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Availability of dietary magnesium to rainbow trout as determined by apparent retention
Aquaculture, 86 (1990) 51-61
Availability of Dietary Magnesium to Rainbow Trout as Determined by Apparent Retention KARL D. SHEARER and TORBJORN ASGARD* U.S. Department of Commerce, National Oceanic and Atmospheric Administration, Northwest Fisheries Center, 2725 Montlake Boulevard East, Seattle, WA 98112 (U.S.A.) (Accepted 27 June 1989)
ABSTRACT Shearer, K.D. and Asgard, T., 1990. Availability of dietary magnesium to rainbow trout as determined by apparent retention. Aquaculture, 86: 51-61. The relative availability of magnesium (Mg) added to semi-purified trout diets was estimated by determining apparent retention. Slightly deficient test diets contained Mg from five inorganic salts or from fish bonemeal. Reference diets containing low and sufficient Mg were also fed. Apparent Mg retentions derived from whole body Mg concentrations indicated that the Mg from the various inorganic salts was of equal availability (apparent retention 76% ) . The availability of Mg from fish bonemeal was significantly less (apparent retention 54% ) . The availability of the Mg sources remained constant over the 6-week experiment, indicating that the fish were unable to increase their intake efficiency from the water or the diet. Mortalities due to Mg deficiency occurred after 4 days in fish fed the low Mg diet. We concluded that apparent retention as determined by whole body analysis of fish fed slightly deficient diets is a sensitive and relatively simple way of determining relative availability.
INTRODUCTION
A recent study (Shearer, 1989) indicated that the magnesium (Mg) requirement of rainbow trout was higher than indicated by earlier studies (Ogino et al., 1978; Knox et al., 1981). The high requirement reported may have been due in part to the high feed efficiency achieved in the study (Shearer, 1988) or the low level of Mg in the water but could also have been the result of low bioavailability of the inorganic Mg source used. Studies examining the bioavailability of essential elements in fish are relatively few and a variety of methods have been used. Each method of determining availability, balance studies, digestibility, and primary or secondary doseresponse indicators has its potential sources of error. *Present address: The Agricultural Research Council of Norway, Institute of Aquaculture Research, N-6600 Sunndalsora (Norway)
0044-8486/90/$03.50
0 1990 Elsevier Science Publishers B.V.
52
K.D. SHEARER AND T. ASGARD
Balance studies failed to adequately predict retention of Mg fed to rats (Heroux and Peter, 1975)) and reviews by Hegsted (1976) and Mertz (1987) concluded that availabilities determined by balance studies must be viewed with skepticism. Studies of this type are even more difficult to conduct in aquatic systems since fish can eliminate some elements by multiple routes - feces, urine, gills (Hardy et al., 1987) - and several elements can be taken up directly from the water. Gastrointestinal absorption has been used as an indicator of availability for some elements in fish (Ogino et al., 1979), but homeostasis of the elements examined by this method must be achieved by elimination rather than intestinal absorption (Mertz, 1987). When an inert marker such as chromic oxide is used, it is assumed that the marker and the element being examined travel through the digestive tract at the same rate (Heroux and Peter, 1975). This may not be the case with Mg which can be absorbed and secreted back into the intestine in bile salts (Aikawa, 1976). Evidence also exists that Mg in some fishes is reabsorbed in the latter part of the intestine (Dabrowski and Schwarz, 1986), so, underestimation of absorption may result if feces are removed for analysis before absorption is complete. Absorption may also be affected by handling stress (Heroux and Peter, 1975). A portion of the element taken up directly from the water may end up in the feces and be indistinguishable from that of dietary sources. In brackish or salt water, it may also be necessary for a fish to retain a portion of the Mg it ingests in the intestine for osmotic purposes (Shehadeh, 1967) introducing an error in digestibility measurements. Finally, the large differences in digestibility observed when Heroux and Peter (1975) fed several levels of a single Mg source to rats are indistinguishable from the results observed when true differences in availability exist between different sources present at the same concentration. Availability can also be determined through dose-response studies where primary (growth, health, performance) or secondary (whole body retention, tissue, hormone, or enzyme level) responses to a substance are measured (Spivey Fox et al., 1981) . When dietary requirements have previously been determined and primary and secondary response indicators have previously been related, absolute nutritional status as well as nutrient availability can be assessed by monitoring a secondary response indicator. The present study was designed to examine the availability of various dietary sources of Mg to rainbow trout. Availability was estimated by measuring apparent Mg retention in rapidly growing fish fed slightly Mg-deficient diets with high feed efficiency. Nutritional status was determined by comparing whole body Mg concentrations of fish fed the experimental diets to levels previously established as normal (Shearer, 1984,1989). MATERIALS AND METHODS
Eight semi-purified casein/gelatin-based diets were prepared and fed to triplicate groups of rainbow trout. Six diets were formulated to contain 550 ppm
AVAILABILITY OF DIETARY MAGNESIUM TO RAINBOW TROUT
53
magnesium, five from reagent grade magnesium sources: MgO, MgC& - 6Hz0, (MgC03)4.Mg(03)2, Mg2Si308, MgHP04*3Hz0, and one from fish bonemeal (Table 1). The fish bonemeal was a byproduct of producing liquefied fish protein concentrate for use in fish diets (Advanced Hydrolyzing Systems, Warrenton, OR). Two additional diets were prepared: one contained no supplemental magnesium, the other contained 1900 ppm Mg from MgO. An additional treatment (treatment 9) was added when food was withheld from one replicate of treatment 1 after mortalities were observed in all three replicates of this treatment on day 4. Each diet was prepared in a single batch and was stored at - 10 oC until fed. Trout, initial weight less than 1 g, were obtained from a commercial producer (Trout Lodge, Sumner, WA). Fish were fed a practical moist diet for 1 week prior to the start of the experiment. Lots of 60 fish were randomly assigned to each of 24 tanks. Each 200-I tank was supplied with 1.2 l/min of dechlorinated municipal water. Temperature was maintained at 13.5 t 0.5”C and a natural photoperiod (October-November) was used. Fish were bulk weighed at the start of the experiment and every 2 weeks thereafter. Fish were fed for 12 consecutive days and then starved for 2 days prior to weighing and sampling for TABLE 1 Composition of experimental diets Ingredients
% dry weight
Casein Gelatin Dextrin Alpha-cellulose“ Vitamin mixb Choline chloride (70% ) Herring oil Amino acid mix” Mineral mixd Ascorbic acid Trace mineral solution”
43.2 8.0 16.0 6.7 2.0 1.0 15.0 4.4 3.5 0.1 -
Mgf “Reduced as Mg containing component increased. ‘Same as reported by Hardy and Shearer (1985). ‘As g/kg diet: arg 11.5, lys 11.0, met 4.0, leu 9.0, va13.2, thr 5.0. dAs g/kg diet: CaHPO,.2H,O, 7.9; KCl, 17.0; NaCl, 0.6; NaH2P0,, 7.4. “As mg/kg diet: CoCl,.GH,O, 4.0; CuS04*5Hz0, 11.8; FeSO,.7H,O, 115; MnSO*-HZO, 32.5; ZnSO,*7H,O, 88; NaHSeO,, 4.2; KI, 1.9. ‘Mgsource added (mg/kg diet): diet 1,O; diet 2, MgO, 829; diet 3, MgO, 2500; diet 4, MgCl,*GH,O, 2920; diet 5, (MgCO,),.Mg(OH),, 1400; diet 6, Mg,Si,O,, 2680; diet 7, MgHPO,*3H,O, 2560; diet 8, fish bonemeal, 95000 (replaced some dextrin in addition to the alpha-cellulose).
