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Availability of phosphorus and trace elements in low-phytate varieties of barley and corn for rainbow trout (Oncorhynchus mykiss)
Aquaculture 170 Ž1999. 285–296
Availability of phosphorus and trace elements in low-phytate varieties of barley and corn for rainbow trout žOncorhynchus mykiss / Shozo H. Sugiura a , Victor Raboy b, Kevin A. Young b, Faye M. Dong a , Ronald W. Hardy c,) a
c
School of Fisheries, UniÕersity of Washington, 3707 Brooklyn AÕe. NE, Seattle, WA 98105, USA b USDA-Agricultural Research SerÕice (ARS), P.O. Box. 307, Aberdeen, ID 83210, USA Hagerman Fish Culture Experiment Station, UniÕersity of Idaho, 3059F, National Fish Hatchery Road, Hagerman, ID 83332, USA Accepted 2 October 1998
Abstract Approximately two-thirds of phosphorus in various grains is present as phytate, which is not well-utilized by fish and other monogastric species. Besides its low availability of phosphorus, phytate is reported to reduce the availability of other dietary nutrients to animals. Single-gene, non-lethal low phytic acid Žlpa. mutations in corn and barley cause the seed to store most of the phosphorus as inorganic phosphorus instead of as phytate phosphorus. Theoretically, using these mutant grains containing lower levels of phytate in animal feeds should reduce phosphorus excretion by the animals, provided that available phosphorus levels in feeds containing these grains are appropriately adjusted downward. This study was conducted as a first step to determine if the biological availability of phosphorus in the low-phytate mutants of barley, dent corn and flint corn differed significantly from that in ordinary grains for fish. Also of interest was the effect, if any, on the availability of other minerals in formulated feeds containing the low-phytate grains. Feeding trials demonstrated that the apparent availability of phosphorus in low-phytate grains was significantly higher than that in ordinary grains when they were combined with low-ash ingredients. Fecal phosphorus content Žon average. decreased 50.2% Žin phytate-phosphorus. or 42.9% Žin total phosphorus. by replacing ordinary grains with low-phytate grains in the low-ash diets. The apparent availabilities of calcium, iron, zinc and strontium also were signifi-
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Corresponding author. Tel.: q1-208-837-9096; Fax: q1-208-837-6047; E-mail: [email protected]
0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 8 . 0 0 4 1 4 - 1
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S.H. Sugiura et al.r Aquaculture 170 (1999) 285–296
cantly higher in the low-ash diet containing low-phytate dent corn than that containing ordinary dent corn. However, no such increase was observed with low-phytate barley or low-phytate flint corn over their counterpart grains in either calcium, iron or zinc. The apparent availabilities of copper, manganese, magnesium, potassium and sodium were not significantly different between ordinary and low-phytate grains. The apparent digestibility of dry matter also was not significantly different between ordinary and low-phytate grains. The results of this study suggest that a substantial reduction of phosphorus discharge from fish, poultry and animal farms could be achieved simply by replacing ordinary grains with low-phytate mutant grains in low-ash feeds. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Phosphorus; Phytate; Corn; Barley; Bioavailability; Sustainability
1. Introduction In many freshwater systems, phosphorus is the first limiting nutrient for the growth of phytoplankton and algae, which reduce dissolved oxygen levels in aquatic ecosystems when they grow excessively ŽMiller et al., 1974; Beveridge, 1984; Boyd, 1990.. This eutrophication thereby alters the local habitat and, in extreme cases, can destroy the environment. An important consideration for freshwater fish farms to reduce their impact on the aquatic environment is therefore to reduce phosphorus levels in effluent water. The ultimate source of phosphorus in aquaculture effluent is feeds. The concentration and availability of phosphorus in the diet are the two most important factors that affect the retention of phosphorus in ingested feeds by the fish. Approximately two-thirds of total phosphorus in various grains is present as phytate or inositol hexaphosphate ŽRaboy, 1997., which is not well-utilized by fish ŽOgino et al., 1979; NRC, 1993. and many other monogastric animals ŽNRC, 1984, 1988.. Besides its low availability of phosphorus, phytate has been shown to interact directly and indirectly with various dietary components to reduce their availability to animals. For example, calcium-bound phytate increases chelation with trace minerals such as zinc to form co-precipitates ŽAnon., 1967.. Phytate may decrease endogenous zinc reabsorption as well as affect availability of dietary zinc ŽMorris, 1986.. Increasing the phytate level from 1.1 to 2.2% in channel catfish diets containing 50 mg zincrkg decreased weight gain, feed efficiency and zinc content in the vertebrae ŽSatoh et al., 1989.. With 1.1% phytate in diets, channel catfish require about 200 mg zincrkg feed, which is 10 times higher than their dietary requirement for available zinc ŽGatlin and Wilson, 1984.. Single-gene, non-lethal low phytic acid Žlpa. mutations in corn and barley cause the seed to store most of the phosphorus as inorganic phosphorus instead of as phytate phosphorus ŽRaboy and Gerbasi, 1996.. Theoretically, using these mutant grains containing lower levels of phytate in fish or animal feeds should reduce phosphorus excretion by the fish or animals without any additional cost or effort. This study was conducted to determine the biological availability of phosphorus in the mutant low phytate grains, and their effect, if any, on the availability of other minerals in formulated feeds using rainbow trout as a model species.
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2. Materials and methods 2.1. Test ingredients (ordinary grains Õs. mutant low-phytate counterparts) Six ingredients Žgrains. were evaluated: three ordinary grains—barley ŽB., dent corn ŽDC. and flint corn ŽFC. —and three low-phytate mutant grains of the same variety— low-phytate barley ŽBLP., low-phytate dent corn ŽDCLP. and low-phytate flint corn ŽFCLP.. The two dent corns were grains produced by a pair of near-isogeneic corn hybrids that are either homozygous non-mutant Žwild-type. or homozygous for the recessive low phytic acid 1-1 Ž1pal-1. allele of the low phytic 1 Ž1pal. gene ŽErtl et al., 1998.. These grains were provided by Pioneer Hi-Bred International. The two flint corns were grains produced by two near-isogeneic lines Žeither homozygous wild-type or homozygous 1pal-1. provided by the USDA, Agricultural Research Service ŽARS., Aberdeen, ID. The barley lines were non-mutant and low phytic 1-1 descendants of the cultivar ‘Harrington’ and were also provided by ARS. The analytical composition of these grains is presented in Tables 1 and 2. Prior to mixing with basal diets or other ingredients, all test ingredients Žgrains. were finely ground using a Wiley mill Ž97%, - 1 mm., weighed and steam-cooked Žwith added water, 1:1 ratio by weight, 0.7 kgrcm2 s ca. 1158C. for 10 min to gelatinize the starch portion of the grains. Previous experiments indicated that this procedure did not affect phytate or inorganic phosphorus concentrations in soybean meal Žunpublished data.. 2.2. AÕailability of minerals in formulated diets I (grain 30% q casein diet 70%) A casein-based semi-purified diet Žbasal diet; Table 3. was mixed with each of the six test ingredients Žbarley or corn. at a 7:3 ratio Žby weight, dry basis., cold-extruded into pellets, dried at room temperature, and stored at 0–48C until fed. The analytical compositions of the casein diet and the formulated diets are given in Table 4. Forty-two rainbow trout Ž Oncorhynchus mykiss . Žinitial mean body weight, 275.3 g; SEM, 3.88. were stocked into seven 145-l fiberglass tanks supplied with spring water Ž14.58C. at ca. 10 lrmin. Each tank of fish was fed the casein diet once daily at 1% of the body weight Ždry basis. for 7 days by the method of Post et al. Ž1965.. On day 8, six of the tanks of
Table 1 Concentration of phytate-phosphorus and inorganic-phosphorus ŽPi. in the test ingredients Žgr100 g dry matter. a
Barley ŽB. Barley, low-phytate ŽBLP. Dent corn ŽDC. Dent corn, low-phytate ŽDCLP. Flint corn ŽFC. Flint corn, low-phytate ŽFCLP. a
Phytate-P
Pi
Phytate-PqPi
0.29 0.12 0.24 0.08 0.24 0.11
0.06 0.16 0.06 0.21 0.08 0.25
0.35 0.28 0.30 0.29 0.32 0.36
Determined on the acid extracts Ž0.65 N–HCl, 3 h.. Each value represents the average of duplicate determinations extracted separately.
