J. Anim. Physiol. a. Anim. Nutr. 85 (2001), 88±100 Receipt of ms.: 4. 8.

2000
Ó 2001 Blackwell Wissenschafts-Verlag, Berlin
ISSN 0931±2439

1
Department of Molecular Biosciences, School of Veterinary Medicine, University of
California, Davis, California 95616, USA, 2Friskies R & D, Nestec, Ltd, St Joseph, MO
64503, USA

Dietary crude protein concentration does not affect the leucine

requirement of growing dogs
By S. J. DELANEY1, A. S. HILL1, R. C. BACKUS1, G. L. CZARNECKI-MAULDEN2 and
Q. R. ROGERS1

Summary

The objective of the present study was to examine the interaction between graded levels of
leucine and dietary crude protein. Dose±response curves were generated using four 3 ´ 3
Latin squares (two dogs/square). Each square represented one of two concentrations of
crude protein (140 or 280 g/kg diet) and one of two combinations of three concentrations
of leucine (5.0, 7.0 and 9.0 g/kg diet or 9.0, 11 and 13 g/kg diet). An additional experiment
was performed by feeding crude protein at 210 g/kg diet with either 7.0 or 11 g leucine/kg
diet. Weight gain, food intake, nitrogen retention, plasma albumin and plasma amino acids
were measured. The requirement was determined to be the minimum leucine concentration
required to maximize weight gain and nitrogen retention. For 8±14-week-old male Beagle
dogs, 140 g crude protein/kg diet in a diet containing 18 kJ metabolizable energy/g does
not appear to support maximal growth. The leucine requirement was not affected by
doubling the dietary crude protein level from 140 to 280 g/kg diet. From these results,
the leucine requirement of 8±14-week-old Beagle dogs appears to be 11 g leucine/kg diet
independent of the level of dietary crude protein, whereas dogs over 14 weeks require only
7 g leucine/kg diet for maximal nitrogen retention.

Introduction

Some essential amino acid (EAA) requirements of chicks (Gallus gallus L.) (GRAU 1948;
BOOMGAARDT and BAKER 1971), pigs (Sus scrofa L.) (BECKER et al. 1957), rats (Rattus
norvegicus L.) (SALMON 1954) and dogs (Canis familiaris L.) (MILNER 1981) are positively
correlated with the level of dietary crude protein (CP). Therefore, in these species, as
dietary CP increases, the requirement for certain essential amino acids (EAA) also
increases. This relationship has been seen in various species for arginine, isoleucine, lysine,
methionine, threonine and tryptophan (ROGERS et al. 1988, 1990). In the dog, using
nitrogen retention as the variable, there is one study that indicates that the lysine
requirement of the growing dog increases signi®cantly when dietary CP is doubled from
140 to 280 g/kg diet (MILNER 1981). Although a weak positive correlation was found for
threonine with dietary protein level in the cat (Felis catus L.) (HAMMER et al. 1996), a weak
negative correlation has been seen for methionine (STRIEKER 1991). We could ®nd no
reports in any species indicating whether the leucine requirement also shows a correlation
with dietary CP level. Since the protein requirement of the growing dog for maximal
nitrogen retention appears to be above 140 g CP/kg diet (ROGERS and MORRIS 1991) and
since the NRC estimated amino acid requirements are based largely on the work of Milner

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 Leucine requirement of growing dogs 89

and co-workers (NRC 1985) using about 140 g CP/kg diet in growing Beagle dogs, it is
important to determine whether amino acid requirements of growing dogs are in¯uenced
by the level of dietary protein.
In examining comparative data from a variety of species, it would appear that the estimated
requirement of several amino acids for the dog may be too low (ROGERS and MORRIS 1991)
but especially that of leucine. Therefore, the following study was carried out to determine
whether dietary crude protein level affects the leucine requirement of the growing dog.

