May | June 2014

Prospects on dietary trace minerals: Aquafeeds & Aquaculture

The International magazine for the aquaculture feed industry

International Aquafeed is published six times a year by Perendale Publishers Ltd of the United Kingdom.All data is published in good faith, based on information received, and while every care is taken to prevent inaccuracies, the publishers accept no liability for any errors or omissions or for the consequences of action taken on the basis of information published. ©Copyright 2014 Perendale Publishers Ltd. All rights reserved. No part of this publication may be reproduced in any form or by any means without prior permission of the copyright owner. Printed by Perendale Publishers Ltd. ISSN: 1464-0058

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Even though, our knowledge in fish nutrition has advanced significantly, the information on trace minerals requirement is still limited and

fragmentary.

Whereas, the sustainability issue has put a new dimension in aquafeed formulation with a wide array of new ingredients and additives, on the other hand, the importance of basic nutrient such as trace minerals is still in sideline.

Substantial investment and integrated sci-entific efforts are warranted to bridge the knowledge gap and further improve our understandings on the significance of dietary

trace mineral in fish nutrition and health at the least cost to environment.

An overview The importance of trace minerals sup-

plementation in fish feed formulation has been well accepted since some of the trace minerals from ambient water and feed itself cannot supply the optimal level required by the cultured aquatic species.

The reluctance among researchers to determine the trace mineral requirement has been partly due to the related difficulty of conducting research on mineral nutrition.

Problems associated with the quantification of mineral requirements include identification

of the potential contribution of minerals from the water, leaching of mineral from the diet prior to consumption, availability of suitable test diets that have a low concentration of the targeted mineral and limited bioavailability (NRC, 2011).

The trace minerals including copper, man-ganese, iron, zinc, selenium, chromium, iodine and fluorine participate in a variety of meta-bolic processes. Some of the vital biochemical processes involving minerals are the formation of skeletal structures and other hard tissues (ex. Fin, rays, scales, teeth and exoskeleton), electron transfer, regulation of acid: base equilibirium, the production of membrane potentials and osmoregulations.

In strict sense, except the osmoregulation, rest of the basic metabolic functions of various elements are same for aquatic and terrestrial animals. Trace minerals or micro-minerals are important components of hormones and

enzymes serve as cofactors and/or activa-tors of a variety of enzymes as well as participate in a wide variety of biochemical processes (NRC, 2011). Only a few scien-tific review including, Davis & Gatlin (1996) and NRC 2011 summarizes the estimate of mineral requirement in few commercially important aquaculture species.

However, the trace mineral require-ments in several other aquaculture species are still unknown and the practical feed formulation follow the general estimation.

Table 1 summarises the function and common deficiency symptoms of trace minerals in aquaculture. The deficiency symptoms depend upon the degree and duration of deprecation of that certain trace mineral in certain species under certain environmental condition.

In strict sense, one cannot define the sub-optimal level of trace mineral for an aquaculture species applicable for all kind of culture environment. The requirement

Prospects on dietary trace minerals: Aquafeeds & Aquaculture by Sungchul Charles Bai, Professor/Director, Deptartment of Marine Bio Materials & Aquaculture, Feeds and Foods Nutritional Research Center (FFNRC), Pukyong National University, Nam-gu, Busan, Korea

Dietary trace minerals must be supplied in adequate quantity to ensure the optimal growth and health of farmed aquatic species. The significance of trace mineral bioavailability has become more important as the aquaculture practice has gone under phenomenal intensification worldwide. Moreover, the composition of majority of commercial fish feed has been changing and there is an increased use of dietary plant protein. As a result, the bio-availability of trace minerals are being adversely affected by the presence of antagonistic factor such as phytic acid in plant protein.

