Quantifying

Nutrient Requirements of Fishl

IsRAHtN.{ H. ZnrrouN, DUINIE E. Ur-l REv, WIr-r-Ierr,t T. MacE.e
Department of Ani mal Husbandry, Mi chi gan State Uni versi ty, East Lansi ng, Mi ch.48824, USA
J o Hu L . Gt l r
Department of Dai ry Sci ence, Mi chi gan State Uni versi ty, East Lansi ng, Mi ch.48824, USA
aNo WsnNen G. BEncEN
Department of Ani mal Husbandry, Mi chi gan State Uni versi ty, East Lansi ng, Mi ch.48824, USA
Zet r ouN, L H. , D. E. Ur - r - ney, W. T. Me. cEE, J. L. Gr Lt - , . cNo W. G. BBncr . N. 1976.
Quant i f yi ng
nutri ent requi rements offi sh. J. Fi sh. Res. Board Can. 33: 167-172.
Four repl i cates of50 rai nbow trout(Sal mo gai rdneri )frngerl i ngs each were assi gned to each
of seven di etary protei n l evel s from 30 Io 60% i n 57o i ncrements. Percentage wei ght gai ns for 10
wk were rel ated to di etary protei n l evel s usi ng an anal ysi s ofvari ance and mul ti pl e compari sons
of the means, a "broken l i ne" anal ysi s, and a pol ynomi al regressi on anal ysi s. When correctl y
used, one ofthese techni ques shoul d l ead to a reasonabl e concl usi on concerni ng di etary protei n
requi rements. However, the pol ynomi al regressi on anal ysi s has the advantage of bei ng conti nu-
ous, l i ke the rel ati on ofgrowth to dose, and shoul d be more accurate than the other methods when
the i nterval s between experi mental di etary nutri ent concentrati ons are wi de. The pol ynomi al
regressi on anal ysi s al so provi des the basi s for maki ng economi c deci si ons rel ati ve to protei n
r equi r ement s f or maxi mum economi c r et ur ns.
Zer r ouN, I . H. , D. E. Ul l nr v, W. T. M, , r cep, , J. L. Gr r - l , . qNo W. G. Bencr N. 1976.
Quant i f yi ng
nutri ent requi rements offi sh. J. Fi sh. Res. Board Can. 33: 167-1' 72.
De s groupes, contenant chacun 50 al evi ns de l a grosseur du doi gt de trui te arc-en-ci el
(S al mo
gai rdneri ), ont 6t6 expos6s dans quatre essai s r6p6t6s i r chacun de sept ni veaux de protei nes
al i mentai res al l ant de 30 d 60%, par gradi ns de 5Va. Les gai ns pond6raux en pourcentage ont 6t6
trai t6s par anal yse de vari ance et comparai sons mul ti pl es de moyennes, ai nsi que par anal yse de
"
l i gne bri s6e
"
et anal yse de r6gressi on pol ynomi al e. Lorsque uti l i s6e correctement, I' une ou
I' autre de ces m6thodes devrai t permettre d' en arri ver d une concl usi on rai sonnabl e quant aux
exi gences en prot6i nes al i mentai res. Cependant, I' anal yse de r6gressi on pol ynomi al e a
I' avantage d' Otre conti nue, tout comme l a rel ati on entre l a croi ssance et l a dose, et devrai t Btre
pl us pr6ci se que l es autres m6thodes l orsque l es extrmes de concentrati on des substances
nutri ti ves du r6gi me exp6ri mental sont espac6s. De pl us, I' anal yse de r6gressi on pol ynomi al e
peut servi r de base d des d6ci si ons 6conomi ques ayant trai t aux prot6i nes requi ses pour des
profi ts maxi mums.