Materials and methods

Dogs
The experimental protocol for this study was approved by the University of California,
Davis, Animal Use and Care Administrative Advisory Committee and was carried out in
accordance with standards of the National Research Council's Dogs: Laboratory Animal
Management (NRC 1994) and the Animal Welfare Act. Twenty-four male growing Beagle
dogs (Marshall Farms, North Rose, NY, USA) were used in the study. The dogs were
weighed upon arrival, placed into three weight blocks and then randomly assigned to dietary
treatments from the blocks for Experiment 1. In Experiment 2, the dogs were divided into
two groups balanced for previous dietary treatment, weight gain and food intake. At the
start of Experiment 1, the mean weight of the dogs ‹ SEM was 2427 ‹ 77 g, and the mean
age ‹ SEM was 55 ‹ 0.7 days. At the start of Experiment 2, the mean weight of the dogs
‹ SEM was 4604 ‹ 231 g, and the mean age ‹ SEM was 97 ‹ 0.7 days. The dogs were
individually housed in stainless-steel metabolism cages (Process Automation, Santa Ana,
CA, USA). The cages were located in temperature-controlled rooms (23° C ‹ 2° C) with a
12-h light cycle.

Diets
Fresh water and food were provided for ad libitum consumption, twice daily between 09.00
and 11.00 h and between 16.00 and 18.00 h. The dogs were acclimatized to their cages and
given pre-test diets for 2 weeks prior to the start of Experiment 1. The pre-test diet given
during the week prior to the start of the study contained 210 g CP/kg diet and 9.0 g leucine/
kg diet. The composition of the basal crystalline L-amino acid diets used in the study is
shown in Table 1. The 280 g CP/kg diet amino acid mixture contained two times the EAA
requirements suggested by ROGERS and MORRIS (1991), while the 140 and 210 g CP/kg diet
mixes, respectively, contained 1.5 and 1.75 times their suggested EAA requirements.
Leucine was added as outlined later with alanine adjusted to make the diets isonitrogenous.
The resulting EAA to dispensable amino acid ratios were 2 : 1 for the 140 g CP/kg diet, 1 : 1
for the 210 g CP/kg diet, and 1 : 1.3 for the 280 g CP/kg diet. The experimental diets had a
calculated metabolizable energy (ME) content of 18 kJ/g diet (based on diet composition
using 16 kJ/g for carbohydrate, 17 kJ/g for protein and 37 kJ/g for fat). The percent of ME
from protein was 14.8% for the 140 g CP/kg diet, 21.4% for the 210 g CP/kg diet, and
29.7% for the 280 g CP/kg diet. The diets had a small amount of dye (Westco, Pico Rivera,
CA, USA) added to help with diet identi®cation and were extruded through a meat grinder
without the blade to create about 2.5 cm long, cylindrical pieces with a diameter of 1.2 cm.

Design experiment 1
The dogs were divided into two groups, with one group given 140 g CP/kg diet and the
other group given 280 g CP/kg diet. Each group was given leucine at 5.0, 7.0 and 9.0 g/kg
diet or 9.0, 11, and 13 g/kg diet for three 2-week periods in a 3 ´ 3 Latin square design
(Table 2).
90 S. J. Delaney et al.

Table 1. Diet composition

Level of dietary crude protein (g/kg)

