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00897 3210

This is to certify that the

thesis entitled

PRETREATMENT 0F SALMONID DIETS WITH PHYTASE
TO REDUCE PHOSPHORUS CONCENTRATIONS
IN HATCHERY EFFLUENTS
presented by

Kenneth Daniel Cain

has been accepted towards fulfillment
of the requirements for

 

 

Master of Science degmmin Fisheries and Wildlife

 

 

[hue March 16, 1993

 

0-7639 MS U is an Afﬁrmative Action/Equal Opportunity Institution

 

 

 

 

 

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PLACE IN RETURN BOX to remove this checkout from your record.
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MSU Is An Affirmative Action/Equal Opportunity Institution
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PRETREATMENT OF SALMONID DIETS WITH PHYTASE TO
REDUCE PHOSPHORUS CONCENTRATIONS IN HATCHERY EFFLUENTS

BY

Kenneth Daniel Cain

A THESIS

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

MASTER OF SCIENCE

Department of Fisheries and Wildlife

1993

 

 

ABSTRACT

PRETREATMENT OF SALMONID DIETS WITH PHYTASE TO
REDUCE PHOSPHORUS CONCENTRATIONS IN HATCHERY EFFLUENTS

BY

Kenneth D. Cain

Performance of rainbow trout fed experimental diets
containing phytase-treated or untreated soybean meal with
graded levels of supplemental phosphorus (P) was compared to
trout fed commercial feeds. Growth rate, feed conversion
and whole carcass P concentrations of fish fed phytase-
treated diets were not significantly different than those of
fish fed commercial diets (ANOVA) at (p<0.05). Pre-
treatment with the enzyme phytase increased P availability
in soybean meal by hydrolyzing phytin P to an available
inorganic form. Converted phytin P replaced supplemental P
in the diet and was utilized effectively by rainbow trout.
The lower dietary P levels resulted in substantial
reductions of P in the effluent, ranging from 9.47 g/kg diet
fed for a commercial feed to 2.20 g/kg feed fed for a diet
containing 0% supplemental P and phytase treatment. Further
P reduction was achieved when routine fecal collection was
implemented (1.88 g/kg diet fed for juvenile fish and 0.34
g/kg diet fed for larger fish). P effluent concentrations
were determined using a mass balance strategy. Whole
carcass P levels of fish indicated that available dietary P

requirements for rainbow trout are between 0.40% and 0.45%.

 

 

ACKNOWLEDGEMENTS

I would like to thank Dr. Thomas Coon, and Dr. Michael
VandeHaar for their review of this manuscript and for
serving as my committee members. I would especially like to
thank Dr. Donald L. Garling, Jr. for providing me with this
research opportunity. His guidance and friendship over the
years will never be forgotten. Also, a special thanks to my
former lab associates and those people who volunteered their
valuable time to help on this project: Andy Westmaas, Jay
Hesse, Chris Starr, Paul Wilbert, Roger Glass, Laurel
Ramseyer, Scott Miller, and Kevin Rathburn.

I would also like to extend my appreciation to the
North Central Regional Aquacultural Center, ALKO
Biotechnologies, and the Michigan Agriculture Experiment
Station for providing support for this project. In addition
I would like to thank Dr. Rick Barrows, Dr. George Ketola,
and Dr. Paul Brown for their participation in this project.

Finally, I would like to thank my fiance Deborah for
her much needed support, encouragement, and patience
throughout the preparation of this thesis.

This thesis is dedicated to my parents Richard and
Karen Cain who have supported me in everything I have done,
and over the years have always been there when I've needed

them.

 

 

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . . . . . . .
LIST OF FIGURES O Q I I O C C O O O O O O O O O I 0
KEY TO SCIENTIFIC NOMENCLATURE . . . . . . . . . .
INTRODUCTION 0 O O O O O C O I I . I O I O O O O I
LITERATURE REVIEW . . . . . . . . . . . . . . . . .
Nutrient Discharge in Hatchery Effluent . . .
Strategies for Effluent Phosphorus Reduction .
Phosphorus Requirements of Fish . . . . . . .
Phosphorus in Feed Ingredients . . . . . . . .
Improvement of Phosphorus Availability Through
use of Phytase . . . . . . . . . . . .
Carbohydrates in Fish Diets . . . . . . . . .
Mass Balance Analysis . . . . . . . . . . . .
MATERIALS AND METHODS . . . . . . . . . . . . . . .
Feeding Trials . . . . . . . . . . . . . . . .
Diet Preparation . . . . . . . . . . . . .
Data Collection and Analysis . . . . . . . . .
RESULTS 0 C O O O O O I O O O I O O O Q I O O O O O
DISCUSS ION C O O O O O O O O O C O O O O O O O I O
SUWY O O O O O O O O O O I O C 0 O O O O C l O 0
CONCLUSIONS . . . . . . . . . . . . . . . . . . . .

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

iv

0 o o I o

O 0 0 t

69

82

84

88

 

10.

LIST OF TABLES

Phytic acid content of plant ingredients that may
be used in feeds. . . . . . . . . . . . . . . . .

Control and semi-purified diets, based on the TZM
formulation of Ketola (Table 4), fed to triplicate
groups of fingerling rainbow trout in experiment

1. 0 o o 0 o o o 0 o o O I o o o o o o o o o o c 0

Control and semi-purified diets, based on the T2M
formulation of Ketola (Table 4), fed to triplicate
groups of fingerling rainbow trout in experiments
2a and b. D O O O I D O C O O O I O O O I O O O 0

Composition of T2M experimental diet (%)3. . . . .

U.S.F.W.S. Vitamin premix No. 30 used in
experimental diets.. . . . . . . . . . . . . . . .

Mineral mixture composition (in mg/kg of diet)
used in experimental diets.. . . . . . . . . . . .

Citrate buffer solution used in the preparation of
phytase-treated soybean meal (Lillie 1948). . . .

Weekly weighing (W), cleaning (C) and randomly
generated (Petersen 1985) fecal sampling (FS)
schedule for experiments 2a and b. . . . . . . . .

Inositol phosphate (IPyé) and inorganic phosphorus
(P1) analysis of phytase-treated and untreated
soybean meal used as diet ingredients in
experiment 1.. . . . . . . . . . . . . . . . . . .

Percent P (dry wight basis) for each of the six
diets fed to fish in experiment 1. . . . . . . . .

Page

. 13

 

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

Performance of triplicate groups of fingerling

rainbow trout fed a commercial diet (Zeigler

salmon starter) and semi-purified experimental

diets with or without phytase treatment and graded
levels of supplemental P in experiment 1. Values

in columns with different superscripts are
significantly different (p<0.05). . . . . . . . . . 42

Body composition‘ analysis of whole rainbow trout
fed semi-purified diets and commercial trout feed
(Zeigler salmon starter) in experiment 1. . . . . . 45

Amount of P fed, retained in fish, and discharged
to the effluent for experiment 1 (10 weeks). . . . . 46

Ino)sitol phosphate (IP3_ ) and inorganic phosphorus
concentrations of phytase-treated and
gt)reated soybean meal and experimental diets
(Tables 2, 3) fed to rainbow trout in experiments
2a and b. . . . . . . . . . . . . . . . . . . . . . 48

Percent P (dry weight basis) and protein for each
of the seven diets fed to rainbow trout in
experiments 2a and b. . . . . . . . . . . . . . . 49

Performance of triplicate groups of fingerling

rainbow trout fed a commercial diet (Zeigler trout
grower) and semi-purified experimental diets with

or without phytase treatment and graded levels of
supplemental P in experiment 2a. Values in

columns with different superscripts are

significantly different (p<0.05). . . . . . . . . . 50

Protein accretion of fingerling rainbow trout
associated with commercial and experimental diets fed
in experiment 2a (10 weeks). . . . . . . . . . . . . 52

Body composition1 analysis of whole rainbow trout

fed semi—purified diets and commercial trout feed
(Zeigler trout grower) in experiment 2a. Values

in columns with different superscripts indicate
significant differences (p<0.05) . . . . . . . . . . 54

Total P (g) recovered in fecal solids collected over a
ten week period in experiment 2a. Values in columns

with different superscripts are significantly different
(p<0.05). 55

Amount of P fed, collected in feces, and

discharged in the effluent, determined upon
termination of Experiment 2a (10 weeks). . . . . . . 57

vi

21.

22.

23.

24.

25.

26.

Performance of triplicate groups of fingerling

rainbow trout fed a commercial diet (Zeigler trout
grower) and semi-purified experimental diets with

or without phytase treatment and graded levels of
supplemental P in experiment 2b. Values in

columns with different superscripts are

significantly different (p<0.05). . . . . . . . . . 59

Protein accretion of rainbow trout associated with
commercial and experimental diets fed in experiment 2b
(10 weeks) I I O O O C O O O C C O O O O O O I I I 60

Body composition‘ analysis of whole rainbow trout

fed semi-purified diets and commercial trout feed
(Zeigler trout grower) in experiment 2b. Values

in columns with different superscripts indicate
significant differences (p<0.05). . . . . . . . . . 63

Total P (g) recovered in fecal solids collected
over a ten week period in experiment 2b. Values
in columns with different superscripts indicate
significant differences (p<0.05). . . . . . . . . . 65

Amount of P fed, collected in feces, and
discharged in the effluent, determined upon
termination of Experiment 2b (10 weeks). . . . . . 66

Feed conversions of juvenile to post juvenile

rainbow trout fed a commercial reference diet

(Zeigler trout grower) and semi-purified

experimental diets. Values with different

superscripts indicate significant differences
(One-sample test of means) at (p<0.05). . . . . . . 67

LIST OF FIGURES

Figure

1.

Tank design with screen and rear standpipe for
routine collection of fecal material to determine
effectiveness of waste management strategies in
reducing P effluent values. Solids accumulated in
front of and behind screen support frame. . . . .

Average weight gain (g/fish) of rainbow trout over
time when fed a commercial reference diet or semi-
purified experimental diets with or without

phytase and graded levels of supplemental P (sup.)
in experiment 1.. . . . . . . . . . . . . . . . .

Average weight gain (g/fish) of rainbow trout over
time when fed a commercial reference diet or semi-
purified experimental diets with or without

phytase and graded levels of supplemental P (sup.)
in experiment 2a.. . . . . . . . . . . . . . . . .

Average weight gain (g/fish) of rainbow trout over
time when fed a commercial reference diet or semi-
purified experimental diets with or without

phytase and graded levels of supplemental P (sup.)
in experiment 2b.. . . . . . . . . . . . . . . . .

viii

Page

 

KEY TO SCIENTIFIC NOMENCLATURE

List of common and scientific names of fishes cited in this

thesis.

Anchovy

Carp, common
grass

Catfish, channel

Drum, red
Eel, Japanese
Mackerel

Salmon, Atlantic
chum

Sand lance

Seabass, Asia

Sea bream, red

Sole

Sunfish, bluegill

Tilapia,

Trout, brook
brown
rainbow

Yellowtail

ix

EmmLLsmrci—ax
ﬁrearm—Sm '
Ctenophagyngodon idella
lgtelurus EEQQ£§£E§
mm
Anguilla japonica
mum
MM
Oncorhynchus get;
Ammodytes s9.
MM
Chrvsophrvs maior
§2l§§ §Ql§§

Lepomis macrochirus
Oreochromis niloticus
Oreochromis mossambicus
Salvelinus fontinalis

Salmo tutta

 

 

Oncorhynchus mykiss

Seriola guinggeradiata

INTRODUCTION

There has been a growing concern over the level of
phosphorus (P) released from fish hatcheries due to its
potential role in increasing eutrophication in receiving
waters (Cowey and Cho 1991). Eutrophication is defined as
the increased production of organic matter in response to an
increased supply of nutrients (Wetzel 1983). P has been
identified as the primary limiting factor in freshwater
aquatic plant growth and high P levels promote
eutrophication (Kendra 1991). Excess P increases
phytoplankton growth, aquatic weed growth, and the
production of algal blooms, which reduce dissolved oxygen
levels through increased respiration from bacterial
decomposition of organic matter (Wetzel 1983). In the
severest cases, eutrophication results in decreased water
quality and fish kills.

