Physiology &Behavior,Vol. 37, pp. 747-758. Copyright©PergamonPress Ltd., 1986. Printedin the U.S.A. 0031-9384/86$3.00 + .

00

Effect of Amino Acid Imbalance

and Deficiency on Dietary Choice
1
Patterns of Rats
P H I L I P M. B. L E U N G z A N D Q U I N T O N R. R O G E R S

Department o f Physiological Sciences, School of Veterinary Medicine

and Food Intake Laboratory, University of California, Davis, CA 95616

R e c e i v e d 26 July 1985

LEUNG, P. M. B. AND Q. R. ROGERS. Effect of amino acid imbalance and deficiency on dietary choice patterns of rats.
PHYSIOL BEHAV 37(5) 747-758, 1986.--Detailed dietary choice patterns were determined with a computerized feeding
monitoring system in groups of Sprague Dawley rats kept on a 12:12 hr light-dark cycle and offered in sequence a series of
dietary choice regimens involving amino acid-imbalanced or deficient diets with threonine as the most limiting amino acid.
Animals established their preference for a threonine-basal diet over a threonine-imbalanced or a threonine-devoid (devoid
of threonine) diet shortly (within 2-3 hr) after the consumption of small quantities of either diet in the beginning of the first
dark-cycle. An intensive sampling process characterized by frequent small bouts was evident throughout the light period.
Both the meal size and the meal frequency of the imbalanced or devoid diet were curtailed after prolonged choices. Animals
preferred the threonine-corrected (imbalance corrected by threonine supplementation) over the threonine-basal diet ini-
tially with an increase in meal frequency. But no clear choice for either diet was observed thereafter. Animals did not
establish their preference for the threonine-corrected diet when paired with the threonine-devoid diet until after 5 days with
a steady decrease in the meal size of the devoid diet but not the meal frequency. When the protein-free diet was introduced
as an alternative for the threonine-imbalanced diet, animals selected the protein-free diet during the first dark-cycle after
consuming a small amount of the imbalanced diet. Initially there was a drastic reduction in meal size of the imbalanced diet
and subsequently a decrease in meal frequency as well. Nevertheless, animals immediately rejected the protein-free diet
and chose the threonine-basal diet when it replaced the imbalanced diet as an alternative. The almost exclusive preference
for the basal diet occurred in the beginning of the first dark-cycle with an increase in meal size but no change in meal
frequency. The sampling bouts of small quantities, which followed the first introduction of the diets in the choice regimens,
may be an inherent investigative behavior whereby the physical or oropharyngeal properties of the diets are recognized.
The establishment of the choices for the alternative diets in the present experiments provides additional information about
the rapid time course of the food intake control mechanisms in rats fed amino acid-imbalanced or deficient diets.

Amino acid imbalance Dietary choice patterns

FOOD intake and growth of animals were depressed when a
substantial quantity of an indispensable amino acid mixture
lacking the growth limiting one was added to a low-protein
diet [2,11]. This diet, which produced the deleterious effect
on food intake and growth was considered to be "imbal-
anced" and could be corrected by supplementing the growth
limiting amino acid (corrected diet). When a single diet was
offered, the animals adapted to the imbalanced diet, after the
initial food intake depression, by slowly increasing their food
intake until it approached normal. More severe food intake
and growth depression with no adaptation occurred in amino
acid deficiency, which included diet devoid of an indispens-
able amino acid [7]. Also, rats rejected an amino acid imbal-
anced or devoid diet in favor of an alternative choice, a diet
containing a relatively balanced amino acid pattern or a
protein-free diet which did not support growth [5]. The de-
gree of preference for a protein-free diet was associated with
the severity of the nature of the imbalance in the added
amino acid mixture which created the imbalance [5]. Plasma
and brain amino acids, as well as certain neural areas, have
been implicated to be involved in the control of food intake
of animals ingesting amino acid imbalanced diets [3, 6, 8, 10,
12, 13, 15-17]. Brain mechanisms and the control of food intake
by macronutrients, including protein and amino acids, have
also been reviewed [1]. There is little information as to when
and how the animals altered their feeding patterns in choos-
ing dietary regimens involving balanced and imbalanced
amino acid diets. The detailed feeding patterns in the present
studies should provide information on the time frame of the
control system for establishing alternative choices in re-
sponses to disproportions of dietary amino acids.
METHOD
Male Sprague Dawley rats weighing 125 g were used for

1This investigation was supported in part by USPHS NIH Grant No. AM13252.
2Requests for reprints should be addressed to Dr. Philip M. B. Leung, Department of Physiological Sciences, School of Veterinary
Medicine, University of California, Davis, CA 95616.

747
 748 I~EUNG AND R()GERS

0.6-
THR BASAL

0.4-

bJ
w' 02-
i-
Z
0.0
0.6" THR BASAL
o
o
0.4-

0.2

0.0 ' ,
III
, ,
I
,
I
,
I,
, ,
I
, , ,
II,

2 4 6 8 I0 12 14 16 18 20 22 24
,,-=
LIGHT - '- DARK "1
Hours

FIG. I. Dietary choice patterns of a representative individual rat (5 days prior to

choice between the threonine-basal diet and the threonine-imbalanced diet) offered 2
identical cups of threonine (thr)-basal diet. Three minutes of non-feeding were used in
all instances as the minimal intermeal intervals.