Table 1: Trace Minerals their functions and deficiency signs in aquatic species

Microminerals/Trace Minerals Functions Deficiency Signs

Copper Metalloenzymes, Fe metabolism

Impaired growth and reduced activity of copper-containing enzymes

Cobalt Vitamin cyanocobalamin ( B12 )synthesis Anemia

Chromium Carbohydrate and lipid metabolism Impaired glucose utilization

Iodine Thyroid hormones, energy production Thyroid hyperplasia

Iron Hemoglobin, enzyme, lipid oxidation Impaired growth, anemia

Manganese Organic matrix of bone Impaired growth, skeletal abnormalities, cataracts

Molybdenum Xanthine oxidase Reduced enzyme activity

Selenium Glutathione peroxidase, component of amino acid selenocysteine

Reduced growth, anemia, exudative diathesis, reduced activity of glutathione peroxidase

Zinc Metalloenzymes, Reduced growth, anorexia, cataracts, skeletal abnormalities,

Source: Adopted and Modified from NRC, 2011 and S. C. Bai, 2011, Nutrient Metabolism & Feeds in Fish, pp. 41

18 | INTERNATIONAL AQUAFEED | May-June 2014

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level and deficiency symptoms for trace minerals under appropriate and stressful environmental con-dition remains to be investigated in aquaculture.

At this point, we must appre-ciate what is known today and try to further improve our under-standing in the significance of trace mineral in fish nutrition, health and environment.

Lower requirementEven though, trace minerals

are typically required in much lower quantity in fish diet but its supplementation at optimum level is a critical issue in fish feed formulation.

Scientific reports gathered over last two decades indicate, few trace mineral are quite sensi-tive and proper care should be taken to ensure their inclusion at optimum level in fish feed formulation. For instance, trace mineral selenium (Se) is an essen-tial micronutrient in animals and is required for normal growth and development.

However, high selenium con-centrations in an animal’s diet can result in toxic effects.

Selenium is similar to sulfur with regard to its basic chemical and physical properties (it has the same valence states, forms, and analogs of hydrogen sulfide, thiosulfate, sulfite, and sulfate) and mammalian studies show that cells do not discriminate well between the two elements as proteins are being synthesized (it is assumed that the mecha-nistic features underlying toxicity are essentially the same for fish, because the resulting pathology and teratogenic features are the same).

When present in excessive amounts, selenium is erroneously substituted for sulfur, result-ing in the formation of a tri-selenium linkage (Se-Se-Se) or a selenotrisulfide linkage (S-Se-S). Either configuration prevents the formation of the necessary disulfide chemical bonds (S-S). The result is distorted, dys-functional enzymes and protein molecules, which impair normal cellular biochemistry (Ganther 1974; Stadtman 1974; Diplock and Hoekstra 1976; Reddy and Massaro 1983; Sunde 1984).

Consequently, there has been public awareness and govern-mental efforts including in the Republic of Korea to establish an upper limit of selenium in aquafeeds to prevent both cul-tured fish and consumers from selenium toxicity.

Consecutive studies conduct-ed in my laboratory, investigated the requirement and toxicity level of dietary Se in few com-mercially important species (Kim et. al., 2003; Lee et al., 2008; Lee et. al., 2010; Arshad et. al., 2010).

Based on our experimental results, we concluded that a dietary Se level above 7.38mg Se/kg is likely toxic and with a long-term feeding trial, a dietary Se level of 4.13mgSe/kg may cause toxic effects in juvenile olive flounder.

While the overall perfor-mance from our preliminary feeding trail, a dietary Se level of 0.21mg NaSeO3/kg diet was concluded to be optimal for proper growth performance and a dietary Se level of 12.3mg Na2 SeO3/kg was anticipated to be toxic to juvenile black seabream.

Likewise, various other stud-ies have reported the toxicity level for Se in different aqua-culture species. Overall obser-vations in our experiments in different species and taking into account other reports, it appears the requirement and toxicity level for Se is species specific and therefore proper care should be taken to avoid its adverse effects.

Further few other trace mineral such as dietary Copper (Cu) toxicity has also been docu-mented by various other authors. Therefore, dietary trace minerals should be supplemented in judi-cious manner, keeping in mind a delicate balance must be main-tained between the ingestion, digestion and absorption of trace minerals.

