Technical Bulletin 678
6 ^ June 1964
c of Protein
e on Gains,
of Beef Heifers
E. Bedrak, A. C. Warnick, J. F. Hentges, Jr.
and T. J. Cunha
Agricultural Experiment Stations
University of Florida, Gainesville
ASE J. R. Beckenbach, Director
INTRODUCTION ....... ...... ....---....- ........- ....- ..... ....- ....-- ..... 3
MATERIALS AND METHODS ......--....---- ..------------.... ....... .... ........ 3
RESULTS AND DISCUSSION ...-...-..-.........----- .....-...------.....-...-- ... 7
Feed Intake and Gains ....................-.............- --- --------.. 7
Reproduction ....-..-....----------- .. .--------- ---...-----------.... 12
Blood Constituents and Liver Protein ....................---- .....----- ...... 19
SUMMARY ----.........---------.......- --...- -..... -....- .................---. 27
LITERATURE CITED ..--..---...-- ...... ....-- ..............---- ..................... .. 28
Effect of Protein Intake on Gains,
Reproduction, and Blood Constituents
of Beef Heifers'
E. BEDRAK, A. C. WARNICK, J. F. HENTGES, JR., AND
T. J. CUNHA2
An emaciated condition and low reproduction in beef cattle
grazing Florida pastures low in protein content during November
to March was observed by Bedrak (2)3 and Warnick et al. (34).
A supplement of cottonseed meal improved the rate of gain
and reproduction, suggesting that a protein deficiency was an
important cause of the poor performance. Since this condi-
tion was similar to that seen on many cattle ranches during
the winter or periods of drought, it seemed important to study
the performance of beef heifers fed varying levels of protein
under these conditions in comparison with the optimum amounts
recommended by the National Research Council (14) and Mor-
rison (27). The purpose of the experiment was to investigate
the effect of varying levels of protein intake (with adequate,
energy, minerals, and vitamins) on feed intake, gains, reproduc-
tion, and blood changes in beef heifers. Several blood consti-
tuents were investigated in an attempt to find some that could
be used for the detection of a protein deficiency in cattle.
MATERIALS AND METHODS
Twenty yearling grade Angus, Hereford, and Angus-Hereford
crossbred heifers averaging 496 pounds in weight and 15 months
in age were divided into four groups and individually fed various
levels of protein with equivalent amounts of total digestible
nutrients (TDN), vitamins, and minerals from March 3 to Sep-
tember 18, 1956. Likewise, in a second trial 20 two-year-old
Hereford heifers (average initial age of 26 months and weight
669 pounds) were divided into four groups and individually
SSupported in part by National Cottonseed Products Association, Inc.,
2 Bedrak, former Research Assistant; present address, Institute for Arid
Zone Research, Beersheva, Israel. Warnick, Animal Physiologist. Hentges,
Associate Animal Nutritionist. Cunha, Animal Nutritionist and Head, Ani-
mal Science Department.
Numbers in parentheses refer to Literature Cited.
4 Florida Agricultural Experiment Stations
fed from February 16 to September 13, 1957. The rations sup-
plied different levels of protein, with similar amounts of TDN,
vitamins, and minerals estimated to be adequate for a daily
gain of 0.75 to 1.00 pound. The rations offered are shown in
TABLE 1.-RATIONS OFFERED DAILY TO YEARLING AND
Yearling Heifers Two-year-old Heifers
No. Ingredients Feed C.P.* TDN Feed C.P.* TDN
Ib. lb. lb. Ib. lb. lb.
I Pangolagrass hay 8.0** 0.19 4.0 9.0: 0.26 4.5
Cottonseed meal, 41% 2.3 0.92 1.7 2.0 0.81 1.5
Carbohydrate 2.8t 2.3 3.5$t 0.27 2.3
Mineral-vitamin mix 0.5tt 0.02 0.1 1.0 0.06 0.5
Total 13.6 1.13 8.1 15.5 1.40 8.8
II Pangolagrass hay 8.0 0.19 4.0 9.0 0.26 4.5
Cottonseed meal, 41% 1.3 0.52 0.9 1.1 0.45 0.8
Carbohydrate 3.6 3.1 4.5 0.35 3.0
Mineral-vitamin mix 0.5 0.02 0.1 1.0 0.06 0.5
Total 13.4 0.73 8.1 15.6 1.12 8.8
III Pangolagrass hay 8.0 0.19 4.0 9.0 0.26 4.5
Cottonseed meal, 41% 0.5 0.20 0.4 0.4 0.16 0.3
Carbohydrate 4.3 3.7 5.3 0.41 3.5
Mineral-vitamin mix 0.5 0.02 0.1 1.0 0.06 0.5
Total 13.3 0.41 8.2 15.7 0.89 8.8
IV Pangolagrass hay 8.0 0.19 4.0 9.0 0.26 4.5
Carbohydrate 4.7 4.0 5.8 0.44 3.8
Mineral-vitamin mix 0.5 0.02 0.1 1.0 0.06 0.5
Total 13.2 0.21 8.1 15.8 0.76 8.8
C.P. = Crude protein.
** Hay contained 2.35% crude protein.
t Grade "C" cane sugar.
tt Mixture included vitamin A, 15,000 I.U.; vitamin D, 2,000 I.U.; vitamin E, 22.03
I.U.; trace mineralized salt, 59.02 gm (trace mineralized salt contained: NaC1, 98.800%; Mn,
.250%; Fe, .270%; Cu, .033%; Co, .010%; I, .007%; and Zn, .005%); sterilized bonemeal
(analyzed 6.27% crude protein), 99.02 gm; and citrus meal, (analyzed 6.24% crude protein),
67.64 gm. Vitamins donated by Dawe's Laboratories, Inc., Chicago, Illinois.
SHay contained 2.84% crude protein.
ft Cane molasses from cane grown on muck soil contained 7.72% crude protein.
Mixture included vitamin A, 22,000 I.U.; vitamin D, 2,000 I.U.; vitamin E, 22.03 I.U.;
trace mineralized salt, 59.02 gm; sterilized bonemeal, 99.02 gm; and citrus meal, (analyzed
6.72% crude protein), 294.64 gm.
Effect of Protein Intake on Beef Heifers 5
Prior to the experiments, these heifers were on feeding
trials where they were offered adequate levels of protein and
TDN over periods of 90 to 120 days and checked for estrus
twice daily. All the heifers were drenched with phenothiazine
and were fed comparable rations for a period of two weeks im-
mediately prior to allotment. They were allotted to their re-
spective groups according to their previous treatment, weight,
and estrual behavior.
The animals were individually fed the concentrates and hay
at 5 p.m. and remained in the barn during the night with access
to water. During the day they were turned out to drylot, where
they had access to fresh water, trace mineralized salt, and bone-
meal, as well as adequate shade. Feed not consumed was
weighed back, and feed consumption data were summarized at
regular intervals. All heifers were weighed initially under uni-
form conditions and every 28 days thereafter, with the final
weight taken approximately one hour before slaughter. The total
time the experimental rations were fed varied from 159 to 207
days depending on reproduction performance.
During the pre-breeding period (first 112 days) the heifers
were checked for estrus three times a day at 6 a.m., 12 noon,
and 6 p.m., using vasectomized bulls. The genital organs of all
heifers were palpated at the start of the experiment and every
14 days thereafter. During the breeding period the yearling
heifers were checked for estrus every 6 hours; whereas, the
two-year-old heifers were observed regularly three times during
the day with a fourth observation at midnight starting two to
three nights prior to the expected date of estrus. The heifers
were recorded as being in estrus when they stood for the bull,
rode the bull, or had a vaginal mucous secretion.
During the breeding period, which began on the 113th day
of the experiment, all heifers were bred at their next estrus to a
fertile bull or were inseminated with freshly collected semen.