1
Ingredients 140 g/kg 210 g/kg2 280 g/kg1

Starch3 400±402 348±353 293±295

Sucrose4 200±201 174±177 146±147
14% CP amino acid mixture5,6 144 0 0
21% CP amino acid mixture5,7 0 222 0
28% CP amino acid mixture5,8 0 0 300
5
L-Leucine 5±13 7±11 5±13
Additional L-Alanine5 0±5.4 1.4±4.1 0±5.4
Animal tallow9 90 90 90
Mineral mixture10 50 50 50
Saf¯ower oil11 30 30 30
Hydrogenated beef tallow12 30 30 30
Celu®l13 20 20 20
Sodium acetate14 14.5 16.9 19.4
Vitamin mixture15 5 5 5
Choline chloride16 3.7 3.7 3.7
1
140 g CP/kg diet and 280 g CP/kg diet were fed in Experiment 1
2
210 g CP/kg diet was fed in Experiment 2
3
Corn Starch, Rykoff-Sexton, Inc., Lisle, IL, USA; Cornstarch added in 2 : 1 with sucrose in varying
amounts due to difference in molecular weight per unit of nitrogen between L-Leucine and L-Alanine.
4
California and Hawaiian Sugar Company, Crockett, CA, USA
5
Ajinomoto Co., Inc., Tokyo, Japan
6
140 g CP/kg diet amino acid composition (g/kg mixture): L-Arginine, 82.4; L-Histidine, 27.1;
L-Isoleucine, 57.4; L-Lysine á HCl, 130.3; L-Cystine, 32.3; L-Methionine, 42.8; L-Tyrosine, 62.6;
L-Phenylalanine, 62.6; L-Threonine, 66.7; L-Tryptophan, 19.8; L-Valine, 72.0; L-Alanine, 56.8; glycine,
47.8; L-Glutamine, 46.6; L-Glutamate, 92.7; L-Asparagine, 21.1; L-Aspartate, 42.4; L-Proline, 36.7
7
210 g CP/kg diet amino acid composition (g/kg mixture): L-Arginine, 62.3; L-Histidine, 20.5;
L-Isoleucine, 43.4; L-Lysine á HCl, 98.6; L-Cystine, 24.5; L-Methionine, 32.3; L-Tyrosine, 47.3; L-Phe-
nylalanine, 47.3; L-Threonine, 50.5; L-Tryptophan, 15.0; L-Valine, 54.4; L-Alanine, 83.1; glycine, 70.1; L-
Glutamine, 68.2; L-Glutamate, 135.8; L-Asparagine, 30.8; L-Aspartate, 62.1; L-Proline, 53.7
8
280 g CP/kg diet amino acid composition (g/kg mixture): L-Arginine, 52.7; L-Histidine, 17.3;
L-Isoleucine, 36.7; L-Lysine á HCl, 83.4; L-Cystine, 20.7; L-Methionine, 27.4; L-Tyrosine, 40.0;
L-Phenylalanine, 40.0; L-Threonine, 42.7; L-Tryptophan, 12.7; L-Valine, 46.0; L-Alanine, 95.8; glycine,
80.8; L-Glutamine, 78.6; L-Glutamate, 156.4; L-Asparagine, 35.5; L-Aspartate, 71.5; L-Proline, 61.9
9
Florin Tallow, Dixon, CA, USA
10
See WILLIAMS et al. (1987)
11
Saffola Quality Foods, Los Angeles, CA, USA
12
Morgan Specialities, Inc., Paris, IL, USA
13
Cellulose, United States Biochemical Corp., Cleveland, OH, USA
14
Sodium acetate á 3H2O, Fisher Scienti®c, Fair Lawn, NJ, USA
15
See Williams et al. (1987)
16
70% Choline chloride, DuPont, Highland, IL, USA

Design experiment 2
The dogs were divided into two groups that were given either 7.0 or 11 g leucine/kg diet.
Both groups were given 210 g CP/kg diet for one 2-week period.

Analytical methods
Food intake and body weight for each dog were measured daily. Food intake recorded
on days 2±13 and body weights from days 1±13 were used for calculations. Urine was
collected daily into a container with hydrochloric acid as a preservative. Faeces were
 Leucine requirement of growing dogs 91

Table 2. Design of Experiment 1

Period

Dog number 1 2 3

140 g 280 g
CP/kg diet CP/kg diet g leucine/kg diet

1 13 5.0 7.0 9.0

2 14 7.0 9.0 5.0
3 15 9.0 5.0 7.0
4 16 5.0 9.0 7.0
5 17 9.0 7.0 5.0
6 18 7.0 5.0 9.0
7 19 9.0 11 13
8 20 11 13 9.0
9 21 13 9.0 11
10 22 9.0 13 11
11 23 13 11 9.0
12 24 11 9.0 13

collected daily and frozen. Pooled urine and faeces collected on days 6±13 and samples of
each diet were analysed for total nitrogen content (Nitrogen Analyzer, LECO, St Joseph,
MO, USA).
Blood was drawn into heparinized syringes from the jugular vein on day 11 of each
period approximately 3.5 h following the addition of fresh food for determination of
plasma amino acid concentrations. Plasma was stored at )80° C until an equal volume of
sulfosalicyclic acid (275 mmol/L) was added to precipitate plasma proteins and then the
deproteinized plasma was analysed (Model 6300 Amino Acid Analyzer, Beckman
Instruments, Palo Alto, CA, USA). Plasma albumin concentrations were also determined
using this frozen plasma sample (Roche Reagent Test Kit for Albumin & Cobas Mira Plus-
CC Chemistry Autoanalyzer, Roche Diagnostic Systems, Inc., Somerville, NJ, USA).
After the ®nal day of faeces and urine collection, the dogs were food and water deprived
from 15.00 to 14.00 h the following day (23 h total) in preparation for general anaesthesia
and for deuterium oxide dilution. General anaesthesia facilitated intravenous injection of
deuterium into the jugular vein, and allowed bioelectrical impedance measurements to be
taken. These data were collected as part of another study on techniques for measuring body
composition. On day 14 of the last period of Experiment 1, 3 mL of blood was drawn once
to determine food-deprived plasma amino acid concentrations. A recovery diet identical to
the pre-test diet was given following food deprivation on day 14.