Fish health managementThe concept of maintain-

ing the health of fish through the best possible nutrition is well-accepted in modern fish farming.

Scientific evidence gathered over the past 30 years indicates that dietary nutrients as well as additives could stimulate the

May-June 2014 | INTERNATIONAL AQUAFEED | 19

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immune system of fish and help to fend off diseases (Kiron, 2014).

Consequently, the last decade has wit-nessed a great deal of attention paid to devel-op safe and cost effective immunostimulants, probiotics, prebiotics, synbiotics and so on.

Worthy to note that ‘functional feed’ has been defined as the feed which can supply the nutrient beyond the basic requirements of a cultured species. Nutritionally well-balanced and properly processed diets are the prime importance in intensive aquaculture, where fishes are frequently reared in stressful envi-ronment.

Deficiency of any nutrients, especially vita-mins and micro minerals could be an ample reason to trigger the disease epidemic in an aquaculture operation.

Trace minerals being the integral part of several metalloenzymes have significantly greater impacts on preventive health manage-ment and success of an aquaculture venture.

For example, Lim et al., (2001a) attributed the imbalances in Iron (Fe) would compro-mise the immune system and the resistance of fish to disease. The effects of dietary zinc on immune response and disease resistance in fish has also been reviewed by Lim et al. (2001b).

Observations reported include enhanced chemotaxis of macrophages, a lower phago-cytic ability, improved or attenuated disease resistance and reduced or negligible effect on antibody production.

Selenium is another important trace ele-ment for fish because it is a constituent of selenoproteins and has structural and enzy-matic roles similar to glutathione peroxidase (the antioxidant enzyme). This mineral modu-lates the immune functions such as inflam-mation and virulence development (Rayman, 2000; Kiron, 2012).

In channel catfish selenoyeast and sele-nomethionine as the source of Se were observed to increase the antibody titer cor-responded to their dietary concentrations (Wang et al., 1997).

Furthermore, Se is an integral part of the

enzyme glutathione peroxidase (GSH-Px), and it has some complementary biochemical functions with vitamin E (Gatlin et. al., 1986a). GSH-Px is part of the cellular defence system against oxidative damage, together with the antioxidant vitamins such as vitamins C and E. Hilton (1989) mentioned that these nutrients do not act independently and are interrelated with other nutrients in terms of function and metabolism.

A few studies on the interactions of these vitamins in fish have been investigated in rainbow trout, Salmo gairdneri (Richardson) (Hung & Slinger 1980) and channel cat fish, Ictalurus punctatus (Rafinesque) (Gatlin et. al.,2003 1986b).

Vitamin E and Se function synergistically in animal tissues to form an important antioxi-dant defence system. The interactions of Se and vitamin E in fish have been reported in channel cat fish (Gatlin III et al. 1986a; Wise, et. al., 1993), Atlantic salmon, Salmo salar L. (Poston et. al., 1976), rainbow trout (Bell et. al., 1985) and chinook salmon, Oncorhynchus tshawytscha (Thorarinsson et. al., 1994).

In addition, high dietary supplementation

of vitamins C and/or E and Se showed posi-tive effects on growth and immune response in brook trout, Salvelinus fontinalis (Mitchill) (Poston & Livingston1969), channel cat fish (Durve & Lovell1982; Li & Lovell 1985; Li et. al., 1993), Atlantic salmon (Salte et. al., 1988; Hardie et. al., 1990) and rainbow trout (Navarre & Halver 1989).

In a study conducted in my laboratory to study the synergetic effects of vitamin C, E and selenium, observation suggested that dietary supplementation of vitamin C and E over required values had positive effects on growth performance, but there were no syn-ergetic effects of dietary vitamin C, E and Se supplementation over minimum requirement levels on the growth performance and disease resistance in fingerlings Nile tilapia.

Altogether scientific evidences suggest, in depth investigations are warranted to explore the opportunities to use the dietary trace mineral in preventive health management in aquaculture.

