Evaluation of zinc and selenium availability in rainbow trout based on organic sources: a review and preliminary investigations of Bioplex® Zn and Sel-Plex®

In recent years there has been particular interest in the role of zinc (Zn) and selenium (Se) in the maintenance and promotion of animal health. Selenium is well known for its anti-cancer and powerful antioxidant properties (Ganther, 1999; Rayman, 2000; Surai, 2002). Zinc is known for its essential role in growth, immunity and DNA replication. Research on all essential elements, including zinc and selenium, has concentrated on higher organisms, with work on fish being rather fragmentary (Watanabe et al., 1997).

Mineral requirements have been determined for only a few commercial fish species, namely catfish and salmonids, and are based on limited and dated research. Establishing optimal fish health is key to maximising aquaculture production, profitability, and minimising environmental impact. However, the roles of trace minerals on certain aspects of fish health, particularly immunity, are largely unknown (Lall, 2000). The recent development of novel organic minerals may prove to be the solution to problems associated with the use of inorganic mineral supplementation, such as toxicity, low bioavailability and excessive wastage.

This paper will review the bioavailability of organic versus inorganic zinc and selenium in fish focusing particularly on commercially farmed salmonids. Some preliminary results are also reported.


Selenium metabolism

UPTAKE, EXCRETION AND STORAGE IN FISH

Selenium occurs inorganically in nature as selenite, selenate and selenide. In feeds and plant materials it is combined with the amino acids cysteine and methionine to form selenocysteine (SeCys) and selenomethionine (SeMet), respectively. Selenium is present as SeCys in around 30 mammalian proteins (Brigelius-Flohe, 1999). SeCys is at the active site in all selenoproteins, of which many are antioxidant enzymes involved in redox reactions (Arthur, 2000).

The metabolism of selenium by fish seems to depend on its route of uptake. Dietary selenium is transported to the liver by the circulatory portal, whereas waterborne selenium is transported to all organs except the liver (Hodson and Hilton, 1983). Selenite is efficiently absorbed by the gills and absorption is a function of the waterborne concentration, not tissue concentrations (Hilton et al., 1980). Excretion of waterborne selenium is directly proportional to exposure levels suggesting excretion is by a passive diffusion process (Hodson and Hilton, 1983).

Dietary absorption of selenite by the small intestine is very efficient, but decreases as dietary level increases (Hilton et al., 1982). This decline is either due to an increased elimination or less efficient uptake at high dietary levels, the former being favoured. As in mammals, the kidney normally has the highest selenium concentration, but at dietary levels above 1.23 mg Se/kg levels are highest in the liver. Therefore the liver may play a role in selenium storage in fish, as well as in metabolism and elimination (Hilton et al., 1980; 1982).

The excretion pathway of selenium is not known in fish but is thought to be through the urinary system via the kidney, the respiratory system via the gills, and a fixed proportion by the faeces, either via bile secretions or release into the caecum (Hilton et al., 1980). Faecal selenium is not affected by dietary level but is affected by injected selenium, suggesting that faecal selenium arises from selenium released into the intestinal tract rather than that which is unabsorbed (Hilton et al., 1982).

Unlike the passive excretion of waterborne selenium, the excretion of dietary selenium increases disproportionately with selenium loading, suggesting it involves an ‘active’ energyrequiring metabolic process (Hodson and Hilton, 1983), which indicates a difference in metabolism between dietary and waterborne forms. Increased liver concentrations at high dietary and waterborne levels may suggest a limit in the methylation of selenium for excretion (Hilton et al., 1982). Like mammals, selenium homeostasis in fish must be regulated by excretion rather than absorption (Hilton et al., 1980).

The highest concentrations of selenium are found in the liver, gill, kidney and intestinal tract, with low levels being found in whole blood. As the bulk of selenium in fish is found in the plasma, this suggests it is a selenium transport medium rather than a storage organ (Hilton et al., 1982). The tissue selenium concentrations of organs change disproportionably with varying dietary selenium status. The kidney has the highest level of all tissues in salmonids (as high as 0.35 mg Se/kg); the liver has the next highest levels, possibly due to the activation of a selenium excretory pathway in this organ (Hilton et al., 1982).

