JOURNAL OF THE WORLD AQUACULTURE SOCIETY

Vol. 24, No. 1 March, 1993

Evaluation of the Dietary Zinc Requirement of Penaeus vannamei and Effects of Phytic Acid on Zinc and

Phosphorus Bioavailability D. ALLEN DAVIS' AND ADDISON L. LAWRENCE

Texas Agricultural Experiment Station, Texas A M University System, P.O. Drawer Q, Port Aransas, Texas 78373 USA

DELBERT M. GATLIN 111 Department of Wildlife and Fisheries Sciences, Texas A&M University System,

College Station, Texas 77843 USA

Abstract Experiments were conducted to determine the dietary zinc requirement of Penaeus vannomri and

evaluate the effects of phytate on zinc bioavailability. Prior to initiation of the growth trial, 2Oday- old P. vonnamei postlarvae (mean weight 0.0032 g) were fed a casein-gelatin based semi-purified diet lacking zinc supplementation but containing 18 mg Z d k g diet for one week. Subsequently, juveniles (mean weight 0.058 g) were fed one of seven diets containing either supplemental zinc (0, 15, 30, 60 mg/kg diet) without phytate or supplemental zinc (0, 60, 200 mg/kg diet) with 1.5% phytate for 33 days. Weight gain was greatest in shrimp fed 15 mg supplemental Zn/kg diet. In the absence of dietary phytate, zinc concentrations in the hepatopancreas of shrimp were maximized when zinc was supplemented at levels greater than or equal to IS mg Zn/kg diet (33 mg total Zn/ kg). Supplementation of 1.5% phytate to the diet did not have a significant effect on growth or zinc concentrations in the carapace; however, it did depress zinc levels in the hepatopancreas. Supple- mentation of 200 mg Zn/kg diet was required to overcome the depressed bioavailability of zinc caused by the presence of dietary phytate and return zinc levels of the hepatopancreas to that observed when phytate was not present. Based on apparent digestibility values phytate phosphorus was unavailable to the shrimp and the presence of phytate depressed the bioavailability of phos- phorus and zinc.

Zinc is required for normal growth, de- velopment, and functioning of all animal species that have been studied (National Research Council 1980). Zinc functions as a cofactor in several enzyme systems and is a component of a large number of metal- loenzymes including carbonic anhydrase, carboxypeptidases A and B, alcohol dehy- drogenase, glutamic dehydrogenase, D-glyc- eraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, malic dehydroge- nase, alkaline phosphatase, aldolase, super- oxide dismutase, ribonuclease, and DNA polymerase (National Research Council 1980).

I Corresponding author's present address: Univer- sity of Texas at Austin, Marine Sciences Institute, P.O. Box 1267, Port Aransas, Texas 78373 USA.

A dietary requirement for zinc has been demonstrated in a variety of freshwater fish- es fed semi-purified diets: 20 mg Zdkg diet for channel catfish Ictalurus punctatus (Gat- lin and Wilson 1983) and blue tilapia Oreo- chrornis aureus (McClain and Gatlin 1988); 15-30 mg Zn/kg diet for red sea bream Cy- prinus major (Ogino and Yang 1979); and 15-30 mg Zdkg diet for rainbow trout On- corhynchus rnykiss (Ogino and Yang 1978).

In addition to dietary sources, a variety of decapod crustaceans have been found to be able to regulate whole-body zinc levels and absorb zinc fiom the water (Bryan 1964, 1968; Renfro et al. 1975). Bryan (1968) sur- veyed 18 species of decapod crustaceans, ranging from freshwater to purely marine species and reported that the amount of un- bound zinc in the blood may be less than that of seawater, which generally contains

0 Copyright by the World Aquaculture Society 1993

40

EVALUATION OF ZINC 41

TABLE 1. Composition of basal diet (% dry weightJa

Digestibility Ingredient Growth trial trial

~

Caseinb Gelatinb Wheat starchb Menhaden fish oilc Lecithin (purified)b Cholesterolb Mineral mixtured Vitamin mixture' s t a y - 0 Chromic oxideb Fish solubles Alpha-cellulod

