Functional foods for aquaculture: benefits of NuPro® and dietary nucleotides in aquaculture feeds

In recent years, the concept of ‘functional foods’ has developed in human and animal nutrition. Functional foods, which first emerged in Japan, have been defined as ‘foods or dietary components that may provide a health benefit beyond basic nutrition’ (IFIC, 2004).

The presence of biologically active components of functional foods can have an impact on health benefits or other desirable physiological effects in addition to their nutritional function. The term ‘functional foods’ covers a broad range of products including, for example, DHA- and selenium-enriched eggs, selenium-enriched pork, stanol- and sterol-enriched margarine, etc. Also included under this category are dietary ‘probiotics’ and ‘prebiotics’.

A probiotic is defined, in the strict sense, as “a viable microbial dietary supplement that beneficially affects the host through its effects in the intestinal tract” (Roberfroid, 2000). It should be noted that the term has also been widely and incorrectly applied in aquaculture, and especially shrimp culture, to include the use of live microbes to beneficially alter the microbial balance in the culture system itself.

Prebiotics, on the other hand, have been described as “non-digestible substances that … provide a beneficial physiological effect on the host by selectively stimulating the favorable growth or activity of a limited number of indigenous bacteria” (Gibson and Roberfroid, 1995).

A ‘functional nutrient’ can be further defined as a dietary ingredient that exerts possible positive effects on health in addition to its direct role as a nutrient. This paper will review some of the work that has been conducted with NuPro®, a proprietary extract produced from a specific strain of yeast, as a ‘functional nutrient’ in various aquaculture species.


NuPro®

Alltech manufactures a range of products from a specific strain of the yeast Saccharomyces cerevisiae. The cell wall of the yeast is removed and processed to yield products such as Bio-Mos® and Mycosorb® while the cell contents are further processed to produce NuPro®.

As a yeast extract, NuPro® is a complex ingredient, since it combines nutritional components, such as protein and vitamins, with more functional components such as nucleotides and free amino acids. Thus, the benefits of using NuPro® in feeds are both nutritional, and as a functional nutrient.


NUPRO® AS A PROTEIN SOURCE

The search for alternatives to fish meal as a source of protein in aquaculture diets has been an important area of research in recent years. Much of this research has focused on increasing the proportion of plant proteins, such as soybean, in feeds for fish and shrimp. However, many sources of vegetable protein have disadvantages, including low nutrient densities, anti-nutritional factors, high carbohydrate content, imbalanced amino acid and fatty acid profiles, low palatability, seasonal variability and potential mycotoxin contamination (Ceulemans et al., 2003; Spring and Fegan, 2005).

Single cell protein (SCP) sources have several advantages, not least in their ability to produce large quantities of heterotrophic SCP through fermentation. However, many SCP have drawbacks such as digestibility of the cell, low protein content, and poor amino acid profile. Nevertheless, many aquaculture feeds contain some SCP, usually yeast, at levels from 1-5% of the diet.

One of the difficulties of using yeast in feeds is that the yeast cell wall reduces digestibility and, since it may represent up to 50% of the cell weight, has a low protein content. The digestibility of the yeast can be increased by enzyme hydrolysis to yield hydrolysed yeast, which breaks down some of the cell wall structure, allowing more effective digestion in the animal intestine. However, since the cell wall material remains, the protein content remains low although overall digestibility is increased.

Complete removal of the cell wall yields yeast extract. The protein in yeast extract is highly digestible and, as the cell wall is removed, has a higher protein content than either whole or hydrolysed yeast. In the case of NuPro®, the crude protein content is around 47-50%, similar to processed soybean meals.

Fish and shrimp, like other animals, do not have a requirement for protein but for a well-balanced mixture of essential and non-essential amino acids from which to construct their own tissue proteins (Wilson, 2002). Crude protein levels, therefore, are less important than the balance of essential amino acids in the diet. A comparison of the essential amino acid profile of NuPro® with fish meal and soybean meal shows that comparatively, it appears to be deficient in most essential amino acids (Figure 1). However, a comparison between NuPro® and typical essential amino requirements for fish and shrimp shows that it provides a close match (Figures 2 and 3).