National attention was focused on this subject when the
Michigan Department of Natural Resources was required to
substantially reduce P discharge from the Platte River
Salmon Hatchery in Honor, Michigan, to limit eutrophication
in Platte Lake (Westers, personal communication). Average P

concentrations in the Platte river were estimated to be 12

2
ug/l upstream from the hatchery and 33 ug/l below (Grant
1979). The Water Quality Division of the Michigan DNR
placed restrictions on the total annual amount of P that
could be released. To meet the target P levels, costly
modifications of the hatchery's settling basin and raceways
were made. Other ways that were explored to minimize P
discharge included reduction in fish production, lowering of
dietary P levels, and switching to less soluble forms of
supplemental inorganic P (Ketola 1985).

High P levels in fish hatchery effluents originate
primarily from the feed. Fish meal serves as the primary
protein source in most commercial trout and salmon diets.
At the levels used in fish feeds, fish meal generally
contains P in excess of the fish's requirements (Lall 1991).
High costs and reduced availability of fish meal and other
animal proteins has caused the development of fish diets
containing plant protein sources. The majority of P
contained in plant feed ingredients is typically found as
organic phytin P (phytic acid). This form of P is poorly
utilized because phytase, the enzyme that hydrolyses phytic
acid, is not present in fish and other monogastric animals
(Lall 1991; Ketola 1975a,b). Inorganic P supplements are
therefore added to fish feeds that are high in plant
proteins in order to meet minimum P requirements. If
phytase could be incorporated into fish diets high in plant

protein then reduced levels of supplemental P would be

3
needed to meet requirements. Nutritionists must develop
fish feeds that provide optimum growth and increased P
bioavailability so that fish diets will contain less total P
and therefore less P will be excreted to the effluent (Lall
1991).

Ketola (1985) showed that reduction in dietary P levels
can markedly reduce effluent P from hatcheries. He also
found that the use of less soluble forms of supplemental
inorganic P in the mineral mix increased retention of P in
fecal material and uneaten feed. Efficient collection of
solid wastes should further reduce P levels discharged from
hatcheries (Westers 1991).

The development of diets that minimize environmental
impact are essential to the future growth and advancement of
the aquaculture industry. Therefore, the objectives of this
study were:

1) To improve the biological availability

of P in soybean meal through the use of
exogenous phytase;

2) To estimate effluent P levels through a

mass balance formula; and

3) To develop low P diets that can reduce P

concentrations in the effluent of fish
culture facilities.
Feeding experiments were designed to evaluate the use

of phytase-treated soybean meal to replace supplemental P

4
while meeting optimum dietary nutritional requirements of
rainbow trout. Growth rates, feed conversions, body
compositions, protein accretion, and fecal P concentrations
of rainbow trout fed experimental diets containing phytase-
treated or untreated soybean meal with graded levels of

supplemental P were compared to those fed commercial feeds.

LITERATURE REVIEW

I. NUTRIENT DISCHARGE IN HATCHERY EFFLUENT

Decreased water quality resulting from increased P
discharge from fish culture facilities has been well
documented (Hinshaw 1973; Bergheim and Selmer-Olsen 1978;
and Korzeniewski et al. 1982; Folke and Kautsky 1989; Kendra
1991). P from fecal wastes and uneaten feed stimulates
increased benthic and planktonic algal blooms and macrophyte
growth in freshwater systems (Lall 1991). Increased
eutrophication has also been observed in salt water systems
from increased nitrogen levels (Persson 1991).

Liao (1970) reported water quality degradation in
receiving streams from the effluent of salmonid hatcheries.
Kendra (1991) showed significant increases in temperature,
pH, suspended solids, ammonia, organic nitrogen, total
phosphorus, and chemical oxygen demand in hatchery effluent
compared to incoming water. He looked at 11 state and 9
commercial salmonid facilities in Washington state during
the summer of 1988. Wiesmann et. al. (1988) analyzed water
entering and leaving nine commercial trout farms of North-
West Germany and showed that concentrations of ortho-

phosphate in the water increased substantially during

6
passage through the farms. He suggested that in order to
limit phosphate pollution of receiving streams, feed
manufacturers should aim to avoid dietary P concentrations
that exceed the requirements of the target fish.

In 1972 the first regulation of fish hatchery waste
discharge was implemented with the creation of the National
Pollutant Discharge Elimination System (NPDES) permit
program (Harris 1981). There are no specific federal
effluent standards established for allowable phosphate
discharges. However, Michigan has established a maximum
allowable phosphate effluent discharge of 1 mg/l, or lower

if necessary, for protection of Great Lakes water quality.

II. STRATEGIES FOR EFFLUENT PHOSPHORUS REDUCTION

Reductions of P effluent levels are needed to minimize
the negative impact of fish farms on the environment. P
reduction can be accomplished with construction of costly
modifications to the hatchery, reformulation of feeds, or
reduction of fish production. Generally, nutritionists
agree that the only long term solution to this problem is
through feed modifications.

In addition to nutritional solutions to P discharge
problems, "low tech" solutions to solid waste management
help reduce P (Westers 1991). In Michigan, state hatcheries
have used baffles and high water flows to efficiently move

fecal and feed wastes to a screened off section at the foot

7
of each raceway. Solids settle out in the foot of the
raceways where they can be removed for disposal (Westers
1991). A sump pump system has also been developed for
solids removal and long term waste storage (Westers 1991).
Fecal and feed waste material are stored until applied to
agricultural fields. Implementation of these techniques can
be expensive; but, for the time being, are the best methods
to effectively prevent the release of solids to the
effluent. When new facilities are built, design
modifications should be incorporated in the plans to reduce
the release of nutrients or solids.

Lall (1991) suggested that immediate strategies to
lower the levels of P discharged from fish culture
operations should include:

1. development of low P feeds that sustain

good growth, feed efficiency, health,
and reproduction.

2. selection of low soluble P supplements,

such as deflourinated rock phosphate

3. selection of feed ingredients and P

supplements with high bioavailability

4. development of diets with better feed

efficiency

5. minimization of feed wastage
Lall (1991) indicated that the use of enzyme technology to
improve the bioavailability of P, such as phytate P, in
feedstuffs must be studied. He also concluded that
reductions in P levels will only be accomplished through

adequate research and planning by fish nutritionists and

feed manufacturers.

8
III. PHOSPHORUS REQUIREMENTS OF FISH

P must be supplied at levels in the diet that meet the
minimal P requirements of fish. Fish have been found to
take up small amounts of P from water but this intake is
insignificant for meeting the P requirements of fish (Coffin
et a1. 1949; Srivastava 1960). Dietary P levels which
promote optimal growth, feed utilization and bone
mineralization have been estimated to range from 0.4% to
0.8% of the dry diet for rainbow trout (Ketola 1975; Ogino
and Takeda 1978), Atlantic salmon (Lall and Bishop 1977),
chum salmon (Watanabe et a1. 1980), carp (Ogino and Takeda
1976) and red sea bream (Sakamoto and Yone 1978). Dietary P
levels of 0.5% were adequate for normal bone mineralization
in Oreochromis aureus reared in calcium-free water (Robinson
et a1. 1987).

Ketola (1975) determined that for young Atlantic
salmon, 0.6% supplemental inorganic P must be added to a
diet containing 0.7% P from plant sources. Diets in these
(experiments were formulated with graded levels of inorganic
£> from 0% to 0.9% and fed to triplicate lots of young
Iktlantic salmon. Feed utilization was significantly
improved at levels of 0.3% or more and was optimum at 0.6%
or'0.75%. He showed inorganic P levels above 0.6% had
, little effect on growth, feed utilization, and bone ash
content. The high content of phytin probably reduced the

available P in the plant source. P requirements of 0.6% for

9
Atlantic salmon held in sea water are similar to other
salmonids (Lall and Bishop 1977).

Ogino and Takeda (1978) estimated P requirements for
rainbow trout to be between 0.7% to 0.8%. Available dietary
P levels that produced maximum growth and normal bone
mineralization for Chum salmon were 0.5% to 0.6% (Watanabe
et a1. 1980).

Available P requirements determined for channel catfish
were lower than values reported for rainbow trout or
Atlantic salmon (Lovell 1978; and Wilson 1982). Lovell
(1978) found that diets containing 0.42% to 0.47% available
P provided adequate levels for optimum growth and bone
mineralization. The signs of deficiency for fingerling
-channel catfish were poor growth and bone mineralization and
a slightly depressed appetite. The P source used in the
experimental diets was monobasic sodium phosphate which was
highly available to catfish (Lovell 1978). Andrews et al.
(1973) reported 0.8% available P was required in the diet
for channel catfish. Lovell (1978) stated that the
difference between the two estimates was apparently caused
by differences in the sources of P fed. Andrews et a1.
(1973) presumed availability of P in feedstuffs was the same
if! fish and poultry. Lovell (1978) estimated that only 25%
of the available P in Andrews' study was supplied by
Slpplemental inorganic P. The amount of P absorbed from

fish meal by catfish was less than one-half the level

10
absorbed by poultry (Lovell 1978).

Available P levels that produced maximum growth for
carp were 0.6% to 0.7% (Ogino and Takeda 1976). Feeding of
low P diets resulted in deficiency signs such as reduced
growth, low feed efficiency and deformity of the head.
Inorganic P was supplied by a mixture of 27 g sodium
phosphate and 73 g potassium phosphate. Both of these
ingredients were determined to be 98% available (from Lall
1991).

Ogino and Takeda (1976) found that changes in dietary
calcium levels did not affect growth of carp or rainbow
trout because fish readily absorbed it from the water (Ogino
and Takeda 1976; Ogino and Takeda 1978). However, Robinson
et a1. (1987) observed that Oreochromis aureus reared in
calcium—free water required 0.7% dietary calcium for optimum
growth. The requirement for dietary calcium may be
important when fish are reared in calcium-free water. Ca:P
ratios have been found to be important in diets of red sea
bream (Sakamoto and Yone 1973) and brook trout (Phillips et
a1. 1958). The importance of these ratios are most likely
related to the calcium content of the diet, the mineral
composition of the water, other dietary components, and
:ﬂlysiological factors (Lall 1991).

Studies on P requirements for other fishes are limited,
bui: some values have been reported. Nose and Arai (1979)

raported available dietary P requirements of about 0.29% for

11
the Japanese eel, which is much lower than reported values
for most other fishes. P requirements for the Asian seabass
have been reported to be 0.65% (cited in Boonyaratpalin
1991). In sole, P retention and growth were optimal at 0.7%
P (Caceres-Martinez 1984, as cited in Guillaume et a1.
1991). Daily P requirements for juvenile grass carp were
found to be 22.1-24.8 mg/100g body wt/day (Huang 1989).
Juvenile red drum were found to require 0.86% dietary P for
maximum tissue mineralization (Davis and Robinson 1986).
Requirements of available P for tilapia were 0.9% of the
diet (Watanabe et a1. 1980). Yellowtail requirements for P
were approximately 0.67% of the diet (Makino 1990, as cited
in Shimeno 1991). These studies provide information on the

P requirements for only a few of the many fishes raised.

IV. PHOSPHORUS IN FEED INGREDIENTS

The percent P levels in fish meal ranges from about
1.5% to 3.2% and is usually highly available to most fish
(Lall 1991). The high cost of fish meal has prompted the
development of feeds that contain protein from ingredients
of plant origin. Plant ingredients contain P; but, a large
portion of this P is present as phytate-P (Simons et al.
1990). Phytates are the salts of phytic acid (inositol
hexaphosphoric acid) which are stored in the seeds (Lall
1991). Phytic acid content of plant ingredients commonly

used in.feeds range from 0.89% to 7.5% (Table 1). The Ca-Mg

12
salt of phytic acid is referred to as phytin and is present
at high levels in some plant sources. The phytin P in these
feed ingredients is not biologically available to fish or
most monogastric animals. These animals lack the enzyme
phytase which hydrolyses phytin to form biologically
available P (Lall 1991).

Processes for lowering P levels in feed ingredients
include removal of bones in fish meal (Shearer and Hardy
1987), removal or hydrolysis of phytic acid in plant
proteins (Lall 1991), and exogenous treatment of plant
proteins with the enzyme phytase (Nelson et al. 1968; Simons
et a1. 1990). The quantity of phytic acid present in a feed
ingredient will markedly affect the bioavailability of P in
that ingredient; and, in many cases, inorganic P
supplementation may be needed. Phytate also binds to some
essential metals, such as zinc, which decreases their
availability in the diet (Maga 1982). These metals must be
added to feeds high in phytin to meet dietary requirements.
The bioavailability of P may also be influenced by other
nutrients, particle size, overall diet digestibility, and
the chemical form of P (Lall 1991).