Plant protein in aquafeed & trace minerals bioavailability

Feed formulations for farmed aquatic ani-mals have historically relied on fishmeal to provide a major part of their nutrient require-ments.

However, economic and sustainability issues have exerted substantial pressure for the reduction of fishmeal in aquafeeds.

Numerous scientific studies in last three decades have investigated the efficacy of different plant protein as an alternative to fishmeal.

Consequently, there has been a massive shift towards the use of plant protein in aquafeed formulation. Plant protein contain a wide array of antinutrients, among them the presence of antagonists factors such as phytic acid has been acknowledges as the major

Table 3 . Efficacy of chelated trace minerals in aquaculture

Minerals Fish/Crustacean Remarks1 Reference

Se Channel catfish Higher Bioavailability Wang & Lovell, 1996

Zn Channel catfish Equivalent M.H.Li & Robinson , 1996

Zn Abalone 2~3 times Tan & Mai, 2001

Cu Grouper 2~4 times Lin et al., 2010

Cu Olive flounder 2 times Mohseni et. al., 2011

Cu Sturgeon 2 times Mohseni et. al., 2011

Cu Pacific white shrimp 3~4 times Bharadwaz et al., 2014

Premix (Cu, Zn, Mn & Fe) Korean rockfish 2~4 times Katya et. al.,2014

Premix (Cu, Zn & Mn)

Pacific White shrimp 4~6 times Katya et. al., 2014 (Unpublished)

Premix (Cu, Zn & Mn) Rainbow trout 2 times M..J.S. Apines et al., 2003, 2004

1 Remarks represent the reported comparative efficiency of chelated trace mineral Vs Inorganic source in respective experiment

Table 2. Growth performance and hematological characterstics of juvenile Black seabream fed different levels of dietary Se for 15 weeks.

Diets1 WG(%) FE(%) SGR(%) PER PCV(%) Hb(g /100ml)

RBC (X106cell/

μl)

Se 0.21 327.4a 93.3a 2.70a 1.72a 42.2 15.3 3.34

Se 0.30 357.5a 94.9a 2.88a 1.79a 38.2 16.5 3.69

Se 0.52 325.6a 91.9a 2.69a 1.72a 38.6 16.1 3.59

Se 1.29 349.7a 94.3a 2.80a 1.78a 32.5 17 3.12

Se 12.3 254.4b 84.2b 2.23b 1.53b 26.6 13.1 2.9

Pooled SEM9 13.0 1.40 0.08 0.03 2.46 0.72 0.16

1Diets Se 0.21, Se 0.30, Se 0.52, Se 1.29, Se 12.3 contained 0.21, 0.30, 0.52, 1.29, and 12.3 mg sodium selenite (Na2Seo3)/kg diet.Note: For more detail, please refer Lee. et. al., 2008

20 | INTERNATIONAL AQUAFEED | May-June 2014

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barrier hindering the bioavailability of trace mineral. Phytic acid (myo-inositol 1,2,3,4,5,6- hexakisphosphate) is the major phosphorus (P) storage compound in plant seeds and can account for up to 80 percent of total phos-phorus. Phytic acid binds with divalent cationic trace minerals rendering them unavailable to the animal and these are consequently lost to the environment as waste (Cheryan, 1980; Davis and Gatlin, 1996; Davis et al., 1993; Li and Robinson, 1997).

The limited bioavilability and potential dietary deficiency of trace minerals are serious concern as adequate trace mineral ensuring the optimum growth and health of cultured species in aquaculture.

An ideal approach to improve the bio-availability of trace mineral has been recom-mended as the inclusion of microbial phytase in fish diet. Phytase is an enzyme chemically known as myo-inositol-hexaphosphate phos-phohydrolase (Class 3: Hydrolases), produced either by microorganisms or present in some plant ingredients.

Monogastric animals cannot produce this enzyme.

Presence of phytase in some animals is of microbial origin. Microbial phytase either as a dry powder or as a liquid is available com-mercially ( Baruah. et. al., 2004).

