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Phosphorus utilization by rainbow trout (Oncorhynchus mykiss): estimation of dissolved phosphorus waste output
Aquaculture 179 Ž1999. 127–140
Phosphorus utilization by rainbow trout žOncorhynchus mykiss / : estimation of dissolved phosphorus waste output D.P. Bureau
a,)
, C.Y. Cho
a,b
a
Fish Nutrition Research Laboratory, Department of Animal and Poultry Science, UniÕersity of Guelph, Guelph, Ontario, Canada N1G 2W1 b Aquatic Ecosystems Research Section, Ontario Ministry of Natural Resources, Guelph, Ontario, Canada N1G 2W1
Abstract Phosphorus ŽP. waste output, notably in the dissolved form ŽDWP., is a major concern for many fish culture operations. Fish are believed to excrete DWP via the urine but this aspect has never been examined in detail. A better understanding of P utilization and renal P handling of fish could aid development of nutritional strategies for the management and reduction P waste. Rainbow trout were fed high corn gluten meal diets, supplemented with dibasic calcium phosphate, containing increasing P levels Ž0.75, 1.15, 1.66 and 2.19%.. P utilization was examined in a 16-week growth trial. A second trial was conducted to determine urinary inorganic P ŽPi. excretion using a non-invasive technique involving the use of a glomerular filtration marker and spot-sampling of urine. A third trial was conducted to measure DWP output through P accumulation in water. Increasing dietary P intake had no significant effect on growth and feed efficiency but significantly increased whole carcass and vertebrae P content. Efficiency of P retention decreased with increasing P intake. DWP represented 25, 47, 63 and 71% of digestible P intake as digestible P increased from 0.29, 0.62, 0.94 to 1.27%. Above a ‘‘threshold’’ plasma inorganic P ŽPi. concentration Ž86 mg Pi ly1 ., urinary Pi excretion was related to plasma Pi in a linear fashion and could be estimated as follows: urinary Pi output wmg kgy1 body weight ŽBW. dayy1 x s y360 q 4.2 plasma Pi Žmg ly1 .; Ž R 2 s 0.82, P - 0.001.. DWP output estimates, based on P
)
Corresponding author. Tel.: q1-519-824-4120, ext. 6688; fax: q1-519-767-0573; 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 9 . 0 0 1 5 7 - X
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accumulation, were 10, 63, 75 and 112 mg kgy1 BW dayy1 for fish fed the diets with 0.29, 0.62, 0.94 and 1.27% digestible P, respectively. The DWP output of these fish, estimated from the difference between digestible P intake and expected P retention, were 7, 29, 58 and 92 mg kgy1 BW dayy1. Urinary Pi excretion rates, estimated based on plasma Pi of the fish, were 0, 21, 94, 101 mg kgy1 BW dayy1. This study suggests that plasma Pi is the main factor determining DWP output of fish and that plasma Pi measurements could help in the estimation of P adequacy of the diet or DWP output. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Phosphorus; Salmonid; Diet; Urine; Plasma; Kidney
1. Introduction Inherent to the practice of intensive aquaculture is the generation of waste and these wastes have immediate and very broad effects on the aquatic environment. There is a growing consensus about the need to reduce waste production in aquaculture to minimize the negative impacts on the environment. Phosphorus ŽP. wastes are a major concern for many aquaculture operations since DWP Žplant-available P. can have very significant eutrophication effects on freshwater or brackish waterbodies ŽPersson, 1991.. Biological approaches in estimating waste outputs of aquaculture operations, based on nutrient intake, digestibility and retention, have been shown to be simple and reliable alternatives to limnological methods relying on direct monitoring of the effluent ŽCho et al., 1991, 1994.. Estimating DWP using biological approaches can be difficult since estimates of apparent P digestibility of diets and ingredients are often variable. In addition, P content of certain ingredients can be quite variable in terms of quantity and relative proportion of the chemical forms of P present. Very significant differences are observed in the digestibility of various forms of P Že.g., bone P vs. phytin P vs. ‘‘organic’’ P. and other factors, such as particle size and feed processing technique, are also known to affect P digestibility ŽLall, 1991.. These factors often make it difficult to derive reliable apparent digestibility coefficients ŽADC. for diets. A number of studies have examined DWP output of salmonids in response to dietary or environmental factors ŽKetola and Harland, 1993; Lanari et al., 1995; Dosdat et al., 1998; Medale et al., 1998. but the relationship between digestible P intake and DWP ´ output has not been completely defined. DWP is believed to be excreted mostly via the urine but renal P handling by fish has received very little attention ŽKaune and Hentschel, 1987; Renfro, 1997.. In mammals, urinary phosphate excretion is determined mostly by plasma phosphate concentration ŽBijvoet, 1980.. A threshold plasma phosphate concentration exists below which phosphate excretion is minimal and above which phosphate excretion is proportional to the increase in plasma phosphate. Because glomerular fish and mammals share similar renal physiology ŽDantzler, 1989., a similar relationship between plasma phosphate and urinary phosphate excretion is likely present in fish. The objectives of this study were to: Ž1. examine the partitioning of dietary P at increasing P intakes, Ž2. determine if the biological method of Cho et al. Ž1991. can be used to estimate DWP output, and Ž3. define the relationship between P intake, plasma Pi and urinary Pi excretion.
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2. Materials and methods 2.1. Diet formulation A high corn gluten meal basal diet was supplemented with increasing amounts of dibasic calcium phosphate ŽCaHPO4 . which replaced calcium carbonate ŽCaCO 3 . in the diet ŽTable 1.. Acid-washed diatomaceous silica ŽCelite AW521, Celite, Lompoc, CA., a source of acid-insoluble ash ŽAIA. ŽAtkinson et al., 1984., was included in all the diets to serve as a digestion indicator. The diets were mixed using a Hobart mixer ŽHobart, Don Mills, ON, Canada. and pelleted using a laboratory steam pellet mill ŽCalifornia Pellet Mill, San Francisco, CA.. The feed pellets were subsequently dried in a current of air at room temperature for 24 h and kept at 48C until used. 2.2. Fish, feeding and experimental conditions Three trials were conducted using rainbow trout Ž Oncorhynchus mykiss . obtained from the Alma Aquaculture Research Station ŽElora, ON, Canada.. The fish were treated in accordance with the guidelines of the Canadian Council on Animal Care ŽCanadian Council on Animal Care, 1984. and the University of Guelph Animal Care Committee. All the trials were conducted in windowless laboratories under a 12 h light:12 h dark
Table 1 Composition of the experimental diets Diet
Ingredients Fish meal, herring Corn gluten meal Brewer’s dried yeast Whey Celite CaHPO4 CaCO 3 Vitamin premix a Mineral premix a Fish oil Total Composition (as is basis) Dry matter Crude protein ŽN=6.25. Lipid Ash Phosphorus Digestible energy ŽDE. DPrDE Žg kJy1 . a
1
2
3
4
15 49 6 7 1 0 6 1 1 14 100
15 49 6 7 1 2 4 1 1 14 100
15 49 6 7 1 4 2 1 1 14 100
15 49 6 7 1 6 0 1 1 14 100
95.1 41.7 17.8 10.2 0.75 19.6 20.5
Composition presented by Bureau et al. Ž1998..