Two fertile bulls were used to breed the yearling heifers, while
three fertile bulls were used for breeding the two-year-old
heifers. Matings were made so that bulls were distributed sim-
ilarly within each group. The number of services per bull
within each dietary group was fairly uniform. Heifers which
did not return in estrus were slaughtered 44 days following
breeding. Those which returned in estrus following the first
mating were rebred a second time, and slaughtered after 44
days if they did not return in heat. Heifers bred at the third
6 Florida Agricultural Experiment Stations
estrous period were killed three days following the last breed-
ing. Heifers which did not show estrus were slaughtered at
random times with the pregnant animals.
At slaughter the reproductive organs were collected for fur-
ther observation of fertilization, embryonic development, and
possible abnormalities. Sections were made and stained using
hematoxylin and eosin stains as described by Kolmer et al. (24).
Pituitary glands were obtained and stored immediately in tightly
stoppered glass vials at a temperature of 0 to 60F. Gonado-
trophin studies were made using day-old White Leghorn male
chicks with a slightly modified procedure of Robinson and Nal-
bandov (32). A section of the non-pregnant uterine horn was
stored in a refrigerator at 0 to 60 F and analyzed for xanthine
oxidase activity by the method described by Axelrod and Elveh-
jem (1) and Dhungat and Sreenivasam (7).
Blood samples were drawn initially when heifers were put
on the experimental ration and every 28 days thereafter, as well
as immediately prior to slaughter. The criteria studied with
yearling heifers were: hemoglobin, by the acid-hematin method
as described by Cohen and Smith (5); packed erythrocyte vol-
ume (hematocrit) as described by Kolmer et al. (24); creatinine
as described by Folin and Wu (11); serum protein, albumin, and
globulin, using the biuret reagent of Gornall et al. (13); electro-
phoretic fractionation of blood serum as described by Durrum
and Gilford (9); inorganic calcium in blood plasma, determined
by a modification of the technique of Cheng and Gray (4); and
inorganic phosphorus in blood plasma by the method of Fiske
and Subbarow (10). The criteria investigated with the two-
year-old heifers were: hemoglobin, hematocrit, blood volume
(using the Evan's Blue T-1824 azo dye), erythrocytes, leuko-
cytes, and differential counts, all as described by Kolmer et al.
(24). Serum protein, albumin and globulin, inorganic calcium,
and phosphorus of blood plasma were also determined. Liver
samples were taken at slaughter from heifers for protein deter-
mination. Rumen filtrates from the two-year-old heifers were
taken at slaughter and were used for the study of in vitro cel-
lulose digestion. Cellulose was determined according to a modi-
fication of the method of Crampton and Maynard (6).
Effect of Protein Intake on Beef Heifers 7
RESULTS AND DISCUSSION
Feed Intake and Gains
Feed Intake.-The average daily intakes of TDN per 100
pounds body weight for the two trials are summarized in Table 2,
and the statistical analyses are shown in Table 3. The multiple
range test proposed by Duncan (8) was used to determine the dif-
ferences among the groups and periods. The TDN intake per 100
pounds body weight of heifers in groups I, II, and III was fairly
similar during the entire experiment, while the amount of TDN
consumed by group IV was 10 to 17 percent less (P<.01) after
the first period. Apparently this was the only group where the
protein deficiency was severe enough to affect appetite signifi-
cantly. The daily intake of TDN for the yearling heifers was
comparable for all groups during the first 28-day period. During
the first 28-day period the consumption of TDN by all groups
was higher (P<.01) than that observed during the other experi-
mental periods. In the two-year-old heifers, groups I, II, and
IV consumed more (P<.01) TDN per 100 pounds of body weight
than group III. During the first 28-day period, the TDN intake
varied among groups, but there was no significant difference due
to period. In comparing the results of the yearlings and two-
year-olds, it should be noted that the energy sources were dif-
ferent and that lower levels of protein were used with the
TABLE 2.-EFFECT OF PROTEIN INTAKE ON TDN CONSUMPTION PER 100
POUNDS OF BODY WEIGHT OF BEEF HEIFERS BY FEEDING PERIODS.
Crude Protein TDN Intake per
Group Daily Intake 28-day Feeding Period
C.P.* C.P.** 1 2 3 4 5 6
lb. lb. lb. lb. lb. lb. lb. lb. lb.
I 1.06 .14 1.18 1.13 1.11 1.11 0.98 1.02 1.09
II 0.65 .09 1.20 1.16 1.23 1.19 1.12 1.08 1.16
[II 0.28 .04 1.11 1.03 1.05 1.10 1.09 1.05 1.07
IV 0.08 .01 1.14 0.93 0.93 0.99 0.88 0.92 0.96
I 1.34 .13 1.24 1.19 1.08 1.12 1.07 1.03 1.12
II 1.06 .10 1.14 1.15 1.08 1.13 1.09 1.07 1.11
III 0.71 .07 0.95 1.06 1.03 1.03 0.98 1.05 1.02
[V 0.62 .06 0.99 1.14 1.07 1.12 1.05 1.11 1.08
Pounds per animal.
** Pounds per 100 pounds body weight.
8 Florida Agricultural Experiment Stations
yearling heifers. Thus, a less severe protein deficiency was
produced with the two-year-old heifers.
TABLE 3.-ANALYSIS OF VARIANCE OF TDN INTAKE AND DAILY GAIN IN
BEEF HEIFERS ON VARIOUS LEVELS OF PROTEIN INTAKE.
Source of TDN intake per Daily gain
Variance d. f. 100 lb. weight
Sub-class 23 0.041 2,061.36
Group 3 0.172** 13,148.63**
Period 5 0.052** 570.99*
G X P 15 0.011 340.70
Error 96 0.020 195.18
Sub-class 19 0.026 2,525.53
Group 3 0.085** 9,607.63**
Period 4 0.022 2,644.52**
G X P 12 0.013 715.34
Error 80 0.009 434.27
Body Weight Changes.-Average daily weight changes for
both trials are summarized in Table 4, and the statistical anal-
yses are shown in Table 3. In both trials a direct relationship
was observed between level of protein intake and gain. The
overall average daily gains for the yearlings heifers were 0.94,
0.70, 0.01, and 0.72 pound per head for groups I through IV, re-
spectively. Groups I and II made greater (P<.01) gains than
groups III and IV. While there was no statistically significant
difference between the gains made by groups I and II, there was
a highly significant difference between the gains of groups III
and IV. The gains made during the second and third 28-day
periods were larger (P<.05) than those made in the first, fourth,
and fifth periods.
The general appearance and thriftiness of yearling heifers
in groups III and IV decreased sharply between 56 and 84 days
on the experiment, and thereafter they appeared to lack vitality
and were slow in their movements. They developed rough hair
coats and generally appeared emaciated (Figure 1). Some ani-
mals lost considerable body weight and showed signs of hyper-
irritability and intermittent muscular twitching.
TABLE 4.-EFFECT OF PROTEIN INTAKE ON AVERAGE DAILY GAIN OF
BEEF HEIFERS BY FEEDING PERIODS.
Protein Av. Gain during Each Gain during Av. Av. Daily Z
Group Intake Initial 28-day Feeding Period Final Final Gain
No. Wt. Period Wt. Throughout
C.P.* 1 2 3 4 5 Experiment z
lb. lb. lb. lb. lb. lb. lb. lb. lb. lb.