Ogino et al. (1979) determined the availability of P
from some inorganic phosphates as well as some feedstuffs
including fish meal. Both carp and rainbow trout were able
tC> utilized P in casein and yeast, while P contained in fish

nueal was utilized more efficiently by rainbow trout than

13

Table 1. Phytic acid content of plant ingredients that may
be used in feeds.

 

 

 

 

 

 

 

 

 

 

 

 

 

Product Phytic Reference
acid (%)

Barley 0.97-1.08 Hartland and
Oberleas, 1977
Corn 0.89 Erdman, 1979
Cottonseed flour (glanded) 2.86-2.9 Erdman, 1979
Cottonseed flour (glandless) 4.29-4.8 Erdman, 1979
Oats 0.84-1.01 Hartland and
Oberleas, 1977
Peanut meal (dehulled) 1.7 Erdman, 1979
Rapeseed protein (conc.) 5.3-7.5 Erdman, 1979
Rapeseed meal 3-5 Uppstrom and
Svensson, 1980
Rice 0.89 Erdman, 1979
Sesame meal 5.18 Erdman, 1979
Soybean meal (dehulled) 1.4-1.6 Erdman, 1979
Soybean protein (isolated) 1.6—2.2 Erdman, 1979

 

Wheat 1.13 Erdman, 1979

 

 

 

 

 

14

carp. Watanabe et a1. (1980) also demonstrated a low
utilization of P from fish meal for tilapia compared to
rainbow trout and chum salmon. The reasons for these
digestibility differences may be the limited amount of
gastric secretions in warmwater species compared to
coldwater salmonid species (Yone and Toshima 1979).

Hayes and Jodrey (1952) examined P utilization in
trout by injection of radioactive P. Small brook trout (12-
20g) were injected intraperitoneally with approximately
12,000 counts per minute of radioactive phosphorus. After
stabilization, the turnover rates of P were the highest in
stomach tissue. Rates in liver, muscle and heart were found
to be dramatically lower, while bone tissue had the lowest
turnover rate. These findings emphasize the functions of
the fish stomach to pass material through its cells. Bone

tissue generally is considered a storage site for P.

V]. IMPROVEMENT OF PHOSPHORUS AVAILABILITY THROUGH THE

USE OF PHYTASE

The incorporation of phytase into diets containing high
levels of plant protein has the potential to increase the
bioavailability of P. The reduced need for P
supplementation in mineral mixes may potentially reduce the
negative environmental impact of agriculture and aquaculture
waStes.

Researchers have shown that phytase produced by a

15
culture of the mold Aspergillus ficugm improved the
utilization of phytate P in diets fed to poultry and swine
(Nelson et a1. 1968,1971; Simons et al. 1990). Nelson et
a1. (1968) observed that chicks utilized phytate P just as
efficiently as inorganic P when fed soybean meal incubated
with phytase from A; ficuum. Best results were obtained
when 1000 g soybean meal was mixed with 50 ml of the
culture, 200 ml warm water, and 250 ml of 1.2 N HCL. The
mixture was incubated for 2-24 h at 50°C and then dried in a
forced air drying oven (Nelson et a1. 1968).

In 1971, Nelson et al. added dried preparations of mold
phytase directly to diets of corn and soybean meal. The
phytase was prepared as an acetone-dried powder and used at
levels from 1-8 g phytase/kg diet. The percent bone ash
increased in chicks fed treated verses untreated diets which
indicated that the enzyme hydrolyzed the phytate to an
available form. It was shown that total hydrolysis of
phytate P occurred with 3 g phytase per kg diet, and that
chicks utilized the hydrolyzed phytate P as well as
supplemental inorganic P. Hydrolysis of phytate occurred
only after ingestion of the diet, indicating that activity
must have occurred in the alimentary tract of the chick.

Based on results from these experiments, Simons et a1.
(1990) studied the use of microbial phytase to improve P
availability in broiler chicken and pig diets. Aspergillus

ficuum was also used in these experiments to hydrolyze

 

 

16
phytate in soybean meal, corn and a liquid compound feed for
pigs. Phytase increased the availability of P by 60% in
broiler diets, while decreasing fecal P concentrations by
50%. In pigs, phytase increased P absorption by 24% and
lowered fecal P concentrations by 35%. Optimum phytase
activity occurred at pH levels of 2.5 and 5.5, while
activity was observed at pH as high as 7.0. The enzyme
remained stable as long as low-temperature pelleting methods
were used. High-temperature pelleting destroyed phytase
activity.

Yeast, bacteria and fungi are known to produce the
enzyme phytase (Simons et a1. 1990). However, yeast phytase
added to diets did not increase phytate P utilization in
pigs (Cromwell and Stahly 1978; Chapple et a1. 1979; Shurson
et a1. 1984).

Studies on the use of phytase in diets used for fish
are very limited. Ketola (personal communication) added
;phytase directly to diets fed to rainbow trout. These diets
icontained soybean meal as the primary plant protein source.
‘UtiJization of phytate P was not observed. Since trout are
cold blooded animals, body temperatures and pH levels were
probably not adequate to activate the phytase enzyme in the

gastrointestinal tract.

17
VI. CARBOHYDRATES IN FISH DIETS

Reducing levels of fish meal and increasing levels of
plant ingredients in commercial fish feeds has led to diets
with higher carbohydrate levels (Shimeno 1982). In nature,
carnivorous fish normally consume a diet low in
carbohydrates. Typically, carnivorous fish utilize
carbohydrates less efficiently than herbivorous or
omnivorous fish. Poor growth and feed efficiency occurs
when carnivorous fish are fed high levels of carbohydrate
(Furuichi and Yone 1980,1982). Carbohydrates that are
digested and absorbed act as an immediate energy source or
are stored as available energy (glycogen) in the liver and
muscle. Omnivorous fish tend to utilize carbohydrates more
efficiently than carnivorous fishes due to increased
intestinal activity of amylase, which hydrolyses starch
(Satoh 1991).

Channel catfish (Garling and Wilson 1976,1977), common
carp (Ogino et a1. 1976; Shimeno et a1. 1977,1981; Furuichi
and Yone 1980), and red sea bream, (Furuichi and Yone
1971,1980) utilize higher levels of carbohydrate than
yellowtail (Shimeno et a1. 1977) and salmonids ( Phillips
and Brockway 1956; Buhler and Halver 1961; Atkinson and
Hilton 1981; Hilton et a1 1982). Likimani and Wilson (1982)
found that the activities of several lipogenic enzymes;
fatty acid synthetase, malic enzyme, glucose-6-phosphate

dehydrogenase, 6-phosphogluconate dehydrogenase, and

 

18
NADP-isocitrate dehydrogenase were stimulated when channel
catfish were fed a high carbohydrate diet. Lipogenic enzyme
activity also was stimulated by feeding common carp a high
carbohydrate (Shimeno et a1. 1981). However, no stimulatory
effect on lipogenic enzyme activity was observed when coho
salmon were fed high levels of carbohydrate (Lin et al.
1977).

Salmonids utilize carbohydrates poorly (Lovell 1989).
Phillips et a1. (1948) reported that trout are able to
digest carbohydrates, but they can utilize only limited
levels. Buhler and Halver (1961) found that growth of
chinook salmon fed high levels of carbohydrate was not
inhibited if vitamins were supplemented adequately. Liver
glycogen concentrations increased in response to increased
dietary carbohydrates. Buhler and Halver concluded that the
nutritive value of the carbohydrate sources was inversely
proportional to the chemical complexity of the carbohydrate
source. Rainbow trout have a limited ability to utilize
dietary carbohydrate in excess of 14% of the diet (Atkinson
and Hilton 1981). Phillips et al. (1966) found that a
carbohydrate concentration as low as 6% in trout diets
resulted in increased liver size and glycogen content.

Dietary carbohydrate use has been studied in a few
other fishes. In the common carp, carbohydrates were found
to be utilized effectively as an energy source (Ogino et al.

1976). Channel catfish used up to 25% dietary carbohydrates

 

19
as efficiently as lipids for energy (Garling and Wilson
1977). Red sea bream exhibited growth retardation and low
feed efficiency when fed diets containing more than 30%
dextrin (Furuichi and Yone 1980). In yellowtail, an
exclusively carnivorous fish, the development of commercial
feeds has been unsuccessful because of their inability to
utilize dietary carbohydrates. Yellowtail grow much more
efficiently when fed frozen fish (Shimeno 1991) and high
intake of dietary carbohydrates in yellowtail was found to
cause metabolic disturbances (Shimeno et al. 1977). In
general, dietary carbohydrates do not seem to be effective

energy sources for salmonids.

VII. MASS BALANCE ANALYSIS

Another potential costly problem for aquaculture that
has received little attention is the methodology used for
hatchery effluent analyses. Values for solids (feces and
uneaten feed), P, and nitrogen from hatchery discharge
waters are generally determined directly on the effluent
water using expensive chemical monitoring techniques (Cho et
al. 1991). These values may significantly overestimate or
'underestimate the level of P being released in hatchery
(effluents due to variation in time of year, rate of flow,
and time of sampling relative to feeding and cleaning times.
Some hatcheries may be required to take as many as 24

samples per year (Cho et al. 1991). Indeed, the Platte

20
River Hatchery is required to sample effluent water more
than 24 times per year (Pecor, personal communication).
This testing can become a substantial added expense for
normal hatchery operating costs.

The values reported from chemical sampling may not
accurately represent nutrient levels discharged from
hatcheries because concentrations are normally measured as
mg of waste product/l of water (Cho et al. 1991). A large
production facility that produces many pounds of fish and
has large flow rates, may discharge low levels of P based on
mg P/l flow. However, the total amount of P discharged
annually from a large fish farm may be very high.
Conversely, a small production facility with limited water
flow may discharge high concentrations of P based on mg P/l
flow; but, the total amount discharged annually may be very
low.

Mass balance calculations of the discharge of P and
other nutrients have the potential to be much more accurate
and much less expensive than chemical methods of effluent
sampling (Cho et al.1991). Total P fed to the fish can be
accurately calculated. In the mass balance method, the
amount of P assimilated by the fish over a specific growth
period is subtracted from the amount fed and the amount
recovered in wastes. This method can be very accurate under
laboratory conditions but feed wastage and poor mortality

estimations introduce error in large-scale adaptation (Cho

21
et a1. 1991). Therefore, strict feed management practices
and accurate growth and mortality records would be needed.
Improved feed management practices also reduce pollution
from uneaten feed.

Cho et al. (1991) compared mass balance and chemical
effluent sampling methods for P output of brown trout reared
at the Harwood Fish Culture Station in Ontario, Canada.
Substantial discrepancies were observed between the two
methods of analysis. Estimations based on the chemical
method had greater variability than mass balance
calculations. Conclusions from this study indicated that
chemical effluent sampling of wastes generated by fish
culture operations can be very unreliable and that a
reliable mass balance approach to the quantification of

waste output could be readily and inexpensively carried out.

MATERIALS AND METHODS

All experiments were conducted at the Michigan State
University Fish Culture Research Laboratory using rainbow
trout of the Shasta strain. Three experiments were
conducted to determine effects of feeding rainbow trout
experimental diets with or without phytase treatment and
with graded levels of supplemental P versus a standard
commercial reference diet on growth rate, fecal P
concentrations, and effluent P levels. Diets that contained
100% inorganic P supplementation provided approximately
0.66% available P. In all three experiments, culture tanks
were supplied with aerated well water at 12.5 i 19C. The
tanks used were 360 liter fiberglass oval tanks filled to
approximately 190 liters. Flow rates for this single pass
flow through system were set at 2.5 liters per minute (1pm).
Fish were maintained on the ambient lightzdark photoperiod
and were fed twice daily on a percent body weight basis

according to recommendations by Piper et al. (1982).

I. FEEDING TRIALS
In Experiment 1, triplicate groups of approximately 20

fingerling rainbow trout (Shasta strain), were fed one of

22

23
five experimental diets or a commercial reference diet
(Table 2) for a period of ten weeks. The diets used were a
commercial reference diet (Zeigler salmon starter), Ketola's
T2M diet (Ketola, personal communication), MSU modified T2M
diet with 100% supplemental P with phytase, MSU modified T2M
diet with 25% supplemental P with and without phytase, and
MSU modified TZH diet with 0% supplemental P with phytase.
Feeding trials were designed to determine if rainbow trout
fed diets containing phytase-treated soybean meal exhibited
any deficiencies or abnormalities. Diets were initially fed
at 3.5% wet body weight/day and adjusted (Piper et al. 1982)
throughout the study to a final rate of 2.0%. Prior to the
start of the experiment, fish were fed the commercial
reference diet at low rations.

