (1994) first observed that dietary supplementation of nucleotides (Ascogen, Chemoforma Basel, Switzerland) had a marked immunopotentiating effect on both humoral and cell-mediated immune responses of tilapia after intramuscular injection or direct immersion with formalin-killed Aeromonas hydrophila. Antibody titers after vaccination as well as mitogenic responses of lymphocytes from fish fed the ascogen-supplemented diet were significantly and tremendously higher than those of fish fed the basal diet. Similar phenomena were reported on other species such as rainbow trout (Burrells et al., 2001b; Leonardi et al., 2003) and hybrid striped bass (Li et al., 2004a). Burrells et al. (2001b) observed that Atlantic salmon fed a nucleotide-supplemented diet for 8 weeks had significantly enhanced specific antibody production compared to fish fed the basal diet. Leonardi et al. (2003) reported a significant enhancement of lymphocyte stimulation in rainbow trout fed a nucleotide-supplemented diet. The antibody titer of hybrid striped bass fed an oligonucleotide-supplemented diet after vaccination with formalin-killed Streptococcus iniae was three times higher than that of fish fed the basal diet (Li et al., 2004a). Low et al. (2003) also discovered that dietary nucleotides enhanced expression of immunoglobulin M and recombinase activating gene in gill and spleen of turbot but reduced their expression in kidney. Although mechanisms of action are practically unknown, these nucleotides may be used as an boral adjuvantQ and therefore enhancement vaccination efficacy. Burrells et al. (2001b) explored this strategy in vaccination and reduced mortality of vaccinated Atlantic salmon from 6% to 2%. Research on modulation of adaptive immunity by exogenous nucleotides has shown consistent results in various

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fish species; therefore, further research on this subject is warranted.

10. Effects of nucleotides on stress responses One of the most accepted hypotheses on the observed beneficial effects of dietary nucleotides in fishes is that the stressors associated with normal aquaculture conditions and practices such as poor water quality, crowding and handling, place additional demands on available nucleotides beyond those provided in typical aquafeeds such that an exogenous supply may result in beneficial effects (Burrells et al., 2001b; Low et al., 2003). One of the possible mechanisms by which dietary nucleotides beneficially influence the fish immune system is by partially offsetting the inhibitory effects of cortisol release associated with stress. Burrells et al. (2001b) first raised the hypothesis that dietary nucleotides could enhance stress tolerance and provided some evidence by comparing osmoregulatory capacity and growth performance of Atlantic salmon fed a nucleotidesupplemented diet and control diet after acute stress by seawater transfer. This hypothesis was not fully proven until Leonardi et al. (2003) observed that dietary nucleotides reduced serum cortisol levels of healthy rainbow trout after 90–120 days feeding and of fish infected with infectious pancreatic necrosis virus. This stress reduction associated with dietary nucleotides resulted in enhanced disease resistance of challenged fish in the same study. However, a recent study in our laboratory with juvenile red drum failed to confirm this phenomenon because of the extreme variation among individual fish (Li and Gatlin, unpublished data). It remains unknown if exogenous nucleotides are involved in signaling pathways associated with stress responses or if various stressors have specific effects on nucleotide metabolism of fishes.

11. Nucleotides and resistance to infectious diseases Because current methodology to comprehensively investigate immunity and disease resistance of fish is still limited, an effective biomarker for disease resistance of fishes has been difficult to identify.

Therefore, survival after challenge with certain pathogens is usually assessed as a measure of disease resistance. It has been reported that dietary nucleotides can enhance resistance of fishes against various pathogens including viral, bacterial and parasitic pathogens, indicating a promising use of these biochemicals for health management in aquaculture. Burrels et al. (2001a) observed that Atlantic salmon fed a nucleotide-supplemented diet for 2 weeks had a cumulative total mortality of 35.7%, compared to fish fed the basal diet (48%) 53 days after initial contact with fish previously injected with infectious salmon anaemia (ISA) virus. The difference in mortality between the two treatments after 39 and 45 days of cohabitation was statistically significant. Leonardi et al. (2003) also reported that all rainbow trout fed a nucleotide-supplemented diet for 60 days survived after injection with infectious pancreatic necrosis (IPN) virus; whereas, all virus-injected fish fed the basal diet died. Enhanced resistance to various pathogenic bacteria also has been reported for several fish species including salmonids (Burrells et al., 2001a), common carp (Sakai et al., 2001) and hybrid striped bass (Li et al., 2004a). Burrells et al. (2001a) reported that after bath challenge with Vibrio anguillarum, rainbow trout fed a nucleotide-supplemented diet had cumulative mortality of 31%, while mortality of fish fed the basal diet and h-glucan-supplemented diet were 49% and 43%, respectively. Although the high variation between replicates of fish fed the nucleotide-supplemented diet and the basal diet may have masked possible significant differences, fish fed the nucleotide-supplemented diet had significantly higher survival than fish fed the h-glucan-supplemented diet. Burrells et al. (2001a) also reported cohabitation of coho salmon with fish infected with Piscirickettsia salmonis, a rickettsia-like intracellular g-proteobacteria resulted in 76.8% mortality in fish fed a basal diet; whereas, only 46.9% mortality was observed in fish fed the nucleotide-supplemented diet. The mechanism(s) contributing to this significant enhancement in survival after P. salmonis challenge was not fully defined. Sakai et al. (2001) intubated nucleotidesuspended saline or an equal amount of dextrin (control group) orally to common carp and injected the fish intraperitoneally with 0.1 ml of 3107 cells ml 1 suspension of A. hydrophila and determined the