95.5 41.5 18.1 10.6 1.15 19.0 20.6
95.8 41.4 18.0 11.0 1.66 18.3 21.1
95.9 41.9 18.1 11.3 2.19 18.6 21.1
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photoperiod. The tanks were individually aerated and water temperature was maintained at 158C by injecting domestic hot water into the incoming water line. 2.2.1. Growth trial Rainbow trout, weighing an average of 8 g body weight ŽBW. each, were stocked Ž53 fish tanky1 . in 12 rectangular fibreglass tanks Ž40 l. supplied with a mixture of well water and city water at a rate of about 1.5 l miny1 . The experimental diets were each allocated to three replicate tanks. The fish were hand fed a predetermined amount of feed based on the theoretical energy requirement of the fish calculated using the method of Cho Ž1992.. This amount was observed to be close to the maximum voluntary feed intake of the fish. Fish were weighed every 4 weeks to calculate live weight gain ŽLWG. and feed efficiency ŽLWG: feed.. In the last 4 weeks of the growth trial, four fecal samples from each diets were collected using the Guelph System according to the method of Cho et al. Ž1982.. At the end of the experiment, five fish from each tank were sampled for chemical analysis of the whole carcass. The pooled fish samples were autoclaved Ž20 min at 1108C., ground into a slurry, lyophilized, reground and stored at y208C until analyzed. Additional pooled samples of five fish were collected from each tank for vertebrae P analysis. The fish were cooked in a microwave for 3 min and the vertebrae isolated, washed, delipidated with chloroform–methanol–water ŽBligh and Dyer, 1959., rinsed with distilled water and dried at 1058C for 24 h. 2.2.2. Urinary excretion trial The urinary Pi excretion was measured in rainbow trout using the non-invasive method of Curtis and Wood Ž1991.. This method involves the determination of the urine flow rate ŽUFR. of non-catheterized fish using a glomerular filtration marker, 1,2 3 H polyethylene glycol 4000 Ž1,2 3 H PEG-4000., and spot-sampling of urine and plasma to measure the concentration of the substance of interest. Rainbow trout, weighing an average of 213 g BW each, were stocked Ž10 fish tanky1 . in 12 rectangular fibreglass tanks Ž50 l. supplied with a mixture of well water and city water at a rate of about 3 l miny1 . The four dietary treatments were each allocated to three replicate tanks according to a complete block design. The four diets were hand fed twice daily to the fish for an acclimation period which lasted 10 weeks. After the acclimation period, 48 fish were distributed to another set of 12 tanks Ž4 fish tanky1 . similar to the acclimation tanks. On the following day, the 48 fish were anaesthetized with tricaine methanesulfonate ŽMS222. and 0.6 ml of Cortland saline ŽWolf, 1963. containing 17 mCi of 1,2 3 H PEG ŽNET-405, Mandel Scientific, Guelph, ON, Canada. was injected into the caudal vein of each fish using a 26 gauge needle. This dose was identical to that used by Curtis and Wood Ž1991.. After the injection, the fish were then returned to their tank. All the animals recovered and were feeding normally within a few hours. The animals were fed to near-satiation as usual for the following 48 h period. Forty-eight hours after injection of the glomerular filtration marker, the water flow was interrupted and each outlet of each tank was sealed with a rubber stopper. Sufficient aeration was continued to maintain dissolved oxygen at an
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appropriate level Ž) 7.5 mg ly1 .. Water samples Ž5 ml. were taken at 0, 1, 2, 3 and 4 h after interruption of water flow to determine the excretion of 1,2 3 H PEG-4000 in the water and permit calculation of UFR. The water samples were diluted with 15 ml liquid scintillation fluid ŽScintiverse II, Fisher Scientific, Fairlawn, NJ.. After the 4-h water sampling period, the fish were anaesthetized with MS 222. Urine was spot-sampled from the urinary bladder using a polyethylene catheter ŽPE-50, Clay Adams, Parsippany, NJ. attached to a 1-ml syringe with a blunted 23 gauge needle. A blood sample Ž500 ml. was then taken from the caudal vein of each fish. The blood was transferred to a plastic microcentrifuge tube containing 100 ml heparinized saline, mixed and immediately centrifuged for 10 min at 13,600 = g. Urine and plasma samples Ž25 ml. from each fish were diluted in 15 ml of liquid scintillation fluid. Urine and plasma samples were rapidly frozen in liquid N2 and kept at y808C until analyzed. The samples were subsequently analyzed for Pi using a colorimetric method with a commercial kit Ž360-UV, Sigma, St. Louis, MO.. 3 H in water, plasma and urine samples was counted Žup to 2 S . after 12 h of dark acclimation using a liquid scintillation counter ŽLS-3801, Beckmann Instrument, Fullerton, CA. with a counting efficiency of about 60%. UFR was calculated for each group of four fish based on the concentration of 1,2 3 H PEG-4000 of the urine and on the accumulation in the tank of 1,2 3 H PEG-4000 attributable to urinary excretion. Approximately 80% of total excretion of 1,2 3 H PEG-4000 is attributable to urinary excretion with the 20% remaining attributable to extra-renal excretion ŽCurtis and Wood, 1991.. Glomerular filtration rate ŽGFR. was calculated based on urine and plasma 1,2 3 H PEG-4000 concentrations. UFR, GFR and urinary Pi excretion rates, derived from 4 h measurements, were expressed on a 24-h basis to facilitate comparison among trials. 2.2.3. Water accumulation trial P accumulation was measured in 12 tanks, each stocked with 4–6 rainbow trout, weighing in average 407 g BW each, acclimated to the experimental diets for 17 weeks. Three days before the water collection period, the fish were weighed and, for the following 3 days, the fish were hand fed twice daily a predetermined amount of the experimental diets based on the theoretical energy requirement of the fish ŽCho, 1992.. A few minutes after the last meal Žthird day., water flow to the tank was interrupted and water was sampled at 0, 2 and 4 h. Water samples Ž100 ml. were stabilized with 0.5 ml concentrated sulfuric acid. Total P content of the water samples was determined by a commercial laboratory ŽPhilips Analytical Services, Burlington, ON, Canada. using the acid hydrolysis–ascorbic acid method ŽAmerican Public Health Association, 1979.. At the end of the water collection period, fish were anaesthetized with t-amyl alcohol, individually weighed and blood Ž900 ml. was sampled from the caudal vein. The blood was transferred to a plastic microcentrifuge tube containing 100 ml heparinized saline, mixed, centrifuged and frozen and analyzed for Pi as describe above. Individual plasma Pi values were used to predict urinary Pi excretion of individual fish using the following equation derived from the urinary excretion trial: Urinary Pi output Ž mg kgy1 BW dayy1 . s y360 q 4.2 = plasma Pi Ž mg ly1 .
D.P. Bureau, C.Y. Cho r Aquaculture 179 (1999) 127–140
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Negative values were disregarded, individual values were averaged per tank and statistical analysis was performed on tank average Žexperimental unit.. This approach is referred to as ‘‘plasmarurinary method’’ to avoid confusion with actual urinary Pi excretion measurements. DWP output of the fish was also calculated using the biological method of Cho et al. Ž1991. ŽDWP outputs Intake P y Fecal P y Retained P. using published ADC values for P ŽLall, 1991; Satoh et al., 1998. and expected P retention of the fish based on the results of the growth trial. 2.3. Chemical analyses Diet, feces and carcass samples were analyzed for dry matter and ash according to AOAC Ž1995., crude protein Ž%N = 6.25. using a Kjeltech auto-analyzer ŽModel a1030, Tecator, Hoganas, ¨ ¨ Sweden. and gross energy using an automated oxygen bomb calorimeter ŽModel a1271, Parr Instruments, Moline, IL.. Digestion indicator was determined using the AIA indicator method as used by Atkinson et al. Ž1984.. P content of diet, fecal material, whole fish carcass and vertebrae was analyzed by a commercial laboratory ŽLabstat, Kitchener, ON, Canada. using AOAC method 4.8.14 ŽAOAC, 1995.. 2.4. Statistical analyses Data were analyzed as a complete random block design or complete random design where appropriate using the general linear model ŽGLM. of the SASrSTAT software ŽSAS Institute, 1988.. Dietary effects were examined using orthogonal contrast ŽSteel and Torrie, 1980.. Regression analysis was performed using the regression function of the Microsoft Excel 7.0 software ŽMicrosoft, Seattle, WA.. 3. Results No significant difference was observed in the LWG, growth rate Žexpressed as thermal-unit growth coefficient; Cho, 1992. and feed efficiency of the fish fed the Table 2 Growth performance of rainbow trout a fed the experimental diets for 16 weeks Gain Žg fishy1 .
Feed Žg fishy1 .
Feed efficiency Žgain:feed.
TGC b
Diet 1 2 3 4 Pooled SEM
66.0 65.9 65.2 67.9 1.6
72.8 72.5 72.4 72.4 0.3
0.91 0.91 0.90 0.94 0.02
0.131 0.130 0.129 0.132 0.002
Contrasts Linear Quadratic
N.S. N.S.
NrAc NrAc
N.S. N.S.
N.S. N.S.
a
Initial live body weight 8.0 g fishy1 . Thermal-unit growth coefficients100=ŽFBW 1r 3 yIBW 1r3 .rÝŽTemp. Ž8C.=day. ŽCho, 1992.. c Not applicable, independent variable. b
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Table 3 Chemical composition of the carcass of rainbow trout fed the experimental diets for 16 weeks Moisture Ž%.
Crude protein Ž%.
Lipid Ž%.
Ash Ž%.
Diet Initial carcass 1 2 3 4 Pooled SEM
74.6 68.3 67.7 68.2 68.5 0.4
11.5 13.8 13.4 13.4 13.9 0.1
5.7 13.5 14.7 13.7 13.0 0.5
2.2 2.0 1.9 1.9 2.2 0.2
Contrasts Linear Quadratic
N.S. N.S.
N.S. - 0.05
N.S. N.S.
N.S. N.S.
P Ž%.
0.40 0.26 0.34 0.39 0.41 0.01
- 0.001 - 0.01
Gross energy ŽkJ gy1 .
Vertebrae P Ž%.
4.9 9.4 9.5 9.1 9.0 0.2
– 5.5 9.1 10.3 10.6 0.4
N.S. N.S.