I 1.06 491 1.49 1.16 1.13 0.61 0.45 0.70 649 0.94
II 0.65 498 0.66 0.84 1.17 0.56 0.71 0.39 625 0.70
III 0.28 496 -.56 0.39 0.23 -.06 -.13 0.26 519 0.01
IV 0.08 499 -1.26 -.66 -.52 -.52 -.60 -.73 383 -.72
I 1.34 654 1.05 1.37 0.20 1.29 0.55 0.09 793 0.75
II 1.06 660 1.01 1.03 0.17 0.19 1.09 -.35 750 0.52
III 0.71 686 -.44 0.00 -.18 0.49 0.19 -.70 674 -.07
IV 0.62 679 -1.23 -.06 -.05 0.01 0.00 -.24 640 -.23
10 Florida Agricultural Experiment Stations
" 1c 1 A.
Figure 1.-Effect of level of protein intake on appearance of yearling heifers.
Group I heifers had a daily intake of 1.06 pounds crude protein; II, 0.65
pounds C.P.; III, 0.28 pounds C.P.; and IV, 0.08 pounds C.P.
The overall average daily gains of two-year-old heifers for
the entire experiment were 0.75, 0.52, -0.7 and -0.23 pound for
groups I through IV, respectively. Gains made by groups I
and II were higher (P<.01) than those made by groups III and
IV. The differences in weight gains between groups I and II
as well as between III and IV were not statistically significant.
The gains made during the third 28-day period were less
(P<.01) than those made during the second, fourth, and fifth
periods. The poor gains exhibited by all groups of two-year-old
heifers during the final period probably resulted from the ex-
cessive handling of the heifers during the blood volume deter-
minations; therefore, these were not included in the statistical
Heifers consuming low levels of protein in both experi-
ments ate more trace mineralized and plain salt, which were
available free-choice as well as in the ration, while bonemeal con-
sumption was comparable for all groups.
A decrease in feed intake observed in yearling heifers on the
lower protein rations is in agreement with the work by Guilbert
and Hart (16). Feed intake was decreased significantly only in
yearling heifers in group IV, where the lowest level of protein
(0.08 pound C.P.) was fed. Yearling heifers in the other three
groups had similar TDN intakes per 100 pounds body weight.
There was a decrease in average daily gain of 0.24 pound as the
Effect of Protein Intake on Beef Heifers 11
intake of crude protein decreased 0.41 pound per day from group
I to group II. However, even though the daily intake of TDN per
100 pounds body weight remained somewhat similar, there was
a decrease in daily gain of 0.69 pound as crude protein intake
decreased another 0.37 pound from 0.65 to 0.28. The differences
in gain between heifers of groups II and III would indicate that
there is a very marked influence of protein intake on gains as
intake approaches a level which only maintains body weight.
The TDN intake per 100 pounds body weight for the two-
year-old heifers was comparable for all groups except III, where
it was significantly decreased. However, the daily gain for
the heifers on the two higher intakes of protein was significantly
higher than heifers on the two lower levels of protein. As the
daily crude protein intake decreased 0.28 pound from 1.34 to 1.06,
there was a decrease in gain of 0.23 pound per day. However,
as the daily intake of crude protein was reduced 0.35 pound from
1.06 to 0.71 pound, there was a decrease in gain of 0.59 pound
per day. Thus, protein intake was again shown to have marked
effect on feed utilization when the ration contained only enough
protein for maintenance of body weight. This is similar to the
results obtained with the yearling heifers. Additional evi-
dence that protein intake is critical in determining gains is the
comparison in gains between groups III and IV of the two-year-
olds. The TDN intake per 100 pounds body weight was consis-
tently higher in heifers of group IV than in heifers of group
III, whereas there was a greater weight loss (-0.23) in group
IV animals compared to group III (-0.07). The latter observa-
tion of increased feed consumption but poor feed utilization
was made by Klosterman et al. (22), who fed cane molasses to
cattle on a ration sub-optimal in protein.
To obtain supplementary information of feed utilization,
rumen filtrates were obtained from two-year-old heifers at slaugh-
ter for determination of the effect of the various levels of
protein and molasses on cellulose digestion. The average per-
cent of cellulose digested by rumen filtrates was 19.1, 12.5, 12.5
and 5.7 percent for groups I through IV, respectively, with large
variability observed within groups.
The results on performance of the protein deficiency produced
in these studies was probably influenced by the amount and
nature of the carbohydrate fed in order to have a ration low in
protein and adequate in energy. In sheep, increased propor-
tions of starch (Head, 20), glucose (Hamilton, 17), and sucrose
12 Florida Agricultural Experiment Stations
and molasses (Briggs and Heller, 3) impaired the apparent
digestibility of protein and crude fiber. Williams (35) reported
similar findings with molasses for cattle.
The findings reported herein show that yearling and two-
year-old heifers consuming the highest levels of protein fed in
this experiment had increased feed utilization as compared to
the lower protein intakes.
The estrual behavior of heifers during the pre-breeding peri-
od of 112 days and the breeding period is shown in Table 5.
TABLE 5.-EFFECT OF PROTEIN INTAKE ON REPRODUCTION IN BEEF HEIFERS
BEFORE AND DURING BREEDING.
(5 animals per group)
Item I II III IV I II III IV
Yearling heifers Two-year-old heifers
mental period, 112 days*
Days to first estrus** 50 76 90 162 10 12 12 18
No. estrual periods 19 13 12 0 26 26 26 23
No. estrual cycles 14 10 9 0 21 21 21 18
Estrual cycles, days 22.9 20.8 20.2 22.5 21.1 21.1 19.6
No. heifers with
corpus luteumt 5 3 2 0 5 5 5 4
Breeding period, 113
days to slaughter
No. animals bred 5 5 2 0 5 5 2 5
Total no. services 5 7 4 10 5 4 9
No. heifers returned in
estrus 0 2 1 3 0 1 2
No. heifers bred at:
Second estrus 2 1 3 1 2
Third estrus 1 2 1 2
Av. cycle length,
days 18.0 20.5 22.5 19.5 16.5
No. normal embryos 44
days postbreeding to
1st or 2nd estrus 5 5 0 0 3 4 1 3
"* Yearling heifers 3/3/56 to 6/23/56; two-year-old heifers 2/15/57 to 6/8/57.
** Heifers not showing estrus were calculated to day of slaughter.
t When breeding began.
Effect of Protein Intake on Beef Heifers 13
Pre-breeding Period.-During the pre-breeding period, all of
the yearling heifers in group I showed estrus, while only three
animals in group II and two in group III showed one or more
regular estrous periods. None of the yearling heifers in group
IV showed any sign of estrus during the pre-breeding or breeding
period. Estrual cycle length was similar for all yearling heifers
showing estrus (Table 5). Rectal palpation of ovaries, per-
formed every 14 days during the experiment, indicated that the
yearling animals which did not show signs of estrus had little
follicular growth and failed to ovulate. Likewise, the rectal
palpation examination on the day the breeding period com-
menced indicated that all the yearling heifers in group I had a
corpus luteum, while groups II and III had only three and two
heifers, respectively, with a corpus luteum. None of the yearling
heifers in group IV had a corpus luteum.
During the pre-breeding period all two-year-old heifers ex-
cept one in group IV showed four to six estrous periods (Table
5). One two-year-old heifer in group IV showed estrus only
once, 11 days following the start of the experiment. She was
in poor physical condition during the first 112 days on experi-
ment and never returned into estrus during this period. How-
ever, she came in estrus during the breeding period and became
pregnant at the first breeding. Rectal palpation studies at 14-
day intervals during the pre-breeding period indicated that all
the two-year-old heifers were ovulating regularly except the one
heifer in group IV. The length of the estrual cycles was similar.