Statistical methods
Statistical analysis was performed using PC-SAS, Version 8.0 (SAS, Cary, NC, USA).
Probability levels £0.05 were considered to be statistically signi®cant. In Experiment 1,
data were analysed by analysis of variance utilizing a general linear model. No signi®cant
interactions among dogs and the sequence of treatments were found. An initial analysis was
run to determine if CP level had an effect on weight gain, food intake, nitrogen retention,
plasma albumin concentration, or plasma leucine concentration. It was determined that CP
level only had a signi®cant effect on nitrogen retention. Therefore, the general linear model
used the data from both levels of CP for all parameters except nitrogen retention. One-
slope broken-line analysis (ROBBINS 1986) of the body weight gain and nitrogen retention
data was used to estimate the dietary leucine requirement. In Experiment 2, data were
analysed by analysis of variance.
92 S. J. Delaney et al.

Results

Experiment 1
Body weight gain, food intake and nitrogen retention all increased (Fig. 1) in response to
increasing concentrations of dietary leucine from 5.0 to 11 g/kg diet dry matter for both CP
levels. Maximal weight gain, food intake, and nitrogen retention occurred at 11 g leucine/kg
diet for both CP levels. The highest mean nitrogen retention values were seen when dogs
were given 11 g leucine/kg diet for both protein levels. For example, nitrogen retention
increased 63 and 51% in dogs given 140 and 280 g CP/kg diet, respectively, when leucine
was given at 11 g/kg diet instead of 9 g/kg diet. It must be noted that no statistical difference
was seen in nitrogen retention between leucine levels in dogs given 140 g CP/kg diet.
No statistically signi®cant effect of protein level was seen for weight gain (p ˆ 0.42),
food intake (p ˆ 0.57), plasma albumin concentration (p ˆ 0.45) and plasma leucine
concentration (p ˆ 0.84). Nitrogen retention, however, was greater for the group receiving
280 g CP/kg diet than it was for the group receiving 140 g CP/kg diet (p ˆ 0.03).
Broken-line analysis of the body weight gain data indicated that the leucine requirement
was 11.0 and 7.4 g/kg diet for dogs given 140 and 280 g CP/kg diet, respectively (SE ˆ 2.91
and 0.52, respectively). While broken-line analysis of the nitrogen retention data indicated
that the leucine requirement was 11.0 and 10.5 g/kg diet for dogs given 140 and 280 g CP/kg
diet, respectively (SE ˆ 5.34 and 1.63, respectively).
The apparent nitrogen digestibility was 77 ‹ 1% and 88 ‹ 1% (mean ‹ SEM) for the
diets containing 140 and 280 g CP/kg diet, respectively. The apparent dry matter
digestibility was 80 ‹ 1% and 86 ‹ 1% (mean ‹ SEM) for the diets containing 140 and
280 g CP/kg diet, respectively.
Plasma albumin concentration was not affected by leucine concentration (e.g. plasma
albumin in dogs given 5.0 g leucine and 13 g leucine were, respectively, mean ‹ SEM,
23 ‹ 2.1 g/l and 23 ‹ 1.7 g/l). Plasma leucine concentration increased in response to
increasing concentrations of dietary leucine from 5.0 to 9 g/kg diet for both CP levels
(Fig. 2). However, plasma leucine levels only slightly increased from 9 to 13 g leucine/kg
diet for both CP levels. Plasma isoleucine and valine concentrations all decreased with
increases in dietary leucine. Plasma arginine and lysine concentrations in the dogs given
140 g CP/kg diet decreased with increases in dietary leucine. Dietary leucine levels did not
affect plasma histidine, methionine, phenylalanine, threonine and tryptophan concentra-
tions (Tables 3 and 4). It should be noted that elevated levels of plasma glutamate (Table 4)
were found in dogs given 280 g CP/kg diet dry matter (mean ‹ SEM, 322 ‹ 45 lmol/l).
Ten of the 12 dogs in this group at some point had elevated plasma glutamate levels ranging
from 278 to 1017 lmol/l. Dogs given 140 g CP/kg diet had plasma glutamate levels ranging
from 34 to 275 lmol/l (mean ‹ SEM, 107 ‹ 10 lmol/l).