However, microbial phytase has been reported to have limited efficiency due to

lower Ph in fish gut apart from its high cost. Deterioration of phytase activity at high tem-perature of fish feed processing, especially in the case of extrusion processing are additional factors, all together limiting the use of phytase in aquafeed formulation.

Another approach to increase the bioavail-ability of trace mineral as highlighted in NRC (2011), “as the aquatic animal feed industry increase its use of plant feedstuffs, the need for mineral supplementation should increase.”

Since environmental pollution due to high rate of mineral excretion by mineral antago-nisms at higher level of dietary inclusion has been a common problem in livestock husbandry. It remains an important research area for scientific community to clearly under-stand the ultimate fate of trace minerals at higher level of dietary inclusion in aquafeed formulation.

Trace mineral form and sources: Critical issue

Inorganic form (sulfate/nitrate) of trace mineral has traditionally been used in aqua-feed formulation.

However, the limited bioavailability of inorganic source of trace mineral due to its higher affinity to antinutrients has hastened the search for alternative form of inorganic trace minerals.

As a result, scientific communities have

attempted to develop more stable and bio-available form of trace minerals suitable for aquaculture. For instance, tri-basic copper chloride (TBCC) has been reported as the more concentrated form of copper than cop-per sulfate (58% vs 25% Cu).

Since it has low hygroscopicity and is insoluble in neutral water, it should be a less reactive and less destructive form of cop-per when combined with vitamins in diets (Cromwell et al., 1998). Shao et. al., (2010) suggested TBCC could be a new dietary cop-per source as more bioavailable than copper sulfate for crucian carp.

However, the supporting information is scanty and needs further research to justify the bioavailability of TBCC in aquaculture.

In last decade, research is increasing shed-ding light on the potential benefit of using organic/chelated form of trace mineral in aquaculture.

Typically, organic trace minerals are more stable in the digestive tract and less prone to interactions and antagonisms as they are bound to organic molecules and less avail-able to interaction and binding. Some of the commonly available organic trace miner-als are metal proteinates, metal amino acid complexes and metal amino acid chelates. Earlier studies have demonstrated improved bioavailability, growth and disease resistance in fish fed metal proteinates (zinc protein-

May-June 2014 | INTERNATIONAL AQUAFEED | 21

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ate) and metal amino acid complexes (zinc methionine) compared to fish fed inorganic sources (Hardy and Shearer, 1985; Paripatananont and Lovell, 1995a,b, 1997).

The glycine chelates of trace minerals have been shown to improve performance, tissue mineral retention, hematology parameters, immune function and disease resistance in the rainbow trout (Apines et al., 2003; Apines-Amar et al., 2004a,b; Satoh et al., 2001) and in red sea bream (Sarker et al., 2005), despite the pres-ence of dietary antagonists such as phytic acid or trical-cium phosphate (Bharadwaz et. al., 2014).

Table 3 summarises the few scientific reports on the efficacy of chelated mineral in aquacul-ture. Nevertheless, a cross com-parison among these reports on chelated trace minerals could be perhaps misleading.

Since, fish species, fish size, diet composition and other experimental condition vary among different experiments.

Further, a fundamental dif-ference among different experi-ment comparing chelated trace mineral and inorganic trace min-erals is the balancing of ligand in all experimental diets has been

overlooked by various research groups.

Since, ligand could be an additional source of nutrients in chelated trace minerals, researchers must give impor-tance to balancing the ligand in inorganic treatment as well, to prevent the bias that difference in results could be due to ligand. Available scientific reports par-ticularly published in last decade evidenced that there is a signifi-cant increase in the efficiency of chelated minerals in aquaculture.

Nevertheless, every manu-facture claim their product to be best and making a right choice is often a challenge. Recently, consecutive studies in my labo-ratory investigated the efficacy of chelated minerals consisted of a range of divalent cationic minerals chelated to two mol-ecules of HMTBa (2-hydroxy-4-methylthiobutanoic acid or hydroxy analog of methionine; Mintrex™, Novus International, St. Louis, USA) claimed to have in an extremely stable configura-tion.