35.0 8.0

18.6 7.0 1 .o 0.5

15.0 8.0 2.9h

4.0

35.0 8.0

19.8 7.0 1 .o 0.5

1 5.0e 8.0 0.2' 0.5 1 .o 4.0

a Basal diet contained 18 mg Zn/kg diet, as deter-

I.C.N. Nutritional Biochemicals Inc., Cleveland,

Zapata Haynie Corp., Reedville, Virginia, USA. * Contains (as g/kg): CaHP04, 500.0; NaCl, 74.0;

K3C6H507.H20, 220.0; K2S0.4, 52.0 MgO, 24.0; MnS04.H20,5.16; CuS04.5H20,0.68; FeS04.7H20, 4.95; KIO3,O.Ol; Na2SeO4,0.0072; KCr(S04)2.12H20, 0.55; cellulose, 118.64.

Supplemented with 1.60 g ZnC03 replacing cellu- lose.

Contains (as g/kg): Vitamin A palmitate, 1.8; Vi- tamin E (250 IU/g), 22.0; Inositol, 180; Choline chlo- ride, 75; Menadione, 2.3; p-Aminobenzoic Acid, 30; Niacin, 26; Riboflavin, 8; Pyridoxine HCl, 3; Thiamine mononitrate, 5; D-calcium pantothenate, 15; Vitamin D3 (40,000 IU/g), 1; Biotin, 1; Folic acid, 5; Vitamin Bl2 crystalline, 1; sucrose, 623.9.

g Vitamin Technologies International, Buhl, Idaho, USA.

mined by atomic absorption spectrophotometry.

Ohio, USA.

3,000 mg active Vitamin C/kg. 300 mg active Vitamin C/kg.

J Sigma Chemical Company, Cleveland, Ohio, USA. ZnCO3 was added at the expense of alpha-cellulose to produce the different dietary zinc concentrations.

5 pg Zn/L. As a result, the concentration gradient should enable zinc to enter the body via absorption across the body surface and gills. This indicated that some zinc could be obtained from the water. However, fresh- water fish maintained in water containing 10-25 pg Z d L were unable to obtain ade- quate zinc from the water for normal growth and mineralization (Ogino and Yang 1979; Gatlin and Wilson 1983, 1984). Since salt- water typically contains 5 pg Z d L (Bryan 1968), a level lower than that of freshwater,

marine species may not be able to meet their physiological needs from water. Although zinc requirements have not been reported for shrimp, the deletion of dietary zinc sup- plements produced a significant depression in tissue mineralization in P. vunnumei in- dicating a dietary effect of zinc (Davis et al. 1992).

Practical diets contain feedstuffs that are relatively rich sources ofzinc (e.g., fish meal); however, for fish the bioavailability of zinc in these feedstuffs is generally very low, making supplementation necessary (Watan- abe et al. 1988). The bioavailability of zinc in various fish meals has been found to be inversely related to the tricalcium phos- phate content of the meal and is thus gen- erally lowest in white fish meal and slightly better in brown fish meals (Watanabe et al. 1988). Practical diets also may contain feed- stuffs that contain relatively high levels of phytate which has been demonstrated to af- fect the bioavailability of zinc in a variety of terrestrial animals (Oberleas et al. 1962; O'Dell et al. 1964; Savage et al. 1964; Lo et al. 1981) and fish (Gatlin and Wilson 1984; Richardson et al. 1985; McClain and Gatlin 1 988). Civera and Guillaume (1 989) examined the availability of sodium phytate as a phosphorus source for shrimp; how- ever, effects on zinc availability were not evaluated. Objectives of this study were to evaluate the dietary zinc requirement of P. vunnumei and to determine effects of phytic acid on zinc bioavailability.

Methods Feeding Trial

The basal diet (Table 1) was formulated to contain 40% protein, gross energy of 3.4 kcaVg and was analyzed to contain 18 mg Zdkg. The basal diet was supplemented with graded levels of zinc (zinc carbonate) and phytate (dodecasodium salt from rice) by replacing cellulose. Four levels of supple- mental zinc (0, 15, 30, and 60 mg Zn/kg diet) were evaluated without supplemental phytate and three levels of zinc (0, 60, and

42 DAVIS ET AL.

200 mg Zn/kg diet) were evaluated with 1 Soh supplemental phytate.

