 

'HESiS

ullllllllmljiiwl

31293 0

This is to certify that the

dissertation entitled '
A/Vrﬂ/ 770A; 191. 5 TﬁﬁTEG/Es R2 47,-
)2 EDUC/xug [baa 77M) 71S //U
”mimic! (/Tuzé’ EFH—U SAWS
presented by

A 6201 f'Q/ 4T?C?Vi /2am5€’7/Pr’

has been accepted towards fulﬁllment
of the requirements for

p; ' b degreein /??7‘

 

 

3/417 9 7

MS U is an Afﬁrmamv Action/Equal Opportunity Institution 0-1

 

LIBRARY

Michigan State
University

 

 

 

v A—-+ +——+ +-A—-v‘~———#+-—* —

5.15.4h

 

 

 

 

PLACE lN RETURN Box
to remove this checkout from your record.
TO AVOID FINES return on or before date due.

I MTE DUE MTE DUE MTE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1!” W14

NUTRITIONAL STRATEGIES FOR REDUCING POLLUTANT S IN
AQUACULTURE EFFLUENTS

By

Laurel J. Ramseyer

A DISSERTATION
Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Fisheries and Wildlife

1997

ABSTRACT

NUTRITIONAL STRATEGIES FOR REDUCING POLLUTANT S 1N
AQUACULTURE EFFLUENTS

By

Laurel]. Ramseyer

A nutritional P mass balance model was constructed for coho and Chinook salmon
as a method of estimating P losses ﬁ'om the Platte River Anadromous State Fish Hatchery.
For the production period of January 1993 through May 1994, the P mass balance model
indicated that 37.7% ofP fed was retained by ﬁsh, 21.0% was discharged in the feces, and
41.3% was discharged in dissolved form. Without any raceway solids removal, a
maximum of 2.8 kg feed P'metric ton‘l ﬁsh produced was discharged into the hatchery’s
stabilization pond. This loss rate was the lowest reported for a salmonid hatchery.
Eﬁcient removal of raceway solids could have reduced hatchery P losses to 1.8 kg
P-metric ton‘l ﬁsh produced.

Improving the digestibility and utilization of nutrients in feeds should reduce
nutrients in aquaculture waste water. Dietary phytate could impair dietary protein and
mineral utilization in ﬁsh by binding zinc (Zn), creating a Zn deﬁciency. Zn deﬁciency
could reduce protein and mineral utilization by reducing insulin secretion, insulin
sensitivity, and the activity of the digestive enzymes carboxypeptidase B and alkaline
phosphatase.

Rainbow trout were fed diets containing untreated or dephytinized soybean meal

and corn gluten meal with or without supplemental Zn to determine if ﬁsh dietary phytate

impairs Zn bioavailability in ﬁsh. Fish fed diets containing untreated soybean meal and
corn gluten meal without supplemental Zn (basal diet) were not Zn deﬁcient after 170 d
based on growth, whole ﬁsh Zn, P and protein content, total bone Zn, and the activity of
alkaline phosphatase and carboxypeptidase B. Although bone Zn concentrations were
reduced in ﬁsh fed the basal diet, total bone Zn increased in all ﬁsh regardless ofdietary
treatment. The basal diet contained enough available Zn to oﬁ‘set any negative eﬁ‘ects of
phytate on Zn bioavailability. Future assessments of Zn status in ﬁsh should be based on

changes in Zn-dependent metabolism or total bone Zn rather than bone Zn concentration.

ACKNOWLEDGMENTS

I would like to thank Drs. Donald Gatling, Jr., Ted Batterson, Gretchen Hill and
Michael Vandehaar for serving on my graduate committee. Thanks and appreciation of
the ﬁrst order go to Dr. Garling for providing me with interesting research opportunities
and 8 years of instruction, guidance and ﬁ'iendship. I sincerely appreciated the laboratory
privileges provided by Dr. Hill and Dr. Batterson, and the tremendous gift of technical
assistance and advice I received from Jane Link and Dr. Pao Ku. I would also like to
acknowledge Dr. Scott Winterstein, Dr. Oliver Schabenberger, Dr. Tempelman, Scott
Miller, Marty Riche, Ibrahim Almohessen, Mike Oetker and Dr. Hill’s graduate students
for thdr contributions to this project.

The Michigan Department of Natural Resources, the Platte River Anadromous
State Fish Hatchery and the Wolf Lake Fish Hatchery generously provided ﬁsh, feeds,
production records and an experimental venue. Feedstuﬁ‘s and feed additives were
provided by the United States Fish and Wildlife Service, Alko Biotechnologies and
International Protein Colloids. This research was supported in part by the Michigan State
University Agricultural Experiment Station and the North Central Regional Aquaculture

Center.

TABLE OF CONTENTS

LIST OF TABLES ................................................... vi
LIST OF FIGURES ................................................... ix
INTRODUCTION .................................................... 1
CHAPTER 1
ESTIMATION OF PHOSPHORUS LOADING FROM THE PLATTE RIVER
ANADROMOUS STATE FISH HATCHERY ............................... 5
Introduction .................................................... 5
Literature Review ............................................... 8
Materials and Methods ........................................... 18
Results ....................................................... 31
Discussion .................................................... 39
Summary and Conclusions ........................................ 44
CHAPTER 2

EFFECT OF DIETARY ZINC SUPPLEMENTATION AND PHYTASE PRE-
TREATMENT OF SOYBEAN MEAL OR CORN GLUTEN MEAL ON GROWTH,
ZINC BIOAVAILABILITY AND PROTEIN UTILIZATION IN

RAINBOW TROUT .................................................. 45
Introduction ..................... . .............................. 45
Literature Review .............................................. 47
Materials and Methods ........................................... 54
Results ....................................................... 66
Discussion .................................................... 73
Summary and Conclusions ........................................ 79

LIST OF REFERENCES .............................................. 80

APPENDD( A

SAMPLE DIGESTION PROCEDURES ................................... 95

APPENDIX B

PHOSPHORUS DETERMINATION PROCEDURES ........................ 97

APPENDIX C
PREDICTING THE NITROGEN AND PHOSPHORUS CONTENT OF WHOLE FISH

FROM FISH WEIGHT ............................................... 101
APPENDIX D
ALKALINE PHOSPHATASE ASSAY PROTOCOL ........................ 114
APPENDIX E
CARBOXYPEPTIDASE B ASSAY PROTOCOL .......................... l 17

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

Table 6.

Table 7.

Table 8.

LIST OF TABLES

StartingpopulationsofcohoandchinooksalmonﬁyatthePlatteRiver
Anadromous State Fish Hatchery beginning each January. Parental stocks
originated ﬁ'om Lake Michigan (MI) and Lake Huron (NY). Fish stock
designations (i.e., Coho ’91) indicate the year eggs were fertilized . . . . 19

Moisture and P content of feeds fed to coho and Chinook salmon at the
Platte River Anadromous State Fish Hatchery between January 1, 1993 and
May 31, 1994 ............................................ 21

Regressions of whole ﬁsh P (mg) on ﬁsh wet weight (g) for coho and
chinook salmon ﬁ'om the Platte River Anadromous State Fish Hatchery
(hatchery), the Michigan State University Fisheries Research Laboratory
(MSU), or both (combined). Values t SE ...................... 32

Monthly P mass balance measurements and calculations ﬁ'om the Platte
River Anadromous State Fish Hatchery ........................ 35

Phosphorus mass balance results for the experimental period of January
1993 through May 1994 at the Platte River Anadromous State Fish
Hatchery ................................................ 38

Composition of experimental diets fed to rainbow trout to determine the
eﬁ‘ects of feedstuﬁ‘ dephytinization and zinc supplementation on ﬁsh zinc
status and zinc bioavailability. Ingredients listed on as-fed basis ...... 57

Mineral and phytic acid composition of experimental diets. All values are
on a dry weight basis ...................................... 59

Weight gain, protein and mineral composition of whole ﬁsh and fat-ﬁee dry
bone, percent Zn retention (PZR)" percent protein retained (PPR)2, speciﬁc
activity of alkaline phosphatase (ALP) and carboxypeptidase B (CPB) and
plasma insulin concentrations of rainbow trout3 fed experimental diets‘
containing untreated or dephytinized ingredients and/or supplemental Zn.
Values :1: SE. ............................................ 68

Table 9.

Table 10.

Table 11.

Table 12.

Results of nonorthogonal contrasts of treatmert means for whole ﬁsh
protein, Zn and P, bone Zn, P and ash, percent Zn retained (PZR), total
bone Zn, percent protein retained (PPR), the speciﬁc activity of alkaline
phosphatase (ALP) and carboxypeptidase B (CPB) and the concentration
of plasma insulin. Signiﬁcant ”contrasts (P < 0.05) were marked ‘X’. . . 70

Regression relationship between log whole ﬁsh N (g) and log wet ﬁsh
weight (g) of selected ﬁsh species. Values :l: SE. ................ 104

Sources ofwholeﬁshNandﬁshwetweightdata, samplesizes(n)andﬁsh
wet weight rangesusedinthestudyofwholeﬁsthontent ........ 105

Regression relationships between whole ﬁsh P (mg) and wet ﬁsh weight (g)
for salmon and trout. Values :l: SE ........................... 107

viii

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

LIST OF FIGURES

Conceptual drawing of water and emuent ﬂow to and ﬁ'om the Platte River
Anadromous State Fish Hatchery .............................. 6

Monthly emuent P loading and fecal P production estimates for the Platte
River Anadromous State Fish Hatchery for the period January 1, 1993
through May 31, 1994 ..................................... 36

RelationshipbetweenPfedandPretainedbycohoandchinook salmonat
the Platte River Anadromous State Fish Hatchery for the period Jamlary l,
1993 through May 31, 1994 ................................. 37

Relationship between percent dietary Zn retained (PZR) and total Zn fed to
rainbow trout receiving diets containing untreated or phytase-treated
ingredients with or without supplemental Zn ..................... 72

Relationship between the concentration of Zn in rainbow trout bone and
the dietary phytic acid:Zn molar ratio .......................... 73

Relationship between bone Zn concentration and the dietary phytic acid:Zn
molar ratio for channel catﬁsh and rainbow trout ................. 78

Relationship between whole ﬁsh P and ﬁsh weight for trout and salmon. 108

INTRODUCTION

Aquaculture facilities have had a signiﬁcant impact on the“ quality and productivity
of their receiving waters by increasing downstream nutrient concentrations (EIFAC 1982;
Iwama 1991; Pillay 1992). Phosphorus (P) and nitrogen (N) from aquaculture wastes
havebeenofspecial concernbecausetheyareusuallytheﬁrstlimitingnutrientsin
freshwater systems, and are therefore potential eutrophicants (Schindler 1977). The
addition of P and N to oligotrophic (nutrient-poor) waters may be desirable in some cases
(Johnston et al. 1990). In the Great Lakes region, where many lakes and streams are
mesotrophic or eutrophic (nutrient-rich), and the addition of nutrients ﬁorn aquaculture
would be potentially damaging. Since the passage of the Federal Water Pollution Control
Act of 19721 and the creation of the National Pollutant Discharge Elimination System2
(NPDES), nutrient inputs ﬁom point sources like aquaculture have been regulated.

In 1988, the Michigan Department of Natural Resources (MDNR) was ordered by
the Ingharn County Circuit Court to reduce P discharged from the Platte River
Anadromous State Fish Hatchery (“Platte Hatchery”, Honor, Michigan) to
421 kg P-year", which was lower than NPDES limits. This action resulted from a lawsuit
brought against the MDNR by the Platte Lake Association. The Platte Lake Association
alleged that P discharged from the Platte Hatchery, located approximately 14 km upstream

ﬁ'om Platte Lake, caused decreases in summer water clarity of Platte Lake. Due to the

 

‘33 U.S.Code §§ 1251 et seq., called the Clean Water Act

US. Public Law 92-500

2
costsoflitigation, theMDNRiscontemplatingtheclosure ofthePlatteHatcheryiffunds
for renovating the hatchery’s emuent management system are not approved by the
Michigan Legislature (Kelley Smith, MDNR, personal communication).

The Platte Hatchery case and others (Axler et al. 1996) have made it clear that
public perception about the environmental impacts of aquaculture may be as important as
sound ﬁsh husbandry methods for the ultimate success of this ‘new’ industry. Finding
ways to minimize the environmental impacts of aquaculture demonstrates not only good
environmental stewardship, but ideally will secure the industry against ﬁrture criticism.

P losses from most hatcheries have been estimated from the diﬁ’erence in the P
concentration of water collected periodically above and below the hatchery multiplied by
thewaterﬂow rate. This chemical methodhasnot accuratelyestimatedtheamountofP
released by most hatcheries evaluated (Cho et al. 1991). Many factors, such as cleaning
schedules, P cycling by algae and bacteria, and changes in husbandry practices aﬁ‘ected
concentrations of eﬁluent P on a daily or even hourly basis. Consequentially, periodic
water analysis has not adequately represented the P dynamics of hatcheries.

Fish feed has been identiﬁed as the primary source of hatchery-added nutrients in
eﬂluents (W esters 1991). A nutritional mass balance model, which partitions the fate of
dietary P into ﬁsh body accretion, recovered wastes, and unrecovered wastes, Should
produce accurate P loading estimates (Cho et al. 1991). Such a model could aid planners
in locating sites for ﬁrture aquaculture facilities based on projected nutrient loadings into
the receiving waters. A nutritional mass balance model should also help hatchery

managers plan annual ﬁsh production without compromising P discharge limits.

3
Maximum possible ﬁsh production can be calculated based on feed P levels, expected P
retention by ﬁsh, and expected P recovered ﬁ'om raceway solids. Finally, the mass balance
approach could be used to evaluate the eﬁciency of P removal ﬁ'om culture systems.

Nutrient loading by hatcheries has been reduced by improving the digestibility of
feed ingredients (Cain and Garling 1995; Rodehutscord and Pfeﬁ‘er 1995). Plant feed
ingredientsalchassoybeanmealhavebeenimportant protein sourcesinﬁshfeeds(NRC
1993). However, soybeans and other seeds contain phytate, a P storage molecule.
Phytate is largely indigestible by ﬁsh because they do not produce phytase, the enzyme
which hydrolyzes phytate. Reduced utilization of dietary P, Zn and protein have been
observed in ﬁsh fed puriﬁed diets supplemented with phytic acid (Spinelli et al. 1983;
Richardson et al. 1985; Gatlin and Phillips 1989), and in practical diets containing native
phytate (Cain and Gatling 1995; Schafer et al. 1995). How phytate reduces protein and
mineral utilization in ﬁsh has not been fully described.

The goal of this research3 was to measure and reduce aquaculture wastes using
nutritional strategies. The text is divided into two chapters with the following objectives:
Chapter 1:

1. Develop a nutritional mass balance model to estimate P retention and losses for
coho and chinook salmon ﬁom ﬁrst-feeding to stocking size.
2. Use the mass balance approach to evaluate the eﬁciency of solids P removal

eﬁ‘orts at the Platte Hatchery.

 

3

Useofﬁshintheresearchdescribedinthis ' 'onwasapprovedbytheAll-UniversityCommitteeon
Animal Use and Care, AUF ii 11/93-420-03.

Chapter 2:

1. Determine rainbow trout fed a plant-based diet high in phytate become Zn deﬁcient
based on growth, protein utilization, plasma insulin concentration, the activity of
carboxypeptidase B and intestinal alkaline phosphatase, and whole ﬁsh and bone
Zn, P or ash content.

2. Determine if dephytinized plant ingredients or a dietary Zn supplement is more
eﬁ‘ective in providing Zn to ﬁsh.

A collateral study was also conducted to determine if whole ﬁsh P and whole ﬁsh

N could be predicted from ﬁsh weight when direct measurements are not possrble. Details

of the collateral study are in Appendix C.

CHAPTER 1
Estimation of Phosphorus Loading ﬁ'om the

Platte River Anadromous State Fish Hatchery

INTRODUCTION

A nutritional P mass balance model was constructed for coho salmon
(Oncorhynchus kisutch) and chinook salmon (0. ashauytrcha) at the Platte River
Anadromous State Fish Hatchery (“Platte Hatchery”, Honor, Michigan) to estimate the
amount ofP discharged from the hatchery into the stabilization pond (Figure 1). Samples
of whole coho and chinook salmon, feeds, and dissected feces were collected monthly
ﬁom May 1993 through April 1994 ﬁom the Platte Hatchery for P analysis. Daily ﬁsh ‘
production, water temperature, feeding, mortality, and monthly removed raceway solids
records for January 1993 through May 1994 were furnished by the hatchery. The
experimental period of January 1993 through May 1994 was chosen so that an entire year-
class of coho salmon could be included. Five individual stocks of ﬁsh were present in the
hatchery during that period, and all were sampled. Monthly P loading estimates based on

water samples collected at the Platte Hatchery’s stabilization pond outlet were furnished

mew—am ease—Sum

 

 

 

Sees: em saw escapees, use as: as
me :5 3e: 2.380 28 85 26: 833 we wage—c EBA—3.80 A oEwE

   

, 83 95952.. 26:50 v93: .8 .835 *
egos coeﬁﬁsa 2.. ease Seas: ®

  

 

 

. 2.. see 323% .eoeao 22? 2a

         
        

Be: 26:50 we :38th A. I I

>5: .833 we 558:9 All
Samoa .

mung .53.:

 

 

 

 

 

 

 

 

 

£3502 coo—3.5

 

 

 

 

 

 

 

 

' 83M 83m

 

 

7

bytheMDNR Maximumand minimumpossibleamountsofP discharged ﬁomthePlatte
Hatchery into the settling basin were calwlated using nutritional mass balance equations.
Live coho and chinook salmon ﬁ'om the Platte Hatchery were brought to the
Michigan State University Fisheries Research Laboratory (MSU) each month for use in
supportingexperimentsto 1) quantifythepercentageoffeedPleavingtherearingunitin
dissolved form, 2) measure fecal P production, and 3) determine if fecal and whole ﬁsh P
values for ﬁsh raised under controlled laboratory conditions were comparable with values
ﬁ'om the Platte Hatchery.
Objectives
1. Develop a nutritional mass balance model to estimate P retention and losses for
coho and chinook salmon from ﬁrst-feeding to stocking size.
2. Use the mass balance approach to evaluate the eﬁciency of solids P removal

eﬂ‘orts at the Platte Hatchery.

LITERATURE REVIEW

1. Wm

Phosphorus has been identiﬁed as the ﬁrst limiting factor in primary productivity in
most ﬁ'eshwater systems (W etzel 1983). Additions of P into freshwater environments
have caused increased productivity and eutrophication (Schindler 1977). Large P inputs
have resulted in a shift of the ﬁrst limiting nutrient ﬁom P to N, carbon, oxygen or light.
Phosphorus loading which decreases the N:P ratio below 10 has precipitated shifts in the
dominant phytoplankton species, favoring nitrogen-ﬁxing bloom-forming bluegreen algae
(Schindler 1977). Blue green algae, or cyanobacteria, have adversely aﬁ‘ected freshwater
productivity by covering the water surface and severely reducing light penetration. The
decay of large algal blooms have in turn reduced dissolved oxygen to concentrations that
are suboptimal or lethal to ﬁsh and other aquatic organisms.

Freshwater primary productivity has been positively correlated with total P
concentration. In a review of experimental lake enrichment studies, Elser et al. (1990)
found that P and P+N enrichment of North American lakes enhanced algal growth
Because primary productivity has been positively correlated with ﬁsh production (Oglesby
1977; Jones and Hoyer 1982), the P enrichment of a lake or river has had a profound

eﬁ‘ect on ﬁsh biomass by expanding the food base. Yurk and Ney (1989) found that the P

9
concentration in 22 southern Appalachian reservoirs could predict 75% of the variation (P
< 0.001) in ﬁsh standing stock In one Virginian reservoir which had been subject to a P
reduction program, total P concentration and ﬁsh biomass decreased together over an 11-
year period, and total P concentration explained 66% of the variation in ﬁsh standing
stock Downing et al. (1990), in a review of lake studies worldwide, found that ﬁsh
production was closely correlated with annual phytoplankton production (r2 = 0.79, P <
0.001), mean total P concentration (r2 = 0.67, P < 0.002) and annual average ﬁsh standing
stock (r2 = 0.67, P < 0.001).

Numerous studies have indicated that ﬁsh hatchery emuents may signiﬁcantly alter
the nutrient status and productivity of receiving waters (Hinshaw 1973; EIFAC 1982;
Bergheim et al. 1984;.Merican and Phillips 1985; Wiesrnann et al. 1988; Kendra 1991).
Szluha (1974) found periphyton production below the Jordan Valley National Fish
Hatchery, Michigan, to be 5 to 7 times greater than above the hatchery. Eloranta and
Palomaeki (1986) found signiﬁcant increases in lake phytoplankton biomass, chlorophyll 0
concentrations, rate of primary productivity, and changes in phytoplankton community
structure as a result of nutrient loading ﬁom a Finnish ﬁsh farm.

The eﬂ‘ects of P loading have been site-speciﬁc and depend on the concentration
and consistency of P inputs, the chemical form of P released, the presence of other
nutrients in the eﬂluent, and the prior nutrient status of the receiving water.

Consequently, P loading may not be detrimental in certain locations. Non-productive
(nutrient-poor) rivers have been deliberately fertilized with N and/or P to increase ﬁsh

production (Johnston et al. 1990). Oberdorﬁ‘ and Porcher (1994) found an increase in the

10
densityandbiomassofmostﬁshspeeiesdownstreamﬁomﬁ'outfarmsinance.

However, sensitive species were often replaced by pollution-tolerant and exotic species.

 

Westers (1991) identiﬁed feed in the form offeces and uneaten feed as the primary
source of P in hatchery emuents (Westers 1991). Reductions in emuent P have been
achieved by feeding experimental diets providing dietary P at near-requirement levels and
in highly digestible forms (Ketola 1985; Ketola et al.1991; Ketola and Richmond 1994;
Cain and Garling 1995). However, minimizing eﬁluent P through feed formulation has
beendiﬁcultinpractice. Substantial quantitiesofnativePoccurinkeyfeedingredients
likesoybeanmeal, corn glutenmeal andﬁshmeal (NRC 1993). Much ofthisP ispresent
in forms that are poorly digested by ﬁsh (Lovell 1978; Satoh et al. 1987). Riche and
Brown (1996) found that P in ﬁsh meal, primarily ﬁ‘om bone, was 21-55% digestible by
rainbow trout. Ogino et al. (1979) observed ﬁsh meal P was even less digestible in ﬁsh
such as the common carp which lack acid digestion. Although most ﬁsh have been shown
to require 5-8 g P-kg'l dietary dry matter (NRC 1993), commercial ﬁsh feeds often contain
10.5-14 g P-kg" dietary dry matter to compensate for poor P digestibility. Low P
commercial feeds (approximately 7 g P-kg“) based on deboned ﬁsh meal are currently
available (i.e., Bioproducts, Warrenton, Oregon). However, the expense of the deboning
process has increased feed costs substantially.

Commercial ﬁsh feeds have traditionally contained ﬁsh meal as the primary protein

source (250-500 g kg"). However, the high cost of ﬁsh meal has prompted an increased

11
useofplantproteinsourcessuchassoybeanmealandcornglutenmeal. Thesefeed

ingredients contain phytate, a P storage molecule which has been shown to be poorly
digested by ﬁsh (Cain and Garling 1995; Richie and Brown 1996). Fifty to 70% of
soybean meal P was bound in phytate (Lolas et al. 1976; Lott 1984). The stability of
phytatewasnot eﬁ‘ectedbyfeedstuﬂ‘processingtechniques suchasdefattingandheating
(Davies and Reid 1979; Clydesdale and Camire 1983).

Phytate can be dephosphorylated by the enzyme phytase (mm-inositol
hexaphosphate phosphohydrolase, EC 3.1.3.8). However, monogastlic ﬁsh like rainbow
trout do not produce intestinal phytase. Additions of P supplements have been required in
ﬁsh feeds which contained untreated soybean meal or other plant md products as major
protein sources (Ketola 1975; Lovell 1978; Cain and Gatling 1995). The bioavailability of
phytate-P in plant feedstuﬁ's has been greatly improved for poultry and swine by pre-
treating the feedstuﬂ‘with fungal phytase (Nelson et al. 1971; Lei et al. 1993b; Qian et al.
1997). Cain and Garling (1995) found that dietary P supplements could be eliminated
from plant-based rainbow trout feeds when soybean meal was pre-treated with phytase.

The addition of microbial phytase or phytase-containing transgenic seeds directly
to the feed has also improved the bioavailability of dietary P for swine (Lei et al. 1993b,c;
Young et al. 1993) and poultry (Nelson et al. 1971; Pen et al. 1993; Qian et al. 1997).
Little or no inorganic P supplementation was required in the phytase-added diets.
Phosphorus availability has also been improved for common carp and rainbow trout fed
diets supplemented with phytase (Rodehutscord and Pfeﬂ‘er 1995; Schafer et al. 1995;

Riche and Brown 1996). In the experiments with ﬁsh, most dephosphorylation of phytate

12
by phytase probably occurred during diet preparation. However, Rodehutscord and

Pfeﬂ'er (1995) found that P utilization (P deposition-P intake" x 100) in phytase-added
dietswasinﬂuencedbyﬁshrearingtemperature. Sincephytaseactivityhasbeenshownto
be temperature dependent (Han et al. 1987), Rodehutscord and Pfeﬁ‘er (1995) concluded
thatsomephytaseactivitymayhaveoccurredintheﬁshgut.