In experiment 2a, the six diets used in experiment 1
and an additional diet with 0% supplemental P without
phytase (Table 3) were fed to juvenile rainbow trout for 10
weeks. The commercial reference, a trout grower diet, was_
used instead of a starter as in experiment 1 because P
concentrations of the grower diet compare favorably to the
experimental diets with 100% P supplementation. Although,
the protein level of the grower diet was lower than
experimental diets. This experiment was designed to confirm
results of experiment 1 and determine deficiency signs
associated with fish fed diets low in available P. Feeding

rates were initially 3.5% wet weight/day and adjusted (Piper

 

24

Table 2. Control and semi-purified diets, based on the TZM
formulation of Ketola (Table 4), fed to triplicate groups of
fingerling rainbow trout in experiment 1.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Diet Type Source Phytase %
treatment supplemental
P
A commercial Zeigler Salmon NA NA
reference Starter
B semi-purified Ketola1 T2M - 100
reference experimental
diet
C Semi-purified MSU Modified + 100
experimental T2M
D Semi-purified MSU Modified - 25
experimental T2M
E Semi-purified MSU Modified + 25
experimental TZM
F Semi-purified MSU Modified + 0
experimental T2M
1 Diet formulation provided by Dr. George Ketola, USFWS,

Tunison Laboratory of Fish Nutrition, Cortland, NY.

 

 

Table 3.

25

Control and semi-purified diets, based on the TZM
formulation of Ketola (Table 4), fed to triplicate groups of
fingerling rainbow trout in experiments 2a and b.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Diet Type Source Phytase %
treatment supplemental
P
A commercial Zeigler (Trout NA NA
reference Grower)
B semi-purified Ketola2 T2M — 100
reference experimental
diet
C Semi-purified MSU Modified + 100
experimental T2M
D Semi-purified MSU Modified - 25
experimental T2M
E Semi-purified MSU Modified + 25
experimental T2M
F Semi-purified MSU Modified + 0
experimental TZM
G Semi-purified MSU Modified - 0
experimental T2M
2 Diet formulation provided by Dr. George Ketola, USFWS,

Tunison Laboratory of Fish Nutrition, Cortland, NY.

 

 

 

26

et al. 1982) throughout the study to a final rate of 2.5%.
Prior to the start of the experiment fish were fed the
commercial reference diet at low rations. At the end of
experiment 2a, 10 fish were removed from each tank. The
removal of these fish served two purposes 1) to compare
growth and proximate analyses to experiment 1, and 2) to
lower densities (kg/m3) for proper growth in experiment 2b.

Experiment 2b was designed to determine effects of long
term feeding of the experimental diets on rainbow trout
approaching market size. The 10 remaining fish in each tank
from experiment 2a were kept in the tanks and fed the same
experimental diets (Table 3) for an additional ten week
period. Feeding rate of diets was adjusted gradually from
2.5% wet body weight/day and adjusted throughout the study

to a final rate of 1.5%.

II. DIET PREPARATION

Ketola (personal communication) provided the
formulation of the TZM diet (Tables 4-6) used in all three
experiments. This diet was prepared along with MSU modified
T2M diets (Tables 2-3) with or without phytase-treated
soybean meal and with graded levels of supplemental P.
Commercial reference diets were used for growth comparisons.
The ingredients in the reference diets were protected by a
closed formula, and were not made available by the feed

manufacturer.

27

Table 4. Composition of T2M experimental diet (%)3.

 

 

 

 

 

 

 

 

 

 

 

Ingredients

Soybean meal 31

Corn gluten meal 30

Blood flour 10 I
Fish oil 11 I
Lysine HCl, L 0.4

Vitamin mix1 1.0

Mineral mix +2
Deflourinated rock +3
phosphate

Herring meal 10
Cellulose +

 

 

 

 

1 Vitamin mix # 30 supplied by USFWS (Table 5).
Mineral mixture (Table 6).

Diet T2M was supplemented with enough finely ground
deflourinated rock phosphate (DRP) to provide a level
of dietary P equal to the difference between 0.66% of
diet and the amount supplied by fish meal. Stated
mathematically: The percentage of dietary P provided
by DRP = 0.66% - the percentage of dietary P supplied
by fish meal (0.27%) (determined by analysis).

 

3 Diet formulation for experimental diets provided by Dr.
George Ketola, USFWS, Tunison Laboratory of Fish Nutrition,
Cortland, NY.

 

28

Table 5. U.S.F.W.S. Vitamin premix No. 30 used in
experimental diets.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

vitamin mg/g premix
D Calcium 26.5
pantothenate
Pyridoxine 7.7
Riboflavin 13.2
Niacinamide 55.1
Folic acid 2.2
Thiamine 8.8
Biotin .0882
Vitamin Bu .0055
Menadione sodium 2.76
bisulfite complex

Vitamin H IU/g
Vitamin E 88.2
Vitamin 03 110 . 25
Vitamin A 1,653,750 USP

 

Note: Choline chloride and ascorbic acid were added at
levels of 0.14 and 0.28 g/kg of diet respectively.

29

Table 6. Mineral mixture composition (in mg/kg of diet)
used in T2M experimental diet.

 

 

 

 

 

 

 

 

Hinerals Amount (mg/kg of diet)

Mn 100
Zn 100
Cu 10

Fe (ferrous) 100
I 5

Se (selenite) 0.1
Mg 990

 

 

 

30

Phytase-treatment of soybean meal followed the
recommendations of Scott Biotechnology Inc.:

1) One liter of water containing citrate buffer at pH
5.0 was prepared at room temperature (Table 7).

2) Two grams of phytase were dissolved in the buffer.

3) One kg of soybean meal was added into the phytase-
buffer mixture and rapidly heated, with constant
stirring, to 50-55°C. The mixture was held at
this temperature for 5 - 6 hours. Dry matter content
was 40% - 50% after the heating period.

4) Once the heating process was complete, treated
soybean meal was cooled and dried at room
temperature in a forced air drying oven for 24
hours to reduce the moisture content to
approximately 10%.

5) Dried, phytase-treated soybean meal was added to
the experimental feeds.

Dry dietary ingredients were mixed in an industrial
food mixer (univex Model M-12B) for 15 minutes. Fish oil
was slowly added and mixed for an additional 10 minutes.
Finally, about 1/2 liter of water was added slowly, to
;prevent clumping, and the ingredients were mixed into a
slightly moist, dough-like material. The material was
passed through an extruder with 1/8 inch die to form

strands. The strands were dried in a forced air oven at

 

Table 7. Citrate buffer solution used in the preparation of

31

phytase-treated soybean meal (Lillie 1948).

 

0.1 M solution of
citric acid (ml)

0.1 M solution of
sodium citrate (ml)

Desired pH level

 

 

20.5

 

29.5

 

5.0

 

Note: Concentration diluted in distilled H20 to a total of

100 ml.

Citric acid solution (0.1 M):

in 1000 ml distilled H20) .

(21.01 g citric acid dissolved

Sodium citrate solution (0.1 M): (29.41 g C6HSO7Na3-2HZO
dissolved in 1000 ml distilled Hg”.

 

 

32
room temperature for 24 hours. Dried strands were crumbled
in a waring blender and passed through U.S. number 4,5 and 6
sieves to separate appropriate pellet sizes.
All feeds were stored at -10W:. Feeds were thawed and

weighed daily before feeding periods.

III. DATA COLLECTION AND ANALYSIS

A. Weight Samples and Feed Conversions

In all Experiments, percent weight gain and feed
conversions were calculated on a wet weight basis at 2 week
intervals. The wet weight of fish was measured by netting
fish from the tank, allowing excess water to drip off, and
transferring them to a pre-weighed, water-filled bucket.
Total fish weight was calculated by subtracting the initial
weight of the bucket and water from the final weight of the
bucket containing water and fish. Fish were not fed the day
prior to weighing to avoid feed in the gut influencing
weights. Feeding resumed on the day after weight samples
were taken. Feed rations were also adjusted (Piper et a1.
1982) at this time.

Feed conversion values were calculated based on the
amount of feed fed for the sample period divided by the
weight increase for the period: PC = feed fed/weight gain.
Methods of sampling and FC determination remained consistent
for each of the three experiments. In experiments 2a and b,

protein accretion and utilization was determined from

 

33

proximate analyses of feed and fish.

B. Fecal Collection and Analysis

In Experiment 2a and b, a fecal sample was collected
based on a pre-determined randomized sampling schedule
(Table 8). All fish were reared in semi-oval tanks modified
for routine fecal collection. A rear standpipe and a screen
were installed in each tank (Figure 1). Fecal solids
accumulated in front of and just behind the screen. Tanks
were cleaned of solids 48 hours prior to sample collection.
Fecal solids and water from each tank were siphoned into
separate 10 liter containers and allowed to settle for a
period of about 15 minutes. After the solids had settled
the water was decanted off until a concentrated slurry of
solids and water remained. The slurry from each tank was
then transferred to separate, pre-weighed, 500 ml plastic
bottles and a total weight for each bottle was taken. The
weight of the empty bottle was subtracted from the total
weight in order to determine the weight of the slurry. Dry
matter and P analyses of the slurry were performed by the
MSU Animal Science Comparative Nutrition Laboratory.

C. Proximate Analysis

Initial samples of 10 fish were collected before the
Start of each experiment for proximate analysis. These fish
were not fed for 72 hours prior to analysis at the start of
the experiment. Upon termination of each experiment, 5 fish

from each tank were randomly removed. The whole fish were

Table 8.

34

Weekly weighing (W), cleaning (C) and randomly
generated (Petersen 1985) fecal sampling (FS) schedule for
experiments 2a and b.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Week Mon. Tues. led. Thurs. Fri. Sat. Sun.
0 W C C C
1 c c C,FS
2 W,C,FS c c c
3 C C C
4 W,C,FS c c c
5 C C C
6 W,C C C C
7 C C C,FS
8 W,C,FS C C C
9 C C C
10 W,C,FS C C C
11 C C C
12 W,C C C C
13 C C,FS C
14 W,C C C,FS C
15 C C C
16 W,C C,FS C C
17 C C C
18 w,c C,FS c c
19 C C C

l 20 W,C

 

 

 

 

35

Figure 1. Tank design with screen and rear standpipe for
routine collection of fecal material to determine

effectiveness of waste management strategies in reducing P

effluent values. Solids accumulated in front of and behind
screen support frame.

V‘m in“ ow \

wt“! '6VQ.‘ —'n- ' - — _- ‘ _ _ - - -- :‘l SMA'ch

 

 

 

 

u o! ' L——!S~ S who
Tan¥.s FF +s_<:: ' ta Aqu—

P ouﬂqou

 

 

 

 

36

stored frozen prior to proximate analysis. At the end of
each experiment, fish were thawed slightly and whole
carcasses were hand ground in a #5 Chop-Rite meat grinder.
Samples were analyzed by the MSU Meat Laboratory for whole
carcass composition. Analysis of fish carcasses included
percent moisture, fat, and protein.

Ash and P content of fish samples were completed at the
MSU Animal Science Comparative Nutrition Laboratory.
All diets were analyzed for total P at the MSU Animal
Science Comparative Nutrition Laboratory, while inorganic P
analyses for treated and untreated soybean meal and diets
were completed by ALKO Biotechnologies in Finland. All
quantitative analyses followed standard A.O.A.C. (1975)
methods.

D. Mass Balance Study (Phosphorus Determination)

P present in hatchery effluents can be described by the

formula:

Pef = Pin + Pho
Pef P in effluent
m P in incoming water source
Pho P from hatchery practices (feeding, solids
collection...)