54
K.D. SHEARER AND T. ASGARD
elemental analysis. Fish were hand-fed several small meals each day until the ration was consumed. The ration level was adjusted weekly, and was based on growth rates and feed efficiencies observed under similar conditions in an earlier study (Shearer, 1989) and predicted growth rates for rainbow trout (Austreng et al., 1987). The initial fish sample for elemental analysis consisted of 12 fish from the common pool. Four fish were randomly selected from each tank after week 2, and five fish were taken from each tank after weeks 4 and 6. Individual fish were dried, ashed at 55O”C, dissolved in acid, and analyses were made using emission spectrophotometry (Shearer, 1984). Visual examinations were made of the gills, kidneys, livers, and spleens from five fish fed each diet at the end of the experiment. A hepatosomatic index (HSI) for these groups was also calculated. Daily feed consumption and mortality were recorded. Apparent retention estimates were calculated at weeks 2,4, and 6. Analyses of variance were performed using StatView 512+ (Brainpower, 1986) and multiple mean comparisons were made using the Student-Newman-Keuls test (Zar, 1974). A significance level of P< 0.05 was chosen. Elemental measurements from replicates were pooled since they were not significantly different. RESULTS
Elemental analysis of the diets indicated that diets 4 through 8 contained approximately the expected level of Mg (537, 545, 540, 537, and 614 ppm). Diets 2 and 3 contained approximately 30% more Mg than they were formulated to contain (716 and 1900 ppm); diet 1, the basal diet, contained about 34 ppm Mg. Diet 8 (bonemeal) contained 2.7 and 2.2% calcium (Ca) and phosphorus (P) while the remaining diets contained 0.3 and 0.9%, respectively, of these two elements. Levels of the other elements met or exceeded currently recognized requirements (NRC, 1981). Protein, fat, and ash levels were 52.2, 15.3, and 4.5 percent, respectively. The rearing water contained 1.4 ppm Mg, 9.6 ppm Ca, and 1.7 ppm Na. Levels of P, Fe, Sr, and Zn were not quantifiable. Mean weights of fish fed diets 2 through 8 were similar at each fortnightly weighing (Fig. 1). Instantaneous daily growth rates for the consecutive 2-week feeding periods were 6.2,4.3, and 3.4% per day. Fish fed diet 1 (no Mg supplement) had growth rates of 3.1,2.5, and 3.2% per day during this period. Fish in treatment 9 (unfed after day 4) showed a weight gain at week 2, but their mean weight declined to slightly below their initial weight by the end of the experiment. Survival of fish fed diets 2 through 7 was 100% (Table 2). Mortality began occurring in fish fed diet 1 on day 4 and survival was 28% in the two tanks which were fed this diet for 6 weeks. Mortality in treatment 9, which was fed diet 1 for 4 days and starved thereafter, stopped once food was withheld and
AVAILABILITY
OF DIETARY MAGNESIUM TO RAINBOW TROUT
55
‘r 6 Treatment
1 0
2
4
6
Time (weeks) Fig. 1. Growth of fish fed experimental diets. Only four treatments are shown; the others lie between diet 6 and diet 3. TABLE 2 Final weight, weight gain, feed efficiency, dry matter, ash, and survival of rainbow trout fed the experimental diets for 6 weeks1 Diet
Final2 weight (g)
Weight gain (%)
Feed efficiency3
Dry matter (% )
Ash in wet wt. (%)
Survival (%)
1 2 3 4 5 6 7 8 9 Pooled S.D.
3.40b 5.99” 5.74” 5.79” 5.69” 6.26” 5.92” 5.73” 1.26” 0.19
162’ 361” 342” 345” 338” 381” 355” 341” -3” 15
0.67b 1.50” 1.40” 1.42” 1.37” 1.57a 1.46” 1.42” - 0.20” 0.06
22.gb 26.1” 26.0” 26.0” 25.9” 25.7” 26.0” 26.4” 18.4” 1.0
2.0b 2.2” 2.1” 2.2” 2.1” 2.3” 2.2” 2.3” 2.4” 0.1
2gd 100” 100” 100” 100” 100” 100” 92b 78” 1
‘Values in the same column with different letters are significantly different (P> 0.5). 21nitialweight 1.3 g. 3Wet weight gain/dry food fed.
overall survival was 78%. Fish fed diet 8 began to die after week 2 and survival to the end of the experimental was 92%. Visual examination of fish livers at the end of the experiment indicated that the livers of all fish were pale, mottled, and slightly enlarged (HSI 2.6, n=5; normal 1.2, Hilton and Hodson, 1983). Gills, kidneys, and spleen appeared normal.
K.D. SHEARER AND T. ASGARD
56
Initial feed acceptance was good in all treatments but fish diet 1 reduced their food consumption coincidental to the start of the mortalities. Fish fed diet 8 (bonemeal) showed behavioral changes during week 4, initially striking at feed but experienced convulsions and feed expulsion immediately after. Tetany often preceded mortality. Overall feed efficiency in groups 2 through 8 (Table 2) was 1.45. As expected, this declined from 1.70 to 1.46 and 1.32 during each 2-week period. Feed efficiencies in fish fed diet 1 were 0.75,0.66, and 0.64 with an overall efficiency of 0.67. Fish fed diet 1 for 4 days (treatment 9) had a feed efficiency of 1.55 for week 1. Whole body Mg concentrations increased with fish growth as expected in treatment 3. The Mg concentrations of fish fed diets 1, 2, and 4 through 8 declined (Fig. 2). In treatment 9 (unfed after day 4)) whole body concentrations returned to normal after feeding was suspended. After 6 weeks, whole body Mg concentrations of fish fed diets 2,4,5,6, and 7 were significantly less than those of fish fed diet 3 (Table 3) and fish fed diet 2 had significantly more Mg than those fed diets 4,6, and 7. The concentration of Mg in fish fed diet 8 was significantly below that of all treatments except treatment 1 which contained no added Mg. Concentrations in fish fed diet 1 dropped considerably in the first 2 weeks but recovered somewhat between weeks 2 and 4 when their growth was slowed by anorexia. It again declined rapidly between weeks 4 and 6 when their feed consumption increased. Whole body Ca and P were elevated in fish fed diet 8 while zinc (Zn) was considerably less than in other groups (Table 3 ) . Manganese was also significantly reduced in fish fed diet 8. Apparent retention of Mg remained constant from period to period, and the
Treatment 1 -2 -3 -4 -5 -6 -7 -8 -*
200 ’ 0
I
I
I
2
4
6
Time (weeks) Fig. 2. Whole body Mg concentrations of Mg from several sources (see text).