Experiment 2
No statistical differences were found between the group given 7.0 g leucine/kg diet and the
group given 11 g leucine/kg diet for body weight gain (p ˆ 0.26), food intake (p ˆ 0.98),
nitrogen retention (p ˆ 0.89), or plasma albumin (p ˆ 0.45) (Table 5). Plasma leucine levels
were higher in the dogs given 11 g leucine/kg diet while plasma isoleucine and valine were
lower. The apparent nitrogen digestibility was 87 ‹ 1% (mean ‹ SEM), and the dry matter
digestibility was 88 ‹ 1% (mean ‹ SEM) for the two diets in this experiment.

Discussion

In this study, the minimum dietary concentration of leucine required to maximize body
weight gain, food intake and nitrogen retention of 8±14-week-old Beagles was found to be
 Leucine requirement of growing dogs 93

11 g/kg diet for diets containing either 140 or 280 g CP/kg diet. The highest mean nitrogen
retention values occurred when dogs were given 11 g leucine/kg diet for both CP levels.
Broken-line analysis of body weight data revealed dogs given 140 g CP/kg diet required
11 g leucine/kg diet, while nitrogen retention data indicated that 11.0 and 10.5 g leucine/kg
diet were required when dogs were given 140 and 280 g CP/kg diet, respectively. Broken-
line analysis of body weight data from dogs given 280 g CP/kg diet that indicated a
requirement of 7.4 was most likely due to an anomalously high weight gain of dogs given
7 g leucine/kg diet that resulted in a shift of the computed break point to the left. Thus, the
leucine requirement of the growing dog was not affected by doubling the dietary CP level.
This is the ®rst published study to our knowledge in an omnivore where a positive
correlation was not found between dietary CP level and the EAA requirement. In the dog,
a positive correlation with lysine can be seen (MILNER 1981). When dogs were given a diet
with 140 g CP/kg diet they retained 0.88 g nitrogen/day with 4.61 g lysine/kg diet and
0.96 g nitrogen/day with 5.77 g lysine/kg diet. Dogs given a diet with 280 g CP/kg diet
retained 1.0 g nitrogen with 4.61 g lysine/kg diet, but retention increased to 1.5 g nitrogen/
day with 5.77 g lysine/kg diet (MILNER 1981).
Previous studies in other species that have found that increases in dietary CP increase the
EAA requirements did not examine leucine, most likely because it is not known to be the
limiting EAA in normal foodstuffs. Therefore, the lack of effect seen in this study may be
unique to leucine, rather than to the growing dog. Arginine, isoleucine, lysine, methionine,
threonine and tryptophan, the EAA where a positive correlation with dietary protein has
been found, are catabolized by enzymes that are induced by high protein diets. Leucine,
however, is rather unique in its metabolism. An excess of a-keto isocaproic acid (a-keto
acid of leucine) inhibits the phosphorylation (inactivation) of branched chain keto acid
dehydrogenase, which is the limiting step in the catabolism of leucine as well as isoleucine
and valine (HARRIS et al. 1995). Thus, plasma isoleucine and valine concentrations
decreased with increases in plasma leucine concentrations. Metabolites of other EAA are
not known to affect the catabolism of leucine, and they did not appear to in this leucine
requirement study. From this study, preventing the activation of branched chain
dehydrogenase (as indicated in vivo from the isoleucine and valine plasma concentrations)
appears to be completely dependent on leucine and independent of excesses of other EAA.
It appears that an amino acid imbalance did occur in the dogs given 280 g CP/kg diet with
5.0 g leucine/kg diet. These dogs had lower values for body weight gain, food intake,
nitrogen retention and plasma leucine concentration than dogs given 140 g CP/kg diet with
5.0 g leucine/kg diet. With EAA studies, using rats and chicks (HARPER et al. 1970 and
ROGERS et al. 1988), when amino acid imbalances occur, a positive correlation between
dietary CP and the EAA requirement is found. However, this did not happen with the
leucine requirement in this study.
The results of this study indicate that the leucine requirement of 8±14-week-old Beagle
dogs appears to be 11 g leucine/kg diet (0.61 mg leucine/kJ), considerably higher than the
6.5 g leucine/kg diet (0.38 mg leucine/kJ) previously reported (BURNS et al. 1984). In
reviewing this previous study, the results could be interpreted to support the requirement
being between 10 and 12 g leucine/kg diet. In the 1984 study, nitrogen retention
maximized at 10 g leucine/kg diet and weight gain maximized at 12 g leucine/kg diet in
dogs 10±12 weeks old. In the 1984 study, no statistical difference in nitrogen retention was
found in dogs given between 6.0 and 12 g leucine/kg diet, and no statistical difference was
found in this study with a similar range of leucine. Physiologically, a statistically signi®cant
difference in nitrogen retention may not be feasible with the number of dogs used with
such a narrow range of leucine levels. The 1984 study established the requirement using a
broad range of leucine levels which provided the basis for this current study to use a more
restricted range. It has been shown that the greater the restriction in the range of dietary
EAA concentrations, the less signi®cant differences become when using mathematical
models (RODEHUTSCORD and PACK 1999). However, the greater than 50% increase in mean
94 S. J. Delaney et al.
 Leucine requirement of growing dogs 95