For this study, chemical analysis of P in the water
source and effluent were not measured based on the
assumption that changes of P in hatchery effluent above
ambient were caused by feeding and solids collection. _The P
concentrations of incoming water were assumed to remain

constant since fish do not take up significant amounts of P

37
from the water (Lall 1991) and water turnover rates were
high (Piper et al. 1982). Total P concentrations in the
effluent that originated directly from hatchery practices
were determined by subtracting P assimilated by the fish
from the P fed. The mass balance equation used in the first

experiment was:

Pho = Pfed — (Pm - Pti)
where:
IR” = P in effluent resulting from hatchery
practices
Pfed = P in feed
Ptm = P in fish at end of experiment
Pti = P in fish at the beginning of experiment

P levels from samples of fish at the beginning and end of
the study were measured. The total amount of P present in
the fish at the start of the experiment (Pﬁ) was determined
by multiplying the percent P initially present in the fish
by the total weight of fish at that time. The total P
present in the fish at the end of the experiment (Pu) was
determined by multiplying the percent P present in the fish
at the termination of the study by the total weight of the
fish. P levels in feeds and fish were determined by the MSU
Animal Science Comparative Nutrition Analytical Lab.
Effluent P concentrations for experiment 2a and b were

determined using the following mass balance equation:

P... = P... - (P. + (P... - PM
where:
Pm. = P in effluent of hatchery origin
PM = P in feed
Pf = P in feces
Pm| = P in fish at end of growth period

 

38

Pti = P in fish at beginning of the growth period

This equation incorporates the amount of phosphorus
present in fecal samples. Since fecal samples were taken
over a 2 day period, total P collected/day was calculated
and then multiplied by the number of days in that feeding
period. This was repeated for each feeding period and total
P recovered was calculated.

E. Statistical Analysis

One-way Analysis of Variance (ANOVA) were conducted on
all growth and whole body composition data to determine if
differences associated with each diet were statistically
significant. Duncans/Newman-Keuls test (p=.05) was
conducted as a multiple comparison on all growth data for
all experiments. One-sample analysis of means were also
conducted to confirm significant differences found with
ANOVA. Feed conversion ratios of fish fed phytase-treated
diets were tested against fish fed non-phytase diets in

experiment 2b (Fisher's LSD test).

RESULTS

EXPERIMENT 1.

In experiment 1, pre-treatment of soybean meal with the
enzyme phytase reduced the concentrations of IP}6 to non-
detectable levels and increased the concentration of
inorganic P (Table 9). These changes indicate that the
phytase-treatment almost completely converted the
unavailable phytin P to the available inorganic P. Total P
concentrations levels of diets ranged from 1.45% for the
commercial diet to 0.66% for the MSU modified T2M diet with
0% P supplementation (Table 10). Because these values are
concentrations of total P, they do not represent the
percentage of P that is biologically available to the fish.

For trout fed the phytase-treated diets and the
commercial reference diet, body weight gains were similar
and no growth abnormalities were noted. Feed conversions
and weight gains for fish fed each diet are summarized in
Table 11. Juvenile rainbow trout that were fed experimental
diet B (100% P supplementation and no phytase treatment) and
diet D (25% P supplementation and no phytase treatment)
exhibited feed conversions that were significantly greater

(ANOVA, p<0.05) than those of fish fed all other

39

40

Table 9. Inositol phosphate (13») and inorganic phosphorus
(Pa analysis of phytase-treated and untreated soybean meal
used as diet ingredients in experiment 1.

 

 

 

Sample IP, 1P, 1P, 1?, P, D)!
umol/g umol/g umol/g umol/q .9/9 (’6)

Soybean meal 25.27’ 3.96 (0.7) 1.65 0.55 100

(untreated)

Soybean meal ND ND ND ND 12:7 100

(Phytase-

treated)‘

 

 

 

 

 

 

 

 

ND = Not Detected
() = Below the limit of quantification
‘ Average of three samples analyzed.

Note: All analysis completed by ALKO Biotechnical Product
Development (Research Report No.: 291073)

 

41

Table 10. Percent P (dry weight basis) for each of the six
diets fed to fish in experiment 1.

 

 

 

 

 

 

 

 

A (Zeigler salmon starter) 1.45 §
B (No phytase, 100% P sup.) 1.11 I
C (Phytase with 100% P sup.) 1.31 I
D (No phytase, 25% P sup.) 0.74

E (Phytase with 25% P sup.) 0.79 I
F;(Phytase with 0% P sup.) 0.66 ?

 

 

42

Table 11. Performance of triplicate groups of fingerling
rainbow trout fed a commercial diet (Zeigler salmon starter)
and semi-purified experimental diets with or without phytase
treatment and graded levels of supplemental P in experiment
1. Values in columns with different superscripts are
significantly different (p<0.05).

 

 

 

 

 

 

 

 

 

 

 

 

 

Initial Final Percent reed
Weight Weight Weight Gain Conversion
(g/fish) (g/fish)
A 3.19 18.69 586' 0.87'
B 3.15 15.03 477b 1.01bl
C 3.42 19.19 561' 0.89'I
D 3.39 16.81 496b 0.99b
E 3.41 18.96 556' 0.90'
F 3.39 18.78 554' 0.92.“
(ANOVA Table)
Source of Degrees of Sum of Mean F Prob.
Variation Freedom Squares Square Ratio
Among groups 5 .2522622 5.045245E-02
3.73 0.004
Within Groups 84 1.137333 1.353968E-02

Adjust. Total 89 1.389596

 

 

43
diets. Figure (2) shows overall growth rates (g/fish) of
trout fed each of the 6 diets for the 10 week growth period.

Body composition of fish is shown in Table 12. Bone
mineralization (as percent ash) ranged from 2.18% for fish
fed the commercial diet (diet A) to 1.55% for those fish fed
diet D which contained no phytase treatment and 25% P
supplementation. Fish fed diet D had significantly lower
percent ash values than fish fed other diets (p<0.05).
Whole carcass P concentration was similar for all fish
except those fed diet D contained significantly less P
(p<0.05) than all others.

Percent moisture was significantly greater while fat
and protein content was significantly lower (p<0.05) for
initial samples of fish than fish 10 weeks later. Fish fed
the commercial diet (diet A) had a higher fat concentration
(p<0.05).

The amount of P discharged to the effluent that
originated directly from the amount of P fed was calculated
as shown in Table 13. Fish that were fed diet F (0%
supplemental P and phytase-treated soybean meal) released
the lowest P levels to the effluent, (2.20 g P/kg diet fed).
Fish fed the commercial diet (diet A) released 9.47 g P/kg
diet fed to the effluent.

There were no mortalities during the course of the

experiment.

44

Figure 2. Average weight gain (g/fish) of rainbow trout
over time when fed a commercial reference diet or semi-
purified experimental diets with or without phytase and
graded levels of supplemental P (sup.) in experiment 1.

2EWEIGHT INCREASE OF TROUT IN EXPERIMENT I

 

Die: A (ZeigIer salmon stats)
+

Die: 3 (No Phytése, 100% sun.)

N
O
'

Die: C (Phytase. with 100% sun. ’
_._ > _ //-

  
    

Diet 0 (No Phytase. 252 sun.
--A--- ' )

_n
n

(

Diet E (Phytase with 252 sun.)

+

Diet F (Phytase. with 02 sup.)
+

 

 

  

 

WEIGHT (g/fish)

 

DI L; ' I I I
O 2 4 6 8 IO

GROWTH PERIOD (Weeks)

 

45

638386 no: u <\z n

.pmu muwﬂo ou osommmuuoo mumppma mamﬁmm ~
.mamwn we mm so so pwmmmumxm F

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

uN¢.o amm.wm uwm.H umH.mH omv.h am¢.¢b h
am¢.o eHm.mm aHO.N omw.mH o¢d.h an.¢b W
nNm.o umm.mm nmm.H coo.mH awa.w abh.¢h D
a¢¢.o umm.mm amH.N omo.mH awm.w www.mb U
aH¢.o cNN.mm abo.m omw.¢H noo.h aNm.mh m
u¢¢.o nom.mm owH.N omn.mH omo.w amm.m> <
amv.o nmm.bm n<\z th.HH non.m uw¢.Nw HMﬁUMCH
usuoomeosm Heuoa not swououm ash ousumwoz
useouom usoouom usoouom useouom useouom useouom Neamaem

 

 

.H unmaﬂuwmxm ca Aumuumum :OEme umamﬂwuv comm usouu Haﬁouwﬁaoo
use human owﬂuﬂusmlﬂﬁmm own usouu Bonsﬂwu macs; mo mamhamsw _coﬂuwmomﬁoo woom

.NH OHQMB

46

l

 

ON.N

 

 

 

 

 

 

 

 

 

 

 

 

 

mO.N mw.H mb.n mm.m h
wm.N QH.M wb.N OH.¢ ww.w ﬂ
mw.m Nm.¢ OH.M HN.N Hm.m Q
Nm.w mm.h m¢.0 NH.¢ hm.OH U
Gm.h mN.h w©.m oo.m mw.w m
wo.w 5v.m Hm.h mm.m om.HH 4
3.8 232. 9:3 88... 9:2 .3 .3 8. .
veuueaoedu m oeuueaouwo m venuenoeuo m vosweuou m can m sewn

 

.Amxmmz oHv H unmaﬂummxm
now usmsauuw ecu ca oooumsomﬂo can .smﬂu as omswmumu .60“ m mo apnoea

.nH OHQMB

 

47
EXPERIMENT 2a.

Inorganic P levels for treated and untreated soybean
meal and diets (Tables 2,3) fed to rainbow trout in
experiments 2a and b are summarized in Table 14. Although
inositol phosphate (IPpé) was not completely converted by
the treatment process, phytase-treated soybean meal had
higher inorganic P levels than untreated soybean meal. No
inositol phosphate was detected in any of the experimental
diets containing phytase-treated soybean meal (diets C, E,
and F). Diet D (25% P supplementation and no phytase) and G
(0% P supplementation and no phytase) had the lowest levels
of inorganic P (P1) available to fish.

Dietary P levels (Table 15) in diets fed to fish in
experiment 2a and b ranged from 1.18% for diet B (100% P
supplementation and no phytase) to 0.69% for diet F (0% P
supplementation with phytase) and G (0% P supplementation
and no phytase). Protein content of all experimental diets
averaged approximately 50% while protein in the commercial
reference diet (Zeigler trout grower) was 43%.

Upon termination of experiment 2a, average feed
conversions (Table 16) for trout fed the 7 experimental and
control diets (Table 3) ranged from 0.79 for diet C (MSU
modified T2M with 100% P supplementation and phytase-treated
soybean meal) to 1.00 for the commercial diet (Zeigler trout
grower). Fish fed diet C had significantly greater weight

gains and lower feed conversions while trout fed diet A had

48

Aomammm ".oz uuoowm soummmmmo usmsooam>oo uooooum Hmoﬂsoomuoﬁm
OMA< an omumamaoo mﬂmzamcm monogamooo oﬁcoouocﬁ can uncommooo Houﬂmosﬁ Has u muoz

.omnzamsm mwamﬁmm mounu no monum>< _

cosumozuouooso no unazz on» zozom I As
owuowumo uoz ﬂ oz

 

 

 

 

 

 

 

 

 

 

 

ooH m.~ oz AC h.H oH A.osm m wo .wwmuzoo ozv o umﬂo
ooH o.m oz oz oz oz A.osm m wo :uwS mmmuznmo z umﬂo
o3. 66 oz oz oz oz T96 o wmm :33 $335 w uoﬁo
o3 In oz 3 «rs To Togo o wmm .mmouzoo ozo o umzo
ooH o.m oz oz oz oz x.osm o woos nus; mmouzzoo o oozo
cos «.5 AC 4.H m.H o.o z.oom o woos .mmmuzzo ozo m uozo
ooH o.m oz oz o.z n.4H zuwzopo poop» uoamzoao a pogo
ooH o.m AC AC om.o H.m _Aowummuulmwmuzsmo Hams cmwnzom
ooH 36 oz oz m.~ R3 83335: Home cmoozom
U\UI U\Hoss O\Hoss u\aoﬁs U\Aoas
Axozo _m no“ .mu non emu oaoaao

 

 

 

 

 

 

 

 

.2 new on musmﬁﬂumoxm :H usonu soncﬂwu
0» new An.~ menoBo muwﬂo Housmﬁﬂummxm one Home now how Umpmwuucs can UOUMOMUIwmmuzoo
no macaumuusmosoo A_mv mouooomoso oﬂswmuocﬂ use Ao.mHv uncommoom HouﬂmosH

.wﬁ OHQMB

49

Table 15. Percent P (dry weight basis) and protein for each
of the seven diets fed to rainbow trout in experiments 2a
and b.

