Group Title: Department of Animal Science mimeograph series
Title: Citrus oil as an intake regulator in a concentrate mixture fed as a supplement to grass hay for cattle
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Permanent Link: http://ufdc.ufl.edu/UF00072994/00001
 Material Information
Title: Citrus oil as an intake regulator in a concentrate mixture fed as a supplement to grass hay for cattle
Series Title: Department of Animal Science mimeograph series - Florida Agricultural Experiment Station ; AN68-6
Physical Description: 16 p. : ; 28 cm.
Language: English
Creator: Hentges, J. F. ( James Franklin ), 1925-
Moore, J. E. ( John E )
University of Florida -- Dept. of Animal Science
University of Florida -- Agricultural Experiment Station
Publisher: Florida Agricultural Experiment Station
Place of Publication: Gainesville, Fla.
Publication Date: 1967
Copyright Date: 1967
 Subjects
Subject: Cattle -- Feeding and feeds -- Florida   ( lcsh )
Citrus oils -- Florida   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
non-fiction   ( marcgt )
 Notes
Statement of Responsibility: James. F. Hentges, Jr. and John E. Moore.
General Note: Caption title.
General Note: "December, 1967."
 Record Information
Bibliographic ID: UF00072994
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 78766733

Full Text



Department of Animal Science Florida Agricultural
Mimeograph Series No. AN68-6 Experiment Station
December, 1967 Gainesville, Florida

CITRUS OIL AS AN INTAKE REGULATOR IN A CONCENTRATE MIXTURE
FED AS A SUPPLEMENT TO GRASS HAY FOR CATTLE

James F. Hentges, Jr. and John E. Moore/2/


One by-product of the citrus fruit processing industry is a vola-
tile essential oil known by various names but composed of 95 percent
or more of d-limonene, a monocyclic terpene. This product is inexpen-
sive, has a pleasing aroma, is exceedingly bitter to the human tongue
and is commercially available in large quantities. This product had
not been studied previously as an animal feed ingredient. The possi-
bility of finding that this inexpensive and available product might
possess a feed intake regulating effect in addition to its pleasing
aroma prompted the research reported in this publication.


General Experimental Procedure

Three experiments were completed, a preliminary feeding trial, an
individual steer-feeding experiment and a rumen fermentation experiment.


TRIAL ONE

Procedure

The preliminary experiment was conducted with 10 steers which
initially weighed 463 kg. All were individually fed grass hay ad
libitum and were offered 4500 grams per day of a concentrate supple-
ment (table 1) plus a 200-gram vitamin-antibiotic mixture (table 2).
Composition of the supplement and vitamin-antibiotic mixture is shown
in table 3. Water intake was measured from automatic drinking cups.
During the first 14 days of the experiment, all 10 steers were offered
a basal supplement shown in table 2. During days 15 to 26, 500 gm.
citrus oil was added to the basal supplement for half of the steers so
that it contained 10% citrus oil by weight. The amount offered each
day was increased to 5000 grams to permit the possible voluntary con-





1/Animal Nutritionist and Associate Animal Nutritionist, Florida
Agricultural Experiment Station, Gainesville.

2/Supported in part by research contract with Animal Husbandry Research
Division, Agricultural Research Service, U. S. Department of Agricul-
ture. The assistance of Dr. R. R. Oltjen is acknowledged.




- 2-


sumptiorB;qf 4500 grams of basal supplement plus citrus oil. During
days;27 to 31,-the level of citrus oil in the supplement for the same
five-steers was reduced to 5% (4500 gm. supplement plus 240 gn. citrus
oil) and 4740 gm. were offered daily to each steer.

TABLE 1

INGREDIENT COMPOSITION OF BASAL SUPPLEMENT


Ingredients. % Kilograms

Soybean oil meal, 44%l' 35 317.52
Corn meal yellow dent #2 51 462.67
Cane molasses, standard 10 90.72
Defluorinated phosphat a 2 18.145
Trace mineralized salt / 2 18.145
100 907.200


a/Guaranteed analysis:

/Guaranteed analysis:


Phosphorus (P) 18.00o Minimum
Calcium (Ca) 31.005o Minimum
Salt (NaCl) not less than 96.0%;
not more than 99.0%