Adding phytase directly to ﬁsh feeds may be beneﬁcial if a stable form of low cost
phytase becomes available. However, pre-treating feedstuﬁ‘s with phytase or preparing
feeds in a way that promoted phytase activity during feed processing would avoid
inconsistent results due to loss of enzyme activity during feed storage or as a result of low
ﬁsh rearing temperatures (lo-15°C).

B. Selidmanasement

Since P is rapidly leached from feed and feces left in water for periods as briefas
5-60 min (“frndell et al. 1978; Smith et al. 1980; Brown et al. 1989; Phillips et al. 1993),
Westers (1991) recommended careful handling of settleable solids-to reduce P in hatchery
eﬂuents. Clark et al. (1985) observed that solids were ﬁ'agile and easily broken into
smaller particles by husbandry practices and ﬁsh movement. Nutrient leaching should be
higher in smaller particles because they have greater surface-to-volume ratios (W esters
1991). Boerson and Westers (1986) designed a baﬂe system to continually move solids
down the raceway to a screened-OE settling area where ﬁsh movement would not
resuspend or break the settling waste. Merican and Phillips (1985) and Asgard et al.
(1991) emphasized that the most eﬁcient method of reducing solids in raceways was to

minimized feed inputs.

13
Pellet binders could improve the eﬁciency of solids removal by increasing the

stability of feces in water (Storebakken 1985; Storebakken and Austreng 1987; Hardy
1989). Westers (1991) reported that intact ﬁsh feces had a rapid settling velocity and
could be eﬁciently removed from raceways, whereas fragmented fecal matter had a
reduced settling velocity and was diﬁcult to collect.

C. v ' v ﬂl

Even well balanced feeds fed appropriately will result in the loss of some P in
dissolved form. Eﬁ‘orts to recapture dissolved P ﬁ'om hatchery eﬁluents have included the
production of economically important species such as the agar-producing algae Gracilaria
spp. in raceway emuents (Buschmann et al. 1994). Mussel and oyster culture conducted
in conjunction with ﬁnﬁsh culture have also beneﬁted ﬁ'om the dissolved nutrients (Folk
and Kautsky 1989; Shpigel et al. 1993). The production and removal of polyphosphate-
accumulating bacteria in‘eﬁluents has been eﬁ‘ective in removing dissolved P, although
such a method may be cost-prohibitive (Kortstee et al. 1994).

In Michigan, some efﬂuent P could be reclaimed by diverting hatchery eﬁluents to
baitﬁsh ponds or hydroponics facilities. However, the perturbation of pond sediments by
baitﬁsh could resuspend particulate P and actually increase P losses. And, the N and P
concentrations in hatchery eﬁluents were too dilute for vegetable production (Rakocy and
Hargreaves 1993). Formal research on the value of secondary production for waste
reduction has been lacking for the northern temperate region.

Constructed wetlands have been used as ‘biological sponges’ for the treatment of

industrial, agricultural and municipal waste (Bavor and Mitchell 1994). Recent research

14

has indicated that constructed wetlands may be useful in treating eﬁluents released
periodically from aquaculture ponds (Schwartz and Boyd 1995). Before constructed
wetlands can be recommended as a treatment for eﬁluents ﬁom ﬂow-through raceway
systems, the issues of large solids loads, high ﬂow rates, large land requirements and
wetland capacity to reduce P must be addressed.
111. MW

Cho et al. (1991) suggested that a dietary mass balance model could be used to
calculate P lost from ﬁsh hatcheries. Mass balance studies have been useful for back-
calculating dietary nutrient concentrations of deer (I-Iowery and Pﬁster 1990), moose
(Leslie et al. 1989), and domestic livestock (Belonje and Van den Berg 1980; Holechek et
al. 1982, 1985). Massbalance models have been used to estimate P and N cycling by ﬁsh
in lakes (Kraft 1992), P turnover from sediments beneath salmon rearing pens (Holby and
Hall 1991) and changes in emuent P resulting ﬁom the dephytinization of dietary soybean
meal (Cain and Garling 1995).

The quantity of P in hatchery eﬁluents can be described by:

Pd=Ph+Ph-Ph (1)

where:

P,f = P in hatchery eﬂluent

P. = P in incoming water

Pb = P of hatchery origin (from dissolved excreta, leachate ﬁ'om feces and

uneaten feed)

P... = P absorbed ﬁom water by ﬁsh

15

Since absorption of P ﬁom water was negligible by ﬁsh at the low P concentrations
of 10-20 ugL" generally present in incoming water (Solomatina and Arsan 1980; Lall
1991), P... may be omitted ﬁ'om the equations under ordinary situations. Therefore P.
should only change as a result of Pb.

Two models used to estimate P. have been the chemical model and the nutritional
model (Cho et al. 1991). In the chemical model, P concentrations were measured in water
samples, and P. was estimated as:

CP,.=(P,.-P.)XW (2)
where:

CP. = P in eﬂluent of hatchery origin estimated using the chemical model

P... = P in hatchery outﬂow

P. = P in hatchery incoming water

W = Water ﬂow rate

TheP content ofﬁsh, feed and fecal matterwereused to estimatePbinthe
nutritional model:

NPho=Pfed'[Pf+(Prn'Pli)+Pm] (3)
where:

NP. = P in efﬂuent of hatchery origin estimated using the nutritional model

PM = P in feed fed

Pf = P in feces and uneaten feed (solids)

P. =Pinﬁshattheendofthegrowthperiod

16

P. =Pinﬁshatthebeginningofthegrowthperiod

P. = P in dead ﬁsh
Equation3 assrrrnesthatﬁsh do not consumefecalmatteroranynon-feed item. Based on
personal observations, this assumption is reasonable for trout or salmon reared in cement
raceways.

Although the chemical method has been the accepted method for determining
hatchery compliance with NPDES regulations in Michigan, it may result in erroneous
estimates ofhatchery P losses. Krom and Neori (1989) and Cho et al. (1991) found the
chemical method to overestimate P loading by 1.1 and 1.8 times, respectively, as
compared to the nutritional method. Krom and Neori (1989) sampled water twice daily
for the duration of their month-long experiment, and Cho et a1. (1991) combined hourly
samples over a weeks time for weekly average P concentrations. These sampling
ﬁequencies were inadequate since many factors like cleaning schedules, uptake by
periphyton in the raceway, or changes in feeds or rearing practices may have aﬁ‘ected day-
to-day P concentrations in eﬁluent water (Bergheirn et al. 1991; Foy and Rosell 1991a).
Since feces left in water have been shown to lose a signiﬁcant proportion of their P
content within 5 min of excretion, even hourly samples could have missed P loading peaks
(Phillips et al. 1993).

In contrast to the conclusions of Cho et al. (1991) and Krom and Neori (1989)
that the chemical model did not accurately measure P losses, Foy and Rosell (1991a)
found close agreement between the two models in‘a year-long survey of efﬂuents at a

Northern Ireland hatchery. The nutritional model they used was:

l7
P..=(FCR x rig-P. (4)

where:

F CR = Feed conversion ratio (kg food fed-kg‘l ﬁsh produced)

P. = Feed P concentration

P.II = P content ofﬁsh (ﬁsh growth x P concentration in 500 g ﬁsh)
To estimate emuent P using the chemical model (Equation 2), they sampled inﬂow and
outﬂow hourly for 24 h about every 5th day.

When calculating P. according to Equation 4, Foy and Rosell (1991a) used the
F CR as an estimate of digestibility, thereby avoiding the need for feces collection.
However, their FCR of 1.8 indicated overfeeding and feed wastage, and they noted that a
FCR of 1.5 or less would have been closer to expectations. They also assumed that the
percent whole body P was the same for 3-500 g trout. Based on data from Shearer
(1984), that assumption was not valid. This error could have resulted in an overestimate

of P. by underestimating the P content of ﬁsh at the beginning of the experiment.

MATERIALS AND METHODS

Fish

Adult coho and chinook salmon were captured between September and December
each year as they returned to Michigan rivers to spawn. Eggs were also collected ﬁom
spawners returning to New York rivers because New York populations were thought to
be ﬁee of bacterial kidney disease which had devastated Michigan populations. Egg
collection data and the condition of return spawners may be found in Pecor (1992). Eggs
were stripped, artiﬁcially fertilized then incubated in Heath trays at the Platte Hatchery
(W esters 1979). Newly hatched coho and chinook salmon (ﬁ'y) were introduced into
indoor 38.2 m3 cement raceways from late December through early February (Table 1).
Chinook salmon remained in the indoor raceways until release the following May or June.
About 9 months after hatching, coho salmon were transferred to outdoor 56.6 m3 cement
raceways where they remained until release in their second spring (May or June),
approximately 17 months after hatching.

Coho and chinook salmon were never placed in the same raceways. Michigan and
New York ﬁsh were kept in separate raceways for 2-12 months, then mixed. Until ﬁsh

were mixed, separate growth curves were developed for Michigan and New York ﬁsh.

18

l9
Averageﬁshweigluwascalallatedmonthlybyhatcherypersonnelusingaﬁsh
growth model based on water temperature history. The model was designed speciﬁcally
for Platte Hatchery echo and chinook salmon (Roger Hubble, MDNR, personal

communication).

Table 1. Starting populations of echo and chinook salmon ﬂy at the Platte River
Anadromous State Fish Hatchery beginning each January. Parental stocks originated ﬁ'om
Lake Michigan (MI) and Lake Huron (NY). Fish stock designations (he, Coho ’91)
indicate the year eggs were fertilized.

 

Number of ﬁy introduced to raceways

 

 

 

Fish stock year introduced MI NY Total

Coho ’91 1992 3,053,268 588,000 3,641,268

Coho ’92 1993 1,936,761 327,600 2,264,361

Coho ’93 1994 2,751,533 351,600 3,103,133

Chinook ’92 1993 ‘ 3,811,961 1,702,484 5,514,445

Chinook ’93 1994 5,087,976 692,640 5,780,616
Platte Hatchery rearing conditions

Fish were reared without supplemental lighting. Natural light intensity was
reduced in the hatchery building (where indoor raceways were located) by green ﬁberglass
windows. Outdoor raceways were subject to full sun.

Three water sources were used by the Platte Hatchery: Brundage Spring,
Brundage Creek, and the Platte River. Eggs were incubated in water ﬁom Brundage

Spring. Mixtures of water from all three sources were used to rear ﬁsh. The proportion

20
of the total ﬂow coming ﬁom each water source was determined by vohlme, temperature
and dissolved oxygen requirements and water clarity. Water ﬂow rates provided 1-4
exchanges-hl depending on ﬁsh density and size. Water temperature ﬂuctuated seasonally
between 6 and 13° C. Standard rearing techniques were employed (Westers 1979).

Platte Hatchery feeds and feeding

All feeds used during the 17 month experimental period were obtained ﬁ'om
Bioproducts, Inc. (W arrenton, Oregon) except for smaller quantities of feed ﬁ'om Zeigler
Bros, Inc. (Gardners, Pennsylvania) used January through March, 1993. Bioproducts,
Inc. feeds were formulated ﬁ'om Kodiak white ﬁsh meal‘, feather meal, cereal tailings
(cooked oats, wheat, barley or rice), spray dried blood meal, herring meal, processed
deboned ﬁsh, vitamin and mineral supplements and preservatives. The P content of the
feeds varied ﬁ'om 6.7-14.8 g P-kg" (as fed basis). Most feeds were “low P”, containing
10 g P-kg‘1 or less. During periods when Kodiak white ﬁsh meal was unavailable to the
manufacturer (usually December through March), feeds contained 10 g P-kg" or more
(Dennis Rolie, Bioproducts, Inc., personal communication).

Most feed lots received by the Platte Hatchery were analyzed for total P by
Lancaster Laboratories, Inc., Lancaster, Pennsylvania (Table 2). However, few of the
medicated feeds were sent to Lancaster Laboratories, Inc for analysis. Feed samples were

digested using nitric and perchloric acids (AOAC 1984) and P was determined using the

 

4
KodiakwﬁkﬁshmlisalowPﬁshprmdnmmmmadebypresdngyumdmwﬁshmﬁw

pouack)mrwghammruwvebom,ﬂdnandsmlsmmnisRoﬁeBiopm1m.,pamnﬂ
communication).

21

Table 2. Moisture and P content of feeds‘ fed to coho and chinook salmon at the
Platte River Anadromous State Fish Hatchery between January 1, 1993 and May 31, 1994

 

 

Feed Designation Moisture P (g-kg" diet
(% as fed)2 as fed)
Bio Diet Starter # 3 20.5 12.0-13.0
Bio Diet Grower 1 mm 20.5 11.0
Bio Dry 1 mm 11.0 11.0
Bio Dry 1.3 mm 11.0 8.0-10.0
Bio Dry 1.5 mm 11.0 8.4-10.0
Bio Dry 2 mm 11.0 8.5-9.2
Bio Dry 3 mm 11.0 7.6-8.7
Bio Dry 1000 LP 1 mm 11.0 8.6-9.2
Bio Moist 1.3 mm 26.0 9.5
Bio Moist 3 mm 26.0 6.7-6.9
Bio Moist + Gallimycin , 1.3 mm 26.0 14.6
Bio Moist + Gallimycin 1.5 mm 26.0 7.5
Bio Moist + Gallimycin 3 mm 26.0 14.8
Bio Moist + Terrimycin 1.5 mm 26.0 07.5
NY 3/32 unknown 12.0
NY 1/8 unknown 12.0

 

‘ All feeds manufactured by Bioproducts, Inc., Warrenton, Oregon except “NY”
diets, which were manufactured by Zeigler Bros, Gardners, Pennsylvania.
2 Based on information provided by the manufacturer.

22
ascorbic acid-molybdenum method (APHA et al. 1985). Feed P concentrations were

conﬁrmed for several feeds at MSU using the method of Gomori~(l942) after digestion in
nitric and perchloric acids (AOAC 1984). Up to eight diﬁ‘erent feeds were in use during
any given month at the hatchery. The use ofmultiple feeds was not communicated to
MSU until after the experiment was concluded. Therefore, the P concentration of several
diets was not conﬁrmed at MSU, but was estimated based on information provided by the
manufacturer (Dennis Rolie, Bioproducts, Inc., personal communication). Sample
digestion and P determination protocols used at MSU are detailed in Appendices A and B,
respectively.

Feeds were stored at approximately 7°C and were generally fed within six months
of receipt. However, feeds for ﬁy and medicated feeds were occasionally fed one year
aﬁer receipt. Individual lots of feed were fed in the order in which they were received.

Feed was oﬁ‘ered at 2% wet body weight-d‘l (as fed basis) to all size classes of
salmon. A portion of the daily ration was distributed every 30 min during the 8 h workday
by an automated pneumatic system. Feed was distributed over about 80% of the raceway
surface. Feed was withheld on days when heavy rain and silt reduced feed visibility to the
ﬁsh. In winter, when low water temperatures resulted in decreased feeding activity, feeds
were oﬁ’ered every second or third day. Daily feed distribution records were maintained

by Platte Hatchery personnel.

23
Platte Hatchery solids maragenrent

Indoor raceways were outﬁtted with a screen and baﬁe system which allowed-
solidstoaccumulateattheendofeachracewayﬁoasonandWesters1986). Anew
vacuum system removed‘water and solids ﬁom the end of indoor raceways every 30 min.
Thevacuumed slurrywasﬁlteredwitha40umscreenandtheresulting sludgerernoved
ﬁom the hatchery system Filtrate was discharged into the hatchery stabilization pond.
Platte Hatchery personnel collected random samples of sludge for P analysis.

Raceways were scrubbed and siphoned once daily when the vacuum system was not in
operation and in raceways not equipped with the system. Siphoned water and solids were
discharged directly into the stabilization pond. Outdoor raceways were scrubbed and

siphoned about every 3 d.

Platte Hatchery ﬁsh and feces sampling procedures

Monthly, 10 coho salmon and 10 chinook salmon were randomly collected ﬁ'om
raceways with a net. Tricaine methane sulfonate (MS-222) was used at 15-30 mg-L'l for
sedation prior to weighing, and at 100-200 mg-L“ (lethal dose) prior to dissection. Feces
were collected by dissection (Brown 1993) for Pf calculations. An incision was made
along the ﬁsh’s left side with a scissors. The intestine was ligated with forceps at the
transition point between the anterior and posterior intestine to prevent movement of
material between the intestinal segments. The posterior intestine was cut away ﬁ'om the
anus, and fecal matter was gently squeezed from the posterior intestine into a pre-weighed

aluminum dish. The emptied intestinal segment was returned to the ﬁsh, and the carcass

24
was ﬂown until processed. The weight of fresh fecal samples was determined by

subtracting the initial weight of the pan ﬁom the weight of the pan containing the sample.
Fecal samples were dried at 105°C for 24 h in a forced air oven. After cooling in a
desiccator, total fecal dry matter was determined by subtracting the initial weight of the
pan from the weight of the pan containing the dry fecal matter. Dried samples were stored
in a desiccator until digestion in nitric and perchloric acids (AOAC 1984). The P
concentration was determined according to APHA et al. (1985).

Frozen whole ﬁsh were sliced into l-cm sections and lyophilized 72 h, dried at
60°C for 12 h in a vacuum oven and cooled in a desiccator. Whole ﬁsh dry matter was
determine by the same method as fecal matter. Dried ﬁsh were ground in liquid nitrogen
using a stainless Steel Waring blender (Waring, New Hartford, Connecticut) , then frozen

in plastic bags until digestion in nitric and perchloric acids (AOAC 1984).

Platte Hatchery water sampling procedures

Water which passed through the hatchery enters a stabilization pond before being
discharged into the Platte River (Figure 1). Platte Hatchery personnel collected composite
24 h water samples beginning every Monday and Thursday morning at the Brundage
Creek water intake site (representing P in water entering the hatchery) and at the
stabilization pond outlets (representing P leaving the stabilization pond). The sample
collection device was a 10 L polypropylene bottle with an air release outlet and water
intake hole in the lid. The rate of water intake was regulated by a 30 gauge needle

attached to the end of the water intake hole.

25

A 250 mL subsample of each 24 h water sample was reﬁigerated in a Teﬂon
topped glass bottle containing 3 mL sulfuric acid. Water samples were analyzed for P
weekly by Great Lakes Environmental Laboratories (Traverse City, Michigan). Unﬁltered
8 mL samples from the glass bottles were digested using the sulﬁuic acid-ammonium
persulfate method (APHA et al. 1985). Total P was quantiﬁed with a Technicon
autoanalyzer using the ascorbic acid-molybdenum method (APHA et al. 1985). The
minimum detection limit was 3 rig-L". A standard curve was made by diluting an external
Environmental Protection Agency standard to 5-60 ugL“. Total P values were used by

the MDNR to calculate monthly discharges of P ﬁ'om the stabilization pond.

Rearing conditions at MSU

Triplicate groups of coho salmon and/or chinook salmon were reared in 41 L or
125 L rectangular plastic tanks. Tanks were supplied with aerated well water at 12 :1: 1°C.
Supplemental light was provided by overhead ﬂuorescent lights on automatic timers
following the natural lightzdark photoperiod. Light intensity was lowered by covering the
light ﬁxtures with 1 mil brown plastic. New ﬁsh were obtained ﬁom the Platte Hatchery
every 4-8 weeks. After an initial 7-10 d acclimation period, ﬁsh were randomly distributed
to tanks and the weight of ﬁsh in each tank was determined. Initial ﬁsh loading rates were
0.5-2 kg-(L per min)“1 (Westers 1979). Each batch of ﬁsh was reared for 4 weeks.
Weekly removal of ﬁsh for fecal collection maintained loading and density at

approximately initial levels.

26
Fish were fed 2% wet body weightd" on a dry matter basis. Feed dry matter was

determinedbythesamemethodasfecaldrymatter. Thedailyrationwasdividedintotwo
feedingsatleast4hapart. Feedwasfedbyhandatarateofl-Zpellets-s". Atthis
feedingrateallfeed should havebeenconsumed (Westers 1987). Fishandfecal samples

were collected weekly using the Platte Hatchery procedures.

Eﬂluent P study at MSU

To measure the proportion of feed P leaving the rearing unit in dissolved form,
emuent water samples were collected ﬁom three 41 L tanks containing 17 coho salmon
each. Average ﬁsh weight (:i: SD) was 6.0 i 0.4 g and total tank weight was 102 :l: 5 g.
Water ﬂow rate was 2.5 L-min". Fish were fed 1.3 mm Bio Dry 1000 which contained 9 g
Pkg“ on a dry matter basis (Bioproducts, Inc.). The eﬂluent experiment began 48 h
before the end of a 28 d growth period. Sampling of eﬂluent water began I h before the
ﬁrst feeding and continued at 30 min intervals for 24 h. Each 50 mL water sample was
stored at 4°C in a polypropylene bottle containing 1 mL concentrated HCl. Water
samples were digested within 24 h of collection using the sulfuric acid-armnonium
persulfate method (APHA et al. 1985). P concentrations were determined according to

APHA et al. (1985). The minimum detection limit was 10 pg P°L'l

Feces production and P content
Acid insoluble ash was measured as an internal marker to calculate feed P

digestibility and fecal P production. Acid insoluble ash was isolated according to Atkinson

27
et al. (1984). Feed and feces were dried for 24 h at 105°C, then ashed for 16 h at 600°C.

Samples were cooled in a desiccator, thenweighed to determine percent ash. Ash was
boiled in 100 mL 2M HCl for 5 min, ﬁltered hot through Whatrnann No.42 ashless ﬁlter
paper, and the cake washed with boiling deionized distilled water. Filter papers were then
dried and ashed for 16 h at 600°C. The residue was considered acid insoluble ash.

Aliteraturesearchwasconductedtoﬁndestimatesofdietarydrymatter
digestibility for juvenile salmonids fed feeds similar to those used at the Platte Hatchery.
Dietary dry matter digestibilities reported by Kim and Kaushik (1992), Shearer et al.
(1992), Gomes et al. (1993) and Oliva-Teles et al. (1994) were averaged, then subtracted
ﬁ'om100togiveanestimateofthepercentofdietarydrymatternotretainedbytheﬁsh.
Monthly Pf values were then calculated as:

(kg dry matter fed) x (loo-dry matter digestibility)-(100)’l x (g P-kg'l dry dissected feces).

Mass balance models

The Platte Hatchery estimated the amount of P released from the stabilization pond
into the Platte River as:

CP.= (Po. - P.) x W (5)

where:

CP. = P exiting the stabilization pond

P... = P in stabilization pond outﬂow

Pill = P in Brundage Spring water entering the hatchery

W = Water ﬂow rate

28

Since fecal P rapidly leaches into water upon excretion, measming P, in feces taken
directly ﬁ'om the posterior intestine (he, before any leaching occurs) will result in a
”minimum possible” Pl. (MINP..). Minimum possrble P. W.) was calculated as:

mu=Par-[Pr+(P.-Ps)+l’.] (6)
where:
MINP. = minimum possible P of hatchery origin in eﬁluents entering the
stabilization pond

P,“ = P in feed

P, = P in dissected feces

P.l =Pinﬁshattheendofthemonth

P,i =Pinﬁshatthebeginningofthemonth

p, =Pindeadﬁsh

Equation 6 was modiﬁed by omitting P, to estimate the maximum possible P. (MA)CP,,,):

Mmm=Per-[(P.-Pt)+P.] (7)

where:

MAXP. = maximum possible P of hatchery origin entering the stabilization pond

A P, represented by P concentrations found in collected raceway solids (P,) should provide
an ”actual” Pb, (AP.,). Actual P. (APb) was calculated as:

APho=Pfed'[Ps+(Pu'Pri)+Pm] (3)

29
where:
AP. = actual amount of P released ﬁ'om the hatchery into the stabilization pond

P, = P concentration in collected raceway solids

The eﬂiciency of P removal ﬁ'om raceways through solids removal was calculated as:
(MlNP./AP.) x 100 (9)

P. was calculated at monthly intervals for each ﬁsh stock or substock (e. g., New
York coho salmon spawned in 1993). P,“ was calculated as (g P-kg'1 feed x kg feed
fed-month"). P, was calculated as (mg P-kg'l dry feces >< kg dry feces produced-month").
P. and PIi were calculated as (mg P-ﬁsh" x number of ﬁsh at the end and beginning of
each month, respectively). Monthly estimates of average ﬁsh weight within each substock
was regressed against day of the year to estimate the weight of dead ﬁsh on any given day.
P. was calculated daily and summed for each month. Estimates of whole ﬁsh P for both
living and dead ﬁsh were made using whole ﬁsh P vs. ﬁsh wet weight regressions
describing ﬁsh sampled at the Platte Hatchery and/or MSU. The percent of feed P
retained in ﬁsh was calculated as (P.- P,i + P.) / P... Separate monthly P. values for
each ﬁsh stock or substock were summed to make monthly hatchery totals. The ratio of P
lost in dissolved form vs. P lost in feces (dzf ratio) was calculated as (% P fed lost in

dissolved form : % P fed lost in feces) using 17 month totals.

30
Statistical analysis
Linear regression relationships were calculated for whole ﬁsh P vs. ﬁsh weight,-
fecal percent dry matter vs. ﬁsh weight, and fecal P concentration vs. ﬁsh weight using the
SAS software General Linear Model procedure (SAS Institute 1997). Data for ﬁsh reared
at MSU and the Platte Hatchery were combined if they regressed homogeneously.