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Table 2 Mineral composition of the test ingredientsa
B BLP DC DCLP FC FCLP
Ash
Ca
Cr
Cu
Fe
K
Mg
Mn
Na
P
Sr
Zn
1.97 1.85 0.99 0.95 1.19 1.20
601.6 582.5 tr.b tr. tr. tr.
1.56 1.51 0.25 0.44 0.19 0.35
23.9 25.1 18.6 21.8 29.2 28.3
71.9 40.6 16.7 14.7 33.5 48.7
3860 3010 1880 1020 1550 1530
1700 1670 1600 1560 2130 2100
17.96 13.89 2.33 1.49 7.62 7.26
532 1060 1170 1180 1760 1430
3290 2800 2570 2820 3390 3370
1.79 1.79 0.30 0.22 0.62 0.50
35.3 49.8 14.6 23.7 34.7 35.0
a
Determined on the ashed samples. Values are expressed as mgrkg dry ingredient Žgr100 g dry ingredient for ash.. b Trace amount Žless than 50 mgrkg dry ingredient..
fish were switched to the formulated diets Žone diet per tank., with the seventh tank continuing to be fed the casein diet Žthe control group., and feeding continued for another 7 days. The feeding level for different dietary treatments was not adjusted although the diets differed in digestible energy levels. After 7 days of feeding the formulated diets, fish were anesthetized with tricaine methane sulfonate ŽMS-222., feces were collected by stripping, pooled by treatment Žsix fish per treatment., and analyzed for mineral content. 2.3. AÕailability of minerals in formulated diets II (grain 30% q fish meal diet 70%) A fish meal-based diet Žbasal diet; Table 3. was mixed with each of the six test ingredients at 7:3 ratio Žby weight, dry basis., cold-extruded into pellets containing approximately 30% moisture, and stored at y208C until fed. The analytical compositions of the fish meal diet and the formulated diets are given in Table 4. A total of 210 rainbow trout Žinitial mean body weight, 167.7 g; SEM, 3.01. were stocked in 21,145-l fiberglass tanks Ž10 fish per tank. receiving spring water Ž14.58C. at ca. 10 lrmin. Three tanks were randomly assigned to each dietary treatment, and fish were fed the fish meal diet once daily as much as they would consume for 7 days. On day 8, the fish meal diet was replaced with one of the formulated diets except for the control group that continued to receive the fish meal diet, and feeding was continued for another 7 days before fecal samples were collected as described above. Samples from each fish were pooled by tank, and analyzed for mineral content. 2.4. AÕailability of minerals in low-ash diets (grain 50% q low-ash ingredients 50%) Test diets were formulated with semi-purified ingredients having low mineral contents except for the test ingredients Žbarley or corn. that comprised 50% of the diets ŽTable 3.. The test diets were prepared as moist pellets and stored at y208C until fed. The analytical compositions of the test diets are given in Table 4. A total of 180 rainbow trout Žinitial mean body weight, 175.4; SEM, 6.4. were stocked in 18,145-l fiberglass tanks Ž10 fish per tank. receiving spring water Ž14.58C. at ca. 10 lrmin and three tanks were randomly assigned for each of six dietary treatments. Fish were fed the test diets
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Table 3 Composition of the basal diets Žcasein diet and fish meal diet. a and the test diets Ingredients
Casein diet Žgr100 g diet.
Fish meal diet Žgr100 g diet.
Test diet Žgr100 g diet.
Barley or Corn Caseinb Herring meal Egg white c Gelatind Dextrin Carboxymethyl cellulosee Celluloseqmarkers Mineral mixture Vitamin mixture Amino acid mixture Herring oil Water Trace mineral solution
– 45 – – 10 12 1 5f 4.5 h 2j 4.5 l 16 Ž30. Ž10. n
– – 60 – 10 10 1 5f – 2j – 12 Ž30. Ž10. o
50 – – 10 10 – – 4g 2i 3k 1m 20 Ž30. Ž10. o
a
The basal diets were fed either as-is or as formulated diets Ž70% basal dietq30% barley or corn.. Vitamin-free casein ŽUnited States Biochemical wUSBx.. c Spray-dried egg white ŽICN.; contained 0.106% P. d From porcine skin, 300 bloom ŽUSB.; contained 0.011% P. e Sodium salt ŽNutritional Biochemicals.. f Celfil ŽUSB. and Y2 O 3 , 0.1 gr100 g dry diet. g Alpha-cellulose ŽSigma Chemical. contained 0.0012% P. Also supplied the following Žgr100 g dry diet.: Cr2 O 3 , 0.5; SiO 2 , 0.5; Y2 O 3 , 0.05. h Provided the following amount Žgr100 g dry diet.: KCl, 1.6; CaHPO4 , 1.2; MgO, 0.2; NaCl, 1.5. i Provided the following amount Žgr100 g dry diet.: KCl, 0.9; MgO, 0.1; NaCl, 1.0. j Provided the following amount Žgr100 g dry diet.: a30 pre-mixture ŽHoffmann-La Roche. 1.5; choline chloride, 0.3; inositol, 0.1; L-ascorbyl polyphosphate, 0.1. k Provided the following amount Žgr100 g dry diet.: a30 vitamin pre-mixture, 2.0; choline chloride, 0.5; inositol, 0.2; L-ascorbyl polyphosphate, 0.3. a30 pre-mixture contained 0.071% P. l Provided the following amount Žgr100 g dry diet.: DL-methionine, 1.0; L-arginine HCl, 1.0; L-histidine, 0.3; L-lysine HCl, 1.0; glycine, 1.0; L-threonine, 0.2. m Provided the following amount Žgr100 g dry diet.: DL-methionine, 0.5; L-lysine HCl, 0.5. n Provided the following amount Žmgrkg dry diet.: KI, 1.9; MnSO4PH 2 O, 40; ZnSO4PH 2 O, 44; Na 2 SeO 3 , 0.9; CoCl 2 P6H 2 O, 4; CuSO4P5H 2 O, 5; FeSO4P7H 2 O, 300. o Provided the following amount Žmgrkg dry diet.: KI, 1.9; MnSO4PH 2 O, 40; ZnSO4PH 2 O, 85; Na 2 SeO 3 , 0.9; CoCl 2 P6H 2 O, 4; CuSO4P5H 2 O, 12; FeSO4P7H 2 O, 300. b
once daily to apparent satiation for 7 days before fecal samples were collected by stripping. Fecal samples from each fish were pooled by tank. A portion of the fecal samples from each tank was analyzed for phytate, and the remaining portion was dried and analyzed for minerals. 2.5. Analyses and calculation Samples of test ingredients and fresh feces were acid-extracted Ž0.65 N-HCl, 3 h at room temperature with continuous shaking., centrifuged Ž1000 = g, 10 min., eluted
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Table 4 Analytical compositiona of the basal diets, formulated diets and the test diets Ash
Ca
Cr
Cu
Fe
K
Mg
Mn
Na
P
Sr
Y
Zn
Basal diets Casein diet 5.00 4190 0.312 29.3 75.5 8150 1110 15.9 8190 6310 2.18 804 64.1 Fish meal diet 11.11 22,800 ND ND 120.0 2300 924 ND 20,900 14,400 40.2 801 ND Formulated diets b (grain 30%q casein diet 70%) B 4.08 3110 0.689 27.7 74.4 BLP 4.05 3110 0.673 28.0 65.0 DC 3.85 2990 0.293 26.3 58.7 DCLP 3.79 2960 0.349 27.1 57.4 FC 3.90 2990 0.277 29.3 63.3 FCLP 3.88 2970 0.323 29.0 67.6
6860 6610 6370 6030 6240 6210
1290 1280 1250 1240 1400 1400
16.5 15.3 12.1 11.6 13.5 13.4
Formulated diets b (Grain 30%q Fish meal diet 70%) B 8.52 16,700 ND ND 103.6 2940 BLP 8.49 16,800 ND ND 99.0 2520 DC 8.10 16,300 ND ND 88.3 2260 DCLP 8.16 16,400 ND ND 84.4 2220 FC 7.88 15,100 ND ND 88.7 2560 FCLP 8.28 16,400 ND ND 91.9 2520
1180 1150 1120 1100 1260 1290
ND ND ND ND ND ND
Test diets c (Grain 50%q low-ash ingredients 50%) B 5.40 841 3320 18.0 86.5 BLP 5.30 800 3330 15.5 76.8 DC 4.81 514 3390 14.1 56.5 DCLP 4.72 509 2530 16.6 69.9 FC 4.97 549 2450 16.8 77.8 FCLP 4.80 528 2210 17.6 81.2
1600 1530 1490 1560 1820 1840
21.6 20.7 14.9 16.5 18.5 19.1
7910 7150 6500 6210 6830 6510
5880 6040 6190 6100 6330 6200
5400 5260 5250 5270 5470 5450
15,200 15,300 15,600 15,300 14,800 15,000
11,500 11,300 11,200 11,200 10,700 11,400
7050 6840 7230 7090 7370 6980
2550 2280 2060 2220 2510 2600
2.06 2.06 1.64 1.59 1.73 1.68
29.4 30.2 29.0 29.5 26.9 29.3
1.80 1.77 1.06 1.00 1.29 1.22
561 562 575 564 572 567
55.4 59.8 50.0 52.1 55.6 55.6
563 566 582 571 547 559
ND ND ND ND ND ND
375 384 384 380 395 386
59.8 39.3 17.2 45.9 54.7 61.6
a
Values are expressed as mg per kg dry diet Žgr100 g dry diet for ash.. ND: not determined. Formulated diets were prepared by mixing one of the test ingredients Žbarley or corn. with the basal diet Žcasein diet or fish meal diet; Table 3. at a ratio of 7:3 Žweight, dry basis.. c Test diets were prepared by mixing one of the test ingredients Žbarley or corn. with low-ash Žsemi-purified. ingredients ŽTable 3. at a ratio of 1:1 Žweight, dry basis.. b
through anion exchange column chromatograph ŽAG1-X4 resin, 100–200 mesh, Bio-Rad Laboratories, Hercules, CA. by the procedure of Harland and Oberleas Ž1986., and analyzed for phytate content according to Latta and Eskin Ž1980.. The concentration of phytate was determined using sodium phytate Ždodecasodium salt, from rice, purity 99%, water 12%, Sigma Chemical, P3168. as the standard. A remaining portion of the acid extract of test ingredients was partially neutralized and analyzed for inorganic phosphorus content ŽTaussky and Shorr, 1953.. Fecal and diet samples were dried Ž1058C, 12 h., ashed Ž5508C, 12 h., and dissolved in a concentrated hydrochloric–nitric acid mixture Ž1:1, vrv. and left at room temperature for 12 h Žor when samples contained added chromium, dissolved in a concentrated sulfuric–perchloric acid mixture, 1:1, vrv, and heated to boiling to oxidize chromium in the samples.. The acidified samples were diluted to an appropriate volume and analyzed for minerals according to Sugiura et al. Ž1998a..