the experiments. They were housed in specially constructed TABLE 1

Plexiglas cages (20× 13x24 in.) with stainless steel screen FOODINTAKE,MEALFREQUENCYAND AVERAGEMEALSIZEOF
bottoms and a round feeding porthole (2.5 in. diameter and RATSOFFEREDCHOICEOF THREONINEBASALVERSUS
2.5 in. length) on either side. A spillage-proof cup with a THREONINE BASALDIET
wide rim was fitted on top of a Mettler Electronic Balance
(PL300) which was positioned underneath the food tunnels Day 18" Day 5*
and interfaced with an on-line computer (PDP-11 Digital
Equipment Corp.). This feeding pattern monitoring device Thr Basal Thr Basal Thr Basal Thr Basal
constituted part of a computerized feeding, drinking and vol-
untary activity system described in more detail earlier [14]. Light
The cages were housed in environmental chambers with in- total (g) 1.50 + 0.55 0.60 _+ 0.15 0.27 _+ 0.13 0.95 +_ 0.37
dividual circulatory and lighting systems (computer con- Meal
trolled 12:12 hr light-dark-cycle, light off from 10:00 p.m. to frequency 14.2 ± 2.4 10.7 _+ 2.6 5.5 +_ 2.3 9.0 + _.2.10
10:00 a.m.). Water was available at all times. Each experi- Meal
mental diet in the two-diet choice regimen was monitored by size (g) 0.10 +_ 0.03 0.06 _+ 0.02 0.05 _+ 0.01 0.10 ± 0.05
a recording station. Since there were only 8 stations avail- Dark
able, 4 animals were employed throughout in the choice ex- total (g) 10.59 _+ 3.26 4.75 _+ 2.83 5.85 _+ 1.60 8.63 ± 1.50
periments. Meal
All experimental diets contained free amino acids as the frequency 18.5 _+ 3.9 10.2 +_ 2.2 12.2 ± 3.6 12,7 ± 3.4
protein source with threonine as the growth limiting amino Meal
acid in the basal diet. The threonine-basal diet contained size (g) 0.52 + 0.12 0.40_+ 0.23 0.52 ± 0.11 0,73 _+ 0.09
8.1% of dispensable amino acids and 4.6% of indispensable
amino acids and was equivalent to a low-protein diet (ap- Values are mean _+ S.E. for 4 rats.
proximately 10.45% crude protein equivalent). The *Indicates days prior to the choice between threonine basal and
threonine imbalanced diets.
threonine-basal diet supported a slow growth rate (approx-
imately 2 g/day). The threonine-imbalanced diet was created
by adding a mixture of indispensable amino acids (9.86%)
lacking threonine to the threonine-basal diet (approximately
16.96% crude protein equivalent). The threonine-devoid diet regimens was to examine the responses of non-naive animals
(approximately 16.82% crude protein equivalent) had the to dietary choices in amino acid imbalance and deficiency.
same composition as the threonine-imbalanced diet except The responses of separate groups of naive rats to similar
that threonine was completely deleted. The threonine- dietary choice regimens have been reported elsewhere [5].
corrected diet (approximately 17.26% crude protein equiv- The avoidance of the amino acid imbalanced diet and prefer-
alent) was similar to the imbalanced diet but had 0.4%, ence for the alternative balanced or protein-free diet also
threonine supplemented to correct the imbalance. The occurred when different indispensable amino acid, other
protein-free diet contained all the necessary nutrients except than threonine used in the present study, was the most limit-
the presence of protein which was replaced by carbo- ing amino acid in the imbalanced diet [2,8]. Animals were
hydrates (starch to sucrose in 2:1 ratio). The complete com- adapted to the threonine-basal diet before they were allowed
position of the amino acid diets used were reported earlier to choose between the threonine-basal diet and either the
[6,13]. threonine-imbalanced or the threonine-devoid diet, Follow-
The purpose of using the same rats in a series of choice ing re-equilibration with the threonine-basal diet, the rats
DIETARY CHOICE PATTERNS IN A M I N O A C I D I M B A L A N C E 749

TABLE 2
FOOD INTAKE, MEAL FREQUENCY AND MEAL SIZE OF RATS OFFERED CHOICES OF THREONINE
BASAL DIET VERSUS THREONINE IMBALANCED OR DEVOID DIET

Day l Day l0 Day I Day 7

Thr Thr Thr Thr Thr Thr Thr Thr
imb basal imb basal devoid basal devoid basal

Light 1.05 1.27 0.18 2.86 1.54 1.36 0.37 1.67

total (g) +__0.20 __.0.94 _+0.03 _+1.19 ___0.29 _+0.91 _+0.21 _+0.81
Meal 22.3* 6.20 5.00 7.50 23.5* 7.20 4.50 7.20
frequency - 1.5 _+1.8 _+0.71 _+0.96 _ 1.04 _+1.6 _+0.87 _+2.4
Meal 0.05 0.14 0.04 0.41 0.07 0.14 0.11 0.19
size (g) _+0.008 _+0.08 _0.001 _+0.21 _+0.01 ___0.08 _+0.07 _+0.07
Dark 1.76 13.6" 0.10 14.82" 1.46 14.58" 0.08 17.20"
total(g) _+0.50 _ 1.64 ---0.05 _+1.27 ___0.38 -+0.43 -+0.04 -+0.63
Meal 12.8 16.5 2.50 16.2" 14.5 15.5 3.70 19.2
frequency -+ 1.2 _+2.4 _+0.65 _+3.2 _+1.8 - 1.9 _+0.13 _+2.5
Meal 0.13 0.85* 0.04 0.97* 0.09 0.99* 0.02 0.93*
size (g) _+0.002 _+0.10 -+0.02 -0.10 _+0.02 -+0.12 _+0.004 _0.11

Values are mean _ S.E. for 4 rats.

*Indicates significant (p<0.05) differences between the two diets in the choice regimens.