Regression relationships were considered signiﬁcant if P s 0.05.

RESULTS

3005* cornposiﬁon

Regression relationships between whole ﬁsh P and ﬁsh weight are summarized for
echo and chinook salmon in Table 3. All regression relationships were signiﬁcant and
highly correlated (r2 > 0.98). Coho salmon reared at the Platte Hatchery and MSU did not
diﬂ’er in the whole ﬁsh P vs. ﬁsh wet weight relationship. Hatchery and MSU data sets for
coho salmon were merged, and the combined regression equation was used to estimate
coho salmon P content in the mass balance study. Regressions of whole ﬁsh P on ﬁsh wet
weight differed signiﬁcantly for chinook salmon reared at the Platte Hatchery and MSU.
The phosphorus content of chinook salmon in the mass balance experiment was estimated

using the hatchery equation (Table 3).

Feces characteristics and production

Rearing location had no eﬁ’ect on fecal percent dry matter or mg P-g‘l dry feces in
coho or chinook salmon. No relationship was found between fecal percent dry matter or
mg Prg‘1 dry feces and the weight of chinook salmon. Chinook salmon feces averaged (i
SE) 16.0 :h 5.6 % dry matter and 5.23 :L- 4.7 mg P-g'1 dry feces. Fecal percent dry matter
and fecal P content were signiﬁcantly eﬁ‘ected by ﬁsh weight for coho salmon:

coho salmon fecal dry matter (%) = -0.0521-[ﬁsh wet weight (g)] + 15.3. (10)

31

32

223338 85.3 3.9: new 23:8 m 55.3 c.0820 0.2.3 038:8 2 to»: 5:35 .

 

 

8.782. em as no a Re. 86 a a: pm: cogs gooeao
2.3:? we 85 85 s Ed- cod a 2 .c cheese. 8.52 access.
melons e2 mod 85 a a . a- 85 a a... mosses 88.8 28
”cloud S and one a 2.. _- So a 3... am: 85.3 2.8
218.0 on and Re a :._- 85 a 2... e222 eases 2.8
Amy owes. Emma)» new a a. 80225 2.2» .5532 36on

 

.mm
a as; .eoseesv 52 .o .596 beseech geese sense sense: 2% someone as ﬁnesse Seen: em 2%
36883.2 8%: ate..— 05 Set :25: 38:20 we: once .8 A3 £303 83 an: =0 3.5 m and 0.9.3 «o meemmaoumox .n 033.

33
mg P-g’l dry coho salmon feces = 0.30[ﬁsh wet weight (g)] + 6.14. (11)
Coho salmon feces averaged 14.4 :1: 3.0% dry matter and 8.76 t 5.4 mg P-g" dry feces.

Measurable amounts of acid insoluble ash were recovered ﬁ'om feeds (0.2-0.7%
dry matter), but not ﬁom the pooled dissected feces of thirty-ﬁve 15 g coho salmon (300
mg dry fecal matter). A literature review and consultation with a Bioproducts, Inc.
nutritionist (Dennis Rolie, personal communication) revealed no data on the dry matter
digestibility of Bioproducts feeds for juvenile salmonids. However, the average dry matter
digestibility of feeds similar to the Bioproducts, Inc. feeds used at the Platte Hatchery was
78.0 :1: 0.9% SD for 14-158 g rainbow trout and Atlantic salmon. Digestibility estimates
should have been more or less the same for juvenile coho and chinook salmon, rainbow
troutandAtlantic salmon, andshould not havebeenaﬂ‘ectedbyﬁshweight oragein
juvenile ﬁsh (Karl Shearer, National Marine Fisheries Service, personal communication).
Based on an assumed retention of 78.0% feed dry matter, 19,016 kg dry feces were
produced over the 17 month experimental period. Total feces were estimated to contain
146.8 kg P.

Since the feeds fed at the Platte Hatchery contained less than 1% ﬁnes, and ﬁsh
were reported to consume all feed offered (Roger Hubble, MDNR, personal
communication), feces should have comprised 98-99% of raceway solids.

Hatchery personnel were unable to provide information on the amount or P
content of raceway sludge (P_) collected by the vacuum system during the experimental
period. The inability to provide this data was not communicated until well after the

experiment ended. Consequently, data on the P content of removed solids (PI) for the

34

experimental period was not available. In addition, the needles which regulated the ﬂow
of water into the Platte Hatchery’s water sampling devices oﬁen either clogged or
admitted too much water into the containers. When operating properly, containers should
have been 50-75% ﬁlll after 24 h (Roger Hubble, MDNR, personal communication).

Approximately 75% of the time, containers were full alter 24 h.

Phosphorus loading

Monthly values for P mass balance parameters are summarized in Table 4 and
Figures 2 and 3. Without P, values, AP. could not be calculated and conclusions could
not be made on the eﬁiciency of the solids P collection system used by the hatchery.
Monthly values for MAXP. varied ﬁ'om -0.24 kg in June 1993 to 103 kg in April 1993
(Table 4). Monthly values for MAXP., MINP..CP., P retained by ﬁsh, and P fed values
followed similar patterns except during March-May 1993 (Figures 2 and 3). CP.
measurements approached or exceeded MAXP. estimates from October through
December 1993 (Figure 2).

The percent of feed P retained by ﬁsh (including mortalities) was 37.7% (Table 5).
MAXP., which represented fwd P lost in feces and dissolved wastes, was 62.3% of P
fed. Feces contained 21.0% of P fed based on dietary dry matter digestibility estimates
and the P concentration in dry feces. Dissolved P entering the stabilization pond was not
measured at the Platte Hatchery. Dissolved P concentrations in efﬂuent water collected
from the efﬂuent study completed at MSU were below the detection limit. Consequently

dissolved waste, or the percentage of feed P not accounted for in ﬁsh and feces, was

35

 

 

 

 

j 3. .e. 2.. a as as raw

 

 

ed. loo... one gel... as new. 23 are. .8... 2.2 8.8 an:
.2 3.2 2.... 83 Zn 8.: S... one as... 8.... 8.9. a...
a... 2... 5.. as... 98 a... ”a.“ :3 .8... 2.8 we... can
«.8 .2. 8.: 82. ca. 8.2 5... as... «ea... 8.... 8.2 an
E. 2...... on... an... 8... 2.: a...” was as... 8.... on... a:
con 5... E... 23 .5. 2.... .e... 9.2 3.... 8.... 2... SN.
3.. :2 can e8... 3: an... 32 e8." .3... an... a... a...
«an 2.3 2.... «8.2 Ex 3.: a..." «an... 2.... an... S..." 2....
.3 3.8 8.: can... «a... 8.2 82. 22. 8.... 8.... 8.2 a...
3.4 3...: 8.: a2... .a. a... $8 8.2 .8... 2.2 .2.“ a...
on... 8... an..." as... a... no... as... .22. 2.... 2o. econ as

2.....- an... 22 3... 3.... no... 2...... 4...... 8.2 3.2 as
a: a..." 3.: 8.2” 2. as... at... :2" 8.... e..." mace 8..
c... 3.8 3.8. $3.. 2. 8.3. as... 3...: 2...... 2e. 3... a...
a: 8.8 2.? 8...“. as" 2.: 32 2.... 2...... .2" 3.2 82
so. 8.... «on 82.. SN 8.... 3.2 8.... Se... :2 on: a."
a: :..... 8.... ”a... 3.. 8.8 E." 2...... 8.... S... on... a:

 

Illlllllll‘ll l!
2.3.2 .o 3.2.2 3.32 .6 3:52 2.... ea 5.. co. .8. 88. .3. can. 532» co. use

seemed... an 2.8...» ﬁle: an...

 

 

 

62.35 .2..— 83m 39:835. .25— 23... o... :8... 8233.8 2... mace—8.58... 02.2.... 3...: m 25:62 .v 0.2....

.33. .3 5,. 5.52. 33. .. .03.... 3.5.. 2.. a.
5.3... a... as... 332.33 .25. 3...... 2.. 8.. 3.58 58?... .. .8... E. 3.3... .. 2582 .N 2.3...

 

 

 

 

 

 

 

 

§>-...__..z
3.5,. 3...: 3...... 3.82 3...»... 3...: 3.5,. 3...... 3......
n p r p b b n P b P P P P p b - ONI
. ..
. .n.
. ..~
6
3
. 8
ea
. 8
. 8
.. .80.. 5o. ..
.— loll
umuo w 8—
2.32: lml
252.2 .. .x. u
. 8.

 

.33. .2 5. 5.2... 33. . 22...... 3...... 2......

20.2.... .3... 2...... 332.25.. .2... 2...... o... 2 8...... .82... .2... 2.8 B .8222 .. .2. .8. .. 283...... 2.222.»... .n 2......

 

 

 

§>-5....2
3:32 3.32 3.5—. 8.52 maﬁom 8...... 8. A“: 8.82 8.5..
p k P .P b D O) b h h L b P o
.x ............ x...
. ,, .x ......
..x. .. . .x . .
.3: .x.. .x. . xx. _ 8
X
7 I 3
3
C I 8 a.
v 8
.. 8—
- B. .TI¢.|
3582.....x... .om.

 

 

 

38
calculated by diﬂ‘erence (62.3% - 21.0% = 41.3%, Table 5). The ratio of P lost as

dissolved waste and fecal-waste was 2:1 (41.3%:21.0%).

Expressing total P losses on a production basis, a maximum of
2.8 kg P°metric ton" of ﬁsh produced was discharged into the stabilization pond. Ifall
fecal P could have been recovered through solids management, the loading rate could have

been as low as 1.8 kg P lost-metric ton" of ﬁsh produced.

Table 5. Phosphorus mass balance results for the experimental period of January 1993
through May 1994 at the Platte River Anadromous State Fish Hatchery.

 

 

 

Variable kg % feed P
P fed 698.67 100
P retained in live ﬁsh 240.15 34.4
P retained in dead ﬁsh 23.01 3.3
P in feces 146.75 21.0
P in dissolved waste 288.76T 41.3”
MAXPh 441.63 62.3
MINPho 288.76

CPi 161.14

 

* Calculated by diﬁ‘erence: 100 - (34.4 + 3.3 + 21.0) = 41.3.

‘ Represents the best case scenario if 100% fecal P could be recovered by raceway solids
removal.

DISCUSSION

Incomplete hatchery records prevented the development of nutritional mass
balance models for the Platte Hatchery based solely on collected data. Hatchery feed and
mortality records were meticulously kept, but ﬁsh inventory, weight distribution and
collected solids information was partially or totally unavailable. For example, 344,418
Coho ’92 were unaccounted for in the Platte Hatchery inventory records June 30, 1993.
Loss of ﬁsh ﬁ'om the records resulted in negative values for MAXP., and MINP.» in June
1993 (Figure 2). There has been little incentive for hatchery personnel to conduct ﬁsh size
inventories because handling stress has increased the risk of disease, and the criterion for
release of salmon ﬁom the Platte Hatchery has been smoltiﬁcation rather than size.
Consequently, certain assumptions had to be made to facilitate mass balance calculations.
It was assumed that average ﬁsh weights estimated ﬁ'om water temperature history were
accurate even though they were not calibrated by sampling. A normal distribution of ﬁsh
weight was also assumed. Both of these assumptions could be easily addressed in the
future by a small scale, relatively non-disruptive sampling program. Ewing et al. (1994)
reported that 7-9 small (2.5 kg) random samples of trout from a raceway provided

estimates of ﬁsh size with only 2-5% error.

39

40
Estimates of dietary dry matter digestibility were used to calculate total fecal P

production. Implicit in this calculation was the assumption that 98-99% of feed fed was
usually consumed by ﬁsh. This assumption may have been invalid March-May 1993, when
values for P fed and P released ﬁom the Platte Hatchery spiked (Figures 2 and 3). During
that period, unusually high amounts of a higher-P medicated feed (14.8 g P-kg'1 as fed)
were fed to Coho ’91 as a treatment for cold water disease. The production offeces was
probably overestimated since more feed was used in the calculation of feces production
than was actually consumed. Further, dissolved P losses were probably underestimated
sincetheywere calculatedbysubtractingPinfeces andPretainedinﬁshﬁ'omPfed.

A 37.7% retention of dietary P by coho and chinook salmon at the Platte Hatchery
compared favorably with retention of P by ﬁsh observed in other studies, especially in light
of the P lost ﬁom overfeeding in March-May 1993. P retention values reported for
salmonids have ranged ﬁ'om 13-50% (Hakanson 1986; Wiesrnann et al. 1988; Cho et al.
1991; Foy and Rosell 1991a; Gomes et al. 1995). In an earlier experiment at the Platte
Hatchery, Ketola et al. (1991) reported that 28.4% and 42.4% dietary P was retained by
coho salmon fed a standard ﬁsh meal-based diet (Oregon Moist Pellets) and a vegetable-
based diet (TZM), respectively. Both diets used by Ketola et al. (1991) contained 10 g
P°kg" (dry matter basis). The wide range in % P retained by ﬁsh probably resulted from
diﬂ‘erences in feeding rates and the digestibility and concentration of dietary P (Foy and
Rosell 1991a; Ketola et al. 1991; Ketola and Harland 1993).

The maximum P loading rate of 2.8 kg P-metric ton" ﬁsh produced during the

experimental period was low compared to P loading rates of 4.8-12.2 kg-metric ton"

41

reported for coho salmon (Ketola et al. 1991) and 4.8 kg-metric ton" as the average for
Nordic ﬁsh farms (Enell 1995). Wiesrnann et al. (1988) fed 90 g rainbow trout an
experimental feed similar in composition to the commercial “low P” feeds used at the
Platte Hatchery’. They reported a loading rate of 3.9 kg-metric ton" ﬁsh produced. In
contrast, Fay and Rosell (1991a) reported a loss of 25.6 kg P-metric ton“ rainbow trout
produced, and attributed the high loading rate to overfeeding.

The ratio of P lost in dissolved vs. fecal form (dzf ratio) was 2:1 for the present
experiment. Data ofKetola et al. (1991) and Ketola and Harland (1993) have shown that
the dzf ratio could be altered through dietary formulation. D:f ratios (calculated using P in
raceway solids instead of feces) for coho salmon fed the TM diet and Oregon Moist
Pellets at the Platte Hatchery were 2:1 and 5:1, respectively. Ketola and Harland (1993)
reduced the d:f ratio ﬁ'om 3:1‘to 1:1 by replacing the highly soluble P supplement,
Cﬂ-IP04-2H20, with less soluble deﬂuorinated rock phosphate in rainbow trout diets.

The P mass balance analysis provided maximum and minimum possible values for
P discharged into the stabilization pond. However, the immediate pollutive potential of
discharged P depends on its chemical form. Orthophosphate, or soluble reactive P, is the
P form most available to aquatic plants, phytoplankton and bacteria (W etzel 1983). Foy
and Rosell (1991b) found that approximately 60% of total P released ﬁ'om a rainbow trout

farm was orthophosphate. Studies to determine the aﬁ‘ect of dietary formulation on the

 

5
DietsusedbyWiesmannetal.(1988)containedlow-ashﬁshmeal,hydmlyzedfeathermeal,and
hydrolyzedwheat Drydietcontained 8.9gP-kg".

42
dzf ratio should also consider how diet aﬂ‘ects the proportion of orthophosphate in

dissolved waste.

CP, (P leaving the stabilization pond as determined by the chemical method)
approached or exceeded MAXP., (P entering the stabilization pond) for October,
NovemberandDecember1993. Duringthatperiod, Pmayhavebeenreleased ﬁomthe
stabilization pond sediments into pond water. Turnover ofP in the sediments ofthe
stabilization pond has been documented during late fall and early winter (Charles Pecor,
MDNR, personal communication). Alternately, ﬂaws in the Platte Hatchery’s water
sampling system may have resulted in overestimates of P discharged ﬁom the stabilization
pond. P losses ﬁom the hatchery were at their lowest during those three months. The
average daily concentrations of P in water discharged each month ﬁ'om the stabilization
pond October through December 1993 were 16 ug PL“, 8 ug PL’1 and 5 ug P-L",
respectively. The November and December concentrations were close to the minimum
detection limit of 3 ug P-L“, suggesting that water P concentrations may have been too
low to accurately measure during those months. The ﬁ'equent malﬁrnctioning of the water
collection devices used by the Platte Hatchery also prevented the collection of
representative 24 h samples.

Representative water samples could be gathered using a more robust device which
continuously delivered water into an acid washed receptacle containing acid and a
preservative. A preservative would prevent the growth of P-absorbing algae or bacteria in
the water sample. Acid treatment of the receptacle would prevent the transfer of P

between the sample and the inner surface of the receptacle (APHA et al. 1985). During

43

periods when water P concentrations were near the lower detection limit (e. g., October
and December 1993 ), relatively large volumes of water would need to be digested for P to
be accurately measured.

Whole body P in Platte Hatchery coho and chinook salmon was comparable to
whole body P in juvenile salmonids fed P-replete diets (Ketola 1975; Watanabe et al.
1980; Shearer 1984; Shearer et al. 1994; Rodehutscord 1996). However, regressions of
whole ﬁsh P on ﬁsh weight diﬂ‘ered for chinook salmon reared at the Platte Hatchery and
at MSU. This may have resulted ﬁom the limited range of ﬁsh sizes reared at the Platte
Hatchery (0.3-3.2 g) or from diﬁ‘erent water temperature regimes, feeds, feeding
ﬁ'equencies and feed eﬁciencies at the Platte Hatchery and MSU (Rodehutscord 1996;
Asgard and Shearer 1997). Consequently, regressions of whole ﬁsh P on ﬁsh weight
developed for coho and chinook salmon at the Platte Hatchery should be used cautiously

to estimate P retention in salmon reared elsewhere.

SUMMARY AND CONCLUSIONS

Based on mass balance estimates ofmaximum possible P losses over a 17 month
period, the Platte River Anadromous State Fish Hatchery was one of the least-polluting
salmonid hatcheries described. During the experimental period, the hatchery discharged
1.8-2.8 kg P-metric ton" ﬁsh produced into the stabilization pond. More precise estimates
of P discharged into the stabilization pond could not be calculated without data on the P
content of sludge removed from the raceways.

Although the P concentration was relatively low in most feeds used at the Platte
Hatchery, the P content of whole coho and chinook salmon was comparable to whole
body P content reported for other salmonids fed diets containing higher concentrations of
P. However, regressions of whole ﬁsh P on ﬁsh weight for coho and chinook salmon
from the Platte Hatchery should be used cautiously to estimate the P content of whole ﬁsh

from other locations.

CHAPTERZ

Eﬂ‘ect of dietary zinc supplementation and phytase pre-treatrnent
ofsoybean meal or corn gluten meal on growth,

zinc bioavailability and protein utilization in rainbow trout
INTRODUCTION

Plant feedstuﬁ's like soybean meal have been important protein sources in ﬁsh feeds
(NRC 1993). However, many plant feedstuﬁ‘s contain phytate, a P storage molecule
which is not digestible by ﬁsh (Cain and Gatling 1995). Phytate has been shown to bind
Zn and other cations in the gut, forming indigestible molecules (Cosgrove 1980; Spinelli et
al. 1983). Reduced protein, P and Zn utilization has been observed in ﬁsh, pigs and
poultry fed diets containing phytate (Gatlin and Phillips 1989; Lei et al. 1993a,b,c; Cain
and Garling 1995; Qian et al. 1997). Ifdietary Zn is bound in an indigestible phytate
complex, a Zn deﬁciency could result. Insulin secretion and the activity proteases and
phosphatases have been shown to be Zn-dependent (Prasad and Oberleas 1971; Prasad
1993). Consequently, reduced protein and P utilization by ﬁsh fed diets containing

phytate could be caused by a Zn deﬁciency.

4S

46
In this experiment, juvenile rainbow trout were fed a combination of diets
containing soybean meal and corn gluten meal either untreated or pre-treated with phytase
with or without Zn supplements for 170 d.
Objectives
1. Determine if rainbow trout fed a plant-based diet high in phytate become Zn
deﬁcient based on growth, protein utilization, plasma insulin concentration, the
activity of carboxypeptidase B and intestinal alkaline phopshatase, and whole ﬁsh
and bone Zn, P or ash content.
2. Determine if dephytinized plant ingredients or a dietary Zn supplement is more

eﬁ‘ective in providing Zn to ﬁsh.

 

Reduced protein (Spinelli et al. 1983), P (Cain and Garling 1995; Riche and Brown
1996) and Zn (Richardson et al. 1985; Gatlin and Phillips 1989) digestibility has been
observed in ﬁsh fed puriﬁed diets containing phytic acid, and in practical diets containing
untreated soybean meal. Protein, P or Zn retention were improved for rainbow trout and
Atlantic salmon when dietary soy products were pre-treated with phytase (Cain and
Garling 1995; Storebakken et al. In press), or when phytase was added directly to the diet
(Rodehutscord and Pfeﬂ‘er 1995; Riche and Brown 1996).

Reduced protein digestibility was observed by Ogino and Yang (1978) in rainbow
trout fed Zn-deﬁcient diets. They reported a protein digestibility of 66% in ﬁsh fed a
semipuriﬁed diet containing 1 mg Zn-kg“ diet for 7 weeks, and hypothesized that Zn
deﬁciency had caused a reduction in carboxypeptidase activity. However, ﬁsh fed diets
with 5 mg Zn-kg" diet exhlhited a normal protein digestibility of 97%. Protein eﬁciency
increased signiﬁcantly in juvenile chinook salmon when dietary Zn was raised from
approximately 50 to 400 mg Zn-kg‘l in diets containing 25.8 g phytic acid-kg" dry diet
(Richardson et al. 1985). Spry et al. (1988) found that severely Zn-deﬁcient rainbow trout
exhibited depressed plasma protein concentrations (2.1 g-dL") compared to Zn-replete

ﬁsh (3.4 g-dL"), even though Zn-deﬁcierlt ﬁsh appeared to have normal feed intakes.

47

48

The possible additive interaction of phytic acid and low dietary Zn concentration
on protein utilization has not been directlyaddressed for ﬁsh Gatlin and Phillips (1989)
reported a signiﬁcant interaction between dietary Zn and phytate on bone Zn
concentration in channel catﬁsh, but not on weight gain or feed eﬁciency.

Oberleas and Prasad (1976) and Oberleas and Harland (1981) suggested that the
bioavailability of dietary Zn could be predicted using dietary phytic acid:Zn and (phytic
acid x Ca) :Zn molar ratios. Reduced Zn absorption and marginal Zn deﬁciency was found
in rats fed diets with phytic acid:Zn molar ratios of 10 or above (Davies and Olpin 1979;
Lo et al. 1981) or with (phytic acid X Ca)'Zn molar ratios above 3.5 (Fordyce et al. 1987).
Dietary phytic acid:Zn and (phytic acid >< Ca):Zn molar ratios have not been tested as
predictors of Zn bioavailability in ﬁsh.

11. Warm

Zn has been identiﬁed as a structural component or cofactor for over 70- enzymes,
including some required for the digestion and synthesis of protein and nucleic acids and for
P digestion (Wallwork and Duerre 1985; Prasad 1993). Zinc is also a structural
component in nucleoproteins and insulin storage crystals (Scott 1934; Greider et al. 1969).
A W

Ogino and Yang (1978) reported that 1.5-3.5 g rainbow trout fed a diet based on
egg albumin, starch and dextrin required 15-30 mg Zn-kg'l dry diet for normal growth rate
(treatment effects were not evaluated statistically). Maage and Julshamn (1993) fed diets
based on cod ﬁllet meal and corn meal to 40 g Atlantic salmon for 12 weeks. Based on

whole body Zn and serum Zn concentrations, they concluded that the dietary Zn

49
requirement of juvenile Atlantic salmon was 37-67 mg Zn-kg" dry diet. Channel catﬁsh,
bluetilapiaand commoncarpfed semipuriﬁed dietshavebeenreported to require 20 mg
Zn-kg" dry diet based on serum or bone saturation with Zn (Ogino and Yang 1979; Gatlin
and Wilson 1983; McClain and Gatlin 1988). Clinical signs of Zn deﬁciency in ﬁsh include
lens cataracts, short-body dwarﬁsm, ﬁn erosion, growth suppression and mortality (Ogino
and Yang 1979; Satoh et al. 1983, NRC 1993).

The diet Maage and Julshamn (1993) fed to Atlantic salmon probably contained
less than 1 g phytic acid-kg“ dry diet, which was approximately 8 times lower than the
phytic acid concentration in a commonly used salmonid diet (Guelph diet, NRC 1993 )‘.
The semipuriﬁed diet fed to rainbow trout by Ogino and Yang (1978) was probably ﬁ'ee of
phytate. The dietary Zn requirements of juvenile salmonids fed most commercial feeds
manufactured in North America maybe higher than the values reported by Ogino and
Yang (1978) and Maage and Julshamn (1993). The higher concentrations of phytic acid
and Ca often found in North American feeds has been shown to reduce Zn availability
(Gatlin and Wilson 1984; Hardy and Shearer 1985; Satoh et al. 1989).

Holcombe et al. (1979) found that ﬁsh absorbed Zn directly ﬁ'om water, probably
via the gills (Joyner 1961; Spry et al. 1988). However, Spry et al. (1988) found that Zn
uptake ﬁ'om the diet was independent of Zn uptake from the water, and that the
concentrations of Zn commonly found in unpolluted water (approximately 10 ug Zn-L")

was not likely to mitigate a dietary Zn deﬁciency. Gatlin and Wilson (1983, 1984)

 

6

Theth salmonid diet (NRC 1993)containsapproximately8gphyticacid-kg‘I drydiet. Estimatesof
phyticacidconcentrationsintheGuelphsalmoniddietandthedietusedbyMaageandJulshamn(l993)
mbasedonthedataofNelsonetal. (1968)andHar1andand0berleas (1987).