- 0.001 - 0.01
experimental diets ŽTable 2.. Increasing dietary P resulted in a significant linear increase in the P content of whole fish carcass and vertebrae, and P gain Žg kgy1 LWG. but this response tended to level off at the highest dietary P intakes since a significant quadratic effect was observed for whole carcass and vertebrae P contents, and P gain Žg kgy1 LWG. ŽTables 3 and 4.. Dietary P level had no effect on nitrogen and gross energy gain or retention efficiency Žresults not shown.. P retention efficiency decreased linearly with
Table 4 Phosphorus of rainbow trout fed the experimental diets for 16 weeks growth trial Phosphorus Žg kgy1 LWG. Intake
Retained
Wastes Total
Solida
Dissolvedb
Diet 1 2 3 4 Pooled SEM
8.3 12.7 18.5 23.4 0.3
2.4 3.3 3.9 4.1 0.1
5.9 9.3 14.6 19.3 0.4
5.1 6.2 8.1 9.4 0.1
0.8 3.1 6.5 9.9 0.2
Contrasts linear quadratic
NrAc NrAc
- 0.001 - 0.05
- 0.001 N.S.
- 0.001 - 0.01
- 0.001 N.S.
a
Based on ADC for P of herring meal Ž52%., brewer’s yeast Ž79%., dibasic calcium phosphate Ž72%. of Lall Ž1991. measured by stripping and ADC for P of corn gluten meal Ž7%. of Satoh et al. Ž1998. measured with TUF column. b By difference total P wasteysolid P waste. c Not applicable, independent variable.
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Table 5 UFR, GFR, plasma and urinary Pi concentrations and excretion of rainbow trout fed the experimental diets in the urinary excretion trial UFR Žml kgy1 BW dayy1 .
GFR Žml kgy1 BW dayy1 .
Plasma Pi Žmg ly1 .
Urine Pi Žmg ly1 .
Urinary Pi excretion Žmg kgy1 % of Pi BW dayy1 . filtered
Diet 1 2 3 4 Pooled SEM
217 201 268 188 63
320 288 487 248 91
56 90 90 104 2
2 33 119 354 36
1 6 33 67 8
3 22 81 257 20
Contrast linear quadratic
N.S. N.S.
N.S. N.S.
P - 0.001 P - 0.01
P - 0.001 P - 0.05
P - 0.001 N.S.
P - 0.001 P - 0.01
increasing dietary P intake. DWP represented 25, 47, 63 and 71% of digestible P intake as digestible P increased from 0.29, 0.62, 0.94, 1.27%. No significant differences were observed for the UFR and GFR of the fish fed the four experimental diets due to large variance ŽTable 5.. Plasma and urine Pi concentrations as well as urinary Pi excretion Žmg kgy1 BW dayy1 and %Pi of filtered by kidney. increased linearly with increasing dietary P intake. A significant quadratic effect was also observed for plasma and urine Pi concentrations and Pi excretion Žexpressed as percent of Pi filtered by kidney.. Pi excretion exceeded the amount of Pi estimated to be filtered through glomerular filtration at the highest dietary P level indicating net renal secretion of Pi by the fish ŽTable 5.. Urine Pi concentrations measured for individual fish were plotted against corresponding plasma Pi concentrations ŽFig. 1.. Fish with low plasma Pi Že.g., - 80 mg ly1 . had very low urine Pi concentration. Fish with high plasma Pi showed, in most cases,
Fig. 1. Urine Pi concentration as a function of plasma Pi concentration of rainbow trout fed the experimental diets in the urinary excretion trial Ž ns 39 fish..
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Fig. 2. The relationship between urinary Pi excretion of rainbow trout and plasma Pi concentration Žtank averages.. This relationship is: y sy360q4.2= Ž P - 0.001, R 2 s 0.82., based on ns9 tank averages Žfish fed diets 2, 3, and 4, fish fed diet 1 excluded from analysis.. Standard error of intercepts 72, standard error of coefficient of x s 0.8.
elevated urinary Pi concentration. Pi excretion rates, calculated for every tank based on the average UFR Ž218 ml kgy1 BW dayy1 ., were plotted against corresponding tank average plasma Pi ŽFig. 2.. Regression analysis using values from tanks Ž n s 9. of fish fed the diet 2, 3, 4 showed a highly significant linear relationship Ž R 2 s 0.82, P - 0.001. between urinary Pi excretion rate and plasma Pi. The plasma Pi threshold concentration for urinary Pi excretion was estimated at 86 mg ly1 .
Table 6 Reactive P accumulation, plasma Pi concentration and predicted urinary Pi excretion of the fish in water accumulation study Dietary P intake
Dissolved phosphorus waste output Žmg kg BWy1 dayy1 . Water accumulation based on 2 h
Water accumulation based on 4 h
Biological methoda
Plasmarurinary methodb
Diet 1 2 3 4 Pooled SEM
75 115 166 219 –
10 63 75 112 13
15 74 64 117 20
7 29 58 92 –
0 21 94 101 17
Contrast Linear Quadratic
NrAc NrAc
- 0.01 N.S.
- 0.05 N.S.
NrAc NrAc
- 0.01 N.S.
a
ŽP intake=theoretical ADC for P.yŽexpected P retention.. See text for details. Plasma Pi averaged 56, 82, 105, 110 mg ly1 for diets 1, 2, 3 and 4, respectively. c Not applicable, independent variable or based on theoretical ADC and P retention values Žno randomization of experimental error.. b
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Table 7 Calculated digestible phosphorus content of the diets based on various methods Total P Ž%.
Diet 1 0.75 2 1.15 3 1.66 4 2.19
Digestible P Ž%. Plasmarurinary a
Water 2 hb
Water 4 hb
ADC c literature
ADC d measured
0.21 0.51 1.29 1.40
0.31 0.93 1.10 1.51
0.36 1.04 0.99 1.56
0.27 0.60 0.93 1.25
0.50 0.39 0.38 0.90
a
Estimated P retention based on data obtained from growth trial ŽTable 4.qestimated urinary Pi based on individual plasma Pi values measured and equation presented in Fig. 2. b Estimated P retentionqmeasured reactive P accumulation in tank water over 2 or 4 h. c Based on ADC for P of herring meal Ž52%., brewer’s yeast Ž79%., dibasic calcium phosphate Ž72%. of Lall Ž1991. measured by stripping and ADC for corn gluten meal Ž7%. of Satoh et al. Ž1998. measured with TUF column. d Based on ADC values measured in this study with the Guelph system.
A linear increase Ž P - 0.05. was observed in the P output of fish in the water accumulation trial ŽTable 6.. Predicted urinary Pi excretion based on plasma Pi Žplasmarurinary method. increased linearly Ž P - 0.01. with increasing dietary P. DWP output estimates from the water accumulation, biological and plasmarurinary methods showed relatively good agreement, especially at low and high P intakes ŽTable 6.. Predicted digestible P of the diets using water accumulation Žbased 2 or 4 h accumulation., plasmarurinary and biological methods and actual apparent digestibility measurements with the Guelph System are presented in Table 7. Estimated digestible P of the diet based on water P accumulation resulted in apparently higher digestible P estimates for the diets 1 and 2 than what was estimated from ADC values from the literature and the plasmarurinary approach.