TABLE 2


COMPOSITION OF VITAMIN-ANTIBIOTIC PREMIXa/


Ingredients Grams

Vitamin A (Dual A 30) 30,000 .U./gm.b/ 200.000
Vitamin D2 (Perma D 64) 64x10 USP/lb.- 5.325
Vitamin E (Perma E 125) 25,000 USP/lb.b/ 0.365
Chlortetracycline (Aurofac 10) 10 gm./lb.i/ 955.CCO
Basal supplement 58,839.310
6O,OCO.CCO


a/Mixture calculated for 300 steer-days supply. Daily allowance of
200 gm. provided 20,000 I.U. vitamin A; 2,500 U.S.P. units vitamin
D2; 15 U.S.P. units vitamin E and 70 mg. chlortetracycline.
/Vitamins provided gratis by Commercial Solvents Corporation, Terre
Haute, Indiana.
/Antibiotic provided gratis by American Cyanamid Co., Princeton, New
Jersey.




-3-


TABLE 3

DRY MATTER AND NUTRIENT COMPOSITION OF BASAL SUPPLEMENT AND HAY


Moisture-free basis, %
Dry Crude Gross energy,
matter Cellulosea/ protein Ash kcal/gm.


Pangolagrass hay 90.6 36.5 4.4 3.3 4518

Basal supplement 85.9 4.3 21.1 9.2 4361

a/-
-'Determined by method of Crampton and Maynard (1938).


Results and Discussion

All steers were gaining in weight during the experiment with the
average gain being 11 kg and the range in individual gains being from
5 to 15 kg during the 31-day period. All were apparently healthy as
judged by blood composition data shown in table 4.

Citrus oil at the 10% level had a limiting effect on intake of the
supplement. See table 5. The average daily intake of.supplement con-
taining 10% citrus oil was 754 gm., compared to 4500 gm. during the
previous 14 days.' The average daily consumption of citrus oil was 75
gm. There was a marked individual variation in acceptance or tolerance
of the supplement containing citrus oil. See table 6. Steer no. 1
consumed nearly the same amount of supplement containing 10% citrus oil
on each of the 12 days but the other four steers offered this supple-
ment showed more day to day variation. The consumption of citrus oil
did not have an evident effect on hay intake. A reduction in average
daily water intake of 13 liters apparently reflected a reduction in
total dry matter intake. Average daily water intake during the pre-
liminary period ranged from 27.8 to 39.9 liters. The average daily
intake of water while consuming the supplement containing 10% citrus oil
ranged from 18.9 to 26.1 liters. Control steers also reduced their
average daily water intake by about 4 liters during this period indicat-
ing that environmental changes may have been contributing factors.

Because 10% citrus oil in the supplement limited intake of supple-
ment to such a low level (754 gm. per day) and because the steers had
exhibited very little increase in tolerance (daily consumption) a
decision was made to reduce the level of citrus oil in the supplement
to 5%.








TABLE 4

VENOUS BLOOD COMPOSITION AT BEGINNING AND END OF. EXPERIMENT BY AINALL


NUMBER AND TREATMENT:


ONE INDICATION OF STATUS OF HEALTH


Steer
No.
Treated ,


Hemoglobin,
gram % ,
IL F


L 15.5
4 13.9
5 13.2
9 12.5
6 13.9
Average 13.8


Controls
3 13.5
6 13.4
11 15.6
14 14.1
57 15.5
Average 14.4


14.9
11.9
13.8
13.3
12.7
13.3


12.3
14.1
13.5
13.0,
14.1
13.4


iHematocrit,

I F


50
42
41
38-
43
43


41
43
48
43
49
45


53
39
*44
-45
44
45


39
48
44'
43
47
'44


/I = initial, F = final.


1

1


RBC
x 106
I F


WBC
x 1000
I F


8.2
7.2
7.3
6.6
7.4
7.2


7.3
7.2
8.2
7.3
8.8
7.5


8.3
5.9
8.0
6.9
6.9
7.2


6.7
8.2
6.7
6.4
7.6
7.1


7.9
8.6
9.7
8.8
9.1
8.8


7.4
6.5
7.2
7.7
8.0
7.4


10,3
8.5
10.0
8.2
10.1
9.4


8.1
7.2
8.8
8.5
10,1
8,5


- m






1APLE 5


AVERAGE DAILY INTAKE OF SUPPLEMENT-


CITRUS OIL, HAY AND WATER BY STEER,


PERIOD A D TREATMENT


Treated Steers Control Steers
Treatment Citrus Citrus
and Steer Supp. oil Hay Water, Steer Supp. oil Hay Water,
period No. gm. gm. gm. liters No. gm. gm. gm. liters