(ppm wet basis) of fish fed diets containing
various levels
* represents normal levels from Shearer (1989).
57
AVAILABILITY OF DIETARY MAGNESIUM TO RAINBOW TROUT
TABLE 3 Mean whole fish Mg, Ca, P, and Zn concentrations’ (ppm wet) and apparent Mg retention (% ) in rainbow trout fed the experimental diets for 6 weeks Diet
Mg cont.
Ca Cont.
P cont.
Zn cont.
Apparent Mg retention (% )”
1 2 3 4 5 6 7 8 9 Pooled S.D.
215” 322b 346” 295” 301b” 281” 300” 250d 284” 13
4323d” 456gcd 4001” 4464cd 4221de 4766” 4555”d 526gb 6171” 398
3752” 4202bc 3909d 4039cd 3911d 4179b” 4088” 4319”s 4426” 203
32.3b 28.0” 26.0” 27.2” 25.2” 27.1” 25.3” 17.ld 43.4” 3.3
307” 67’ 26d 77b 75b 80b 81b 54” 0” 5
In= 15. 2Apparent retention = [ (final Mg burden-initial
Mg burden) /Mg fed] X ( 100). n = 3.
overall retention was 76% for the diets containing the inorganic salts, 26% for diet 3, and 54% for diet 8. The fish fed diet 1 had an apparent retention of 307%, indicating that they obtained Mg directly from the water. The fish in treatment 9 ended the experiment with less than their initial burden of Mg leading to a negative retention. DISCUSSION
Primary dose-response indicators (growth, health, etc. ) (Spivey Fox et al., 1981) have been used to assess availability in terrestrial animals and fish, but are often insensitive (Baker, 1986). In the present study, equivalent growth occurred in fish fed diets 2 through 8 even though whole body Mg concentrations of all but group 3 (1900 ppm dietary Mg) were less than normal and mortalities occurred in group 8 (bonemeal). Satoh et al. (1987a) also observed normal growth but reported cataracts and low vertebral zinc in trout fed diets with low zinc availability. However, they reported conflicting results when they were able to relate reduced growth to differences in availability in an additional study (Satoh et al., 1987b). Dabrowska et al. (1989a) attributed reduced growth in tilapia (Oreochromis niloticus ) to low availability of Mg, but they observed the highest carcass Mg concentrations in fish receiving the diet reported to have the lowest availability. In fact, body Mg concentrations in fish reported to be Mg-deficient did not differ from levels they reported as normal in an additional study (Dabrowska et al., 1989b). Secondary indicators of response (whole body retention; tissue, hormone, or
58
K.D. SHEARER AND T. ASGARD
enzyme levels) appear to be the most useful means of comparing availabilities, especially when these secondary indicators have previously been correlated with primary indices (Spivey Fox et al., 1981). Normal whole body Mg concentrations in rainbow trout have previously been reported (Shearer, 1984) and have been related to primary indices such as growth (Shearer, 1989). Feeding rapidly growing fish diets deficient in Mg requires them to make full use of this element from the diets. If uptake of Mg from the water is timedependent, the rapid growth also prevents them from meeting their requirement from the water. The equivalent rates of Mg retention observed when the five magnesium salts were fed indicates that they had similar availabilities. The slightly higher Mg concentration observed in fish fed diet 2 appears to be a consequence of the slightly higher Mg concentration in this diet. The lower whole body levels in the fish fed the bonemeal diet indicate that it was less available than the inorganic sources. When excess levels are fed (diet 3,190O ppm ) , retention can not be used as an indicator of availability since the percent retained is an artifact of the amount fed. This will continue to decline as the level in the feed is increased. We can confirm that diets 2 and 4 through 9 were not in excess since the body concentrations were less than normal (Shearer, 1984 ) . The results observed in the present study confirm that the dietary Mg requirement reported earlier (Shearer, 1989) is correct and can be higher than stated by Ogino et al. (1978) or Knox et al. (1981). The level reported by Shearer (1989) (1300-1400 ppm) may also be an underestimate since a portion of the requirement may have been met by direct uptake from the water. Whether Mg is taken from the water when adequate Mg exists in the diet remains unknown. In addition, it is possible to achieve faster growth and higher feed efficiency than achieved in that study, further increasing the fish’s dietary requirement. The critical whole body Mg concentration for fish of this size appears to be about 80% of normal. At this level, mortalities were observed in fish fed diets 1 and 8. The fact that fish fed diet 8 still showed normal growth and feed efficiency at 6 weeks despite the occurrence of mortalities contrasts with the results observed from treatment 1 where growth was reduced before week 2. Some adaptation to low Mg availability was made in treatment 8 fish; perhaps they were able to mobilize enough Mg from the bones to maintain normal growth and enzymatic activity. The onset of Mg deficiency in fish fed the Mgfree diet (diet 1) was apparently too rapid for an adjustment to occur. The fact that apparent availability remained constant for each diet during consecutive fortnightly periods indicates that fish are unable to improve the efficiency of uptake of this element from the water or the diet. The higher Ca and P concentrations in Mg-deficient fish have been reported previously (Shearer, 1989). Diets 1,2, and 4 through 7 contained much lower levels of Ca and P than diet 8, so these fish would not have the opportunity to increase
AVAILABILITY
OF DIETARY
MAGNESIUM
TO RAINBOW
TROUT
59