The use of NuPro® as a protein source has been investigated for a number of fish and shrimp species including tilapia, cobia, black tiger prawn and Pacific white shrimp. In a series of trials to develop organically certifiable feeds for fish and shrimp, it has been shown that complete replacement of fish and soybean meal with NuPro® is possible, although cobia, a marine carnivore, showed a reduced growth rate at levels of 50% replacement and higher (Craig and McLean, 2005).


NUPRO® AS A FUNCTIONAL NUTRIENT

Considered solely as a protein source, NuPro® represents an excellent nutrient given its high digestibility, availability and, as a result of the control of the manufacturing process, consistency. However, in addition to the purely nutritional aspects, NuPro® has also been shown to have significant benefits as a functional nutrient. NuPro® represents a rich source of nucleotides, estimated to be around 5% of the dry weight. Most of the nucleotides in NuPro® are soluble, making them much easier to assimilate than nucleotides bound in insoluble forms such as nucleoproteins (D’Souza, personal communication).




Figure 1.Comparison of the EAA profile of NuPro®, fish meal and soybean meal (Note: fish meal and soybean meal methionine values are given for cystine and methionine combined).





Figure 2.Comparison of typical EAA requirement of fish and EAA profile of NuPro®.





Figure 3. Comparison of typical EAA requirement for shrimp and EAA profile of NuPro®.



Nucleotides

Nucleotides are among the basic building blocks of life. They are low molecular weight biological compounds consisting of a nitrogenous base (either a purine or a pyrimidine) linked to a pentose sugar with at least one phosphate group attached (Figure 4). If the phosphate group is absent, the compound is referred to as a nucleoside. Pyrimidine bases comprise uridine (U), cytosine (C), and thymine (T) whereas purine bases comprise adenine (A), guanine (G), and hypoxanthine, a breakdown product from either inosine or adenosine that is converted into inosine 5'monophosphate (IMP), a purine-based nucleotide. A group of nucleotides linked together forms a nucleic acid, either RNA (ribonucleic acid) when the sugar is ribose or DNA (deoxyribonucleic acid) if the sugar is 2'-deoxyribose.




Figure 4.Generalised structure of nucleotides (from Mateo and Stein, 2004).



In the form of nucleic acids, nucleotides are of fundamental importance as the basis of the genetic code. Genetic information is stored in DNA (except in the case of RNA viruses) providing the basic information coding for all the proteins produced in the body whereas RNA acts as a chemical messenger relaying the information stored in DNA from the nucleus to other parts of the cell.

Other than their role in genetics and protein production, nucleotides also play major roles in almost all biological processes including:

• Storage of energy, mainly through adenosine tri-phosphate (ATP).

• As components of several important coenzymes such as nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), flavin adenine dinucleotide (FAD) and coenzyme A, all of which are involved in carbohydrate, protein and fat metabolism (Mateo, 2005).

• Mediation of important cellular processes through messengers such as cyclicadenosine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP).

• Control of several enzymatic reactions.

• Serving as intermediates in biosynthetic reactions, especially in glycogen and glycoprotein synthesis and synthesis of polyunsaturated fatty acids (Gill et al., 1985).

Nucleotides have been recognised as important elements in mammalian nutrition especially during periods of rapid growth or physiological stress as well as appearing to play a key role in efficient immune system function (Uauy, 1989; Barness, 1994; Van Buren, 1994). They can be synthesised directly or scavenged by salvage pathways in the body although it appears that exogenous dietary sources are preferentially used (Uauy, 1994). However, immune cells and intestinal cells cannot synthesise nucleotides and depend on nucleotides from other sources (Quan, 1992).

Synthesis and salvage of nucleotides are thought to be energy intensive in metabolic terms and dietary nucleotides may reduce the metabolic cost of de novo nucleotide synthesis. Dietary sources of nucleotides may also benefit rapidly dividing tissues, such as those of the immune system, especially under a challenge, and the term ‘conditionally essential’ has been used to describe their role in nutrition (Carver and Walker, 1995).