Preliminary 1 4500 0 3719 27.8 3 4500 0 4682 35.3
Day 1-14 4 4500 0 3865 34.2 6 4500 0 4621 35.0
5 4500 0 6195 39.9 11 4500 0 4180 --
9 4500 0 5673 39.4 14 4500 0 5124 35.0
16 4500 0 5087 35.6 57 4500 0 6814 43.9
Lot average 1500 -0 7909 35.4 4500 0 5-0 37.3

10% 1 380 42 2864 18.9 3 4500 0 5918 29.6
Citrus oil 4 1045 116 4287 20.1 6 4500 0 5171 29.9
Day 15-26 5 1071 119 4874 26.1 11 4500 0 3825
9 418 47 7521 26.1 14 4500 0 5067 31.8
16 480 53 5042 23.3 57 4500 0 6971 40.9
Lot average 679 75 4918 22.9 450- 0 5390 33.1

5% 1 1450 76 4752 21.1 3 4500 0 5616 37.7
Citrus oil 4 2386 129 4378 27.4 6 4500 0 4942 34.0
Days 27-31 5 2876 151 6016 33.4 11 4500 0 3582 --
9 1474 78 5260 31.7 14 4500 0 4966 34.0
16 1824 96 5208 29.2 57 4500 0 6370 43.0
Lot average 2002 1 5123 26 i500 0 5095 37.2


a/Does not include 200 gm. vitamin-antibiotic premix per day. All figures are on air-dry basis.







- 6 -


TABLE 6


VARIATION


IN DAILY INTAKE OF SUPPLEMENT CONTAINING CITRUS OIL
BY ANIMALS AND TREATMENTS'


Animal Average daily Standard
Treatment No. intake deviation Range
gm. g gm. gm.


Control




Lot average
Gm. offered



10% ,
Citrus oil



Lot average
Gm. offered



5%
Citrus oil



Lot average
Gm. offered


1
4
5
9.
16


4500
4500
4500
4500
4500
4500
4500


1
5
9
16


422
1161
. 1190.
465-
533
750
5000


1526
2585
3027
1552
1920
2122
S4740


22
381
614
258
348
357-


14o
522
792
575
381
T2-


790
1370
2070
8CO
1240


280
1150
1700
1290
900


-





-7


Average daily intake by individual steers of supplement containing
5% citrus oil averaged 2122 gm. compared to 754 gm. when the supplement
contained 10% citrus oil and 4500 gm. when none was added to the supple-
ment. The average daily intake of citrus oil was 106 gm. as compared to
75 gn. when the supplement contained 10% citrus oil. The variation in
intake among individual steers was wide and ranged from 1526 to 3027
grams of the supplement containing 5%o citrus oil. Again, there was
little effect of the citrus oil on average daily hay intake. Average
daily water intake of steers offered the supplement containing 5% citrus
oil was increased by 5.7 liters over the intake of 22.9 liters when they
were fed the supplement containing 10% citrus oil. The average daily
water intake of control steers was 4.1 liters higher; consequently, other
factors may have affected water intake.

In sum, citrus oil limited intake of the supplement in which it was
mixed. The limiting effect seemed to be related to the concentration of
citrus oil in the supplement rather than the actual amount consumed.
Larger amounts of the supplement were eaten at the 5% than 10% level of
citrus oil. Citrus oil in the supplement had no apparent effect on hay
intake. Citrus oil in the supplement affected water intake insofar as
it regulated dry matter intake; however, environmental factors seemed to
have an effect on water intake.


TRIAL TWO

Procedure

This experiment was conducted with 20 yearling steers which averaged
385.kg (850 lb.) of initial weight. They were individually fed and han-
dled like those in Trial 1. The steers were randomly allotted to four
lots and were offered supplements containing the following percentages of
citrus oil: none, 2.5, 5.0 and 7.5. See table 7 for basal supplement
composition. All steers were offered grass hay ad libitum. A vitamin
premix was added to each supplement to insure a daily intake of at least
30,000 International units vitamin A and 3500 U.S.P. units vitamin D2.
An intramuscular injection of one million I.U. vitamin A had been admin-
istered to each steer prior to the experiment to provide for liver reserves
of this vitamin.

