NITROGEN METABOLISM OF G R A V I D GILTS FED PURIFIED DIETS
DEFICIENT IN EITHER LEUClNE OR TRYPTOPHAN
Robert A. Easter I and David H. Baker
University o f Illinois, Urbana 618012
SUMMARY
INTRODUCTION
Three nitrogen metabolism assays using
first-litter, crossbred gilts averaging 80 days
postcoitum were conducted to evaluate the
quantitative leucine and tryptophan requirements of the gravid gilt. Each assay consisted of
three dietary treatments fed at 2.0 kg per day
in three consecutive 9-day periods (4-day pretest, 5-day quantitative urine collection). Completely purified diets (12.8% protein equivalent) were formulated with crystalline L-amino
acids included at the known minimum levels
needed for pregnancy in swine. Nitrogen for
dispensable amino acid biosynthesis was provided by a mixture of glutamic acid and
glycine. In assay 1, reduction of leucine from
.58 to.34% of the diet did not depress nitrogen
retention, however, retention was depressed in
a subsequent assay when levels less than .34%
were fed. In assay 3, complete deletion of
tryptophan resulted in absolute diet refusal
within 4 days in two of three gilts observed.
Nitrogen retention was reduced by feeding
.03% tryptophan compared with .07%, the
N.R.C. requirement. Negative nitrogen balance
( - 1 . 6 g/day) was obtained for the single gilt
consuming a tryptophan-free diet. Plasma urea
nitrogen tended to be elevated when deficient
levels of tryptophan were fed. Thus, feeding
less than .34% available leucine (6.8 g/day) or
.07% available tryptophan (1.4 g/day) to gravid
gilts during the last one-third of gestation
appears unwarranted at this time.
(Key Words: Tryptophan, Leucine, Pregnancy,
Swine, Amino Acids.)
In a series of nitrogen balance assays, Rippel
et al. (1965) defined the quantitative isoleucine, lysine, methionine, cystine, tyrosine, threonine and valine requirements of gravid gilts.
Because of the pattern of amino acids contained in the intact protein used in their diet,
minimum requirements could not be established for arginine, histidine, leucine, tryptophan or phenylalanine. However, the minimum
concentrations of these amino acids present in
their diet have been adopted as requirements
(N.R.C., 1973) for pregnancy, although the
actual needs are unknown. We have recently
demonstrated that the gravid gilt requires a
dietary histidine concentration of .12% (Easter
et al., 1974a), while arginine is clearly dispensable (Easter et al., 1974b; Easter and Baker,
1976) for pregnancy in swine.
It appeared desirable to also evaluate the
leucine and tryptophan needs of gravid swine.
Both of these amino acids are considered
indispensable for growth in nonruminant species, including the human (Nakagawa et aL,
1960), pig (Mertz et al., 1952) and rat (Rose et
al.,, 1948). Moreover, these species also require
both tryptophan and leucine for adult maintenance (Rose et al., 1954, 1955; Baker and
Allee, 1970; Wissler et al., 1948). In view of
this evidence the gravid, nonruminant mammal
would be expected to require a dietary source
of these amino acids, although, we are unaware
of data to support this contention.
Nitrogen metabolism assays were conducted
in the studies described herein in order to
determine the qualitative and quantitative need
of gravid firstqitter gilts for leucine and tryptophan.
Part of a thesis submitted to the Graduate C o l l e g e
EXPERIMENTAL
PROCEDURE
of the University of Illinois in partial fulfillment of
The nitrogen metabolism assays, employing
the requirements for the Ph.D. degree in Animal
Science.
first-litter crossbred gilts, were initiated when
2Animal Science Department.
the gilts averaged 80 days postcoitum and had
417
JOURNAL OF ANIMAL SCIENCE, Vol. 4.4, No. 3,1977
418
EASTER A N D B A K E R
been shown gravid by the procedure o f Lindahl
et al. (1972). The three diets used in each assay
were fed to individual gilts in three consecutive
9-day nitrogen balance periods. Each balance
period consisted of a 4-day pretest (dietary
adaptation period) followed by 5 days of
quantitative urine collection. Early work in
man demonstrated that the effect of either
leucine or t r y p t o p h a n deprivation on urinary
nitrogen excretion achieves stability within 4 to
5 days o f the initial feeding (Rose et al., 1951,
1954). Thus the 4-day adaptation used herein
was considered adequate. A single 10-day composite fecal collection was made during each
assay and the average daily fecal nitrogen used
in calculating nitrogen balance. Procedures for
excreta collection, storage and nitrogen analysis
have been described previofisly (Easter et al.,
1974b).