50

reported that channel catﬁsh reared in water containing 10-25 pg Zn-L'1 were not able to
obtain adequate Zn from water for normal growth and mineralization.

Fish sequester Zn in skeletal tissue in amounts relative to the dietary Zn
concentration up to a saturation point (Ogino and Yang 1978; Satoh et al. 1987; McClain
and Gatlin1988). Adult sea trout were reported to use Zn deposited in scales during
spawning migration (O'Grady 1981), and it is conceivable that bone Zn may also be
available during such a major physiological event. However, O'Grady and Abdullah
(1985) concluded that Zn deposited in bone was not available to juvenile brown trout
during periods of low food (Zn) intake. Hatchery-phase rainbow trout are juveniles and
do not undergo the physiological changes associated with maturation.

B. W

Carboxypeptidase B [EC 3.4.17.2] is a pancreatic exopeptidase which contains Zn
as an essential structural element (Folk and Schirmer 1963; Yoshinaka et al. 1985).
Carboxypeptidase B (CPB) has been shown to hydrolyze the peptide bond adjacent to the
carboxy terminal of a peptide chain, preferentially cleaving the basic amino acids arginine,
lysine and ornithine (Folk and Gladner 1958). Since trypsin is an endopeptidase with a
speciﬁcity for arginine and lysine, CPB is important in the degradation of products of
tryptic digestion (REF).

Carboxypeptidases have been identiﬁed, isolated and characterized for several
ﬁshes including churn salmon (Uchida 1970), catﬁsh (Y oshinaka et al. 1984a, 1985),
lungﬁsh (Reeck and Neurath 1972), dogﬁsh (Prahl and Neurath (1966), cod (Overnell

1973), and Atlantic halibut (Glass et al. 1987). Fish CPB was thought to be a

5 1
metaﬂoenzymebecauseitwassnonglymhrbitedbythemetalchdatorsEDTAand 1, 10—
phenanthrolin (Kishimura and Hayashi 1992). Yoshinaka et al. (1985) hypothesized that
Zn is the integral metal ion in ﬁsh carboxypeptidases since chelator-induced inhibition was
reversed by the addition of ZnCl2 to a carboxypeptidase A preparation. Mammalian
carboxypeptidases contain one Zn atom per molecule (Folk 1971).

Ogino and Yang (1978) hypothesized that the decreased protein digestibility they
observed in Zn-deﬁcient rainbow trout was a result of reduced carboxypeptidase activity.
However, the eﬁ‘ect of Zn deﬁciency on carboxypeptidase activity has not been
determined in ﬁsh. Signiﬁcant reductions in the activity of pancreatic carboxypeptidase in
Zn-deﬁcient rats have been reported (Hsu et al. 1966; Prasad and Oberleas 1971).

Intestinal alkaline phosphatase (ALP) [EC 3.1.3.1] has been identiﬁed in ﬁsh as a
non-speciﬁc phosphomonoesterase catalyzing transphosphorylation reactions and the
hydrolysis of phosphomonoesters and pyrophosphates (Whitmore and Goldberg 1972a).
Based on the observation that ALP spanned the apical membrane in rainbow trout, Gasser
and Kirschner (1987a) suggested that it ﬁrnctioned as a transport mechanism between the
intestinal lumen and cytoplasm for an undetermined substance.

Fosset et al. (1974)'reported that mammalian and microbial ALP contained four Zn
atoms per dimer. Each subunit contained one Zn atom for structural integrity and another
for catalytic activity (nomad and Greenwood 1971). Yora and Sakagishi (1986) found
that ﬁsh intestinal ALP was inhibited by EDTA, and considered this as evidence that ﬁsh
intestinal ALP was also a metalloenzyme. Methods of isolation and characteristics of ﬁsh

intestinal ALP have been described for carp and eel (Y era and Sakagishi 1986), rainbow

52
trout (Prakash 1960; Noda and Tachino 1965a,b; Noda 1967a,b,c; Colin et al. 1985; Yora
and Sakagishi 1986; Gasser and Kirschner 1987a,b; Whitrnore and Goldberg 1972a,b),
Atlantic salmon (Johnston et al. 1994), pike, bream and roach (Kuz’mina and Smirnova
1991), sea bass (Bogé et al. 1993), tilapia (Gelman et al. 1992) and sterlet (Kuz’mina and
Kuz’mina 1991).

ALPactivitywasreduced inthepancreasandintestineonn-deﬁcientratsand
mice (Prasad et al. 1967; Prasad and Oberleas 1971; Taneja and Arya 1992). However,
Taneja and Arya (1992) have questioned whether the changes in enzyme activity were
caused by insuﬁcient Zn for enzyme activity or by general malnutrition associated with
Zn deﬁciency. The eﬁ'ect of Zn deﬁciency on ﬁsh intestinal ALP activity has not
previouslybeenaddressed. SerumALPhasbeenusedasanindicatoronn statusor
physiological state in channel catﬁsh (Gatlin and Wilson 1983), rainbow trout (Hille 1984)
and Atlantic salmon (Maage and Julshamn 1993; Johnston et al. 1994).

C. m

Zn status may aﬁ‘ect protein (amino acid) utilization in ﬁsh through its relationship
with insulin. Ince and Thorpe (1977) reported that insulin secretion was stimulated in ﬁsh
by increases in plasma amino acids. Insulin has been shown to facilitate the uptake of
amino acids by ﬁsh tissues (Ince and Thorpe 1978) and stimulate muscle protein synthesis
(Millward 1989). Zn has been shown to have a role in insulin storage (Scott 1934;
Greider et al. 1969) and insulin sensitivity (Park et al. 1986). Droke et al. (1993) reported

that serum insulin concentrations were reduced in growing lambs fed a severely Zn-

53
deﬁcient diet (1 mg Zn-kg" dry diet) but were normal in lambs fed a Zn-marginal diet (5

ms Zn'ks" dry diet).

MATERIALS AND METHODS
Fish
Juvenile rainbow trout (Oncorlrynchm mkiss) were obtained from the Michigan

Department of Natural Resources Wolf Lake Hatchery. The ﬁsh had been artiﬁcially
spawned ﬁom mature rainbow trout returning to the Little Manistee River, Michigan
From ﬁrst feeding in June 1995 to the start ofthe experiment in December 1995, ﬁsh were
fed Biodry 10007 (Bioproducts, Inc., Warrerrton, Oregon). One day prior to the beginning
of the experiment, 9-11 ﬁsh were randomly distributed to tanks. Initial ﬁsh weight (:1: SD)

was 118* 1.1 g. Totalweight ofﬁshinatankwas 115:2 12g.

Culture practices

Triplicate groups of rainbow trout were reared in 125 L rectangular plastic tanks
for 170 d. Tanks were supplied with aerated well water at 12 : 1°C. Well water
contained 10 pg Zn-L“. Flow rates for the single pass, ﬂow-through system were 2.5
L-m". Fish were maintained on the natural lightzdark photoperiod with no supplemental
lighting. Fish were periodically treated for ﬁlngal infection with static formalin baths (250

ppm formalin for 25 min followed by 50% water replacement).

 

7
Biodry 1000 met NRC (1993) recommendations for protein (450 g~kg“ diet as fed), fat (165 gkg“ diet as
fed) and Zn (133 mg-ltg‘ dry diet).

54

55

Phytase activity and application tofeedstuﬂs

The activity of fungal phytase (Finase lot # 500033, Alko Ltd., Rajanraki, Finland)
was determined colorimetrically (Han et al. 1987). Enzyme activity was approximately
500,000 PU-g". One phytase unit (PU) was deﬁned as the amount of enzyme which
liberated 1 nmol of inorganic P from sodium phytate per minute at pH 5 and 37°C.

Defatted soybean meal (49% protein) and corn gluten meal (60% protein) were
dephytinized using a procedure modiﬁed ﬁ'om Cain and Gatling (1995). Three g phytase
was dissolved in 1.5 L SOmM citrate buﬁ‘er, pH 5.0. The buﬂ‘ered phytase was mixed with
1.5 kg soybean meal or corn gluten meal, loosely covered, and allowed to stand for 24 h at
25°C. Treated meal was dried in a forced-air oven at 27 i 1°C for 28 h The dried
products were stored in plastic containers at room temperature until used. Soybean meal

not treated with phytase was sham-treated in buﬁ'er.

Phytate minis

Feed ingredients and a commercial salmonid feed used as the reference diet
(Biodry 1000, Bioproducts, Inc., Warrenton, Oregon) were analyzed for phytic acid
according to Latta and Eskin (1980). Approximately 0.1 g sample was placed in a
scintillation vial with 5 mL of 2.4% HCl. Vials were gently agitated on a shaker table
overnight. The samples and 20 mL deionized distilled (DDI) water, used to rinse the vial,
were ﬁltered through Whatrnan #1 ﬁlter paper and the liquid was collected in a ﬂask. A
column containing 200-400 mesh AGl-X4 anion exchange resin was prepared with 0.5 g

resin in approximately 5 mL 0.05 NaCl, charged with 15 mL 0.7 M NaCl and rinsed with

56

15 mLDDIwater. Aﬁertheliquid samplewasadded tothecolumn, itwaswashedwith
15 mL DDI water, then with 15 mL 0.05 M NaCl. The column was then eluted with 15
mL 0.7 M NaCl, and the eluate was collected into a beaker for colorimetric analysis using
sodium phytate as the standard. One mL Wade Reagent (0.03 g FeC13-6HZO and 0.3 g
sulfosalicylic acid diluted to 100 mL with DDI water) was added to 3 mL of standard or
sample within 30 min ofelution ﬁom the column. Absorbance was read at 500 nm using
DDI water as a blank

The phytic acid content of individual ingredients was used to calculate the phytic
acid content of the experimental diets. Dietary phytic acid:Zn molar ratios were calculated

based on molecular weights of 660.08 g phytic acid-mole'l and 65.38 g Zn-mole".

Feeds and feeding

The composition of the experimental diets is presented in Table 6. Diets contained
530 g protein-kg" dry diet. Diets designated NT (‘not treated’) and T (‘treated’)
contained sham-treated and dephytinized soybean meal, respectively. The numbers 0 and
50 in the diet labels indicated mg supplemental Zn-kg" dry diet added as ZnSO.°7H20.
Diets labeled TC contained dephytinized corn gluten meal as well as dephytinized soybean
meal. The mineral and phytic acid composition of the reference and experimental diets are
presented in Table 7. Diets containing sharn-treated soybean meal were supplemented

with P ﬁom deﬂuorinated rock phosphate (18% P) to equal total P in soybean meal

(0.74% P by analysis).

 

andn end“ andn 3.5.. 3.: 825:8 a

 

 

 

.. 2... .. 2... .. 2...... 0...N

.. .. .. S. 2. 22.8.... .8. 8.2.5.8..

n. n. 2 n. n. .8... .28....
2... a... a... .2. 2... 2.... 63.8....
2... z... z... z... z... 8.8.... 8......o
c. e. c. c. c. .2... can...»

4 e e e e .0: 8....-.
2. 2. o: 2. 2. .83.? 5 ..o a...

.tl. 8. 8. 8. 8. 8. .8... 8.26..
8. 8. 8. 8. 8. .8: .8...
8. .. .. .. .. .8... 8...... 28 8.2.5.8..

.. 8. 8m 8m 8.. .8... =2...» ...8
8... Sn 2 .. .. .. .8... 28...... 88.5.2.8

.. .. .. c. n e. n .8... 28......

8.. on... a. 8.2 .82 8.88%
.mﬁu

 

an... 8...... 8 8.... 28:29.. .e.........§8... an .2.
8...... an a... 8 8388...... an ...... 8.3.3.2.... 8.3.8.... .88 u... 8.2.2.... e. 3.... 33...... o. 8. .8... 382:8... .o 8288.8 .e 2......

58

. , .8... 88...... 88.5.2.2.
.... ..2. ... .8... 8...... ...8 888...... 858.8 up 8.2.... .8... ...8..N ...... ...... .... 19.5 8:328... a... 8.8.8. 2...... .8... 0..

... 8 ...... .. 23...... ...... 8888. ..8... 88.... 8823...... ...... 8.88.5... 8......8 .88.... .. ...... .88.. .2... ..z 8.88.. ...... .
8...... .82....8... .38... .08.... .8... ..o... .8...

.82.... .... 6.39.8.6 ...... 6.88.... a... 6.3.8.. .8. 6.5.8.... ”8.8... ......s 22...... .22.... 8.....5..&...... ...... .....N .

.8: 85...... .< ......s... 8...... .... ......s... .2... ... ......s... ......a.

a...” 8......8 35...... ......8. 80.88... .88... .... ..........> .88... .....o... ...... .88.... .... ....8 ...... .. .2 68.5.3.8... a... 838......
.5. 83...... ...... 6.8.8.8.. ......28 a ......8... ......a. 8......8 8...... 8.83 ...... ...... 8.8 8...... ... 8......3 8. ...... .....a..> .
...... ...... 22.238.80.88... .28 8....8... o... 8.83.8.0... .882... _

......8. o ......F

9
5

5030030. .308 50980 385530“. 9.0 3.8.0.55 00:5ch @030: v a. 2.: v0.00: 8: v HZ “0085.00“. 805

000m 00:20.02 05 83 Acowﬂo
£95.33 :0:— aﬁavoaoﬁ .82 >625 a .05 ..005 50.18 0333.33 8 =03 3 .008 :32» E3 Egan—0.0
3.3.8 up 332 305 Aswan 5 8:. 5 ..3...N 352233 me 382 32.2 as o... s on .05 o 2322 2:

60$ v0.7.8: 05 co cons—:82. 83

88— >693 #— 30.. 00:050. 05 .«o 5:82.350 28 05.3.. 05. “gag—000.“ no 530.8088 v.00 0.23; 05 :0 @003

noun—3.00 0.83 80:. 3808598 mo 80:05:02.8 300 £53 .306 v0.7.3. 8.. Bums—.80.. 0.03 8235:0280 .8032 .

n

 

:4 2.8 3.2: cNﬁoa 0:3.—

 

 

83 a? 2 a 3..» 3:
ES :4. 3+ «.3 a.» 03 3:. .5 .3.» . 28 25:
a: :0 n: 2 .a 3.0 2 3.2 6% .5 ..mme . 0
3n 2: in m: 2w 5.3 8:. 5 70.01 cm
80. on... 8 on: 8.2 x as :08qu 5.05
«.05

 

.28.. £303 .3. a so 03 82.3 =<

..306 3:08.596 00 550.888 200 0203.. 98 3052

.5 035.

60

Diets were made in 2-kg batches. Dry ingredients for individual batches were pre-
mixed in a liquid-solids blender for 2 hours (Patterson-Kelley Co., Inc., East Stroudsburg,
Pennsylvania). Fish oil was blended into the premixed dry ingredients using a Univex
mixer (Univex Corp., Salem, New Hampshire), then DDI water was added to create a
moist dough. Dough was extruded without heat through a 3 mm die. The resulting
spaghetti-like strands were dried at ambient temperatures in a forced-air oven. After
drying, the dry matter content of diets was 89 - 94%. Dry matter of feed was determined
at 2 wk intervals throughout the experimental period in order to calculate daily rations on
a dry matter basis. Dried strands were chopped in a Waring blender (Waring, New
Hartford, Connecticut) and screened to retain pellets 1-2 mm in diameter. Pellets were
stored ﬂown in plastic containers until the day of use.

A commercial salmonid feed (Biodry 1000; Bioproducts, Inc., Warrenton, Oregon)
from a single manufacturing lot was used as the reference feed. Biodry 1000 was
formulated with ﬁsh meal from which bones, skin and scales had been removed, feather
meal, cereal tailings (cooked oats, wheat, barley or rice), spray dried blood meal, herring
meal, processed deboned ﬁsh, vitamin and mineral supplements and preservatives.

The daily ration (dry matter basis) was calculated as a percentage of total weight
of ﬁsh per tank determined every 14 d (Piper et al. 1982). Daily rations were divided into
two feedings fed at least 4 h apart. Minor adjustments to the daily ration were made
between tank weighings to account for ﬁsh that died. Feed was fed by hand. To prevent
gut contents ﬁ'om aﬁ‘ecting ﬁsh weights, feed was withheld one day prior to weighing.

Feeding resumed the day after weighing.

62

Tame collection

On the ﬁnal day of the experiment (day 170), morning feed was oﬁ‘ered to one
randomly selectedtankeveryBOmirL Samplingbeganapproxirnately 1 haﬁerfeeding.
Elapsed time between feeding and blood sampling (60-80 min) was recorded to the
nearest minute.

Fish were emersed in a tank containing 15-30 mg-L'l of the anaesthesia MS-222
(tricaine methane sulfonate). After each ﬁsh was screened for clinical signs of Zn
deﬁciency (opaque lens of the eye and eroded ﬁns), a 1 mL blood sample was drawn ﬁom
thecaudalveinwitha l mLtuberculinsyringewhichhadbeenrinsedinaZOOU-mL"
solution of Zn-ﬁee sodium heparin. Blood samples were placed into tubes containing 20
unitsonn-free sodiumheparin. Wholebloodwaskeptinanicebathuntil all samples
were collected. Whole blood was centrifuged for 10 min at room temperature and 13,600
x g, and the plasma pipetted into mierotubes and stored at -20°C.

Aﬁer blood sampling, ﬁsh weight was recorded, and the pyloric caeca and intestine
were dissected and weighed. The pyloric caeca was the portion of the digestive tract
found between the pyloric region of the stomach and the posterior-most cecal sac, and
contained the duodenum. The ‘intestine’ consisted of the remainder of the intestine,
including anterior and posterior intestine and the rectum. Because pancreatic cells are
diﬁ‘used throughout the mesentery surrounding the pyloric caeca in rainbow trout
(Y asutake and Wales 1983), tissue homogenates for CPB analysis were prepared from the

entire pyloric caeca and related connective and fatty tissue. Dissected tissues and

62

Tissue collection

On the ﬁnal day of the experiment (day 170), morning feed was oﬁ‘ered to one
randomly selected tank every 30 min. Sampling began approximately 1 h after feeding.
Elapsed time between feeding and blood sampling (60-80 min) was recorded to the
nearest minute.

Fish were emersed in a tank containing 15—30 mg-L" of the anaesthesia MS-222
(tricaine methane sulfonate). After each ﬁsh was screened for clinical signs of Zn
deﬁciency (opaque lens of the eye and eroded ﬁns), a 1 mL blood sample was drawn ﬁom
theeaudalveinwitha l mLtuberculinsyringewhichhadbeerrrinsedina200U°mL'l
solution of Zn-ﬁ'ee sodium heparin. Blood samples were placed into tubes containing 20
unitsonn-free sodiumheparin. Wholebloodwaskeptinanicebathuntilallsamples
were collected. Whole blood was centrifuged for 10 min at room temperature and 13,600
x g, and the plasma pipetted into microtubes and stored at -20°C.

After blood sampling, ﬁsh weight was recorded, and the pyloric caeca and intestine
were dissected and weighed. The pyloric caeca was the portion of the digestive tract
found between the pyloric region of the stomach and the posterior-most cecal sac, and
contained the duodenum. The ‘intestine’ consisted of the remainder of the intestine,
including anterior and posterior intestine and the rectum Because pancreatic cells are
diﬁirsed throughout the mesentery surrounding the pyloric caeca in rainbow trout
(Y asutake and Wales 1983), tissue homogenates for CPB analysis were prepared ﬁ'om the

entire pyloric caeca and related connective and fatty tissue. Dissected tissues and

63
carcasses were ﬂown separately in Whirl-Pak" (Nasco, Fort Atkinson, Wisconsin) bags
on dry ice, then stored at -20°C.

Carcasses were later thawed, and a 2-cm section of the spinal column from
betweenthebaseofthe head andthe anterior edge ofthe dorsalﬁnwasrernoved forbone
mineralanalysis. The spinal samplewaslightlyserapedoftissueusingastainlesssteel
scalpel, and only the centra were retained for bone mineral analysis. Bone samples were

dried at 105°C for 24 h and stored in Whirl-Paks.

Mineral anabIsis

After the removal of bone samples, carcasses were sliced into l-cm sections and
lyophilized 72 h. Lyophilized ﬁsh were ground in liquid nitrogen using a stainless steel
Waring blender, then frozen in Whirl-Paks. Ground ﬁsh, feedstuﬂ‘, feed samples and a
bovine liver standard (National Institute of Standards and Technology, United States
Department of Commerce) were dried in a forced-air oven 24 h at 105 °C in pre-weighted
aluminum pans, then cooled in a desiccator. Dry matter content was determined by
subtracting the initial weight of the pan ﬁ'om the weight of the pan containing the dried
sample. Samples were wet ashed using nitric and perchloric acids (AOAC 1984) as
described in Appendix A Mineral residues were dissolved in DDI water.

Oven—dried bone samples were individually wrapped in Whatrnan #1 ﬁlter paper,
dehydrated in 500 mL absolute ethyl alcohol for 8 h and de-fatted in ethyl ether for 8 h

The resulting samples of fat-ﬁee dry bone were weighed, then ashed at 600°C for 16 h in

64
a muﬁe furnace. Bone ash was dissolved in concentrated (69-70%) nitric acid prior to

dilution and analysis.

Fish, feedstuﬂ‘ and feed digests and bone ash were assayed for P using visible light
spectroscopy (Gomori 1942) as described in Appendix B. Zn in sample digests and
rearing water was determined by atomic absorption spectroscopy. Instrument parameters
wa'evaiﬁedbyanalysisofthebovineﬁverstandudandconﬁmedwithanatomic
spectral standard (Baker Instra-Analyzed", J. T. Baker Chemical Co., Phillipsburg, New
Jersey). Carcass Zn values were corrected for Zn removed in bone samples. Zn
concentrations were not determined for digestive tissues removed for enzyme analysis.
The carcass constituted approximately 91% total body weight.

Total bone weight per ﬁsh was estimated using a total wet bone weight vs.ﬁsh
weight regression for Atlantic salmon (Rottiers 1993). Total bone Zn was calculated for
each ﬁsh as (pg Zn-g" wet bone) x (g wet boneﬁsh" ).

The N content of whole ﬁsh and diets was determined using the micro Kjeldahl

method (AOAC 1984). Crude protein was calculated as NX6.25.

We and insulin assays

DetailedALPandCPB assayprotocolsarepresentedinAppendicesDandE.
Brieﬂy, ALP and CPB activities were determined spectrophotometrically in the
supernatants of tissue homogenates using the artiﬁcial substrates p-nitrophenyl phosphate
and hippurl-L-arg, respectively (SIGMA Chemical Co., St. Louis, Missouri). Enzyme

activity is reported based on supernatant protein content (speciﬁc activity), which was

65
determined according to Bradford (1976) using a Sigma Total Protein Kit 610-A. Porcine

or bovine enzymes (SIGMA Chemical Co., St Louis, Missouri) were used as standards in
all assays.

The concentration of insulin in plasma was determined by radioimmunoassay with
skipjack tuna (class Osteichthyes, family Scombridae, Katsuwonas pelamis) insulin as the

standard and a rabbit anti-skipjack insulin antibody (Gutierrez et al. 1984).

Zinc and protein eﬂiciency
Percent Zn retained (PZR) was calculated as (mg Zn gained-mg'l Zn fed) X 100.

Percent protein retained (PPR) was calculated as (g protein gained-g" protein fed) x 100.

Statistical analysis

WeiglrtgainofaﬂﬁshinatankPZR, PPRandmortalitydatawere subjected toan
analysis of variance (ANOVA). ALP and total bone Zn were subjected to a nested
ANOVA, where each ﬁsh per tank was considered a subsarnple. Analysis of covariance
was used to detect treatment effects on plasma insulin concentration, whole ﬁsh Zn, P and
protein content and bone Zn and P concentrations. The covariate for insulin was
postprandial time (Navarro et al. 1993). The covariate for whole ﬁsh Zn, P and protein
content and bone Zn and P concentration was ﬁsh weight (Shearer 1994).

Signiﬁcant differences between means (P < 0.05) were identiﬁed in nonorthogonal

contrasts using the Bonferroni t statistic (Gill 1978). Statistical tests were performed

66

using the SAS software Mixed procedure, with tank(treatment) as a random variable (SAS

Institute 1997).

The nonorthogonal contrasts were designed to answer the following questions:

1. Did dephytinized soybean meal improve ﬁsh performance (contrast NTO v T0)?

2. Does a diet containing both dephytinized soybean meal and dephytinized corn
gluten meal improve ﬁsh performance (contrast NTO v TCO)?

3. Did dephytinized soybean meal with 50 mg ang‘1 diet improve ﬁsh performance
(contrast NTO v T50)?

4. Is ﬁsh performance improved by using dephytinized corn gluten meal in a diet
which already contains dephytinized soybean meal (contrast T0 v TCO)?

5. Do ﬁsh fed a diet containing dephytinized soybean meal plus 50 mg Zn-kg" diet
perform as well as ﬁsh fed a diet containing both dephytinized soybean meal and
dephytinized corn gluten meal (contrast T50 v TCO)?