b
Fig. 1. Food intake (a), weight gain (b) and nitrogen retention (c) of dogs given diets with ®ve levels
of dietary leucine and two levels of dietary crude protein. Each point represents means ‹ SEM for
six dogs except for the points at 9.0 g leucine/kg diet which represent 12 dogs. Different letters
represent signi®cant differences between dietary leucine levels, p £ 0.05. (m), 140 g CP/kg; (d), 280 g
CP/kg

Fig. 2. Concentrations of plasma isoleucine, leucine, lysine, phenylalanine and valine on day 11 in dogs
given diets with ®ve levels of dietary leucine and two levels of dietary crude protein: (a) 140 g CP/kg,
(b) 280 g CP/kg. Each point represents means ‹ SEM for six dogs except for the points at 9.0 g
leucine/kg diet which represent 12 dogs. Different letters represent signi®cant differences between
means for plasma leucine concentration, p £ 0.05. (m), Leucine; (h), isoleucine; (´), phenylalanine;
(j), valine; (d), lysine
 96 S. J. Delaney et al.

4,5 Table 3. Plasma amino acid concentrations (lmol/l)1

140 g crude protein/kg

g leucine/kg
Food-deprived
5.0 7.0 9.0 11 13 mean2

Alanine 747 805 629 484 330 340

SEM 157 154 92 63 58 51
Arginine 234 287 248 236 183 138
SEM 47 57 46 31 35 15
Asparagine 68 62 54 48 35 50
SEM 11 13 7 8 7 2
Aspartate 20 24 18 18 17 15
SEM 2 4 3 1 2 3
Citrulline 171 183 135 158 141 157
SEM 20 18 15 16 16 23
Cystine 36 43 36 51 50 63
SEM 8 6 6 10 8 8
Glutamate 101 123 113 84 106 112
SEM 26 34 19 18 23 70
Glutamine 559 629 510 555 494 517
SEM 80 71 44 38 61 109
Glycine 534 462 416 423 288 373
SEM 61 71 50 72 34 45
Histidine 65 97 81 80 76 42
SEM 12 16 11 6 11 7
Hydroxyproline 56 54 52 56 45 67
SEM 5 9 5 6 2 4
Isoleucine 113 162 97 79 58 38
SEM 29 31 17 12 11 3
Leucine 42 44 85 97 100 68
SEM 9 8 8 15 16 5
Lysine 517 577 505 454 348 212
SEM 81 127 93 63 77 39
Methionine 59 110 109 122 111 24
SEM 14 26 25 27 11 4
Ornithine 75 100 96 77 74 34
SEM 20 25 24 16 22 6
Phenylalanine 52 72 66 68 62 48
SEM 9 11 7 6 7 4
Proline 190 229 215 180 137 135
SEM 37 44 33 25 19 6
Serine 173 117 109 123 87 149
SEM 18 11 7 18 5 8
Taurine 150 139 140 138 132 146
SEM 13 10 14 18 17 21
Threonine 639 672 602 707 615 252
SEM 67 147 98 93 92 71
Tryptophan 51 89 74 81 89 44
SEM 11 22 10 7 17 8
Tyrosine 112 105 81 96 102 31
SEM 22 17 14 19 14 6
Valine 285 359 265 231 170 89
SEM 61 104 51 41 44 7
1
Samples collected on day 11 of each experimental period; n = 6/group except for the groups fed
9.0 g leucine/kg diet and that were food deprived which represent 12 dogs
2
Samples collected on day 14 of the last period of Experiment 1
 Leucine requirement of growing dogs 97

