Aquaculture, 76 (1989) 109-118 109

Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

Utilization of Fish Silage by Rainbow Trout

(Salmo gairdneri)

FREDERICK E. STONE, RONALD W. HARDY, KARL D. SHEARER and THOMAS M.

SCOTT
U.S. Department of Commerce, NOAA, National Marine Fisheries Service, Northwest and
Alaska Fisheries Center, Utilization Research Division, 2725 Montlake Boulevard East, Seattle,
WA 98112 (U.S.A.)
(Accepted 8 June 1988)

ABSTRACT

Stone, F.E., Hardy, R.W., Shearer, K.D. and Scott, T.M., 1989. Utilization of fish silage by rain-
bow trout (Salmoguirdneri). Aquaculture, 76: 109-118.

The apparent digestibility and utilization of fish protein subjected to the ensilaging process
were compared with fish meal in dry diets fed to rainbow trout (Salmo gairdneri). During the
ensilaging process, a majority of the proteins were converted to peptides, a portion of which was
further hydrolyzed to free amino acids. Although apparent digestibility values were higher for the
fish silages than for fish meal, the fish silages were not as efficiently utilized for growth. Whole
fish and fish processing wastes were found to be equivalent sources of nitrogen provided the degree
of autolysis was the same and the diets were otherwise nutritionally balanced. Autolysis was re-
duced and growth rates enhanced when the ingredients were stored at - 5’ C prior to ensiling.

INTRODUCTION

Fish silage is produced when sufficient acid is added to minced whole fish or
fish processing wastes to activate endogenous proteolytic enzymes while inhib-
iting microbial growth. The cumulative effect of numerous digestive and cath-
eptic enzymes produces a liquefied product which is rich in proteins, poly-
peptides, and free amino acids (Stone and Hardy, 1986). Fish silage may be
stored without refrigeration, dried with other feed ingredients, or pelleted to
form moist or dry diets (Hardy et al., 1983; Hardy et al., 1984; Stone et al.,
1984 ) .
There is increasing evidence that the nutritional value of fish silage can be
increased significantly by limiting the extent to which proteins and polypep-
tides are hydrolyzed to free amino acids. Studies have shown that the majority
of the protein nitrogen ingested by humans (Mathews, 1972; Silk, 1980; Silk
et al., 1985) is absorbed by the intestinal mucosa as di- and tripeptides while

0044-8486/89/$03.50 0 1989 Elsevier Science Publishers B.V.

110

a lesser portion is absorbed as free amino acids. Similarly, plasma levels of

essential amino acids remain at elevated levels for longer periods when trout
(Yamada et al., 1981) and carp (Plakas et al., 1980; Plakas and Katayama,
1981) are fed a diet containing intact protein (casein) rather than an equiva-
lent diet containing free amino acids as the protein source. Rainbow trout had
higher weight gains, protein efficiency ratios (PER) (Hardy et al., 1983), net
protein utilization (NPU), and apparent digestibility coefficients (ADC)
(Hardy et al., 1984) when fed silages in which autolysis was terminated after
3-7 days or restricted by temperature and pH (Hardy et al., 1983; Stone and
Hardy, 1986) than when autolysis was permitted to continue until high levels
of free amino acids appeared in the silage.
This study was conducted to evaluate the effect of replacing fish meal in a
dry trout diet with fish silage made from fresh and frozen whole fish and fish
processing wastes. The effects of protein autolysis in fish silage upon final
weights, food conversion values, PERs, NPUs, and protein digestibilities of
these diets are reported.

MATERIALS AND METHODS

Sample preparation

Fresh whole fish and fish processing wastes (FPW) consisting of the heads,
frames, and viscera of Pacific whiting (Merluccius productus) were obtained
from commercial sources near Astoria, Oregon. These materials were obtained
less than 24 h after the fish were caught and were kept on ice until use. A
portion of the fresh ingredients was converted to fish meal in a steam-jacketed
vacuum dryer at 635 mm of mercury (maximum temperature was 70 oC ) , an-
other portion ensiled immediately, and the remainder stored in polyethylene-
lined fish boxes at - 5 5 2 ’ C. Samples (1 kg) of fish silage were prepared from
frozen material at the end of 0, 520, and 40 days. The remaining ingredients
were converted into silage after 40 days of frozen storage. Since Pacific whiting
is a low-fat fish ( < 2.5% ), no antioxidants were added to the materials before
or after processing.
Fish silage was made from ground ingredients (9.5 mm) using 2% sulfuric
and 0.75% propionic acids as the acidulents with adjustments made to pH 4.0.
Batches for the feeding trials (40 kg) were held in covered polyethylene con-
tainers at ambient temperature (18-22 o C ) with occasional stirring. After 40
days of ensiling, the batches for feeding were neutralized with 1.6% Ca (OH),
to pH 6.5 and dried in the vacuum dryer.