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Apparent availability Ždigestibility. of minerals in diets was determined as a fractional net absorption of each mineral from diets based on yttrium as the non-absorbable indicator in all experiments ŽHardy, 1989.. The use of yttrium in nutrient absorption studies, as an inert nonabsorbable dietary marker, has been established previously with higher animals ŽMarcus and Lengemann, 1962; Hurwitz and Bar, 1966; Sklan et al., 1975.. Apparent availability of minerals in ordinary and low-phytate grains was compared using a two-tailed t-test. Differences were considered significant at P - 0.05. Fish were handled and treated in accordance with the guidelines approved by the Animal Care and Use Committee of the University of Idaho.
3. Results and discussion In formulated diets Žwith casein or fish meal., there was little or no effect of addition of low-phytate grains on the apparent availabilities of minerals ŽTable 5.. The basal casein diet had similar values to the formulated diets in mineral availabilities. The fish meal diet had lower availability of iron and phosphorus than did the formulated diets. The apparent digestibility of dry matter was similar in all dietary treatments. In test diets Žlow-ash., however, low-phytate grains markedly increased the apparent availability of phosphorus and strontium ŽTable 5; Fig. 1.. Fecal phytate-phosphorus and total-phosphorus were significantly lower in fish fed low-phytate grains than ordinary grains in the low-ash test diets ŽTable 6; Fig. 1.. Fecal phosphorus content decreased ca. 50.2% on average for phytate-phosphorus or 42.9% on average for total phosphorus when ordinary grains were replaced with low-phytate grains in the low-ash diets. This finding confirms the results found with poultry fed diets containing low-phytate grains ŽErtl et al., 1998.. The content of phosphorus Žand other minerals. in the grains was considerably lower than that in the basal diets used to produce the formulated diets, making it nearly impossible to detect differences associated with the low-phytate grains. Also, the phosphorus content of the fish meal-based diets was above the dietary requirement of trout due to high phosphorus content in fish meal. Even low-ash fish meals, e.g., herring meal, deboned fish meal, supply more than enough phosphorus in practical trout feeds to meet the requirement of the fish. The availability of minerals in fish meal Žmainly associated with the bone fraction. is normally low, ranging from 15 to 60% ŽRichie and Brown, 1996; Nordrum et al., 1997.. However, when the level of fish meal or fish bone in the diet is reduced, availability Ž%. of phosphorus Žand some other minerals. increases ŽSugiura, 1998.. This indicates that the bone phosphorus is ‘conditionally available’ depending on the concentration in the diet. This pattern was observed with the fish meal-based diets, where the availability of phosphorus in the basal fish meal diet was lower than that in the formulated diets. This is primarily due to the dilution effect Žof phosphorus. in the formulated diets, which was apparently more important than the inherent availability of phosphates in the grains. By reducing the phosphorus concentration in the diet to the minimum levels required by fish, differences in the inherent availability of phosphorus in the grains should become more evident. At present, because most commercial salmonid feeds still contain abundant phosphorus from fish meal, the advantage of using low-phytate grains in salmon or trout feeds will not be
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Table 5 Apparent availability a of minerals in the basal diets, formulated diets and the test diets Ca
Cu
Fe
K
Mg
60.8
19.0
97.6
85.6
ND
61.9
ND
ND
48.1
ND
ND
Formulated diets b (grain 30% q casein diet 70% ) B 18.5 47.1 16.7 99.5 BLP y2.6 40.9 12.5 97.3 DC 32.0 44.7 y20.5 98.4 DCLP 29.7 55.8 y11.2 98.3 FC 11.9 54.3 6.2 98.9 FCLP 34.0 55.2 24.6 99.4
81.3 80.2 82.3 81.1 73.1 79.3
12.3 y5.2 36.3 21.5 15.4 12.2
y40.2 y57.4 y25.1 y11.1 y17.2 y1.8
77.9 77.9 83.6 89.3 78.3 85.3
y4.8 y38.9 y6.3 y10.6 y15.4 9.9
45.1 49.0 66.9 73.3 46.2 55.2
Formulated diets c (grain 30%q fish meal diet 70% ) B 14.6 ND 17.6 ND BLP 11.4) ND 14.1) ND DC 13.0 ND 16.7 ND DCLP 12.8 ND 15.5 ND FC 10.6 ND 24.2 ND FCLP 13.4 ND 20.2 ND Pooled 2.4 – 1.9 – SEM
62.9 61.3 63.7 63.0 59.7 59.5 3.0
ND ND ND ND ND ND –
ND ND ND ND ND ND
54.0 53.2 52.3 52.1 53.1 54.8 1.8
ND ND ND ND ND ND
ND ND ND ND ND ND –
Test diets d (grain 50%q low-ash ingredients B y162 8.9 y6.7 BLP y137 y 8.7 y15.4 DC y269 y80.6 y32.7 DCLP y178)) y33.4 2.6) FC y340 y33.7 3.8 FCLP y219 y26.2 8.7 Pooled 24 13.7 7.7 SEM
65.6 75.5 64.1 68.3 62.3 71.1 4.2
Basal diets Casein 32.9 diet b Fish 12.4 meal diet c
ND
6.99
50% ) 100 100 101 100 100 100 0.3
Mn 28.2
13.4 7.3 12.9 18.4 19.8 20.0 3.3
Na
P
10.2
87.1
–
y23.3 y25.4 y55.1 y29.4 y34.6 y26.5 17.6
Sr 1.5
–
Zn 67.9
47.1 y69 56.0 64.9)) y20)) 35.6) 36.7 y143 y70.1 69.0))) y70))) 48.8)) 36.3 y136 42.3 65.6) y70))) 51.1 3.5 4.6 7.5
a
Expressed as the fractional net absorption per intake Ždiet.. ND: not determined. Each value represents pooled sample of six fish fed 1% BW for each fish daily. Dry matter digestibility Ž% . of casein diet and the formulated diets was 82.4, 76.9, 75.2, 77.7, 82.1, 79.7, 80.8% for casein diet, B, BLP, DC, DCLP, FC, FCLP diets, respectively. c Each value represents the average of three tanks. Values with asterisk are significantly different from those of the ordinary grain Ž): P - 0.05, )): P - 0.01, ))): P - 0.001 .. Dry matter digestibility Ž% . of fish meal diet and the formulated diets was 79.2, 75.7, 73.3, 74.5, 73.8, 74.7, 74.7% for fish meal diet, B, BLP, DC, DCLP, FC, FCLP diets, respectively. d Each value represents the average of three tanks. Values with asterisk are significantly different from those of the ordinary grain Ž): P - 0.05, )): P - 0.01, ))): P - 0.001 .. Dry matter digestibility Ž% . was not significantly different between ordinary and low-phytate grains; 61.8, 60.6, 40.7, 46.8, 45.0, 48.2% for B, BLP, DC, DCLP, FC, FCLP, respectively. b
fully realized. Conversely, for catfish, tilapia and various species of carps, fish meal is a minor feed ingredient and grains such as corn often comprise more than 50% of the diets, which is an ideal formulation to take full advantage of the low-phytate grains. Because of its strong chelating property, phytate has been known to interact with other dietary components, especially with divalent cations such as calcium and stron-
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293
Fig. 1. Fecal content and apparent availability of phosphorus in low-ash diets containing either ordinary grain or low-phytic-acid-mutant-grain. The low-ash diets contained one of the following test ingredients Žgrains. at 50% in diets Ždry basis.: B Žordinary barley.; BLP Žlow-phytate barley.; DC Žordinary dent corn.; DCLP Žlow-phytate dent corn.; FC Žordinary flint corn.; FCLP Žlow-phytate flint corn.. Columns or plots of low-phytic-acid-mutant grains with asterisks are significantly different from those of the ordinary grains by the two-tailed t-test Ž): P - 0.05; )): P - 0.01; ))): P - 0.001.. ns 3 Žtanks. per treatment.