their utilization of these elements. Low whole body Zn in fish fed diet 8 appears to be the result of the complexing of this element with Ca and P in the diet (Hardy and Shearer, 1985 ) . True dietary retention in fish fed diets 2 and 4 through 8 can be estimated by subtracting the amount of Mg taken up from the water by fish fed diet 1 from the apparent retention observed in these diets. If it is assumed that 100% of the Mg fed to fish in group 1 was retained, then these fish were able to obtain 94 mg of Mg/g of fish from the water. If we assume that fish fed the deficient diets were able to accumulate an equivalent amount from the water, then they would have taken up 339 mg/fish. Retention from the feed then becomes 57% from diets 2,4,5,6, and 7 and 36% from diet 8. Cook (1973) found 58% of the Mg from inorganic sources was available to rats. Our figure for retention from the diet may still be overstated since some loss of feed was experienced in fish fed diet 1. Assuming a feed efficiency similar to the other groups, retention would be reduced by a further 5%. True availability will, of course, be higher than true retention by the amount of endogenous loss. For Mg, this appears to be small, less than 1% of body burden per day (Shearer, unpublished). Despite the fact that the true availability of the diets can only be estimated, the relative availability of these sources remains unchanged. In the study by Dabrowska et al. (1989a), it appears that the reduced growth reported in fish fed Mg-sulfate may be due to a sulfate effect. The fact that fish fed a diet containing this Mg source had normal carcass Mg concentrations supports this conclusion. Anorexia, a sign of Mg deficiency (Shearer, 1989), was not observed. It is unlikely that a Mg deficiency developed with the low feed efficiency, slow growth rates, and high water-borne Mg reported in their experiment. In fact, the low Mg deposition appears to indicate that the diets contained an excess of available Mg. In summary, this study shows that a secondary indicator of Mg status (whole body Mg concentration) is useful in determining the relative availability of Mg to rainbow trout from various dietary sources. The apparent retention of Mg from inorganic salts was found to be less than 76% and this was higher than apparent retention of Mg from fish bonemeal (54% ) . Fish fed Mg deficient diets were unable to increase Mg uptake efficiency either from feed or water. Under conditions of moderate deficiency they were able to maintain normal growth and feed efficiency, apparently by maintaining adequate concentrations for specific functions. ACKNOWLEDGEMENTS
We thank Erich J. Gauglitz, Jr., and Joanne Hudson for mineral analysis and Dr. Ron Hardy and Dr. Barbara Grisdale-Helland for criticism of the manuscript. Participation of Torbjorn Asgard in this project was made possible by
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K.D. SHEARER AND T. ASGARD
a grant from the Agricultural Research Council of Norway and its Institute of Aquaculture Research. REFERENCES Aikawa, J.K., 1976. Biochemistry and physiology of magnesium. In: A.S. Prasad (Editor), Trace Elements in Human Health and Disease, Vol. II. Academic Press, New York, NY, pp. 47-48. Austreng, E., Storebakken, T. and Asgard, T., 1987. Growth rate estimates for cultured Atlantic salmon and rainbow trout. Aquaculture, 60: X7-160. Baker, D.H., 1986. Problems and pitfalls in animal experiments designed to establish dietary requirements for essential nutrients. J. Nutr., 116: 2339-2349. Brainpower, 1986. Statview 512 +. 24009 Ventura Blvd., Calabrasa, CA 91302,108 pp. Cook, D.A., 1973. Availability of magnesium: balance studies in rats with various inorganic magnesium salts. J. Nutr., 103: 1365-1370. Dabrowska, H., Gunther, K.-D. and Meyer-Burgdorff, K., 1989a. Availability of various magnesium compounds to tilapia (Oreochromis niloticus). Aquaculture, 76: 269-276. Dabrowska, H., Meyer-Burgdorff, K. and Gunther, K.-D., 1989b. Interaction between dietary protein and magnesium level in tilapia (Oreochromis niloticus). Aquaculture, 76: 277-291. Dabrowski, K.R. and Schwarz, F.J., 1986. Mineral content in the digestive tract of stomachless fish. Zool. Jahrb. Physiol., 90: 193-200. Hardy, R.W. and Shearer, K.D., 1985. Effect of dietary calcium phosphate and zinc supplementation on whole body zinc concentration of rainbow trout. Can. J. Fish. Aquat. Sci., 42: 181184. Hardy, R.W., Sullivan, C.V. and Koziol, A.M., 1987. Assessment of absorption, body distribution, and excretion of dietary zinc by rainbow trout (Salmo guirdneri). Fish Physiol. Biochem., 2: 133-143. Hegsted, D.M., 1976. Balance studies. J. Nutr., 106: 307-311. Heroux, 0. and Peter, D., 1975. Failure of balance measurements to predict actual retention of magnesium and calcium by rats as determined by direct carcass analysis. J. Nutr., 105: 11571167. Hilton, J.W. and Hodson, P.V., 1983. Effect of increased dietary carbohydrate on selenium metabolism and toxicity in rainbow trout (Salmo guirdneri). J. Nutr., 113: 1241-1248. Knox, D., Cowey, C.B. and Adron, J.W., 1981. Studies on the nutrition of salmonid fish. The magnesium requirement of rainbow trout (Salmo guirdneri). Br. J. Nutr., 45: 137-148. Mertz, W., 1987. Use and misuse of balance studies. J. Nutr., 117: 1811-1813. NRC (National Research Council), 1981. Nutrient requirements of coldwater fishes. National Academy Press, Washington, DC, 102 pp. Ogino, C., Takashima, F. and Chiou, J.Y., 1978. Requirement of rainbow trout for dietary magnesium. Bull. Jpn. Sot. Sci. Fish., 45: 137-148. Ogino, C., Takeuchi, L., Takeda, H. and Watanabe, T., 1979. Availability of dietary phosphorus in carp and rainbow trout. Bull. Jpn. Sot. Sci. Fish., 45: 1527-1532. Satoh, S., Takeuchi, T. and Watanabe, T., 1987a. Availability to rainbow trout of zinc in white fish meal and of various zinc compounds. Nippon Suisan Gakkaishi, 53: 595-599. Satoh, S., Izume, K. and Takeuchi, T., 1987b. Availability to rainbow trout of zinc contained in various types of fish meals. Nippon Suisan Gakkaishi, 53: 1861-1866. Shearer, K.D., 1984. Changes in elemental composition of hatchery-reared rainbow trout, Sulmo guirdneri, associated with growth and reproduction. Can. J. Fish. Aquat. Sci., 41: 1592-1600. Shearer, K.D., 1988. Dietary potassium requirement of juvenile Chinook salmon. Aquaculture, 73: 119-129. Shearer, K.D., 1989. Whole body magnesium concentration as an indicator of magnesium status in rainbow trout (Sulmo guirdneri). Aquaculture, 77: 201-207.
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OF DIETARY
MAGNESIUM
TO RAINBOW
TROUT
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Shehadeh, A.H., 1967. The role of intestine in salinity adaption of the rainbow trout (S&no gairdneri). Zoology Ph.D. Thesis. University of California, Los Angeles, CA, 81 pp. Spivey Fox, M.R., Jacobs, R.M., Jones, A.O.L., Fry, B.E., Jr., Raksowa, M., Hamilton, R.P., Harland, B.F., Stone, C.L. and Tao, S.-H., 1981. Animal models for assessing bioavailability of essential and toxic elements. Cereal Chem., 58( 1): 6-11. Zar, J.H., 1974. Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, NJ, 620 pp.