The citrus oil was obtained from Florida Citrus Oil Co., Bartow,
Fla. and was mid-season orange oil which had more aroma than those used
in Trials 1 or 2. The cost of this product varied from 25 to 35 cents
per pound during the period of thesp experiments.








TABLE .7

INGREDIENT COMPOSITION OF BASAL. SUPPLEMENTS
OFFERED TO STEERS IN TRIAL,.2


Ingredients

Soybean oil meal, 50% 40

Corn meal, yellow 45

Cane molasses, std. 10

Phosphorus source!/ 2

Trace mineralized salt 2

Vitamin premix-/ 1


1/Defluorinated rock phosphate, min. 18% P.
2/Perma-Dual 30A and Perma-D 16D2 provided gratis
by Commercial Solvents Corporation, Terre Haute,
Indiana.


Results and Discussion

All steers gained weight, averaging 726 gm. (1.6 lb.) of gain per
day. All were in apparent good health at the termination of the 42-day
trial.

Voluntary consumption of supplement, as shown in table 8, varied
from the consumption of all the 4000 gm. offered in lot 1 to 3950, 2589
and 2279 gm., respectively, for lots 2, 3 and 4. The 2.5% level of
citrus oil did not effectively reduce voluntary intake of supplement
but the 5.0% level limited average intake to 1141 gm. less than the
amount offered. A further average reduction of 310 gm. per day was
recorded when the citrus oil level was increased to 7.5%.

As in the previous trial, considerable variation among steers was
observed. At the 5% citrus oil level, one steer consumed only 1813 gmn
of the 4000 gm. of supplement offered to him daily while the remainder
consumed 2586, 2633, 2815 and 3097 gm., respectively. At the 7.5% level
of citrus oil, the magnitude of individual variation was similar, ranging
from 1819 to 2877 gm.

The supplement containing 5% citrus oil was consumed in larger
quantities in this trial (2589 gm.) than in Trial 1 (2122 gm.) in spite
of the larger average weight of steers in Trial 1 (463 vs. 385 kg).
Whether this differential in response reflected a difference in chemical




-9-


TABLE 8

EFFECT OF THREE LEVELS OF CITRUS OIL IN A SUPPLEMENT ON THE AVERAGE
DAILY INTAKE OF SUPPLEMENT, HAY AND WATER BY INDIVIDUAL STEERS


Av. Daily Intake
Animal Supplement, Hay, Water,
Lot No. and Treatment No. gm. gm. gal.


1. Basal supplement,
without citrus oil







2. Basal + 2.5% citrus oil








3. Basal + 5.0o citrus oil







4. Basal + 7.5% citrus oil


1
5
9
17
18
Treatment
Means


2
4
8
12
14
Treatment
Means


6
7
10
11
13
Treatment
Means


3
15
16
19
20.
Treatment
Means


4000
4ooo

4000
4000
4000
4000



3970
3979
3821
3982
3950


1813
2633
3097
2586
2815
2589


1819
1836
2005
2859
2877
2279


8280
4652
6901
4984
5639
6091


6549
5299
5309
5968
5501
5725


5147
5932
8192
6876
6325
6494


4326
5806
6624
6880
5820
5891


11.6
8.7
11.2
9.5
9.6
10.0


11.8
9.1
9.0
9.4
10.0
9.9


7.4
10.8
11.7
9.5
9.6
9.8


6.8

7.6
10.3
13.5
9.6




- 10 -


properties of the midseason orange oil used in Trial 2 and the stripper
oil, largely D-limonene, used in Trial 1 is not known. The latter was
a by-product of citrus molasses processing, was not as aromatic and had
no color. The variation of supplement consumption among steers was
about the same for each trial, ranging from 1526 to 3027 gm. in Trial
1 and 1813 to 3097 gm. in TriE'2 2.

. Hay consumption was not consistently affected by the consumption
of citrus oil. The larger average hay intake of lot 3 might have
reflected to a small extent their smaller supplement intake but the
unusual appetite of steer number 10 for hay throughout the experiment
appeared to be a contributing factor.

Water consumption data were recorded and are reported in table 8
*but no significance is attached to the small differences among lots.
Individual steer variation was large. Some meters were damaged during
freezing weather causing the loss of some data.