Recei ved June 4, 1975
Accepted October 2l, 1975
EsrIlraerEs of quantitative nutrient requirements
are influenced not only by the criteria used but by
the statistical methods chosen to evaluate criterion
response to differing dietary nutrient concentra-
tions. In studies of protein or amino acid require-
ments of fish, confusion is evident regarding the
appropriate method to use
(Delong et al. 1958,
1962; Halver 1960, 1965; Halver et al. 1964;
Chance et al. 1964; Cowey et aL. 1972; Zeitow
et al. 1973. 1974). This matter is of considerable
Regu l e 4j ui n 1975
Accept d l e 2l oct obre 1975
significance due to the substantial growth of fish
cul t ure
(Bardach
et al . 1972; Huet 1973) and t he
observation that nutritional deficiency diseases are
common wherever intensive flsh culture is prac-
ticed (Halver
1972). The qualitative nutrient re-
quirements of various fish species have been
previously reviewed (Halver 1969, 1970, 1972;
Phi l i ps 1969; Cowey and Sargent 1972). When
estimating nutrient requirements to achieve bio-
logical potential, the consequences of using various
statistical techniques should be considered, and
several of these techniques will be discussed.
Weight gain is a common parameter used in
evaluating different dietary levels of an essential
nutrient and in estimating the quantitative require-
ments of that nutrient for fish. While weight gain
'Michigan
Agricultural Experiment
Article No. 6929.
Pri nt ed i n Canada (I 3861)
Imprim6 au Canada (J3861)
Station Journal
167
J
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r 68
J. FI SH. RES. BOARD CAN. , VOL. 33( I ) , 1976
Tanrr 1. Surnmary of various responses of rainbow trout (Salmo gairdneri) fingerlings to different levels of dietary
protein concentration (derived from Zeitoun et al 1973).
Dietary protein concentration (%,)
SEMg 60 55 50 4s 40 35 30
Initial weight (s')"
Final weight (g)"
Weight
eain
(:Z)^
Weight gain (f)b
Protein retention (g)"
6. 5"
14. 2.
1 1 9 . 3 "
119. 3'
t . z -
6. 8" o
7 7 . Od
1 5 0 . 7 d
I 50. 7d
1 . 6 0
6. 2.
1 8 . 5 "
197 . 3{
197 . 3"
2. 0.
6. 3'
19. 2d
204 . s"
204.5"
2 . 7 " f
6. 4'
79. 6d
208. 9"
208.9'
2 . 2 f
6 . 3 , 7 . 2 d 0 . 1 5
t 9. 4d 20. 5t 0. 39
2 0 8 . 1 " 1 8 4 . l f 7 . l
2 0 8 . 1 " 1 8 4 . 1 " 7 . l
2 . 2 d 2 . 3 f 0 . 0 6
uDuncan' s
multiple range test was used to compare the means.
bTukey' s
Honestly Significant Difference (HSD) test was used to compare the means.
"' d' "' rMeans
on the same line followed by different superscripts are significantly (P<0.05) different.
gsrN,r
:
standard error of the mean
:
fMsnln;
n
:
+.
and growth are not necessarily equivalent, in
young animals the former is a good measure of
the latter. Growth in higher vertebrates is the
summation of the continual and coordinated
chemical and biological processes, starting with
egg fertilization and terminating when the animal
achieves adult physical conformity (Mitchell
1962). Growth in fish proceeds in a diminishing
pattern throughout life (Lagler et al. 1962) if
food is available and environmental conditions
are favorable.
Generally, the use of weight gain for estimating
quantitative nutrient requirements is based on the
assumption that weight gain (and hence, growth)
will be impaired if an essential nutrient is inade-
quately supplied or absent from the diet. The
lowest dietary nutrient level at which maximum
weight gain is achieved is commonly considered
the minimum requirement of that nutrient for
growth. While weight gain can be easily measured
and used as a criterion of growth, dietary nutrient
levels used in a given experiment are necessarily
discontinuous, so a statistical tool is needed to
establish where growth is maximum.