Diets were prepared by mixing the dry ingredients in a twin shell dry blender (Pat- terson-Kelley Co., East Strausburg, Penn- sylvania). The menhaden fish oil and an ap- propriate amount of hot deionized water (required for pelleting) were then added to the dry ingredients and homogenized in a food mixer (Hobart Corp., Troy, Ohio). Each diet was pelleted using a meat grinder and a 3 mm die. After pelleting, the diets were dried by forced air at 60 C for 3 h followed by forced ambient air for 12 h to a moisture content of 8-10%. Portions, as needed for feeding, were mechanically crumbled and sieved to the desired size and then frozen at - 10 C until fed.

The feeding trial was conducted in 19 L culture chambers (bottom surface area of 0.06 m2) which were part of a 50 metric ton semi-closed recirculating system designed to maintain a constant environmental tem- perature (28 k 1 C) and salinity (30 f 1 ppt). The photoperiod was set for a 12: 12 h light : day cycle. To ensure that adequate water quality parameters were maintained, the system’s temperature, dissolved oxygen, and salinity were measured daily. Ammo- nia nitrogen and nitrite were measured weekly utilizing spectrophotometric meth- ods (Spotte 1979).

One week prior to initiation of the feeding trial, 20-day-old postlarval P. vannamei (mean weight of 0.0032 g) were maintained in a holding tank and fed the basal diet lack- ing zinc supplementation but containing 18 mg Zn/kg. At the start of the growth trial, conditioned juvenile shrimp were hand graded to a uniform size and stocked at a density of 10 shrimp per culture chamber. To estimate the initial weight of the shrimp, four groups of shrimp (1 0 shrimp per group) were randomly selected during stocking and set aside. These shrimp were then towel- dried and weighed individually to deter- mine the mean initial weight and standard deviation of the shrimp (0.058 +. 0.027 g).

Seven dietary treatments were each fed to

eight replicate groups of shrimp over a 33 d growth trial. Shrimp were fed 12 times a day in excess for the duration of the exper- iment. At the midpoint of the experimental period, the density of shrimp was reduced to six per culture chamber. At the conclu- sion of the growth trial, all shrimp were enu- merated, weighed and then immediately frozen for subsequent mineral analyses.

Frozen shrimp were rinsed with deion- ized water and then the carapace and he- patopancreas were removed by dissection. Tissue samples of shrimp from two culture chambers were pooled to produce four com- posite samples per dietary treatment. The samples were oven-dried and wet-ashed ac- cording to procedures described by the As- sociation of Official Analytical Chemists (1984). Zinc was analyzed by atomic ab- sorption spectrophotometry (Association of Official Analytical Chemists 1984). All data were subjected to analysis of variance (ANOVA) to determine significant (P < 0.05) differences between means due to the main effects (dietary zinc and phytate) and their interaction. Regression analysis was then utilized to determine effects of the di- etary treatments on the dependent vari- ables. All data were analyzed using the Sta- tistical Analysis System (SAS Institute Inc. 1988).

Digestibility Trial To determine effects of phytate on zinc

availability, an apparent digestibility trial was conducted utilizing adult P. vannamei (20 g). Digestibility diets (with 1.5% phytate and without phytate) were prepared utiliz- ing previously described procedures and the formulation presented in Table 1. To eval- uate the availability of minerals in the basal diet, a third diet was prepared by replacing the mineral premix with wheat starch.

The shrimp were maintained in 136 L fiberglass tanks at a density of 10 shrimp per tank. Exuviae and feces were removed from the tanks before the shrimp were given an initial feeding. To ensure that previously consumed material (feces and exuviae) was

EVALUATION OF ZINC 43

cleared of the digestive system, feces from the first feeding were discarded. Thirty min- utes after feeding, feces were collected and the uneaten feed removed. To limit mineral contamination from saltwater, feces si- phoned from tanks were immediately rinsed with distilled water. Once fecal samples were collected, the feeding procedure was re- peated. Three pooled samples from 16 tanks (two feeding cycles per pooled sample) were collected for each dietary treatment. Chro- mium, zinc and phosphorus contents of feed and feces were determined by inductively coupled plasma spectrometry (Association of Official Analytical Chemists 1984). Ap- parent digestibility of zinc was then deter- mined as described by the National Re- search Council (1983).