The basal diet used herein was identical to
that used earlier (Easter and Baker, 1976)
except that both leucine and tryptophan were
deleted. The diet contained crystalline L-amino
acids patterned after the known requirements
for the gravid gilt (Rippel et al., 1965). Glutamic acid, an efficacious source of nitrogen for
dispensable amino acid synthesis (Rose et al.,
1949; Rippel et al., 1965) and glycine were
added to bring total dietary nitrogen to a level
of 2.05% (12.8% protein equivalent). Vitamin
and mineral requirements were met by the
addition of purified supplements. Additions of
leucine in assays 1 and 2 and t r y p t o p h a n in
assay 3 were made at the expense of cornstarch.
Glutamic acid levels were varied as needed to
keep the diets isonitrogenous.
Three levels of dietary leucine were assessed
in each of the first two assays using three
littermate gilts in a Latin square arrangement.
Following each 27-day assay, blood samples
were obtained from all gilts by vena cava
puncture.
The effect o f either 0, .03 or .07% dietary
t r y p t o p h a n on nitrogen metabolism was studied
in assay 3, Preliminary observations in our
laboratory had suggested that voluntary feed
consumption would be very sensitive to deficient levels of tryptophan. Therefore, with the
expectation of possible unequal numbers of
gilts successfully completing the assay, three
littermate sisters were subjected to each of the
three diets in a randomized complete block.
F o l l o w i n g the assay, two gilts were fed each
diet on three consecutive days and blood
samples were obtained 3 hr after the morning
meal.
Duplicate 20/A aliquots of blood were taken
immediately after exsanguination for hemoglobin determination (Crosby et al., 1954) and the
remainder was heparinized and centrifuged at
3,000 X g for 10 minutes. The resulting plasma
was analyzed for blood urea nitrogen by the
method of Fawcett and Scott (1960).
Balance and blood data were subjected to
appropriate analysis of variance procedures
(Steel and Torrie, 1960). Single degree-of-freedom comparisons and pooled standard errors
were calculated where appropriate.
RESULTS
Leucine. Reduction o f dietary leucine from
.58 to .34% (table 1) did not affect (P>.10)
nitrogen retention. However, when crystalline
leucine was reduced from .26 to .18%, retained
nitrogen declined (P<.10) from 10.1 to 7.3 g
per day. The difference between .26 and .34%
was n o t statistically significant (P>.10). Retention, however, was consistently improved by
feeding the latter level. Individual observations
of plasma urea nitrogen and hemoglobin (table
2) were within normal ranges.
T r y p t o p b a n . Observations were successfully
made for only two o f three gilts used in assay 3,
TABLE 1. NITROGEN METABOLISM BY
GRAVID GILTS FED GRADED
LEVELS OF DIETARY LEUCINE
Leucine, %
Mean daily nitrogen, ga
Urinary
Fecal
Retained
.58
.46
.34
26.9
25.8
26.9
Assay 1b
1.7
1.7
1.7
12.5
13.6 c
12.5
Assay 2 d
.34
.26
.18
27.8
28.3
31.1
2.7
2.7
2.7
10.6
10.1
7.3
aDaily nitrogen intake was 41.0 grams. Each mean
represents three observations.
bpooled standard error of N retention for Assay 1
was .27.
CA missing value was calculated (Steel and Torrie,
1960).
dpooled standard error of N retention for Assay 2
was .22.
LEUCINE AND TRYPTOPHAN FOR GRAVID GILTS
TABLE 2. HEMOGLOBIN AND PLASMA
UREA-NITROGEN CONCENTRATIONSOF
GRAVID GILTS FED GRADED LEVELS
OF LEUCINEa
Leucine, %
Plasma
urea-nitrogen
mg/100 ml
.58
.46
.34
9.6
10.5
10.7
.34
8.4
12.0
.26
8.6
11.8
.18
8.0
10.8
Hemoglobin
g/lO0 ml
Assay 1
12.7
12.3
12.9
Assay 2
asingle observations made for each treatment at
the conclusion of each assay.
and of these, only one consumed the tryptophan-free diet for a sufficient duration to
obtain acceptable data. Feeding either 0 or
.03% tryptophan resulted in reduced (P<.05)
nitrogen retention relative to .07% tryptophan
(table 3). In fact negative nitrogen balance was
achieved in the gilt receiving the diet devoid of
tryptophan. The tryptophan-free diet was extremely anorexigenic, resulting in absolute diet
refusal within 4 to 5 days of initial consumption in two of the three gilts. Complete
restoration of appetite was achieved only after
feeding the corn-soybean meal pre-trial diet for
several days. In view of this it was somewhat
surprising that one gilt continued to eat the
419
tryptophan-free diet for the 9-day collection
period. Plasma urea nitrogen (table 4) tended to
be greater, although not significantly (P>.10),
for the two lower levels of tryptophan. An
amino acid deficiency typically results in an
elevated level of urea in blood (Brown and
Cline, 1974).