In many biochemical processes, primary nucleotides such as 5'AMP, 5'CMP, 5'GMP, 5'IMP and 5'UMP are used to produce a number of intermediate metabolites through a series of enzymatic reactions. Supplementation of primary nucleotides in the diet provides a ready source of nucleotides for use in the synthesis of intermediate nucleotides when required (Mateo, 2005, personal communication).


SOURCES OF NUCLEOTIDES

Any ingredients of animal and plant origin containing cellular material are potential sources of nucleotides, usually in the form of nucleoproteins. The nucleotide content is particularly high in ingredients such as fish solubles, animal protein solubles, fish meal, legumes (adenine content is particularly high in black-eyed peas), yeast extracts and unicellular organisms such as yeasts and bacteria that are rich in RNA or DNA.

The content, proportion and availability of nucleotides differs among ingredients. Muscle protein is a poor source of nucleotides as they are mainly in the form of actin-myosin protein. Oilseeds, such as soybeans, grains, fruits, vegetables and processed milk products are also poor sources of nucleotides (Barness, 1994; Devresse, 2000; Mateo, 2005).

Among marine protein sources, anchovies and sardines, for example, have much higher guanine levels than squid, clams or mackerel. Availability and digestibility are also important issues. Whole yeast is much less digestible than yeast extract, possibly due to the need to digest the yeast cell wall and yeast extract having much higher levels of soluble protein. Fish and animal protein solubles are highly digestible but they leach easily, affecting overall availability (Devresse, 2000).


FEEDING STIMULANT/ATTRACTANT PROPERTIES

Aquaculture feeds need to be both attractive and palatable, especially for slow feeding species such as shrimp. Feeds that are less attractive will be located and consumed more slowly resulting in a loss of their nutritional value due to leaching of water soluble components.

However, in addition to being attractive, feeds must also be palatable to avoid being rejected. Although typical diets containing marine meals and oils such as those from fish and squid are thought to be sufficiently attractive and palatable, this does vary and, as more plant proteins and oils are used in feeds, attractability and palatability can become an issue.

In such cases, feed stimulants, attractants and palatability enhancers may provide benefits. Such compounds include free amino acids, betaine, nucleotides, nucleosides, amines, sugars and other hydrocarbons, organic acids, alcohols and aldehydes, and their mixtures (Kasumyan and Døving, 2003) NuPro® is a rich source of compounds such as nucleotides and free amino acids that are known to be potent feeding stimulants and attractants. NuPro® contains significant amounts of glutamic acid, glutamate and active nucleotides such as 5'-IMP and 5'-GMP that are known to enhance flavour (Diehl, 2004).

Inosine monophosphate, for example, increased feeding in a number of fish species, including mackerel, turbot and largemouth bass and specific receptors for nucleotides are found in the olfactory organs of fish (Mackie and Adron, 1978; Ishida and Hidaka, 1987; Ikeda et al., 1988, 1991; Kubitza et al., 1997; Kiyohara et al., 1975). Synthetic nucleotide mixtures were also found to be highly attractive to crustaceans, including shrimp, crabs and lobster (Mackie, 1973; Carr et al., 1984). Takeda and Takii (1992) later reported that diet supplementation with amino acids and nucleotides stimulated feed intake in the Japanese eel (Anguilla japonica) as well as enhanced growth performance.

Free amino acids have also been shown to be powerful attractants and feeding stimulants for a range of fish species. Kasumyan and Døving (2003) reported on the response to common free amino acids for 21 fish species and found that most fish species showed a stimulant response to a number of amino acids. The response appears to be highly species-specific and also depends on the conformity of the amino acids since L-isomers were usually more highly palatable.

Free amino acids are highly water soluble and easily diffused in water, an important consideration in aquaculture situations. Several non-essential amino acids such as L-alanine, L-glutamic acid and glycine have been reported to have dietary attractant properties. However, it should be noted that the individual amino acids are less effective than mixtures, especially with synergistic compounds such as glycine, betaine or inosine (Polat and Beklevik, 1999).

Although there are no detailed studies to date on the specific effects of NuPro® as an attractant or palatability enhancer, shrimp fed diets containing NuPro® have better feed conversion ratios than those fed conventional feeds. In one trial, carried out with Litopenaeus vannamei in tanks, the average feed conversion ratio using a diet containing 2% NuPro® was significantly lower than the control diet after 90 and 120 days of culture (Figure 5).