      

—‘

[HM ‘ - H- ”A A |
l—_u*

   
 

 

 

 

 

 

 

 

 

 

A (Zeigler trout grower) 1.12 43.0 ‘
B (No phytase, 100% P sup.) 1.15 49.6

C (phytase with 100% P sup.) 1.18 50.9

D (No phytase, 25% P sup.) 0.82

E (Phytase with 25% P sup.) 0.81

F (Phytase with 0% P sup.) 0.69

G (No phytase, 0% P sup.) 0.69

 

50

Table 16. Performance of triplicate groups of fingerling
rainbow trout fed a commercial reference diet (Zeigler trout
grower) and semi-purified diets with or without phytase
treatment and with graded levels of supplemental P in
experiment 2a. Values in columns with different
superscripts are significantly different (p<0.05).

       
   
   

Initial
Weight
(glitch)

 

  
 
 
  

 
 
 
 

Percent
Weight Gain

 

 

 

 

 

 

 

 

 

 

 

 

 

A 11.20

B 1.88 12.97 690'

C 1.85 15.45 835c

D 1.91 13.43 703'

E 1.97 15.37 780'

F 1.95 14.77 757'

G 1.88 _ 12.15 __ 646'

(ANOVA Table)
£23222"; """ B;;;;;;'SE"§;;'SE"'§;;; """" £3 """" SEES?
Variation Freedom Squares Square Ratio
Among groups 6 .435333 7.192222E-02
6.80 0.000

Within Groups 98 1.036587 1.057742E-02

Adjust. Total 104 1.46812

51
statistically higher average feed conversions (p<0.05).
Protein accretion and utilization was calculated to
determined protein gain of fish fed each diet (Table 17).
The highest degree of protein utilization (39%) was observed
in fish fed diet C (100% P supplementation and phytase-
treated soybean meal). Figure (3) illustrates average
weight gain (g/fish) for fish fed each of the 7 diets
throughout experiment 2a.

Body composition analysis (Table 18) of rainbow trout
showed significant differences (p<0.05) for moisture, fat,
protein, ash, and P in initial samples compared to samples
analyzed at the end of the experiment. Values for moisture,
ash, and P were statistically higher, while fat and protein
levels were lower (p<0.05) for fish at the end of the
experiment compared to fish sampled at the beginning of the
experiment. Significantly lower percent ash and P levels
(p<0.05) were observed in fish that were fed diets D (25% P
supplementation and no phytase) and G (0% P supplementation
and no phytase) when compared to fish fed any of the other
diets.

Initial samples of fish were lower (p<0.05) in percent
protein than fish fed any of the other diets. Moisture
content was higher (p<0.05) in both the initial samples and
in fish fed the commercial diet when compared to fish fed
experimental diets.

Total P recovered in solids (Table 19), of fish fed

Table 17.

52

Protein accretion of fingerling rainbow trout
associated with commercial and experimental diets fed in
experiment 2a (10 weeks).

 

 

 

 

 

 

 

 

 

 

 

 

Diet Protein fed Protein Protein Protein
(g) assimilated accretion utilization
(9) (sprotem (%)
galned/kg
diet fed)
A 239.7 83.3 149.4 35
B 293.7 100.3 150.7 34
C 328.1 127.0 155.6 39
D 305.7 108.4 156.8 36
E 336.3 123.7 153.9 37
F 333.4 119.9 155.9 36
G 291.7 96.4 156.4 33

 

 

53

Figure 3. Average weight gain (g/fish) of rainbow trout
over time when fed a commercial reference diet or semi-
purified experimental diets with or without phytase and
graded levels of supplemental P (sup.) in experiment 2a.

WEIGHT INCREASE OF TROUT IN EXPERIMENT 20

 

    
   
     

 

 

 

 

 

18'
p I. Diet A (Zeigler trout grower)
IC :- —ii‘-—
I .
I. Duet 3 (No Phytﬂcse. 100: sun.) 1";
14 — “”""““ /./-
A I_ Diet C (Phytase with 1002 sun.) X?" A
5: z + 35" x73
.9 14 - Diet 0 (No Phytase. 257. sun.) __,{-'}’
H— _ --.;’\_.-- .'
E 10 _ Din E (Phytase with 252 sun.)
V _ +
f_ Diet F (Phytase with 0: sun.)
:1: a " +
Q " Diet G (No Phylcse. 0% sun.)
Lu 6I _
E .
4 Q-
2 .
I
3' .
O I I I

 

0 2 4 6 8 10
GROWTH PERIOD (Weeks)

54

.oaoozzm>a no: u «\z n
.60“ mumwo ou osoommuuoo mumuuma mamﬁom ~
.mwmmn no mm on so owmmmuoxm F

 

 

 

 

 

 

 

 

 

 

oomo nluuom.sa .mm.a .om.ma .mn.a .om.~e o

F .am.o .nm.ea .ea.a .om.ma .mn.a .mm.~e m
.me.o .oa.oa .mo.~ .aa.mz .so.a .eo.~s m

.mn.o .am.>a .sm.a .me.mz .mo.a . .mm.~e o

.e¢.o .ee.sa .o~.~ .on.mH .4o.o .ea.ms o

.mq.o .ms.oa .sa.~ .ao.es .sm.o .s~.ms m

.oo.o .n>.oa .mm.~ .ms.vs .ma.o nao.ms o

836 n«\z nos.~ .eo.ms 84>.n aeo.oe Hmzpch

eouosoaonm Heuoa nus sqououm new ousaeaox

HQOOHOQ HQOOHOA HQOOROQ HQOOHOQ #QOOHOQ HQOOHOQ ~0dmﬁdm

 

 

 

 

 

 

 

 

.Amo.ovov acoummuﬂo zHOGMOﬂuﬂsmﬂm who muoﬁuomzoosm usmuwmuﬂo
ouw3 msssaoo :ﬂ mosam> .om usmaﬂzmoxm :w Aum3ouo uoouu umaoﬂmwo comm anon» Hoﬂozmsaoo
tam mumwo omwmwusolwaom new uses» Bonsﬂou maosz no mﬂmzamso zEQﬂuHmooﬁoo zoom .wa manna

55

Table 19. Total P (g) recovered in fecal solids collected
over a ten week period in experiment 2a. Values in columns
with different superscripts are significantly different
(p<0.05).

    

1___________ed in solids .

         

 

 

 

 

 

 

 

 

 

1 Sample letters correspond to diets fed.

56
each of the seven diets ranged from a high of approximately
1.28 g for trout fed diet 8 (100% P supplementation and no
phytase) to a low of 0.44 g for trout fed diet E (25% P
supplementation with phytase). Rainbow trout that were fed
diets E and F (0% P supplementation with phytase) showed
lower fecal P concentrations throughout the experiment than
fish fed other diets. Total P recovered from fecal solids
was determined from routine fecal samples taken at random
two week intervals.

P discharged to the effluent after fecal collection
ranged from 4.80 g P/kg diet fed for fish fed diet B, to a
low of 1.88 g P/kg diet fed for fish fed diet F (Table 20).
The lowest P effluent levels were again observed in tanks
where fish were fed diet F.

There were no mortalities over the course of the

experiment.

 

 

 

 

 

 

 

 

 

 

W hm.~ mm.~ wv.H mo.H mm.n 0
Hw.H mm.H vN.H o¢.o mm.¢ m

mH.N mm.m nh.H vv.o H¢.m m

w.N mm.m ow.H mN.H oo.m Q

mh.m oh.¢ wo.m Ho.H Ho.> O

bN.¢ om.v ww.N wN.H H&.@ m

wm.¢ em.v mm.~ wH.H vm.o d

2:86 332. am; 83 93.. my. .3 :3 12

oooueoouao m venuesonqo m oouuusonﬂo A uooou a“ u now m Howe

 

 

 

 

 

 

 

 

.Amxmm3 oHv on usmsﬂuwmxm mo coauosﬂﬁzmu com:
necesumumo .usmoamum on» as ommumsomﬂo one .mmomu cw omvomaaoo .68“ m uo usoosd

.ON UHQMB

58
EXPERIMENT 2b.

Feed conversions were more variable in experiment 2b
when compared to variations observed in experiments 1 and
2a. Average feed conversions of rainbow trout in experiment
2b (Table 21) ranged from high of 1.15 for fish fed diet G
(0% P supplementation and no phytase) to a low of 0.83 for
those fed diet C (100% P supplementation with phytase).
Feed conversions of trout fed diets C and F (0% P
supplementation with phytase) were significantly better than
feed conversions of fish fed other diets. Fish fed diet G
grew less (p<0.05) and were observed to be less aggressive
during feeding periods. When growth of fish fed phytase-
treated diets were tested against non-phytase diets
(Fisher's LSD test), significantly lower growth was
exhibited by fish fed the non-phytase diets (p<0.05).
Protein accretion and utilization were calculated (Table
22), and protein utilization by fish fed phytase-treated
diets and the commercial feed was observed to be greater
than fish fed non-phytase diets. Average weight gain
(g/fish) of trout fed each of the diets is illustrated in
Figure (4).

Fish fed diet E were not fed for a period of 4
consecutive days between weeks 6 and 8 because of a feed
shortage. Normal growth resumed, and feed conversions were

not affected.

59

Table 21. Performance of triplicate groups of rainbow trout
fed a commercial diet (Zeigler trout grower) and semi-
purified diets with or without phytase treatment and with
graded levels of supplemental P in experiment 2b. Values in
columns with different superscripts are significantly
different (p<0.05).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Diet Initial Final Percent Feed
Weight ‘Weight Weight Gain Conversion
(SI!) (9/1)
A 12.17 40.17 330' 1.008
B 14.47 42.69 295' 1.06'
C 15.93 62.53 392° 0.83°
D 14.23 42.90 301' 1.08'
E 16.37 52.45 320‘ 0.90'
F 16.97 63.70 375° 0.85°
G 12.70 35.10 276b 1.15b
(ANOVA Table)
Source of Degrees of Sum of Mean F Prob.
Variation Freedom Squares Square Ratio
Among groups 6 1.395025 .2325042
6.16 0.000
Within Groups 96 3.625975 3.777057E-02

Adjust. Total 102 5.021

 

60

 

 

 

 

 

 

 

 

Table 22. Protein accretion of rainbow trout associated
with commercial and experimental diets fed in experiment 2b
(10 weeks).
Diet Protein Protein Protein Protein
fed (g) assimilated accretion utilization
(9) (9_prote1n (%)
galned/kg
diet fed)
A 361.2 132.2 157.4 37
B 288.0 90.1 155.2 31
C 590.6 235.6 203.1 40
D 461.7 137.5 148.0 30
E 348.2 127.6 184.7 37
F 601.8 234.4 196.7 39
G 393.4 105.8 136.8 27

 

 

 

 

 

 

 

 

 

61

Figure 4. Average weight gain (g/fish) of rainbow trout
over time when fed a commercial reference diet or semi-
purified experimental diets with or without phytase and
graded levels of supplemental P (sup.) in experiment 2b.

7c:NEIGHT INCREASE OF TROUT IN EXPERIMENT 2b

0)

O

(n
O

WEIGHT (g/fish)

 

 

O ' I I I I I
- 6 8 10
GROWTH PERIOD (Weeks)

MAE-ﬁrm“) Dinamomfmxao.) DiaC{P!yu-uihtmao.) DietOOmeeam23mH
....3... —e—- -

C)
N
p.

*

 

WEO’MIDGZEXaD.) Oqu(thn--ih02u.) oucmomaxan.)
-—+— —a— —_7.—

Note: Reduces growth for diet E due to ﬁsn
being off feed for 4 days.

62

Initial proximate analysis samples of rainbow trout
used in experiment 2b corresponded to the body compositions
of fish at the termination of experiment 2a. These values
are represented in parentheses in Table 23.

Ash and P values did not change in fish grown to larger
size by the termination of experiment 2b when compared to
values observed in fish at the termination of experiment 2a.
However, P levels fell slightly in fish fed diets D (25% P
supplementation and no phytase) and G, 0.32% to 0.31% and
0.28% to 0.26% respectively. Whole carcass ash and P values
were also significantly lower in trout fed diets D and G.
These two diets (D and G) contained very low levels of
available inorganic P (Table 15) and fish that were fed
these diets exhibited typical P deficiencies signs. Fish
fed diet G had both poor bone mineralization and poor
growth. Fish that were fed diet F used more dietary P in
experiment 2b as compared to experiment 2a. Whole carcass
concentrations of P were 0.45% for fish fed diet F in this
experiment as compared to 0.39% for the juvenile fish fed
diet F in experiment 2a.