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bacterial number in blood, liver and kidney 2, 4, 8, 12 h after injection. In the blood, kidney and liver of fish treated with nucleotides, no A. hydrophila cells were detected 12 h after challenge; however, the number of bacteria in the blood and liver of control fish reached 1103 cfu ml 1. Juvenile hybrid striped bass fed a nucleotide-supplemented diet for 7 or 8 weeks prior to exposure to S. iniae had reduced mortality (3% trial 1, 13% trial 2) compared to fish fed the basal diet (20% trial 1, 40% trial 2). The mortality of fish fed the nucleotide-supplemented diet after re-exposure to S. iniae was 52% and significantly lower than the 83.8% mortality experienced by fish fed the basal diet (Li et al., 2004a). Because pathogenic bacteria may have various infection routes and induce numerous immune responses of the host (Ellis, 1999), the protective mechanism(s) of exogenous nucleotides might be very diverse. Besides viral and bacterial pathogens, dietary nucleotides significantly reduced the number of sea lice infecting Atlantic salmon (Burrells et al., 2001). Burrells (2001) also reported that dietary supplementation of nucleotides in conjunction with cypermethrin affected the development potential of early chalimus stages of sea lice, thereby reducing the numbers of mobile preadult lice available to cross-infest other fish. Thus, rather consistent results from various experiments have indicated that dietary nucleotides enhance resistance of fishes against numerous different pathogens. This phenomenon may have important application for disease control in aquaculture.

12. Concerns about dose, administration timing and types of dietary nucleotides Dose is a primary consideration in administration of immunostimulants (Sakai, 1999). As for nucleotides, the effort to explore optimization of doses of nucleotides has been limited. Sakai et al. (2001) reported a dose-dependent effect of exogenous nucleotides on macrophage phagocytic activity. Terrestrial monogastric animals generally can not tolerate high levels of dietary nucleotides because of high serum uric acid from purine metabolism and associated toxicity, as well as adverse effects on the metabolism of other nutrients (Rumsey et al., 1992). For example, 4 g of purines and pyrimidines/day is

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regarded as the safe limit to prevent excessive uric acid and gout production (Devresse, 2000). However, fish such as salmonids and sea bass may be able to tolerate a high dietary level of nucleic acids and/or yeast by virtue of their active liver uricase (Kinsella et al., 1985; Rumsey et al., 1992; Oliva-Teles and Goncalves, 2001). Rumsey et al. (1992) reported that yeast RNA extract up to 4.1% of diet did not depress growth of rainbow trout. An earlier study showed the growth-depressing effects of bacterial RNA extract (10% of diet) on rainbow trout associated with increased serum urea and carcass ash content, while RNA extract at 2.5% and 5% of diet did not reduce growth (Tacon and Cooke, 1980). However, Adamek et al. (1996) reported that 0.62 and 2.5 g Ascogen kg 1 diet increased growth and feed efficiency of rainbow trout, while 5 g Ascogen kg 1 diet led to growth depression of rainbow trout and goldfish after 37 days of feeding. Although the conclusiveness of this study was somewhat limited by statistical considerations, it aroused attention to dose-optimization of nucleotide products for aquaculture. It appears very difficult to explain the inconsistency concerning growth-depressing effects of dietary nucleotides on rainbow trout in the preceding studies (Tacon and Cooke, 1980; Adamek et al., 1996), although sources, forms, levels and bioavailability of nucleotides employed in the studies were different. Future progress in identifying molecular mechanisms of nucleotide signaling may provide useful information to address these issues including optimal dosage. Administration regimes for optimum responses are another important issue surrounding application of immunostimulants in aquaculture. In some instances, prolonged administration of immunostimulants such as peptidoglycan and levamisole may cause undesirable side effects on growth and disease resistance of cultured fish (e.g., Matsuo and Miyazano, 1993; Li et al., 2004b). At this time, there is no definitive evidence that efficacy of dietary nucleotides is strictly associated with administration duration. However, Leonardi et al. (2003) observed that mitogenic response of B lymphocytes from rainbow trout was influenced by dietary nucleotide after 60 days of feeding, but not after 120 days. Hybrid striped bass fed an Ascogen PR-supplemented diet for 16 weeks failed to show any enhancement of innate immune responses including blood neutrophil oxidative radical