tium, reducing their availability to animals and humans. Although no animal species has been reported to require strontium ŽNRC, 1980., it appears to be a useful indicator for phytic acid content in diets since the different content of phytic acid in the grains directly influences the absorption of strontium. In the present study, the apparent availabilities of calcium, iron and zinc also were significantly higher in the test diet containing low-phytate dent corn than that containing ordinary dent corn ŽTable 5.. However, no such increase was observed with low-phytate barley or low-phytate flint corn over their counterpart grains in either calcium, iron or zinc. The apparent availabilities of copper, manganese, magnesium, potassium and sodium were not significantly different between ordinary and low-phytate grains. The apparent availabilities of calcium, sodium and strontium were negative in all treatments with low-ash test
Table 6 Fecal contents of phytate-phosphorus and total-phosphorus from fish fed test diets of low-ash content a
B BLP DC DCLP FC FCLP Pooled SEM a
Phytate-P
Total P
0.34 0.16)) 0.21 0.11)) 0.30 0.15) 0.019
0.36 0.20)) 0.22 0.13)) 0.30 0.17) 0.017
Determined on the acid extracts of fresh feces, duplicate determinations on a single extract. Expressed as the percentage in dried feces. Each value represents the average of three tanks. Values with asterisk are significantly different from those of the ordinary grain Ž): P - 0.05, )): P - 0.01..
294
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diets, and those of copper and iron were either negative or close to zero. Negative availability values indicate that fish excreted minerals in feces in an amount higher than the dietary intake, presumably due to endogenous loss and the excretion via the intestine of water-borne minerals absorbed via the gills, but it is most importantly associated with the low dietary intake of minerals per se, which amplified these effects. Phytate has been shown to strongly reduce zinc availability when added to salmon or catfish diets ŽGatlin and Wilson, 1984; Richardson et al., 1985; Satoh et al., 1989.; however, in the present study, the relationship between dietary phytate level and zinc availability was not consistent among the grains tested. In past studies, phytate is often added to diets fed to fish for long periods, and zinc availability assessed by measuring tissue zinc levels. The present study simply measured zinc availability by measuring fecal loss and thus might not reflect the effects of long-term studies with feeds containing ordinary and low-phytate grains. The apparent digestibility of dry matter also was not significantly different between ordinary and low-phytate grains; however, the values of barley were higher than those of corn. No significant difference associated with the inert dietary marker in the calculated availability Ždigestibility. values of any minerals was found when yttrium or acid-insoluble ash were used, except for manganese, which had a higher Ž P - 0.05. availability value when acid-insoluble ash was used as the indicator than when yttrium was used. Apparent availability values calculated based on chromium frequently gave lower values Ž P - 0.05. than those based on yttrium or acid-insoluble ash Ždata not given., as observed in an earlier study ŽSugiura et al., 1998b.. This work suggests that allelic variation at a single gene of a feed grain species can potentially have a major impact on reducing phosphorus flow through fish farms, and likely other forms of livestock production. Feeding trials, both at the laboratory and farm-scale, are needed to confirm the potential of low-phytate grains to achieve this reduction and support production expectations. Further work is also needed to demonstrate that the low-phytate grains will be successful in commercial crop production; however, with dent corn, large-scale production is already occurring. Even if the economics of production of low-phytate grains are less favorable than ordinary grains, the use of low-phytate grains in fish and animal feeds may remain advantageous because reducing phosphorus into the environment is rapidly increasing in priority as a part of global environmental awareness. The present study, using rainbow trout as a model fish species, demonstrates that using low-phytate grains can reduce fecal excretion of phosphorus compared to using ordinary grains in feeds. Thus, substantial reductions of fecal phosphorus from fish, poultry and animal farms may be possible simply by replacing ordinary grains with low-phytate mutant, at least in species which are fed grain-based feeds.
Acknowledgements We are grateful to Jo Ann K. Lund, National Marine Fisheries Service, NOAA, Seattle, WA, for her technical assistance in mineral analysis, and to Pioneer Hi-Bred International, for providing the dent corn samples. This research was supported by the
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Cooperative State Research Service, U.S. Department of Agriculture to the Western Regional Aquaculture Center under agreement No. 95-38500-1458.
References Anon., 1967. Zinc, calcium, and phytate. Nutr. Rev. 25, 215–218. Beveridge, M.C.M., 1984. Cage and pen fish farming. Carrying capacity models and environmental impact. FAO Fisheries Technical Paper No. 255. FAO, Rome, Italy, 131 pp. Boyd, C.E., 1990. Water Quality in Ponds for Aquaculture. Alabama Agricultural Experiment Station, Auburn University, Auburn, AL, 482 pp. Ertl, D.S., Young, K.A., Raboy, V., 1998. Plant genetic approaches to phosphorus management in agricultural production. J. Environ. Quality 27, 299–304. Gatlin, D.M. III, Wilson, R.P., 1984. Zinc supplementation of practical channel catfish diets. Aquaculture 41, 31–36. Hardy, R.W., 1989. Diet preparation. In: Halver, J.E. ŽEd.., Fish Nutrition, 2nd edn. Academic Press, NY, pp. 473–546. Harland, B.F., Oberleas, D., 1986. Anion-exchange method for determination of phytate in foods: collaborative study. J. AOAC 69, 667–670. Hurwitz, S., Bar, A., 1966. Rate of passage of calcium-45 and yttrium-91 along the intestine, and calcium absorption in the laying fowl. J. Nutr. 89, 311–316. Latta, M., Eskin, M., 1980. A simple and rapid colorimetric method for phytate determination. J. Agric. Food Chem. 28, 1313–1315. Marcus, C.S., Lengemann, F.W., 1962. Use of radioyttrium to study food movement in the small intestine of the rat. J. Nutr. 76, 179–182. Miller, W.E., Maloney, T.E., Greene, J.C., 1974. Algal productivity in 49 lake waters as determined by algal assays. Water Res. 8, 667–679. Morris, E.R., 1986. Phytate and dietary mineral bioavailability. In: Graf, E. ŽEd.., Phytic Acid: Chemistry and Applications. Pilatus Press, Minneapolis, pp. 57–76. NRC ŽNational Research Council., 1980. Mineral Tolerance of Domestic Animals. National Academy Press, Washington, DC, 577 pp. NRC ŽNational Research Council., 1984. Nutrient Requirements of Poultry, 8th rev. edn. National Academy Press, Washington, DC, 71 pp. NRC ŽNational Research Council., 1988. Nutrient Requirements of Swine, 9th rev. edn. National Academy Press, Washington, DC, 93 pp. NRC ŽNational Research Council., 1993. Nutrient Requirements of Fish. National Academy Press, Washington, DC, 114 pp. Nordrum, S., Asgard, T., Shearer, K.D., Arnessen, P., 1997. Availability of phosphorus in fish bone meal and inorganic salts to Atlantic salmon Ž Salmo salar . as determined by retention. Aquaculture 157, 51–61. Ogino, C., Takeuchi, L., Takeda, H., Watanabe, T., 1979. Availability of dietary phosphorus in carp and rainbow trout. Bull. Jpn. Soc. Sci. Fish. 45, 1527–1532. Post, G., Shanks, W.E., Smith, R.R., 1965. A method for collecting metabolic excretions from fish. Prog. Fish-Cult. 27, 108–111. Raboy, V., 1997. Accumulation and storage of phosphate and minerals. In: Larkins, B.A., Vasil, I.K. ŽEds.., Cellular and Molecular Biology of Plant Seed Development, Vol. 4. Kluwer Academic Publishers, Dordrecht, pp. 441–477. Raboy, V., Gerbasi, P., 1996. Genetics of myo-inositol phosphate synthesis and accumulation. In: Biswas, B.B., Biswas, S. ŽEds.., Subcellular Biochemistry, Vol. 26, myo-Inositol Phosphates, Phosphoinositides, and Signal Transduction. Plenum Press, New York, pp. 257–285. Richardson, N.L., Higgs, D.A., Beames, R.M., McBride, J.R., 1985. Influence of dietary calcium, phosphorus, zinc and sodium phytate level on cataract incidence, growth, and histopathology in juvenile chinook salmon Ž Oncorhynchus tshawytscha.. J. Nutr. 115, 553–567.