Percent moisture was significantly higher for fish fed
diet A than for fish fed other diets (p<0.05). Percent fat
of fish fed diet D were significantly higher than samples of
fish fed other diets (p<0.05). Percent protein present in
whole fish samples was significantly higher for fish fed

diets C and F (p<0.05).

 

.maosow some you mmoao> Heavens oumoﬂosﬂ Av mmmwousmuoo so muonsoz "muoz

.oou humao ou oncomwunoo mumuuwa mHQEcm ~
.mamon we we so so ommmmuoxm _

 

 

 

 

 

 

 

 

Ao~.o in.sao Am.so Aon.mzo Amm.ao zom.mso
0one .os.sa .mn.a .oo.mz .ao.a .ao.as o

Aan.o .~.sao Ao.~o Aom.mao Amm.ao Amm.~so
.me.o .am.sa .na.~ son.oz .mo.a ssH.Hs z

Ame.o Aa.omo Ao.~o Aaz.mso Aso.ao zeo.mso
.44.o .mz.sa .na.~ .oo.oz .oo.o .mm.~s m

m Amn.o Ao.sao Ao.ao Ame.mso Amo.ao Amm.mso
.Hm.o no~.oa .sm.a .oo.mH ass.os .ms.Hs o

A44.o Az.sao Am.mo zon.mzo zeo.oo Aes.mso
.ev.o .Hm.oa .na.~ ase.os .Ho.o .mo.ss o

Ame.o zo.oao x~.~o zao.vao Asm.oo Asm.nso
.me.o .ma.oa .om.~ .os.mz .an.o .os.~s m

zoo.o As.oao in.~o Ams.eso Ama.oo Aso.mso
.me.o .oa.oa .s~.~ .ev.ma .HH.» .me.es a

WEOAQQOBQ HIHOB and RaouOHm Huh ONSHW%OS
useouom useouom asoouom useowom usoouom usouuom Neuoaem

 

 

 

 

 

 

 

 

 

.Amo.ovmv mmocwumuuﬂo UCMOHmacon wuoOﬂocﬂ mooﬂuomnwosm ucwumwmﬁo
ouﬂ3 mssoaoo :ﬂ mosao> .on usmﬁﬂumoxw :ﬁ Aumsouo uoouu Hoaoﬂmso omou uoouu deﬂoumssoo
oso wuwﬁo omﬁuﬂusmnﬂawm oou noon» soasﬂou mHooa mo mﬂmzaoco F:oﬁuﬁmoosoo zoom .mm wanna

64

Total fecal P recovered over the 10 week period from
tanks of fish in experiment 2b ranged from a high of 3.79 g
for tanks of fish fed diet A to a low of 1.27 g for fish fed
diet F (Table 24). Trout which were fed diets E and F
produced lower P concentrations in fecal solids throughout
the study. As in experiment 2a, fish fed diets E and F
excreted the lowest fecal P concentrations.

Effluent P concentrations were determined using the
mass balance formula: PM = Pfed - [Pf + (Pm - Pti)]. P
discharged to the effluent was very low for fish that
were fed diet F, 0.34 g P/kg feed fed. Solids discharged
from tanks containing fish fed diet C equaled 3.73 g P/kg
feed fed (Table 25). These values were lower than those
observed in experiment 2a.

Trends began to appear in fish fed experimental diets
over the additional 10 week period. The fish fed diets that
were not treated with phytase showed an increase in feed
conversions (decreased growth rates) from juvenile to post
juvenile stages (Table 26). Protein utilization of non-
phytase-treated diets was significantly lower than phytase-
treated diets (p<0.05).

In experiment 2b, 6 fish from a tank fed diet E and 7
fish from a tank fed diet 8 died. The mortalities were

caused by an inadvertent blockage of incoming water which

65

Table 24. Total P (g) recovered in fecal solids collected
over a ten week period in experiment 2b. Values in columns
with different superscripts indicate significant differences
(p<0.05).

Total P collected in solids

3.79.
2.69'
3.21'

3.15'
1.33b
1.27b
2.29'

 

1 Sample letters correspond to diets fed.

Note: Fish fed diets B and E had 7 and 6 fewer fish
respectively than other diets.

 
 
 

 

 

 

 

 

 

 

 

 

 

« mag; 2...». 8.3 a~.~ 8.... o
mN.o vm.o H¢.o bN.H N~.w h
o¢.H Hm.a HN.H mm.H m¢.o m
mo.N om.a om.H mH.m No.5 o
oa.n nh.m mm.¢ Hm.m mo.MH U
mm.m n¢.m mm.~ mo.m om.h m
m¢.~ ov.~ wo.~ oh.m Ho.o d
15.6 232. 9:2 83 our. 9.8. .2 32.8.3 .3 .2 .
oeouenoeao m oeuaesoowo m o» oomuenoewo m money as m oou u mean

 

 

 

 

 

 

.Amx003 oHo om unmawumoxm mo coauocﬂsumu some
omsﬂsuoumo .usosamum ms» cw odouosomﬂo oco .mmomu ca omuooaaoo .owm o no 9:50ﬁ4 .mm manna

67

Table 26. Feed conversions of juvenile to post juvenile
rainbow trout fed a commercial reference diet (Zeigler trout
grower) and semi-purified experimental diets. Values with
different superscripts indicate significant differences
(One-sample test of means) at (p<0.05).

___ 1 ____1__1._. _111 1___1_ ---~~)
I Feed Conversion Feed Conversion Difference
(Exp. 2a) (Exp. 21:) __

 

 

 

 

 

 

 

 

 

 

 

 

A 1.00 1.00 0.00'
B 0.89 1.06 -0.17b
C 0.79 0.83 -0.04'
0 0.89 1.08 -0.19b
E 0.83 0.90 -0.07'
F 0.86 0.85 0.01'
G 0.93 1.15 -0.22b
Note: Negative values indicate reduced growth rates

fish.

 

68
resulted in decreased oxygen levels. Fish in each tank were
weighed and feeding rates were adjusted to compensate for

the reduced number of fish.

DISCUSSION

This study was designed to evaluate the potential use
of rainbow trout diets containing phytase-treated soybean
meal to reduce P released from fish hatcheries. Since the
primary source of P in the effluent comes from the feed, it
is generally accepted (Lall 1991; Cho et al. 1991; Ketola
1985) that the most effective way to limit discharge
problems is to develop fish feeds that provide optimum
growth and increased P bioavailability with the least
possible amount of P in the diet.

In all experiments, deflourinated rock phosphate was
used as the P supplement because of its low solubility in
water. The source of supplemental P is important in
relation to fecal P concentrations. Some common inorganic P
supplements, such as dicalcium phosphate, are highly soluble
in water and allow greater leaching of P from feed (Ketola
1985). The ideal diet would contain no P supplement and
still meet the fish's requirements.

Experiment 1

A preliminary experiment was designed to: 1) determine
if rainbow trout fed diets containing phytase-treated
soybean meal exhibited any deficiencies or abnormalities. 2)

compare growth rates and body compositions of fish fed

69

 

70
phytase-treated or untreated diets with graded levels of
supplemental P to those fed a commercial reference diet
(Zeigler salmon starter) and 3) compare the amount of P lost
from tanks of fish fed these diets.

Work with poultry and swine diets has shown the
potential of using phytase to increase P availability in
soybean meal (Nelson et al. 1968,1971; Simons et a1. 1990).
The use of phytase in fish diets has been limited. Ketola
(personal communication) supplemented fish diets with the
enzyme phytase; however, no significant increase in phytate
P utilization by the fish was observed. The optimal
temperature and pH for wheat phytase to actively hydrolyze
phytin P is 55°C and 5.15, respectively (Sigma Chemicals, St
Louis, MO). In contrast, the optimum temperature for
growing rainbow trout is 10°C - 15.6°C (Piper et a1. 1982) .
Because trout are cold-blooded, internal and ambient
temperatures are similar, and internal body temperature
probably inhibited enzyme function. Ketola (1985) found
that the use of low phytin soybean meal in salmonid diets
had the potential to reduce P effluent levels.

In our experiments, Ketola's T2M diet was used as the
test diet because it contained soybean meal as the primary
protein source. Cho et al. (1974) and Ketola (1975a) showed
that soybean meal could be used as an alternate protein
source to fish meal. We found growth rates and feed

conversion ratios of rainbow trout fed the T2M diet compared

71
well to fish fed commercial trout and salmon diets. Feed
conversion ratios were used as comparison values because
they relate the conversion of nutrients into flesh and are
useful indicators of feed utilization. High feed
conversions represent low feed efficiency. In our study,
growth rates and feed conversion ratios of rainbow trout fed
MSU modified T2M diets containing soybean meal treated with
phytase were not significantly different than those of fish
fed a commercial diet (Zeigler salmon starter) over a 10
week growth period. Treatment with phytase increased P
availability of soybean meal and decreased the need for
supplemental P.

Fish actively consumed all of the diets tested;
however, weight gain was slightly lower for fish fed diets B
and D. Diet B (TZM experimental diet with 100% P
supplementation) had poor pellet stability and produced many
fine particles. Observations indicated that fish tended not
to consume these fine particles. This resulted in higher
feed conversion ratios. Diet D was not phytase-treated and
contained only 25% P supplementation. Fish appeared to
consume all of diet D fed, but were much less aggressive in
feeding. The low inorganic P level in this diet appeared to
reduce fish growth.

Lower fat and protein levels were observed (Table 12)
from samples of fish collected at the beginning of the'

eXperiment (initial samples) compared to levels in fish

72
samples taken at the end of the experiment. This was
probably related to the change in size of fish over the 10
week growth period or the low feeding rate of fish before
the start of the experiment. Small fish tend to have varied
body compositions. Energy is utilized more for size
increase instead of storage when intake is limited (Shearer
1992; Gardiner and Geddes, 1980). At the end of experiment
1, samples of fish were taken approximately 24 hours after
final feeding in order to clear the digestive track (Fange
and Grove 1979). The higher percentage of fat found in fish
fed the commercial diet compared to fish fed the
experimental diets was most likely attributed to the use of
a starter feed (Zeigler salmon starter) for this experiment.
Starter feeds typically have higher levels of fat than
grower diets. Excess fat in the feed can cause fatty
infiltration of the liver (Piper et al. 1982). Liver
composition was not determined in these experiments.

Low percent ash values, whole carcass P levels, and
reduced growth rates of fish fed diet D indicated typical P
deficiency signs for fish (Lall 1991). Percent ash and P
levels of fish fed the other experimental diets were not
significantly different from levels in fish fed the
commercial feed. Therefore, the average of these values,
approximately 2.1% ash and 0.43% P (as is basis), should be
near optimum levels for rainbow trout. Results show that

fish fed diets low in supplemental P with phytase-treated

73
soybean meal performed as well as fish fed the commercial
feed or better than diets with 100% P supplementation.
Inorganic P converted from phytin by phytase in the soybean
meal was utilized by rainbow trout as well as supplemental
inorganic P in the diets tested.

The dietary deficiency signs caused by low P include
depressed weight gain, high feed conversions and poor bone
mineralization (Lall 1991). Fish fed diet D (25% P
supplementation without phytase) were the only fish from
experiment 1 to exhibit these deficiency signs. Diet E and
F contained phytase-treated soybean meal with 25% and 0%
supplemental P, respectively. Fish fed diets E and F did
not exhibit P deficiency signs or reduced growth rates.
Consequently, we conclude that enzyme pretreatment with
phytase improved P availability from soybean meal to the
point that supplementation was not needed, and that phytase-
treated soybean meal could be used in fish feeds to reduce
or eliminate the need for P supplement in the diet.

P effluent concentrations

P effluent levels calculated in experiment 1 (Table
13) reflected total P discharged and consisted of P bound in
fecal and uneaten feed solids and that which leached from
solids. Fish fed the diet with phytase-treated soybean meal
and no P supplementation (diet F) released much less P to
the effluent (2.2 g/kg feed fed) than fish fed any other

diets. Indeed, diet F resulted in 77% less P discharge than

 

74

the commercial diet. The total P concentration of diet F
was only 0.66%, but the phytase treatment increased the P
availability. Therefore, 60% of the P present in diet F was
retained by the fish.