Breeding Period.-All the yearling heifers in group I and
three heifers in group II were pregnant from the first service
and had normal embryos. The remaining two heifers in group II
returned in estrus 18 days later, were rebred, and had normal
embryos. Neither of the two heifers bred in group III became
pregnant at first or subsequent breeding (Table 5). One heifer
in group III appeared to have settled at the first breeding, while
the other one was bred at three consecutive estrous periods and
was killed three days following the last breeding. The heifers
which showed no estrus were slaughtered at random times with
the pregnant animals, and none had evidence of luteal tissue on
The reproductive performance of the two-year-old heifers
varied considerably within the groups. Of those in group I,
only two animals were pregnant at first breeding; another be-
came pregnant at the second service, while the remaining two
14 Florida Agricultural Experiment Stations
heifers required a third breeding and were killed three days
following the last service. Of the latter heifers, one had a fer-
tilized ovum as well as a regressed embryo, while the other failed
to ovulate (Table 6). All two-year-old heifers in group II be-
came pregnant at first service as determined by rectal palpation
and cessation of estrus, although one heifer had a regressing
embryo and recent corpus luteum at time of slaughter (Figure
2). One animal of two bred in group III settled at first service
and had a normal embryo. The other heifer in group III re-
quired a third breeding, and when she was killed three days
following the last service, an ovum with a ruptured zona pellucida
was recovered. The remaining three heifers in this group had
ovulated without showing estrus as determined by rectal palpa-
tion and examination of the ovaries at slaughter. Three of the
two-year-old heifers in group IV became pregnant at first serv-
TABLE 6.-EFFECT OF PROTEIN INTAKE ON EMBRYOS AND OVARIES
OF BEEF HEIFERS AT SLAUGHTER.
(5 animals per group)
Item I II III IV I II III IV
Yearling heifers Two-year-old heifers
No. heifers bred 5 5 2 0 5 5 2 5
No. normal embryos
at 44 days 5 5 0 0 3 4 1 3
No. regressed embryos
before or at 44 days 0 0 0 0 1** 1t 0 Itt
Weight, gm 2.2 2.1 2.6 2.2 1.7 2.4
Length, mm 28.2 27.6 28.1 27.4 24.0 26.0
Ovary weight, gm 9.1 8.6 5.0 4.2 9.8 10.8 10.2 9.5
Follicular index* 32.3 26.6 37.5 32.8 28.2 27.8 36.0 32.0
No. heifers 3 days
Fertilized ovum 1 1
Unfertilized ovum 1$ 1$ 1 1
Follieular index = (number of follicles 4 mm and larger) x (size in mm).
** Returned in heat after 18 days with regressed embryo in uterine body and a fertilized
t Recent Quiet ovulation with regressed embryo.
tt Bred at 3rd estrous period, had regressed embryo and unfertilized ovum.
t No ovulation.
Ruptured zona pellucida.
Effect of Protein Intake on Beef Heifers 15
ice and had normal embryos, while the remaining two heifers
each had three breeding. When these two heifers were slaugh-
tered three days following the third service, one heifer had a
fertilized ovum, while the other heifer had a non-fertilized
ovum as well as a regressed embryo (Figure 2). The short
estrous cycle of the two-year-old heifers in group IV was due
to one heifer that had a cycle of eight days. There is no ap-
parent explanation for the increased number of services in the
Figure 2.-(1) Pregnant uterus, at 44 days, of yearling heifer of Group II
fed 0.65 pound C.P. daily. Infantile uterus of yearling heifer of Group IV
fed 0.08 pound C. P. daily. (2) Uteri from yearling heifers. No. 190,
Group IV, infantile uterus; No. 194, Group III, not pregnant at 44 days but
increased vascularity and size of uterus indicating fetal resorption; No.
408 of Group I pregnant at 44 days. (3) Uterus of two-year-old heifer
of Group II. Note increased size of uterus and recent corpus luteum.
Heifer had a regressing embryo. (4) Uterus of two-year-old heifer of
Group IV. Arrow points to a regressed embryo, and animal had an un-
fertilized ova three days after third breeding.
All the yearling heifers in groups I and II had a normal em-
bryo, while none of the yearling heifers in groups III and IV had
either an embryo or a fertilized ovum at slaughter (Table 6).
Heifer 194 from group III (Figure 2), which was bred once
16 Florida Agricultural Experiment Stations
and appeared pregnant on palpation, did not have a normal em-
bryo, although there was an increased vascularity and size of
one uterine horn. It seems, therefore, that the heifer might
have been pregnant, but embryonic death occurred. A case
of estrus without ovulation three days after breeding was ob-
served in one yearling heifer of group III which was bred at
three different estrous periods. She had a 14 mm follicle cov-
ered with a thick connective tissue-like material on the right
ovary. This ovary also had a 16 mm yellowish-orange corpus
The average weight of the ovaries of the yearling heifers
was heavier in groups I and II than in groups III and IV, due to
the presence of the active corpus luteum (Table 6). The ovarian
activity, as measured by the follicular index, indicated that the
stimulus for follicular growth was approximately the same for
all yearling heifers. There was, however, a large difference in
size of the follicles present in the ovaries. Groups I and II had
larger follicles, whereas groups III and IV had a larger number
of small follicles as well as a large number of atretic follicles.
The absence of a corpus luteum and the presence of only small
follicles in the yearling heifers showing no estrus in groups III
and IV confirmed data obtained by rectal palpation throughout
The variations in weight and measurement of embryos among
the two-year-old heifer groups did not follow any particular
pattern (Table 6). The combined ovarian weight of the two-
year-old heifers in groups II and III was slightly heavier than
that of groups I and IV. In the two-year-old heifers there were
no significant differences that could be attributed to the various
dietary protein levels in the histology of the non-pregnant
uterine horns, the various zones of the adrenal glands, and the
ovaries. Likewise, the variation in number and size of follicles
in the thyroid gland was not statistically significant among the
four dietary groups.
Anterior Pituitary Assay.-The anterior pituitary assay for
gonadotrophin activity showed no statistically significant dif-
ferences in the weight of testes and combs of chicks injected with
anterior pituitary material from either yearling or two-year-old
heifers on the various levels of dietary protein (Table 7). How-
ever, the pituitary powder from the pregnant yearling heifers
(groups I and II) gave heavier (P<0.01) chick comb weights
than powder from the non-pregnant yearling heifers (groups
Effect of Protein Intake on Beef Heifers 17
III and IV). It is impossible to determine if the above difference
was due to variation in pregnancy or variation in level of protein
intake, since these two variables could not be separated. Like-
wise, reproductive stage and level of protein in the two-year-old
heifers could not be separated, but the pituitary powder from
heifers on the two higher levels of protein intake gave some-
what heavier chick comb weights compared to those from the
heifers on the two lower levels (Table 7).
TABLE 7.-EFFECT OF LEVEL OF PROTEIN INTAKE ON ANTERIOR
PITUITARY GONADOTROPHIN ACTIVITY OF BEEF HEIFERS.
Source No. Total Dose/ Weight in mg of Chick
of Assay Pitui- No. Chick in 2 ml
Materials taries Chicks of Solution Testes Comb
I 1.06 4 3 18 mg* 49.1 82.7
II 0.65 5 4 18 mg* 51.5 68.4
III 0.28 5 5 18 mg* 37.4 49.7
IV 0.08 5 6 18 mg* 48.8 52.3
I 1.34 5 7 18 mg* 45.4 67.6
II 1.06 4 7 18 mg* 49.6 68.1
III 0.71 4 7 18 mg* 50.6 60.7
IV 0.62 5 6 18 mg* 41.7 64.0
Unfractionated 10 250 I.U.** 47.7 66.3
sheep anterior 10 500 I.U. 57.7 79.9
pituitary, Abbott 10 1000 I.U. 61.0 67.8
Control 10 36.7 51.3
All anterior pituitary glands dessicated for 72 hours.
"** Equivalent to international units of chorionic gonadotrophin.