Availability of Dietary Magnesium to Rainbow Trout as Determined by Apparent Retention KARL D. SHEARER and TORBJORN ASGARD* U.S. Department of Commerce, National Oceanic and Atmospheric Administration, Northwest Fisheries Center, 2725 Montlake Boulevard East, Seattle, WA 98112 (U.S.A.) (Accepted 27 June 1989)
ABSTRACT Shearer, K.D. and Asgard, T., 1990. Availability of dietary magnesium to rainbow trout as determined by apparent retention. Aquaculture, 86: 51-61. The relative availability of magnesium (Mg) added to semi-purified trout diets was estimated by determining apparent retention. Slightly deficient test diets contained Mg from five inorganic salts or from fish bonemeal. Reference diets containing low and sufficient Mg were also fed. Apparent Mg retentions derived from whole body Mg concentrations indicated that the Mg from the various inorganic salts was of equal availability (apparent retention 76% ) . The availability of Mg from fish bonemeal was significantly less (apparent retention 54% ) . The availability of the Mg sources remained constant over the 6-week experiment, indicating that the fish were unable to increase their intake efficiency from the water or the diet. Mortalities due to Mg deficiency occurred after 4 days in fish fed the low Mg diet. We concluded that apparent retention as determined by whole body analysis of fish fed slightly deficient diets is a sensitive and relatively simple way of determining relative availability.
INTRODUCTION
A recent study (Shearer, 1989) indicated that the magnesium (Mg) requirement of rainbow trout was higher than indicated by earlier studies (Ogino et al., 1978; Knox et al., 1981). The high requirement reported may have been due in part to the high feed efficiency achieved in the study (Shearer, 1988) or the low level of Mg in the water but could also have been the result of low bioavailability of the inorganic Mg source used. Studies examining the bioavailability of essential elements in fish are relatively few and a variety of methods have been used. Each method of determining availability, balance studies, digestibility, and primary or secondary doseresponse indicators has its potential sources of error. *Present address: The Agricultural Research Council of Norway, Institute of Aquaculture Research, N-6600 Sunndalsora (Norway)
0044-8486/90/$03.50
0 1990 Elsevier Science Publishers B.V.
52
K.D. SHEARER AND T. ASGARD
Balance studies failed to adequately predict retention of Mg fed to rats (Heroux and Peter, 1975)) and reviews by Hegsted (1976) and Mertz (1987) concluded that availabilities determined by balance studies must be viewed with skepticism. Studies of this type are even more difficult to conduct in aquatic systems since fish can eliminate some elements by multiple routes - feces, urine, gills (Hardy et al., 1987) - and several elements can be taken up directly from the water. Gastrointestinal absorption has been used as an indicator of availability for some elements in fish (Ogino et al., 1979), but homeostasis of the elements examined by this method must be achieved by elimination rather than intestinal absorption (Mertz, 1987). When an inert marker such as chromic oxide is used, it is assumed that the marker and the element being examined travel through the digestive tract at the same rate (Heroux and Peter, 1975). This may not be the case with Mg which can be absorbed and secreted back into the intestine in bile salts (Aikawa, 1976). Evidence also exists that Mg in some fishes is reabsorbed in the latter part of the intestine (Dabrowski and Schwarz, 1986), so, underestimation of absorption may result if feces are removed for analysis before absorption is complete. Absorption may also be affected by handling stress (Heroux and Peter, 1975). A portion of the element taken up directly from the water may end up in the feces and be indistinguishable from that of dietary sources. In brackish or salt water, it may also be necessary for a fish to retain a portion of the Mg it ingests in the intestine for osmotic purposes (Shehadeh, 1967) introducing an error in digestibility measurements. Finally, the large differences in digestibility observed when Heroux and Peter (1975) fed several levels of a single Mg source to rats are indistinguishable from the results observed when true differences in availability exist between different sources present at the same concentration. Availability can also be determined through dose-response studies where primary (growth, health, performance) or secondary (whole body retention, tissue, hormone, or enzyme level) responses to a substance are measured (Spivey Fox et al., 1981) . When dietary requirements have previously been determined and primary and secondary response indicators have previously been related, absolute nutritional status as well as nutrient availability can be assessed by monitoring a secondary response indicator. The present study was designed to examine the availability of various dietary sources of Mg to rainbow trout. Availability was estimated by measuring apparent Mg retention in rapidly growing fish fed slightly Mg-deficient diets with high feed efficiency. Nutritional status was determined by comparing whole body Mg concentrations of fish fed the experimental diets to levels previously established as normal (Shearer, 1984,1989). MATERIALS AND METHODS
Eight semi-purified casein/gelatin-based diets were prepared and fed to triplicate groups of rainbow trout. Six diets were formulated to contain 550 ppm
AVAILABILITY OF DIETARY MAGNESIUM TO RAINBOW TROUT
53
magnesium, five from reagent grade magnesium sources: MgO, MgC& - 6Hz0, (MgC03)4.Mg(03)2, Mg2Si308, MgHP04*3Hz0, and one from fish bonemeal (Table 1). The fish bonemeal was a byproduct of producing liquefied fish protein concentrate for use in fish diets (Advanced Hydrolyzing Systems, Warrenton, OR). Two additional diets were prepared: one contained no supplemental magnesium, the other contained 1900 ppm Mg from MgO. An additional treatment (treatment 9) was added when food was withheld from one replicate of treatment 1 after mortalities were observed in all three replicates of this treatment on day 4. Each diet was prepared in a single batch and was stored at - 10 oC until fed. Trout, initial weight less than 1 g, were obtained from a commercial producer (Trout Lodge, Sumner, WA). Fish were fed a practical moist diet for 1 week prior to the start of the experiment. Lots of 60 fish were randomly assigned to each of 24 tanks. Each 200-I tank was supplied with 1.2 l/min of dechlorinated municipal water. Temperature was maintained at 13.5 t 0.5”C and a natural photoperiod (October-November) was used. Fish were bulk weighed at the start of the experiment and every 2 weeks thereafter. Fish were fed for 12 consecutive days and then starved for 2 days prior to weighing and sampling for TABLE 1 Composition of experimental diets Ingredients
% dry weight
Casein Gelatin Dextrin Alpha-cellulose“ Vitamin mixb Choline chloride (70% ) Herring oil Amino acid mix” Mineral mixd Ascorbic acid Trace mineral solution”
43.2 8.0 16.0 6.7 2.0 1.0 15.0 4.4 3.5 0.1 -
Mgf “Reduced as Mg containing component increased. ‘Same as reported by Hardy and Shearer (1985). ‘As g/kg diet: arg 11.5, lys 11.0, met 4.0, leu 9.0, va13.2, thr 5.0. dAs g/kg diet: CaHPO,.2H,O, 7.9; KCl, 17.0; NaCl, 0.6; NaH2P0,, 7.4. “As mg/kg diet: CoCl,.GH,O, 4.0; CuS04*5Hz0, 11.8; FeSO,.7H,O, 115; MnSO*-HZO, 32.5; ZnSO,*7H,O, 88; NaHSeO,, 4.2; KI, 1.9. ‘Mgsource added (mg/kg diet): diet 1,O; diet 2, MgO, 829; diet 3, MgO, 2500; diet 4, MgCl,*GH,O, 2920; diet 5, (MgCO,),.Mg(OH),, 1400; diet 6, Mg,Si,O,, 2680; diet 7, MgHPO,*3H,O, 2560; diet 8, fish bonemeal, 95000 (replaced some dextrin in addition to the alpha-cellulose).