6. Did the addition of 50 mg Zn-kg'l to the basal diet improve ﬁsh performance
(contrast NTO v NTSO)?

7. Do 50 mg Zn-kg" diet and dephytinized soybean meal equally aﬁ‘ect ﬁsh
performance (contrast NTSO v T0)?

8. Does adding 50 mg Zn-kg“ diet to a diet containing dephytinized soybean meal
improve ﬁsh performance (contrast T0 v T50)? . I

9. Did ﬁsh fed the basal diet perform as well as ﬁsh fed the reference feed (contrast

NTO v R)?

RESULTS

Final values for weight gain, whole ﬁsh protein, Zn and P content, bone Zn, P and
ashconcentration, PZIL PPR speciﬁcactivityofALPandCPB andplasmainsulin
concentration are summarized for each diet in Table 8. Results of the nonorthogonal
contrasts are summarized in Table 9. Dietary treatment signiﬁcantly aﬂ‘ected all variables
except whole ﬁsh protein and PPR Signiﬁcant diﬂ‘erences between treatments were not
found for weight gain or plasma insulin concentration using the pre-planned contrasts
(Table 9). Fish fed the experimental diets averaged a 4.5-fold weight increase during the
experimental period. No clinical signs of Zn deﬁciency were observed.

Substituting dephytinized soybean meal for untreated soybean meal in the basal
diet did not affect ﬁsh performance (contrast NTO v T0; Table 9). Fish fed diets
containing both dephytinized soybean meal and dephytinized corn gluten meal had higher
bone Zn concentrations and total bone Zn than ﬁsh fed the basal diet (contrast NTO v
TCO). Bone Zn and ash concentrations and total bone Zn were also increased when ﬁsh
were fed the diet containing dephytinized soybean meal and 50 mg Zn-ltg’l diet (contrast
NTO v T50).

Using dephytinized corn gluten meal in diets containing dephytinized soybean meal
increased whole ﬁsh P, bone ash concentration and/or bone P concentration, but did not

aﬁ'ect whole ﬁsh or bone Zn (contrast T0 v TCO and T50 v TCO).

67

68

 

 

 

 

.«2 «an. .3. an: 3." ...«2 .\. ......

3: a: ...: .3 is. «an .x. «N.
8. ..." Sn 8. ... o: 8 a «a 8. a .2 a a .2 a. a n: a: ..N 2.3 .58.
.... a «.3 3. a 2k ...N a ...m 2 a a: ...N a 2m o." a 2n 25.. .\. .3.
S. a 2n 3 a a...“ ...n a 3n 3 a 3n ...n a an an a ...a 2.8 .3... ..
an a 2.. .... a 8. an a on. S. a a. 9. a 8. R a s. 2.3 3.»: ..N

26m
2... a a... an... a 2.... 3... a 92 a... « ...... S... a. a... a... a 8+ in ..maa ..
8... .... 2.. 8... a 8.. 3.... a 82. ”2. a 8.. 8... a. «a... an... a 2.. ..e. ..mae ..N
a... « ...... 3.... a 3. a... a 3.. z... a S. a... a he. a"... a 3. a... ..m.» 5.2..

...... 0.2.3

a... « ...... ... a as. ... a S... ... « ...: .... a is ... a 92. a a...» 2303

8... any 8 an: 8.2 ... ...5 2.35

.mm « mos—.3

...N 35539:» SE5 35:53.: wedge—5.. ..o 5.55.... 5.38:8 ....omv 3.58598 5.. mac: 3358 ..o 80.5.5050
....a... .52... as .88 m 83.5338 ..5 Es gases... 8.3... ..o 5.6. uses was... 8.52 5.2..
.598.— ..AmNB 5:32 ..N .528 .28.. be 858.. v5 ﬁe 0.23 we 526883 355:. v5 .585 dim =30?

... 0.3.

69

.5... 502.8 53.25% 8 :03 8 .5... 5.33 Eco 52:535..
3......8 0.. 3.2.... ...»... 2.8.5 ...... ...... .... .. .....N 3552...... m... 8.8.... 22.... ...... 2.. ... o. ...... o 2852..
2... ....o>..8..§ .88 82...... 8.2.3.3.. ...... 3.8.25... 852:8 2.28.... a ...... $28.. .2... ..2 8.3.8.. ......

 

v
.56» .05 .58....qu 2.3 2.3 ..m.w3. S. .25 2.3 3...: 799.. 2.2. .25 .3. 22.3 ”w w... .2303 ”82.3 ...c 53.: .
.8. x AB. 55... F... 3.25m .565 m. u on... .3555... .585 .
do. x 60.. ..N 38.55% ..N me. n .5355. 25 2.85m .
on a a... m... a an. a... ”m ..o. .... a ...n. v... a 5.. ... « 3. 7.5.9. 5.8...
2 .o a. 3nd 2 .c a. Rte 36 ... 8nd and ﬂ 3:. S .c ... «and :6 n." 3m... £22.. 79...: GAO
36 a. wand 86 ... 3nd 8.: an «56 86 a 8nd mod n.. emu... «ed a. Sad .58...— .ME.D a

 

8.2.8. M. m......

 

£3»:—

 

 

 

x m8
x .3<
Em
x x x «Na
x - x - . x x - x 59318...
x x x x .3.
x m
x x x x x 5
m as
x x m
x ..N
£32m
.2. 223
- . . - - . - . 53 29»?
«>32 2852. 8.2.,8. .52: 28.8 2855 3:82 855 8.51:: 033:5
:33
..x. Bias 3 God v 5 ”55:8 .5835 .5.35 £83 a. Sagasas 2.. as :38 m 0233335
2.. $3 3329*23 2.3.. he £58 058% 2.. 2.5 3%.». €35 282 ...N 83 .52 .2an 8532
..N 88.8.. .53 v5 .— ...N 2.3 .m 93 =N £085 ﬁg 0.9.3 8.. 88:. E0882. no mach—.8 Some—taco: «o 2.33— .a 03:.

7 1

Supplementing the basal diet with 50 mg Zn-kg" diet resulted in increased bone Zn
concentration and total bone Zn (contrast NTO v NTSO). Adding 50 mg Zn-kg" basal diet
wasmoreeﬁ‘ectivethanusingdephytinized soybeanmealinincreasingtheboneln
concentration and total bone Zn (contrast NTSO v T0). Adding 50 mg Zn-kg" diet to a
diet containing dephytinized soybean meal increased bone ash concentration, but not bone
Zn concentrations (contrast T0 v T50).

Whole ﬁsh Zn, bone Zn concentration, total bone Zn and the speciﬁc activity of
ALPandCPB weregreaterinﬁshfedthereferencefeedthaninﬁshfedthebasaldiet
(contrast NTO v R).

PZR was highest in ﬁsh fed diets without supplemental Zn (Table 8 and Figure 4).
ThePZRofﬁshfedthebasaldietwasincreasedwhen dephytinized soybeanmealand
dephytinized corn gluten meal were used (contrast NTO v TCO).

Bone Zn concentration was negatively correlated with dietary phytic acid:Zn molar
ratios (Figure 5). Total bone Zn was signiﬁcantly lower in ﬁsh fed diet NTO than in ﬁsh
fed NTSO, T50, TCO, and R (Table 8). However, total bone Zn increased during the
experiment in all ﬁsh regardless ofdietary treatment. Total bone Zn in ﬁsh fed diet NTO
increased ﬁom 80.0 pg at the beginning of the experiment to 231 ug at the end, a 293%
increase.

A 16% mortality rate was observed over the course of the experiment. The
distribution of the mortalities was not related to dietary treatment. Examination of
moribund ﬁsh by the MSU Animal Health Diagnostic Laboratory revealed that ﬁsh were

infected with the fungus Saprolegnia spp.

.82 beam as a. as 88E». 2:. as .5 we. .2.
eN 3580—38 an. 3.8%.: an Ea a £38.... 2:. .16.: 5238 cacobomszﬁ 3 :03 8 .8:— =82w E8 3.8.5.033.—
wogeeo c8. 85 ..nozuoonma .12: 5238 vsgbéﬁ 23 3.8.25 355:8 L: v5 .92. 33563 “”85 .303

BEEF?» $382 as. 335.: a E ..N .53 2.. 25: 8552 ..N 52.. .522. 8252. @2829: e 2%:

 

 

3.5 B. ..N .35
9; . c2 8— 8 8 9. am o
P P L P “I
o
a I o
n .
4 4 O r n
n u 4 o
m
I o—
x
M .. D I
08 + + D T W—
omb o
8. x Ia
03.2 4
8.2 0 ++

 

 

 

 

.nN

73

@—

  

.ozﬁ Ben. 56%: 03.3.. .083“. 05 23 0:3 :5... 3358 E =N we 533.8050 2.. e833 manages—ox .n 23E

one 3.2: 56.8 cub—q E05

3 N— 2 w e v N

88... u «x
92: + 52.. a a

 

T59 be 03.5 3»: c

 

8—

to:

.. 3—

1 On—

1c:

u on—

.8—

.. ch—

18—

.8—

 

r 8N

auoq Km 9013-191 3N2 8n

DISCUSSION

Fish fed the basal diet were not Zn deﬁcient after 170 d. The ﬁnal concentration of
Zninbonewaslowerinﬁshfed dietNTOthaninﬁshfedtheotherdiets, but normalPPR
and speciﬁc activities of ALP and CPB indicated there was no functional deﬁciency.
Clinical signs of absolute Zn deﬁciency such as cataracts or ﬁn erosion (Ogino and Yang
1978) were not observed. Further, increases in total bone Zn of ﬁsh fed diet NTO
indicated that a Zn deﬁciency would not have developed even if ﬁsh were fed diet NTO for
an extended period. Therefore, enough Zn was available in the high-phytate, plant-based
diet to meet the metabolic requirements of juvenile rainbow trout.

Weight gain and body composition of ﬁsh fed the experimental diets were similar
to values reported for rainbow trout (Shearer 1984; Cain and Garling 1995). Although
ALP and CPB activities were reduced in ﬁsh fed the basal diet (NTO) relative to ﬁsh fed
the commercial reference diet (R), weight gain, whole body protein content and PPR were
not signiﬁcantly diﬁ‘erent. Possibly, phytic acid in diet NTO directly interfered with
enzyme activity in the lumen. Phytic acid has been shown to interfere with the activation
of trypsinogen and reduce the stability of trypsin (Caldwell 1992).

Blood samples were drawn approximately 1 h after feeding, when concentrations

of plasma insuﬁn should have been at their highest (Navarro et al. 1993). The

74

75
concentrations of plasma insulin observed in the present experiment were comparable to
values observed for rainbow trout l h after being fed commercial feeds to satiation
(Sundby et al. 1991; Navarro et al. 1993). Based on growth, ALP and CPB activity, total
boneZn, arrdnormal concentrations ofplasmainsulin, ﬁshinthepresentexperirnentwere
not Zn deﬁcient. However, since decreases in insulin secretion and insulin sensitivity (i.e.,
clearance) have both been observed in Zn-deﬁcient animals (Kirchgessner and Roth 1983;
Park et al. 1986), it may have been possible to observe normal plasma insulin
concentrations in Zn-deﬁcient ﬁsh.

CPB activities have not been previously reported for rainbow trout. The speciﬁc
activity ofCPB foundinthepresent studywassimilartothespeciﬁcactivityofCPB in
extracts of the pancreas and pyloric caeca of cod (Overnell 1973), carp (Cohen et al.
1981) and catﬁsh (Yoshinaka et al. 1984b). Thespeciﬁc activity ofALP in intestinal
homogenates was similar to the speciﬁc activity of ALP in scrapings of intestinal mucosa
in rainbow trout (Gasser and Kirschner 1987a).

Adding 50 mg Zn-kg" diet was a more eﬁ‘ective way of increasing bone Zn
concentration and total bone Zn than using dephytinized soybean meal (contrast NTSO v
T0, Table 9). Both dephytinized soybean meal and dephytinized corn gluten meal were
required in the diet to increase bone Zn concentration and total bone Zn relative to values
inﬁshfedthebasaldiet. Thesoybeanmealusedinthisexperimentcontainedwmg
Zn°kg". Consequently, dephytinized soybean meal only contributed 12 mg Zn-kg“ diet.
Dephytinized soybean meal together with dephytinized corn gluten meal (29 mg Zn-kg“)

contributed a total of 21 mg Zn-kg‘l diet.

76

PZR decreased as total Zn fed increased (Figure 4). The observed trend supported
an earlier ﬁnding by Spry et al. (1988). They reported that as dietary Zn concentration
increased, theconcentrationonninwholerainbowtroutalsoincreased, butata
diminishing rate.

Znstatusinﬁshhasbeenassessedusingmanycriteriaincludinggrowth, protein
eﬁciency, feed eﬁciency, and the concentration of Zn in liver, kidney, plasma, scales,
bone, or the whole ﬁsh. On rare occasions, serum ALP activity or protein digestibility
have been used as indicators onn status (Ogino and Yang 1978; Gatlin and Wilson 1983;
MaageandJulshamn 1993). The dietaryanequirementsofﬁshhavebeenbasedonthe
dietary Zn concentration resulting in bone Zn saturation because bone Zn concentration
was very sensitive to increasing dietary Zn concentrations (Gatlin and Wilson 1983;
Maage and Julshamn 1993). For most ﬁsh fed puriﬁed diets, 15-30 mg kag" dry diet
was the minimum concentration which resulted in bone Zn saturation (NRC 1993).
Although normal growth, feed eﬁciency, protein digestibility, and serum ALP activity
were observed in ﬁsh fed diets containing 5-10 mg Zn-kg" dry diet, decreased bone Zn
concentrations in ﬁsh fed the lower-Zn diets was interpreted as a trend toward deﬁciency
(Gatlin and Wilson 1983; Maage and Jushsamn 1993).

The dietary Zn requirement may be overestimated if it is equated with the dietary
Zn concentration that saturates bone. Since bone Zn is probably not available for
metabolic use in juvenile ﬁsh (O'Grady and Abdullah 1985), a high concentration of bone

Zn indicates excessive Zn intake. The present study demonstrated that it was possible for

77
diets to provide adequate Zn for metabolic requirements without maximum Zn deposition
in bone. Similar results have been reported for rats fed low-Zn diets (Zhou et al. 1992).

To determine if decreased concentrations of Zn in ﬁsh bone really represents a
deﬁciency or a trend toward deﬁciency, changes in total bone Zn should be measured
(Zhou et al. 1992; Bobilya et al. 1994). For example, Maage and Julshamn (1993) fed
Atlantic salmon a practical diet (17 mg native Zn-kg")' supplemented with
0-80 mg Zn-kg'l dry diet. Based on dietary Zn concentrations that saturated bone and the
whole body with Zn, they recommended supplements of 20-40 mg kag" for that diet.
However, when their bone Zn concentration data was recalculated and expressed as total
bone Zn, it was revealed that total bone Zn increased in all ﬁsh, including those fed the
unsupplemented diet. Increases in total bone Zn implied that the diets provided Zn in
excess of metabolic requirements. Assessing Zn status using total bone Zn may have lead
to the conclusion that native dietary Zn was adequate.

The phytic acid:Zn molar ratio has not been used as an indicator of Zn availability
in ﬁsh diets. However, data ﬁom the present study and recalculated data from Gatlin and
Phillips (1989) and Satoh et al. (1989) suggested that the concept could be applicable to
ﬁsh (Figure 6). The concentration of Zn in ﬁsh bone had an apparent negative relationship
with the dietary phytic acid :Zn molar ratio. The dietary phytic acid:Zn molar ratio could
be an important tool for formulating diets once its impact on ﬁsh performance is more

clearly deﬁned.

 

8

DietsofMaageandJulshamn(l993)containedlSOgcornmeal-ltg"drydict Basedonapproximately9
sphyﬁcacid-kg'commeal (HarlandandOberleas 1987),thedietscontained 1.4 gphyﬁcacid-kg'dry
diet.

.32. 335.: E! 5.58 352—0 .8.“ e38 Sea :8an 23.3 30% as. couches—.8 :N veep e853 Baa—.233— .e 25E

 

 

2.8 8.2: .565. 03.3." .9305
8 R 8 an 2. on 8 2 o
P k p b p p p p c
T a
X
o x
. 8.
n o o x
X
o 1 c2
0
9
0
e o o
o . 8N
. an
o
o
3...: Ease. 33.3 o o . 8m
88. a. .0 ..eé £2.28 35.20 x
38. .35.: 2.. £38 3.8 .355 o

 

 

r can

woq mp 9:19-19} 3/"2 3n

SUMMARY AND CONCLUSIONS I

Feeding plant-based diets containing untreated soybean meal and no supplemental
Zn to rainbow trout for 170 (1 did not result in a Zn deﬁciencybased on growth, whole
ﬁsh Zn and protein composition, total bone Zn, and the activity of ALP and CPB. The
opposite conclusion may have been drawn ion status had been assessed using the
traditional indicator, the concentration of Zn in bone.

No beneﬁt in terms of weight gain or ﬁsh protein content was realized from
dephytinizing dietary ingredients or adding supplemental Zn to the plant-based diets.
However, some Zn supplementation may be required for other feed formulations. The
dietary Zn requirements of salmonids fed standard commercial forrmilations need to be
reassessed based on changes in Znodependent metabolism or total bone Zn, possibly in

relation to the dietary phytic acid :Zn molar ratio.

80

SUMMARY AND CONCLUSIONS .

Feeding plant-based diets containing untreated soybean meal and no supplemental
Zn to rainbow trout for 170 d did not result in aZn deﬁciency based on growth, whole
ﬁsh Zn and protein composition, total bone Zn, and the activity of ALP and CPB. The
opposite conclusion may have been drawn if Zn status had been assessed using the
traditional indicator, the concentration of Zn in bone.

No beneﬁt in terms of weight gain or ﬁsh protein content was realized from
dephytinizing dietary ingredients or adding supplemental Zn to the plant-based diets.
However, some Zn supplementation may be required for other feed formulations. The
dietary Zn requirements of salmonids fed standard commercial formulations need to be
reassessed based on changes in Zn-dependent metabolism or total bone Zn, possibly in

relation to the dietary phytic acid :Zn molar ratio.

80

LIST OF REFERENCES

Anderson,R.O.andS.J.Gutreuter.1983.1.ength,weight,andassociatedstructmalindices.Pams283-
300inL.A.NielsenandD.L.Johnson,editors Fisheriestechniques.AmericanFisheries
Society,Bethesda,Maryland

AOAC(AssociationofOﬁcialAmlylicalChemisls). 1984. Oﬁcialmethodsofanalysis. 14thedition.
AssodaﬁmofOﬁdﬂAnﬂyﬁcalChuniﬂgAﬂmvaSA.

ARIA(Amedeathc}hahhAsmdaﬁm),AmuimnWatanksAmodaﬁomandWdehmm
ControlFederation. 1985. Standardmethodsfortheercaminationofwaterandwastcwater, 16th
edition. American Public Health Association Washington, DC.

AsgardT.andK. D. Shearer. 1997. DietaryphowhorusrequirementofjuvenileAﬂanticsalmomwmo
salarL. Aquaculture Nutrition 3:17-23.

Asgﬁrd, T., Langaker, RM, Shearer, KD., Austreng, E. and A Kittelsen. 1991. Rationoptimintion to
reducepotentialpollutants-preliminaryremlts. AmericanFisheriesSoeietySymposium 10:410-
416.

Atkinson, J.L., Hilton. J.W. and SJ. Slinger. 1984. Evaluation «acid-insoluble ash as an indicator of
feeddimstibilityinrainbowtrwﬂSabnogairdnerl). CanadianJournalofFisheriesanquuatic
Science 41:1384o1386.

Axler,R,Larsen,C.,Tikkanen, C.,McDonald,M.,Yolrom, S.andP. Aas. 1996. Waterqmlityissues
assodawdwimmmezamxsnnymmimpithkesWatuEnvimmReswchﬂzws-
1011.

Bai, S. C., Nematipour, G. R, Perera, R P., Jaramillo, F., Jr., Murphy, B. R. and D. M. Gatlin Ill 1994.
Tmalbodydecuimlmnmwﬁvityformndesmmwmeammmentofbodymmposiﬁmofred
drum. Progressive Fish-Culturist 56:232-236.

Bavor, HI. and D.S. Mitchell (editors). 1994. Wetland systems in water pollution control Proceedings
ofthe IAWQ 3rd International Specialist Conferenceon Wetland Systems in WaterPollution
Control, Sydney, Australia 23-25 November 1992. Water Science and Technology 29:1-336.

BelonjeP.C.andA.VandenBerg 1980.1'heuseoffemlanalysistoestimatetbephosphomsintakeby

yazingsheep.n.Thempambﬂitydthetthqmandmemﬂmofvarymgphosphons
imake.0nderstepoortJoumalofVeterinaryResearch47:l69-172.

Bergheim,A.,Aabel,J.P.andE.A. Seymour. 1991. Pastandpresentapproachestoaquamlmrewaste
mnagementinNorwegiannetpenculnn'eoperationsPaged117-136inC.B.CoweyandC.Y.
Cho, editors Nutritional StrategiesinManagementoquuaculmre Wastes

81

82

Bergheim,A.,Hustveit.I-I.,Kittelsen,A.andA.R. Selma-Olsen. 1984. Estimatedpollutionloadings
from Norwegian ﬁsh farms II. Investigations 1980-1981. Aquaculture 36:157-168.

Bobilya, DJ., Jolnnning, GI... Veum, TL. and BL. O’Dell. 1994. Chronological loss ofbone zinc
during dietary zinc deprivation in neonatal pigs. American Journal of Clinical Nutrition 59:649-
653.

Boerson, 6. audit Westers 1986. Waste solidsin lutchery raceways. Progress’veFish—Culnnist
48:151-154.

3096, G.,Balocco, C.,Roche, H. andG. Brichon. 1993. Theinﬂuenceofcalciumandmagnesimnonaea
bass (Dicenrmrclms labrax) intestinal brush border membrane puriﬁcation and activity.
Comparative Biochemistry and Physiology 106A:227-232.

Bradford, M. M. 1976. A rapid and sensitive method for the quantitation ofmicrogram quantities of
protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248-254.

Brown, PB. 1993. Comparison of fecal collection methods for determining phosphorus absorption in
rainbow trout. Pam 443-447 in S. J. Kaushik and P. Luquet, editors. Fish nutrition in practice.
[NRA Paris.

Brown, P.B.,Robinson,EH andG. Finne. 1989. Nutrientconcentrationsot‘catﬁshfewsandpractical
diets alter immersion in water. Journal of the World Aquaculture Society 20:245-249.

Buschmann,A.I-I.,Mora,0. A.,Gomez,P.,B6ttger,M,Buitano,S.,Retamales,C.,Vergara,P. Aand
A. Gutierrez 1994. Gracilaria chilensis outdoor tank cultivation in Chile: use of land-based
salmon culture efﬂuents. Aquamltural Engineering 13:283-300.

Cain,KD.andD.L.Garling 1995. Pretreatmentofsoybeanmealwithphytaseforsalmoniddietsto
reduce phosphorus concentrations in hatchery eﬁluents. Progressive Fish-011m 57:114-119.

CaldwelLRA. 1992. Eﬁ‘ectofcalciumandphyticacidontheactivationoftrypsinogenandthestability
of trypsin Joumal of Agricultural and Food Chemistry 40:43-47.

Carlander, K. D. 1969. Handbook of freshwater ﬁshery biology, volume 1. Iowa State University Press,
Ames, Iowa.

Carlander, K. D. 1977. Handbook of ﬁeshwater ﬁshery biology, volume 2. Iowa State University Press,
Ames, Iowa.

Cho, C.Y., Hynes, J.D., Wood, RR. and HK Yoshida. 1991. Quantitation of ﬁsh culture wastesby
biochemical (nutritional) and chemical (limnological) methods, the development of high nutrient
dense (I-IND) diets Pages 37-50 in CB. Cowey and CY. Cho, editors. Nutritional Strategies in
Management of Aquaculture Wastes.

Cho, C.Y., Slinger, SJ. and H.S.Bay1ey. 1982. Bioenergeties of salmonid ﬁshes Energy intake
expenditure and productivity. Comparative Biochemistry and Physiology 73B:25-41.

83

Clark,E.R, Harman,J.P. andJRMForster. 1985. Productionofmetabolicwasteproductsby
intensivelyfarmedrainbowtrout, Sabnogairdun' Richardson JournalofFish Biology 27: 381-393.

C1ydesdale,F.MandAL.Camire. 1983. Eﬁ’ectoprandheatingonthebindingofironcalcimn,
magnedmandzincandthelossofphyticacidinsoyﬂour.JournalofFoodScienoe48:1272.

Cohen,T.,Gert1er,AandY.Birk 1981. Pancreaticproteolyticenzymesﬁomcarp(Cypr-imrscarpio)-
Lmandphyawpmascruypmchymmdmmmpema.
ComparativeBiochemistryand Physiology 69B:639-646.

Colin,D.A., Nonnotte,G., Leray, C. andL. Nonnotte. 1985. Natransportandenzymeactivitiesinthe
Mesﬁneoftheﬁeshwaterandm-wateradapteduorn(Sabnogair¢breﬁiR). Comparative

Biochemistry and Physiology 81A. 695-698.

Cosgrove, DJ. 1980. Inositol phosphates: their chemistry, biochemistry, and physiology. Elsevier, New
York

Davies, N. T.andS. E. Olpin 1979. Smdiesonthephytatezzincmolaroontentsindietsasadeterminant
onn availabilitytoyotmg rats. BritishJom'nalofNutritim 41:591-603.