4. Discussion The lack of effect of P level on growth and feed efficiency, but very significant effect on whole carcass and vertebrae P contents and P gain observed in this study is in agreement with several other studies that have shown that the requirement for P is lower for growth than for maximum P gain, at least over relatively short-term determination periods ŽRodehutscord, 1996; Asgard and Shearer, 1997.. Diets used in this experiment may have been marginally deficient in lysine which resulted in suboptimal performance; this was indicated by the poorer nitrogen retention efficiencies and growth rates than observed with the same strain of fish fed other practical diets. The present study is, to our knowledge, the first report of a clear threshold relationship between plasma Pi concentration and urinary Pi excretion in fish. This relationship is very similar to what is observed in mammals ŽBijvoet, 1980.. At similar plasma Pi, urinary Pi excretion rates measured in the present study appear comparable to what was measured by Kaune and Hentschel Ž1987. with Crucian carp, Carassius
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137
auratus. Fish fed the diet with the highest digestible P level Ždiet 4. exhibited net renal Pi secretion, e.g., Pi excretion greater than what was estimated to enter the renal tubules by glomerular filtration. This phenomenon has been shown to occur in fish, amphibians and birds ŽSchneider et al., 1980; Kaune and Hentschel, 1987; Renfro, 1997. but this study is, perhaps, the first demonstration of net renal secretion of P in fish in which plasma Pi was modulated through dietary means. Estimated UFR of the fish in this study averaged 218 ml kgy1 BW dayy1 . This value is two to three times higher than reported in most studies on urine production of freshwater fish ŽHunn, 1982; Curtis and Wood, 1991.. An intermediate UFR value was observed in a recent trial in our laboratory with rainbow trout ŽUFR s 144 ml kgy1 BW dayy1 . using the same method ŽBureau, 1997.. The much higher UFR and GFR measured in the present study compared to other studies may be explained, at least in part, by the fact that most studies have used fish that were fasted for several days prior to the measurement which may result in a significantly reduced urine production ŽHunn, 1982.. Oxygen consumption Žmetabolic rate. may have a determinant effect on GFR and UFR. Changes in surface area and blood perfusion may be factors influencing the flux of water through the gills ŽHunn, 1982; Isaia, 1984.. UFR increases with temperature with a Q10 of about 2, which is consistent with expected increase in oxygen consumption with temperature ŽHunn, 1982; Cho, 1992.. It is possible that the high UFR measured in this study was the result of stress. However, the fish recovered after the injection and were feeding well on the day of the measurement and appeared relatively calm during the radioactivity accumulation period when water flow was interrupted. The UFR, GFR and urinary Pi output estimates obtained in this study were, nevertheless, highly variable. This variability is probably a compromise related to the use of free-swimming and actively feeding fish, which is essential to obtain meaningful results ŽCho et al., 1982.. Fish fed the diet with no P supplementation excreted very little DWP, indicating that digestible P intake of the fish was directed almost completely toward deposition. Increasing digestible P intake resulted in a significant increase in DWP output. These results are in agreement with those of Rodehutscord Ž1996. who observed a decrease in the efficiency of P utilization Žretention. at increasing digestible P intake. There is still question about the necessity of maximizing mineralization of the skeleton of fish for long term maintenance of health and performance ŽRodehutscord, 1996; Asgard and Shearer, 1997; Baeverfjord et al., 1998.. The results from the present study and that of Rodehutscord Ž1996. indicate that maximum mineralization cannot be achieved without significant DWP output. Since a well-defined plasma Pi threshold for urinary Pi excretion was observed in this study, it would be of interest to verify the effect of long term feeding diets with digestible P levels producing plasma Pi close to this threshold level Žin order to minimize DWP. on health and performance of the fish. Estimates of DWP obtained with the various methods used in this study were in relatively good agreement at low and high digestible P intakes. Disagreement between the water accumulation method and the biological and plasmarurinary methods were observed at intermediate digestible P levels. DWP output estimates obtained in the water accumulation trial were highly variable and occasionally aberrant ŽTable 6, 4 h based estimates for diet 2 and 3.. Interference by fecal material contamination may have
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D.P. Bureau, C.Y. Cho r Aquaculture 179 (1999) 127–140
contributed to this variability and resulted in higher estimates of DWP that what was excreted by the fish. Filtration and stabilization of the water sample with chloroform would have been preferable ŽDosdat et al., 1994.. Calculated digestible P content of the experimental diets using the plasmarurine method or ADC for P of feed ingredients of Lall Ž1991. and Satoh et al. Ž1998. were similar. Actual measurements of apparent P digestibility with the Guelph System resulted in aberrant values in the present study. Lall Ž1991. observed that, for P digestibility, collection of fecal material by stripping yielded more reliable estimates than the Guelph System. Relatively high variability of ADC for P and ash can be obtained with the Guelph System for certain diets probably as a result of feces breakup and differential recovery of mineral fecal components. Results from this study suggest that reliable ADC are available in the literature for the feed ingredients used in this study and that the biological method of Cho et al. Ž1991. is appropriate to estimate DWP output. The results suggest that plasma Pi could, for certain purposes, be used to determine P adequacy of diets and estimate DWP output in lieu of expensive and time-consuming growth and digestibility trials. Plasma Pi measurements are easily performed using simple colorimetric assays and could be especially valuable under field conditions. Studies have, however, shown plasma Pi or DWP output to be highly variable over the whole day ŽDosdat et al., 1998; Medale et al., 1998; Sugiura, 1998.. This may ´ complicate use of plasma Pi measurements to estimate DWP output of fish. However, the pronounced post-prandial pattern or hourly variability of plasma Pi or DWP output observed in certain studies may be related, in part, to the experimental design used. A pronounced post-prandial pattern was observed for the oxygen consumption Ža good indicator of nutrient absorption and utilization. of rainbow trout upon refeeding following fasting or severe feed restriction periods. This post-prandial pattern was minimized when the fish fed normally to near-satiation for 2 to 4 days ŽCho et al., 1982.. More research is required to define the effects of biological, nutritional and environmental factors on plasma Pi, renal handling of P, and DWP output.
Acknowledgements Sincere thanks to Greg Ardnt, Stephen Gunther, Choi-Lan Ha, Andrew Harris, Fardin Pourkhataei and Ursula Wehkamp for their assistance and the Alma Aquaculture Research Station for donating the fish used in this study. This study was made possible by the financial support of the Ontario Ministry of Natural Resources ŽOMNR. and the Ontario Ministry of Agriculture, Food and Rural Affair ŽOMAFRA..
References American Public Health Association ŽAPHA., 1979. Standard Methods for the Examination of Water and Waste Water, 14th edn. APHA, Washington, DC. AOAC, 1995. Official Methods of Analysis of AOAC International: Vol. I. Agricultural Chemicals; Contaminants, Drugs, 16th edn. AOAC International, Arlington, VA.
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Asgard, T., Shearer, K., 1997. Dietary phosphorus requirement of juvenile Atlantic salmon Salmo salar L. Aquacult. Nutr. 3, 17–23. Atkinson, J.L., Hilton, J.W., Slinger, S.J., 1984. Evaluation of acid-insoluble ash as an indicator of feed digestibility in rainbow trout Ž Salmo gairdneri .. Can. J. Aquat. Sci. 41, 1384–1386. Baeverfjord, G., Asgard, T., Shearer, K.D., 1998. Development and detection of phosphorus deficiency in Atlantic salmon, Salmo salar L., parr and post-smolts. Aquacult. Nutr. 4, 1–11. Bijvoet, O.L.M., 1980. Indices for the measurement of the renal handling of phosphate. In: Massry, S.G., Fleisch, H. ŽEds.., Renal Handling of Phosphate. Plenum Medical Book, New York, NY, pp. 1–37. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Bureau, D.P., 1997. The partitioning of energy from digestible carbohydrates by rainbow trout Ž Oncorhynchus mykiss .. PhD Thesis, University of Guelph, Guelph, Ontario, Canada. Bureau, D.P., Harris, A.M., Cho, C.Y., 1998. The effects of purified alcohol extracts from soy products on feed intake and growth of chinook salmon Ž Oncorhynchus tshawytscha. and rainbow trout Ž Oncorhynchus mykiss .. Aquaculture 161, 27–43. Canadian Council on Animal Care ŽCCAC., 1984. Guide to the Care and Use of Experimental Animals, Vol. 2. CCAC, Ottawa, Ontario. Cho, C.Y., 1992. Feeding systems for rainbow trout and other salmonids with reference to current estimates of energy and protein requirements. Aquaculture 100, 107–123. Cho, C.Y., Slinger, S.J., Bayley, H.S., 1982. Bioenergetics of salmonid fishes: energy intake, expenditure and productivity. Comp. Biochem. Physiol. 