54
K.D. SHEARER AND T. ASGARD
elemental analysis. Fish were hand-fed several small meals each day until the ration was consumed. The ration level was adjusted weekly, and was based on growth rates and feed efficiencies observed under similar conditions in an earlier study (Shearer, 1989) and predicted growth rates for rainbow trout (Austreng et al., 1987). The initial fish sample for elemental analysis consisted of 12 fish from the common pool. Four fish were randomly selected from each tank after week 2, and five fish were taken from each tank after weeks 4 and 6. Individual fish were dried, ashed at 55O”C, dissolved in acid, and analyses were made using emission spectrophotometry (Shearer, 1984). Visual examinations were made of the gills, kidneys, livers, and spleens from five fish fed each diet at the end of the experiment. A hepatosomatic index (HSI) for these groups was also calculated. Daily feed consumption and mortality were recorded. Apparent retention estimates were calculated at weeks 2,4, and 6. Analyses of variance were performed using StatView 512+ (Brainpower, 1986) and multiple mean comparisons were made using the Student-Newman-Keuls test (Zar, 1974). A significance level of P< 0.05 was chosen. Elemental measurements from replicates were pooled since they were not significantly different. RESULTS
Elemental analysis of the diets indicated that diets 4 through 8 contained approximately the expected level of Mg (537, 545, 540, 537, and 614 ppm). Diets 2 and 3 contained approximately 30% more Mg than they were formulated to contain (716 and 1900 ppm); diet 1, the basal diet, contained about 34 ppm Mg. Diet 8 (bonemeal) contained 2.7 and 2.2% calcium (Ca) and phosphorus (P) while the remaining diets contained 0.3 and 0.9%, respectively, of these two elements. Levels of the other elements met or exceeded currently recognized requirements (NRC, 1981). Protein, fat, and ash levels were 52.2, 15.3, and 4.5 percent, respectively. The rearing water contained 1.4 ppm Mg, 9.6 ppm Ca, and 1.7 ppm Na. Levels of P, Fe, Sr, and Zn were not quantifiable. Mean weights of fish fed diets 2 through 8 were similar at each fortnightly weighing (Fig. 1). Instantaneous daily growth rates for the consecutive 2-week feeding periods were 6.2,4.3, and 3.4% per day. Fish fed diet 1 (no Mg supplement) had growth rates of 3.1,2.5, and 3.2% per day during this period. Fish in treatment 9 (unfed after day 4) showed a weight gain at week 2, but their mean weight declined to slightly below their initial weight by the end of the experiment. Survival of fish fed diets 2 through 7 was 100% (Table 2). Mortality began occurring in fish fed diet 1 on day 4 and survival was 28% in the two tanks which were fed this diet for 6 weeks. Mortality in treatment 9, which was fed diet 1 for 4 days and starved thereafter, stopped once food was withheld and
AVAILABILITY
OF DIETARY MAGNESIUM TO RAINBOW TROUT
55
‘r 6 Treatment
1 0
2
4
6
Time (weeks) Fig. 1. Growth of fish fed experimental diets. Only four treatments are shown; the others lie between diet 6 and diet 3. TABLE 2 Final weight, weight gain, feed efficiency, dry matter, ash, and survival of rainbow trout fed the experimental diets for 6 weeks1 Diet
Final2 weight (g)
Weight gain (%)
Feed efficiency3
Dry matter (% )
Ash in wet wt. (%)
Survival (%)
1 2 3 4 5 6 7 8 9 Pooled S.D.
3.40b 5.99” 5.74” 5.79” 5.69” 6.26” 5.92” 5.73” 1.26” 0.19
162’ 361” 342” 345” 338” 381” 355” 341” -3” 15
0.67b 1.50” 1.40” 1.42” 1.37” 1.57a 1.46” 1.42” - 0.20” 0.06
22.gb 26.1” 26.0” 26.0” 25.9” 25.7” 26.0” 26.4” 18.4” 1.0
2.0b 2.2” 2.1” 2.2” 2.1” 2.3” 2.2” 2.3” 2.4” 0.1
2gd 100” 100” 100” 100” 100” 100” 92b 78” 1
‘Values in the same column with different letters are significantly different (P> 0.5). 21nitialweight 1.3 g. 3Wet weight gain/dry food fed.
overall survival was 78%. Fish fed diet 8 began to die after week 2 and survival to the end of the experimental was 92%. Visual examination of fish livers at the end of the experiment indicated that the livers of all fish were pale, mottled, and slightly enlarged (HSI 2.6, n=5; normal 1.2, Hilton and Hodson, 1983). Gills, kidneys, and spleen appeared normal.
K.D. SHEARER AND T. ASGARD
56
Initial feed acceptance was good in all treatments but fish diet 1 reduced their food consumption coincidental to the start of the mortalities. Fish fed diet 8 (bonemeal) showed behavioral changes during week 4, initially striking at feed but experienced convulsions and feed expulsion immediately after. Tetany often preceded mortality. Overall feed efficiency in groups 2 through 8 (Table 2) was 1.45. As expected, this declined from 1.70 to 1.46 and 1.32 during each 2-week period. Feed efficiencies in fish fed diet 1 were 0.75,0.66, and 0.64 with an overall efficiency of 0.67. Fish fed diet 1 for 4 days (treatment 9) had a feed efficiency of 1.55 for week 1. Whole body Mg concentrations increased with fish growth as expected in treatment 3. The Mg concentrations of fish fed diets 1, 2, and 4 through 8 declined (Fig. 2). In treatment 9 (unfed after day 4)) whole body concentrations returned to normal after feeding was suspended. After 6 weeks, whole body Mg concentrations of fish fed diets 2,4,5,6, and 7 were significantly less than those of fish fed diet 3 (Table 3) and fish fed diet 2 had significantly more Mg than those fed diets 4,6, and 7. The concentration of Mg in fish fed diet 8 was significantly below that of all treatments except treatment 1 which contained no added Mg. Concentrations in fish fed diet 1 dropped considerably in the first 2 weeks but recovered somewhat between weeks 2 and 4 when their growth was slowed by anorexia. It again declined rapidly between weeks 4 and 6 when their feed consumption increased. Whole body Ca and P were elevated in fish fed diet 8 while zinc (Zn) was considerably less than in other groups (Table 3 ) . Manganese was also significantly reduced in fish fed diet 8. Apparent retention of Mg remained constant from period to period, and the
Treatment 1 -2 -3 -4 -5 -6 -7 -8 -*
200 ’ 0
I
I
I
2
4
6
Time (weeks) Fig. 2. Whole body Mg concentrations of Mg from several sources (see text).