In sum, citrus oil was effective as a limiter of supplement intake
when added at 5 and 7.5% levels to a concentrate mixture offered ad
libitum to long yearling steers. While.either 5 or 7.51 citrus oil was
effective on the average, a large individual steer variation was observed
with each level. This variation was not as marked as that reported earlier
(Animal Science Mimeo Series No. AE68-4 ) when various levels of sodium
chloride were studied as limiters of supplement intake. The observed
limiting of supplement intake without limiting intake of hay may justify
further sturdy of citrus oils for the regulation of intake of supplements
for grazing ruminants under pasture conditions. Problems could arise
from the volatile nature of citrus oil if it were subjected to feed
processing methods which cause high temperatures, namely steam pelleting
or block compression. -Before commercial use of citrus oils could be
advocated in ruminant animal feeds, studies would have to prove that
there were no detrimental effects of d-limonene on the bacterial and
protozoal populations of the forestomach (ruminoreticulum).


TRIAL THREE

Procedure

This trial was divided into two parts, the first being an in vivo
study of the effects of citrus oil on rumen fermentation in fistulated
steers and the second part being an in vitro study of the effects of
citrus oil on rumen fermentation of cellulose and hay.

In the first part, four rumen fistulated Hereford steers were fed
low-quality pangolagrass hay ad libitum and a concentrate supplement
(table 9).





- 11 -


TABLE 9

INGREDIENT COMPOSITION OF BASAL SUPPLEMENT


Ingredient

Soybean meal, 50% 30
Corn meal 56
Cane molasses, std. 10
Trace mineralized salt 2
Defluorinated phosphate 2
Aureomycin and Vitamins A, D, E +
100


After a preliminary period of 14 days, 2 of the 4 steers were
changed to a 10% citrus oil supplement prepared by mixing 9 parts of
basal supplement with 1 part of citrus oil (orange oil). Supplements
were offered to each steer in amounts to provide 8 gm. of basal sup-
plement per kg body weight (8 lb./1000 lb.). Because the 10% citrus
oil supplement was unpalatable, it was mixed with equal parts of basal
supplement to make a 5% citrus oil supplement. Each steer consumed
nearly all of the 5% supplement.

After 14 days of access to the 5% supplement, samples of rumen
fluid were obtained at 0, 2, 4 and 18 hours post-feeding. It was
observed that steers fed the basal supplement consumed all that was
offered within one hour while those offered the 5% citrus oil supple-
ment had consumed only 170 to 210 gm. by the end of the 4-hour (after
feeding) rumen sampling period. This made proper comparisons between
treatments impossible.

In order to compare.the fermentation of the two supplements during
comparable time periods, the basal and 10% citrus oil supplements were
administered through the rumen fistulas. This was done for only 3 days
because the two steers receiving the citrus oil supplement sharply
reduced their feed intake. These steers consumed 252 to 283 gm. citrus
oil per day.

All steers were then returned to 2.5 kg basal supplement per day
for the remaining four days of the experiment. Rumen fluid samples
were taken 18 hr. after the last force-feeding of citrus oil and 18
hr. after feeding for four additional days.


Results and Discussion

Feed intakes for individual steers throughout the trial are shown
in tables 10 and 11.




- 12 -


TABLE 10

DAILY HAY INTAKE PER STEER, KG

Day Citrus oil Basal supplement Citrus oil suppo
of in supp,, Steer Steer Steer Steer
trial'/ #1 #2 #3 #4

1 5 6.6 7.0 7.0 7.0
2 5 7.4 7.7 7.2 7.6
3 5 6.8 7.4 6.9 6.7
4 5 7.3 7.7 6.6 7.6

5 10/ 7.3 7.7 1.7 2.9
6 lo1 5.0 7.7 -- 1.2 3.1
7 102/ 4.9 7.7 0.9 1.8
8 0 6.3 7.5 0.1 2.0
9 .0 6.0 7.5 0.5 0.6
10 0 6.3 7.5 1.2 2.9
11 0 7.5 7.5 .6.0 6.0

1/Preliminary period preceded day 1 shown here.
2/Supplement administered through rumen fistula.