Absorption of SeMet is more efficient than selenite and as with mammals SeMet is incorporated non-specifically into skeletal muscle proteins (Bell and Cowey, 1989; Wang and Lovell, 1997). There is no evidence that SeCys is incorporated non-specifically in salmonids (Bell and Cowey, 1989). Similarities in selenium metabolism between mammals and fish suggest that two selenium pools are present in fish, with the skeletal muscle also acting as a long-term selenium pool (Lorentzen et al., 1994). Slight differences may occur in selenium metabolism between fish species, since Gatlin and Wilson (1984a) found an increase in catfish muscle selenium when fed selenite, however this does not occur in salmonids (Lorentzen et al., 1994).


MEASUREMENT OF SELENIUM STATUS IN FISH

There are few methods currently used to measure selenium status. Glutathione peroxidase (GSH-Px) activity has been widely used to measure selenium status for over 30 years.

However, only a minor proportion of an organism’s selenium content is present as GSH-Px (Behne and Wolters, 1983). Future studies should include other seleno-enzymes that have been more recently identified and may require higher levels of dietary selenium for optimal activity. A direct relationship between GSH-Px activity and selenium requirement for all tissues cannot be assumed, and solely relying on this enzyme to assess selenium status may no longer be appropriate (Brown and Arthur, 2001).

GSH-Px activity correlates well between adequate and marginal nutritional selenium levels, but plateaus above requirement levels in mammals (Neve, 2000). This plateau also occurs in fish and may be due to the possible homeostatic role of cytosolic GSHPx. In salmonids, plasma and liver GSH-Px activity plateaus around the selenium requirement (Hilton et al., 1980). In fish this may be dependent on the tissue type and the species. In catfish Gatlin and Wilson (1984a) found a plateau at 0.5 mg Se/kg in liver but not in the plasma, which increased with dietary selenium up to 5 mg/kg. Contrary to several previous studies, Felton et al. (1996) found maximal GSH-Px activity in salmonids at a dietary level of 11.1 mg Se/kg; far above the plateau found in any previous study.

GSH-Px activity:tissue Se ratios have been used in salmonids as a measure of the proportion of tissue selenium used in active proteins. These ratios are higher in the liver than in plasma in fish (the higher the ratio, the greater the proportion of selenium as GSH-Px), indicating that more selenium is used in active selenoproteins in the liver, which being an organ of selenium metabolism, is expected (Bell and Cowey, 1989). It may also be caused by the fact that plasma GSH-Px was measured rather than erythrocyte or whole blood GSH-Px.
Studies in animals and cell lines suggest thioredoxin reductase (TrxR) may also be an indicator of selenium uptake. Levels of this enzyme have been found to increase in supra-nutritional dietary levels of selenium but may decrease with continued high dietary selenium (Neve, 2000). As there are two redox centres in TrxR, insulin is used as a substrate if assessing selenium status, so only activity from the SeCys residue is measured (Ganther, 1999). There are no published data on TrxR activity in fish to date.


THE SELENIUM REQUIREMENT OF SALMONIDS

The dietary selenium requirement has been determined for rainbow trout based on maximal plasma GSH-Px activity, using sodium selenite supplementation and a purified diet. The requirement was established as 0.35 mg Se/kg (Hilton et al., 1980; Gatlin and Wilson, 1984a).

However, a requirement based on only one enzyme is questionable, as it does not take into account the other functional roles of selenium (e.g., immunomodulation or genomic stabilisation), which may require higher selenium status. Moreover, selenium requirements have not been determined for organic selenium sources. In catfish, based on growth the requirement for selenite is 0.28 mg Se/kg, but for SeMet and selenoyeast this requirement is reduced to 0.09 and 0.11 mg Se/kg, respectively, highlighting the importance of assessing and comparing sources (Wang and Lovell, 1997).

The measurement of a requirement should include a suite of biochemical markers involving several active proteins and should include functional markers, such as effects on genetic stability, antioxidant status and immune status. In addition, selenium requirements in salmonids may not be rigid, and may possibly decrease with age (Hilton et al., 1980) and could depend on previous dietary selenium intake (Lorentzen et al., 1994). No studies have quantified these effects in fish.