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Richie, M., Brown, P.B., 1996. Availability of phosphorus from feedstuffs fed to rainbow trout Oncorhynchus mykiss . Aquaculture 142, 269–282. Satoh, S., Poe, W.E., Wilson, R.P., 1989. Effect of supplemental phytate andror tricalcium phosphate on weight gain, feed efficiency and zinc content in vertebrae of channel catfish. Aquaculture 80, 155–161. Sklan, D., Dubrov, D., Eisner, U., Hurwitz, S., 1975. 51 Cr-EDTA, 91 Y and 141 Ce as nonabsorbed reference substance in the gastrointestinal tract of the chicken. J. Nutr. 105, 1549–1552. Sugiura, S.H., 1998. Development of Low-Pollution Feeds for Sustainable Aquaculture. PhD Dissertation, University of Washington, Seattle, WA, 252 pp. Sugiura, S.H., Dong, F.M., Rathbone, C.K., Hardy, R.W., 1998a. Apparent protein digestibility and mineral availabilities in various feed ingredients for salmonid feeds. Aquaculture 159, 177–202. Sugiura, S.H., Dong, F.M., Hardy, R.W., 1998b. Effects of dietary supplements on the availability of minerals in fish meal; preliminary observations. Aquaculture 160, 283–303. Taussky, H.H., Shorr, E., 1953. A microcolorimetric method for the determination of inorganic phosphorus. J. Biol. Chem. 202, 675–685.
Availability of phosphorus and trace elements in low-phytate varieties of barley and corn for rainbow trout žOncorhynchus mykiss / Shozo H. Sugiura a , Victor Raboy b, Kevin A. Young b, Faye M. Dong a , Ronald W. Hardy c,) a
c
School of Fisheries, UniÕersity of Washington, 3707 Brooklyn AÕe. NE, Seattle, WA 98105, USA b USDA-Agricultural Research SerÕice (ARS), P.O. Box. 307, Aberdeen, ID 83210, USA Hagerman Fish Culture Experiment Station, UniÕersity of Idaho, 3059F, National Fish Hatchery Road, Hagerman, ID 83332, USA Accepted 2 October 1998
Abstract Approximately two-thirds of phosphorus in various grains is present as phytate, which is not well-utilized by fish and other monogastric species. Besides its low availability of phosphorus, phytate is reported to reduce the availability of other dietary nutrients to animals. Single-gene, non-lethal low phytic acid Žlpa. mutations in corn and barley cause the seed to store most of the phosphorus as inorganic phosphorus instead of as phytate phosphorus. Theoretically, using these mutant grains containing lower levels of phytate in animal feeds should reduce phosphorus excretion by the animals, provided that available phosphorus levels in feeds containing these grains are appropriately adjusted downward. This study was conducted as a first step to determine if the biological availability of phosphorus in the low-phytate mutants of barley, dent corn and flint corn differed significantly from that in ordinary grains for fish. Also of interest was the effect, if any, on the availability of other minerals in formulated feeds containing the low-phytate grains. Feeding trials demonstrated that the apparent availability of phosphorus in low-phytate grains was significantly higher than that in ordinary grains when they were combined with low-ash ingredients. Fecal phosphorus content Žon average. decreased 50.2% Žin phytate-phosphorus. or 42.9% Žin total phosphorus. by replacing ordinary grains with low-phytate grains in the low-ash diets. The apparent availabilities of calcium, iron, zinc and strontium also were signifi-
)
Corresponding author. Tel.: q1-208-837-9096; Fax: q1-208-837-6047; E-mail: [email protected]
0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 8 . 0 0 4 1 4 - 1
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cantly higher in the low-ash diet containing low-phytate dent corn than that containing ordinary dent corn. However, no such increase was observed with low-phytate barley or low-phytate flint corn over their counterpart grains in either calcium, iron or zinc. The apparent availabilities of copper, manganese, magnesium, potassium and sodium were not significantly different between ordinary and low-phytate grains. The apparent digestibility of dry matter also was not significantly different between ordinary and low-phytate grains. The results of this study suggest that a substantial reduction of phosphorus discharge from fish, poultry and animal farms could be achieved simply by replacing ordinary grains with low-phytate mutant grains in low-ash feeds. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Phosphorus; Phytate; Corn; Barley; Bioavailability; Sustainability
1. Introduction In many freshwater systems, phosphorus is the first limiting nutrient for the growth of phytoplankton and algae, which reduce dissolved oxygen levels in aquatic ecosystems when they grow excessively ŽMiller et al., 1974; Beveridge, 1984; Boyd, 1990.. This eutrophication thereby alters the local habitat and, in extreme cases, can destroy the environment. An important consideration for freshwater fish farms to reduce their impact on the aquatic environment is therefore to reduce phosphorus levels in effluent water. The ultimate source of phosphorus in aquaculture effluent is feeds. The concentration and availability of phosphorus in the diet are the two most important factors that affect the retention of phosphorus in ingested feeds by the fish. Approximately two-thirds of total phosphorus in various grains is present as phytate or inositol hexaphosphate ŽRaboy, 1997., which is not well-utilized by fish ŽOgino et al., 1979; NRC, 1993. and many other monogastric animals ŽNRC, 1984, 1988.. Besides its low availability of phosphorus, phytate has been shown to interact directly and indirectly with various dietary components to reduce their availability to animals. For example, calcium-bound phytate increases chelation with trace minerals such as zinc to form co-precipitates ŽAnon., 1967.. Phytate may decrease endogenous zinc reabsorption as well as affect availability of dietary zinc ŽMorris, 1986.. Increasing the phytate level from 1.1 to 2.2% in channel catfish diets containing 50 mg zincrkg decreased weight gain, feed efficiency and zinc content in the vertebrae ŽSatoh et al., 1989.. With 1.1% phytate in diets, channel catfish require about 200 mg zincrkg feed, which is 10 times higher than their dietary requirement for available zinc ŽGatlin and Wilson, 1984.. Single-gene, non-lethal low phytic acid Žlpa. mutations in corn and barley cause the seed to store most of the phosphorus as inorganic phosphorus instead of as phytate phosphorus ŽRaboy and Gerbasi, 1996.. Theoretically, using these mutant grains containing lower levels of phytate in fish or animal feeds should reduce phosphorus excretion by the fish or animals without any additional cost or effort. This study was conducted to determine the biological availability of phosphorus in the mutant low phytate grains, and their effect, if any, on the availability of other minerals in formulated feeds using rainbow trout as a model species.