4,5 Table 4. Plasma amino acid concentrations (lmol/l)1

280 g crude protein/kg

g leucine/kg
Food-deprived
5.0 7.0 9.0 11 13 mean2

Alanine 1141 923 1057 810 603 358

SEM 202 140 135 86 89 49
Arginine 152 167 189 143 133 121
SEM 29 46 23 20 14 27
Asparagine 124 126 134 107 78 52
SEM 23 26 19 15 15 6
Aspartate 44 65 61 54 47 14
SEM 11 27 16 16 18 3
Citrulline 127 167 140 130 111 133
SEM 21 41 13 16 12 25
Cystine 36 36 46 41 38 55
SEM 7 10 8 11 10 4
Glutamate 240 368 315 345 347 97
SEM 62 160 79 104 129 59
Glutamine 726 667 695 598 451 493
SEM 51 38 63 68 95 85
Glycine 688 772 814 725 618 380
SEM 126 136 93 63 67 39
Histidine 89 99 97 96 82 49
SEM 14 15 10 9 11 10
Hydroxyproline 34 41 47 41 55 70
SEM 5 5 4 1 7 7
Isoleucine 302 241 250 193 95 42
SEM 69 48 46 25 22 5
Leucine 18 55 95 103 107 80
SEM 3 14 14 11 18 8
Lysine 374 395 423 330 307 213
SEM 60 86 50 35 37 41
Methionine 73 79 99 82 59 27
SEM 16 16 15 12 11 5
Ornithine 71 84 79 57 51 26
SEM 14 30 13 6 12 4
Phenylalanine 62 68 73 62 59 52
SEM 10 8 5 5 6 5
Proline 424 417 517 446 288 129
SEM 93 97 87 66 70 10
Serine 189 163 164 148 135 141
SEM 27 16 11 8 13 12
Taurine 151 165 180 159 191 156
SEM 12 12 18 13 31 10
Threonine 435 460 486 479 356 171
SEM 79 125 76 57 70 61
Tryptophan 83 81 86 90 71 43
SEM 19 17 10 5 6 8
Tyrosine 103 85 84 84 53 32
SEM 19 18 9 10 5 4
Valine 575 506 540 461 242 121
SEM 93 57 90 56 53 18
1
Samples collected on day 11 of each experimental period; n = 6/group except for the groups
fed 9.0 g leucine/kg diet and that were food deprived which represent 12 dogs
2
Samples collected on day 14 of the last period of Experiment 1
98 S. J. Delaney et al.

Table 5. Food intake, weight gain, nitrogen retention, plasma albumin and plasma branched chain
amino acids of 24 14-week-old dogs fed 210 g crude protein/kg diet and either 7 or 11 g leucine/kg
diet (Experiment 2)

210 g crude protein/kg

Measure 7 g leucine/kg 11 g leucine/kg Pooled SEM

Food intake, g/day 245 246 12

Weight gain, g/day 102 115 8
Nitrogen retention, g/7 days 11.5 11.8 1.2
Plasma albumin, g/l 21.6 22.1 0.09
Plasma leucine, lmol/l 59.5 97.7* 8.0
Plasma isoleucine, lmol/l 303 124* 22
Plasma valine, lmol/l 640 352* 48
Each point represents means ‹ SEM for 12 dogs. Asterisks represent signi®cant differences
between means for the given variable, p £ 0.05