0"61 , THR IMBALANCED

0.4

*,.o 0.2
,.gd
10
llll lllfl | flllfl I I I I I O • Ji n Jl J t n I fl
0.0

"0
0 o.e | THR BASAL
u.0

0.2

o.o . .,. i ! ., . ,. . I . . . . . i . . . . . . .
2 4 6 8 10 12 14 16 18 20 22 24
I LIGHT = I ,, DARK "I
Hours

FIG. 2. Dietary choice patterns of a representative individual rat (day 1) offered the choice
between thr-imbalanced and thr-basal diets. Upper and lower panels represent the portions of
intakes from the diets indicated above. The intakes of thr-imb vs. thr-basal diet in light and dark
phases were 1.00 vs. 2.72 g (light), and 1.46 vs. 14.71 g (dark), respectively.

w e r e offered the c h o i c e b e t w e e n the t h r e o n i n e - b a s a l a n d t h e lasted at least 8 days. E a c h c h o i c e e x p e r i m e n t w a s c a r r i e d

t h r e o n i n e - c o r r e c t e d diet. T h e n t h e a n i m a l s w e r e e q u i l i b r a t e d
w i t h t h e t h r e o n i n e - c o r r e c t e d diet b e f o r e t h e y w e r e s u b j e c t e d
to t h e c h o i c e b e t w e e n t h e t h r e o n i n e - c o r r e c t e d a n d t h e
t h r e o n i n e - d e v o i d diets. A n i m a l s w e r e f u r t h e r e q u i l i b r a t e d
w i t h t h e t h r e o n i n e - b a s a l diet b e f o r e t h e y w e r e o f f e r e d t h e
c h o i c e s b e t w e e n t h e p r o t e i n - f r e e diet a n d e i t h e r t h e
t h r e o n i n e - i m b a l a n c e d diet o r t h e t h r e o n i n e - b a s a l diet p r i o r to
the termination of the experiment. Equilibration with the
a p p r o p r i a t e d i e t s j u s t p r i o r to c h a n g e s in c h o i c e r e g i m e n s
o u t for at least 6 days. L o n g e r c h o i c e p e r i o d s w e r e allowed
for the t w o diets, t h a t w e r e relatively b a l a n c e d b u t differed in
a m i n o acid c o n t e n t s a n d t h o s e t h a t w e r e similar in e v e r y
a s p e c t e x c e p t the p r e s e n c e o f one i n d i s p e n s a b l e a m i n o acid.
B o d y w e i g h t s o f a n i m a l s w e r e r e c o r d e d j u s t p r i o r to e a c h
c h a n g e in d i e t a r y c h o i c e r e g i m e n so as to a v o i d u n t o w a r d
i n t e r r u p t i o n s d u r i n g t h e c o n t i n u o u s feeding p a t t e r n m o n i t o r -
ing p r o c e s s . T h e following f l o w c h a r t r e p r e s e n t s the m e n -
tioned sequential choice regimens.
750 LEUNG AND ROGERS

o.s I THR DEVOID

o.3~ t

1[ .tt
0,1
OO IIIIIII Ill III III Ig J I I II I I I 1 I ~. I , I I I (] I

o 05"~ THR BASAL

o'3 i
0.1

O0 ', I jl . . . . U. . . . , , ,'
2 4 8 8 10 12 14 16 18 20 22 24

I- LIGHT " I" DARK t

Hours

FIG. 3. Dietary choice patterns of a representative individual rat (day 1) offered the choice
between thr-devoid and thr-basal diets. Upper and lower panels represent respectively the por-
tions of intakes from the diets indicated above. The intakes of the thr-devoid vs. thr-basal diet in
light and dark phases were 1.52 vs. 0.14 g (light) and 1.53 vs. 15.01 g (dark), respectively.

Threonine basal provided more detailed feeding patterns than those of longer
vs. (18 days) intervals (5, l0 or 15 minutes), which were examined, with-
Threonine basal out altering the results.
The food cups containing the experimental diets were
Threonine basal changed at the beginning of the light-cycle to minimize inter-
vs. (10 days) ference in the remainder of the light-dark cycle, since in the
Threonine imbalanced dark feeding period, rats fed single diets containing dispro-
Threonine basal portionate amounts of dietary amino acids consumed most of
vs. (7 days) their daily rations in the dark-cycle [16]. Changes in dietary
Threonine devoid regimens in the beginning of the light-cycle also would limit
the occurrence of whatever small feeding bouts or sampling
Threonine basal activities in the process of recognition of the physical or
vs. (10 days) other characteristics of the diets to the non-feeding light-
Threonine basal phase. This should aid in the determination of the sequence
of events induced by the metabolic or nutritional conse-
Threonine basal quences from the usual consumption of larger quantities of
vs. (21 days)
Threonine corrected the diets in the dark-cycle. It has also been reported that
when rats allowed to choose between a 6(~ casein and a
Threonine corrected protein-free diet, the composition of selected meals follows a
vs. (11 days) daily rhythm with protein concentration decreasing from
Threonine corrected light to dark meals [4]. All food cups were rotated daily from
side to side and among the animals at random to discourage
Threonine corrected side preference and individual markings (if any).
vs. ( 13 days) To test if rats given a particular choice showed significant
Threonine devoid increase or decrease in food intake, meal frequency and av-
erage meal size of one diet rather than the other, the amounts
Threonine basal
vs. (8 days) of food and the number of meals as well as the average size
Threonine basal of meals eaten from both diets were compared with a paired
t-test.
Protein-free
vs. (8 days)
Threonine imbalanced RESULTS
The patterns of food intake, meal frequency and average
Protein-free meal size of rats offered the choice between 2 identical
vs. (6 days)
Threonine basal threonine-basal diets during the basal diet pre-feeding ~ r i o d
on the 18th and 5th days prior to the dietary switchinginvolv-
End of experiment ing threonine-basal and the threonLne-imbalanc~ diet are
shown in Table 1. The animals ate randomly from the 2 food
Detailed choice patterns are presented in certain critical cups containing the identical threonine-basal diet. There
periods, as indicated. Three-minute intervals (without feed- were no significant differences in food intake, meal fre-
ing) were used as the intermeal interval criteria, since they quency or average meal size of the diets offered on the days
DIETARY CHOICE PATTERNS IN AMINO ACID IMBALANCE 751

90 Thr Imb
72 40
Thr Imb
54

36 20

18 10-
E E
'-I 0 I"1
z 0 , rl i i i i I I I ! I
Z
IU m
0 t~
¢D

Basal
12
211118 Thr
8 Thr Basal
4 ._n
O" ~ , ,I-I Q , , , 0 I I I I i
0.5 1.5 21.5 3'.5 4.5 0.5 1.5 2.5 3.5 4.5
Meal Size (g) Meal Size (g)
FIG. 4. Light-cycle meal size and total meal number distributions FIG. 5. Dark-cycle meal size and total meal number distributions
(day 1) of 4 rats offered simultaneously the thr-imbalanced (imb) and (day 1) of 4 rats offered simultaneously thr-imb and thr-basal diets.
thr-basal diets. Upper and lower panels represent respectively the Upper and lower panels represent respectively the distribution pat-
distribution patterns of the 2 diets offered. terns of the 2 diets offered.