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2. Materials and methods 2.1. Test ingredients (ordinary grains Õs. mutant low-phytate counterparts) Six ingredients Žgrains. were evaluated: three ordinary grains—barley ŽB., dent corn ŽDC. and flint corn ŽFC. —and three low-phytate mutant grains of the same variety— low-phytate barley ŽBLP., low-phytate dent corn ŽDCLP. and low-phytate flint corn ŽFCLP.. The two dent corns were grains produced by a pair of near-isogeneic corn hybrids that are either homozygous non-mutant Žwild-type. or homozygous for the recessive low phytic acid 1-1 Ž1pal-1. allele of the low phytic 1 Ž1pal. gene ŽErtl et al., 1998.. These grains were provided by Pioneer Hi-Bred International. The two flint corns were grains produced by two near-isogeneic lines Žeither homozygous wild-type or homozygous 1pal-1. provided by the USDA, Agricultural Research Service ŽARS., Aberdeen, ID. The barley lines were non-mutant and low phytic 1-1 descendants of the cultivar ‘Harrington’ and were also provided by ARS. The analytical composition of these grains is presented in Tables 1 and 2. Prior to mixing with basal diets or other ingredients, all test ingredients Žgrains. were finely ground using a Wiley mill Ž97%, - 1 mm., weighed and steam-cooked Žwith added water, 1:1 ratio by weight, 0.7 kgrcm2 s ca. 1158C. for 10 min to gelatinize the starch portion of the grains. Previous experiments indicated that this procedure did not affect phytate or inorganic phosphorus concentrations in soybean meal Žunpublished data.. 2.2. AÕailability of minerals in formulated diets I (grain 30% q casein diet 70%) A casein-based semi-purified diet Žbasal diet; Table 3. was mixed with each of the six test ingredients Žbarley or corn. at a 7:3 ratio Žby weight, dry basis., cold-extruded into pellets, dried at room temperature, and stored at 0–48C until fed. The analytical compositions of the casein diet and the formulated diets are given in Table 4. Forty-two rainbow trout Ž Oncorhynchus mykiss . Žinitial mean body weight, 275.3 g; SEM, 3.88. were stocked into seven 145-l fiberglass tanks supplied with spring water Ž14.58C. at ca. 10 lrmin. Each tank of fish was fed the casein diet once daily at 1% of the body weight Ždry basis. for 7 days by the method of Post et al. Ž1965.. On day 8, six of the tanks of
Table 1 Concentration of phytate-phosphorus and inorganic-phosphorus ŽPi. in the test ingredients Žgr100 g dry matter. a
Barley ŽB. Barley, low-phytate ŽBLP. Dent corn ŽDC. Dent corn, low-phytate ŽDCLP. Flint corn ŽFC. Flint corn, low-phytate ŽFCLP. a
Phytate-P
Pi
Phytate-PqPi
0.29 0.12 0.24 0.08 0.24 0.11
0.06 0.16 0.06 0.21 0.08 0.25
0.35 0.28 0.30 0.29 0.32 0.36
Determined on the acid extracts Ž0.65 N–HCl, 3 h.. Each value represents the average of duplicate determinations extracted separately.
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Table 2 Mineral composition of the test ingredientsa
B BLP DC DCLP FC FCLP
Ash
Ca
Cr
Cu
Fe
K
Mg
Mn
Na
P
Sr
Zn
1.97 1.85 0.99 0.95 1.19 1.20
601.6 582.5 tr.b tr. tr. tr.
1.56 1.51 0.25 0.44 0.19 0.35
23.9 25.1 18.6 21.8 29.2 28.3
71.9 40.6 16.7 14.7 33.5 48.7
3860 3010 1880 1020 1550 1530
1700 1670 1600 1560 2130 2100
17.96 13.89 2.33 1.49 7.62 7.26
532 1060 1170 1180 1760 1430
3290 2800 2570 2820 3390 3370
1.79 1.79 0.30 0.22 0.62 0.50
35.3 49.8 14.6 23.7 34.7 35.0
a
Determined on the ashed samples. Values are expressed as mgrkg dry ingredient Žgr100 g dry ingredient for ash.. b Trace amount Žless than 50 mgrkg dry ingredient..
fish were switched to the formulated diets Žone diet per tank., with the seventh tank continuing to be fed the casein diet Žthe control group., and feeding continued for another 7 days. The feeding level for different dietary treatments was not adjusted although the diets differed in digestible energy levels. After 7 days of feeding the formulated diets, fish were anesthetized with tricaine methane sulfonate ŽMS-222., feces were collected by stripping, pooled by treatment Žsix fish per treatment., and analyzed for mineral content. 2.3. AÕailability of minerals in formulated diets II (grain 30% q fish meal diet 70%) A fish meal-based diet Žbasal diet; Table 3. was mixed with each of the six test ingredients at 7:3 ratio Žby weight, dry basis., cold-extruded into pellets containing approximately 30% moisture, and stored at y208C until fed. The analytical compositions of the fish meal diet and the formulated diets are given in Table 4. A total of 210 rainbow trout Žinitial mean body weight, 167.7 g; SEM, 3.01. were stocked in 21,145-l fiberglass tanks Ž10 fish per tank. receiving spring water Ž14.58C. at ca. 10 lrmin. Three tanks were randomly assigned to each dietary treatment, and fish were fed the fish meal diet once daily as much as they would consume for 7 days. On day 8, the fish meal diet was replaced with one of the formulated diets except for the control group that continued to receive the fish meal diet, and feeding was continued for another 7 days before fecal samples were collected as described above. Samples from each fish were pooled by tank, and analyzed for mineral content. 2.4. AÕailability of minerals in low-ash diets (grain 50% q low-ash ingredients 50%) Test diets were formulated with semi-purified ingredients having low mineral contents except for the test ingredients Žbarley or corn. that comprised 50% of the diets ŽTable 3.. The test diets were prepared as moist pellets and stored at y208C until fed. The analytical compositions of the test diets are given in Table 4. A total of 180 rainbow trout Žinitial mean body weight, 175.4; SEM, 6.4. were stocked in 18,145-l fiberglass tanks Ž10 fish per tank. receiving spring water Ž14.58C. at ca. 10 lrmin and three tanks were randomly assigned for each of six dietary treatments. Fish were fed the test diets
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Table 3 Composition of the basal diets Žcasein diet and fish meal diet. a and the test diets Ingredients
Casein diet Žgr100 g diet.
Fish meal diet Žgr100 g diet.
Test diet Žgr100 g diet.
Barley or Corn Caseinb Herring meal Egg white c Gelatind Dextrin Carboxymethyl cellulosee Celluloseqmarkers Mineral mixture Vitamin mixture Amino acid mixture Herring oil Water Trace mineral solution
– 45 – – 10 12 1 5f 4.5 h 2j 4.5 l 16 Ž30. Ž10. n
– – 60 – 10 10 1 5f – 2j – 12 Ž30. Ž10. o
50 – – 10 10 – – 4g 2i 3k 1m 20 Ž30. Ž10. o
a
The basal diets were fed either as-is or as formulated diets Ž70% basal dietq30% barley or corn.. Vitamin-free casein ŽUnited States Biochemical wUSBx.. c Spray-dried egg white ŽICN.; contained 0.106% P. d From porcine skin, 300 bloom ŽUSB.; contained 0.011% P. e Sodium salt ŽNutritional Biochemicals.. f Celfil ŽUSB. and Y2 O 3 , 0.1 gr100 g dry diet. g Alpha-cellulose ŽSigma Chemical. contained 0.0012% P. Also supplied the following Žgr100 g dry diet.: Cr2 O 3 , 0.5; SiO 2 , 0.5; Y2 O 3 , 0.05. h Provided the following amount Žgr100 g dry diet.: KCl, 1.6; CaHPO4 , 1.2; MgO, 0.2; NaCl, 1.5. i Provided the following amount Žgr100 g dry diet.: KCl, 0.9; MgO, 0.1; NaCl, 1.0. j Provided the following amount Žgr100 g dry diet.: a30 pre-mixture ŽHoffmann-La Roche. 1.5; choline chloride, 0.3; inositol, 0.1; L-ascorbyl polyphosphate, 0.1. k Provided the following amount Žgr100 g dry diet.: a30 vitamin pre-mixture, 2.0; choline chloride, 0.5; inositol, 0.2; L-ascorbyl polyphosphate, 0.3. a30 pre-mixture contained 0.071% P. l Provided the following amount Žgr100 g dry diet.: DL-methionine, 1.0; L-arginine HCl, 1.0; L-histidine, 0.3; L-lysine HCl, 1.0; glycine, 1.0; L-threonine, 0.2. m Provided the following amount Žgr100 g dry diet.: DL-methionine, 0.5; L-lysine HCl, 0.5. n Provided the following amount Žmgrkg dry diet.: KI, 1.9; MnSO4PH 2 O, 40; ZnSO4PH 2 O, 44; Na 2 SeO 3 , 0.9; CoCl 2 P6H 2 O, 4; CuSO4P5H 2 O, 5; FeSO4P7H 2 O, 300. o Provided the following amount Žmgrkg dry diet.: KI, 1.9; MnSO4PH 2 O, 40; ZnSO4PH 2 O, 85; Na 2 SeO 3 , 0.9; CoCl 2 P6H 2 O, 4; CuSO4P5H 2 O, 12; FeSO4P7H 2 O, 300. b