73B, 25–41. Cho C.Y., Hynes, J.D., Wood, K.R., Yoshida, H.K., 1991. Quantitation of fish culture wastes by biological Žnutritional. and chemical Žlimnological. methods; the development of high nutrient dense ŽHND. diets. In: Cowey, C.B., Cho, C.Y. ŽEds.., Nutritional Strategies and Aquaculture Waste. Proceedings of the First International Symposium on Nutritional Strategies in Management of Aquaculture Waste, University of Guelph, Ontario, Canada, pp. 37–50. Cho, C.Y., Hynes, J.D., Wood, K.R., Yoshida, H.K., 1994. Development of high nutrient-dense, low pollution diets and prediction of aquaculture wastes using biological approaches. Aquaculture 124, 293–305. Curtis, B.J., Wood, C.M., 1991. The function of the urinary bladder in vivo in the freshwater rainbow trout. J. Exp. Biol. 155, 567–583. Dantzler, W.H., 1989. Comparative Physiology of the Vertebrate Kidney. Zoophysiology, Vol. 22. SpringerVerlag, Berlin, Germany. Dosdat, A., Gaumet, F., Chartois, H., 1994. Marine aquaculture effluent monitoring: methodological approach to the evaluation of nitrogen and phosphorus excretion by fish. Aquacult. Eng. 14, 59–84. Dosdat, A., Metailler, R., Desbruyeres, E., Huelvan, C., 1998. Comparison of brown trout Ž Salmo trutta. ´ ` reared in fresh water and sea water to fresh water rainbow trout Ž Oncorhynchus mykiss .: II. Phosphorus balance. Aquat. Living Resour. 11, 21–28. Hunn, J.B., 1982. Urine flow rate in freshwater salmonids: a review. Prog. Fish-Cult. 44, 119–125. Isaia, J., 1984. Water and nonelectolyte permeation. In: Hoar, W.S., Randall, D.J., Brett, J.R. ŽEds.., Fish Physiology, Vol. 10. Academic Press, New York, NY, pp. 1–37. Kaune, R., Hentschel, H., 1987. Stimulation of renal phosphate secretion in the stenohaline freshwater teleost: Carassius auratus Gibelio Bloch. Comp. Biochem. Physiolo. 87A, 359–362. Ketola, H.G., Harland, B.F., 1993. Influence of phosphorus in rainbow trout diets on phosphorus discharges in effluent water. Trans. Am. Fish. Soc. 122, 1120–1126. Lall, S.P., 1991. Digestibility, metabolism and excretion of dietary phosphorus in fish. In: Cowey, C.B., Cho, C.Y. ŽEds.., Nutritional Strategies and Aquaculture Waste. Proceedings of the First International Symposium on Nutritional Strategies in Management of Aquaculture Waste, University of Guelph, Ontario, Canada, pp. 21–36. Lanari, D., D’Agaro, E., Ballestrazzi, R., 1995. Dietary N and P levels, effluent water characteristics and performance in rainbow trout. Water Sci. Technol. 31, 157–166. Medale, F., Boujard, T., Vallee, ´ ´ F., Blanc, D., Mambrini, M., Roem, A., Kaushik, S.J., 1998. Voluntary feed intake, nitrogen and phosphorus losses in rainbow trout Ž Oncorhynchus mykiss . fed increasing dietary levels of soy protein concentrate. Aquat. Living Resour. 11, 239–246. Persson, G., 1991. Eutrophication resulting from salmonid fish culture in fresh and salt waters: Scandinavian
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experiences. In: Cowey, C.B., Cho, C.Y. ŽEds.., Nutritional Strategies and Aquaculture Waste. Proceedings of the First International Symposium on Nutritional Strategies in Management of Aquaculture Waste, University of Guelph, Ontario, Canada, pp. 163–185. Renfro, J.L., 1997. Hormonal regulation of renal inorganic phosphate transport in the winter flounder, Pleuronectes americanus. Fish Physiol. Biochem. 17, 377–383. Rodehutscord, M., 1996. Response of rainbow trout Ž Oncorhynchus mykiss . growing from 50 to 200 g to supplements of dibasic sodium phosphate in a semipurified diet. J. Nutr. 126, 324–331. SAS Institute ŽSAS., 1988. SASrSTAT User’s Guide, Release 6.03 edn. SAS Institute, Cary, NC. Satoh, S., Takanezawa, M., Watanabe, T., 1998. Changes of phosphorus absorption from several feed ingredients in rainbow trout during growing stages. VIII International Symposium on Nutrition and Feeding of Fish, 1–4 June 1998, Las Palmas, Spain, p. 136 ŽAbstract.. Schneider, E.G., Hanson, R.C., Childers, J.W., Fitzgerald, E.M., Gleason, S.D., 1980. Is phosphate secreted by the kidney. In: Massry, S.G., Fleisch, H. ŽEds.., Renal Handling of Phosphate. Plenum Medical Book, New York, NY, pp. 59–78. Steel, R.G.D., Torrie, J.H., 1980. Principles and Procedures of Statistics. McGraw-Hill, New York, NY. Sugiura, S.H., 1998. Development of low-pollution feeds for sustainable aquaculture. PhD Thesis, University of Washington, Seattle. Wolf, K., 1963. Physiological salines for freshwater teleosts. Prog. Fish-Cult. 25, 135–140.
Phosphorus utilization by rainbow trout žOncorhynchus mykiss / : estimation of dissolved phosphorus waste output D.P. Bureau
a,)
, C.Y. Cho
a,b
a
Fish Nutrition Research Laboratory, Department of Animal and Poultry Science, UniÕersity of Guelph, Guelph, Ontario, Canada N1G 2W1 b Aquatic Ecosystems Research Section, Ontario Ministry of Natural Resources, Guelph, Ontario, Canada N1G 2W1
Abstract Phosphorus ŽP. waste output, notably in the dissolved form ŽDWP., is a major concern for many fish culture operations. Fish are believed to excrete DWP via the urine but this aspect has never been examined in detail. A better understanding of P utilization and renal P handling of fish could aid development of nutritional strategies for the management and reduction P waste. Rainbow trout were fed high corn gluten meal diets, supplemented with dibasic calcium phosphate, containing increasing P levels Ž0.75, 1.15, 1.66 and 2.19%.. P utilization was examined in a 16-week growth trial. A second trial was conducted to determine urinary inorganic P ŽPi. excretion using a non-invasive technique involving the use of a glomerular filtration marker and spot-sampling of urine. A third trial was conducted to measure DWP output through P accumulation in water. Increasing dietary P intake had no significant effect on growth and feed efficiency but significantly increased whole carcass and vertebrae P content. Efficiency of P retention decreased with increasing P intake. DWP represented 25, 47, 63 and 71% of digestible P intake as digestible P increased from 0.29, 0.62, 0.94 to 1.27%. Above a ‘‘threshold’’ plasma inorganic P ŽPi. concentration Ž86 mg Pi ly1 ., urinary Pi excretion was related to plasma Pi in a linear fashion and could be estimated as follows: urinary Pi output wmg kgy1 body weight ŽBW. dayy1 x s y360 q 4.2 plasma Pi Žmg ly1 .; Ž R 2 s 0.82, P - 0.001.. DWP output estimates, based on P
)
Corresponding author. Tel.: q1-519-824-4120, ext. 6688; fax: q1-519-767-0573; 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 9 . 0 0 1 5 7 - X
128
D.P. Bureau, C.Y. Cho r Aquaculture 179 (1999) 127–140
accumulation, were 10, 63, 75 and 112 mg kgy1 BW dayy1 for fish fed the diets with 0.29, 0.62, 0.94 and 1.27% digestible P, respectively. The DWP output of these fish, estimated from the difference between digestible P intake and expected P retention, were 7, 29, 58 and 92 mg kgy1 BW dayy1. Urinary Pi excretion rates, estimated based on plasma Pi of the fish, were 0, 21, 94, 101 mg kgy1 BW dayy1. This study suggests that plasma Pi is the main factor determining DWP output of fish and that plasma Pi measurements could help in the estimation of P adequacy of the diet or DWP output. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Phosphorus; Salmonid; Diet; Urine; Plasma; Kidney
1. Introduction Inherent to the practice of intensive aquaculture is the generation of waste and these wastes have immediate and very broad effects on the aquatic environment. There is a growing consensus about the need to reduce waste production in aquaculture to minimize the negative impacts on the environment. Phosphorus ŽP. wastes are a major concern for many aquaculture operations since DWP Žplant-available P. can have very significant eutrophication effects on freshwater or brackish waterbodies ŽPersson, 1991.. Biological approaches in estimating waste outputs of aquaculture operations, based on nutrient intake, digestibility and retention, have been shown to be simple and reliable alternatives to limnological methods relying on direct monitoring of the effluent ŽCho et al., 1991, 1994.. Estimating DWP using biological approaches can be difficult since estimates of apparent P digestibility of diets and ingredients are often variable. In addition, P content of certain ingredients can be quite variable in terms of quantity and relative proportion of the chemical forms of P present. Very significant differences are observed in the digestibility of various forms of P Že.g., bone P vs. phytin P vs. ‘‘organic’’ P. and other factors, such as particle size and feed processing technique, are also known to affect P digestibility ŽLall, 1991.. These factors often make it difficult to derive reliable apparent digestibility coefficients ŽADC. for diets. A number of studies have examined DWP output of salmonids in response to dietary or environmental factors ŽKetola and Harland, 1993; Lanari et al., 1995; Dosdat et al., 1998; Medale et al., 1998. but the relationship between digestible P intake and DWP ´ output has not been completely defined. DWP is believed to be excreted mostly via the urine but renal P handling by fish has received very little attention ŽKaune and Hentschel, 1987; Renfro, 1997.. In mammals, urinary phosphate excretion is determined mostly by plasma phosphate concentration ŽBijvoet, 1980.. A threshold plasma phosphate concentration exists below which phosphate excretion is minimal and above which phosphate excretion is proportional to the increase in plasma phosphate. Because glomerular fish and mammals share similar renal physiology ŽDantzler, 1989., a similar relationship between plasma phosphate and urinary phosphate excretion is likely present in fish. The objectives of this study were to: Ž1. examine the partitioning of dietary P at increasing P intakes, Ž2. determine if the biological method of Cho et al. Ž1991. can be used to estimate DWP output, and Ž3. define the relationship between P intake, plasma Pi and urinary Pi excretion.