95
Thr Devoid 40
76 Thr Devoid
57
30
2 0 ~[i_i
38
.¢= 10
19-
.0 E
0
E 0 ""~ ~
i , , , , , , z
z m

30
¢D
24
Thr Basal 16

B
D
12 Thr Basal
18
t~
12 8

6 4

0 "t-I ra r ' t I'l ('I , , , , 0 ,rf-nt- , n

I i i i
0.5 15 2 5 3.5 - 4.5 0.5 1.5 2 5 315 ' 4~5

Meal Size (g) Meal Size (g)

FIG. 6. Light-cycle meal size and total meal number distributions FIG. 7. Dark-cycle meal size and total meal number distributions
(day 1) of 4 rats offered simultaneously thr-devoid and thr-basal (day 1) of 4 rats offered simultaneously thr-devoid and thr-basal
diets. Upper and lower panels represent respectively the distribution diets. Upper and lower panels represent respectively the distribution
patterns of the 2 diets offered. patterns of the 2 diets offered.

indicated. The same random choice held true throughout the
threonine-basal pre-feeding period. Examples of choice pat-
terns from a representative individual rat selecting the same
threonine-basal diet during the light or dark-cycle are shown
in Fig. 1. There was clearly no preference for either food
cups containing the threonine-basal diet.
The initial and final patterns of meal frequency and meal
size of rats offered the choices between threonine-basal and
either the threonine-imbalanced or the threonine-devoid diet
following the basal diet pre-feeding are shown in Table 2.
There was a significant increase in meal frequency of the
threonine-imbalanced'diet with frequent small feeding bouts
compared to the basal diet during the first light-cycle, al-
though the food intake of both diets remained similar (Table
2). The establishment of the preference for the threonine-
basal diet and the avoidance of the threonine-imbalanced
diet occurred during the first dark-cycle with marked reduc-
tion in meal size but not the meal frequency of the imbal-
anced diet. The portion of the imbalanced diet ingested dur-
ing the first dark-cycle constituted only 13% of the combined
752 L E U N G AND ROGERS

TABLE 3
FOOD I N T A K E , MEAL F R E Q U E N C Y AND MEAL SIZE OF RATS OFFERED CHOICES OF T H R E O N I N E
CORRECTED DIET VERSUS T H R E O N I N E DEVOID OR BASAL DIET

Day 1 Day 4 Day I Day 6

Thr Thr Thr Thr Thr Thr Thr Thr
corr basal corr basal corr devoid corr devoid

Light 1.52 0.13 0.35 1.03 0.53 1.03 0.95 0.57

total (g) +_0.46 +_0.04 +_0.11 _+0.45 +_0.24 +_0.35 +_0.81 _+0.20
Meal 12.8" 3.70 6.25 4.75 4.00 8.50 6.75 6.75
frequency _+1.90 _+1.10 _+1.55 +_0.75 _+0.91 +_1.71 _+1.55 _+0.47
Meal 0.12 0.03 0.05 0.19 0.15 0.12 0.10 0.09
size(g) _+.03 +_0.004 _+0.006 _+0.07 _+0.04 +_0.04 _+0.07 +_0.03
Dark 12.98" 4.45 7.74 9.80 11.51 8.64 14.69" 1.31
total (g) +_2.50 -+2.56 _+2.76 -+2.79 +_0.49 _+2.37 _+1.77 _+0.27
Meal 14.2" 3.50 9.75 9.50 10.0 11.0 14.25" 10.25
frequency _+1.30 _+1.80 _+2.39 _+4.27 +-1.47 _+0.58 _+2.63 +-2.66
Meal 0.92 0.73 0.68 1.24 1.25 0.78 1.11" 0.15
size(g) +-0.16 _+0.43 +-0.21 _+0.17 _+0.24 +_0.20 _+0.29 _+0.05

Values are mean --_ S.E. for 4 rats.

*Indicates significant (p <0.05) differences between the two diets in the choice regimens,

THR CORR
0.6

0.4

.II
0.2

i10
0.0
0.8
nrnnn00 0 rill ti n It
THR BASAL
06

0.4

0.2

0.0
2 4 6 8 10 12 14 16 18 2 o ;h i4
I- LIGHT " I" DARK "-I
Hours

FIG. 8. Dietary choice patterns of a representative individual rat (day I) offered the choices
between thr-corrected (corr) and thr-basal diets. Upper and lower panels represent respectively
the portions of intakes from the diets indicated above. The intakes of thr-corr vs. thr-basal diet in
light and dark phases were 1.74 vs. 0.02 g (light), and 12.57 vs. 8.66 g (dark), respectively.

total of 15.4 g in the choice regimen. Prolonged choices
caused a reduction in dark meal frequency as well as light
and dark-cycle meal size of the imbalanced diet as the
animals selected almost exclusively the threonine-basal diet
(Table 2). The subsequent introduction of the threonine-
devoid diet in place of the imbalanced diet produced similar
initial and final pattern changes in meal size and meal fre-
quency. Intensive sampling activity characterized by small
feeding bouts was also observed during the light-cycle fol-
lowing the first introduction of the devoid diet. The devoid
diet comprised only 9% and 0.5% of the combined total food
intake in the first and the f'mal (day 7) dark-cycles of the
choice regimen, respectively. Examples of the detailed initial
choice pattern of a representative individual rat allowed to
choose between the threonine-imbalanced and the threo-
nine-basal diet are shown in Fig. 2. The intense sampling
process was evident shortly after the introductionof the im-
balanced diet during the beginning of t h e light-cycle. The
almost complete rejection of the i ~ a n c e d diet occurred
shortly after the initial consumption of a small quantity of the
imbalanced diet during the first dark-cycle (Fig, 2), Heavier
sampling activities were also a s s o c i a t ~ w i ~ the first intro-
duction of the threonine-devoid diet(Fig, 3). The avoidance
of the devoid diet w a s also rapid during the first dark-cycle
after the ingestion of a minute quantity of the diet :(Fig. 3).
The first light and dark-cycle distribution of meal size and
D I E T A R Y C H O I C E P A T T E R N S IN A M I N O ACID I M B A L A N C E 753