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production, serum lysozyme and extracellular and intracellular superoxide anion production of head kidney cells, which was not consistent with the results after 8 weeks of feeding the same diet (Li et al., 2004a). These reports make us suspect that administration length or regime should be taken into consideration in the use of dietary nucleotides; however, comprehensive research is needed to clarify the possible effects of prolonged feeding of nucleotides on immunity and disease resistance of fishes. Research to date on dietary nucleotides has focused on a mixture of nucleotides, rather than specific types of nucleotides except inosine and IMP for feeding stimulation research. It is known that the free-base form of adenine in the diet (1.54%) is undesirable because it severely depressed feed intake, weight gain, nitrogen retention and increased mortality of rainbow trout; whereas, other purine bases such as guanine (1.85%), xanthine (2.17%) and hypoxanthine (1.94%) did not show obvious adverse effects (Rumsey et al., 1992). The toxicity of adenine to cells might be related to increased intracellular cAMP levels. Additional research to explore specific effects of various nucleotides is needed to gain a better understanding of this aspect of nucleotide nutrition.

13. Future research The selection of appropriate sources of nucleotide should be the primary consideration to study in the future. To date, most publications on nucleotide supplementation for fishes have used patented or registered commercial products; therefore; information pertaining to concentration and ratio of various types of nucleotides are generally unavailable. Therefore, it is very difficult to quantitatively estimate or compare the effects of supplemented nucleotides on the immune responses of various fish species. For example, yeast such as Saccharomyces cerevisiae is an important source of nucleotides. However, lack of processing procedures or detailed information about these products makes it very difficult to determine if the nucleotides are present in the form of mononucleotides or polynucleotides (oligonucleotides). Based on information from terrestrial animals, mononucleotides and polynucleotides may have different influences on the subject organism. The immunosti-

mulatory effect of microbial oligonucleotides or oligodeoxynucleotide primers also has been shown in vitro (Laing et al., 1999; Meng et al., 2003) as well as in vivo (Tassakka and Sakai, 2002) with fish, although these oligonucleotides were not from yeast. Although early attempts at exploring nucleotide supplementation for fishes have been encouraging, an improvement in methodology, especially the selection of nucleotide sources, is essential to future advancements in this area. Although nucleotides can be experimentally used as alternative nitrogen sources and feeding stimulants, based on the current knowledge in this area, health management is the most promising use of dietary nucleotides for the aquaculture industry. However, current research on dose, administration regime, even nucleotide type is rather limited to ensure the efficacy of these biochemicals under various physiological and ecological situations. Research on possible age/size related responses of fish, especially sub-adult fishes, to dietary nucleotides is not sufficient. In addition, the digestion and absorption processes of nucleotides should be studied in greater detail. At least the form(s) of nucleotides most readily absorbed by fish should be screened for development of dietary supplements. Grimble (2001) addressed concerns about the sensitivity of various genotypes to immunonutrients, as genetic polymorphism influencing pro-inflammatory cytokine production has been discovered. To the best of our knowledge, this type of information is not available for fish, although it is well established that genetic polymorphisms influence growth and some physiological responses such as lysozyme production of fishes. Although this topic is difficult to explore, genetic variation might contribute to inconsistencies in research findings on nucleotide nutrition of fishes. Exogenous nucleotides may be involved in cell signaling pathways as well as serve as nutrients for biosynthesis. The influences of nucleotide availability on gene transcription rate have been reported in murine intestinal epithelial cells (Walsh et al., 1990, 1992) and intestine (Valde´s et al., 2000; Sa´nchez-Pozo and Gil, 2002). The specific responsible cis-acting element of genes such as hypoxanthine phosphoribosyl transferase as well as the binding protein has been characterized to be sensitive to nucleotide availability (Walsh et al., 1990, 1992). To precisely and compre-

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hensively detect various alterations in expression of specific genes in various tissues and organs of fish, development and application of novel tools in molecular biology will be necessary to further support research on nucleotide nutrition of fish. Low et al. (2003) set an example for future research in this area. Progress in deciphering genomes of fishes has assisted in promoting the development of powerful analytical tools such as microarrays that may shed light on the various influences of nucleotides. However, tremendous efforts in this area are needed to establish a reference knowledge base.

Acknowledgements The funding for P. Li was provided in part by a Graduate Merit Fellowship, by a Tom Slick Fellowship and by a Graduate Research Scholarship awarded through Texas A&M University’s College of Agriculture and Life Sciences, and by a Billy Cooper Graduate Research Scholarship awarded by the Texas Aquaculture Association.

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