Protein autolysis

Periodically, subsamples were withdrawn, neutralized to pH 6.5 with cal-

cium hydroxide, frozen to -80°C and freeze dried. Total Kjeldahl nitrogen
 111

(TKN) was measured as described by the AOAC (1975). Nonprotein nitrogen

(NPN), mol. wt. < 8-10 000, was determined by homogenizing samples for 1
min with IO volumes of 10% trichloroacetic acid (TCA) and measuring the
TKN of the filtrate (Haard et al., 1985). Free amino acids (FAA) and am-
monia were measured using a Beckman1 Model 118CL amino acid analyzer
after extracting with 4% sulphosalicylic acid (SSA) and filtering through a
0.45 pm filter (excludes mol. wt. > 500). Peptide nitrogen was calculated by
subtracting FAA and ammonia nitrogen from the TCA-soluble NPN. The au-
tolysis of proteins to TCA-soluble peptides and free amino acids was reported
as the ratio (% ) of each fraction to TKN.

Feeding studies

Six dry diets were prepared based on Abernathy dry formulation A19-2 (86)
(Table 1) . Minor adjustments were made to keep the diets approximately iso-
nitrogenous. The diets all contained 45% protein, 15% fat, and 7.5% ash, on
an as-fed basis. Each diet was hand fed three times per day to triplicate 250-1,

TABLE I

Composition of experimental diets-modified Abernathy formulation A19-2 (86)

Ingredients Percent in diet

Fish meal or silage 50

Dried whey 6
Wheat germ meal 5
Wheat middlings 15.2
Dried blood meal 10
Poultry by-product meal 1.5
Vitamin premix’ 1.5
Trace mineral premix’ 0.1
Choline chloride (70% liquid) 0.6
Ascorbic acid 0.1
Lignin sulfonate binder 2
Herring oil 9

‘Vitamin premix supplied the following (mg or IV/kg dry diet): Vitamin A palmitate or acetate,
6600 IU; Vitamin Da, 441 IU; ol-tocopherol acetate, 502 IU; menadione sodium bisulfite complex,
28: thiamin mononitrate, 47; riboflavin, 53; pyridoxine HCl, 38; dicalcium pantothenate, 115;
niacin, 220; biotin, 0.6; Vitamin B12, 0.06; myo-inositol, 132; and folic acid, 12.7.
‘Trace mineral premix supplied the following (mg/kg diet): ZnSO,, 185; MnSO,, 55; CuSO,, 3.9;
KIOa, 16.9.

Use of trade names in this publication does not imply endorsement by the National Marine
Fisheries Services.
112

outdoor tanks containing 125 rainbow trout (initial average weight 16 g; pro-
tein 15.7%). The tanks were supplied with 4 l/min of constant temperature
(15’C) dechlorinated water. Each week the bulk weight of the fish was mea-
sured, the feed conversion ratios2 calculated, and the feeding levels adjusted
by the Buterbaugh and Willoughby method (1967) using a hatchery constant
of 16. All feed was eaten by the fish at each feeding. At the end of 12 weeks,
three fish from each tank (nine fish per dietary treatment) were pooled for
protein analysis using the Kjeldahl method (AOAC, 1975) and the protein
efficiency ratios ( PER)3 calculated.
At the end of the feeding trial, the ingredients were combined in a 1:l ratio
with the Oregon Test Diet (OTD ) described by the National Research Council
(1973)) 0.5% chromic oxide was added, and the diets fed to groups of 30 larger
fish (400-500 g) for 1 week. The Oregon Test Diet containing 0.5% chromic
oxide was also fed. Feces were collected by fecal stripping on 3 consecutive
days. The feces were dried, and analyzed for chromium by perchloric acid di-
gest (Furakawa and Tsukahara, 1966), and protein by the micro-Kjeldahl
method (AOAC, 1975). Apparent digestibility coefficients (ADC)* were used
to calculate the net protein utilization (NPU)5 for the ingredients. Analysis of
variance was conducted on the fish weights, feed conversion rates, and diges-
tibilities to test for significance among dietary treatments (Steel and Torrie,
1960). Duncan’s New Multiple Range Test was used to compare treatment
mean values.