Manometric determination for xanthine oxidase did not de-
tect any enzyme activity in the uterus of the pregnant and non-
The data reported herein are in agreement with those of
Guilbert and Hart (16) and Palmer et al. (29), who found that
a low protein intake affects the reproductive performance of
growing heifers. The most pronounced adverse effect on repro-
duction was observed in the yearling heifers consuming 0.08 and
18 Florida Agricultural Experiment Stations
0.28 pound crude protein daily. These heifers, which developed
a severe protein deficiency with low energy (Figure 1), showed
delayed puberty, a long anestrous period, weak manifestation
of estrus, and short estrous periods. Likewise, a case of early
embryonic resorption and estrus without ovulation occurred with
the yearling heifers consuming 0.28 pound crude protein daily.
The degree of protein deficiency produced with the two-year-
old heifers was not as severe as with the yearling heifers. This
was due to the fact that the protein levels fed the two-year-old
heifers were higher. Also, the older and heavier animals prob-
ably had a lower protein requirement and or a larger reserve of
protein and could, therefore, tolerate a lack of protein for a long-
er period without showing as adverse effects as the yearling
heifers. Thus, the regularity of estrus in the two-year-old heif-
ers during the pre-breeding period was influenced less by level
of protein intake. Several cases of weak manifestation of estrus
were observed with the two-year-old heifers consuming 0.62 and
0.71 pound crude protein daily. These heifers showed interest
in the bull and had a clear vaginal secretion, yet would not stand
for mating. Rectal palpation of these heifers indicated a poor
tonus of the uterine muscle, which was not characteristic of
estrus. The weak manifestation of estrus and poor muscle tonus
of uterus suggests that low levels of dietary protein may reduce
estrual activity, which could result from an inadequate level of
estrogen production or a lack of proper sensitization of the ner-
The embryos studied from the yearling and two-year-old
heifers pregnant at 44 days following breeding indicated no gross
abnormalities that could be attributed to level of dietary protein.
The embryos appeared normal in size and weight, and no abnor-
malities were observed in the extra-embryonic membranes. Al-
though early embryonic development was not affected by low
levels of protein intake in this study, it is possible that fetal
changes might have occurred at later stages of gestation. More
experimental data are required to study this possibility.
All yearling heifers consuming 0.28 or 0.08 pound of crude
protein daily that failed to show estrus had some follicular
growth, but the follicles never reached the mature stage and
mostly measured less than 10 mm in diameter. These ovaries
had a large number of atretic follicles, but a recent corpus luteum
was not found. The follicular index which was determined at
slaughter time indicated that the stimulus for follicular growth
Effect of Protein Intake on Beef Heifers 19
was comparable in all groups of heifers. The output of the fol-
licle-stimulating hormone by the anterior pituitary glands was
probably similar in all the heifers on the various levels of dietary
protein; this is indicated by testes weights from the pituitary
assay for gonadotrophin. The data obtained suggest that ex-
tremely low intakes of protein in yearling heifers may reduce
the luteinizing hormone activity of the pituitary as measured
by comb growth.
The data reported herein confirmed the observation made
by various other investigators that low dietary protein adver-
sely affected the ovarian activity and manifestation and reg-
ularity of estrus in laboratory animals (Guilbert and Goss, 15;
Nelson and Evans, 28; Pearson et al. 30) and in ruminants
(Hart and Miller, 19; Palmer et al., 29). These phenomena
may be due in part to the lack of luteinizing hormone rather
than to levels of total gonadotrophin as proposed by Guilbert
and Hart (16) and Sutton (33). The physiological stress ex-
erted on the heifers as a result of protein deficiency led to clinical
anemia and proteinemia. These conditions undoubtedly adver-
sely affected the entire metabolism of the animal and resulted
in poor reproductive performance in growing beef heifers. These
data confirm those from cattle on pasture experiments by Bed-
rak (2) in which low protein intake reduced reproductive per-
There was a high correlation between reproductive activity
and rate of gain in the yearling heifers, but this was not true
of the two-year-olds. However, there were seven normal em-
bryos from the 10 heifers on the rations showing gains and only
four embryos on the rations showing a loss of weight in the two-
year-old heifers. The main reason for the smaller number of
embryos in the latter two groups was a failure of estrus rather
than differential embryonic mortality. The two-year-old heifers
were probably not on a low enough protein intake to markedly
influence reproduction; also yearlings may have a higher protein
requirement than two-year-old cattle.
Blood Constituents and Liver Protein
Hemoglobin.-The analysis of variance for the various blood
constituents is shown in Table 9. Yearling heifers in groups I
and II maintained their hemoglobin concentration within the
normal range, varying between 10.8 and 11.9 percent during the
20 Florida Agricultural Experiment Stations
entire experiment, but the animals fed the low levels of dietary
protein showed borderline symptoms of anemia (Table 8). Ac-
cording to Duncan's (8) multiple range test, groups I and II had
higher (P<.01) hemoglobin levels than groups III and IV, and
the heifers in group III had higher (P<.01) values than group
IV; but there were no statistically significant differences between
groups I and II. Group III yearling heifers showed a decline
to 9.4 during the third 28-day period, and this level was main-
tained throughout the remainder of the trial. Likewise, there
was a marked reduction in hemoglobin concentration of the
heifers in group IV during the third period and a further decline
to a concentration of 7.2 at the final bleeding, a reduction of
30 percent below the initial bleeding. This lag period before
hemoglobin levels fall, even in severe protein deficiency, may be
TABLE 8.-EFFECT OF PROTEIN INTAKE ON BLOOD HEMOGLOBIN, HEMATOCRIT,
CALCIUM, PHOSPHORUS, AND LIVER PROTEIN OF BEEF HEIFERS.
(5 animals per group)
Item Group Number
I II III IV I II II IV
gr /100 ml
Initial 11.0 11.7 12.0 10.3 11.5 12.6 12.0 13.6
1 10.8 12.1 10.9 11.4 10.3 11.9 10.8 11.7
2 11.2 11.1 10.3 10.3 11.4 11.8 11.5 11.4
3 10.9 11.1 9.4 8.8 11.2 12.3 11.5 10.9
4 10.8 11.2 9.9 7.5 12.4 12.7 11.7 11.4
5 10.8 10.7 9.3 7.6 11.7 12.6 11.2 10.7
Final 11.4 11.8 9.4 7.2 11.7 12.5 10.5 10.9
Initial 44.6 48.8 45.6 41.4 41.7 45.7 43.1 45.8
1 44.7 42.9 46.4 45.9 38.8 44.6 43.9 45.1
2 48.6 43.6 41.4 40.7 41.8 43.5 43.5 40.0
3 45.5 43.6 36.7 32.6 40.4 43.1 40.2 38.5
4 42.7 41.5 36.8 30.0 44.0 44.1 39.6 38.6
5 42.2 41.8 37.0 28.8 38.6 40.8 38.0 34.8
Final 44.6 43.5 37.0 28.4 37.3 40.1 34.6 34.7
Calcium** mg % 11.1 11.1 10.8 11.1 11.1 10.6 11.0 11.3
Phosphorus** mg % 5.0 4.8 4.9 5.4 5.1 5.4 4.9 5.0
Liver protein, % 13.6 12.7 10.8 10.1 14.4 14.3 13.4 12.8
Periods 1 to 5 were 28 days apart.
** Average of three different bleedings taken initially, at second and fourth period in
yearlings and at first, fifth, and final period in two-year-olds.
Effect of Protein Intake on Beef Heifers 21
related to the life of the erythrocytes and to the labile protein
stores of the animal.
The two-year-old heifers in group II had a higher (P<.01)
hemoglobin concentration than groups I, III, and IV; whereas the
differences observed among groups I, III, and IV were not signi-
ficant. Animals on the lowest protein intake (group IV) ex-
hibited a slight decrease in hemoglobin as the experiment
progressed (Table 8).
