Aquaculture 179 1999.

127140

Phosphorus utilization by rainbow trout Oncorhynchus mykiss / : estimation of dissolved phosphorus waste output
D.P. Bureau
a

a, )

, C.Y. Cho

a,b

Fish Nutrition Research Laboratory, Department of Animal and Poultry Science, Uniersity of Guelph, Guelph, Ontario, Canada N1G 2W1 b Aquatic Ecosystems Research Section, Ontario Ministry of Natural Resources, Guelph, Ontario, Canada N1G 2W1

Abstract Phosphorus P. waste output, notably in the dissolved form DWP., 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 0.75, 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 Pi. 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 Pi. concentration 86 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 BW. dayy1 x s y360 q 4.2 plasma Pi mg ly1 .; 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: dbureau@aps.uoguelph.ca 0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 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 P. wastes are a major concern for many aquaculture operations since DWP plant-available P. can have very significant eutrophication effects on freshwater or brackish waterbodies Persson, 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 Cho 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 e.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 Lall, 1991.. These factors often make it difficult to derive reliable apparent digestibility coefficients ADC. for diets. A number of studies have examined DWP output of salmonids in response to dietary or environmental factors Ketola 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 Kaune and Hentschel, 1987; Renfro, 1997.. In mammals, urinary phosphate excretion is determined mostly by plasma phosphate concentration Bijvoet, 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 Dantzler, 1989., a similar relationship between plasma phosphate and urinary phosphate excretion is likely present in fish. The objectives of this study were to: 1. examine the partitioning of dietary P at increasing P intakes, 2. determine if the biological method of Cho et al. 1991. can be used to estimate DWP output, and 3. 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 CaHPO4 . which replaced calcium carbonate CaCO 3 . in the diet Table 1.. Acid-washed diatomaceous silica Celite AW521, Celite, Lompoc, CA., a source of acid-insoluble ash AIA. Atkinson et al., 1984., was included in all the diets to serve as a digestion indicator. The diets were mixed using a Hobart mixer Hobart, Don Mills, ON, Canada. and pelleted using a laboratory steam pellet mill California 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 Oncorhynchus mykiss . obtained from the Alma Aquaculture Research Station Elora, ON, Canada.. The fish were treated in accordance with the guidelines of the Canadian Council on Animal Care Canadian 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 1 Ingredients Fish meal, herring Corn gluten meal Brewers dried yeast Whey Celite CaHPO4 CaCO 3 Vitamin premix a Mineral premix a Fish oil Total Composition (as is basis) Dry matter Crude protein N=6.25. Lipid Ash Phosphorus Digestible energy DE. DPrDE g kJy1 .
a

2 15 49 6 7 1 2 4 1 1 14 100

3 15 49 6 7 1 4 2 1 1 14 100

4 15 49 6 7 1 6 0 1 1 14 100

15 49 6 7 1 0 6 1 1 14 100

95.1 41.7 17.8 10.2 0.75 19.6 20.5

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

Composition presented by Bureau et al. 1998..

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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 BW. each, were stocked 53 fish tanky1 . in 12 rectangular fibreglass tanks 40 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 1992.. 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 LWG. and feed efficiency LWG: 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. 1982.. 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 20 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 chloroformmethanolwater Bligh 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 1991.. This method involves the determination of the urine flow rate UFR. of non-catheterized fish using a glomerular filtration marker, 1,2 3 H polyethylene glycol 4000 1,2 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 10 fish tanky1 . in 12 rectangular fibreglass tanks 50 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 4 fish tanky1 . similar to the acclimation tanks. On the following day, the 48 fish were anaesthetized with tricaine methanesulfonate MS222. and 0.6 ml of Cortland saline Wolf, 1963. containing 17 m Ci of 1,2 3 H PEG NET-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 1991.. 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 ) 7.5 mg ly1 .. Water samples 5 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 Scintiverse 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 PE-50, Clay Adams, Parsippany, NJ. attached to a 1-ml syringe with a blunted 23 gauge needle. A blood sample 500 m l. was then taken from the caudal vein of each fish. The blood was transferred to a plastic microcentrifuge tube containing 100 m l heparinized saline, mixed and immediately centrifuged for 10 min at 13,600 = g . Urine and plasma samples 25 m l. 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 360-UV, Sigma, St. Louis, MO.. 3 H in water, plasma and urine samples was counted up to 2 S . after 12 h of dark acclimation using a liquid scintillation counter LS-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 Curtis and Wood, 1991.. Glomerular filtration rate GFR. 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 46 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 Cho, 1992.. A few minutes after the last meal third day., water flow to the tank was interrupted and water was sampled at 0, 2 and 4 h. Water samples 100 ml. were stabilized with 0.5 ml concentrated sulfuric acid. Total P content of the water samples was determined by a commercial laboratory Philips Analytical Services, Burlington, ON, Canada. using the acid hydrolysisascorbic acid method American Public Health Association, 1979.. At the end of the water collection period, fish were anaesthetized with t-amyl alcohol, individually weighed and blood 900 m l. was sampled from the caudal vein. The blood was transferred to a plastic microcentrifuge tube containing 100 m l 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 mg kgy1 BW dayy1 . s y360 q 4.2 = plasma Pi mg ly1 .