(ppm wet basis) of fish fed diets containing
various levels
* represents normal levels from Shearer (1989).
57
AVAILABILITY OF DIETARY MAGNESIUM TO RAINBOW TROUT
TABLE 3 Mean whole fish Mg, Ca, P, and Zn concentrations’ (ppm wet) and apparent Mg retention (% ) in rainbow trout fed the experimental diets for 6 weeks Diet
Mg cont.
Ca Cont.
P cont.
Zn cont.
Apparent Mg retention (% )”
1 2 3 4 5 6 7 8 9 Pooled S.D.
215” 322b 346” 295” 301b” 281” 300” 250d 284” 13
4323d” 456gcd 4001” 4464cd 4221de 4766” 4555”d 526gb 6171” 398
3752” 4202bc 3909d 4039cd 3911d 4179b” 4088” 4319”s 4426” 203
32.3b 28.0” 26.0” 27.2” 25.2” 27.1” 25.3” 17.ld 43.4” 3.3
307” 67’ 26d 77b 75b 80b 81b 54” 0” 5
In= 15. 2Apparent retention = [ (final Mg burden-initial
Mg burden) /Mg fed] X ( 100). n = 3.
overall retention was 76% for the diets containing the inorganic salts, 26% for diet 3, and 54% for diet 8. The fish fed diet 1 had an apparent retention of 307%, indicating that they obtained Mg directly from the water. The fish in treatment 9 ended the experiment with less than their initial burden of Mg leading to a negative retention. DISCUSSION
Primary dose-response indicators (growth, health, etc. ) (Spivey Fox et al., 1981) have been used to assess availability in terrestrial animals and fish, but are often insensitive (Baker, 1986). In the present study, equivalent growth occurred in fish fed diets 2 through 8 even though whole body Mg concentrations of all but group 3 (1900 ppm dietary Mg) were less than normal and mortalities occurred in group 8 (bonemeal). Satoh et al. (1987a) also observed normal growth but reported cataracts and low vertebral zinc in trout fed diets with low zinc availability. However, they reported conflicting results when they were able to relate reduced growth to differences in availability in an additional study (Satoh et al., 1987b). Dabrowska et al. (1989a) attributed reduced growth in tilapia (Oreochromis niloticus ) to low availability of Mg, but they observed the highest carcass Mg concentrations in fish receiving the diet reported to have the lowest availability. In fact, body Mg concentrations in fish reported to be Mg-deficient did not differ from levels they reported as normal in an additional study (Dabrowska et al., 1989b). Secondary indicators of response (whole body retention; tissue, hormone, or
58
K.D. SHEARER AND T. ASGARD
enzyme levels) appear to be the most useful means of comparing availabilities, especially when these secondary indicators have previously been correlated with primary indices (Spivey Fox et al., 1981). Normal whole body Mg concentrations in rainbow trout have previously been reported (Shearer, 1984) and have been related to primary indices such as growth (Shearer, 1989). Feeding rapidly growing fish diets deficient in Mg requires them to make full use of this element from the diets. If uptake of Mg from the water is timedependent, the rapid growth also prevents them from meeting their requirement from the water. The equivalent rates of Mg retention observed when the five magnesium salts were fed indicates that they had similar availabilities. The slightly higher Mg concentration observed in fish fed diet 2 appears to be a consequence of the slightly higher Mg concentration in this diet. The lower whole body levels in the fish fed the bonemeal diet indicate that it was less available than the inorganic sources. When excess levels are fed (diet 3,190O ppm ) , retention can not be used as an indicator of availability since the percent retained is an artifact of the amount fed. This will continue to decline as the level in the feed is increased. We can confirm that diets 2 and 4 through 9 were not in excess since the body concentrations were less than normal (Shearer, 1984 ) . The results observed in the present study confirm that the dietary Mg requirement reported earlier (Shearer, 1989) is correct and can be higher than stated by Ogino et al. (1978) or Knox et al. (1981). The level reported by Shearer (1989) (1300-1400 ppm) may also be an underestimate since a portion of the requirement may have been met by direct uptake from the water. Whether Mg is taken from the water when adequate Mg exists in the diet remains unknown. In addition, it is possible to achieve faster growth and higher feed efficiency than achieved in that study, further increasing the fish’s dietary requirement. The critical whole body Mg concentration for fish of this size appears to be about 80% of normal. At this level, mortalities were observed in fish fed diets 1 and 8. The fact that fish fed diet 8 still showed normal growth and feed efficiency at 6 weeks despite the occurrence of mortalities contrasts with the results observed from treatment 1 where growth was reduced before week 2. Some adaptation to low Mg availability was made in treatment 8 fish; perhaps they were able to mobilize enough Mg from the bones to maintain normal growth and enzymatic activity. The onset of Mg deficiency in fish fed the Mgfree diet (diet 1) was apparently too rapid for an adjustment to occur. The fact that apparent availability remained constant for each diet during consecutive fortnightly periods indicates that fish are unable to improve the efficiency of uptake of this element from the water or the diet. The higher Ca and P concentrations in Mg-deficient fish have been reported previously (Shearer, 1989). Diets 1,2, and 4 through 7 contained much lower levels of Ca and P than diet 8, so these fish would not have the opportunity to increase
AVAILABILITY
OF DIETARY
MAGNESIUM
TO RAINBOW
TROUT
59