Procedures
Data (Table
l) gathered by Zeitoun et al. (1973)
in a determination of the protein requirement of
rainbow trout (Salmo gairdneri) fingerlings were
adapted for this presentation. In this study, four
replicates of 50 fish each were assigned to each
dietary protein level. Seven dietary protein levels
from 30 to 60Vo in 5Vo increments were fed for
10 wk. Preparation of the diets and weighing tech-
niques were identical to those described by Halver
( r e57
)
.
ANALYSTS oF VARTANcE eNo MutrrprE CoMpARrsoN
oF TnsArI\dpNT MEANS
Gr i mi nger et al . ( 1955) ,
Mor r i son et al . ( 1956)
and Fi sher et al . (1957)
have used thi s procedure
to
establish the minimum amino acid requirements of
birds. Recently, Zeitolun et al.
(1973, 1974) applied
this concept to determine the minimum dietary pro-
tein level for maximum
growth of rainbow trout and
coho salmon
(Oncorhynchus
nerka). This method
presumes that gain in weight is linearly related to
increasing dietary levels of an essential nutrient as
long as it is below the required level. At the require-
ment, weight gains plateau and then decline as the
dietary nutrient concentration surpasses the animal' s
t ol erance of t hat nut ri ent .
This procedure requires that differences in gain,
effected by dietary nutrient level, be evaluated by
analysis of variance techniques. Multiple comparisons
of treatment means are commonly made to approxi-
mate the location of substantial differences. Two
methods frequently used in this comparison are
Duncan' s multiple range test (Steel and Torrie 1960)
and Tukey' s Honestly Significant Difference (HSD)
test (Snedecor 1.956). To compare these methods,
both tests were conducted on the data in Table 1, and
a first order regression line (Y =
a I bX) was fitted
to the means of percent weight gain that increased
significantly with increasing levels of dietary protein
(from
30 to 40%). Using Tukey' s HSD test, the
percentage gains at and beyond 40Vo d,ietary protein
concentration were not significantly different, and the
average of these means was used to determine the
point of interception of the regression line with the
horizontal line purported to demonstrate the biologi-
cal capacity of the living organism to withstand
higher levels of nutrients than required. As shown in
Fig. I, the lines intersected at a mean (1 sE) per-
centage weight gain of 200.6
+
4.6 ar' d a dietary pro-
tein concentration of 4O.7Vo.
Using Duncan's multiple range test, the percentage
gains from 40 to 55% dietary protein were not sig-
nificantly different, whereas 60Vo dietary protein de-
pressed gain. As a consequence, this latter value was
excluded when the interception point was calculated,
giving a mean percentage of 41.3%. These differences,
resulting from application of Tukey's and Duncan's
test, were small, and may largely be accounted for
by the inclusion or exclusion of response at 60Vo
dietary protein. However, the philosophical basis for
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ZEITOUN ET AL.:
QUANTIFYING
NUTRIENT REQUIREMENTS
Tantn 2. Mean square errors
(MSE) of different regression lines for percentage
weight gain of rainbow trout fingerlings.
Range of dietary protein
levels tested (f) Regression equation
169
MSE
3V40.7"
30-40. 8.
30-40.9^
30-41 .0"
30-60b
Y:
- 120. 9+7. 91X
Y:
- 118. 6+7. 84X
Y
: - 116. 3+7. ' 7' 7X
Y:
- 114. 0+7. 70X
Y
: -
397 .96 + 24.58X- 0.2476X2
310. 63
310.47
3 10. 46
3 10. 60
278.20
uFirst
order polynomial. Values above each "breakpoint" equated to the "break-
point" value. "Breakpoint" values above and below those listed were tested, and as
the "breakpoint" deviated further from 40.9, the mean square error increased.
bSecond
order polynomial.