1.o I THR CORR

0.8"

0.6-

0.4"

0.2"

0,0 0
IL I1t J
"0 1.0- THR DEVOID
0
~o 0.8-

0.6"

0.4- 1
0.2"

0.0 I1 0 n ti ,
2 4 6 8 10 12 14 18 18 20 22 :;4
L" ' LIGHT i, ] • DARK --- I
Hours

FIG. 9. Dietary choice patterns of a representative individual rat (day 1) offered the choices
between thr-corr and thr-devoid diets. Upper and lower panels represent respectively the por-
tions of intakes from the diets indicated above. The intakes of thr-corr vs. thr-devoid diet in light
and dark phases were 1.36 vs. 1.02 g (light), and I 1.29 vs. 7.08 g (dark), respectively.

40 24
Thr Corr
Thr C o r r
30 18

2O 12

J~ 6
E E
, p 7P i n! nI n n ! I"I
z
! ! ! i i ! i I Z o i i ! !

m I
CO t~

16
Thr Basal m
"~ 12

4 Thr Basal
2

0 nq I I
n! !
0 I
o'.s ' l'.s ' ' 3:5 ' 4.5
' 015 1.5 2.5 3.5 4.5

Meal S i z e (g) Meal S i z e (g)

FIG. 10. Light-cycle meal size and total meal number distributions FIG. I I. Dark-cycle meal size and total meal number distributions
(day 1) of 4 rats offered simultaneously thr-corr and thr-basal diets. (day l) of 4 rats offered simultaneously thr-corr and thr-basal diets.
Upper and lower panels represent respectively the distribution pat- Upper and lower panels represent respectively the distribution pat-
terns of the 2 diets offered. terns of the 2 diets offered.

total meal number (using the three minute non-feeding as the diet, the animals were given a choice between the
criteria) for all the animals (n=4) in the threonine- threonine-corrected and the threonine-basal diet. After the
imbalanced versus threonine-basal dietary choice are shown initial extensive sampling of the threonine-corrected diet dur-
in Figs. 4 and 5, respectively. Most meals ingested during the ing the first light-cycle, the animals consumed more of the
light-cycle, especially those for the imbalanced diet, were 0.1 corrected diet during the first dark-cycle (Table 3). The ini-
g or less (Fig. 4). During the first dark-cycle, a majority of tial increase in the dark-cycle intake of the threonine-
the ingested basal diet was composed of meals of 0.5 g or corrected diet was mainly through an elevation in meal fre-
more. However, meal size remained mostly small (0.1 g or quency while meal size remained similar to that of the
less) for the imbalanced diet (Fig. 5). Similar initial light and threonine-basal diet (Table 3). However, by day 4 no clear
dark meal size distribution was observed for all animals in difference between the choice patterns of the 2 diets was
choices involving the threonine-devoid and the threonine- observed, and these patterns persisted until the end of the
basal diet (Figs. 6 and 7). choice period (Table 3).
Following a second equilibration with the threonine-basal During the initial choice between the threonine-corrected
 754 L E U N G A N D R()GERS

12.5]
lO.O-~ Thr Corr 16 Thr Corr
12

h,. 57."°5 t l 8

J¢l 4
E
I I I
Z 0 ! @
nnnnm l !
BFI n I~ r--
I I
I ' ,n, , ,
3°1.
I
2oJ
Devoid
=[ 2411 Thr Devoid 16

181t
il
12 Thr

'211
0 I i r ' ~ r l f'l ,
0.5
n I
1.5
i
215
I I
3.5
I I
4.5 (1.5
l
F~
@
1.5
nn
I
nn
!
2.5
I
3.5
~-~
! I
4.5
Meal Size (g) Meal Size(g)
FIG. 12. Light-cycle meal size and meal number distributions (day 1) FIG. 13. Dark-cycle meal size and meal number distributions (day I)
of 4 rats offered simultaneously thr-corr and thr-devoid diets. Upper of 4 rats offered simultaneously thr-corr and thr-devoid diets.
and lower panels represent respectively the distribution patterns of Upper and lower panels represent respectively the distribution pat-
the 2 diets offered. terns of the 2 diets offered.

TABLE 4
FOOD I N T A K E , M E A L F R E Q U E N C Y A N D SIZE O F RATS O F F E R E D C H O I C E S O F P R O T E I N - F R E E DIET
VERS US T H R E O N I N E I M B A L A N C E D OR B A S A L DIET

Day 1 Day 8 Day 1 Day 6

Thr Protein Thr Protein Thr Protein Thr Protein

imb free imb free basal free basal free

Light 0.68 2.78 0.11 2. l0 8.37* 0.20 0.92 0.26

total (g) _+0.35 _+1.39 _+0.01 _+1.10 _+0.88 _+0.12 +_0.49 +_0. I1
Meal 13.33 9.00 7.50 4.00 10.5 7.25 6.50 8.50
frequency _+1.86 _+1.15 _+5.52 +_0.91 +_1.04 _+3.04 _+0.05 +_2.99
Meal 0.05 0.29 0.48 0.03 0.60* 0.03 0.15 0.03
size (g) +_0.07 +_0.11 +_0.22 _+0.004 _+0.11 _+.006 +_0.08 +_0.008
Dark 4.67 12.74' 3.28 1 3 . 7 6 " 17.79" 0.86 16.86' 2.43
total (g) +_1.30 _+1.45 +_1.39 +_0.97 _+1.75 +_0.61 _+1.23 _+1.39
Meal 17.0 11.67 6.50 1 3 . 2 5 " 14.35 8.75 12.25 9.00
frequency +_3.0 +_2.03 _+0.64 _+2.46 +_1.70 _+2.78 _+1.70 _+2.70
Meal 0.27 1.22" 0.46 1.14" 1.29* 0.15 1.43* 0.22
size (g) _+0.05 _+0.26 +_0.17 +_0.11 _+0.18 _+0.13 +_0.14 ±0.11

Values are meal - S.E. for 4 rats.