TABLE 11

DAILY SUPPLEMENT INTAKE PER STEER, KG

Day Citrus oil Basal supplement Citrus oil supp,
of in supp., Steer Steer Steer Steer
trial #1 #2 #3 #4

1 5 2.43 2.58 2.52 2.03
2 5 2.43 2.58 2.52 2.68
3 5 2.43 2.58 2.52 2.31
4 5 2.43 2.58 2.52 2.26

5 10/ 2.43 2.58 2..2 2.68
6 10.. 2.43 2.58 2.66 2,83
7 102/ 2.43 2.58 2.66 2.83
8 0 2.50 2.50 0. :5 2,50
9 0 2.50 2.50 1.o00 .10
10 0 2.50 2.50 2.50 1.53
11 0 2.50 2.50 2.50 2.50

-/Preliminary period preceded day 1 shown here.
E Supplement ac'ninis-'er-d roughh rumen fistula.





- 13 -


These data indicate that 5% citrus oil in a supplement fed normally
had no effect on hay or supplement intake. Fistula administration of thc
basal supplement depressed hay intake only slightly in one steer. However,
citrus oil force-fed depressed hay intake markedly and immediately. Good
recovery of hay and supplement intake was achieved in 4 days after dis-
continuing citrus oil. The amount of citrus oil force-fed at one time
was approximately double the amount previously consumed slowly over an
18-hour period.

Rumen fluid composition after 14 days on basal versus 5% citrus oil
supplements is shown in table 12.


TABLE 12

RUMEN FLUID COMPOSITION IN STEERS FED BASAL AND 5% CITRUS OIL
SUPPLEMENTS (AVERAGE OF 2 STEERS)


Hours Supp. pH TVFA3/ C2/C3/ NH3-N2/
(mM/ iT) (mg%)

02/ Basal 6.35 89 3.81 8.88
Citrus oil 6.17 96 3.96 7.17

2 Basal 5.98 111 3.78 16.85
Citrus oil 6.15 96 3.97 9.70

4 Basal 5.89 113 3.72 14.52
Citrus oil 6.29 96 3.98 9.59

18 Basal 6.01 117 3.73 7.04
Citrus oil 5.99 107 3.94 10.70


i/After feeding
2/Pre-feeding
3Total Volatile Fatty Acids
.Acetate:propionate ratio
2/Ammonia-Nitrogen


These data indicate very little difference between diets at 18
hours after feeding or pre-feeding (0 hours). However, when the basal
supplement was fed, there was a rapid decrease in pH and increases in
TVFA and NH3. These changes indicate rapid fermentation of the supple-
ment. The lack of such changes in steers fed citrus oil is due to a
lack of supplement intake during the 4-hour post-feeding period.

Rumen fluid composition 18 hours after the third administration
of supplements by fistula and for 4 consecutive days thereafter is
shown in table 13.




- 14 -


TABLE 13.

RUMEN FLUID COMPOSITION AFTER FISTULA ADMINISTRATION OF SUPPLEMT
WITH AND WITHOUT CITRUS OIL

Dayl .Supp. -pH TVFA/, C2/C3I 1-H-

(mM/1) (5)

7 Basal 6.39 103 4.11 1.53
Citrus oil 6.53 128 2.65 44.67

8 Basal 6.09 115 3.87 3.38
Citrus oil5/ 6.75 62 3.85 17.43

9 Basal 6.09 115. 3.81 6.76
Citrus oil 6.10 76 4.07 12.17

10 Basal 6.37 105 3.91 O1.CO
Citrus oi5l 5.64 98 3.90 18.4'

11 Basal 6.02 113 3.80 7.59
Citrus oil5- 5.66 95 3.58 18.22


l/Corresponds to days in


tables 10 and 11; samples taken 18 hours pos-


feeding.
iTotal Volatile Fatty Acids
.3Acetate:propionate ratio
V/^Ammonia-Nitrogen
5/Citrus oil discontinued after day 7;
previously fed citrus oil.


these averages are for those steers


On day 7, the rumen fluid composition of steers fed citrus oil was
characterized by higher TVFA concentration, lower C2/C3 ratios and higher
rumen NH3 than that of steers fed the basal supplement. Hay intake had
been decreased by the citrus oil, so only the grain supplement was avail-
able for fermentation. The composition of the rumen fluid indicated that.
the corn meal and soybean meal were fermented. The decrease in TVFA on
day 8 reflects the lower supplement intake after returning to normal
feeding. Rumen composition gradually.returned to:that of the basal-fed
steers, except for pH. The decreased pH on days 10 and 11 in steers
previously fed citrus oil may have :been due to a lower hay:concentrate
ratio since less hay was offered these steers.