D.P. Bureau, C.Y. Cho r Aquaculture 179 (1999) 127–140
129
2. Materials and methods 2.1. Diet formulation A high corn gluten meal basal diet was supplemented with increasing amounts of dibasic calcium phosphate ŽCaHPO4 . which replaced calcium carbonate ŽCaCO 3 . in the diet ŽTable 1.. Acid-washed diatomaceous silica ŽCelite AW521, Celite, Lompoc, CA., a source of acid-insoluble ash ŽAIA. ŽAtkinson et al., 1984., was included in all the diets to serve as a digestion indicator. The diets were mixed using a Hobart mixer ŽHobart, Don Mills, ON, Canada. and pelleted using a laboratory steam pellet mill ŽCalifornia Pellet Mill, San Francisco, CA.. The feed pellets were subsequently dried in a current of air at room temperature for 24 h and kept at 48C until used. 2.2. Fish, feeding and experimental conditions Three trials were conducted using rainbow trout Ž Oncorhynchus mykiss . obtained from the Alma Aquaculture Research Station ŽElora, ON, Canada.. The fish were treated in accordance with the guidelines of the Canadian Council on Animal Care ŽCanadian Council on Animal Care, 1984. and the University of Guelph Animal Care Committee. All the trials were conducted in windowless laboratories under a 12 h light:12 h dark
Table 1 Composition of the experimental diets Diet
Ingredients Fish meal, herring Corn gluten meal Brewer’s dried yeast Whey Celite CaHPO4 CaCO 3 Vitamin premix a Mineral premix a Fish oil Total Composition (as is basis) Dry matter Crude protein ŽN=6.25. Lipid Ash Phosphorus Digestible energy ŽDE. DPrDE Žg kJy1 . a
1
2
3
4
15 49 6 7 1 0 6 1 1 14 100
15 49 6 7 1 2 4 1 1 14 100
15 49 6 7 1 4 2 1 1 14 100
15 49 6 7 1 6 0 1 1 14 100
95.1 41.7 17.8 10.2 0.75 19.6 20.5
Composition presented by Bureau et al. Ž1998..
95.5 41.5 18.1 10.6 1.15 19.0 20.6
95.8 41.4 18.0 11.0 1.66 18.3 21.1
95.9 41.9 18.1 11.3 2.19 18.6 21.1
130
D.P. Bureau, C.Y. Cho r Aquaculture 179 (1999) 127–140
photoperiod. The tanks were individually aerated and water temperature was maintained at 158C by injecting domestic hot water into the incoming water line. 2.2.1. Growth trial Rainbow trout, weighing an average of 8 g body weight ŽBW. each, were stocked Ž53 fish tanky1 . in 12 rectangular fibreglass tanks Ž40 l. supplied with a mixture of well water and city water at a rate of about 1.5 l miny1 . The experimental diets were each allocated to three replicate tanks. The fish were hand fed a predetermined amount of feed based on the theoretical energy requirement of the fish calculated using the method of Cho Ž1992.. This amount was observed to be close to the maximum voluntary feed intake of the fish. Fish were weighed every 4 weeks to calculate live weight gain ŽLWG. and feed efficiency ŽLWG: feed.. In the last 4 weeks of the growth trial, four fecal samples from each diets were collected using the Guelph System according to the method of Cho et al. Ž1982.. At the end of the experiment, five fish from each tank were sampled for chemical analysis of the whole carcass. The pooled fish samples were autoclaved Ž20 min at 1108C., ground into a slurry, lyophilized, reground and stored at y208C until analyzed. Additional pooled samples of five fish were collected from each tank for vertebrae P analysis. The fish were cooked in a microwave for 3 min and the vertebrae isolated, washed, delipidated with chloroform–methanol–water ŽBligh and Dyer, 1959., rinsed with distilled water and dried at 1058C for 24 h. 2.2.2. Urinary excretion trial The urinary Pi excretion was measured in rainbow trout using the non-invasive method of Curtis and Wood Ž1991.. This method involves the determination of the urine flow rate ŽUFR. of non-catheterized fish using a glomerular filtration marker, 1,2 3 H polyethylene glycol 4000 Ž1,2 3 H PEG-4000., and spot-sampling of urine and plasma to measure the concentration of the substance of interest. Rainbow trout, weighing an average of 213 g BW each, were stocked Ž10 fish tanky1 . in 12 rectangular fibreglass tanks Ž50 l. supplied with a mixture of well water and city water at a rate of about 3 l miny1 . The four dietary treatments were each allocated to three replicate tanks according to a complete block design. The four diets were hand fed twice daily to the fish for an acclimation period which lasted 10 weeks. After the acclimation period, 48 fish were distributed to another set of 12 tanks Ž4 fish tanky1 . similar to the acclimation tanks. On the following day, the 48 fish were anaesthetized with tricaine methanesulfonate ŽMS222. and 0.6 ml of Cortland saline ŽWolf, 1963. containing 17 mCi of 1,2 3 H PEG ŽNET-405, Mandel Scientific, Guelph, ON, Canada. was injected into the caudal vein of each fish using a 26 gauge needle. This dose was identical to that used by Curtis and Wood Ž1991.. After the injection, the fish were then returned to their tank. All the animals recovered and were feeding normally within a few hours. The animals were fed to near-satiation as usual for the following 48 h period. Forty-eight hours after injection of the glomerular filtration marker, the water flow was interrupted and each outlet of each tank was sealed with a rubber stopper. Sufficient aeration was continued to maintain dissolved oxygen at an
D.P. Bureau, C.Y. Cho r Aquaculture 179 (1999) 127–140
131
appropriate level Ž) 7.5 mg ly1 .. Water samples Ž5 ml. were taken at 0, 1, 2, 3 and 4 h after interruption of water flow to determine the excretion of 1,2 3 H PEG-4000 in the water and permit calculation of UFR. The water samples were diluted with 15 ml liquid scintillation fluid ŽScintiverse II, Fisher Scientific, Fairlawn, NJ.. After the 4-h water sampling period, the fish were anaesthetized with MS 222. Urine was spot-sampled from the urinary bladder using a polyethylene catheter ŽPE-50, Clay Adams, Parsippany, NJ. attached to a 1-ml syringe with a blunted 23 gauge needle. A blood sample Ž500 ml. was then taken from the caudal vein of each fish. The blood was transferred to a plastic microcentrifuge tube containing 100 ml heparinized saline, mixed and immediately centrifuged for 10 min at 13,600 = g. Urine and plasma samples Ž25 ml. from each fish were diluted in 15 ml of liquid scintillation fluid. Urine and plasma samples were rapidly frozen in liquid N2 and kept at y808C until analyzed. The samples were subsequently analyzed for Pi using a colorimetric method with a commercial kit Ž360-UV, Sigma, St. Louis, MO.. 3 H in water, plasma and urine samples was counted Žup to 2 S . after 12 h of dark acclimation using a liquid scintillation counter ŽLS-3801, Beckmann Instrument, Fullerton, CA. with a counting efficiency of about 60%. UFR was calculated for each group of four fish based on the concentration of 1,2 3 H PEG-4000 of the urine and on the accumulation in the tank of 1,2 3 H PEG-4000 attributable to urinary excretion. Approximately 80% of total excretion of 1,2 3 H PEG-4000 is attributable to urinary excretion with the 20% remaining attributable to extra-renal excretion ŽCurtis and Wood, 1991.. Glomerular filtration rate ŽGFR. was calculated based on urine and plasma 1,2 3 H PEG-4000 concentrations. UFR, GFR and urinary Pi excretion rates, derived from 4 h measurements, were expressed on a 24-h basis to facilitate comparison among trials. 2.2.3. Water accumulation trial P accumulation was measured in 12 tanks, each stocked with 4–6 rainbow trout, weighing in average 407 g BW each, acclimated to the experimental diets for 17 weeks. Three days before the water collection period, the fish were weighed and, for the following 3 days, the fish were hand fed twice daily a predetermined amount of the experimental diets based on the theoretical energy requirement of the fish ŽCho, 1992.. A few minutes after the last meal Žthird day., water flow to the tank was interrupted and water was sampled at 0, 2 and 4 h. Water samples Ž100 ml. were stabilized with 0.5 ml concentrated sulfuric acid. Total P content of the water samples was determined by a commercial laboratory ŽPhilips Analytical Services, Burlington, ON, Canada. using the acid hydrolysis–ascorbic acid method ŽAmerican Public Health Association, 1979.. At the end of the water collection period, fish were anaesthetized with t-amyl alcohol, individually weighed and blood Ž900 ml. was sampled from the caudal vein. The blood was transferred to a plastic microcentrifuge tube containing 100 ml heparinized saline, mixed, centrifuged and frozen and analyzed for Pi as describe above. Individual plasma Pi values were used to predict urinary Pi excretion of individual fish using the following equation derived from the urinary excretion trial: Urinary Pi output Ž mg kgy1 BW dayy1 . s y360 q 4.2 = plasma Pi Ž mg ly1 .