SELENIUM BIOAVAILABILITY IN FISH

Ingested selenium is ultimately required for important metabolic processes. However, selenium deposited in the tissues may not necessarily be available for utilisation in active proteins. SeMet effectively increases apparent selenium status as it is nonspecifically incorporated into proteins, e.g., albumin and haemoglobin, but it has no catalytic activity and must be mobilized and converted to hydrogen selenide before being used for selenoprotein synthesis (Rayman, 2000). Organic selenium, especially SeMet, is reportedly more bioavailable than inorganic selenium in both mammals and fish.

Selenium bioavailability in fish has been assessed using digestibility, GSH-Px activity, tissue selenium levels, growth, and responses to bacterial challenge. Bioavailability studies in fish come to different conclusions, but this may be due to differences in methods. SeMet was found to be significantly more digestible than both selenite and SeCys in salmonids. Yet, based on GSH-Px activity alone, selenite would appear to have the greatest bioavailability. In salmonids lower plasma GSH-Px:Se ratios result from SeMet-supplemented and fish meal-based diets than from selenite or SeCyssupplemented diets. Selenium bioavailability studies in fish at sub-optimal dietary levels show differences in growth and GSH-Px activity, but not at higher levels.

In salmonids dietary SeMet, but not selenite, is incorporated into muscle. Selenoyeasts also contribute to selenium deposition in the muscle, which is expected as selenoyeasts contain predominantly selenomethionine (Lorentzen et al., 1994). In catfish selenite can however accumulate in the muscle, but to a much lesser extent than organic selenium sources (Wang and Lovell, 1997). An elevated selenium level in fish fillets would provide an additional source of selenium to the consumer, which is particularly important in regions where diets are selenium deficient (Rayman, 2004).

The benefits of SeMet may be due to selenoproteins other than GSH-Px. Thus, the outcomes of bioavailability studies are dependent on the parameters measured. In chicks, SeMet was four times more efficacious in the prevention of pancreatic atrophy than selenite or SeCys, but the latter were more effective at preventing pancreatic diathesis (Cantor et al., 1975).

In fish SeMet was more effective than selenite in preventing mortality from an E. ictaluri challenge, with SeCys being intermediate in its protective effects. This was also accompanied with an improved immune response and weight gain for organic sources (Wang et al., 1997).

To be bioavailable selenium must first be effectively digested. The absorption of selenium may be affected by the following: forms of selenium present in fish feed; interactions with other nutrients, e.g., the antagonist effects of copper (Lorentzen et al., 1998); chelation effects; and the adsorption of elements on organic and inorganic surfaces. Selenium availability in chickens, assessed as the ability to prevent exudative diathesis, was 86% for corn, 60% for soybean meal and only 16-25% for fish meal (Cantor, 1975).

These findings show that although plant-derived feeds contain lower levels of selenium than fish meal, the selenium within plant materials is more digestible. Since animal-based feedstuffs are generally more digestible than plant-based, this would suggest overall diet digestibility is not responsible for low selenium digestibility in fish meal. The low digestibility of selenium in fish meal has also been found in fish: 47% for a fish meal diet compared with a purified diet with selenium added as selenite (63%), SeCys (52%) and SeMet (91%) (Bell and Cowey, 1989). Future digestibility studies should assess organic sources such as selenium yeast in practical diets using the yttrium oxide method, which has proved to be more effective and accurate than the chromic oxide method used in most selenium digestibility studies to date (Ward et al., 2005).


Zinc metabolism

UPTAKE, EXCRETION AND STORAGE IN FISH

Unlike mammals, fish have two routes of zinc uptake, since waterborne zinc can be absorbed via the gills through apical calcium channels in the chloride cells. Drinking of freshwater by trout is low and freshwater zinc levels are low, typically <10 μg/L, so the highest possible contribution by drinking water would only be 17% of available dietary intake. Therefore uptake via the gills is the main uptake of waterborne zinc (Spry et al., 1988). Only high waterborne zinc levels can contribute significantly to body zinc content and be at a sufficient level to avoid severe deficiency (Spry et al., 1988).

In aquatic systems most zinc is sequestered in sediments and is almost entirely adsorbed onto dissolved organic and inorganic compounds in the water column. It is only in the gastrointestinal (GI) tract that zinc is desorbed from food and ingested particulate matter, thus making it available. Consequently, uptake of dietary zinc via the GI tract is of much greater importance to body zinc status (Glover and Hogstrand, 2002a; Spry et al., 1988). The gut has a much greater capacity for zinc uptake, with a maximal uptake rate of 933 nmol/kg/h, compared with 240-410 nmol/kg/h for branchial uptake (Bury et al., 2003). There appears to be no interaction between the uptake of waterborne and dietary zinc (Spry et al., 1988).