Davies,N.T.andH.Reid. 1979. Anevaluationofthephytate,zinc,copper,ironandmanpnese
Motandlnwﬁhbmtyﬁomwya-basedmxmred-wgaabb-mdnmemwumsm
meaterctenders BritishJournal of Nutrition 41:579-589.

Downing,J.A,Plante,C.andS.Lalonde. 1990. Fishproductionoorrelatedwithprimaryproductivity,
namemomhoedaphicmdmaCanadimennﬂofFishaiesanquuaﬁchm47zl929-
1936.

Droke, E.A., Spears, J.W., Armstrong, J.D., Kegley, E.B. andR.B. Simpson 1993. Dietary zinc aﬁects
serumconcenuaﬁonsifinsmmandmsdm-ﬁkegrowthfactorlingroadnglambs Journalof
Nutrition 123213-19.

EIFAC(EuropeanInlandFisheriesAdvisoryCommission). 1982. ReportoftheEII-‘ACmrltshoponﬁsh
farm emuents, Silkeborg. Denmark, 26-28 May 1981. EIFAC Tech. Paper 41.

Eloranta,P.andA.Palomaeki. 1986. PhytoplanktoninlakeKonnevesiwithspecialreferenceto
eutrophicationofthelakebyﬁshfarming. AquaFennica 16:37-45.

E1ser,J.J.,Marzolf, ER andC.R Goldman 1990. Phosphorusandnitrogenlimitationofphytoplankton
mmmeﬁeshmmofNonhAmuimzamvkwanduiuqmofexpuimmlmﬁchmenm.
CanadianJournalofFisheriesanquuatic Sciences-17214684477.

EnelLM. 1995. Environmentalimpactofmm'ientsfromNordicﬁshfarming WaterScienceand
Technology 31:61-71.

Ewing, RD., Walters, TR, Lewis, MA. and JE. Sheahan 1994. Evahration of inventory procedures for

hatcheryﬁsh LEstimatingweightsofﬁshinracewaysandﬁanspontruckstgrersiveFish-
Culturist 56:153-159.

84

FergusonE.L.,Gibson,RS.,‘I'hompson,L.U.andS.Ounp|m.1989.Dietaryca1cium,phytate,and
zimmmkesandthecaldmphymteandzimmdmmﬁmofthediasofaselededgrmmof
EastAfricanchildren AmericanJournalofCIinical Nutrition 50:1450-1456.

Folk, C. andN. Kautsky. 1989.1hem1eofecosysternsforamstainabledevelopmernofaquamlune.
Ambio 18:234-243.

Folk, J. 1971. CarboxypeptidaseB. Pages57-79in P.D. Boyer, editor.TheEnzymes,vohrme3.
HydrolysiszpeptidebondsAcademicpress,“

Folk,J.AndJ.Gladner. 1958. CarboxypeptidaseBlPuriﬁationd’thezymomandspeciﬁcityofthe
enzyme. Journal of Biological Chemistry 231:379-391.

Folk, J. E. andE. W. Schirmer. 1963. ‘I'heporcinepancreaticarboxypeptidaseAsystern Journals!
Biological Chemistry 238:3884-3894.

Fondyce, E.J., Forbes, RM, Robbins, KR. and J.W. Erdnmn, Jr. 1987. Phytate x calcium/zinc molar
ratios: areﬂreypredicﬁwofzimbioavailabiﬂtyﬂournalofFoodeence52z440444.

Fosset, M., Chappelet-Tordo, D. and M. Lazdrmski. 1974. Innstirnl alkaline phosphatase: physical
properties and quaternary structure. Biochemistry 13:1783-1787.

Foy,RH.andR.Rose11. 1991a. loadingsofnitrogenandphosphorusﬁomaNorthernlrelandﬁshfarm.
Aquaculture96zl7-30.

Foy,R.H.andRRose11. 1991b. FractinationofphosphonsandnitrogenloadingsﬁomaNonhern
Irelandﬁshfarm. Aquaculture 96:31-42.

Gasser, KW. andL.B. Kirscher. 1987a. ‘I‘heinhmitionanddispositionofintestinalalkaline
phosphatase. Journal of Comparative Biochemistry and Physiology 1573:461467.

Gasser, KW. andL.B. Kirscher. 1987b. 'I'heresponseofalkalinephosphatasetoosmoreguhtory
changesintheuouLSalmogairdner-i. Janualot‘ComparativeBiochemistryandPhysiology
157Bz461-467.

GatlinDM. IIIandHF.Phi11ips 1989. Dietarycalcium,phytateandzincinteractionsinchanne1
catﬁsh Aquaculture 79:259-266.

GatlinDM. andRP. Wilson 1983. Dietaryzincrequirementofﬁngerlingchannelcatﬁsh. Journalof
Nutrition 113:630-635.

GatlinD.M.II1andR.P.Wilson 1984. Zincsupplementationofpracticalchannelcatﬁshdiets.
Aquaculune41:3l-36.

GelmanA,Cogan,U.andS.Mokady. 1992.1‘hethermalpropertiesofﬁshenzymesasapossible
indicatmofmetmnpaanmadaptaﬁonpmenﬁﬂoftheﬁshCompamﬁwBiochamMand
Physiology 1018:205-208.

Gill, J.L. 1978. Designandanalysisofanimaleuperiments IowaStateUniversityPress,Ames,Iowa.

85

G1ass,I-1.J.,MacDonald,L.andJRStark. 1987. Warninmarineﬂatﬁsh-N.Carbohydrateand
protein dimstion in Atlantic halibut (Hippoglams hippoglossus L.) ComparativeBiochemistry
andPhysiology86B:281-289.

Gomes.E.F.,Corraae,G.andS.Kaushik1993. Eﬁ'ectsofdietaryincorporationofaco-errtrudedplant
prudn(mpeseedandpas)ongrowth,mmiemmilinﬁmandmxbfanyaddmmpodﬁmof
rainbowtrout(0neorhynchus nrykr'szr). Aquaculture 113:339-353.

GomeaEF.,Rema,P.,Gurveia,AandA.Oliva-Teles1995.Rep1acementofﬁshmealbyplant
prudmmdiasfmminbowm(0nwrhynahun9vﬂs)zeﬁeadtheqmlhydtheﬁshmeal
hasedmmoldiasmdigsﬁbihtyandmniemwamWdeemeandehmhgynzms-
211.

Gomori,G. 1942. Amodiﬁcaﬁonofthecolorimetricphosphcrusdeterminaﬁonforusewiththe
photoelectriccolorimeter. Journaloflaboratoryand ClinicalMedicine 27:955-960.

Greider,M.H,Howell,SL.andP.E. Lacy. 1969.1solationandpropertiesofsecretorygrannlesﬁomrat
isletsofLangerhans. II. ultrastructure ofthebetagramrle. JournalofCell Biology 41:162-166.

Gutierrez,J., Carillo,M., Army, 8., andJ.P1anas. 1984. Dailyrhytthd‘insulinandglucoselevelsin

theplasrnaofseabassDiceno-archuslaboraraﬁerexperimentalfeeding GeneralComparative
Endocrinology55: 393-397.

I-Iakanson,L. 1986. Fishcagearlnne-mwhichcoanalenvironmmn7Pays52-57rnNaﬁonaISwedish
BoardofFisheries InstituteofFreshwaterResearch,ReportNo. 63.

Han, Y.W., Gallagher, DJ. and A.G.Winﬁed. 1987. Phytaae production byAszmgilIrrsﬁcum on
semisolid substrate. Journal oflndustrial Microbiology 2:195-200.

Hardy, RW. 1989. Diet preparation. Pages 475-547 in J. E. Halver, editor. Fish mrtrition Academic
Press, New York.

Hardy,R.W.andK.D. Shearer. 1985.Eﬁ‘ectofdietarycalciumphosphateandzincsupp1emeutationon
wholebodyzincconcentrationofrainhowtrout(8ahnogalrdreri). CanadianJournalofPisheries
anquuaticScienws42:18l-184.

Hardy,RW., Sullivan, C. V.andA.MKoziol. 1987. Ahsmpﬁmbodydisuibutionandertcretionof
dietaryzinchyrainbowtrout (Salmo gairdnen'). FishPhysiologyandBiochemistry 3:133-143.

MB.F.andD.Grerleas. 1987. Phytateinfoods. WorldRcviewot'NtmiﬂonandDietetics52235-
259.

I-Iecht, S. 1916. Form and growth in ﬁshes. Journal of Morphology 27:379-400.

HeinenJ.M.,Hankins,J. AandM. Subramanyan 1993. Evaluationoffourcommeroialdietsfor
rainbowtrout. Progressive Fish-Culturist 55:265-269.

Hines. 1984.11neﬂ‘eaofenvimnmemﬂandurdogmmfaaasmbhodmnsﬁumofmiﬂrow
trout(Salmogairdren)-I. Foodeomentofstomachandintestine. ComparativeBiochemistryand
Physiology77Az311-3l4.

86

Hinshaw, RN. 1973. Pollution as a result of ﬁsh culture activities. USEPA Report EPA-R3-73-009,
Washington. DC

Holby,O. andP.O.J.I-Ia11.1991.Chemica1ﬂuxesandmassbalancesinamarineﬁshcagefarnr IIP.
Marine Ecology Progress Series 70:263-272.

Holcombe, G.W., Benoit, DA and EN. Leonard 1979. Long-term effects ofzinc exposures on brook
trout (Sahelinusﬁmrinalis). Transactiom of the American Fisheries Society 108:76-87.

Holechek, J.L., Galyean, J.D., Wallace, JD. and H. Woﬂ'ord. 1985. Evaluation of fecal indices for
predicting phosphorus status of cattle. Grass and Forage Science. 40:489-492.

Holechek,JL.,Vavra,M.andP. Arthrn. 1982. Relationshipsbetweenperformance,intake,dietnutritive
qrmhtyandfecalmmiﬁwquahtyofcaukmmmmminmngeJmnnﬂofRangeMamgemem
35:741-744.

Howery,L.D. andJA Pﬁster. 1990. Dietaryandfecalconcentrationsofnitrogenandphosphonrsin
penned white-tailed deer does. Journal of Wildlife Management 54:383-389.

Hsu,J.M., Anilane,J.K. andD.E. Scalon 1966. Pancreaticcarboxypeptidaseactivitiesinn‘nc-deﬁcient
rats. Science 153:882-883.

Ince,B. W. andA. Thorpe. 1977.6hrcoseandaminoacid-stimulatedinsulinre1easeinvivointhe
European silver eel (Anguilla anguilla L.). General Comparative Endocrinology 31:249-256.

Ince,B. W.andA.Thorpe. 1978. Theeﬁ'ectsofinsulinonplasmaaminoacidlevelsintheNorthern
pikeEsox lucius L. Journal of Fish Biology 12:503-506.

1wama,G.K. 1991.1meractiombetweenaquaaﬂmmandtheenvimnmentCriﬁcalReviewsin
EnvironmentalControlZl: 177-216.

Johnston, C.E., Horney, B.S.,De1uca, S.,MacKenzie, A., Bales, J.G. andR Angus. 1994. Changesin
MmphosphammisxnzymeacﬁﬁtymﬁsnnsandphsmaofAthnﬁcmlmoMSabuomla)
bdomandduﬁngsnohiﬁcaﬁmandgonadalmahnaﬁonFishPhyﬁobgyandedunisuy
12:485-497.

Johnston, N.T., Perrin, C.J., Slaney, P.A. and B.R Ward. 1990. Inaeased juvenile salmonid growth by
whole-river fertilization Canadian Journal of Fisheries and Aquatic Sciences 47:862-872.

Jones,J..RandM.V Hoyer. 1982. Sportﬁshharvestpredictedbysummerchlorophyllaconcentrationin
midwesternlalnesandreservoir. TransactionsoftheAmericanFisheriesSocietylll: 176-179.

Joyner, T. 1961. Exchangeofzincwithenvironmentalsolutionsbythebrownbullhead. Transactionsof
theAmericanFisheriesSociety90:444-448.

Kendra, W. 1991.Qualityofsalmonidhatcheryeﬁ1uentsduring summerlow-ﬂowseason Transactions
of the American Fisheries Society 120:43-51.

Ketola,H.G. 1975. RequirementofAtlanticsalmonfordietaryphosphorus Transactionsofthe
American Fisheries Society. 104:548-551.

87

Ketolnl-LG. 1985. Mineralmrtrition: Eﬁectsd‘phosphorusintroutandsalmonfeedsonwaterquality.
Pages465-473inC.B.Cowey,A.M.Mackie,andJ.G.BelLeditorsNutritionandFeedingin
Fish AcademicPress,London

Ketola,H.G. andB. F. Harland. 1993. Inﬂuencesfphosphorusinraiﬁtroutdietson
dischargesineﬂluentwater. TransactionsoftheAmericanFisheriesSociety12221120-1126.

Ketola,I-LG.andM.E.Richmond. 1994. Requirunentofrainbowtroutﬁordietaryphosphorusandits
rdaﬁonshipmtheammmdischargedmlntchaydrmTramcﬁomoftheAmmican
FisheriesSociety123:587-594.

KetolnHG.,Westers,I-I.,I-Ioughton,W.andC.Pecor.1991.Eﬁectddietongromhandsurvivald

mhomlmnandmpmsphmusdischarpsﬁomaﬁshhnchay.Amm-icaanheriesSoday
Symposium 10:402-409.

Kim,JD.andS.J.Kaushik. 1992. Contributionofdipstibleenergyfromcarbohydramsandestimation

ofprdeinlenergquﬁmmemﬁorgrowmwminbowﬁuuwnwrhwchusmykiss). Aqrmculture
160:161-169.

Kirchpssner,M.andH.-P.Roth 1983.Nutritiona1inﬂuenceofzincontheactivityofenzymesand
hormones M363414inHSigeLedimr.Maa1iommbiologicalsymms,vohnn15.Zinc
anditsroleinbiologyandnutritionMaroelDekker,NewYork.

KisﬁmrnHaMKIhyashi.1992.PuﬁﬁaﬁmandpropaﬁadmmoxypepddmeB4ikemzyme
from the starﬁshAsrerias masts. Memoirsofthe Faculty ofI-Iokkaido University 43:169-176.

KortsteeG.J.J.,Appe1doorn,K.J.,Bonting,C.F.C.,vanNie1,E.W.J.andH.W.vanVeen. 1994.
B'loflll l'l "llinl ll'l'lll
removal. FEMS Microbiology Reviews 15:137-153.

Kraﬁ,C.E. 1992. Esﬁmﬂesofphosphonsandmuogencycﬁngbyﬁshmingabioenerydaappmach
CanadianJournalot‘FisherhsanquuaticScienws4922596-2604.

Krom,M.D. andANeor-i 1989.Atota1mrtrientbudgetforanexperimentalintensiveﬁshpondwith
circularlymovingseawater. Aquaculurre83:.345-358

Kuz’mina, V.V.andYe.G.an’mina. 1991. Characteristicsofaomedipstivetractenzymesinthe
sterlet,Acipenser ruthenrrs. Journal of Ichthyology 31:306-313.

Kuz’mina, V.V. andYe.G. Smirnova 1991. Distributionofalkalinephosphataseactivityalongthe
kngthrftheMesﬁneofﬁeshmmleoststnmld'ICMhyohgy3lz989-995.

Lall, SP. 1991. Digestibility, metabolismandexcretionofdietaryphosphorusinﬁshPagele-36in
CB. Cowey,A.MMadtie,andJ.G.Bell,editors NutritionandFeedinginFish. Academic
Press,London

launMandMEddn1980.Anmpkandmpidmlonmeuicmahodforphymtedaaminaﬁm.
JournalongriculturalandFoodChemistry28zl313-l315.

88

Lei, X.G., Kn, P.K, Miller, ER, Ullrey, DE. and MT. Yokoyann. 1993a. Supplemental microbial
phytaseimprovesbioavailabilityofdietaryzinctoweanlingpigs JourmlofNutrition 123:1117-1123.

Lei, X.G., Kn, P.K, Miller, ER and Mil“. Yokoyam. 1993b. Supplementing corn-soybean meal diets
with . l'lpl 1' I . l l l '1' . by 1' pig
Journal of Animl Science 71:3359-3367.

Lei,X.G.,Ku,P.K, Miller, ER, Yokoyama, M.T.andD.E. Ullrey. 1993c Supplementingcorn-aoybean
mealdietswithmicrobral. ' phytasemarnmrns’ ' phytatephosphorusutrlrzatron" ' byweanlmg’ pigs.
Journal of Animal Science 71:3368-3375.

1eslie,D.MJr.,Jenks,J.A.,Chi1e11i,M andG.RLavigne 1989.1«frtrogenanddiaminopimelicacidin
deerandmoosefeces JournalofWildlifeManagemeru532216-218.

Lo,..,GS Settle,..,SL Steinke,F..HandD.T1-lopkins. 1981. Eﬂ'ectofphytatezzincmolarratioand
isolatedsoybeanproteinonzincbioavailability. JourrralofNutritionlll: 2223-2235.

Lolas,G.M,Palamidis, N.andP.Markakis. 1976. Thephyﬁcaddmtalphosphormrelationshipin
barley, oats, soybeans, andwheat. CerealChemistry53:867-871.

LOnnerdaLB.,Bell,J.G.,1-Iendrid0t,A.G.,Brnns,RA.andC.L.Keen. 1988. Eﬁectofphytate
removalonn’ncabsorptionfromsoyformula. AmericanJournald’ClinicalNutrition48:1301-
1306.

Lott,J.N.A 1984. AccumuhﬁonofseedresmvesofphosphmusandothermineralsPagesl39-l66in
D.RMnrray,editor. SeedPhysiology,Volume1.AcademicPress,NewYork.

Lovell, RT. 1978. Dietary phosphorus requirement of clumnel catﬁsh (Ictalrrrus prmcratus).
Transactions of the American Fisheries Society 107:617-621.

Maage,AandKJmshamn1993.AsswanemofdmmmjumﬂeAdmdcmlmn(Sabnome)
bymeasurementofwholehodyandtissuelevelsofzinc.Aquaculmre 117:179-191.

McClainW.RandD.M.Gatlin,III. 1988.Dietaryzincrequirementof0reochromisaroerrsandeﬁects
ofdiaarymldumandphymmmﬁmbimvaihbiﬁtmemoftheWodqumﬂmreSoday
19:103-108.

MericanZHO andMJ. Phillips 1985. Solidwasteproductionﬁornrainbowtrout,Sabuagairdneﬁ
Richardsoncageculture. AquaaﬂmmandFishuiesMamgement16z.55-69

Millward,D. J. 1989. Thenutritionalregulationot'musclegrowthandproteinnnnover. Aquaculture
79:1-28.

Navarro, 1., Cameiro,M N.,Parrims,M,Maestro,J.L.,P1anas,J. andJ. Gutierrez 1993. Post-feeding
levelsot'insulinandglucagonintroutwalmorrurrafa'io). ComparativeBiochemistryand
Physiology 104Az389-393.

Nelson,T. S.,Ferrara,L. W. andN. L. Storer. 1968. Phytatephosphoruscontentoffeedingredients
derived from plants. Porrltry Science 47:1372-1374.

89

Nelson,T.S., Shieh,T. R, Wodzinski,RJ.andJ.H.Ware. 1971. Eﬁ'ectofsupplementalphytaseonthe
utilizationofphytatephosphorusbychicks. JornnalofNutrition 101:1289-1293.

Noda,H. 1967a. Snrdiesonvarimrsphosphatmesoftheﬁshes-MChanysintheactiviﬁesof
phosphahnsmrnngdewbpmemd'rainhowhmnSahuoiﬁdeustmald'Faamyd
Fisheries, Prefectural University ofMie 7:57-64.

Noda,H. 1967b. Sudiesmvariarsphospwansoftheﬁshes-N.Eﬂ‘eaofgromhupmphosphamses
acﬁviﬁuofminhomeabmmm.JmnmldFamhyd‘FkhaieaPreﬁecnnalUmvanty
ofMie7:65-7l.

Noda,H. 1967c. Smdiesonvariousphosphatasesoftheﬁshes-V. Variationinfourphosphatases
acﬁviﬁesmdngmsnngofminbowﬁmnSammdeustnnﬂowatyofFishuies,
PrefecnrralUniversityofMie7z73-80.

Noda,H.anrlS.Tachino.1965a. Surdiesonvariousphosphatasesot'theﬁshes-LEnzymemeasmements
Jourmlot'Faculty ofFisheriesPrefecnualUniversity ofMie 6:291-301.

Noda,H.andS.Tachino. 1965b. Strrdiesonvariorrsphosphatasesot‘theﬁshes-RDistributionof

phosphatasesintheﬁsh-organs JorunalofFacultyofFisheries,PrefectnralUniversityd‘Mie
6:303-311.

NRC (NatiomlResearch Council). 1993.Nutritiona1requirementsofﬁsh. National AcadernyPress.
Washington, DC.

Oberdorﬁ,T.andJ. P. Porcher. 1994. Aninderrofbioticintegritytoamssbiologimlimpactsof
salmonidfarmemuentsonreceivingwaters. Aquaculture 119:219-235.

Ober'leas,D.andB.F.Harland. 1981. Phytatecontentoffoods:eﬂ'ectondietaryzincbioavailability.
JournaloftheAmericanDieteticAssociation79z433-436.

Oberleas,D. andAS. Prasad.1976.Factorsaﬁ‘ectingzinchomeostasis. Pages155-162MAS. Prasad,
editor. Traceelementsinhumanhealthanddisease,Volume1. Zincandcopper. Academic
Press,NewYor1L

Ogino, C. andG-Y. Yang 1978. Requirementofrainbowtroutfordietaryzinc. NipponSuimn
Gakkaishi 44:1015-1018.

Ogino,C.andG-Y.Yang1979.Requirernemdcarpfordietaryzinc.BulleﬁnoftheJapaneseSociety
for Scientiﬁc Fisheries 45:967-969.

Ogino, C., Takerrchi, T., Takeda. H and T. Watanabe. 1979. Availability of dietary phosphorus in carp
and rainbow trout. Bulletin of the Japanese Society for Scientiﬁc Fisheries 45:1527-1532.

Oglesby, RT. 1977. Relationshipofﬁshyieldtolakephytoplanktonstandingcrop, production, and
morphoedaphic factors Journal of the Fisheries Resources Board of Canada 34:2271-2279.

O'Grady,R. T. 1981. 'I'heresorptionofzincfronrscalesofwatrout(Sabno outmduringtheupstream
spawningmigration Freshwater Biology 11:561-565.

90

O'Grady,K.T.andM.I.Abdullah. 1985. MobilityandresidencedlninbrowntoutSalnrao-rma:
Reantsofenvnonmenmllymdrwedchmgemrmghum.EnvironmenmlPoﬂmion 38A:109-
127.

Oliva-Teles, A.,Gouveia,A.J.,Gomes,E.andP.Rema. 1994.1'heeﬁ7ectofdiﬁ’erentprocessing

OvernelLJ. 1973. Digesﬁveenzymesofthepyloﬁccaecaandofthdrassodatedmesentuymthecod
(Gadrrsmorhua).ComparativeBiochemistryandPhysiology4GB:519-531.

Park, J.H.Y., Grandjean, C.T., Hart, M.H., Erdman, S.H., Pour, P. and LA. Vanderhof. 1986. Eﬁ‘ect of
prnezincdeﬁciencyonglucosetoleranceandinsulinandglucagonlevels AmericanJournalof
Physiology 251:E273-278.

ParipatananontT. andRT. Lovell. 1995. Chelatedzincredrmsthedietaryzincrequirernentot‘channel
catﬁsh, Ictalurus pram-rams. Aquaculture 133:73-82.

Pecor,C.H. 1992. PlatteRiverharvestweirandcohosalmonegg-takereport,1991. Michigan
DeparﬁnmtofNahnalResmrcesFishuiesDivisimTechﬁcalReponNumber92-3.

Pen,J., Verwoerd,T..C, vanParidonP.A., Beudeker,.,RF vandenElzen,P..,JM. GeerseK, vander
Klis,J..,D Versteegh,I-I.A.J., vanOoyenAJJ. andAHoekema. 1993. Phytase-containing

uansgenicseedsasanovelfeedaddiﬁveforinrprovedphosphorusunﬁzaﬁon Bio/technology
11:811-814.

Phillips,M.J., ClarkeR andAMowaL1993.PhosphorusleachingfromAtlanticsalmondiets.
Aquacultural Engineering 12:47-54. ‘

Pillay, T.V.R 1992. Aquacultureandtheenvironment. HalstedPress, John Wileyand Sons, Inc, New
York

Piper, RG., McElwain, I.B., Orme, L.E., McCraren, J.P., Fowler, L.G. and JR Leonard 1982. Fish
hatcherymanagement. United StatesDepartmentoftheInteriorFishandWildlife Service,
Washington, D.C., USA

Podoliak,H.A 1961. Relaﬁonbetweenwatertempuamreandmetabolismofdietaryphosphorusby
WhmokMTmnsacﬁonsofﬂreAmmicanFisheriesSoddyNﬁMl

Prahl, J.W. andH. Nernath. 1966. PancreaticenzymesofthespinyPaciﬁcdogﬁsh II.
Procarboxypeptidase B and carboxypeptidase B. Biochemistry 524137-4145.