D.P. Bureau, C.Y. Cho r Aquaculture 179 (1999) 127–140
132
Negative values were disregarded, individual values were averaged per tank and statistical analysis was performed on tank average Žexperimental unit.. This approach is referred to as ‘‘plasmarurinary method’’ to avoid confusion with actual urinary Pi excretion measurements. DWP output of the fish was also calculated using the biological method of Cho et al. Ž1991. ŽDWP outputs Intake P y Fecal P y Retained P. using published ADC values for P ŽLall, 1991; Satoh et al., 1998. and expected P retention of the fish based on the results of the growth trial. 2.3. Chemical analyses Diet, feces and carcass samples were analyzed for dry matter and ash according to AOAC Ž1995., crude protein Ž%N = 6.25. using a Kjeltech auto-analyzer ŽModel a1030, Tecator, Hoganas, ¨ ¨ Sweden. and gross energy using an automated oxygen bomb calorimeter ŽModel a1271, Parr Instruments, Moline, IL.. Digestion indicator was determined using the AIA indicator method as used by Atkinson et al. Ž1984.. P content of diet, fecal material, whole fish carcass and vertebrae was analyzed by a commercial laboratory ŽLabstat, Kitchener, ON, Canada. using AOAC method 4.8.14 ŽAOAC, 1995.. 2.4. Statistical analyses Data were analyzed as a complete random block design or complete random design where appropriate using the general linear model ŽGLM. of the SASrSTAT software ŽSAS Institute, 1988.. Dietary effects were examined using orthogonal contrast ŽSteel and Torrie, 1980.. Regression analysis was performed using the regression function of the Microsoft Excel 7.0 software ŽMicrosoft, Seattle, WA.. 3. Results No significant difference was observed in the LWG, growth rate Žexpressed as thermal-unit growth coefficient; Cho, 1992. and feed efficiency of the fish fed the Table 2 Growth performance of rainbow trout a fed the experimental diets for 16 weeks Gain Žg fishy1 .
Feed Žg fishy1 .
Feed efficiency Žgain:feed.
TGC b
Diet 1 2 3 4 Pooled SEM
66.0 65.9 65.2 67.9 1.6
72.8 72.5 72.4 72.4 0.3
0.91 0.91 0.90 0.94 0.02
0.131 0.130 0.129 0.132 0.002
Contrasts Linear Quadratic
N.S. N.S.
NrAc NrAc
N.S. N.S.
N.S. N.S.
a
Initial live body weight 8.0 g fishy1 . Thermal-unit growth coefficients100=ŽFBW 1r 3 yIBW 1r3 .rÝŽTemp. Ž8C.=day. ŽCho, 1992.. c Not applicable, independent variable. b
D.P. Bureau, C.Y. Cho r Aquaculture 179 (1999) 127–140
133
Table 3 Chemical composition of the carcass of rainbow trout fed the experimental diets for 16 weeks Moisture Ž%.
Crude protein Ž%.
Lipid Ž%.
Ash Ž%.
Diet Initial carcass 1 2 3 4 Pooled SEM
74.6 68.3 67.7 68.2 68.5 0.4
11.5 13.8 13.4 13.4 13.9 0.1
5.7 13.5 14.7 13.7 13.0 0.5
2.2 2.0 1.9 1.9 2.2 0.2
Contrasts Linear Quadratic
N.S. N.S.
N.S. - 0.05
N.S. N.S.
N.S. N.S.
P Ž%.
0.40 0.26 0.34 0.39 0.41 0.01
- 0.001 - 0.01
Gross energy ŽkJ gy1 .
Vertebrae P Ž%.
4.9 9.4 9.5 9.1 9.0 0.2
– 5.5 9.1 10.3 10.6 0.4
N.S. N.S.
- 0.001 - 0.01
experimental diets ŽTable 2.. Increasing dietary P resulted in a significant linear increase in the P content of whole fish carcass and vertebrae, and P gain Žg kgy1 LWG. but this response tended to level off at the highest dietary P intakes since a significant quadratic effect was observed for whole carcass and vertebrae P contents, and P gain Žg kgy1 LWG. ŽTables 3 and 4.. Dietary P level had no effect on nitrogen and gross energy gain or retention efficiency Žresults not shown.. P retention efficiency decreased linearly with
Table 4 Phosphorus of rainbow trout fed the experimental diets for 16 weeks growth trial Phosphorus Žg kgy1 LWG. Intake
Retained
Wastes Total
Solida
Dissolvedb
Diet 1 2 3 4 Pooled SEM
8.3 12.7 18.5 23.4 0.3
2.4 3.3 3.9 4.1 0.1
5.9 9.3 14.6 19.3 0.4
5.1 6.2 8.1 9.4 0.1
0.8 3.1 6.5 9.9 0.2
Contrasts linear quadratic
NrAc NrAc
- 0.001 - 0.05
- 0.001 N.S.
- 0.001 - 0.01
- 0.001 N.S.
a
Based on ADC for P of herring meal Ž52%., brewer’s yeast Ž79%., dibasic calcium phosphate Ž72%. of Lall Ž1991. measured by stripping and ADC for P of corn gluten meal Ž7%. of Satoh et al. Ž1998. measured with TUF column. b By difference total P wasteysolid P waste. c Not applicable, independent variable.
D.P. Bureau, C.Y. Cho r Aquaculture 179 (1999) 127–140
134
Table 5 UFR, GFR, plasma and urinary Pi concentrations and excretion of rainbow trout fed the experimental diets in the urinary excretion trial UFR Žml kgy1 BW dayy1 .
GFR Žml kgy1 BW dayy1 .
Plasma Pi Žmg ly1 .
Urine Pi Žmg ly1 .
Urinary Pi excretion Žmg kgy1 % of Pi BW dayy1 . filtered
Diet 1 2 3 4 Pooled SEM
217 201 268 188 63
320 288 487 248 91
56 90 90 104 2
2 33 119 354 36
1 6 33 67 8
3 22 81 257 20
Contrast linear quadratic
N.S. N.S.
N.S. N.S.
P - 0.001 P - 0.01
P - 0.001 P - 0.05
P - 0.001 N.S.
P - 0.001 P - 0.01
increasing dietary P intake. DWP represented 25, 47, 63 and 71% of digestible P intake as digestible P increased from 0.29, 0.62, 0.94, 1.27%. No significant differences were observed for the UFR and GFR of the fish fed the four experimental diets due to large variance ŽTable 5.. Plasma and urine Pi concentrations as well as urinary Pi excretion Žmg kgy1 BW dayy1 and %Pi of filtered by kidney. increased linearly with increasing dietary P intake. A significant quadratic effect was also observed for plasma and urine Pi concentrations and Pi excretion Žexpressed as percent of Pi filtered by kidney.. Pi excretion exceeded the amount of Pi estimated to be filtered through glomerular filtration at the highest dietary P level indicating net renal secretion of Pi by the fish ŽTable 5.. Urine Pi concentrations measured for individual fish were plotted against corresponding plasma Pi concentrations ŽFig. 1.. Fish with low plasma Pi Že.g., - 80 mg ly1 . had very low urine Pi concentration. Fish with high plasma Pi showed, in most cases,
Fig. 1. Urine Pi concentration as a function of plasma Pi concentration of rainbow trout fed the experimental diets in the urinary excretion trial Ž ns 39 fish..
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Fig. 2. The relationship between urinary Pi excretion of rainbow trout and plasma Pi concentration Žtank averages.. This relationship is: y sy360q4.2= Ž P - 0.001, R 2 s 0.82., based on ns9 tank averages Žfish fed diets 2, 3, and 4, fish fed diet 1 excluded from analysis.. Standard error of intercepts 72, standard error of coefficient of x s 0.8.
elevated urinary Pi concentration. Pi excretion rates, calculated for every tank based on the average UFR Ž218 ml kgy1 BW dayy1 ., were plotted against corresponding tank average plasma Pi ŽFig. 2.. Regression analysis using values from tanks Ž n s 9. of fish fed the diet 2, 3, 4 showed a highly significant linear relationship Ž R 2 s 0.82, P - 0.001. between urinary Pi excretion rate and plasma Pi. The plasma Pi threshold concentration for urinary Pi excretion was estimated at 86 mg ly1 .
Table 6 Reactive P accumulation, plasma Pi concentration and predicted urinary Pi excretion of the fish in water accumulation study Dietary P intake
Dissolved phosphorus waste output Žmg kg BWy1 dayy1 . Water accumulation based on 2 h
Water accumulation based on 4 h
Biological methoda
Plasmarurinary methodb
Diet 1 2 3 4 Pooled SEM
75 115 166 219 –
10 63 75 112 13
15 74 64 117 20
7 29 58 92 –
0 21 94 101 17
Contrast Linear Quadratic
NrAc NrAc
- 0.01 N.S.