Figure 5.Comparison of feed conversion ratio of Litopenaeus vannamei fed on a standard shrimp diet and a similar diet containing NuPro® (2%).



Similar results have been noted in commercial farm trials in which the use of NuPro® at 2% in the feed improved the FCR by 14% compared to control ponds given standard feed. This represents a considerable economic benefit given that feed generally accounts for around 50% of the production cost of shrimp.

Although NuPro® as a protein source will contribute to improved growth, the improvement in FCR may be at least partly due to improved attractability and palatability of the feed. Anecdotal reports from farmers suggest that shrimp consume NuPro®-enriched feeds more quickly than standard feeds. If this is indeed the case, then there will be less time for water-soluble components of the feed to leach and the feed as consumed will be more nutrient-dense, contributing to the improved feed conversion ratio.


IMMUNE-STIMULANT PROPERTIES

The ability of NuPro® to increase the level of immune response has been demonstrated in shrimp. Sritunyalucksana et al. (2005) found that NuPro® (incorrectly identified as Netrepro™ in the publication) fed at 2% and 4% in the diet for 4 weeks resulted in an increase in total hemocyte count as well as significantly increasing the number of granular hemocytes, from 2.8 x 105/mL to over 7.0 x 105/mL. This increase was also reflected in an improved rate of bacterial clearance from the hemolymph following an injection challenge using an elevated dose (109 CFU/mL) of the shrimp pathogen Vibrio harveyi.

Interestingly, no difference was found between the granular hemocyte count or bacterial clearance between the 2% and 4% NuPro® treatments although the initial bacterial clearance rate may have been faster at the higher concentration (Figure 6).




Figure 6.Comparison of bacterial clearance rate from the hemolymph in shrimp fed NuPro® at 0, 2 and 4% of the diet (from Sritunyalucksana et al., 2005).



Similar results were also reported by Ancieta-Pröbstl et al. (2005) for P. monodon and P. merguiensis in diets supplemented with nucleotides. They reported that for both species, increases in numbers of total hemocytes and granular hemocytes as well as an increase in prophenoloxidase activity were noted in the nucleotide-supplemented treatments.

Commercial trial data (Mendoza et al., 2001, unpublished report) from a farm in Ecuador also suggested that NuPro® may play a role in reducing the severity of shrimp viral diseases such as White Spot Syndrome Virus (WSSV). In a series of three pairs of ponds, all of which had been exposed to WSSV, ponds in which NuPro® was added to the feed at 1% showed consistently higher average body weight, survival and yield as well as reduced feed conversion (Table 1).


Table 1.Comparison of production parameters in WSSV-exposed shrimp in paired ponds given control and NuPro® (1%) diets.




Although the precise mechanism is as yet unknown, it is likely that nucleotides account for at least some of the increased immune response. Research has shown that dietary nucleotide supplementation has been associated with improved humoral and cellular immunity in animals (Stein and Mateo, 2005).

In mammals, dietary nucleotide supplementation has been shown to influence immune function and appears to facilitate phagocytosis, increase natural killer cell activity and cytokine production (e.g., interleukin (IL)-2) (Carver, 1994). In fish, an elevated immune response was seen in tilapia vaccinated with Aeromonas hydrophila vaccine (Ramadan et al., 1994) and increased growth rates of rainbow trout (Adamek et al., 1996) were achieved with diets supplemented with nucleotides.

Burrells et al. (2001a) found that supplementation of the diet with exogenous nucleotides could have a positive influence on resistance of Atlantic salmon, coho salmon and rainbow trout to bacterial (Vibrio anguillarum), viral (Infectious Salmon Anemia), rickettsial (Piscirickettsia salmonis) and ectoparasitic (sea lice – Lepeophtheirus salmonis) infection. The nucleotide diet also appeared to be more effective than a diet supplemented with ß-glucan. ß-glucans primarily enhance the non-specific immune system and the authors speculated that the additional benefit of dietary nucleotides might be through enhancement of the potential of the specific immune system to mount greater and more rapid responses.