30 35 40 45 50 55 60
olrfory Piotlin Concanfrolion, 96
Ftc. 1. Effect of Duncan' s and Tukey' s tests on the
point of interception of the horizontal line and the
percentage weight gain of rainbow trout
(Salmo
gairdneri) fingerlings. Yp is the average percentage
when Duncan' s test was used to compare the means
and Yr is the average percentage gain when Tukey' s
test was applied.
the two methods is different, and one can expect to
obtain different results in some sets of data.
BnorpN LrNe Axlrysrs
Baker et al. (1.971) used the broken line analysis
to estimate the requirements of tryptophan for baby
pigs. Thiq procedure was developed by H. W. Norton,
University of Illinois, Urbana, 1972, personal com-
munication, and assumes a positive linear relatron
between growth (Y) and the dietary level of the
essential nutrient
(X)
at or below the minimum re-
quirement. At the minimum requirement level
(R),
the ascending line which represents the relation be-
tween growth and diet nutrient concentration breaks
instantly to horizontal. Assuming that the breakpoint
represents R, then Y values for X at and greater
than R are estimates of the same response.
A1l values of X greater than the breakpoint are
equated to the breakpoint R for purposes of comput-
i nga regressi on l i ne, Y
=
a* bX. Several regressi ons
are attempted using different breakpoints. Concur-
rently, the deviation sum of squares for each regres-
sion is estimated. The regression line that gives the
least mean square error
(MSE) is considered the best
fitted line to represent the linearity between dose
levels and responses and which is the least squares
estimate of the requirement.
Using the growth data of rainbow trout fingerlings
from Table 1 for this analysis, and using arbitrary
values of X at O.lVo dietary protein intervals, the
regression line that fit the percentage weight gain with
a breakpoint at 40.9Vo protein in the diet gave the
least MSE if compared to other lines (Table 2).
Que.one,uc
RtcnpssroN on Sr.coNo Onosn Porvvo-
UIAI- RECNISSION ANALYSIS
The second order polynomial regression analysis is
represented by the equation Y
:
Bo * B.X * B"X' .
This curve is characterized by having a unique maxi-
mum point (Y-*) along its range. The value of X-"*
that corresponds to Y-.* is defined as the maximum
concentration of the dietary nutrient that produces
optimum growth, and beyond which growth is de-
pressed. The advantage of this procedure is that it
often provides a better empirical fit to the growth
responses of living organisms which do not exhibit
an abrupt change from linearity as postulated in the
previous two analyses.
Cowey et al.
(t972)estimated the protein require-
ments of the plaice (Pleuronectes platessa) by apply-
ing the quadratic regression analysis. The minimum
dietary protein level was defined as the level that pro-
duced the highest point on the curve. To apply the
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170
J. FI SH, RES. BOARD CAN. . VOL. 33( I \ . 1976
same concept to rainbow trout fingerling growth data
(Table
1), a quadratic equation was calculated (Y :
-397.96
+ 24.58X
-
0.2476X" which showed that
the maximum response of these fish was achieved at
50Vo dietary protein (Fig. 2). In addition, the profile
of the data, which exhibited a substantial growth
depression at both extremes of dietary protein con-
centration, favored the use of this analysis. The cal-
culated MSE of the curve was noticeably lower
(278.20)
than those estimated by the broken line
procedure (Table
2).
Although this curve would best represent the rela-
tion of
$owth
to dose, it does not reflect the practi-
cally insignificant differences in percentage gain
below and beyond the maximum point, nor does it
consider the ability of the animal to adapt to a range
of dietary protein levels between a deficiency on the
left side of the curve and toxicity on the right. That
is, there are minimum and maximum levels of intake
within which the animal is able to store, excrete, or
adapt to the level of nutrient supplied without sub-
stantial changes in metabolic processes. A statistical
approach could be adapted to determine the level of
nutrients in the diet that can yield a response that is
within a certain confidence range. of Y^"". The
selection of the confidence level is dependent on the
researcher and his objectives. In the case of the rain-
bow trout fingerlings, confidence limits of 95Vo of
all estimated responses of the curve
(?)