Discussion
As anticipated, leucine was f o u n d to be an
indispensable amino acid for pregnancy in
swine. The requirement would appear to be no
greater than .34% and no less than .26%.
Although we were unable to detect a significant
difference between these two levels, the consistent reduction in nitrogen retention when feeding the .26% leucine diet is justification for
selecting . 34% as a requirement until additional
evidence to the contrary can be obtained. In
swine there does not appear to exist the 1 to
threefold differences in individual leucine requirements f o u n d in adult man (Rose et al.,
1955), woman (Leverton et aL, 1956) and the
human infant (Snyderman et al., 1961).
Both nitrogen balance and blood urea nitrogen data strongly support the contention that
.03% L-tryptophan or less is inadequate for
gravid gilts. This finding is in agreement with
the work of Alice and Baker (1970) who
showed that a lysine-fortified corn diet containing .05% total tryptophan would n o t support
optimal nitrogen retention when fed to gravid
gilts during late gestation. If the biological
availability of corn tryptophan is only 60 to
70%, then the actual available tryptophan in
TABLE 3. NITROGEN METABOLISM OF GRAVID GILTS FED
THREE LEVELS OF TRYPTOPHAN (ASSAY 3)
Dietary
tryptophan, %
Gilt
no.
Urine
.
0
1
2b
40.5
.
.
.03
1
2
33.4
31.0
2.1
3.1
5.5
6.9
.07
1
2
29.3
27.9
2.1
3.1
9.6
10.0
.
9
Mean daily nitrogen, ga
Feces
.
.
2.1
.
Retained
-1.6
.
.
aDaily nitrogen intake was 41.0 grams.
bDue to anorexia, no observation was made for this gilt when fed the tryptophan-free diet.
.
420
EASTER AND BAKER
TABLE 4. PLASMA UREA-NITROGEN OF
GRAVID GILTS FOLLOWING CONSUMPTION
OF INDIVIDUAL MEALS CONTAINING
DIFFERENT TRYPTOPHAN LEVELS
(ASSAY 3)
Dietary
tryptophan,
%
Gilt 1
Gilt 2
Mean a
0
.03
.07
12.2
11.3
7.8
7.7
8.4
6.8
9.9
9.8
7.3
apooled standard error was .87.
the Alice and Baker (1970) diet was very
similar to the .03% c o n t a i n e d in o u r diet. In
their report, the a d d i t i o n o f .04% t r y p t o p h a n
to the diet c o m p l e t e l y restored nitrogen balance. Thus, feeding less than .07% available
t r y p t o p h a n to gravid gilts seems to be unjustified at this point.
The anorexia associated with the t r y p t o phan-free diet was particularly striking in t h a t
diet refusal, with only one e x c e p t i o n , was
c o m p l e t e and w i t h o u t remission. The a b n o r m a l
pattern of plasma a m i n o acids associated with
the c o n s u m p t i o n o f a diet devoid o f an indispensable a m i n o acid is t h o u g h t to be a m a j o r
factor causing reduced feed intake (Munro and
Allison, 1964). In addition, the plasma concentration of t r y p t o p h a n directly m o d u l a t e s the
brain level o f serotonin, a m o n a m i n e neurotransmitter p o s t u l a t e d to be involved in behavioral control and m e n t a l depression ( F e r n s t r o m
and Wurtman, 1971). Moreover, t r y p t o p h a n
deficiency has been f o u n d to u n i q u e l y cause
rapid (1 to 3 hr) p o l y s o m e disaggregation
(Wunner e t al., 1966; Staehelin e t al., 1967), a
physiological lesion impairing protein biosynthesis. Thus, in a g r e e m e n t with Harper e t al.
(1970) the animal system seems to have elected
to avoid the i m m e d i a t e physiological consequences of ingesting the t r y p t o p h a n - d e v o i d diet
by fasting.
The findings o f this study suggest that gravid
gilts fed 2.0 kg per day require no m o r e than
.34% leucine for m a i n t e n a n c e of acceptable (12
g / d a y ) nitrogen r e t e n t i o n . The .03% t r y p t o p h a n
level was clearly inadequate. It should be n o t e d
that all amino acids in this diet can be
considered c o m p l e t e l y available and thus the
requirements are n o t c o n f o u n d e d by this largely u n k n o w n factor. Conversely, e x t r a p o l a t i o n
o f the r e q u i r e m e n t s d e t e r m i n e d herein to use in
diets b a s e d on intact proteins m u s t include
adjustments for the biological availability of the
specific a m i n o acid in the particular f e e d s t u f f
being used.
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