RESULTS AND DISCUSSION

Protein autolysis in fish silage

Fish silage made from fresh whole fish (Fig. 1) contained a variety of active
proteases which converted intact proteins to TCA-soluble (mol. wt.<8000-
10 000) peptides (Mukundan et al., 1986). Endopeptidase activity predomi-

Food fed (g dry)

‘Feed conversion ratio =
Weight gain (g wet)
3PER = Weight gain (wt wet)
Protein fed (wt dry)
4ADC protein in ingredient + ADC protein in OTD = ADC combined protein =
% Cr in feed % protein in feces
loo- x
% Cr in feces % protein in feed ’ loo

ADCprotei~ in _
ADC~~~j~d - (ADC protein in OTD) X (% OTD in diet)
ingredient in diet
mgred,ent-
%

Protein gain
5NPU =
Protein fedx ADC protein
 113

80

20

0
Days ensiled 0 5 20 40 0 5 2040 0 5 2040 0 5 2040

Days frozen
prior to ensiling 0 5 20 40

Fig. 1. Protein autolysis of fish silage made from whole fish frozen at - 5 ’ C for up to 40 days; ( 0 )
TCA ( 10% ) insoluble proteins; ( ) TCA soluble peptides; ( n ) free amino acids.

nated during the first few days after which the accumulation of free amino
acids was accelerated. The essential amino acids (EAA) were released more
rapidly than the nonessential amino acids (NEAA) resulting in a ratio of EAA
to NEAA in the free form of 1.38 compared with 0.78 initially. After 40 days,
autolysis occurs more slowly and is mainly limited to the degradation of certain
free amino acids such as arginine (Stone and Hardy, 1986) and tryptophan
(Backhoff, 1976) accompanied by the accumulation of ammonia. Ammonia
represented 0.7%, 1.6%, and 1.5% of the TKN in the fish meal, fresh silage,
and frozen silage, respectively.
Fish silage made from ingredients stored at -5°C contained more protein
(TCA-insoluble) and fewer amino acids than silages made from fresh ingre-
dients (Fig. 1) . The ratio of EAA to NEAA in the free form was similar to that
of silage made from fresh ingredients. Further changes in proteolytic activity
were not observed after 20 days of storage. Although changes in enzyme activ-
ity associated with protein solubility are particularly rapid in gadoid fish stored
at - 5 ‘C (Dingle and Hines, 1975)) silages made from ingredients stored at or
below-20°C are similar to those made from fresh ingredients (Haard et al.,
114

8C

20

0
123456

Ingredients Complete diet

Fig. 2. Nitrogen distribution in ( 1) whole fish meal; (2 ) fish processing waste fish meal, (3 ) whole
fish silage; (4) FPW fish silage; (5) whole fish silage from frozen ingredients; (6) FPW fish silage
from whole frozen ingredients. ( q ) TCA (10% ) insoluble proteins; ( •j ) TCA soluble peptides;
( n ) free amino acids.

1985). Short-term storage of fish or fish processing wastes at temperatures

slightly below freezing could be an alternative method for reducing the free
amino acid content of fish silage.
After 40 days, silages made from fish processing wastes contained similar
amounts of proteins, peptides, and free amino acids as silages made from whole
fish (Fig. 2). This is not surprising since Backhoff (1976) observed a syner-
gistic relationship between the proteolytic enzymes in silages made from the
flesh, viscera, and heads of cod (Gadus morhua). These similarities remained
after the silages were dried and formulated with the other diet ingredients (Fig.
2). Fish meal (Fig. 2) contained more protein (TCA-insoluble) and less free
amino acid than silages made from comparable ingredients.

Feeding study

Rainbow trout fed dry diets containing fish meal grew faster than those fed
diets containing dried fish silage (Fig. 3; Table 2 ) . Palatability of the diets was
 115

Fish meal

60 -

Silage
(frozen ingredients)

Silaga
(fresh ingredients)

0’
I I 1
4 8 12

Weeks of feeding

Fig. 3. Growth rate of rainbow trout fed diets containing fish meal and fish silage made from fresh
and frozen ingredients.