Results Mean f standard deviation total am-

monia nitrogen, nitrite nitrogen and dis- solved oxygen were 0.08 k 0.03 mg NIL, 0.13 f 0.01 mg N/L and 6.4 f 0.2 mg 02/ L, respectively. Based on the water quality parameters reported for Penaeid shrimp (Wickins 1976; Chin and Chen 1987; Chen and Chin 1988), the water quality observed in the current experiment should not have adversely affected the growth rate and sur- vival of the shrimp.

Percent survival during the first 16 days significantly increased with the supplemen- tation of phytate but did not respond to the zinc content of the diet (Table 2). Percent survival from Day 17-33 did not respond to the presence of phytate or dietary zinc levels. The final weight gain of shrimp fed the various dietary treatments did not re- spond significantly to the presence of phy- tate or the interaction between phytate and zinc (Table 2). Although weight gain was significantly affected by zinc, regression analysis of the data did not indicate a dose response to increasing levels of dietary zinc. However, among diets without phytate sup- plementation, shrimp fed diets supple- mented with 15 mg Zn/kg grew significantly better than shrimp fed the other dietary

treatments. The removal of this treatment from the data set resulted in no significant differences in the treatment means for final weight gain due to dietary zinc, possibly in- dicating that the response of shrimp fed di- ets containing 15 mg Zn/kg diet was an anomaly in the data. This conclusion is sup- ported by the results of a preliminary 28 d study in which there were no significant dif- ferences in growth rates of shrimp fed diets containing similar levels of zinc.

The zinc content of the carapace from shrimp fed diets without phytate ranged from 27.7 pg Zdg for shrimp fed diets con- taining 18 mg Zdkg to 24.0 pg Z d g for shrimp fed diets containing 33 mg Zn/kg. The zinc content of the carapace from shrimp fed diets with phytate ranged from 2 1.9 pg Zn/g for shrimp fed diets containing 18 mg Zdkg to 25.9 pg Zn/g for shrimp fed diets containing 200 mg Zn/kg. There were no significant differences between the treat- ment means due to the main effects or their interaction.

The zinc content of the hepatopancreas was significantly affected by dietary zinc and phytate. Although the interaction between phytate and zinc supplementation was not significant, the data sets (with and without phytate) were separated to allow a clearer description of the shrimp responses (Fig. 1). Zinc levels of the hepatopancreas were de- pressed when supplemental zinc was' not present and reached a plateau after the sup- plementation of 15 mg Zdkg diet, which provided a total of 33 mg Zdkg diet. In the presence of 1.5% phytate in the basal diet, zinc mineralization of the hepatopancreas was positively correlated with dietary zinc supplementation. In the presence of 1.5% phytate, the supplementation of 200 mg Zn/ kg diet (total 2 18 mg Zn/kg diet) appeared to be required for hepatopancreatic zinc concentrations to reach levels near those observed for shrimp fed diets without phy- tate supplementation.

Apparent dry-matter digestibility (mean k SD) of the complete diet without and with phytate was 83.1 f 0.81% and 83.4 f

44 DAVIS ET AL.

TABLE 2. Percent survival and weight gain of juvenile P. vannamei fed diets containing various levels of supplemental zinc with and without phytate for 33 days.a

Supplemental Zn mg/kg (total zinc)

Weight Percent survivalb Phytate pain, g

(%) Day CL16 Day 17-33 (%)

0 (18)

15 (33) 30

(48) 60

(78) 0

(18) 60

(78) 200

(2 1 8) ANOVA (P > F)

Dietary zinc Phytate Interaction

Pooled standard error

0.0 80.0

0.0 75.0

0.0 87.5

0.0 78.8

1.5 86.3

1.5 92.5

1.5 88.8

0.008 1 0.0490 0.0056 0.2822 3.98

75.0

89.6

79.2

77.1

72.9

81.2

89.6

0.2706 0.1280 0.8588 0.5940 6.72

0.99 (1,671)

1.54 (2,573)

1.18 (1,953)

1.05 (1,757)

1.22 (2,037)

1.15 (1,952)

1.35 (2,278) 0.0306 0.0 194 0.1557 0.5639 0.134

a Mean of eight replicates. Number of shrimp per replicate was reduced to 6 on Day 17.