Dietary uptake may be under intestinal regulation in rainbow trout (Glover and Hogstrand, 2002a). Dietary zinc is excreted via the gills, which is supported by the fact that zinc is actively transported from the blood to the gills (Hardy et al., 1987; Maage and Julshamn, 1993). Similar to mammals, zinc is not excreted via the urine in fish to any significant extent. Only 0.12-1% is lost via the urine, and thus although zinc is found at relatively high levels in the kidney, it does not imply an excretory regulatory role for this organ (Hardy et al., 1987). No studies have considered the possibility of intestinal zinc excretion in fish, despite this being the main zinc excretory pathway in mammals. The study by Hardy et al. (1987) measured only the total amount of zinc absorbed and excreted via the urine and gills; the faecal zinc was presumed to be unabsorbed dietary zinc. This study reported that the GI tract contained 40% recovered zinc, and thus postulated an important regulatory and storage role for this organ.

It is suggested that in fish, as in mammals, the GI tract regulates plasma zinc (Hardy et al., 1987). A storage and regulatory role for the GI tract in salmonids has also been reported by Maage and Julshamn (1993), who showed that the intestine can mobilise zinc when dietary levels are low. The GI tract may be involved in zinc excretion, as in mammals. The integument and mucus may also play a role in excretion, as high levels of zinc are found in the integument (Spry et al., 1988). Fish plasma zinc content also appears to be under homeostatic control over a wide range of waterborne and dietary zinc concentrations (Spry et al., 1988).

Between 35 and 60% of dietary zinc is retained by rainbow trout and turnover is estimated to be around 1% per day. In descending order, the highest levels of zinc are found in the intestine, followed by the liver, gill, kidney and the skin. The bone, eye, blood, muscle and gonad all have significantly lower levels than the aforementioned tissues (Hardy et al., 1987). Wekell et al. (1986) found similar results but noted that the eye has by far the highest zinc concentration, which is consistent with studies in mammals.

Fish, including salmonids, do not store zinc to any great extent. Unlike mammals, zinc is not stored in the liver. In rainbow trout, the zinc concentration in the liver remains constant for dietary concentrations ranging from 4 to 90 mg Zn/kg, but increases for high inorganic levels (440-1700 mg Zn/kg). Plasma zinc shows the same trend (Wekell et al., 1983; 1986).

Other visceral tissues including the kidney (Overnell et al., 1988), spleen and hepatopancreas are also not responsive to dietary zinc (Jeng and Sun, 1980). However, Maage and Julshamn (1993) report that kidney zinc levels may range between 17 and 97 mg Zn/kg. The response of the kidney to dietary zinc therefore needs to be assessed. Muscle zinc content also does not change significantly with dietary levels (Wekell et al., 1986; Overnell et al., 1988); and zinc is known to increase in the skeletal tissues before the muscle (Jeng and Sun, 1980).

Whole body zinc increases with increasing inorganic supplementation (17-97 mg Zn/ kg, Maage and Julshamn, 1993; 67-140 mg Zn/kg, Lorentzen and Maage, 1999). Other studies have also reported that whole body zinc increases as dietary levels increase (Hardy and Shearer, 1985; Wekell et al., 1983; 1986). The gills also positively respond to dietary supplementation, probably due to their excretory role (Wekell et al., 1983; 1986; Maage and Julshamn, 1993).

Likewise, the intestine responds significantly to dietary supplementation, due to its zinc storage role in fish (Wekell et al., 1986; Maage and Julshamn, 1993; Hardy and Shearer, 1985; Ogino and Yang, 1978). Bone and vertebrae also respond to dietary supplementation; it appears that fish can mobilise zinc from the vertebrae, particularly during deficiency (Ogino and Yang, 1978; Maage and Julshamn, 1993; Do Carmo E Sa et al., 2004). Unlike mammals the bone may be regarded as a storage organ in fish.

As with mammals, zinc in fish is under homeostatic control with the GI tract playing a major role. Zinc is stored in the intestine and bone; the visceral tissues do not store zinc to any great extent. If zinc is not supplied at a sufficient dietary level, whole body, intestinal and bone zinc is not maintained. A constant supply of zinc through the diet is therefore required.