nitrogen retention that was seen when dogs were given leucine at 11 g/kg diet instead of
9.0 g/kg diet is nutritionally important.
Results from Experiment 2 of this current study appear to support the lower
requirement proposed by the 1984 study. However, it appears that age began to play a
role in diminishing the EAA requirement as has been seen in dogs previously (CZARNECKI
and BAKER 1982). While no age effect was seen in Experiment 1 when the dogs were
8±14 weeks old, a sudden increase in food intake by all dogs (from a mean of about 170 g/day
to a mean of about 240 g/day) during the last 3 days of Experiment 1 was observed and
continued into Experiment 2. The timing of this apparent decrease in EAA requirement
was very similar to the timing seen by CZARNECKI and BAKER (1982). In that study, the
requirement for tryptophan in growing English Pointer dogs decreased 25% when the dogs
were 12±14 weeks old as compared to when they were 10±12 weeks old. Therefore, the
lack of a difference between the two groups given either 7.0 or 11 g leucine/kg diet appears
to be the result of the dogs being 14±16 weeks old. It should be noted that, in Experiment
2, a higher plasma leucine concentration and lower plasma isoleucine and valine
concentrations were seen in dogs given 11 g leucine/kg diet as compared to the dogs
given 7 g leucine/kg diet. This indicates that the dietary leucine required to normalize
branched chain amino acid metabolism was not being met with 7.0 g leucine/kg diet.
Plasma albumin concentrations were not affected in this study, most likely because
2-week periods were too short to allow for suf®cient depletion of plasma albumin which
has a half-life of 8 days in the dog. The low apparent nitrogen and dry matter digestibilities
probably are due to the starch in the diet apparently not being heat-processed long enough
to maximize starch digestion. Incompletely cooked powdered cornstarch is known to cause
loose stools in young dogs (CZARNECKI and BAKER 1982), and caused loose stools in the
dogs of this study. It has been reported that substantial carbohydrate fermentation in the
lower gut results in a shift of nitrogen from urine to faeces that does not affect nitrogen
retention (DE SCHRIJVER et al. 1999). However, the shift of nitrogen from urine to faeces
does lower the apparent nitrogen digestibility.
The elevated plasma glutamate levels seen in the dogs given 47 g glutamate/kg diet or
2.6 mg glutamate/kJ (amount contained in the diet with 280 g CP/kg diet) were greater
than 10 times the normal values seen in normal growing and adult dogs given commercial
diets (STROMBECK and ROGERS 1978; BLAZER-YOST and JEZYK 1979; HANSEN et al. 1992),
yet no apparent adverse effects were seen in any of the dogs. These extremely high levels
have not been reported before and have not been seen in this laboratory before. However,
these are the ®rst complete plasma amino acid concentrations reported in growing dogs
 Leucine requirement of growing dogs 99

given puri®ed amino acid diets. Only plasma phenylalanine and tyrosine concentrations
have been reported previously (MILNER et al. 1984).
The leucine requirement in many species has been found to be similar to that found in
this study. The growing chicken requires 12 g leucine/kg diet (MORI and OKUMURA 1984),
the pig requires 12.5 g leucine/kg diet early in life (EGGERT et al. 1954), the growing rat
requires 10.7 g leucine/kg diet (BENEVENGA et al. 1994), and the kitten requires between
10.6 and 12 g leucine/kg diet (ANDERSON et al. 1980; HARGROVE et al. 1984).
The protein requirement of the dog has been determined to be between 150 and 200 g
CP/kg diet when puri®ed protein diets are given (HEIMAN 1947; GESSERT and PHILLIPS
1956; BURNS et al. 1982), and between 230 and 275 g CP/kg diet when mixed-protein diets
are given (CASE and CZARNECKI-MAULDEN 1990). In this study, 140 g CP/kg diet appeared
to support near maximal growth (not signi®cantly different). Nitrogen retention, however,
was greater for the group receiving 280 g CP/kg diet than it was for the group receiving
140 g CP/kg diet. Therefore, it would appear that the CP requirement of young growing
dogs is greater than 140 g/kg diet when protein-free amino acid diets are given.
Interestingly, the leucine requirement does not appear to be affected by the low CP level
of the diet with 140 g CP/kg diet. Previously, most of the EAA requirements of dogs were
determined with amino acid diets containing 140±150 g CP/kg diet (NRC 1985), raising
the possibility that dietary nitrogen could have been limiting which may have resulted in
some of the EAA requirements being underestimated.
In conclusion, results from this study indicate that the leucine requirement for growing
dogs prior to 14 weeks of age is 11 g/kg diet when dietary crude protein is either 140 g/kg
diet or 280 g/kg diet, whereas the leucine requirement appears to be met with 7 g/kg diet
for Beagle dogs over 14 weeks of age. Further work needs to be performed with leucine in
other species, and with tryptophan, lysine and threonine in the dog, to ascertain whether
the dog is unique or whether the unique metabolism of leucine causes the lack of positive
correlation between dietary CP and the leucine requirement.

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Authors' address: Q. R. ROGERS, VM-Molecular Biosciences, University of California Davis, Davis,

CA 95616, USA. tel.: +1 530 752 3086; fax: +1 530 752 4698; e-mail: qrrogers@ucd-
avis.edu