Further trials with Atlantic salmon (Salmo salar) indicated that fish fed supplementary nucleotides at a combined inclusion level of 0.03% (equivalent to 300 g per tonne of feed) significantly increased antibody titres following vaccination against Aeromonas salmonicida (the causative organism of furunculosis) and reduced mortality following a subsequent challenge. An increase in red blood cells and a significant increase in growth rate following feeding with the nucleotide-supplemented diets, possibly due in part to an increase in gut mucosal surface area through a significant enhancement in intestinal fold morphology (Burrells et al., 2001b), was also observed.

Low et al. (2003) also found that a diet supplemented with cytidine-5'-monophosphate (CMP), disodium uridine-5'-monophosphate (UMP), adenosine-5'-monophosphate (AMP), disodium inosine-5'-monophosphate (IMP), disodium guanidine-5'- monophosphate (GMP) and RNA to give a combined nucleotide inclusion level of 0.03% significantly increased the expression of key immune genes in the gill and spleen of turbot as well as an increase in expression of interleukin-1h in the kidney.


OTHER BENEFITS OF NUCLEOTIDE SUPPLEMENTATION

Nucleotides may be especially important during periods of rapid growth or cell replication such as early development of larvae, reproduction or in times of stress or disease when blood cell production increases rapidly. It has been shown that the requirement for exogenous nucleotides can increase considerably in mammals during periods of rapid growth or physiological stress (Carver and Walker, 1995). Activation of lymphocytes, for example, causes a rapid increase in the synthesis of nucleotides, which are required first for the increase in energy metabolism and later as precursors for nucleic acid synthesis.

Aquaculture practices often place a great deal of physiological stress on the animals, which can result in immune suppression, reduced growth rate and increased susceptibility to disease (Anderson, 1996). Under such conditions, a nucleotide-supplemented diet may provide benefits. For example, salmon fed a diet supplemented with nucleotides appeared to have a greatly enhanced capacity for osmoregulation (Burrells et al., 2001b).

This suggests that nucleotide supplements may be useful in specific diets for salmon smolts prior to transfer into seawater.

There is a high requirement for nucleic acid and protein synthesis during reproduction and egg development and it has been suggested that adding nucleotides to broodstock diets may have a beneficial effect on egg development. Gonzalez-Vecino (2002; 2003; 2004) investigated the effect of nucleotide-enriched diets for broodstock nutrition in Atlantic halibut (Hippoglossus hippoglossus) and haddock (Melanogrammus aeglefinus).

In halibut, feeding nucleotide-enriched diets (0.5% for haddock and 0.8% for halibut) to broodstock resulted in a 20% increase in relative fecundity (no. of eggs/kg female body weight). A similar trend was observed in haddock with some slight differences noted between the 2002 and 2003 seasons. Hatching rate of eggs was also significantly higher in both species as was the larval quality and survival at the end of the yolk sac stage. Experiments on first feeding with both species also showed that larvae from broodstock fed nucleotide-enriched feeds had better intestinal development, first feeding success (FFS) and larval performance. These results suggest that nucleotide supplementation of broodstock diets may be one way to improve reproductive and larval performance.


Feed formulation with NuPro®

Current recommendations are to use NuPro® at between 1% and 2% in commercial feeds (10-20 kg/mT). NuPro® can be added in feed formulations either ‘over the top’ or, more cost-effectively, as a replacement for existing protein sources in the diet such as fish meal or soybean meal. Other ingredients, such as immune stimulant compounds and specific attractants may also be reduced or removed when using NuPro® in the feed.

Alternatively, NuPro® can be included in formulations as an additive to provide a given level of nucleotide. Many of the benefits of nucleotides can be seen when adding nucleotides at 0.02 – 0.03%. Supplying these through the use of NuPro® would require between 4 – 6 kg NuPro®/mT of feed.

A nucleotide-enriched NuPro®, NuPro® 15, has also been developed specifically for use in aquaculture as a nucleotide supplement. The nucleotide content of NuPro® 15 is, as the name suggests, 15%. In addition to the typical benefits of NuPro®, NuPro® 15 has a high concentration of 5'AMP and 5'IMP, both of which have documented biological and physiological benefits (C. D. Mateo, 2005, personal communication).

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