MEASURING ZINC STATUS IN FISH

As yet there are few reliable ways to determine zinc status in mammals (Wood, 2000) and the same is true of fish. In mammals, proposed methods to assess zinc status are the measurement of plasma or serum zinc concentrations; leukocyte, neurophil or erythrocyte zinc concentrations; activity of enzymes, such as 5'-nucleotidase and alkaline phosphatase; zinc transporter levels; and plasma metallothionein concentrations (Salgueiro, 2000; Failla, 1999).

In mammals plasma metallothionein is described as the best indicator of zinc status (Thompson, 1991), but this does not appear to be the case with fish. Liver and kidney metallothionein levels in flounder do not respond to dietary levels from 100 to 1000 mg Zn/kg (Overnell et al., 1988). Alkaline phosphatase has been used in fish and may be a good indicator of zinc status. In Atlantic salmon activity of this enzyme was weakly but significantly increased after 8 weeks using diets with 17-97 mg Zn/kg (Maage and Julshamn, 1993). Increased serum alkaline phosphatase activity has also been found in tilapia (Do Carmo E Sa et al., 2004). There may be a plateau of activity for this enzyme in rainbow trout as has been recorded in tilapia, but the dietary zinc level at which this may occur has not been determined. In mammals levels of this enzyme are conserved during deficiency but this does not seem to occur in carp (Do Carmo E Sa et al., 2004).

Plasma zinc is frequently used to measure zinc status in fish since there is a much higher zinc concentration and range in fish than mammals. In fish decreases in plasma zinc levels are found from 1 to 90 mg Zn/kg, but from 90 to 590 mg Zn/kg there is no change in plasma levels (Spry et al., 1988). Whole body zinc is a good measure of zinc status in fish (Ogino and Yang, 1978; Maage and Julshamn, 1993; Wekell et al., 1986) and is also affected by the source of zinc (Hardy and Shearer, 1985).

However, whole body zinc may stabilise at dietary levels above 40 mg Zn/kg (Maage and Julshamn, 1993). Since zinc is mobilised from both the GI tract and the vertebrae during low zinc intake, a good measure of zinc requirement may be to determine the dietary levels required to keep the zinc levels of these tissues constant. At low dietary levels, below 30 mg Zn/kg, growth may be used to assess zinc status (Ogino and Yang, 1978). Above this growth is not affected (Wekell et al., 1983). Similar findings were noted in Atlantic salmon; dietary levels from 17 to 97 mg Zn/kg did not affect growth (Maage and Julshamn, 1993).


THE ZINC REQUIREMENT OF SALMONIDS

The zinc requirement for rainbow trout has been determined to be 15-30 mg Zn/kg (Ogino and Yang, 1978; NRC, 1993) using an 8-week trial with purified egg white-based diets supplemented with 1-30 mg Zn/kg as Zn sulphate. The requirement was determined based on maximum growth rate, and is the only study specific to the zinc requirement of rainbow trout. It should be noted that 30 mg Zn/kg was the highest level used in this study.

Since this study, several studies using fish meal have shown that zinc requirement is higher in fish meal-based diets than purified diets. Therefore, the zinc requirement for practical diets will be higher, especially for whitefish meals (Ketola, 1979; Satoh et al., 1987a,b). The reduced bioavailability of zinc in fish meal-based diets, particularly whitefish meal diets, is due to increased levels of hydroxyapatite in fish meal, mainly in the form of tricalcium phosphate (Satoh et al., 1987a,b). Although a fish meal diet will contain around 65 mg Zn/kg, due to low bioavailability, this is insufficient to sustain whole body zinc (Lorentzen and Maage, 1999 in Atlantic salmon). Similar results were found in Atlantic salmon by Maage and Julshamn (1993). In rainbow trout, inorganic supplementation at 40 mg Zn/kg to whitefish meal diets was required to obtain normal growth (Satoh et al., 1987a). In Atlantic salmon a practical LT94 fish meal-based diet without zinc supplementation did not maintain optimum zinc status. The same diet supplemented with 68 mg Zn/kg, giving a total dietary zinc level of 140 mg Zn/kg, achieved a normal zinc status (Lorentzen and Maage, 1999).