2.05%, respectively. The basal diet con- tained 18 mg Zn/kg, 0.3 g P/100 g and 0.03 g Ca/ 1 00 g and was supplemented with 1 25.2 mg Zn/kg as zinc carbonate; 1.7 g P/lOO g and 2.2 g Ca/lOO g as calcium phosphate secondary dibasic. Diets supplemented with phytate had an additional 0.2 g P/lOO g originating from the sodium phytate. The apparent zinc availability of the complete semi-purified diet without phytate and with phytate was 67.5 k 7.93% and -40.0 f 7.6%, respectively. The apparent zinc avail- ability of the basal diet (without mineral supplements) was determined to be 5 1 .1 k 20.2%.

The apparent phosphorus availability of the complete semi-purified diet without and with supplemental phytate was 4 1.4 k 1.5% and 25.1 k 10.Ooh, respectively. The ap- parent phosphorus availability of the basal diet (without mineral supplements) was de- termined to be 86.3 f 7.1%. Based on these results, the apparent phosphorus availabil- ity of calcium phosphate dibasic from the

complete diet was 33.5%. Phytate phospho- rus was not available and the presence of phytate reduced the bioavailability of phos- phorus from other sources.

Discussion In a preliminary study concerning the es-

sentiality of dietary minerals for P. vunna- mei, the deletion of zinc from the mineral premix did not affect growth significantly, but did depress tissue zinc content (Davis et al. 1992). In the current study, the final weight gain of shrimp fed diets without phytic acid was highest at 15 mg supple- mental Zn/kg diet (total of 33 mg Zn/kg diet); however, regression analysis of the data did not indicate a typical dose response that would be characteristic of a dietary defi- ciency, and toxicity of zinc at levels greater than 33 mg Zn/kg diet appeared unlikely. Gatlin and Wilson (1 983) found that dietary zinc supplementation as high as 100 mg/kg diet had no adverse effects on channel cat- fish when fed in a semi-purified diet. Ad-

EVALUATION OF ZINC

H 200- e P a

150 - 0

P a n c 100- r e a t i 50- C

z

45

Without Phytate Ttasue Zn - 8182.07 ( - l f (Zn artpplement++ + 1 4 6 3 _.-

.-- -.

-t/ ,...-.-

+ With Phytate Tiasue Zn 17.40 log(Zn aupplement) + 28.83

Adj R 2 = 0.7216

0 ' I I I I I

n 0 50 100 150 200 250 C

FIGURE 1. Relationship [predicted value k 95% confidence interval for the expected value (mean) of the dependent Total dietary zinc (mg/kg)

variable] between dietary zinc and hepatopancreatic zinc (pg Z d g dry weight).

ditionally, in terrestrial animals, adverse physiological effects of zinc were not ob- served at dietary concentrations lower than 600 mg Zdkg diet (National Research Council 1 980). Consequently, the increased weight gain observed at 33 mg Zdkg diet may be an anomaly.

In the current experiment, mineralization of the hepatopancreas increased when shrimp were fed diets containing 15 mg sup- plemental Zn/kg diet and then plateaued. The hepatopancreas has been identified as a site of zinc excretion and absorption for a variety of decapod crustaceans (Bryan 1964, 1967, 1968). If the hepatopancreas was simply functioning as a storage site for the excretion of zinc, a linear increase would be expected. The disproportionate increase in concentrations of hepatopancreatic zinc indicates that tissue stores were not satisfied without dietary zinc supplementation and that zinc stores andor absorption may be regulated. Similar responses of tissue zinc have been found in fish (Ogino and Yang 1978, 1979; Gatlin and Wilson 1983; Satoh et al. 1987) and eel (Park and Shimizu 1989)

with the depletion of tissue zinc character- istic of a zinc deficiency. Additionally, the need for a dietary supplement is supported by the positive availability of zinc from the basal diet without mineral supplementation (Le., if adequate zinc was obtained from non- dietary sources, excretion into the feces would be expected to occur).