were calcu-
l at ed usi ng t hc f ol l owi ng expressi on:
Y
+
t . " . . L
n
+ ( X- X) ' V( b , ) +
( Z- Z) ' V( b " )
+ 2(X
-
Xl tZ
_
71corl b,b,1f""
where MSE is the mean square error of the means,
n is the number of observations on Y, X is the treat-
ment level, X is the average of treatments, Z is the
square of X, 2 i s t he average of Z, and V(b, ), V(0, ),
llo l2o l3o t4o t5o 160 r7o rao r9o 2oo 2to 22o 2fi
W.ight coin, %
Frc. 3. Relation between percentage weight gain
and protein retention of rainbow trout fingerlings.
and cov(brDr) are variances and covariance of esti-
mated parameters of the polynomial curve.
Levels of confidence were plotted (Fig. 2) on either
side of the quadratic line, and a straight line parallel
to the abscissa and passing through the maximum
level of the lower line of the confidence limit was
drawn. Moving left from Y-.., this horizontal line
first crosses the polynomial curve at a point Xr. Then,
continuing to the left, the horizontal line intersects
the upper line of the confidence limit at a point Xo.
The value X' is the estimated level of dietary protein
concentration expected to produce a growth response
equal to the lower bound on the interval estimate of
Y-"" in the initial study. The value Xo is the estimated
level of protein concentration for which an upper
bound on predicted growth response is equal to the
lower bound on Y-"*. Strictly speaking one cannot
say that the response at X (say Yr) is "not statistically
significantly different from Y^""," because the dis-
tribution of (Y*"*
-
Y') is not known exactly and
the sampling variance is a complex nonlinear func-
tion. But statistical significance is somewhat irrelevant
for regressions with significant parameters (Williams
1959). What is relevant is a difference that has prac-
tical importance. When economics dictates, dietary
levels of protein ranging between Xo and Xr may be
taken as a rough estimate of the concentration which
minimizes cost while maintaining adequate growth
response. If estimates of cost per unit dose (C) and
economic income per unit of yield (I) are available,
one can base a decision on those rather than the
somewhat arbitrary range, & to X' . Savings from
reduced dose are C(X-"-
-
X"), where the economic
dose (X") is to be determined. Loss in return is
IAY-,", where yield expressed at the economic dose
is (1
-
A)Y---. If one maximizes net return with
respect to A, then
Xu
=
X-"*
-
{A[X' 9-"*
-
(bo/b")l]' /'
and
A
-
(c/2r),[x,^^*
-
(bo/ b")]/Y,^^*.
Letting C-"*
=
CX-"* and I*""
:
IY-"*, one obtains
X"
=
X*"-[1
-
C^""/2I^^*)
+ c-""/ 2I -"-X ^, *)
(b, / b")l
as the estimated economic dose. Whereas it is possible
2. 4
' 6
I A
220
zto
200
t20
l o
roo
t90
8 l @
.E tzo
_
160
I
r5o
t 40
t30
30 35 40 xo xr 45 r@r
55 60
Di al ory Prol l i n Concant rol i on, . [
Frc. 2. The second order polynomial relation (solid
curved line) of percentage weight gain and dietary
protein
concentration for rainbow trout fingerlings,
with 0.95 confidence limits (dashed curved lines).
O
Estimated mean of Y (Y);
Observed mean of
Y.
J
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ZEITOUN ET AL.:
QUANTIFYING
NUTRIENT REQUIREMENTS
t 7l
to set fiducial limits on X*"* (Finney 1964), which
is economically irrelevant, the complexity of X" makes
computation of limits intractable.