*Indicates significant (p<0.05) differences detween the two diets in the choice regimens.

and the threonine-devoid diet after the equilibration with the
threonine-corrected diet, the animals showed no preference
for either diet (Table 3). Nevertheless, the animals
endeavored to distinguish between the 2 diets after 5 days
and discriminated against the devoid diet, drastically de-
creasing their dark-cycle intake by markedly curtailing their
meal size (Table 3).
Examples of the initial choice patterns of a representative
individual rat selecting the threonine-corrected and the
threonine-basal or subsequently the threonine-devoid diet
are shown in Figs. 8 and 9. The frequent sampling o f the
threonine-corrected diet immediately following its introduc-
tion during the first light-cycle did not lead to an exclusive
preference or rejection of the alternative threonine-basal diet
in the beginning of the first dark-cycle (Fig. 8). The lack of
obvious avoidance for the threonine-devoid diet was also
evident in the initial choice between the corrected and the
devoid diet although the animals sampled both diets fre-
quently during the first light-cycle (Fig, 9).
The initial light and dark-cycle meal distribution of meal
D I E T A R Y CHOICE P A T T E R N S IN A M I N O A C I D I M B A L A N C E 755

THR I M B A L A N C E D
0.6-

0.4"

¢D 0.2-

0.0 In
"O 0.8-
O
,P PROTEIN- FREE
0.6"

0.4-

0.2-

O0 , 0 , i, p ,I Q 0 t . . . . . .
2 4 6 8 10 ' 12 14 16 18 20 22 24
I- UGHT - I- DARK -I
Hours
FIG. 14. Dietary choice patterns of a representative individual rat (day 1) offered the choice
between thr-imb and protein-free diets. Upper and lower panels represent the portions of intakes
from the diets indicated above. The intakes of thr-imb vs. protein-free diet in light and dark
phases were 0.29 vs. 0.89 g (light), and 2.24 vs. 14.58 g (dark), respectively.

o.6 THR BASAL

"~ 0.2
0

0.0 , . . ,l , I .I .n , g . I ,I , .h . . . . . . . . . . . .

"~ PROTEIN- FREE

i~O 0,6

0.4

0,2

0.0 . .fltl . . . . . [I . . . . . n.I] . n . . . . . . . . . .

2 4 6 8 10 12 14 16 18 20 22 24
I LIGHT "[ " DARK I
Hours

FIG. 15. Dietary choice patterns of a representative individual rat (day 1) offered the choice
between thr-basal and protein-free diets. Upper and lower panels represent the portions of in-
takes from the diets indicated above. The intakes of the thr-basal vs. protein-free diet in light and
dark phases were 7.84 vs. 0.07 g (light), and 17.64 vs. 0.05 g (dark), respectively.

size and total meal frequency for all animals in the protein-flee or the threonine-imbalanced diet was observed
threonine-corrected versus threonine-basal choice are during the first light-cycle, the imbalanced diet was over-
shown in Figs. I0 and 11, respectively. Intake of the 2 diets whelmingly rejected during the first dark-cycle with a
offered was composed of meals of 0.1 g or less during the marked reduction in its meal size but not its meal frequency
first light phase (Fig. 10) but of much larger meals of 1.0 g or (Table 4). By day 8, the dark meal frequency of the imbalaneed
more during the first dark-cycle (Fig. 11). Similar initial meal diet was also significantly curtailed while the animals main-
size distribution with small meals for most of the light-cycle tained their preference for the protein-free diet and decisively
intake (Fig. 12) and larger meals for most of the dark-cycle rejected the imbalanced diet throughout the choice period (Ta-
intake (Fig. 13) was observed in the threonine-corrected ver- ble 4). However, when the imbalanced diet was substituted by
sus threonine-devoid choice when no distinct preference for the threonine-basal diet, the preference for the protein-free diet
either diet was apparent (Table 3). was immediately reversed in favor of the basal diet during
The patterns o f the initial and final choices between the the first light-cycle accompanied by abrupt increase in its
protein-free and the threonine-imbalanced diets or subse- light-cycle intake and meal size (Table 4). The choice for the
quently the threonine-basal diet following the re- basal diet was established in the beginning of the first dark-
equilibration with the threonine-basal diet are shown in cycle with a significant increase in meal size but not the meal
Table 4. Although no distinct preference for either the frequency (Table 4). The animals maintained their choice
 756 L E U N G AND ROGERS

40
40 Thr Imb Thr Imb
30
30
20
L 20-
® .O 10
J= 10-
E
E r"i 0
0 n, Z
! i i ! I
Z I I I

q)
16
m tO 12
Protein-Free
o= 30-
Protein - F r e e
20- 8

10- 4

0 :~n i ,n, rI , , n, 0 i i
0 15 2.5 315 4.5
0.5 1.5 2.5 3.5 4.5
Meal Size(g) Meal Size (g)
FIG. 16. Light-cycle meal size and meal number distributions (day 1) FIG. 17. Dark-cycle meal size and meal number distributions (day 1)
of 4 rats offered simultaneously thr-imb and protein-free diets. of 4 rats offered simultaneously thr-imb and protein-free diets.
Upper and lower panels represent respectively the distribution pat- Upper and lower panels represent respectively the distribution pat-
terns of the 2 diets offered. terns of the 2 diets offered.