Prakash,A. 1960. Someobsuvaﬁomonthedisuihrﬁonandedependenceofthemtesﬁnalalkalim
phosphatase of steelheadtrout(Salmo gambler-ii gairdneﬁr).CanadianJournalof Zoology
38:228-229.

Prasad,A.S. 1993. Biochemistryofzinc.P1enumPress,NewYork.

Prasad,A.S.andD.Ober1eas. 1971. Changesinactivitiesofzinc-dependentenzymesinzinc-deﬁcient
tissuesofrats.JournaloprpliedPhysiology3l:842-846.

91

Prasad,A S.,Obedeas,D.,Wolf,P.andJ.P.Horwitz. 1967. StudiesonZn-deﬁciencymhanysintrace
demmSandenzymeacﬁvniesmﬁsansonn-deﬁdemmJommlofChmcalhvsﬁgaﬁm
46:549-557.

Qian,H.,Kornegay,E.T.andD.M Derrbow. 1997. Utilimtionofphytatephosphonrsandcalciumas
inﬂumdbymiaobiﬂphyhsechobmkifaohandthemldmﬂdﬂphosphommﬁombrdhr
diets.PoultryScience76:37-46.

Rakocy,JE. andJ.A Hargreaves 1993. Megrationofvegetablehydroponicswithﬁshculmre: areview.
Pages 112-136 in J.-K. Wang, editor. Techniques formodernaquaculture. Proceedingsof the
Aquacultural Engineering Conference, Spokane, Washington 21-23 ere 1993. American
SocietyongriarltrualEngineers, St Joseph,Michigan

Reeck, GRandHNeurath. 1972.1solaﬁonandcharacterizationofpancreaticprocarboxypeptidase8
andcarboxypeptidase B of the African lungﬁsh. Biochemistry 11:3947-3955.

RichardsonN.L., Higgs,D.A, Beames,RM, andJ..RMcBride 1985.1nﬂuenceofdietaryca1cium,

phosphoruszinc, andsodiumphytateoncatamctincidemgromhandhistopathologyin
juvenilechinooksalmon (Oneal-lonelier rshawtscha). JournalofNutrition 115: 553-567.

Riehe,M andP.B. Brown 1996. Availabilityot‘phosphomsﬁomfeedstuﬂ’sfedtorainbowtrout,
Onoorhymchus Irwin's. Aquamlture 142:269-282.

Rodehutscord,M 1996. ResponseofminbowtorMOncorlnmdmsmyIdn-nrowingﬁomSOtOZOOgto
ampkmentsofdibasicsodiumphosphatemasunipruiﬁeddinlmnnalofNuuiﬁm 126:324-
331.

Rodehutscord,M andE.Pfeﬁ‘er.1995.Eﬁ‘ectsofamplemenm1miaobialphymseonphosphoms
digesﬁbﬂityandrrﬁhmﬁonianuorMOnoor-Imchusmykiss). WaterScienceand
Technology 31:143-147.

Rottiers, D.V. 1993. Relationshipbetweentheammrmdbone,maiorcaﬁonsandbodysizeinAﬂanﬁc
salmonSabnosalar.Copeia1993:440-446.

SAS Institute. 1997. SAS/STAT‘ Soﬁware: Changes and enhancements through Release 6.12. SAS
Institute, Inc, Cary, North Carolina

Satoh, S., Poe, W. E. and R P. Wilson 1989. Eﬁ'ect of supplemental phytate and/or tricalcium
phosphamonweigMgainfeedeﬁidencyandzimwnmmmvutebraeofchmnelcatﬁsh
Aquaculture 80:155-161.

Satoh, S.,Takeuchi, T.andT.Watanabe.1987.Avai1abilitytorainbowtroutofzincinwhiteﬁshmeal
and of various zinc compormds Nippon Suisan Gakkaishi 53:595-599.

Satoh,S.1-L,Yamamoto,H.,Takeuchi, T.andT.Watanabe. 1983. Eﬁ‘ectongrowthandmineral
composiﬁmofminbowtmuofdeleﬁmofuaceekmemsormgnesiumﬁomﬁshmealdia.
Bulletinot' the Japanese Societyfor ScientiﬁcFisheries 49:425-429.

Schat'er, A, Koppe, W.M, Meyer-Burgdorﬁ', K.-H. andK.D. Gunther. 1995. Eﬂ‘ects ofamicrobial

phymseonmeuﬁliaaﬁonofmﬁwphosphonnbycarpmadiabasedonsoybeanmeal. Water
Science and Technology 31:149-155.

92
Schindler, D.W. 1977. Evolrrtion of phosphorus limitation in lakes Science 195:260-262.

Schwartz, MF. andC.E. Boyd. 1995. Constructedwetlandsfa'treatmentofchannelcatﬁshpond
eﬁluents. Progressive Fish-Culturist 57:255-266.

Scott, D. A. 1934. Crystalline insulin. Biochemiml Journal 28:1592-1606.

Shearer,K.D. 1984. Changesinelernentalcomposiﬁonoflntchery-rearedminbowMSalmo
gammamodaedegrthandremomwﬁonCmadianJmnmldFkhuiesanqumﬁc
Sciences41:1592-1600.

Shearer,K.D. 1994. Faetorsaﬁeaingtheprordmatecomposiﬁonofarlnnedﬁsheswithemphasison
salmonids.Aquaculture119:63-88.

Shearer, KD.,Asﬁrd,T., Andorsddttir, G.andG.H Aas. 1994. Wholebodyelementalandproximate
wmpodﬁonofAdanﬁcmlmn(Sahuowlw)mningmehfecycleJmuMofFishBiology
44:785-797.

Shearer,K.D.,MaageA,Qrstvedt,J.AndHMrmdheim.1992. Eﬁ‘ectsofhigh-ashdietsongrowth,
ﬁeedeﬁciency,andzincstamsofjrwenileAtlanticsalmon(Sabnosalar). Aquaarlture106:345-
355.

ShpigeL M,Lee,J., Soohoo,B.,Fridman,RandI-I. Gordin 1993. Useofeﬂuentwaterfromﬁsh-
pondsasafoodsouroeforthePaciﬁcoyster,Crassoso-eagigas1‘hunberg. Aquamltureand
Fisheries Management 24:529-543.

Sievers,D.M1971.Carbon-phosphonrsavailabilityandeurtophication Doctoraldissertation.
UniversityofMissouriColumbia

Smith,RR,Peterson,M.C.andAC.Allred. 1980.Elfectof1eachingonapparentdigestion
coeﬁ'rcientsot‘ feedsurﬁ‘sforsalmonidsProgressiveFrsh-Culmrist42z195-199.

Solomatina,V.D.andO.MArsan 1979.11reinﬂuenceofenvironmentalphoqrhorusonsomephasesin
the metabolism of the carp, Oprinus caplo. Journal of Ichthyology 19:131-136.

Spinelli,J.,Houle,C.RandJ.C.Wekell. 1983. Theeﬂ'ectofphytatesonthegrowthot‘rainbowtrout
(Sabaogdrcbreﬁﬁedprﬁﬁeddiasmmainmgwryingqmndﬁadcaldumandmgnedum
Aquaculture 30:71-83.

Spry,D.J.,Hodson,P. V.andC.MWood 1988. Relativecontributionofdietaryandmterbornezinc
intherainhowMSalmogairdrrer-i. CanadianJourmlofFisheriesanquuaticSciences
45:32-41.

Storebakken,T.1985.Bindasmfeeds.I.Effeaofa1ginmeandgmrgrmmgrowth,digesﬁbihty,feed
intakeandpassagethroughthegastrointestinaltractofrainbowtrout. Aquacultrne47:11-26.

Storebakken,T.andE.Austreng 1987. BindersinﬁshfeedsH.Eﬁ’ectofdiﬁ‘erentalginamsonthe
digestibility ofmacronutrientsinrainbowtrout. Aquacultrne 60:121-131.

93

StorebakkenT.,Shearer,K.D.andAJ.Roem. Inpress. Availabilityofproteinphosphorusandother
demenmmﬁshmeaLsoypradnmnmeandphymsenemdwyprotdnmncenumebased
ondietstoAtlanticmlmonSalruosalar.AquacultureNutrition

Sundry,A,Eliassen,K.A,Blom,AKandT.As¢rd1991.P1asrminsulin,g1ucagon,glucagon-like
pepﬁdeandglwwekvdsmrespmsemfeedingaawaﬁmandhfehngreanaedfeedmﬁmm
mlmonids Fish PhysiologyandBiochemistry 9:253-259.

Szluha, AT. 1974. Potomologicaleﬁ‘ectsofﬁshhatcherydischarge. Transactionsot‘theAmer-ican
Fisheries Society 103:226-234.

TanejnS.K.andP.Arya 1992.1naniﬁonmayredrrceaﬂm1inephosphataseacﬁvityinﬁverandimesﬁne
d zinc-deﬁcient mice.Journalof Nutrition 122:1744-1747.

Trotrnan, C.N.A andC. Greenwood 1971. Eﬁ‘ectsot‘zincandothermetalionsonthestabilityand
activity of Escherichia coli alkaline phosphatase. Biochemical Journal 124225-30.

Uchian. 1970. Biochemicalstudiesonproteolyticenzymesinpyloriccaecaofﬁsh- 1.Fundamental

suﬂymthepmpanesofprmwlydcmzymesmcmmmlmonpybricmeaNipponsmm
Gakkaishi20:313-319.

Wallwork,J.C.andJ.ADuerre. 1985. Eﬁ'eaofzincdeﬁcientymnrethioninemetabohmmethylation
reacdonsandprmeinsymhedsinisolatedpafusedratﬁveernnalofNuuiﬁon115:252-262.

WatanabeT.,Murakami,A,Takeuchi,L.,Nose,T.andC.Ogino.1980.Requirementofchmnsalmon
heldmﬁeshwwfmdiaaqphosphmusBmkﬁnofmeJapanseSodayodemﬁﬁchhuies
46:361-367.

Westers,H. 1979.FishmmnepracﬁcesformeSmofMichiganMichiganDepamnunofNamal
Westers,H. 1987. Feedinglevelsforﬁshfedformulateddiets.ProgressiveFish-Culmrist 49:87-92.

WestersH 1991.0paaﬁona1wastemanagenremmaqrmnuneemuerusPages231-238InCB.
CoweyandC.Y.Cho,editmsNrmiﬁomlSumegiesmMmagununoquuaannneWastes

Wetzel, RG. 1983. Limnology, Secondedition Sarmders CollegePublishing, NewYork.

Whitmore,DJ-1. andE.Goldberg 1972a Troutintestinalalkalinephosphatast. Somephysical-
chemicalcharacteristics. JournalofExperimentalZoology182:47-58.

Whitmare,D.H.andE.Gol®erg 1972b. Troutintestinalalkalinephosphatases. H.1‘heeﬁ'ectot‘

temperatrueuponenzymaticactivityin viooandinvivo. JorrrnalofExperimentalZoology
182:59-68.

Wiesrnann, D., Scheid, H andE. Pfeﬂ‘er. 1988. Waterpolhrtionwithphosphorusofdietaryoriginby
intensively fed rainbow trout (Salmo gairdnen’ Rich). Aquaculture 69:263-270.

Windell, J.T.,Folz, J.W. andJ.A. Sarokon 1978. Eﬂ'ectofﬁshsiae, temperatureandamountfedon
mmierudigesﬁbihtyofapeneteddietbyminbomealmogairdneﬁ. Transactionsofthe
American Fisheries Society 1072613-616.

94

Yasutake, W.T. andJ.H. Wales 1983. Microscopicanatomyot‘salmcnids: anatlas UnitedStates
DepartmentofFisheriesanderdlifeResourcePublication 150.

YomT.andY.Sahgism.1986.CompamﬁwbiochmialaudydahmlimpMspWMymsm
ﬁsh,amphibians,reptiles,birdsandmammals ComparativeBiochemistryandPhysiology
858:649-658.

Yoshinaka,J.,Sato,M,Morishita,J.andS.lkeda. 1984a. Enzymaticcharacterizationoftwo .
' BﬁomthecatﬁshpanamBuueﬁnoftheSocietyofScientiﬁcFisheries
50:1723-1727.

Yoshimh,J.,Sam,M,Monst,J.andS.nreda.1985.Enzymaﬁcclnraaainﬁonof
° AﬁornthecatﬁshpancreasBulletinoftheJapaneseSocietyofScientiﬁc
Fisheries 51:113-116.

Yoshinaka, J., Sato, M, Morishita, J. Itoh, Y., Hujita, M and S. Ikeda. 1984b. Puriﬁcationand some
properﬁesoftwocarboxypeptidasestromthecatﬁshpancreas. BulletinoftheJapaneseSociety
of Scientiﬁc Fisheries 50:1717-1722.

Yormg, L.G.,Leunissen,M andJ.L. Atkinson 1993. Additionofmicrobialphytasetodietsofyormg
pigs. Journal of Animal Science 71:2147-2150.

Yurk, J.J. andJ.J. Ney. 1989. Phoqrhorus-ﬁsh communitybiomassrelationshipsinsorrthern
Appalachianreservoirs: Canlakesbetoocleanforﬁsh?LakeandReservoirManapment5:83-
90.

Zhou, LR, Fordyce, E.J., Raboy, V., Dickinson, D.B., Wong, M-S., Burns, RA and J.W. Erdman, Jr.
1992. Wmofphyﬁcaddmsoybeanpromrasimprwesnmbioavailabihtymmlmnnﬂ
of Nutrition 122:2466-2473.

APPENDICES

95

APPENDIX A

Sample Digestion Procedures

The following procedures were used to digest ﬁsh, feed, fecal and water samples in

experimentsdescribedinChapterslandZ.

I.

A

Eunmlrloncaaidnnhml - for ﬁsh, feed and fecal 881119168-
Each sample was placed in a 250 mL Phillips beaker. Feeds and feces were

digested in 10 mL concentrated nitric acid and 3 mL perchloric acid. Fish samples
were digested in 20—40 mL concentrated nitric acid and 5-10 mL perchloric acid.
Blanks were digested in the same manner as the samples. Beakers were covered
with watch glasses. Additional nitric acid was added as needed.

Samples were boiled on a hot plate until chemical reactions were complete and the
perchloric acid created a thick white smoke. Watch glasses were then removed to
allow excess acid to time until about 1 mL remained.

Beakers were allowed to cool, and the inside beaker walls were rinsed down with
deionized distilled water (DDI). Cooled samples were covered with plastic wrap if
not immediately stored in test tubes.

Bovine liver and citrus leaves (Standard Reference Materials 1577a and 1572,
respectively, National Institute of Standards and Technology, United States

Department of Commerce) were digested as internal standards.

Reference:

AOAC (Association of Oﬁcial Analytical Chemists). 1984. Ofﬁcial methods of analysis, l4th
edition Association ofOﬁcial Analytical Chemists, Arlington, Virginia, USA

 

._ - for water samples.

One mL 11N H280, and 0.4 g ammonium persulfate were added to a 125 mL
Erlenmeyer ﬂask containing 50 mL unﬁltered water or standard. Samples were
boiled downto about 10mLonahot plateinafumehood.

Samples were allowed to cool. Flasks were capped with aluminum foil if samples
were not processed immediately.

Reference:

APHA(American Public Health Association), AmericanWater WorksAssociation, andWater
Pollution ControlFederation 1985. Standardmethodsfortheenminationofwater
andwastewater,16thedition AmericanPrrblicI-lealthAssociationWashington, D.C.

Notes: Preliminary experimentation revealed that fecal samples and the bovine
liver standard were not completely digested using the sulﬁrric acid-ammonium
persulfate digestion method (APHA et al. 1985). Experimental fecal, feed and ﬁsh
samples were therefore digested using nitric and perchloric acids (AOAC 1984).
Sample sizes for dry ﬁsh and feed were approximately 1 g. Because fecal samples
were very small (0.2-28 mg dry matter), the entire sample was digested.
GlasswareusedinwaterPanalysiswassoakedina6NHClbathforat
least 15 min. GlasswareusedintheanalysisofPinfeed, fecesandﬁshwas
soaked in a 5 I-INO3 acid bath for 12 h Acid-washed glassware was rinsed in DDI
water, dried in an inverted position, and capped with plastic wrap or aluminum

foil.

97
APPENDIX B

Phosphorus determination procedures

These procedures were used to determine the P content of digested ﬁsh, feed, fecal

and water samples ﬁom experiments described inChapters 1 and 2.

I.

A.

W - for whole ﬁsh, feed and bone samples.

Reagents

W: Dissolve 5.0 g Na2M004-HZO into 200 mL DDI water. Add 14 mL
concentrated H2804. Bring volume to 1 L.

W: Dissolve 3 g NaHSO3 into 50 mL DDI water. Dissolve 1 g Elon’
into the NaHSO, solution. Bring vohrme up to 100 mL.

Standards

W: A 60 mg P-L" certiﬁed commercial phosphate standard
(Banco"‘ phosphate standard, Anderson Laboratories, Inc, Ft. Worth, TX) was
diluted to the following concentrations for whole ﬁsh, feed and bone samples (mg
PL"): 0, 10, 20, 30, 40, 50, 60.

Sample treatment

Five mL MS and 0.5 mL Elon solution were added to 1 mL sample or standard.
Color was allowed to develop for 45 minrrtes. Absorbance was read using visible

light at 700 nm on a Beckrnan spectrophotometer using a 1 cm light path. Note:

 

’ ElonT“(KodakCo.)isacommeroia1nameformethyl-p-aminophenolsrmate.

 

98
sample and reagent volumes could be divided in halfwithout altering results.
Phosphorus was expressed as elemental P.
Final dilution
Final concentrations were approximately: 97.3% water, 1.4% Na,MoO,-H,O, 1%
11280,, 0.2% NaHSO3, and 0.1% Elon.
Reference: Gomori (1942).
MM - for fecal and water samples.
Reagents
W: 140 mL concentrated HZSO4 to was added to approximately 500
mL DDI water. After cooling, the solution was brought to a ﬁnal volume of 1 L.
WM: 2743 s K(Sb0)C.H.0a°1/2H20 was
dissolved in DDI water, brought to a ﬁnal volume of 1 L and stored in dark glass
bottle at 4°C.
Ammonium”: 40 s (NIL)6M01027'4H20 was dissolved in DDI
water, brought to a ﬁnal volume of 1 L and stored in polypropylene bottle at 4°C.
W: 8.80 g CJLO, was dissolved in DDI water, brought to a
ﬁnal volume of 500 mL, and stored at 4°C. Solution was stable for about 1 week.
Wm: Reagents were mixed in the following order for a total volume
of 500 mL: 250 mL 5N sulfuric acid, 25 mL potassium antimony] tartrate solution,
75 mL ammonium molybdate solution, 150 mL ascorbic acid solution.

Concentrated reagent was stored in a dark glass bottle for up to 4 h.

Standards

WM): 0.2197 g potassium phosphate monobasic
(KI-12PO, dried at 105°C) was dissolved in DDI water. One mL concentrated
H2SO, and 1 mL chloroform (CHC13) were added as preservatives. The solution
wasbroughttoaﬁnalvolumeofl Landstoredinaglassbottleat4°C.

h stan l ' n 1 " : 20 mL of phosphorus stock solution
was diluted to 1 L with DDI water. The solution was prepared ﬁesh daily.
W: The 1 ppm standard solution was diluted to concentrations of
0.01, 0.03, 0.05, 0.10, 0.30, 0.50 mg'L". Working standards were made ﬁesh
daily.

Sample treatment

One drop of phenolphthalein indicator solution was added to each sample and
standard digest. While swirling the digest, 6N NaOH was added dropwise until
the solution just turned pink. The solution was brought to 50 mL ﬁnal volume
with DDI water using a volumetric ﬂask, then transferred back to the 125 mL
digestion ﬂask. Eight mL of combined reagent was added. Color was allowed to
develop for at least 10 min but no longer than 30 min. Absorbance was measured
at 880 nm using a spectrophotometer. A reagent blank was the reference solution.

P was expressed as elemental P.

100
References:
APHA(AmericanPublicl-halthAssociation), AmericanWaterWorksAssociation, andWater
PollutionControlFederation 1985. Standardmethodsfortheerraminationofwater
and wastewater, 16th edition American Public Health Association Washington DC

GomorLG.1942.Amdiﬁmﬁmofthecdonmeuicphosphmusdaaminaﬁonforuxwimme
photoelectriccolorimeter. Journalof 1aboratoryandC1inica1Medicine27z955-960.

Limnological ResearchLaboratory. 1990. Unpublishedmemorandumforthedeterminationof
tmaldissohedreacﬁvemmldissdvenandmlphosphorusDepmmdFisheries
and Wildlife, Michigan State University, East Lansing

Notes: Total phosphorus was determined in whole ﬁsh and feed digests according

to Gomori (1942) and in fecal and water digests according to APHA et al. (1985).

The P determination method was selected based on laboratory availability at MSU.

Based on preliminary tests using digested fecal matter, bovine liver and phosphate

standards, the two P determination procedures provided identical results.

101
APPENDIX C

Predicting the Nitrogen and Phosphorus content of whole ﬁsh ﬁ'om ﬁsh weight _

INTRODUCTION

ThemardmumpossibleamountfeedNandeischargedintoﬁshhatchery
eﬂluentscanbecalculatedifthequantitiesofNandeedtoandretainedbyﬁshare
known (Chapter 1). To calculatethe amount ofNandP retainedbyﬁsh, theNandP
composition ofﬁshatthebeginningandendofthegrowthperiodmustbelmown
Collecting body composition data for each ﬁsh species cultured and for every production
period represents an enormous task. For more commonly cultured species, a growing
amount of published body composition data is available. Ifwhole ﬁsh N- or P vs. ﬁsh
weight regression equations were established for commonly cultured species, considerable
resources could be saved by predicting these constituents ﬁom body weight.

The objective of this study was to describe the mathematical relationships between

whole ﬁsh N and P and ﬁsh weight for several species ofﬁsh important to the aquaculture

industry.

 

102
METHODS

DatasetscontainingwholeﬁshN, wholeﬁshP and correspondingﬁshwetweight
values were located using the Aquatic Biology, Aquaculture, and Fisheries Resources
NISC database (National Information Services Corp., Baltimore, Maryland) using
combinations of the key words proximate composition body composition nitrogen,
phosphorus and protein. Database entries covered 1974 through 1996. Additional data
sets were found through citations within papers, personal communication with researchers
and by manually searching the journals Aquaculture and Progressive Fish-Culturist ﬁ'om
approximately 1987-1996. The literature search was not exhaustive. Any omission of
pertinentpaperswasaccidental andinnowayconstituted ajudgement oftheirmerits.
Tabulated data sets with at least a 10-fold weight difference between the smallest and
largestﬁshwerepreferred. However, intheinterest ofgatheringdataonasmanyspecies
as possible, smaller data sets or data ﬁ'om ﬁgures was occasionally used. Nitrogen was
calculated ﬁom crude protein values as: (crude protein -=- 6.25). Data sets, including those
from this dissertation were collected for bluegill sunﬁsh, green sunﬁsh, longear srmﬁsh,
yellow perch, muskellunge, tiger muskellunge, largernouth bass, seabass, red drum,
sunshine bass, rainbow trout, brook trout, chinook salmon, coho salmon and sockeye
salmon

Linear regression relationships were calculated using the SAS software GLM
procedure (SAS Institute 1997). Data sets for a species or genus were combined if
evidence against homogeneous regression was not strong (P 2 0.05). Regression

relationships were considered signiﬁcant if P s 0.05.

103
RESULTS

Whole ﬁsh N data were collected for 14 ﬁsh species (Table 10). Whole ﬁsh N
content was signiﬁcantly correlated with ﬁsh wet weight in all cases (r2 2 .89). Values
ﬁom individual data sets could be combined into a single data set for each species except
yellow perch Data could also be combined to produce equations for the genera Lepomis
spp. (sunﬁshes) and Oncorhynchus spp. (salmonids). Although the assumption of
homogeneous regression was not met for yellow perch data sets or all species combined,
regressions were computed for those combinations for illustrative purposes. Table 11
contains details on individual data sets.

WholeﬁshP and ﬁshwet weight datawerefound for4 speciesofsalmonids
(Table 12). All data sets regressed homogeneously. Data were combined to create
regression equations for trout, salmon and trout+salmon (Table 12). The degree of data

scatter(YresponsetoanX)increasedwithincreasingﬁshsize(Figure7).

104

.8683»... ascoeomoﬁogéoe 8.9.0.. 8:388 203 88 8% 3323.8... .

 

 

$78.. as a... 8... .... 8. 7 8... ... 8.. _ 8.8% 8:388
98-... .N o. «no 8... a 2.7 no... a 8.. 85.9.3...
.373. 8 8o 8... a on..- 8... a 8.. =.2.U 8.
.....-mg o. a... «a... ... 8.7 m. .c a a. .. was. 58.53...
31.8 a 3... m. .o a 8.7 o. .c a ... .. 338m
83.... a 8.. 8... a 8..- 8... a 8.. as. 2.22.8
31.2 8 3... 8... a 8.7 8... a 8.. _ 5...... 32.9.
87...: on .3... 3... a 8..- 8... a 8.. 85......»
2.7%.. ..N. 8.. S... s 8.7 8... ... 3.. 85.8 as 38.
Am. ems: 2.303 .03 ...... .. m 2.38.... one.» 9.2m 8 8.03m

 

.mm a 33.5 .862... ..m... .8823 ..o 3. 2303 ..m... 83 mo. ...... Am. 2 ...»... 0.2.3 no. 50252. 3.7.5522 3.8033.