- 0.05 N.S.
NrAc NrAc
- 0.01 N.S.
a
ŽP intake=theoretical ADC for P.yŽexpected P retention.. See text for details. Plasma Pi averaged 56, 82, 105, 110 mg ly1 for diets 1, 2, 3 and 4, respectively. c Not applicable, independent variable or based on theoretical ADC and P retention values Žno randomization of experimental error.. b
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Table 7 Calculated digestible phosphorus content of the diets based on various methods Total P Ž%.
Diet 1 0.75 2 1.15 3 1.66 4 2.19
Digestible P Ž%. Plasmarurinary a
Water 2 hb
Water 4 hb
ADC c literature
ADC d measured
0.21 0.51 1.29 1.40
0.31 0.93 1.10 1.51
0.36 1.04 0.99 1.56
0.27 0.60 0.93 1.25
0.50 0.39 0.38 0.90
a
Estimated P retention based on data obtained from growth trial ŽTable 4.qestimated urinary Pi based on individual plasma Pi values measured and equation presented in Fig. 2. b Estimated P retentionqmeasured reactive P accumulation in tank water over 2 or 4 h. c Based on ADC for P of herring meal Ž52%., brewer’s yeast Ž79%., dibasic calcium phosphate Ž72%. of Lall Ž1991. measured by stripping and ADC for corn gluten meal Ž7%. of Satoh et al. Ž1998. measured with TUF column. d Based on ADC values measured in this study with the Guelph system.
A linear increase Ž P - 0.05. was observed in the P output of fish in the water accumulation trial ŽTable 6.. Predicted urinary Pi excretion based on plasma Pi Žplasmarurinary method. increased linearly Ž P - 0.01. with increasing dietary P. DWP output estimates from the water accumulation, biological and plasmarurinary methods showed relatively good agreement, especially at low and high P intakes ŽTable 6.. Predicted digestible P of the diets using water accumulation Žbased 2 or 4 h accumulation., plasmarurinary and biological methods and actual apparent digestibility measurements with the Guelph System are presented in Table 7. Estimated digestible P of the diet based on water P accumulation resulted in apparently higher digestible P estimates for the diets 1 and 2 than what was estimated from ADC values from the literature and the plasmarurinary approach.
4. Discussion The lack of effect of P level on growth and feed efficiency, but very significant effect on whole carcass and vertebrae P contents and P gain observed in this study is in agreement with several other studies that have shown that the requirement for P is lower for growth than for maximum P gain, at least over relatively short-term determination periods ŽRodehutscord, 1996; Asgard and Shearer, 1997.. Diets used in this experiment may have been marginally deficient in lysine which resulted in suboptimal performance; this was indicated by the poorer nitrogen retention efficiencies and growth rates than observed with the same strain of fish fed other practical diets. The present study is, to our knowledge, the first report of a clear threshold relationship between plasma Pi concentration and urinary Pi excretion in fish. This relationship is very similar to what is observed in mammals ŽBijvoet, 1980.. At similar plasma Pi, urinary Pi excretion rates measured in the present study appear comparable to what was measured by Kaune and Hentschel Ž1987. with Crucian carp, Carassius
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137
auratus. Fish fed the diet with the highest digestible P level Ždiet 4. exhibited net renal Pi secretion, e.g., Pi excretion greater than what was estimated to enter the renal tubules by glomerular filtration. This phenomenon has been shown to occur in fish, amphibians and birds ŽSchneider et al., 1980; Kaune and Hentschel, 1987; Renfro, 1997. but this study is, perhaps, the first demonstration of net renal secretion of P in fish in which plasma Pi was modulated through dietary means. Estimated UFR of the fish in this study averaged 218 ml kgy1 BW dayy1 . This value is two to three times higher than reported in most studies on urine production of freshwater fish ŽHunn, 1982; Curtis and Wood, 1991.. An intermediate UFR value was observed in a recent trial in our laboratory with rainbow trout ŽUFR s 144 ml kgy1 BW dayy1 . using the same method ŽBureau, 1997.. The much higher UFR and GFR measured in the present study compared to other studies may be explained, at least in part, by the fact that most studies have used fish that were fasted for several days prior to the measurement which may result in a significantly reduced urine production ŽHunn, 1982.. Oxygen consumption Žmetabolic rate. may have a determinant effect on GFR and UFR. Changes in surface area and blood perfusion may be factors influencing the flux of water through the gills ŽHunn, 1982; Isaia, 1984.. UFR increases with temperature with a Q10 of about 2, which is consistent with expected increase in oxygen consumption with temperature ŽHunn, 1982; Cho, 1992.. It is possible that the high UFR measured in this study was the result of stress. However, the fish recovered after the injection and were feeding well on the day of the measurement and appeared relatively calm during the radioactivity accumulation period when water flow was interrupted. The UFR, GFR and urinary Pi output estimates obtained in this study were, nevertheless, highly variable. This variability is probably a compromise related to the use of free-swimming and actively feeding fish, which is essential to obtain meaningful results ŽCho et al., 1982.. Fish fed the diet with no P supplementation excreted very little DWP, indicating that digestible P intake of the fish was directed almost completely toward deposition. Increasing digestible P intake resulted in a significant increase in DWP output. These results are in agreement with those of Rodehutscord Ž1996. who observed a decrease in the efficiency of P utilization Žretention. at increasing digestible P intake. There is still question about the necessity of maximizing mineralization of the skeleton of fish for long term maintenance of health and performance ŽRodehutscord, 1996; Asgard and Shearer, 1997; Baeverfjord et al., 1998.. The results from the present study and that of Rodehutscord Ž1996. indicate that maximum mineralization cannot be achieved without significant DWP output. Since a well-defined plasma Pi threshold for urinary Pi excretion was observed in this study, it would be of interest to verify the effect of long term feeding diets with digestible P levels producing plasma Pi close to this threshold level Žin order to minimize DWP. on health and performance of the fish. Estimates of DWP obtained with the various methods used in this study were in relatively good agreement at low and high digestible P intakes. Disagreement between the water accumulation method and the biological and plasmarurinary methods were observed at intermediate digestible P levels. DWP output estimates obtained in the water accumulation trial were highly variable and occasionally aberrant ŽTable 6, 4 h based estimates for diet 2 and 3.. Interference by fecal material contamination may have
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contributed to this variability and resulted in higher estimates of DWP that what was excreted by the fish. Filtration and stabilization of the water sample with chloroform would have been preferable ŽDosdat et al., 1994.. Calculated digestible P content of the experimental diets using the plasmarurine method or ADC for P of feed ingredients of Lall Ž1991. and Satoh et al. Ž1998. were similar. Actual measurements of apparent P digestibility with the Guelph System resulted in aberrant values in the present study. Lall Ž1991. observed that, for P digestibility, collection of fecal material by stripping yielded more reliable estimates than the Guelph System. Relatively high variability of ADC for P and ash can be obtained with the Guelph System for certain diets probably as a result of feces breakup and differential recovery of mineral fecal components. Results from this study suggest that reliable ADC are available in the literature for the feed ingredients used in this study and that the biological method of Cho et al. Ž1991. is appropriate to estimate DWP output. The results suggest that plasma Pi could, for certain purposes, be used to determine P adequacy of diets and estimate DWP output in lieu of expensive and time-consuming growth and digestibility trials. Plasma Pi measurements are easily performed using simple colorimetric assays and could be especially valuable under field conditions. Studies have, however, shown plasma Pi or DWP output to be highly variable over the whole day ŽDosdat et al., 1998; Medale et al., 1998; Sugiura, 1998.. This may ´ complicate use of plasma Pi measurements to estimate DWP output of fish. However, the pronounced post-prandial pattern or hourly variability of plasma Pi or DWP output observed in certain studies may be related, in part, to the experimental design used. A pronounced post-prandial pattern was observed for the oxygen consumption Ža good indicator of nutrient absorption and utilization. of rainbow trout upon refeeding following fasting or severe feed restriction periods. This post-prandial pattern was minimized when the fish fed normally to near-satiation for 2 to 4 days ŽCho et al., 1982.. More research is required to define the effects of biological, nutritional and environmental factors on plasma Pi, renal handling of P, and DWP output.
Acknowledgements Sincere thanks to Greg Ardnt, Stephen Gunther, Choi-Lan Ha, Andrew Harris, Fardin Pourkhataei and Ursula Wehkamp for their assistance and the Alma Aquaculture Research Station for donating the fish used in this study. This study was made possible by the financial support of the Ontario Ministry of Natural Resources ŽOMNR. and the Ontario Ministry of Agriculture, Food and Rural Affair ŽOMAFRA..
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