their utilization of these elements. Low whole body Zn in fish fed diet 8 appears to be the result of the complexing of this element with Ca and P in the diet (Hardy and Shearer, 1985 ) . True dietary retention in fish fed diets 2 and 4 through 8 can be estimated by subtracting the amount of Mg taken up from the water by fish fed diet 1 from the apparent retention observed in these diets. If it is assumed that 100% of the Mg fed to fish in group 1 was retained, then these fish were able to obtain 94 mg of Mg/g of fish from the water. If we assume that fish fed the deficient diets were able to accumulate an equivalent amount from the water, then they would have taken up 339 mg/fish. Retention from the feed then becomes 57% from diets 2,4,5,6, and 7 and 36% from diet 8. Cook (1973) found 58% of the Mg from inorganic sources was available to rats. Our figure for retention from the diet may still be overstated since some loss of feed was experienced in fish fed diet 1. Assuming a feed efficiency similar to the other groups, retention would be reduced by a further 5%. True availability will, of course, be higher than true retention by the amount of endogenous loss. For Mg, this appears to be small, less than 1% of body burden per day (Shearer, unpublished). Despite the fact that the true availability of the diets can only be estimated, the relative availability of these sources remains unchanged. In the study by Dabrowska et al. (1989a), it appears that the reduced growth reported in fish fed Mg-sulfate may be due to a sulfate effect. The fact that fish fed a diet containing this Mg source had normal carcass Mg concentrations supports this conclusion. Anorexia, a sign of Mg deficiency (Shearer, 1989), was not observed. It is unlikely that a Mg deficiency developed with the low feed efficiency, slow growth rates, and high water-borne Mg reported in their experiment. In fact, the low Mg deposition appears to indicate that the diets contained an excess of available Mg. In summary, this study shows that a secondary indicator of Mg status (whole body Mg concentration) is useful in determining the relative availability of Mg to rainbow trout from various dietary sources. The apparent retention of Mg from inorganic salts was found to be less than 76% and this was higher than apparent retention of Mg from fish bonemeal (54% ) . Fish fed Mg deficient diets were unable to increase Mg uptake efficiency either from feed or water. Under conditions of moderate deficiency they were able to maintain normal growth and feed efficiency, apparently by maintaining adequate concentrations for specific functions. ACKNOWLEDGEMENTS
We thank Erich J. Gauglitz, Jr., and Joanne Hudson for mineral analysis and Dr. Ron Hardy and Dr. Barbara Grisdale-Helland for criticism of the manuscript. Participation of Torbjorn Asgard in this project was made possible by
60
K.D. SHEARER AND T. ASGARD
a grant from the Agricultural Research Council of Norway and its Institute of Aquaculture Research. REFERENCES Aikawa, J.K., 1976. Biochemistry and physiology of magnesium. In: A.S. Prasad (Editor), Trace Elements in Human Health and Disease, Vol. II. Academic Press, New York, NY, pp. 47-48. Austreng, E., Storebakken, T. and Asgard, T., 1987. Growth rate estimates for cultured Atlantic salmon and rainbow trout. Aquaculture, 60: X7-160. Baker, D.H., 1986. Problems and pitfalls in animal experiments designed to establish dietary requirements for essential nutrients. J. Nutr., 116: 2339-2349. Brainpower, 1986. Statview 512 +. 24009 Ventura Blvd., Calabrasa, CA 91302,108 pp. Cook, D.A., 1973. Availability of magnesium: balance studies in rats with various inorganic magnesium salts. J. Nutr., 103: 1365-1370. Dabrowska, H., Gunther, K.-D. and Meyer-Burgdorff, K., 1989a. Availability of various magnesium compounds to tilapia (Oreochromis niloticus). Aquaculture, 76: 269-276. Dabrowska, H., Meyer-Burgdorff, K. and Gunther, K.-D., 1989b. Interaction between dietary protein and magnesium level in tilapia (Oreochromis niloticus). Aquaculture, 76: 277-291. Dabrowski, K.R. and Schwarz, F.J., 1986. Mineral content in the digestive tract of stomachless fish. Zool. Jahrb. Physiol., 90: 193-200. Hardy, R.W. and Shearer, K.D., 1985. Effect of dietary calcium phosphate and zinc supplementation on whole body zinc concentration of rainbow trout. Can. J. Fish. Aquat. Sci., 42: 181184. Hardy, R.W., Sullivan, C.V. and Koziol, A.M., 1987. Assessment of absorption, body distribution, and excretion of dietary zinc by rainbow trout (Salmo guirdneri). Fish Physiol. Biochem., 2: 133-143. Hegsted, D.M., 1976. Balance studies. J. Nutr., 106: 307-311. Heroux, 0. and Peter, D., 1975. Failure of balance measurements to predict actual retention of magnesium and calcium by rats as determined by direct carcass analysis. J. Nutr., 105: 11571167. Hilton, J.W. and Hodson, P.V., 1983. Effect of increased dietary carbohydrate on selenium metabolism and toxicity in rainbow trout (Salmo guirdneri). J. Nutr., 113: 1241-1248. Knox, D., Cowey, C.B. and Adron, J.W., 1981. Studies on the nutrition of salmonid fish. The magnesium requirement of rainbow trout (Salmo guirdneri). Br. J. Nutr., 45: 137-148. Mertz, W., 1987. Use and misuse of balance studies. J. Nutr., 117: 1811-1813. NRC (National Research Council), 1981. Nutrient requirements of coldwater fishes. National Academy Press, Washington, DC, 102 pp. Ogino, C., Takashima, F. and Chiou, J.Y., 1978. Requirement of rainbow trout for dietary magnesium. Bull. Jpn. Sot. Sci. Fish., 45: 137-148. Ogino, C., Takeuchi, L., Takeda, H. and Watanabe, T., 1979. Availability of dietary phosphorus in carp and rainbow trout. Bull. Jpn. Sot. Sci. Fish., 45: 1527-1532. Satoh, S., Takeuchi, T. and Watanabe, T., 1987a. Availability to rainbow trout of zinc in white fish meal and of various zinc compounds. Nippon Suisan Gakkaishi, 53: 595-599. Satoh, S., Izume, K. and Takeuchi, T., 1987b. Availability to rainbow trout of zinc contained in various types of fish meals. Nippon Suisan Gakkaishi, 53: 1861-1866. Shearer, K.D., 1984. Changes in elemental composition of hatchery-reared rainbow trout, Sulmo guirdneri, associated with growth and reproduction. Can. J. Fish. Aquat. Sci., 41: 1592-1600. Shearer, K.D., 1988. Dietary potassium requirement of juvenile Chinook salmon. Aquaculture, 73: 119-129. Shearer, K.D., 1989. Whole body magnesium concentration as an indicator of magnesium status in rainbow trout (Sulmo guirdneri). Aquaculture, 77: 201-207.
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OF DIETARY
MAGNESIUM
TO RAINBOW
TROUT
61
Shehadeh, A.H., 1967. The role of intestine in salinity adaption of the rainbow trout (S&no gairdneri). Zoology Ph.D. Thesis. University of California, Los Angeles, CA, 81 pp. Spivey Fox, M.R., Jacobs, R.M., Jones, A.O.L., Fry, B.E., Jr., Raksowa, M., Hamilton, R.P., Harland, B.F., Stone, C.L. and Tao, S.-H., 1981. Animal models for assessing bioavailability of essential and toxic elements. Cereal Chem., 58( 1): 6-11. Zar, J.H., 1974. Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, NJ, 620 pp.