Plant-based feeds have a lower zinc content than fish meal (NRC, 1993) and contain phytates, which reduce the availability of zinc to fish (Satoh, 1997a; Storebakken et al., 2000). The trend to increase plant-based ingredients in fish diets will therefore further increase the requirement for zinc. In rainbow trout a high soybean-based diet required 150 mg Zn/kg to obtain optimum growth (Satoh, 1997a).


ZINC DIGESTIBILITY IN DIFFERENT FEEDS AND ZINC SOURCES

It is evident that one of the most important factors in zinc bioavailability is dietary zinc digestibility/absorption. The type of diet and the chemical form of the element can affect the digestibility of metals by formation of insoluble complexes, interactions with other elements, and adsorption to organic and inorganic surfaces. There are several zinc absorption activators including picolinic acid (secreted by the pancreas), vitamin B6 (increases picolinic acid secretion), citrate and amino acids such as glycine, histidine (also found in fish; Glover and Hogstrand, 2002b), lysine, cysteine and methionine. However, there are also a number of important absorption inhibitors including phytic and oxalic acids, tannins, fibre, selenium, iron and calcium. Calcium and iron only affect zinc absorption when present at higher than natural concentrations (Salguerio et al., 2000). The absorption of both organic and inorganic zinc is decreased with increasing dietary zinc in fish (Apines et al., 2001). At 590 mg Zn/kg in diet, apparent absorption is only 2% (Spry et al., 1988).

In fish the availability of inorganic zinc is significantly lower for practical diets than purified diets (Ketola, 1979; Gatlin and Wilson 1984b; Hardy and Shearer, 1985; Satoh, et al. 1987a,b). This lower availability is a result of reduced zinc digestibility due to the high level of tricalcium phosphates originating from hard tissues, such as bones in fish meal and phytic acid in plant-derived products. Expectedly, the absorption of zinc is lower in practical fish meal-based feeds than practical soy-based feeds. This difference is largely due to the higher levels of phytic acid in soya- than fish meal-based feeds, 8.1 and 0.9 g/kg phytic acid, respectively (Storebakken et al., 2000).

Organic sources of zinc differ in digestibility compared with inorganic sources. In rainbow trout, amino acid chelates are digested more efficiently than zinc sulphate, zinc methionine and glass-embedded zinc. The chelation of zinc with amino acids protects the metal from forming insoluble complexes and thus facilitates its transport into the mucosal cells (Apines et al., 2001).


ZINC BIOAVAILABILITY IN FISH

Based on growth, at a dietary level of 20 mg Zn/kg, the bioavailability of zinc compounds in descending order is: ZnSO4, ZnNO3, ZnCl2, and at 40 mg/kg the least bioavailable form is 5ZnO•2CO3. At 20 mg Zn/kg, vertebrae zinc content was lowest for ZnCl2 (Satoh et al., 1987b).

In rainbow trout organic forms of zinc are generally considered more bioavailable than inorganic zinc, however this depends on the species, diet, and the parameter measured. Using 40 mg Zn/kg in a practical diet, Apines et al. (2003) found that in rainbow trout amino acid-chelated zinc (Zn-AA) had no significant effect on growth, feed conversion and plasma or liver zinc compared with inorganic zinc (ZnSO4). However, the activity of alkaline phosphatase was significantly higher, and the bone deposition of zinc marginally higher for Zn-AA, indicating a higher availability. Similarly, in rainbow trout fed practical diets, growth and feed conversion were not affected by supplementing dietary zinc as Zn-AA, zinc methionine (Zn-Met) or ZnSO4 at 55-85 mg Zn/kg, but at approximately 85 mg Zn/kg, bone deposition, whole body zinc and alkaline phosphatase were all higher for Zn-AA. Bioavailabilities of ZnSO4, Zn-Met and glass-embedded zinc were not significantly different (Apines et al., 2001).

In catfish growth, feed conversion, and bone zinc indicated equal bioavailability of zinc sulphate and zinc methionine using dietary levels from 50 to 140 mg Zn/kg. This study also showed that based on bone zinc levels, dietary Zn proteinate at 45 mg Zn/kg was not equivalent to 200 mg Zn/kg ZnSO4 in practical diets (Li and Robinson, 1996). In rainbow trout fed diets containing low to medium levels of calcium and phosphate, zinc in the form of a proteinate had increased bioavailability over ZnSO4. However, the bioavailability of both Zn proteinate and zinc sulphate was the same in diets containing high levels of calcium and phosphate (Hardy and Shearer, 1984), suggesting that high levels of dietary zinc may mask the differences in bioavailability between organic and inorganic zinc sources.