Phytate has been shown to affect the bio- availability of zinc in a variety of terrestrial animals (Oberleas et al. 1962; O'Dell et al. 1964; Savage et al. 1964; Lo et al. 198 1) and fish (Gatlin and Wilson 1984; Richardson et al. 1985; McClain and Gatlin 1988). In the previous studies with terrestrial animals and fish, increased levels of zinc were re- quired to overcome nutritional deficiencies caused by the inhibitory effects of phytate. In the present experiment, phytate signifi- cantly decreased zinc levels of the hepato- pancreas, indicating a negative effect on bio- availability of zinc. The supplementation of 200 mg Z a g appeared to be required to overcome the depressed bioavailability of zinc and return zinc levels of the hepato- pancreas to levels near those observed when

46 DAVIS ET AL.

phytate was not present. Although tissue mineralization was affected by the presence of dietary phytate, there were no adverse effects on growth of the shrimp.

The addition of phytate did not affect dry weight digestibility but did reduce the ap- parent availability of zinc and phosphorus. Diets supplemented with phytate had a neg- ative zinc availability (i.e., excretion ex- ceeded absorption), indicating a complex- ing of phytate with zinc from dietary and nondietary sources. The observed negative availability of zinc in the presence of phy- tate and the positive availability of zinc in the basal diet without mineral supplements would indicate that although zinc may be excreted by the digestive system, it appears to be reabsorbed if a complexing agent is not present.

Under the experimental conditions, phy- tate phosphorus appeared unavailable and inhibited the availability of phosphorus from other sources. Similarly, Civera and Guillaume (1 990) reported that the appar- ent digestibility of total phosphorus was lower in diets containing phytate than in diets containing disodium phosphate. How- ever, the apparent availability of phytate phosphorus was reported to be 8.4% for P. vannamei and 47.3% for P. japonicus. How- ever, in that study, phytate was the primary source of phosphorus, whereas in the pres- ent study, calcium phosphate was the pri- mary source. The difference in phosphorus sources and levels may account for the slight difference in the observed phosphorus availability from phytate.

Conclusion Hepatopancreatic zinc appeared to be a

sensitive indicator of dietary zinc status, whereas carapace mineralization and growth were not affected by dietary zinc intake or the presence of phytic acid. Under the con- ditions of this study, the presence of 1 Soh phytate in the diet did not result in a sig- nificant depression in growth; however, it did depress tissue mineralization and zinc bioavailability. Furthermore, the presence

of 1.5% phytate in the diet decreased the apparent phosphorus availability. Based on these results, as well as those reported for fish, the supplementation of up to 200 mg Zn/kg diet as zinc carbonate appears safe and warranted in the presence of dietary phytate. Under the current experimental conditions and when phytate was not pres- ent in the diet, 15 mg of supplemental Z d kg diet fiom zinc carbonate appeared to sat- isfy the zinc requirement of P, vunnumei when the basal diet contained 18 mg Zdkg dry weight.

Acknowledgments The authors would like to express their

thanks to K. Hall for her technical assis- tance. This research was supported by the Texas Agricultural Experiment Station un- der project H-6 3 2 5 .

Literature Cited Associationof Official AnalyticalChemists. 1984. Of-

ficial methods of analysis. Association of Official Analytical Chemists, Arlington, Virginia, USA.

Bryan, G. W. 1964. Zinc regulation in the lobster Homarus vulgaris I. Tissue zinc and copper con- centrations. Journal of the Marine Biology Asso- ciation of the United Kingdom 57:621-667.

Bryan, C. W. 1967. Zinc regulation in the freshwater crayfish (including some comparative copper anal- yses). Journal of Experimental Biology 46:281- 296.

Bryan, G. W. 1968. Concentrations of zinc and cop- per in the tissues of decapod crustaceans. Journal of the Marine Biology Association of the United Kingdom 48:303-321.

Chen, J.-C and T.-S. Chin. 1988. Acute toxicity of nitrite to tiger prawn, Penaeus monodon, larvae. Aquaculture 69:253-262.

Chin, T.S. and J . 4 . Chen. 1987. Acute toxicity of ammonia to larvae of the tiger prawn, Penaeus monodon. Aquaculture 66: 247-2 5 3.

Civera, R. and J. Cuillaume. 1989. Effect of sodium phytate on growth and tissue mineralization of Penaeus japonicus and Penaeus vannamei juve- niles. Aquaculture 77:145-156.