Proximate analysis of fish suggested that requirements
for available P of rainbow trout in this system were 0.40%
to 0.45% of the diet. These values support recent findings
by Ketola (personal communication); but, are slightly less
than previously reported values for rainbow trout (Ogino
1978; Reinitz 1978). The phytase-treated soybean meal
provided sufficient available P in diet F to meet the P
requirement of trout without the addition of supplemental
inorganic P. The results of experiment 1 demonstrated the
potential to increase P utilization in fish diets and
effectively reduce levels of P in the effluent without
sacrificing growth rates of fish.
WW

Experiments 2a and b were designed to confirm results
of preliminary experiment 1 and determine long term effects
of feeding phytase-treated experimental diets on growth,
feed conversion ratios, body composition, and fecal P
concentrations of rainbow trout. Ten fish from were removed
from each tank after 10 weeks, this enabled us to compare
results of experiment 2a to experiment 1 and reduce loadings
on remaining fish. The effects of routine fecal collection

on effluent P levels were evaluated at the end of experiment

 

75
2a and b.
mm

In addition to the 6 diets fed in experiment 1, an
additional diet (diet G) without supplemental P and without
phytase treatment was fed during this 10 week experimental
period. Results from experiment 2a supported results of
experiment 1. Fish fed diet F (phytase-treated with 0%
supplemental P) fed very well and no deficiency signs or
abnormalities were observed. Feed conversion ratios (Table
16) of fish fed the commercial diet were higher than fish
fed any of the other diets. This reduced growth might be
attributed to a lower protein concentration (43%) in the
commercial diet than in the experimental diets which
contained approximately 50% protein (Table 15). Recommended
levels of dietary protein for optimum growth of rainbow
trout are between 40-46% (NRC 1981). Fish fed diet C
(phytase-treated with 100% supplemental P) may have grown
better than fish fed the other diets due to the elevated
levels of available inorganic P, but this was not evident by
higher P concentrations in the body.

Whole-body fat and protein concentrations from initial
samples of fish were lower than those of fish killed at the
end of the experiment. Perhaps, the feeding rate before the
start of the experiment or body composition changes related
to body size were responsible for the lower fat and protein

of the younger fish (Shearer 1992). Juvenile fish generally

76
have higher protein requirements than fish in later phases
of growth (Lovell 1989). No differences in whole-body fat
or protein concentrations were observed in fish fed any of
the experimental diets or the commercial diet.

Ash and P values were significantly higher in initial
samples of fish compared to fish fed experimental or
commercial diets. This suggests that juvenile fish may have
higher requirements for P than larger fish. Calcium and P
concentrations in the body decline as fish grow from
juvenile stages to adult stages of life (Shearer 1984).

Since phytic acid accounts for approximately 60% to 70%
of the P present in plant ingredients (Lovell 1989),
available inorganic P (P5) in diet G was lower (0.29%, as
estimated from Table 14) than other diets fed. Fish fed
diet G along with those fed diet D (0.34% F5) were expected
to exhibit typical P deficiency signs. Fish fed diet G (no
phytase and 0% supplemental P) and diet D (no phytase and
25% supplemental P) had very low whole carcass ash and P
concentration when compared to all other samples analyzed.
This indicated, as expected, P deficiency signs due to poor
bone mineralization and low carcass P levels.
Interestingly, growth rates of fish fed diets D and G were
not significantly lower than growth rates of fish fed the
other diets which suggests that P may be more important in
bone mineralization and development than in muscle growth.

Ketola (1991) also found dietary P to have a greater effect

77
on body ash and P content than on body weight gain. Fish
fed diets D and G appeared to consume most of the feed fed,
but were lethargic and fed poorly.

Differences in body composition (Tables 12,17) of
initial samples of fish compared to fish fed experimental
and commercial diets in experiments 1 and 2a may have been
related to 1) different requirement and metabolism levels at
early life stages, 2) feeding rates prior to time of
sampling, and 3) the diet fed from fry to juvenile stage.
Mira—tit»!

P released to the effluent varied depending on the
amount recovered by fecal collection. Total fecal
collection was not possible because of leaching and loss of
fine solids. Based on personal observations of various
methods of solids removal at fish hatcheries, the proportion
collected in these experiments should fall well within the
range that could be collected on a routine basis from most
hatcheries. Concentrations of P in fecal solids were lowest
in fish fed diet E (phytase-treated with 25% supplemental P)
and diet F, which contained phytase—treated soybean meal and
no supplemental P (Table 18). Lower dietary P
concentrations and increased P availability of diets E and F
probably resulted in the reduction of P concentrations in
fecal material.

P concentrations released to the effluent were found to

be lowest (1.88 g/kg feed fed) from fish fed diet F (Table

 

78
19). Effluent P consisted of fine particles that could not
be recovered by routine collection of feces and soluble P
which leached from the solids prior to collection. Starr
(Personal communication) found that, for periods of up to 7
days, no appreciable leaching was observed from solids.
However, Brown (1985, 1991) found that during the first hour
a portion of soluble P was lost to the effluent due to
leaching. Data collected by Windell et al. (1978) clearly
indicated that most nutrient leaching occurs during the
first hour after defecation. Fecal collection further
reduced levels of P released from tanks of fish fed the same
diets in experiment 1. Therefore, it is evident that lower
P effluent concentrations can be achieved by implementation
of waste management strategies along with reduced levels of
P in the feeds.

Experiment 2a confirmed results of our preliminary
experiment 1. The use of phytase-treated soybean meal in
rainbow trout diets improves P bioavailability, eliminates
the need for supplemental P, and decreases P effluent
levels.

Experiment 2b

Experiment 2b was a continuation of experiment 2a and
was designed to determine long term feeding effects
associated with experimental diets. Ten fish were removed
from each tank at the completion of experiment 2a. The

remaining fish were grown out for an additional 10 weeks.

79
Diets fed to fish in this experiment were the same as in
experiment 2a.

Six fish died in one of the tanks being fed diet E and
seven fish died in one of the tanks being fed diet B due to
a depletion of dissolved oxygen caused by an obstruction in
the water inlet valve. No differences were observed in
weight gain (g/fish) and feed conversion ratios of surviving
fish in these tanks when compared to average weight
gain/fish fed the same diet in other tanks. These
mortalities did not appear to influence results.

By continuing to grow fish for an additional 10 weeks,
trends began to appear in fish fed experimental diets. The
fish fed diets that were not treated with phytase showed an
increase in feed conversions (decreased growth rates) from
juvenile to post juvenile stages (Table 24). From
observations of feeding behavior, the fish fed treated diets
and the commercial diet were much more aggressive and
consumed all feed fed, while fish that were fed the non-
phytase-treated diets were less aggressive and did not
always actively feed. This would be expected with diets
that did not contain adequate levels of supplemental P, but
was not eXpected from diet B, which met requirement levels
of fish with supplemental inorganic P. Protein utilization
(Table 22) of fish fed diets containing phytase-treated and
non-treated soybean meal indicated that perhaps the enzyme

itself or the heating of the soybean meal during the enzyme

 

80

treatment process improved the nutritional quality of this
ingredient.
P effluent concentration

One of the most interesting findings from experiment 2b
was the substantial reduction of P released to the effluent
of fish fed diet F (0.34 g P/kg diet fed). Whole carcass P
concentrations increased in fish fed diet F, 0.39% for
juveniles in experiment 2a to 0.45% for the larger fish in
experiment 2b. A higher proportion of P was recovered from
the feces and greater utilization of P by these larger fish
was observed. Fish fed all diets exhibited a reduction in P
discharged to the effluent in experiment 2b compared to
experiment 2a (Table 23). The larger fish in this
experiment were observed to have denser fecal material,
which simplified collection of solids.

Factors that may have affected fecal collection in
experiments 2a and b include:
1) Size of fish: small fish had smaller and less

dense fecal material, which may have allowed

increased leaching of P and a decreased efficiency

of fecal collection;
2) Activity of fish: fish in some tanks were much

more active, which resulted in the break up of

fecal solids and further loss of particles to the

effluent;

3) Disturbance of fish: when fish density (kg

81
fish/m?) increased with age, fecal collection
caused a greater disturbance of fish and fecal

material.

 

SUMMARY

Results of a 10 week preliminary experiment showed that
the incorporation of phytase-treated soybean meal into
rainbow trout diets can markedly improve P availability in
soybean meal and reduce P effluent levels dramatically.
Growth rates of fish fed semi-purified experimental diets
containing phytase-treated soybean meal were not
significantly different than growth rates of fish fed a
commercial reference diet. Whole body P levels were
statistically lower in fish fed diet D (25% P
supplementation and no phytase) than P levels in fish fed
any of the other diets. P released to the effluent was
reduced by 77% (2.20 g P/kg feed fed) for fish fed diet F
(0% P supplementation with phytase-treated soybean meal)
than for fish fed a commercial reference diet (9.47 g P/kg
feed fed). Whole carcass compositions of fish fed diet F
were not significantly different than fish fed the
commercial diet.

Experiments 2a confirmed findings of experiment 1 and
showed that routine collection of fecal solids along with
reduced P levels in diets further reduced P effluent levels
(1.88 g P/kg feed fed (diet F)). Fish fed diets E and F

(containing phytase-treated soybean meal and low levels of

82

83
supplemental P, 0% and 25% respectively) had consistently
lower concentrations of P in fecal material than fish fed
any of the other diets.

Long term feeding of experimental diets containing
phytase-treated soybean meal had no adverse effects on
rainbow trout. Larger fish that had been fed diet F for a
period of 20 weeks retained a higher whole body percentage
of P than juvenile fish fed this diet over a 10 week period.
Protein utilization of fish fed phytase-treated diets and
the commercial diet were observed to be greater than those
fed non-phytase diets. A higher percentage of solids was
collected from tanks containing larger fish than from tanks
containing juvenile fish because of larger and denser fecal
material. P released to the effluent for larger fish fed
diet F was minimal (0.34 g P/kg feed fed). Routine solids
collection and the use of low P diets indicated that proper
waste management practices have the ability to substantially
reduce P levels in the effluent. Proximate analysis of fish
in these experiments indicated that P requirement for
rainbow trout are approximately 0.40% to 0.45% available

dietary P.

 

CONCLUSIONS

Our results indicated that if cost effective phytase
treatments of plant feedstuffs can be developed, P levels in
trout feeds and P effluent concentrations from trout
hatcheries can be significantly reduced. The use of phytase
in salmonid diets was shown to substantially improve the
bioavailability of P in soybean meal. Improving P
availability eliminated the need for the addition of
supplemental P to rainbow trout diets. In all experiments,
fish fed diet F (0% P supplement and phytase-treated soybean
meal) had excellent feed conversion ratios and discharged
levels of P to the effluent dramatically lower than fish fed
commercial feeds. Feed acceptance of diet F was very good
throughout all experiments.
Future research

In order to effectively reduce P discharge from fish
hatchery effluents, future research needs to be conducted on
all life stages of rainbow trout and other fish raised for
aquaculture purposes. P requirement levels should be
determined for different life stages. Diet formulations at
different life stages may need to be adjusted to meet
different requirement levels.

Mass balance estimation of P effluent levels can

84

 

85
improve accuracy of measurements over traditional methods of
chemical analysis of effluent. This method of analysis
would also be less costly than traditional chemical
analysis. Future research needs to make direct comparisons
of chemical and nutritional analysis on a large scale level.

Potential problems have recently been found with diets
that contain corn gluten meal as a feed ingredient (Barrows
and Nelson, personal communication). These problems consist
of poor flesh taste and color of rainbow trout fed feeds
containing corn gluten meal. This becomes a major concern
if fish are raised commercially for food markets. Future
research should be aimed at replacing this ingredient with
an alternative ingredient that contains similar nutritional
characteristics.

Further work must be conducted in the area of P
effluent reduction. These experiments have shown that
incorporation of phytase in the diet increases P
bioavailability in soybean meal and reduces P effluent
levels through increased utilization of dietary P by rainbow
trout. Continued research of these diets should be aimed at
1) Cost evaluation: Is it economically feasible to

produce this diet on a large commercial scale?
2) P regpirement levels of trout and salmon at

different life stages: Do P requirements change in

response to life stage changes?

3) Corn gluten meal replacement: Can alternative

86
ingredients be found that do not cause poor flesh
taste or off color?
4) Large scale phytase treatmen : Can treatments be
incorporated with existing feed processes, such as
heat treatment of soybean meal, on a large scale

while keeping cost low?

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