The stability of these mol-ecules renders chelated trace minerals less available to binding to phytic acid and to interfer-ence from other dietary antago-nists.

These molecules are thus

Figure 1: Average mortality (Mean ± SD) of juvenile olive flounder exposed to dietary selenium for 10 weeks. Diets Se0.61, Se4.13, Se7.38, Se18.6, Se35.9, Se66.0, and Se146, contained 0.61, 4.13, 7.38, 18.6, 35.9, 66.0, and146 mg Se/kg diet respectivelyNote: For more detail, please refer Lee. et. al., 2010

Figure.3. Survival rate (%) of Olive flounder fed different levels of dietary chelated Cu for 12 weeks. Diets contained 7 (Cu0), 10.4 (CuM5), 15.8 (CuM10), 24.9 (CuM20), 43.4 (CuM40), 82.1 (CuM80), 158 (CuM160), 308 (CuM320), 658 (CuM640),and 1267 (CuM1280) mg Cu/kg diet Note: For more detail please refer, Mohseni et. al., 2012.

22 | INTERNATIONAL AQUAFEED | May-June 2014

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able to reach the receptors in the gut epithelium where they are absorbed into the circulation of the animal (Eide, 2004; Wang and Zhou, 2010; Yi et al., 2007).

Overall performance observed in our experiments vouched the potential benefit of using che-lated trace mineral, Mintrex Cu in marine fish, Olive flounder and fresh water fish white sturgeon (Mousheni et. al., 2011), Mintrex Cu, Zn, Mn & Fe premix in Marine fish Korean rockfish (Katya et. al., 2014) and Mintrex Cu, Zn & Mn premix in marine shrimp, Pacific white shrimp (unpublished).

Worthy to note that, chelated trace minerals and their premix should also be supplemented at optimum level, high level of die-tary inclusion could also be toxic.

Table. 4. shows the toxic effects of chelated mineral pre-mix observed in Olive flounder (Mohseni et. al., 2012).

Overall trend shows, chelated trace minerals and premix is hold-ing a great potential as safe and effective alternative to traditional inorganic source of trace miner-als in aquaculture. Even though despite of potential benefit, the high cost of chelated trace min-eral often acknowledged as the major factor limiting its use in aquafeed industry.

Thus, complementary inclu-

sion of chelated with inorganic trace minerals could be logical step to encourage the inclusion of alternative dietary mineral source in aquafeed formulation.

Conclusion The significance of dietary

trace minerals should get due recognition, for the vision of aquaculture sustainability.

The true value of any high quality fish feed is non-existent, if the basic nutrient requirement has been compromised in feed formulation.

We need to have a clear understanding on the species specific trace minerals require-ments in aquaculture.

As the aquafeed formulation has been shifting to dietary plant protein from fishmeal, emphasis should also be placed to revise the inclusion level of as well as efficacy of new perspective to ensure the bioavailability of trace mineral.

At this point, we must appreci-ate what is known today and try further improving our knowledge on the significance of dietary trace minerals in fish nutrition and health.

Acknowledgement:

I wish to thank my PhD student, Kumar Katya for his input in articulating this article.

Figure 2: Cumulative mortality rate (%) of Nile tilapia challenged by E. tarda. Diet Control: (150mg AA,100mgTAand 0.2mg Se per kg diet)Excessive ascorbic acid (eAA): (2000 mg AA, 100mg TA and 0.2mg Se per kg diet)Excessive a-tocopheryl acetate (eTA): (150mg AA, 240mg TA and 0.2mg Se per kg diet) Excessive selenium (eSe): (150 mg AA, 100 mg TA and 0.5 mg Se per kg diet)Excessive all (eALL): (2000 mg AA, 240 mg TA and 0.5 mg Se per kg diet)Note: for more detail please refer, Kim et. al., 2003

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