30 Thr Basal 20
Thr Basal
24
16
18
12
12
8

6
.O
4
E 0 1-11"I
i I
m ,ran I I
nI no
l i i
E
=I 0 I ! i v i
Z Z

30- Protein-Free 35
¢D Protein-Free
24- 28
B
18- 21

12-

6- 7

0 Mra
! i
rl i i
0 015 I 1'5 i 215 ' 3~S ! 4~5 o.s 1'.5 2'.5 ' 3'.5 4'.s
Meal Size (g) Meal Size (g)
FIG. 18. Light-cycle meal size and meal number distributions (day 1) FIG. 19. Dark-cycle meal size and meal number distributions (day 1)
of 4 rats offered simultaneously thr-basal and protein-free diets. of 4 rats offered simultaneously thr-basal and protein-free diets.
Upper and lower panels represent respectively the distribution pat- Upper and lower panels represent respectively the distribution pat-
terns of the 2 diets offered. terns of the 2 diets offered.

patterns with almost exclusive preference for the basal diet
to day 6 (Table 4) and beyond until the end of the experi-
ment.
Examples of the detailed mode of selection by a represen-
tative individual rat which avoided the threonine-imbalanced
diet in favor of the protein-free diet and subsequently re-
jected the protein-free diet in favor of the threonine-basal
diet are shown in Figs. 14 and 15. Following alternative
sampling bouts involving the imbalanced and the protein-free
diet during the first light-cycle, the animals selected the
protein-free diet after ingesting small amounts of the imbal-
anced diet, shortly after the beginning of the dark-cycle (Fig.
14). When the threonine-basal diet was introduced as an al-
ternative to the imbalanced diet, a large quantity o f the basal
diet was immediately consumed and the protein-free diet was
decisively rejected in the beginning of the first dark-cycle
(Fig. 15). The first light- and dark-cycle meal distribution
during the imbalanced versus the protein-free choice for all
the animals are shown respectively in Figs. 16 and 17. The
majority of the meals consumed during the first light-cycle
DIETARY CHOICE PATTERNS IN AMINO ACID I M B A L A N C E
were 0.1 g or less with occasional large meals (0.5 to 2.5 g)
for the protein-free diet (Fig. 16). Meal sizes of 1.5 g or more
for the protein-free diet were evident as the animals estab-
lished their choice for this diet during the first light-cycle
(Fig. 17). The substitution of the imbalanced diet by the basal
diet, with the protein-free diet as an alternative choice,
evoked light-cycle consumption of large meals (over 3.0 g) of
the basal diet while meal size for the protein-free diet re-
mained small (0.1 g or less) (Fig. 18). The animals almost
exclusively selected the basal diet over the protein-free diet
during the first dark-cycle with a majority of the meals rang-
ing from 1.0 to 4.0 g for the basal diet and 0.1 g or less for the
protein-free diet (Fig. 19).
DISCUSSION
It has been previously reported that when naive rats were
allowed to choose between a basal diet, a corrected diet, or a
protein-free diet and an imbalanced diet, they invariably re-
jected the imbalanced diet in favor of the alternative choices
[5,17]. The studies confirmed these findings in non-naive
rats, and they also provided information as to the mode
whereby the choices were established and the course of
events in which the animals initiated their selection process.
Under normal circumstances, in a single diet situation in-
volving dietary amino acid disproportions, rats ate in dis-
crete meals without frequent small bouts which were charac-
teristic of sampling activities [16]. In the present studies, the
frequent small bouts which interspersed between one diet
and its alternative choice soon after their introduction during
the first light-cycle, appeared to be an integral part of the
selection process for establishing subsequent dietary prefer-
ence during the first dark feeding period. Since the diets
were introduced immediately after the beginning of the
light-cycle, which was the non-feeding period for the rats, it
was not apparent whether similar frequent small sampling
bouts would be evident if the diets were introduced in the
beginning of the dark-cycle. The introduction of any diets at
any time may induce untoward feeding activities in rats. It
would be reasonable to assume that introduction of the diets
at the beginning of the light-cycle may prevent the interrup-
tion or induction of untoward interference in the start of the
dark-cycle when the feeding activity was usually intense.
The consumption of small "sampling" bouts of minute
quantities of the imbalanced or the devoid diet during the
first light-cycle did not evoke physiological and/or biochemi-
cal responses to reject such a diet. The animals continued to
ingest relatively larger amounts of imbalanced or devoid diet
in the beginning of the first dark-cycle before altering their
choice patterns in favor of the alternative basal diet (Figs. 2
and 3). Similar phenomena were observed in the initial
choice between the imbalanced and the protein-free diet as
the animals continued to consume the imbalanced diet in the
beginning of the first dark-cycle before selecting the protein-
free diet (Fig. 14). Thus the avoidance of the imbalanced or
devoid diet and the selection of the basal or protein-free diet
appeared to be initiated during the first dark-cycle shortly
after the ingestion of a small amount of the imbalanced or
devoid diet. It is not clear, however, if the small "sampling"
bouts facilitated the selection process in the beginning of the
dark feeding period by providing information for the physical
and oral-pharyngeal properties of the 2 diets offered.
The introduction of the unfamiliar threonine-corrected
diet also evoked intense "sampling" activities during the
first light-cycle, but no preference was established for either
757
the corrected or the basal diet in the beginning of the first
dark-cycle (Fig. 8). This is probably due to the lack of ad-
verse physiological and/or biochemical responses from the
ingestion of either diet. The failure of the animals to favor-
ably select the corrected diet over the devoid diet in the
initial choice period was probably due to the pre-feeding of
the corrected diet in the equilibration period. The similarity
in physical characteristics and the metabolic self-correction
from alternate consumptions of these 2 diets apparently pre-
vented the animals from fully realizing the metabolic charac-
teristics of each diet (Fig. 9). However, prolonged ingestion