Bioavailability study of Sel-Plex® selenium and Bioplex® Zn

METHODS

A 10-week feeding trial using rainbow trout was conducted at Plymouth University to determine the bioavailability of organic versus inorganic selenium and zinc in practical diets. As diet is known to affect mineral bioavailability, practical diets containing whitefish meal and soybean meal were used. This diet also ensured the relevance of the study to practical aquaculture conditions. A control diet was formulated to contain minimal background selenium as many fish feed ingredients contain high selenium levels. Selenium and zinc were supplemented to approximately double that of the control diet to form organic and inorganic selenium and zinc treatments with totals of approximately 2 mg Se/kg and 200 mg Zn/kg. The organic treatment was supplemented with Bioplex® Zn and Sel-Plex® (Alltech Inc.) and the inorganic treatment was supplemented with sodium selenite and zinc sulphate.

Bioavailability was measured by assessing selenium and zinc levels of various tissues, digestibility using the inert marker yttrium oxide, whole body selenium and zinc, and Se- and Zn-dependent enzymes. Se-dependent enzymes included hepatic GSH-Px and thioredoxin reductase; Zn-dependent enzymes included intestinal carboxypeptidase B and intestinal and plasma alkaline phosphatase.


RESULTS

Initial results, of which only enzyme data are reported in this paper, indicate that organic selenium and zinc have a greater bioavailability compared with inorganic forms.

Of the Zn-dependent enzymes, only plasma alkaline phosphatase was affected by selenium and zinc supplementation (Figure 1). Activity of this enzyme is significantly higher in fish given Bioplex® Zn than the inorganic zinc supplement (Mann-Whitney P=0.0051). Intestinal alkaline phosphatase and carboxypeptidase B were not significantly affected by organic or inorganic zinc supplementation (Figures 2 and 3).

Both TrxR and GSH-Px activities were affected by selenium supplementation. Thioredoxin reductase was increased by Sel-Plex® supplementation only (Mann-Whitney P=0.0341; Figure 4). GSH-Px activity was increased by both the organic and inorganic sources (Figure 5), but was significantly different only for the organic source (Mann- Whitney P=0.0089).
















CONCLUSIONS

The enzyme part of this bioavailability study, the interpretation of which will be extended when data for other measurements are obtained, demonstrates that Sel-Plex® selenium and Bioplex® Zn have a different bioavailability than inorganic forms. The results show that enzymes respond differently and these responses are tissue dependent. These findings highlight the importance of studying multiple enzymes and tissue types when conducting a bioavailability study.


SUMMARY

As with mammals, zinc and selenium have an important role in fish health. Although requirements for these minerals have been determined, they are based on limited and outdated research. In addition to this, much of the previous research has concentrated on inorganic compounds, namely sodium selenite and zinc sulphate, and purified diets.

The use of inorganic minerals may not be best practice, as research in both mammals and fish shows that organic minerals may be far superior in terms of bioavailability and performance. Organic minerals have been shown to have several positive effects on animal health, such as increased disease resistance, growth and feed conversion. Such effects may have significant benefits in aquaculture, including decreased incidence of disease, better fish production, increased fillet quality and increased water quality due to decreased mineral wastage. Organic minerals may therefore play a key role in maximizing fish production and its profitability.

More research is needed on zinc and selenium bioavailability, the roles of these minerals in fish health and on requirements when organic mineral sources are used. Studies on the role of minerals in fish nutrition need to be designed in such a way as to ensure findings are relevant to the aquaculture practices of today and the future.
The production of novel, high quality organic minerals, may play a key role in the aquafeed industry. Supplementing Sel-Plex® and Bioplex® Zn to fish feeds may provide many benefits over and above the current practice of inorganic mineral supplementation. In mammalian nutrition these products have already shown to play a major role in optimizing animal health; the same might be expected in fish.


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Authors: SEBASTIEN A. RIDER and SIMON J. DAVIES
Marine Institute, University of Plymouth, Devon, UK