Civera, R. and J. Guillaume. 1990. Digestive utili- zation of phytate phosphorus in two species of shrimp: Penaeus japonicus and Penaeus vanna- mei. Abstract. World Aquaculture Meeting, Hal- ifax, Canada.

Davis, D. A., A. L. Lawrence and D. M. Getlin 111. 1992. Mineral requirements of Penaeus vanna-

EVALUATION OF ZINC 47

mei: a preliminary examination of the dietary es- sentiality for thirteen minerals. Journal of the World Aquaculture Society. 23:8-14.

Gatlin, D. M. 111 and R. P. Wilson. 1983. Dietary zinc requirement of fingerling channel catfish. Journal of Nutrition 1 13:63C-635.

Gatlin, D. M. I11 and R. P. Wilson. 1984. Zinc sup- plementation of practical channel catfish diets. Aquaculture 41:3 1-36.

Lo, G. S., S. L. Settle, F. H. Steinke and D. T. Hopkins. 198 1. Effect of phytate: zinc molar ratio and iso- lated soybean protein on zinc bioavailability. Journal of Nutrition 11 1:2223-2235.

McClain, W. R. and D. M. Gatlin 111. 1988. Dietary zinc requirement of Oreochromis aurea and effects of dietary calcium and phytate on zinc bioavail- ability. Journal of the World Aquaculture Society

National Research Council. 1980. Mineral tolerance of domestic animals. National Academy Press, Washington, D.C., USA.

National Research Council. 1983. Nutrient require- ments of warmwater fishes and shellfishes. Na- tional Academy Press, Washington, D.C., USA.

Oberleas, D., M. E. Muhrer and B. L. O’Dell. 1962. Effects of phytic acid on zinc availability and para- keratosis in swine. Journal of Animal Science 21: 57-61.

O’Dell, B. L., J. M. Yohe and J. E. Savage. 1964. Zinc availability in the chicken as affected by phy- tate, calcium and ethylenediaminetetraacetate. Poultry Science 43:45419.

Ogino, C. and G Y . Yang. 1978. Requirement ofrain- bow trout for dietary zinc. Bulletin of the Japanese Society of Scientific Fisheries 44:1015-1018.

Ogino, C. and GY. Yang. 1979. Requirement ofcarp

19: 103-108.

for dietary zinc. Bulletin of the Japanese Society of Scientific Fisheries 45:967-969.

Park, C. W. and C. Shimizu. 1989. Suitable levels of zinc supplementation to the formulated diets in young eel. Nippon Suisan Gakkaishi 55:2 137- 2141.

Renfro, W. C., S. W. Fowler, M. Heyraud and J. LaRosa. 1975. Relative importance of food and water in long-term accumulation by marine biota. Journal Fisheries Research Board Canada

Richardson, N. L., D. A. Higgs, R. M. Beames and J. R. McBride. 1985. Influence of dietary calcium, phosphorus, zinc and sodium phytate level on cat- aract incidence, growth and histopathology in ju- venile chinook salmon Oncorhynchus tshawyts- cha. Journal of Nutrition 1 I5:553-567.

SAS Institute, Inc. 1988. SASISTAT user’s guide, release 6.03 edition. Cary, North Carolina, USA.

Satoh, S., K. Tabata, K. Izume, T. Takeuchi and T. Watanabe. 1987. Effect of dietary tricalcium phosphate on availability of zinc to rainbow trout. Nippon Suisan Gakkaishi 53:1199-1205.

Savage, J. E., J. M. Yohe, E. E. Picket and B. L. O’Dell. 1964. Zinc metabolism in the growing chick. Tissue concentration and effect of phytate on absorption. Poultry Science 43:420-426.

Spotte, S. 1979. Fish and invertebrate culture: water management in closed systems, 2nd edition. Wi- ley, New York, New York, USA.

1988. Availability of minerals in fish meal to fish. Asian Fisheries Society 1:175-i95.

Wickins, J. F. 1976. The tolerance. of warm-water prawns to recirculated water. Aquaculture 9: 19- 37.

32~1339-1345.

Watanabe, T., S. Satoh and T. Takeuchi.