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Negative values were disregarded, individual values were averaged per tank and statistical analysis was performed on tank average experimental 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. 1991. DWP output s Intake P y Fecal P y Retained P. using published ADC values for P Lall, 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 1995., crude protein %N = 6.25. using a Kjeltech auto-analyzer Model a1030, Tecator, Hoganas, Sweden. and gross energy using an automated oxygen bomb calorimeter Model a1271, Parr Instruments, Moline, IL.. Digestion indicator was determined using the AIA indicator method as used by Atkinson et al. 1984.. P content of diet, fecal material, whole fish carcass and vertebrae was analyzed by a commercial laboratory Labstat, Kitchener, ON, Canada. using AOAC method 4.8.14 AOAC, 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 GLM. of the SASrSTAT software SAS Institute, 1988.. Dietary effects were examined using orthogonal contrast Steel and Torrie, 1980.. Regression analysis was performed using the regression function of the Microsoft Excel 7.0 software Microsoft, Seattle, WA.. 3. Results No significant difference was observed in the LWG, growth rate expressed 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 g fishy1 . Diet 1 2 3 4 Pooled SEM Contrasts Linear Quadratic
a b

Feed g fishy 1 .

Feed efficiency gain:feed. 0.91 0.91 0.90 0.94 0.02

TGC b

66.0 65.9 65.2 67.9 1.6

72.8 72.5 72.4 72.4 0.3 NrAc NrAc

0.131 0.130 0.129 0.132 0.002

N.S. N.S.

N.S. N.S.

N.S. N.S.

Initial live body weight 8.0 g fishy1 . Thermal-unit growth coefficient s 100=FBW 1r 3 yIBW 1r 3 .rTemp. 8C.=day. Cho, 1992.. c Not applicable, independent variable.

D.P. Bureau, C.Y. Cho r Aquaculture 179 (1999) 127140 Table 3 Chemical composition of the carcass of rainbow trout fed the experimental diets for 16 weeks Moisture %. Diet Initial carcass 1 2 3 4 Pooled SEM Contrasts Linear Quadratic Crude protein %. 11.5 13.8 13.4 13.4 13.9 0.1 Lipid %. Ash %. P %. Gross energy kJ gy1 . 4.9 9.4 9.5 9.1 9.0 0.2 Vertebrae P %.

133

74.6 68.3 67.7 68.2 68.5 0.4

5.7 13.5 14.7 13.7 13.0 0.5

2.2 2.0 1.9 1.9 2.2 0.2

0.40 0.26 0.34 0.39 0.41 0.01

5.5 9.1 10.3 10.6 0.4

N.S. N.S.

N.S. - 0.05

N.S. N.S.

N.S. N.S.

- 0.001 - 0.01

N.S. N.S.

- 0.001 - 0.01

experimental diets Table 2.. Increasing dietary P resulted in a significant linear increase in the P content of whole fish carcass and vertebrae, and P gain g 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 g kgy1 LWG. Tables 3 and 4.. Dietary P level had no effect on nitrogen and gross energy gain or retention efficiency results not shown.. P retention efficiency decreased linearly with

Table 4 Phosphorus of rainbow trout fed the experimental diets for 16 weeks growth trial Phosphorus g kgy1 LWG. Intake Retained Wastes Total Diet 1 2 3 4 Pooled SEM Contrasts linear quadratic
a

Solida 5.1 6.2 8.1 9.4 0.1

Dissolvedb 0.8 3.1 6.5 9.9 0.2

8.3 12.7 18.5 23.4 0.3 NrAc NrAc

2.4 3.3 3.9 4.1 0.1

5.9 9.3 14.6 19.3 0.4

- 0.001 - 0.05

- 0.001 N.S.

- 0.001 - 0.01

- 0.001 N.S.