TABLE 9.-ANALYSIS OF VARIANCE OF HEMOGLOBIN, HEMATOCRIT, SERUM
PROTEIN, SERUM ALBUMIN AND GLOBULIN, ERYTHROCYTES, AND LEUKOCYTES
OF BEEF HEIFERS ON VARIOUS LEVELS OF DIGESTIBLE PROTEIN INTAKE.
of df Hb. Hemat. Protein Albumin Glob. R.B.C. W.B.C.
Variance % % % % %
Subclass 27 8.32 159.3 1.17 1.33 1.10 -
Group 3 36.60** 619.1** 4.73** 2.93** 0.83 -
Period 6 18.83** 243.6** 1.20** 3.21** 2.46** -
G X P 18 3.24** 54.6** 0.47* 0.51 0.70 -
Error 112 0.78 16.6 0.23 0.32 0.48 -
Subclass 27 2.74 54.5 1.19 1.79 2.64 1.01 5,960,276
Group 3 7.80** 82.8* 3.10** 1.42* 0.31 2.46** 7,352,313
Period 6 3.68* 143.9** 3.12** 7.12** 12.56** 1.80** 10,254,437**
G X P 18 1.74 20.0 2.36** 0.20 0.49 1.90** 4,296,883*
Error 112 1.26 22.1 0.29 0.50 0.52 0.43 1,983,516
Hematocrit.-Yearling heifers of groups I and II maintained
their hematocrit volume with small fluctuations, whereas groups
III and IV exhibited a marked decline which was comparable to
the decline in their hemoglobin values (Table 8). The heifers
in group III maintained normal hematocrit levels until the third
28-day period, when there was a sharp decline to 36.7 percent;
levels were maintained in this range during the remainder of
the trial. The yearling animals in group IV showed a decline
in hematocrit from 41.4 percent at the initial bleeding to 28.4
percent at the final bleeding, with the greatest decline occurring
at the end of the third period. Groups I and II had a higher
(P<.01) hematocrit value than groups III and IV. No differ-
22 Florida Agricultural Experiment Stations
ence was exhibited between groups I and II, while group III was
higher (P<.01) than group IV. The values for all yearling heif-
er observed during the initial bleeding and first two 28-day peri-
ods were higher (P<.01) than those obtained during the subse-
The overall decline in hematocrit for the two-year-old heifers
during the entire experiment was 4.4, 5.6, 8.5, and 11.1 percent
for groups I through IV, respectively (Table 8). However, sta-
tistical analysis indicated that while there was a significant dif-
ference between the hematocrit levels of groups II and IV
(P<.01), there were no differences among groups I, II, and III
and among groups I, III, and IV. There was a decrease (P<.01)
in hematocrit values with periods as the experiment continued.
Hematocrit levels of yearling and two-year-old heifers in
groups IV declined 13 and 11 percent, respectively, during the
trial. These decreases were larger than the 5 percent reported
by Kehar and Murty (21) with cattle, probably because the ex-
periments reported herein lasted longer.
Inorganic Calcium and Phosphorus.-Calcium and phosphorus
in the blood plasma were determined at three times during the
experiment in both the yearling and two-year-old heifers. There
were no significant dietary or period differences, and all values
were within the normal range (Table 9). Thus, it is evident
that the mineral requirements of the heifers were amply met,
and that response in gains, reproduction, and blood was due to
differences in protein intake rather than mineral deficiencies.
Liver Protein.-On a wet weight basis, yearling heifers in
group I and II had larger (P<.01) amounts of protein in their
livers at slaughter than groups III and IV (Table 8). Likewise,
two-year-old heifers of groups I and II had a higher (P<.05)
liver protein content than heifers in group IV. The animals
having the lowest liver protein also. had the lowest hemoglobin
and serum protein.
Erythrocytes and Leukocytes.-The two-year-old heifers in
groups I, II, III, and IV showed an absolute decline in erythro-
cytes of 0.20, 0.40, 1.60, and 1.50 million per cubic millimeter, re-
spectively, as calculated from Table 10. Group II had a higher
(P<.01) erythrocyte count than groups I and IV; whereas the
differences between groups II and III as well as I, III, and IV
were not significant. The data from all heifers showed a de-
crease (P<.01) in cell count as the experiment progressed. The
decline in erythrocytes, which followed the decrease in hemo-
Effect of Protein Intake on Beef Heifers 23
globin in group IV of the two-year-old heifers, was not accom-
panied by abnormalities in the morphology of the cells.
TABLE 10.-EFFECT OF LEVEL OF PROTEIN INTAKE ON ERYTHROCYTES
OF TWO-YEAR-OLD BEEF HEIFERS.
(5 animals per group)
Bleeding period Group Number
I II III IV
Erythrocytes x 10" per cu. mm
Initial 6.2 6.9 7.2 6.4
1 6.2 6.4 6.2 6.2
2 6.0 6.6 6.6 6.3
3 6.1 6.6 6.5 5.6
4 6.2 7.0 6.4 5.9
5 6.4 6.4 5.9 5.8
Final 6.0 6.5 5.6 4.9
The leukocyte counts in group II were higher (P<.01) than
in the other groups of two-year-old heifers on the different
Blood Volume.-Determinations of total blood volume in the
two-year-old heifers 140 days after the experiment began showed
an average of 5.1, 4.7, 4.5, and 4.8 percent of the body weight
for groups I through IV, respectively. When the hematocrit
volume was related to body weight, there were 21.2, 19.4, 17.4,
and 16.1 ml per kg for heifers in groups I through IV, respec-
tively. Neither total blood volume nor hematocrit per kilogram
of weight showed significant statistical differences due to pro-
tein intake. The volume of plasma in milliliters per kilogram of
body weight was 27.1, 25.0, 25.5, and 29.7 in groups I through
IV, respectively. The values in groups I and IV were greater
(P<.05) than groups II and III, but this indicates no direct rela-
tionship with level of protein intake.
The percent blood volume of the dietary groups of two-year-
old heifers was essentially unchanged. However, when the ratio
of plasma to packed cells was calculated by dividing the milliliters
of plasma by the milliliters of hematocrit per kilogram body
weight, the group consuming the low dietary protein appeared
to have the most diluted blood. The ratios obtained for groups
I, II, III, and IV were 1.28, 1.28, 1.47, and 1.85, respectively.
This may be partly explained by the fact that the heifers in
groups III and IV consumed more of the trace mineralized and
24 Florida Agricultural Experiment Stations
common salt than groups I and II, which in turn should have
increased their water consumption, resulting in an increase of
plasma per unit of live weight. The plasma volume observed in
this study is lower than previously reported for cattle by Hansard
et al. (18) and agreed more closely with the value obtained by
Serum Protein and Blood Creatinine.-There was essentially
no change in serum protein in groups I and II of the yearling
heifers, but group III decreased from 7.0 to 6.3 percent, and
group IV declined from 6.7 to 5.4 percent (Table 11). Groups
III and IV had lower (P<.01) serum protein content than groups
I and II. While there was no difference between groups I and
II, group IV was lower (P<.01) than group III. There was an
overall decrease (P<.01) in serum protein during the later peri-
ods; this decrease occurred primarily in groups III and IV. In
the two-year-old heifers, groups I and II had higher (P<.01)
values than groups III and IV.
A prolonged protein deficiency in other species has been
shown to induce anemia and leukopenia because the hemopoietic
organs are deprived of the amino acids necessary for cell forma-
tion and hemoglobin production (Yamamoto, 37, and Whipple
and Madden, 35). The yearling heifers in this study showed a
decrease of 30 percent in hemoglobin concentration and only 20
percent in serum protein.