once daily to apparent satiation for 7 days before fecal samples were collected by stripping. Fecal samples from each fish were pooled by tank. A portion of the fecal samples from each tank was analyzed for phytate, and the remaining portion was dried and analyzed for minerals. 2.5. Analyses and calculation Samples of test ingredients and fresh feces were acid-extracted Ž0.65 N-HCl, 3 h at room temperature with continuous shaking., centrifuged Ž1000 = g, 10 min., eluted
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Table 4 Analytical compositiona of the basal diets, formulated diets and the test diets Ash
Ca
Cr
Cu
Fe
K
Mg
Mn
Na
P
Sr
Y
Zn
Basal diets Casein diet 5.00 4190 0.312 29.3 75.5 8150 1110 15.9 8190 6310 2.18 804 64.1 Fish meal diet 11.11 22,800 ND ND 120.0 2300 924 ND 20,900 14,400 40.2 801 ND Formulated diets b (grain 30%q casein diet 70%) B 4.08 3110 0.689 27.7 74.4 BLP 4.05 3110 0.673 28.0 65.0 DC 3.85 2990 0.293 26.3 58.7 DCLP 3.79 2960 0.349 27.1 57.4 FC 3.90 2990 0.277 29.3 63.3 FCLP 3.88 2970 0.323 29.0 67.6
6860 6610 6370 6030 6240 6210
1290 1280 1250 1240 1400 1400
16.5 15.3 12.1 11.6 13.5 13.4
Formulated diets b (Grain 30%q Fish meal diet 70%) B 8.52 16,700 ND ND 103.6 2940 BLP 8.49 16,800 ND ND 99.0 2520 DC 8.10 16,300 ND ND 88.3 2260 DCLP 8.16 16,400 ND ND 84.4 2220 FC 7.88 15,100 ND ND 88.7 2560 FCLP 8.28 16,400 ND ND 91.9 2520
1180 1150 1120 1100 1260 1290
ND ND ND ND ND ND
Test diets c (Grain 50%q low-ash ingredients 50%) B 5.40 841 3320 18.0 86.5 BLP 5.30 800 3330 15.5 76.8 DC 4.81 514 3390 14.1 56.5 DCLP 4.72 509 2530 16.6 69.9 FC 4.97 549 2450 16.8 77.8 FCLP 4.80 528 2210 17.6 81.2
1600 1530 1490 1560 1820 1840
21.6 20.7 14.9 16.5 18.5 19.1
7910 7150 6500 6210 6830 6510
5880 6040 6190 6100 6330 6200
5400 5260 5250 5270 5470 5450
15,200 15,300 15,600 15,300 14,800 15,000
11,500 11,300 11,200 11,200 10,700 11,400
7050 6840 7230 7090 7370 6980
2550 2280 2060 2220 2510 2600
2.06 2.06 1.64 1.59 1.73 1.68
29.4 30.2 29.0 29.5 26.9 29.3
1.80 1.77 1.06 1.00 1.29 1.22
561 562 575 564 572 567
55.4 59.8 50.0 52.1 55.6 55.6
563 566 582 571 547 559
ND ND ND ND ND ND
375 384 384 380 395 386
59.8 39.3 17.2 45.9 54.7 61.6
a
Values are expressed as mg per kg dry diet Žgr100 g dry diet for ash.. ND: not determined. Formulated diets were prepared by mixing one of the test ingredients Žbarley or corn. with the basal diet Žcasein diet or fish meal diet; Table 3. at a ratio of 7:3 Žweight, dry basis.. c Test diets were prepared by mixing one of the test ingredients Žbarley or corn. with low-ash Žsemi-purified. ingredients ŽTable 3. at a ratio of 1:1 Žweight, dry basis.. b
through anion exchange column chromatograph ŽAG1-X4 resin, 100–200 mesh, Bio-Rad Laboratories, Hercules, CA. by the procedure of Harland and Oberleas Ž1986., and analyzed for phytate content according to Latta and Eskin Ž1980.. The concentration of phytate was determined using sodium phytate Ždodecasodium salt, from rice, purity 99%, water 12%, Sigma Chemical, P3168. as the standard. A remaining portion of the acid extract of test ingredients was partially neutralized and analyzed for inorganic phosphorus content ŽTaussky and Shorr, 1953.. Fecal and diet samples were dried Ž1058C, 12 h., ashed Ž5508C, 12 h., and dissolved in a concentrated hydrochloric–nitric acid mixture Ž1:1, vrv. and left at room temperature for 12 h Žor when samples contained added chromium, dissolved in a concentrated sulfuric–perchloric acid mixture, 1:1, vrv, and heated to boiling to oxidize chromium in the samples.. The acidified samples were diluted to an appropriate volume and analyzed for minerals according to Sugiura et al. Ž1998a..
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Apparent availability Ždigestibility. of minerals in diets was determined as a fractional net absorption of each mineral from diets based on yttrium as the non-absorbable indicator in all experiments ŽHardy, 1989.. The use of yttrium in nutrient absorption studies, as an inert nonabsorbable dietary marker, has been established previously with higher animals ŽMarcus and Lengemann, 1962; Hurwitz and Bar, 1966; Sklan et al., 1975.. Apparent availability of minerals in ordinary and low-phytate grains was compared using a two-tailed t-test. Differences were considered significant at P - 0.05. Fish were handled and treated in accordance with the guidelines approved by the Animal Care and Use Committee of the University of Idaho.
3. Results and discussion In formulated diets Žwith casein or fish meal., there was little or no effect of addition of low-phytate grains on the apparent availabilities of minerals ŽTable 5.. The basal casein diet had similar values to the formulated diets in mineral availabilities. The fish meal diet had lower availability of iron and phosphorus than did the formulated diets. The apparent digestibility of dry matter was similar in all dietary treatments. In test diets Žlow-ash., however, low-phytate grains markedly increased the apparent availability of phosphorus and strontium ŽTable 5; Fig. 1.. Fecal phytate-phosphorus and total-phosphorus were significantly lower in fish fed low-phytate grains than ordinary grains in the low-ash test diets ŽTable 6; Fig. 1.. Fecal phosphorus content decreased ca. 50.2% on average for phytate-phosphorus or 42.9% on average for total phosphorus when ordinary grains were replaced with low-phytate grains in the low-ash diets. This finding confirms the results found with poultry fed diets containing low-phytate grains ŽErtl et al., 1998.. The content of phosphorus Žand other minerals. in the grains was considerably lower than that in the basal diets used to produce the formulated diets, making it nearly impossible to detect differences associated with the low-phytate grains. Also, the phosphorus content of the fish meal-based diets was above the dietary requirement of trout due to high phosphorus content in fish meal. Even low-ash fish meals, e.g., herring meal, deboned fish meal, supply more than enough phosphorus in practical trout feeds to meet the requirement of the fish. The availability of minerals in fish meal Žmainly associated with the bone fraction. is normally low, ranging from 15 to 60% ŽRichie and Brown, 1996; Nordrum et al., 1997.. However, when the level of fish meal or fish bone in the diet is reduced, availability Ž%. of phosphorus Žand some other minerals. increases ŽSugiura, 1998.. This indicates that the bone phosphorus is ‘conditionally available’ depending on the concentration in the diet. This pattern was observed with the fish meal-based diets, where the availability of phosphorus in the basal fish meal diet was lower than that in the formulated diets. This is primarily due to the dilution effect Žof phosphorus. in the formulated diets, which was apparently more important than the inherent availability of phosphates in the grains. By reducing the phosphorus concentration in the diet to the minimum levels required by fish, differences in the inherent availability of phosphorus in the grains should become more evident. At present, because most commercial salmonid feeds still contain abundant phosphorus from fish meal, the advantage of using low-phytate grains in salmon or trout feeds will not be
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Table 5 Apparent availability a of minerals in the basal diets, formulated diets and the test diets Ca
Cu
Fe
K
Mg
60.8
19.0
97.6
85.6
ND
61.9
ND
ND
48.1
ND
ND
Formulated diets b (grain 30% q casein diet 70% ) B 18.5 47.1 16.7 99.5 BLP y2.6 40.9 12.5 97.3 DC 32.0 44.7 y20.5 98.4 DCLP 29.7 55.8 y11.2 98.3 FC 11.9 54.3 6.2 98.9 FCLP 34.0 55.2 24.6 99.4
81.3 80.2 82.3 81.1 73.1 79.3
12.3 y5.2 36.3 21.5 15.4 12.2
y40.2 y57.4 y25.1 y11.1 y17.2 y1.8
77.9 77.9 83.6 89.3 78.3 85.3
y4.8 y38.9 y6.3 y10.6 y15.4 9.9
45.1 49.0 66.9 73.3 46.2 55.2
Formulated diets c (grain 30%q fish meal diet 70% ) B 14.6 ND 17.6 ND BLP 11.4) ND 14.1) ND DC 13.0 ND 16.7 ND DCLP 12.8 ND 15.5 ND FC 10.6 ND 24.2 ND FCLP 13.4 ND 20.2 ND Pooled 2.4 – 1.9 – SEM
62.9 61.3 63.7 63.0 59.7 59.5 3.0