of large quantities of the devoid diet apparently produced
eventual untoward metabolic consequences and thus they
avoided the devoid diet after 5 days (Table 3). It was uncer-
tain how the animals distinguished the corrected diet from
the devoid diet. The fact that olfactory bulbectomized rats
discriminated against the imbalanced or devoid diet in favor
of the protein-free diet indicated that olfactory properties
were not the sole guide for diet recognition in a free choice
situation [9].
Despite the differences in physical and sensory charac-
teristics of the imbalanced and the protein-flee diet, the
animal alternately consumed small amounts of the 2 diets
throughout the first light-cycle indicating non-preference for
either diet (Fig. 14). Overwhelming selection of the protein-
free diet occurred only after the animal experienced
postabsorptive metabolic consequences from consuming
larger amounts of the imbalanced diet in the early part of the
first dark-cycle (Fig. 14). The abrupt consumption of large
quantities of the basal diet, shortly after its introduction in
place of the protein-flee diet, was due apparently to at-
tempted protein repletion by the animal after the prolonged
preference for the protein-free diet (Fig. 15). The consump-
tion of large amounts of the basal diet in the first light-cycle
apparently initiated the abandonment of the protein-free
diet, and thus, the rejection of the protein-flee diet from the
start of the first dark-cycle (Fig. 15 and Table 4). Under the
present choice situations, the small "sampling" bouts, the
prior nutritional status of the animals, and their initial re-
sponses to the nutritional and metabolic characteristics of the
diets, are the major determinants in establishing dietary prefer-
ences. It also appears that the quantities of the diets con-
sumed in the sampling process influenced the rapidity of
selections.
From the results in meal size distribution patterns, it is
apparent that the initial response to dietary imbalance or
deficiency was a depression in meal size, and the curtailment in
meal frequency occurred only after complete recognition of
the adverse metabolic consequence of the imbalanced or de-
void diet.
The rejection of the imbalanced diet or the devoid diet
was rapid and evident within 2-3 hours after the ingestion of
relatively large meals of diets containing the imbalanced or
deficient amino acid mixtures during the beginning of the
first dark feeding period. Thus it appears that the time frame
required for the control system to establish the choices for
the alternative diets is similar to that which is required for
the curtailment of intake of a single diet in responses to
disproportion of dietary amino acids [ 16]. Also, the choice of
a protein-free diet or a relatively balanced amino acid diet
over a diet with an imbalance of amino acids appears to be
the most sensitive measure of a dietary amino acid balance.
It is not clear how the dietary selection involving amino acid
imbalance relates to "learned aversion" or "learned prefer-
ence" as occurs in rats ingesting poison or vitamin deficient
758
diets [18]. A n i m a l s w i t h lesions in the a n t e r i o r p r e p y r i f o r m
c o r t e x s e l e c t e d the i m b a l a n c e d diet o v e r the p r o t e i n - f r e e diet
I8], indicating the i n v o l v e m e n t o f c e r t a i n neural a r e a s in the
c o n t r o l o f food intake o f a m i n o acid i m b a l a n c e d diets. T h e
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2. Harper, A. E., N. J. Benevenga and R. M. Wohlhueter. Effect of
ingestion of disproportionate amounts of amino acids. Ph~,siol
Rev 50: 428-558, 1970.
3. Harper, A. E., N. J. Benevenga and R. M. Wohlhueter. Effect of
intake. In: Hunger: Basic Mechanisms and Clinical Implica-
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Different diurnal rhythms of protein and non-protein energy in-
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5. Leung, P. M. B., Q. R. Rogers and A. E. Harper. Effect of
amino acid imbalance on dietary choice in the rat. J Nutr 95:
482-492, 1%8.
6. Leung, P. M. B. and Q. R. Rogers. Food intake: Regulation by
plasma amino acid pattern. Life Sei 8: 1-9, 1969.
7. Leung, P. M. B. and Q. R. Rogers. Effect of pituitary extract on
food intake of intact and hypophysectomized rats. Nutr Rep Int
4: 207-215, 1971.
8. Leung, P. M. B. and Q. R. Rogers. Importance of prepyriform
cortex in food intake response of rats to amino acids. Am J
Physiol 221: 92%935, 1971.
9. Leung, P. M. B., D. M. Larson and Q. R. Rogers. Food intake
and preference of olfactory bulbectomized rats fed amino acid
imbalanced or deficient diets. Physiol Behav 9: 553-557, 1972.
10. Leung, P. M. B. and Q. R. Rogers. Effect of amygdaloid lesions
on dietary intake of disproportionate amounts of amino acids.
Physiol Behav 11: 221-226, 1973.
[ . E U N G A N D R()(IER,%
e x a c t m e c h a n i s m w h e r e b y the a n i m a l s initiate the accep-
t a n c e or rejection o f diets c o n t a i n i n g d i s p r o p o r t i o n a t e
a m o u n t s o f dietary a m i n o acid a w a i t s elucidation.
1 I. Leung, P. M. B. and Q. R. Rogers. Disturbances in amino acid
balance. In: Tota! Parenteral Nutrition, edited by H. Ghadimi.
New York: John Wiley & Sons, 1975, pp. 25%284.
12. Leung, P. M. B. and Q. R. Rogers. Effects of hippocampal
lesions on adaptive intake of diets with disproportionate
amounts of amino acids. Physiol Behav 23: 12%136, 1979.
13. Leung, P. M. B. and Q. R. Rogers. Hyperphagia alter ventral
tegmental lesions and food intake responses of rats fed dispro-
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457-464, 1980.
14. Leung, P. M. B., Q. R. Rogers, J. S. Stern and V. E. Mendel.
An on-line computerized feeding, drinking and activity patterns
analysis system. Proc Soe Neurosci 7: 873. 1981.
15. Meliza, L. L., P. M. B. Leung and Q. R. Rogers. Cingulate
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on the neuroregulation of food intake. Fed Proe 32: 170%1719.
1973.
17. Rogers, Q. R. and P. M. B. Leung. The control of food intake:
When and how are amino acids involved? In: The Chemical
Senses and Nutrition. edited by M. R. Kare and O. Mallet. New
York: Academic Press, 1977, pp. 213-249.
18. Rozin, P. and J. W. Kalat. Specific hungers and poison
avoidance as adaptive specializations of learning. Psycho1 Rev
78: 459-486, 1971.