Based on ADC for P of herring meal 52%., brewers yeast 79%., dibasic calcium phosphate 72%. of Lall 1991. measured by stripping and ADC for P of corn gluten meal 7%. of Satoh et al. 1998. 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 ml kgy1 BW dayy1 . Diet 1 2 3 4 Pooled SEM Contrast linear quadratic GFR ml kgy 1 BW dayy 1 . Plasma Pi mg ly 1 . Urine Pi mg ly 1 . Urinary Pi excretion mg kgy 1 % of Pi BW dayy1 . filtered 1 6 33 67 8 3 22 81 257 20

217 201 268 188 63

320 288 487 248 91

56 90 90 104 2

2 33 119 354 36

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 Table 5.. Plasma and urine Pi concentrations as well as urinary Pi excretion mg kgy1 BW dayy 1 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 expressed 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 Table 5.. Urine Pi concentrations measured for individual fish were plotted against corresponding plasma Pi concentrations Fig. 1.. Fish with low plasma Pi e.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 n s 39 fish..

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Fig. 2. The relationship between urinary Pi excretion of rainbow trout and plasma Pi concentration tank averages.. This relationship is: y sy360q4.2= P - 0.001, R 2 s 0.82., based on n s 9 tank averages fish fed diets 2, 3, and 4, fish fed diet 1 excluded from analysis.. Standard error of intercept s 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 218 ml kgy1 BW dayy 1 ., were plotted against corresponding tank average plasma Pi Fig. 2.. Regression analysis using values from tanks n s 9. of fish fed the diet 2, 3, 4 showed a highly significant linear relationship 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 mg kg BWy1 dayy 1 . Water accumulation based on 2 h Diet 1 2 3 4 Pooled SEM Contrast Linear Quadratic
a b

Water accumulation based on 4 h 15 74 64 117 20

Biological methoda

Plasmarurinary methodb

75 115 166 219 NrAc NrAc

10 63 75 112 13

7 29 58 92 NrAc NrAc

0 21 94 101 17

- 0.01 N.S.

- 0.05 N.S.

- 0.01 N.S.

P intake=theoretical ADC for P.yexpected 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 no randomization of experimental error..

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Table 7 Calculated digestible phosphorus content of the diets based on various methods Total P %. Digestible P %. Plasmarurinary a Diet 1 0.75 2 1.15 3 1.66 4 2.19
a

Water 2 hb 0.31 0.93 1.10 1.51

Water 4 hb 0.36 1.04 0.99 1.56

ADC c literature 0.27 0.60 0.93 1.25

ADC d measured 0.50 0.39 0.38 0.90

0.21 0.51 1.29 1.40

Estimated P retention based on data obtained from growth trial Table 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 52%., brewers yeast 79%., dibasic calcium phosphate 72%. of Lall 1991. measured by stripping and ADC for corn gluten meal 7%. of Satoh et al. 1998. measured with TUF column. d Based on ADC values measured in this study with the Guelph system.

A linear increase P - 0.05. was observed in the P output of fish in the water accumulation trial Table 6.. Predicted urinary Pi excretion based on plasma Pi plasmarurinary method. increased linearly 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 Table 6.. Predicted digestible P of the diets using water accumulation based 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 Rodehutscord, 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 Bijvoet, 1980.. At similar plasma Pi, urinary Pi excretion rates measured in the present study appear comparable to what was measured by Kaune and Hentschel 1987. with Crucian carp, Carassius

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auratus. Fish fed the diet with the highest digestible P level diet 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 Schneider 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 Hunn, 1982; Curtis and Wood, 1991.. An intermediate UFR value was observed in a recent trial in our laboratory with rainbow trout UFR s 144 ml kgy1 BW dayy1 . using the same method Bureau, 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 Hunn, 1982.. Oxygen consumption metabolic 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 Hunn, 1982; Isaia, 1984.. UFR increases with temperature with a Q10 of about 2, which is consistent with expected increase in oxygen consumption with temperature Hunn, 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 Cho 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 1996. who observed a decrease in the efficiency of P utilization retention. 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 Rodehutscord, 1996; Asgard and Shearer, 1997; Baeverfjord et al., 1998.. The results from the present study and that of Rodehutscord 1996. 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 in 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 Table 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 Dosdat et al., 1994.. Calculated digestible P content of the experimental diets using the plasmarurine method or ADC for P of feed ingredients of Lall 1991. and Satoh et al. 1998. were similar. Actual measurements of apparent P digestibility with the Guelph System resulted in aberrant values in the present study. Lall 1991. 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. 1991. 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 Dosdat 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 a 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 Cho 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 OMNR. and the Ontario Ministry of Agriculture, Food and Rural Affair OMAFRA..

References
American Public Health Association APHA., 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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