ND ND ND ND ND ND –
ND ND ND ND ND ND
54.0 53.2 52.3 52.1 53.1 54.8 1.8
ND ND ND ND ND ND
ND ND ND ND ND ND –
Test diets d (grain 50%q low-ash ingredients B y162 8.9 y6.7 BLP y137 y 8.7 y15.4 DC y269 y80.6 y32.7 DCLP y178)) y33.4 2.6) FC y340 y33.7 3.8 FCLP y219 y26.2 8.7 Pooled 24 13.7 7.7 SEM
65.6 75.5 64.1 68.3 62.3 71.1 4.2
Basal diets Casein 32.9 diet b Fish 12.4 meal diet c
ND
6.99
50% ) 100 100 101 100 100 100 0.3
Mn 28.2
13.4 7.3 12.9 18.4 19.8 20.0 3.3
Na
P
10.2
87.1
–
y23.3 y25.4 y55.1 y29.4 y34.6 y26.5 17.6
Sr 1.5
–
Zn 67.9
47.1 y69 56.0 64.9)) y20)) 35.6) 36.7 y143 y70.1 69.0))) y70))) 48.8)) 36.3 y136 42.3 65.6) y70))) 51.1 3.5 4.6 7.5
a
Expressed as the fractional net absorption per intake Ždiet.. ND: not determined. Each value represents pooled sample of six fish fed 1% BW for each fish daily. Dry matter digestibility Ž% . of casein diet and the formulated diets was 82.4, 76.9, 75.2, 77.7, 82.1, 79.7, 80.8% for casein diet, B, BLP, DC, DCLP, FC, FCLP diets, respectively. c Each value represents the average of three tanks. Values with asterisk are significantly different from those of the ordinary grain Ž): P - 0.05, )): P - 0.01, ))): P - 0.001 .. Dry matter digestibility Ž% . of fish meal diet and the formulated diets was 79.2, 75.7, 73.3, 74.5, 73.8, 74.7, 74.7% for fish meal diet, B, BLP, DC, DCLP, FC, FCLP diets, respectively. d Each value represents the average of three tanks. Values with asterisk are significantly different from those of the ordinary grain Ž): P - 0.05, )): P - 0.01, ))): P - 0.001 .. Dry matter digestibility Ž% . was not significantly different between ordinary and low-phytate grains; 61.8, 60.6, 40.7, 46.8, 45.0, 48.2% for B, BLP, DC, DCLP, FC, FCLP, respectively. b
fully realized. Conversely, for catfish, tilapia and various species of carps, fish meal is a minor feed ingredient and grains such as corn often comprise more than 50% of the diets, which is an ideal formulation to take full advantage of the low-phytate grains. Because of its strong chelating property, phytate has been known to interact with other dietary components, especially with divalent cations such as calcium and stron-
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Fig. 1. Fecal content and apparent availability of phosphorus in low-ash diets containing either ordinary grain or low-phytic-acid-mutant-grain. The low-ash diets contained one of the following test ingredients Žgrains. at 50% in diets Ždry basis.: B Žordinary barley.; BLP Žlow-phytate barley.; DC Žordinary dent corn.; DCLP Žlow-phytate dent corn.; FC Žordinary flint corn.; FCLP Žlow-phytate flint corn.. Columns or plots of low-phytic-acid-mutant grains with asterisks are significantly different from those of the ordinary grains by the two-tailed t-test Ž): P - 0.05; )): P - 0.01; ))): P - 0.001.. ns 3 Žtanks. per treatment.
tium, reducing their availability to animals and humans. Although no animal species has been reported to require strontium ŽNRC, 1980., it appears to be a useful indicator for phytic acid content in diets since the different content of phytic acid in the grains directly influences the absorption of strontium. In the present study, the apparent availabilities of calcium, iron and zinc also were significantly higher in the test diet containing low-phytate dent corn than that containing ordinary dent corn ŽTable 5.. However, no such increase was observed with low-phytate barley or low-phytate flint corn over their counterpart grains in either calcium, iron or zinc. The apparent availabilities of copper, manganese, magnesium, potassium and sodium were not significantly different between ordinary and low-phytate grains. The apparent availabilities of calcium, sodium and strontium were negative in all treatments with low-ash test
Table 6 Fecal contents of phytate-phosphorus and total-phosphorus from fish fed test diets of low-ash content a
B BLP DC DCLP FC FCLP Pooled SEM a
Phytate-P
Total P
0.34 0.16)) 0.21 0.11)) 0.30 0.15) 0.019
0.36 0.20)) 0.22 0.13)) 0.30 0.17) 0.017
Determined on the acid extracts of fresh feces, duplicate determinations on a single extract. Expressed as the percentage in dried feces. Each value represents the average of three tanks. Values with asterisk are significantly different from those of the ordinary grain Ž): P - 0.05, )): P - 0.01..
294
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diets, and those of copper and iron were either negative or close to zero. Negative availability values indicate that fish excreted minerals in feces in an amount higher than the dietary intake, presumably due to endogenous loss and the excretion via the intestine of water-borne minerals absorbed via the gills, but it is most importantly associated with the low dietary intake of minerals per se, which amplified these effects. Phytate has been shown to strongly reduce zinc availability when added to salmon or catfish diets ŽGatlin and Wilson, 1984; Richardson et al., 1985; Satoh et al., 1989.; however, in the present study, the relationship between dietary phytate level and zinc availability was not consistent among the grains tested. In past studies, phytate is often added to diets fed to fish for long periods, and zinc availability assessed by measuring tissue zinc levels. The present study simply measured zinc availability by measuring fecal loss and thus might not reflect the effects of long-term studies with feeds containing ordinary and low-phytate grains. The apparent digestibility of dry matter also was not significantly different between ordinary and low-phytate grains; however, the values of barley were higher than those of corn. No significant difference associated with the inert dietary marker in the calculated availability Ždigestibility. values of any minerals was found when yttrium or acid-insoluble ash were used, except for manganese, which had a higher Ž P - 0.05. availability value when acid-insoluble ash was used as the indicator than when yttrium was used. Apparent availability values calculated based on chromium frequently gave lower values Ž P - 0.05. than those based on yttrium or acid-insoluble ash Ždata not given., as observed in an earlier study ŽSugiura et al., 1998b.. This work suggests that allelic variation at a single gene of a feed grain species can potentially have a major impact on reducing phosphorus flow through fish farms, and likely other forms of livestock production. Feeding trials, both at the laboratory and farm-scale, are needed to confirm the potential of low-phytate grains to achieve this reduction and support production expectations. Further work is also needed to demonstrate that the low-phytate grains will be successful in commercial crop production; however, with dent corn, large-scale production is already occurring. Even if the economics of production of low-phytate grains are less favorable than ordinary grains, the use of low-phytate grains in fish and animal feeds may remain advantageous because reducing phosphorus into the environment is rapidly increasing in priority as a part of global environmental awareness. The present study, using rainbow trout as a model fish species, demonstrates that using low-phytate grains can reduce fecal excretion of phosphorus compared to using ordinary grains in feeds. Thus, substantial reductions of fecal phosphorus from fish, poultry and animal farms may be possible simply by replacing ordinary grains with low-phytate mutant, at least in species which are fed grain-based feeds.
Acknowledgements We are grateful to Jo Ann K. Lund, National Marine Fisheries Service, NOAA, Seattle, WA, for her technical assistance in mineral analysis, and to Pioneer Hi-Bred International, for providing the dent corn samples. This research was supported by the
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Cooperative State Research Service, U.S. Department of Agriculture to the Western Regional Aquaculture Center under agreement No. 95-38500-1458.
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