Blood creatinine values were determined on the yearling
heifers at each bleeding. The average values at slaughter of
all heifers varied from 1.4 to 1.6 mg per 100 ml of whole blood,
and they were not influenced by protein intake or periods. In
these studies a marked decline in the serum protein concentra-
tion occurred without an increase in the creatinine level of whole
Serum Albumin and Globulin.-The yearling heifers in group
IV had less (P<.01) albumin in the serum than heifers in the
other three dietary groups. The initial concentration of 3.2
percent in group IV decreased to 2.3 and 2.9 percent at the fifth
and final periods, respectively (Table 11). For some unexplained
reason, the overall values for the first and second periods were
higher (P<.01) than those for the initial, third, and subse-
quent periods. Likewise, the concentrations of serum albumin
observed in groups I and II of the two-year-old heifers were
higher (P<.01) than those observed in group IV. There were
no significant differences among the albumin concentrations of
Effect of Protein Intake on Beef Heifers 25
group I, II, and III and between groups III and IV with the
two-year-old heifers. The albumin concentration of all groups
of two-year-old heifers was higher (P<.01) during the initial
bleeding than in the fourth and final periods.
TABLE 11.-EFFECT OF LEVEL OF PROTEIN INTAKE ON SERUM PROTEIN,
ALBUMIN, AND GLOBULIN IN HEIFERS.
(5 animals per group)
I II III IV I II III IV
Yearling heifers Two-year-old heifers
Serum Protein, gm/100 ml
Initial 6.7 6.6 7.0 6.7 7.4 7.5 7.4 7.2
1 7.0 6.9 6.9 6.8 6.9 6.9 6.7 6.8
2 6.7 7.1 6.5 6.4 6.9 6.9 7.0 6.4
3 6.9 6.8 6.4 5.7 7.4 7.4 6.6 6.4
4 7.4 7.4 6.3 6.3 7.7 7.9 7.3 7.2
5 6.7 6.9 6.2 5.8 7.7 8.0 7.1 7.1
Final 6.7 6.8 6.3 5.4 8.3 8.2 7.4 7.4
Albumin, gm per 100 ml
Initial 3.5 3.3 3.5 3.2 5.1 5.2 5.0 4.7
1 4.0 4.3 4.2 4.0 -
2 4.4 4.4 4.2 4.1 -
3 3.8 4.0 3.8 3.1 -
4 3.5 3.4 3.7 3.1 4.8 4.6 4.0 3.7
5 3.3 4.5 3.4 2.3 -
Final 3.3 3.5 3.2 2.9 4.0 3.9 3.8 3.5
Globulin, gm per 100 ml
Initial 3.2 3.3 3.5 3.5 2.4 2.3 2.4 2.5
I 3.1 2.6 2.8 2.8 -
2 2.4 2.7 2.5 2.3 -
3 3.0 2.8 2.7 2.6 -
4 4.0 4.1 2.6 3.2 2.9 3.3 3.3 3.5
5 3.4 2.4 2.8 3.5 -
Final 3.5 3.3 3.1 2.4 4.3 4.4 3.6 4.0
Periods 1 to 5 were 28 days apart.
Apparently the globulin levels were least influenced by the
dietary protein deficiency. Data presented by Garner (12),
Kehar and Murty (21) and Klosterman et al. (22) were in gen-
eral agreement with the present findings in which the protein
deficient heifers showed a marked decline in the albumin con-
centration while the total globulin remained relatively un-
changed. Apparently, the serum albumin is the most labile
protein which can be depleted from the body stores.
26 Florida Agricultural Experiment Stations
Electrophoretic Fractionation of Blood Proteins.-The elec-
trophoretic investigations of blood serum protein in yearling
heifers confirmed the marked decline in albumin content as
observed with the chemical method as the level of dietary protein
decreased. While there was no significant difference in the total
serum globulin of yearling heifers fed the four levels of protein,
there were some differences with regard to various fractions
of the globulin. The values of alpha2 and beta globulin were
not influenced by diet (Table 12). Group II had less (P<.01)
gamma globulin than dietary groups I, III, and IV. Alpha1
globulin level increased as the dietary protein level decreased
in the yearling heifers-a result similar to findings of Zeldis
et al. (38) with protein-depleted dogs. Likewise, the gamma
globulin level showed a significant increase at the lower levels
of dietary protein.
TABLE 12.-EFFECT OF PROTEIN INTAKE ON PERCENT OF ALPHA, ALPHAs,
BETA, AND GAMMA GLOBULIN IN BLOOD SERUM OF YEARLING HEIFERS.
(5 animals per group)
Bleeding Period I II III IV
Percent** alpha1 globulin
2 19.4 18.7 19.2 19.4
3 16.4 16.4 19.4 19.5
4 12.9 14.0 17.8 17.5
5 17.2 18.2 18.0 23.1
Final 16.0 19.7 21.2 19.8
Percent alpha. globulin
2 13.5 12.6 10.9 10.7
3 13.6 14.6 14.4 13.6
4 14.8 15.0 16.6 15.5
5 14.3 15.5 15.0 12.6
Final 13.8 18.9 15.7 15.5
Percent beta globulin
2 15.8 19.0 18.5 20.9
3 18.5 16.6 17.8 19.0
4 18.6 14.8 16.6 19.0
5 13.4 23.5 16.2 22.9
Final 17.9 13.5 13.7 16.4
Percent gamma globulin
2 10.1 7.5 9.1 11.8
3 11.6 9.2 10.9 11.4
4 12.6 13.0 14.1 12.5
5 11.2 3.8 12.2 11.0
Final 13.8 11.0 15.3 16.3
Determined by electrophoresis.
** Percent of total serum protein.
Effect of Protein Intake on Beef Heifers 27
Twenty yearling Angus, Hereford, and Angus-Hereford
crossbred heifers and 20 two-year-old Hereford heifers were
individually fed rations consisting of different levels of protein
but similar amounts of energy, minerals, and vitamins. Five
heifers of each age were allotted per group and fed for a period
of 159 to 207 days, depending on reproduction performance.
Yearling heifers, initially weighing about 500 pounds, with a
daily consumption of 1.06, 0.65, 0.28, and 0.08 pound crude
protein, gained 0.94, 0.70, 0.01, and -0.72 pound per day, re-
spectively. Two-year-old heifers, initially weighing 675 pounds,
consuming 1.34, 1.06, 0.71, and 0.62 pound crude protein daily,
gained 0.75, 0.52, -0.07, and -0.23 pound per day, respectively.
The differences obtained in the performance between the year-
ling and two-year-old heifers were due in part to age and weight
differences, as well as source of energy and level of TDN and
protein intake. The nutritional stress due to a low protein
intake decreased appetite and rate of gain.
At 44 days post-breeding, there were 10 normal embryos
from 10 yearling heifers fed the two highest levels of protein,
while there were no normal embryos from the 10 heifers on the
two lower levels of protein. Eight of these latter heifers did
not ovulate. Yearling heifers that consumed 0.28 pound crude
protein daily or less exhibited delayed puberty, long anestrous
periods, failure to show estrus and ovulate, and weak expres-
sion of estrus.
Two-year-old heifers on the two higher intakes of protein
had seven normal embryos from 10 heifers bred, while there were
only four normal embryos from seven heifers bred that were
on the lower protein intakes. Three of the five two-year-old
heifers fed 0.71 pound crude protein daily ovulated without
showing estrus and could not be bred. The explanation for
the large number of regressed embryos and of increased services
per normal embryos in the two-year-old heifers is not apparent.
Heifers on the protein-deficient rations had low hemoglobin
and hematocrit values in the blood. The anemia was largely
due to a decreased number of erythrocytes, while no apparent
change occurred in blood volume. The number and type of leu-
kocytes was not apparently influenced, and, likewise, calcium
and phosphorus were not affected by the protein deficiency.