.2 033.

 

 

 

 

 

....-. 5......
....78w N. 2...... sea. ..22. 32.9.

2.78... o. .32. .225 .85 2&3. ..22. 32.9.

2.1.3 2 .22. 822.3%... 3.02:... 222. 32.2.

22.2 o. 8...... 3.82 ...... 22.28.39 222. 32.9.

872. m .82. m22s.. 2223 have.2

23.... 4 an... 9.2.5 22...... :8.»

1.3.... a as... 9.28.. 22...... ...mg...

5 .272... 8 as... .... a so... .5........ 9628..
w canned a. 88.. 83:0 .88.: 9.0269.
@2722 v .82. .... e 820.. Se. 33......

2.8.. .A.. a $3.. ... e 2.233. .32. 32......-

v.$.-8.~ 8 23.... see... 58.382...

«87...... a .5.... 5.23. so: 33......

9.8-. 2 a .88.. .... s 2.2.2.. as. 28:.

Am. 09.... 2.903 e 858...: 8.8mm

 

2:828 Z
...... 22.3 a 3.3.. 2.. 2 .83 ass: 223 .3 a... ...s .... an... «ass .32. 2283 .3 ...... 2.. z ...... 22.3 .o 828m ... 2......

106

82:6 ...... use... .2 82.3 _

 

 

 

 

9.36.3 m Gee: .... .0 ...—85 09.2.8.3... 8a..
......1... .N m .32. ... e 3.8... 22.2.2.2
22-2.. .... .32. .... .0 .... =5... ..e
.2332 ... .32. .... a 9.8... 3.. 58.5.2
”.212 a .32. 828 ...... 3.826 238..
83... w .32. ... s .2325 as. 2.22.3
37$. ... .82. .... e .32.. as. 822:...

Am. ems... 2.903 .. 02.9.0.8 8.8mm

 

.....28. .. 22....

107

 

 

 

 

8.73... mm 5.3.08... ...... .. .0320 58.3 .0220
3.3.5... 9.. 5.3.003... «.... .. .8920 58.8 9.8
8.23... m 68.. .... .o .802 32. .8...
8.4.3. 3.. 8.3.8»... ...... .u 82.2.0 :5.. 33......
20.8.. 2 .32. 05:5 ...... ...8 so: 33......

A8 09.8 x .. 02.0.0.0. 8.00am

m 83...... a? a... a... a 8... 8... a 8... 8:588 .8... ...... 85......
m 82...... ....u 3... .0... « .3. 8... a ...... so: 32...... ...... .8...
... $34.2... «an a... on... “.n .... .- 8... a 8... 88.3 .3820 ...: 2.8
0?“. x c N.. 2.080.... 0......» 00.00%

 

.Mm n." 82.5 .58. ...... 56...... 3.. Am. ...».03 ...... .03 ...... 3.5 m ...... 0.9.3 503.2. 2.2823? 5.9.030..—

.N. 030,—.

.853 as. 32. 3.. :32, a... a... m a... 22.3 8023 gigging s 25E

 

 

 

3 =32: .25 in
8. 8. a: as 8_ 8 8 3 8
8 k, ,
w
§ MM x x“
X X» X
x x
X wavw X
X X ﬂ XX
x
x
x x x 2 332.8 :95: U32.5 +
x 9 .8829 :95: 2.00 0
GR. 3 .0 502V 38. €95 x
a 3.3.9 .25 33:3. x
£2 «£5 Ea 56V :5.. 32.5. o

 

 

 

Icon

rs?

.8n

 

(3w) 4

109
DISCUSSION

The high degree of correlation found between whole ﬁsh N and ﬁsh weight
indicated that whole ﬁsh N analyses are probably not necessary for most species already
characterized. Considerable resources could be saved by estimating whole ﬁsh N with
regression equations calculated ﬁ'om existing data.

Although not identical, parameters ﬁ'om the whole ﬁsh N vs. ﬁsh weight
regressions were similar for the diﬁ‘erent species. Parsons (1888) discovered over a
cenmwagothatvaﬁousspedesofﬁshmdaquaﬁcmmnmalsuesuildnglysimiluin
three-dimensional morphology. Hecht (1916) further reported a “general similarity of
form” for seven marine ﬁshes based on the regression relationships of several body
dimension combinations. He attributed the regularity of form to the demands of life in
water, which is dense relative to air. Since approximately 70% of wet ﬁsh mass was
muscle (Krumholz 1956), and ﬁsh share similarities in form, it was not surprising that the
ﬁsh species summarized here, which were all ﬁ'om the taxonomic subdivision Teleostei,
shared similarities in N content.

In 1956, Krumholz found that eight species of freshwater ﬁsh contained
approximately the same percentage of muscle. When Krumholz’ data is expressed as g
ﬁsh muscle vs. 3 ﬁsh weight, the r2 = 1.00. Groves (1970) reported that the relationship
between ﬁsh length and protein content was predictable and nearly identical for several
salmonid species, provided that the ﬁsh were not fasting or starving. After a non-
statistical comparison of protein-ﬁsh weight relationships for several ﬁsh species, Shearer

(1994) hypothesized that the protein-body'weight relationship was species speciﬁc. To

110
ourhrowledgethepresent studywastheﬁrststatisticalanalysisofwholeﬁsths.ﬁsh

weight data for several species.

Certain limitations of the whole ﬁsh N vs.ﬁsh weight regressions should be
addressed. First, equations based on narrow ﬁsh weight ranges should be used with
caution Error is greater in parameters calculated ﬁ'om somewhat clustered data This
wudanonstruedmthepmmaaesﬁmuesforseabassandhrganouthbasswhichhad
the narrowest ﬁsh weight ranges, the lowest r2 values, and the highest standard errors of
parameter estimates. Secondly, diﬁ‘erences between slopes or intercepts for most species
were small yet signiﬁcant. The importance of even minor diﬁ‘erences can be seen through
the following example. If the combined equation was used to predict whole ﬁsh N for
salmonids, with salmonid data making up 37% of the combined data, whole ﬁsh N
estimateswouldbeoﬁ‘byabout 1%. If thissmalldiﬂ‘erencewassummedacrossa
standing stock of several tons, the cumulative error could be quite signiﬁcant. Despite
these limitations, even rough estimates calculated ﬁ'orn the combined equation or an
equation for a related species could be of value in production situations where no other
means of estimating N retention or loss are available.

Most literature values for whole ﬁsh N content were determined on pooled
samples of 2 or more ﬁsh of approximately the same size. Pooling could artiﬁcially reduce
data scatter, thereby falsely inﬂating the coeﬁcient of determination (r2). However,
regressions of whole ﬁsh N values determined for individual ﬁsh by Groves (1970), Bai et
al. (1994) and Brown et al. (1993) yielded r2 values of 0.99-1.00. Thus it seems fair to

conclude that the coeﬁcients reported were not inﬂated by sample pooling.

111

Thevariabilityinwholeﬁsthetweenindividuals ofagivenweightincreasedwith
ﬁsh size. Phosphorus concentrations in growing salmonids have been shown to vary with
dietary P concentration (Ketola and Richmond 1994; Rodehutscord 1996), dietary P form
(Cain and Garling 1995), feed emciency (Asgard and Shearer 1997), and feed intake. Any
diﬁ‘erencesinindividual ﬁshP causedbythesefactors couldbecompoundedbytime.

Undernormal circumstances, bonePbutnot ﬁestharieswithPintake. Bone
and whole ﬁsh P plateau in most growing salmonids when at least 5-6 g available P-kg"
dry diet is fed (Ketola 1975; Watanabe et al. 1980; Ketola and Richmond 1994;
Rodehutscord 1996; Asgérd and Shearer 1997). Depending on dietary P intake, a ﬁsh
may contain a range of whole ﬁsh P concentrations up to and including an amount
resulting ﬁ'om bone P saturation Therefore, the increased scatter seen in whole ﬁsh P
with increasing ﬁsh weight may have been due to the increasing contribution of bone P to
whole ﬁsh P with increasing ﬁsh size. Skeletal P accounted for 10% whole ﬁsh P in 5 g
chum salmon fed a 1.15% P diet for 7 weeks (Watanabe et al. 1980). However, bone may
account for 35-44% of whole ﬁsh P in 0.4-2.8 kg rainbow trout”. Therefore, any
variability in dietary P intake should have less of a measurable eﬁ‘ect on small ﬁsh whole
body P than on large ﬁsh whole body P.

Because whole ﬁsh P was responsive to dietary P independent of ﬁsh weight,

regressions should be used cautiously for ﬁsh fed diets with very low concentrations (< 5

 

ContributionofbonePtowholeﬁsthasestimatedusingdrybone%Pdatafor0.4-2.8kgrainbowtrout
fromPersson(1988, citedinLall 1991), whole AtlanticsalmonbonecompartmentdatafromRottiers
(1993)andwholerainbowtroutPcontentdataﬁ'omShearer(l984). Sinceribsandotherbonesareless
mineralizedthanvertebralcentrainsomeﬁshmaserandHarvey1982),theﬁgurespresentedshouldbe
consideredmaximumestimates.

l 12
gkg") of available P. Finally, whole ﬁsh N or P should only be predicted for ﬁsh within

the weight range speciﬁed for each species in Tables 10 through 12.

1 13
CONCLUSIONS

WholeﬁshN and P were highly predictable ﬁomﬁshwetweight forall species.
Regression parameters were similar for all ﬁsh species examined. With the exception of
yellow perch, individual data sets for each species could be combined. The regression
equations presented for predicting whole ﬁsh N and P ﬁom ﬁsh weight may be used to

estimate ﬁsh N and P retention when direct measurements are not possible.

1 14
LIST OF REFERENCES

AsgariTmndK. D. Shearer. 1997. DietaryphosphorusrequirementofjuvenileAtlanticsalmon,SaImo
salarL.AquacultureNutrition3:l7-23.

Bai, S. C., Nermtipour, G. R, Perera, R. P., Jammillo, F., Jr., Murphy, 8. R. andD. M. GatlinIIl. 1994.
Tmlbodyekcuimlwnmmﬁtyformndesuucﬁwmrememofbodycmnpodﬁmcfmd
drum. ProgressiveFish-Oulnuist 56:232-236.

Bredra,B.J.,IrIooe,ML.andD.I-1.Wahl1995.Comparisonofgrowth,survival,andbodyccmposition
dmuskdlmgeandﬁgermmkdhmgefedfwmmmdaldmmwFish-Crunuin
57:37-43.

Brecka.B.J.,Wahl,D.I-1.andM.L.Hooe.1996.Growth,survival,andbodycompositioncflarmrth
bassfedwﬁmmmudaldiasandprudnmncenmﬁmaPmMFish-Cﬂnniusszlw
110.

Brett]. R,Shelboum,J. E.andC. T. Shoop. 1969. Growthrateandbodycompositionofﬁnmrling
sockeyesalmoaOnwﬂonchusmrka,mn1aﬁmmwmpaanneandraﬁmdzeJannﬂofﬂn
FisheriesResearchBoardof Canada 26:2362-2394.

Brown,M.L. Gadian,D.M.andB.R.Mmphy. 1993. Nondestructivemeasurernentofamshinebass,
Moron: drown (Raﬁnesque) xMoraine mulls (Walbaum),bodycompositionnsingelactriml
conductivity. Aquaculture and Fisheries Management 24:585-592.

Cain,K.D.andD.L.Gar-ling 1995. Pretreatmentot‘soybeanmealwithphytaseforsalmoniddietsto
mphosphoruscomuaﬁominlntchuyeﬂluenmﬁogressiveFish-Crﬂmﬁst 57:114-119.

Cataanan,MRandR.MColoso. 1995. Eﬂ'ectofdietaryproteintoenergyratiosonyowrthsurvival,
andbodycomposition of juvenﬂeAsianwabasaLates calcarifer. Aquaculture 131:125-133.

Fraser , CC and HH. Harvey. 1982. Elemental composition ofbone ﬁomwhite sucker (Catastomus
commemmﬁnrelationtolakeacidiﬁcation. CanadianJomnalofFisheriesanquuaticScienms
39:1289-1296.

Garber,KJ.1981.Gasuicevacuaﬁonandproteinenergeticsinyeﬂowperch MSThesiaUniversityof
W‘rsconsiaMadison.

Gerking,S.D. 1952.1heprmdnmaaboﬁsmofmnﬁshesofdiﬂ'aemagesPhysiologicalZodogy
25:358-372.

Gerking, S. D. 1955. mﬂmnceofmteoffeedingonbodycomposiﬁonandprmeinmetabdismofbhwgin
mnﬁsh Physiological Zodlogy 28:267-282.

Groves,T. D. D. 1970. BodycompositionchangesduringgrowthinyormgsockeyHOncorhmm
nerka) infreshwater. Journal of the FisheriesResearchBoardof Canada 27:929-942.

Kaushik, S. J., Cravedi, J. P.,Lalles, J. P., Sumpter, 1., Fauconneau, B.andM Laroche. 1995. Partialor
mulmplwememdﬁshmcalbywybcanprmdnongrmhprmdnuﬁlimﬁoapmenﬁal

115

Wummwmmmmmmm
Ms. Aquaculmre 133:257-274.

Ketola,H.G. 1975. Requirernanot'AﬂanticmlmonfordietaryphosphorusTnnsactionsofﬂie
AmericanI-‘isheries Society. 104:548-551.

KetolaHG.andM.E.Richmond 1994. Requirementdrainbowtmutfordietaryphosphorusandits
rdadmshipmtheammmdischmgedinmhayeﬁlmmoftheAmerican
Fisheries Society 123:587-594.

Krumholz,L.A. 1956. Oservaﬁonsontheﬁshpopulaﬁonofahkecomaminatedbyradioacﬁvewastes.
BulletinoftheAmericanMuseumofNaturalI-Iistory1102326-33l.

Lall, SP. 1991. Digestibility,metabolisrnandexcretionofdietaryphosphorusinﬁsh. Pages21-36 in
CB. Cowey, AM Mackie, and 1.6. Bell, editors. Nutrition and Feeding inFish. Academic
Press,London.

McCay, C. M,Tunison,A.V., Crowel,M.andP. Henry. 1936. 'Ihecalciumandphosphomscontentof
mebodyofbmokumninnlaﬁonmageJrowmandfoodhnmld’BidogicalChanm
114:259-263.

mudeB.1888.1hedisplmunemsandthem-mwesofﬁshTmnmcﬁomdmeAmmhn
SocietyofMechanicalEngineers9zé79-695.

WG.1988.RehﬁonsMpsbawemfee¢mmﬁqawwuuﬁonmtmhmingdhrgeMnbow
truut(Salmo gairrbren'). SwedishEnvironmentalProtectionAgency, Stockholm,Pl\B534.

Phillips,A.M,Livingston,D.L.andRI-‘.Dumm.1960.Eﬁ’eetofstarvationandfeedingonthe
chernicalcomposition of brooktrout Progresa’veFish-Culmrist 22:147-154.

Reinitz,G. 1983a mﬂueweofdiaandfeedingratemtheperformanceandpmmpﬁoncostofminbow
trout. Transactions of theAmericanFisheriesSociety 112:830-833.

Reinitz, G. 1983b. Relaﬁveeﬁeaofagedietandfeedingrateonthebodycomposiﬁonofyoung
rainbow trout (Salmo gairdneri). Aquaculture 35:19-27.

Rodehutscord,M 1996. Responseofrainbowﬁorn(0ncorhynchusnolkiss)growingﬁom50to200gto
supplements ofdibasicsodium phosphateinasemipuriﬁeddiet. Jorn'nalofNutrition126:324-
331.

Rodgers, D. W. and S. U. Qadri. 1982. Growthand mercuryaccumulation inyearlingyellowperch,
Percaﬂaveseens, in the Ottawa River, Ontario. Environmental Biology of Fishes 7:377-383.

Rottiers,DN. 1993. Relationshipbetweentheamormtofbone, majorcationsandbodysizeinAtlantic
salmon, Salmosalar. Copeia 1993:440446.

Shearer,KD. 1984. Changesinelementalcomposiﬁonofhatchery-rearedrainbowMSalmo
gdréreﬁmmdﬂedwﬂyoﬂhaﬁmprﬁonCamdianermlofFisheriesandAmmﬁc
Sciences 41:1592-1600.

116

Shearer,K.D. 1994. Faaorsaﬁ'ecﬁngthepmidmatecomposiﬁonofculnnedﬁsheswithemphasison
salmonidsAquaculturell9zé3-88.

Tanasichiuk,RW.andW. C. Mackay. 1989. Quanﬁtativeandqualitativecharacwristicsofsomaticand
gonadalgrowthd‘ydhwpachWerwﬂawsaenﬂﬁunIxSteAmAlbauCamdim
Janna]ofFisheriesanquuuaticScience46:989-994.

WatanﬂueT.,Muurakami,A.,Takeuchi,L.,Nose,T.andC.Ogino. 1980. Requirementofchumsalmon
hewmﬁeshmfwdiamyphosphausBunednoftheJapameSodadedmﬁﬁeFishais
462361-367.

Webster, C. D.,Tiu,L. G.,Tidwell,1. H, VanWyk,P.arudR D.Howerton. 1995. Eﬂ'ectsifdietary

prdeinandhpidkvdsmgrowthandbodycompodﬁmd‘amshimhasstblmduwops xM.
saxatilis) rearedincages. Aquaculnure 1312291-301.

1 17
APPENDIX D

Alkaline Phosphatase Assay Protocol

1155mm
Theintestinewasslitlongitudenallyto errposethemucosa, rinsedinapproximately
10 mL ice-cold 0.7% NaCl (ﬁsh physiological saline), blotted dry and weighed to
the nearest 0.1 mg. The blotted tissue was chopped with scisors and homoginized
for 30 s in 3 mL ice-cold deionized distilled (DDI) water using a Turtox ﬁssure
homogenizer on minimum speed. The homogeruizer probe was pre-chilled in ice
water.
Tissue homogenates were centrifuged at 17,300 x g for 20 min at 4°C.
Supernatants were aspirated and stored on ice until assayed. Supernatants were
diluted 1:1 with DDI water prior to analysis. A 100 [.LL aliquot of the ﬁnal
supernatant dilution was reserved for protein analysis.
Randal:
p-Nitrophenyl phosphate+H20+alkaline phospluitase ~

p-Nitrophelon-linorganic phosphate
Conditions
T=37°C, pH=9.2, A=410, light path=1 cm
Method
Stopped spectrophotometric rate determination

smears

118
100 mM glycine buﬁ‘er with 1.0 mM magnesium chloride, pH 9.2 at 37°C.

To prepare 50 mL, combine 0.375 g glycine and 0.010 g magnesium chloride in
approximately 30 mL DDI water. Heat to 37°C and adjust pH with 0.5 M NaOI-L
Bring volume up to 50 mL with DDI water. Prepare ﬁ'esh daily. Glycine, ﬁ'ee
base, FW 75.07. MgClz-6Hzo, FW 203.3.

15.2 mM p-Nitrophenyl phosphate solution (PNPP).

To prepare 5 mL, dissolve 0.020 g PNPP in 5 mL DDI water. Prepare ﬁ'esh daily.
PNPP Sigma phosphatase substrate stock no. 104-0, FW 263.1.

20 mM sodium hydroxide solution (NaOH).

To prepare 1 L, dissolve 0.8 g NaOH in 1 L DDI water. NaOH, FW 40.0

Procedure
Pipette (in mL) the following reagents into 14 mL tubes:

 

 

Reagent Test Blank
glycine buﬁ‘er 0.5 0.5
PNPP 0.5 0.5

 

Mix and equih’brate to 37°C. Then add (mL):

 

Reagent Test Blank
supernatant solution 0.1 --
deionized distilled water -- 0.1

 

 

119

Addsupernatant samplesandblanksatBOsintervalsusingastopwatch Vortex
aftereachaddition Aﬁerexactally 10minadd 10mL20mMNaOHtotubeand
vortex. Record the absorbance at A 410 nm using the visible lamp.

VII. (Aquatics:
Based on the Bouguer-Lambert-Beer law:

Abs = (e)(c)(d)

where: e=extinction coeﬁicient; c=concentration; d=light path (cm). Rearranged,
c=(Abs)-(e><d)". Accounting for the absorbance of the blank dilution factors,
assay time and volume, and sample volume:

A Units-mL’l supernatant solution =
(Abs. - Abs,qu1.1><d.£)(18.3><0.l><10><1)°‘.
where: 11.1= assay ﬁnal volume in mL; df= dilution factor, inthe case ofmost
supernatants, 2; 18.3 = millimolar extinction coeﬁcient ofPNPP at 410 nm; 0.1=
volume of supernatant solution used in mL; 10 = time of assay (in min) as per Unit
deﬁnition; 1 = light path (cm).

B. Units-tissue " = (U°mL" supernatant solution) x 3
where: 3 = mL water the intestine was homogenized in.

C. Units-g" tissue = (U -tissue")-(tissue weight)‘l

D. Speciﬁc activity =
(U -mL'l supernatant solution)-(mg protein-mL" supernatant solution)".

120
One unit will hydrolyze 1.0 pmole of p=nitrophenyl phosphate per min at pH 9.2 at

37°C.

121
IX. Mamas:

Anonymous 1994. SIGMAqnslitycontroltestprocediueEnzymaticassaycfphosphatase,
alkaﬁne(EC3m13l).Glydmamy.SIGMAChanicalCompany,St1miaMimi.

NodaHandS.Tachiruo. 1965. Studiesonvariousphosphatasesoftheﬁshes-L Enzyme
measurements. JournalofFacuultyofI-‘isheries, PrefecturalUniversity ofMie6:291-301.

Fl

Pass

122
APPENDIX E

Carboxypeptidase B Assay Protocol

I. .
Pyloric caecae were thawed at 4°C. Tissues were chopped with a scisors and
homoginized inanicebathfor 30 secinDDIwater. Thehomogenizerprobewas
pre-chilled in an ice bath.

Tissue homogenates were centrifuged at 5495 x g for 60 min at 4°C.
Supernatants were aspirated and stored on ice until assayed. Supernatants were
diluted with DDI water prior to analysis. A 100m aliquot of the ﬁnal dilution was
reserved for protein analysis.

was

I-lippuiyl-Irarginine+H,O-l-carboxypeptidase A - hippuric acid-+L-arginine
Commons: T=25°C, pH=7.65, A=254 , light path=1 cm

W: Continuous spectrophotometric rate determination

met—its:

25 mM Tris HCl buffer with 100 mM sodium chloride, pH 7.65 at 25°C.

To prepare 1L, add 3.94 g Trizma-HCl and 5.844 g NaCl to water. Adjust pH to
7.65 using NaOH. Transfer to a 1L volumetric ﬂask andbring to volume with

water.

123
1.0 mM Hippuryl-L-arginine solution

To prepare 50 mL, dissolve 16.77 mg H-L-Arg in 50 mL water using a volumetric
ﬂask. Must be prepared ﬁ'esh daily. SIGMA Hipputyl-L-Arginine, product No.
H-2508.

W: The following volumes (mL) were added to 3-mL cuvettes

 

 

Reagent Test Blank
H-L-A solution 2.9 ‘ 2.9
Deionized distilled water -- 0.10
Sample or standard 0.10 --

 

Equilibrate H-L-A solution to 25 °C. Add sample, blank, or standard and mix
immediately by iversion. Record changes in absorbance.
Calculatians:
Based on the Bouguer-Lambert-Beer law:

Abs = (e)(c)(d)
where: e=extinction coeﬁcient; c=concentration; d=light path (cm)
Rearranged, c = (Abs)-(e><d)'l
Accounting for the absorbance of the blank, dilution factors, assay time and
volume, and sample volume:
Units-mL‘l supernatant solution =

[(e Abs'niin" .., - A Absmin" ,.,) (3th)]-(0.36><0.1 x 1)

124

where: 3 = assay ﬁnal volume (mL), df= dilution factor, 0.36 = millimolar
extinction coeﬁcient of H-L-A at 254 nm, 0.1 = volume of sample or standard
used (mL). 1= light Path (cm)
Units-tissuel = (U -mL" supernatant solution) x VB
where: VB = volume of buﬁ‘er in which pyloric caeca was homogenized (mL)
Units-g tissiie’l = (U -tissue")-(tissue weight)“
Speciﬁc activity =

(U-mL" sample solution)(mg protein-mL" supernatant solution)"
Won: One unit will hydrolyze 1.0 pmole of hippuryl-L arginine per min
at pH 7.65 at 25°C.
W:

Anonymous 199411. SIGMAqualitycontroltestprocedure. Enzymaticassayof
carboxypeptidase B (EC 3.4.17.2). SIGMA Cluemical Company, St. Louis, Missouri.

Yoshinaka,1., Sato,M.,Morishita.1.andS. lkeda. 1985. Enzymaticcharacterizationof
carboxypepﬁdaseAfromthecatﬁshpanaeasBuﬂleﬁnofﬂueJapaneseSodetyof
ScientiﬁcFisheries 51:113-116.

Yoshinaka, 1., Sato, M, Morishita, 1.1toh, Y., Hujita, M. and S. lkeda. 1984. Puriﬁcationand
somepropertiesoftwocarboxypeptidasesBﬁomthecatﬁshpancreas. Bulletinofthe
Japanese Societyof Scientiﬁc Fisheries 50:1717-1722.

"lllllllllllllllilllll