TABLE 2

Final mean weights, feed conversion ratios, PER, NPU, and apparent digestibility coefficient
(ADC) of protein for trout fed the experimental diets for 12 weeks1

Dietary Final mean Feed conv. PER4 NPU’ ADC

ingredients weight (g) ratios3

Whole fish meal 68.5 + 4.5A 1.014 0.09c 2.19 0.41 85.8 + 0.8~
FPW* fish meal 63.9 + 2.9A 1.01+ 0.04c 2.07 0.39 85.612.2~
Silage (fresh
whole fish) 42.7? 1.5C 1.71+ 0.05A 1.28 0.22 94.0 + 0.5a
Silage (fresh
FPW’) 43.6* l.lC 1.66 + 0.05A 1.33 0.24 90.0? 1.2b
Silage (frozen
whole fish) 52.5 +2.7B 1.34 + 0.06B 1.73 0.30 93.7+ 1.7a
Silage (frozen
FPW2) 53.5 + 0.6B 1.33 + 0.04B 1.65 0.30 90.9? 2.lab

‘Mean values 1 SD followed by the same letter are not significantly different (P> 0.01 for capitals;
P> 0.05 for small letters). Values are the average of three tanks. Initial weight was 16.0 g.
‘FPW = fish processing wastes.
3Feed conversion ratios = feed fed (dry wt. ) /weight gain (wet wt. ) .
4PER (protein efficiency ratio) = wet weight gain (g) /protein fed (g) .
5NPU (net protein utilization) =protein gain/protein fedx ADC protein.
116

not a factor in this growth difference, since the groups of fish consumed all of
the feed that was offered. Silage preserved with sulfuric acid and propionic acid
has been shown to have a lower acceptability to Atlantic salmon than silage
preserved with formic acid (Austreng and Asgard, 1986). In this study, the
acid-preserved silages were neutralized before drying to eliminate this poten-
tial problem. Digestion and absorption of amino acids from fish meal begins
in the stomach, is highest in the pyloric caeca, and continues in the middle and
posterior intestine (Ash, 1980; Dabrowski and Dabrowski, 1981). Protein syn-
thesis proceeds most efficiently when optimum concentrations of all of the
amino acids are simultaneously available to the tissues (Geiger, 1947). When
dietary amino acids were properly balanced, rainbow trout grew nearly as well
when fed a crystalline amino acid diet as when fed a high quality protein diet
(Aoe et al., 1970; Yamada et al., 1981). The gradual liberation of amino acids
from fish meal by intestinal enzymes ensures that amino acids are available
for growth over a prolonged time period.
Fish protein was readily digested and absorbed from the silage but was uti-
lized less efficiently for growth than protein from fish meal (Fig. 3; Table 2).
Fish silage may be inferior because it contains high levels of essential amino
acids in the free form which are available for immediate absorption. The re-
maining amino acids are gradually absorbed as digestion progresses. The es-
sential amino acids, if prematurely absorbed, may be irreversibly further me-
tabolized and not available for protein synthesis (Geiger, 1947).
The nutritional value of fish silage was improved by storing the ingredients
at - 5°C prior to ensiling. Diets made with these silages contained more intact
protein and less free amino acids than diets made with conventional silage
(Fig. 2). These diets may have been utilized more efficiently because the ab-
sorption of peptides and free amino acids was prolonged as digestion pro-
gressed in the gastrointestinal tract, resulting in more efficient conversion of
dietary protein to fish flesh. The silage diets, however, were inferior to those
containing fish meal in supporting fish growth in part because they contained
a higher proportion of essential amino acids in the free form. It is clear from
these results that the autolysis of fish silage should be restricted to preserve
nutritional value.
Although silage made from whole fish is somewhat more digestible than sil-
age made from fish processing wastes, this difference did not affect final weight,
feed conversion ratios, protein efficiency ratios, and net protein utilization
values (Fig. 3; Table 2). The digestibility of fish meal made from these ingre-
dients was also similar. It appears that whole fish and fish processing wastes
are equivalent sources of protein nitrogen for trout diets provided the degree
of hydrolysis is the same and the diets are otherwise nutritionally balanced.

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