Discussion
An examination of the consequences of using
the three described statistical procedures reveals
that growth maxima may be identified in a range
of dietary protein concentration from 40 to 50%.
llowever, selection of the upper figure assumes
that the maximum percent weight gain deflned
by application of the second order polynomial
regression represents a response which is different
biologically from the lower values defined by use
of the polynomial limits. Statistically, the limits
of response expected with sOEo
(X-u*) dietary
protein include the mean response expected with
44Vo (Xr)
dietary protein. Economically, this
difference in dietary protein can be very impor-
tant, particularly if the saving is greater than the
fall in returns (Carpenter L97l). Dietary protein
is generally one of the most expensive compo-
nents of artificial fish diets, and if increasing
increments of dietary protein concentration do
not result in concomitant increase in value, the
economic success of fish culture is in
jeopardy.
The selection of Xo to X1 on the polynomial
regression as the minimum range of protein re-
quirement is an economic decision; and while
this decision may be economically conservative,
the selection of X-o- is more physiologically con-
servative. Using local prices of dietary protein
and fish, average initial weight of the fish, and
estimated efficiency of feed conversion, it was
possible to estimate the ratio, C-o,/2I,,,u", which
equal s 0. 1534. 2 Then, t he est i mat ed economi c
prot ei n requi rement i s X"
-
50(1- 0. L534) +
( 0. 1534/
50) ( - 397. 96/
- 0. 25)
=
47. 2%. For
the current data, this result is above Xt
-
44Vo.
However, if the cost of dietary protein rises
above the figure assumed in these calculations, or
if the selling price of fish declines, then the esti-
mate of the economic protein requirements
(or
economic dose, X,) would be lower. Decreased
efficiency of conversion of dietary protein to gain
' Protein
was derived from Purina Trout Chow
(40% minimum crude protein), at a price of $9.85/
22.7 kg or $1.08/kg of protein. Local prices of
farm-reared rainbow trout averaged approximately
$2.64/kg. Estimated efiiciency of feed conversion to
gain was 1.5. Cost of protein per kilogram gain at the
max i mum ( X- , * :
5O%)
: ( 1. 5) ( 0. 5) ( $1. 08) :
0.81; gain at maximum rate (average initial weight)
l Y^- " / 100)
-
l l
= ( 0. 2) 1212/ 100)
-
1l
=
0. 224ke;
maxi mum cost (C, ", ")
-
(0. 81)(0. 224) =
0. 1, 874;
I -"-
-
(2. 64) (0. 224) :
0. 5914.
will have the same effect. Estimation by Xn is to
be preferred, but when economic considerations
are strong, and information on costs and income
are difficult to obtain, one may choose as a
simple substitute the range between Xe and X1.
Certainly an increase in the number of observa-
tions used to determine the polynomial curve
would narrow the confidence limits and tend to
shift the range of Xn to Xr to the right. Whether
this shift is important must be answered by the
researcher himself. If the criterion of response
were badly chosen
(in representing the effect of
increasing dietary nutrient levels), such a shift
may be very important.
Weight gain has been criticized
(Phillips et al.
1957; Allison 1959; Maynard and Loosli 1969)
for inaccuracy as a measure of growth, since gain
in weight may result from deposition of fat rather
than from true growth. The potential for growth
may be considered identical with maximum pro-
tein retention, and when values for the latter
(Table 1) were regressed against percent weight
gain (Fig. 3
),
the correlation coelncient was
0.93. It is apparent that for rainbow trout finger-
Iings, weight gain was a good measure of true
growth. Analysis of variance and multiple com-
parison of treatment means, "broken line" anal-
ysis, and second order polynomial regression
analysis may lead to similar conclusions concern-
ing dietary protein requirements. Flowever, the
polynomial approach has the advantage of being
continuous, like the relation of growth to dose,
and should be more accurate than the other
methods if the intervals between experimental
dietary nutrient concentrations are wide. Also,
the polynomial method is well adapted to eco-
nomic analysis if information is available on
costs and returns. One should remember, how-
ever, that the polynomial is only a smooth, sym-
metric approximation to the real relation of gain
and intake and may be in some cases better than
others.
Acknowledgments
We thank Drs P. L Tack and E. R. Miller for
reviewing the manuscript.
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