Marked declines occurred in liver and blood serum protein levels
28 Florida Agricultural Experiment Stations
on the low protein intake with no apparent change in blood
creatinine. The lower serum protein was due to a decline in
albumin concentration, with no appreciable change in total
globulin. Electrophoretic fractionation of the serum protein
revealed increased concentration of alpha and gamma globulin
but no apparent change in alpha2 or beta globulin of heifers on
low protein intake.
The daily protein need for yearling beef heifers weighing 500
pounds with daily gains of 0.70 pound was met by feeding 0.65
pound crude protein daily under the conditions of this experi-
ment. A level of 1.06 pound crude protein daily was required
to achieve a somewhat similar gain of 0.75 pound daily with
two-year-old heifers weighing 675 pounds.
1. Axelrod, A. E., and C. A. Elvehjem. The xanthine oxidase content of
rat liver in riboflavin deficiency. J. Biol. Chem. 140:725. 1941.
2. Bedrak, E. The effect of wintering protein supplementation upon
reproductive phenomena in beef cattle on pasture. M. S. Thesis,
University of Florida, Gainesville, Florida. 1955.
3. Briggs, H. M., and V. G. Heller. The effect of adding blackstrap
molasses, potassium salts, sucrose and corn syrup to a lamb-fatten-
ing ration. J. Agr. Res. 67:359. 1943.
4. Cheng, K. L., and R. H. Bray. Determination of calcium and mag-
nesium in soil and plant material. Soil Science 72:449. 1951.
5. Cohen, B., and A. H. Smith. The colorimetric determination of hemo-
globin. J. Biol. Chem. 39:489. 1919.
6. Crampton, E. W., and L. A. Maynard. The relation of cellulose and
lignin content to the nutritive value of animal feeds. J. Nutr. 15:383.
7. Dhungat, S. B., and A. Sreenivasam. The use of phyrophosphate
buffer for the manometric assay of xanthine oxidase. J. Biol. Chem.
8. Duncan, D. B. Multiple range and multiple F. tests. Biometrics
9. Durrum, E. L., and S. R. Gilford. Recording integrating photoelectric
and radioactive scanner for paper electrophoresis and chromotogra-
phy. Review of Scientific Instruments. 26:51. 1955.
10. Fiske, C. H., and Y. Subbarow. The colorimetric determination of
phosphorus. J. Biol. Chem. 66:375. 1925.
11. Folin, 0., and H. Wu. A system of blood analysis. J. Biol. Chem.
12. Garner, R. J. Variation in serum protein in cattle. J. Comp. Path.
and Therp. 62:279. 1952.
Effect of Protein Intake on Beef Heifers 29
13. Gornall, A. G., C. J. Bradwill, and M. M. David. Determination of
serum protein by means of the biuret reaction. J. Biol. Chem. 17:751.
14. Guilbert, H. R., P. Gerlaugh and L. L. Madsen. Recommended nutrient
allowances for domestic animals. IV. Recommended nutrient allow-
ances for beef cattle. National Research Council, Washington, D. C.
15. Guilbert, H. R., and H. Goss. Some effect of restricted protein intake
on estrous cycle and gestation in the rat. J. Nutr. 5:251. 1932.
16. Guilbert, H. R., and G. H. Hart. California Beef Production. A pub-
lication of the College of Agriculture, University of California,
Manual 2. 1951.
17. Hamilton, T. S. The effect of added glucose upon the digestibility of
protein and fiber in ration for sheep. J. Nutr. 23:101. 1942.
18. Hansard, S. L., W. O. Butler, C. L. Comar, and C. S. Hobbs. Blood
volume of farm animals. J. Animal Sci. 12:402. 1953.
19. Hart, G. H., and R. F. Miller. Relation of certain dietary essentials
to fertility in sheep. J. Agr. Res. 55:47. 1937.
20. Head, M. J. The effect of quality and quantity of carbohydrate and
protein in the ration of sheep on the digestibility of cellulose and
other constituents of the ration, with a note on the effect of adding
vitamins of the B-complex on the digestibility and retention of the
nutrients of a hay ration. J. Agr. Res. 43:281. 1953.
21. Kehar, N. D., and V. V. S. Murty. Blood changes in cattle fed on a
protein deficient diet. Indian J. Vet. Sci. and Animal Husb. 15:183.
22. Klosterman, E. W., O. G. Bently, A. L. Moxon, and L. E. Kunkle.
Relationship between level of protein, molasses, trace minerals and
quality of hay in ration for fattening cattle. J. Animal Sci. 15:456.
23. Klosterman, E. W., M. L. Buchanan, and D. W. Bolin. The effect of
amount and kind of protein fed upon blood serum proteins of
pregnant ewes and their lambs. J. Animal Sci. 15:456. 1948.
24. Kolmer, J. A., E. H. Spaulding, and H. W. Robinson. Approved Lab-
oratory Technic. Appleton-Century-Crofts, Inc., New York. 1951.
25. Lofgreen, G. P., J. K. Loosli, and L. A. Maynard. Comparative study
of conventional protein allowance and theoretical requirements of
growing Holstein heifers. J. Animal Sci. 10:171. 1951.
26. Loosli, J. K., R. B. Becker, P. H. Phillips, C. F. Huffman, and J. C. Shaw.
Nutrient requirements of dairy cattle. National Academy of Sciences,
National Research Council Publication 464. 1956.
27. Morrison, F. B. Feeds and Feeding, 22nd Edition. (The Morrison
Publishing Co., Ithaca, New York). 1956.
28. Nelson, M. M., and H. M. Evans. Relation of dietary protein level to
reproduction in the rat. J. Nutr. 51:71. 1953.
29. Palmer, L. S., T. W. Gulickson, and W. L. Boyd. The effect of ration
deficient in phosphorus and protein on ovulation, estrus and repro-
duction of dairy heifers. J. Dairy Sci. 24:199. 1941.
30. Pearson, P. B., E. B. Hart, and G. Bohstedt. The effect of the quality
of protein on the estrous cycle. J. Nutr. 14:329. 1937.
30 Florida Agricultural Experiment Stations
31. Reynolds, M. Plasma and blood volume in the cow using the T-1824
hematocrit method. Am. J. Physiol. 173:421. 1953.
32. Robinson, G. E., Jr., and A. V. Nalbandov. Changes in the hormone
content of swine pituitaries during the estrual cycle. J. Animal
Sci. 10:469. 1951.
33. Sutton, T. S. The role of nutrition in reproduction. J. Am. Vet. Med.
Assn. 98:17. 1941.
34. Warnick, A. C., E. Bedrak, M. Koger, A. G. Lewis, and T. J. Cunha.
Reproduction performance of cattle grazing grass versus clover-
grass pasture as influenced by cottonseed meal supplementation. J.
Animal Sci. 14:1259. 1955.
35. Whipple, G. H., and S. C. Madden. Hemoglobin, plasma protein and
cell protein: Their interchange and construction in emergencies.
Medicine 23:215. 1944.
36. Williams, P. S. The effect of cane molassees on the digestibility of
a complete ration fed to dairy cows. J. Daily Sci. 8:94. 1925.
37. Yamamoto, M. Nutritional role of the blood protein. II. The behavior
of the blood protein as reserve protein. J. Physiol. Soc. Japan.
13:462. (from Chem. Abst. 46:2142.) 1951.
38. Zeldis, L. J., E. L. Ailing, A. B. McCoord, and J. P. Kulka. Plasma
protein metabolism-electrophoretic studies. Chronic depletion of
circulating protein during low-protein feeding. J. Exp. Med. 82:157.