The differences observed in these studies may be attributed to two
factors:

1. Five percent citrus oil added to a supplement depressed the
rate of consumption of the supplement without reducing the
total amount of supplement consumed.




15 -


2. Citrus oil added to the rumen by fistula decreased hay consump-
tion but did not prevent fermentation of the supplement.

The second part of this study was done to further investigate the
effects of citrus oil. on rumen fermentation. Two in vitro experiments
were conducted, one with purified cellulose and the other with bermuda-
grass hay as the substrate. Treatments were graded levels of citrus
oil (orange oil) or d-limonene (major constituent of citrus oil). The
highest concentration used, 10 mg/ml, was equivalent to the estimated
rumen concentration in a steer with 50 1. rumen fluid and consuming
500 gm. of citrus oil per day. The additives were mixed with the dry
substrate in fermentation tubes and 30 ml. media were added. The
inoculum was 20 ml. of strained and centrifuged rumen fluid. After
24 hr. fermentation, residual cellulose and volatile fatty acids were
determined. The percent of initial cellulose digested and the synthesis
of individual and total volatile fatty acids were calculated. Table 14
presents the results of the two fermentations.

TABLE 14

EFFECTS OF D-LIMONENE AND ORANGE PEEL OIL ON IN VITRO RUMEN
FERMENTATIONS (24 HOURS; 30 ML MEDIA PLUS 20 ML INOCULUM)


Cellulose TVFA Molar % (Synthesized)
Treatment Digested Synthesized Acetate Propionate Butyra.e
mg/ml mM

-------------------------- Bermudagrass-----------------------------

Control 0.00 65.5 4.57 68.3 25.2 6.6
d-limonene 0.01 66.2 4.84 68.0 25.6 6.5
0.10 64.9 4.09 68.2 25.8 6.1
1.00 15.8 1.34 49.7 18.7 31.6
10.00 4.7 1.09 39.6 47.0 13.4
Orange Oil 0.01 65.9 4.31 67.9 25.9 6.3
0.10 65.1 4.69 69.5 25.3 5.3
1.00 24.1 1.74 54.0 18.8 27.2
l0.o0 3.7 0.42 94.1 0.0 5.9

----------------------------ellulose/-------------------------------

Control3/ 0.00 78.3 5.00 43.7 54.9 1.3
d-limonene 0.01 71.2 4.65 44.1 54.9 1.0
0.10 67.7 3.90 42.7 53.9 1.0
1.00 7.2 0.08 78.2 0.0 21.9
10.00 8.5 0.00
Orange Oil 0.01 73.8 4.66 43.4 55.6 1.1
0.10 85.8 2.56 47.1 51.9 0.9
1.00 7.5 0.02
10.00 6.3 0.01


I/Ground to pass 40 mesh sc:.een; 1.000 gm/tube; 2'-.'l cellulose.
2/Sol.ka- loc WJ 40; 1.000 gE/tube; 84.7/o cellulose.
3J/One tu,;e only, the duplicate wai lost.






16 -



The digestion of bermuhagrass and purified cellulose was unaffected
by 0.01'and 0.1 mg/ml of citrus oil and d-limonene. The 1.0 mg/ml level
of both additives inhibited bermudagrass cellulose digestion, but less
than did the 10.0 mg/mi level..1 Both higher levels were equally inhibitcr;
to purified cellulose digesti6n.

Total volatile fatty acid synthesis during bermudagrass fermenta-
tion was unaffected by 0.01 and 0.1 mg/ml of either additive, while the
0.1 mg/ml level inhibited TVFA synthesis during purified cellulose fer-
mentation. While TVFA synthesis during bermudagrass fermentation was
inhibited by 1.0 and 10.0 mg/ml of either additive, there were some
TVFA's synthesized even when cellulose fermentation was almost completely
inhibited. This would indicate that citrus oil and d-limonene did not
completely inhibit fermentation of the non-cellulose material cf bermuda-
grass, such'as soluble carbohydrates, hemicellulose and proteins. This
is consistent with the continued fermentation of grain previously observed
in fistulated steers administered citrus oil by fistula.

There were no changes in the molar proportions of the volatile
fatty acids synthesized due to the two lower levels of d-limonene and
citrus oil additions. With the two higher levels, bermudagrass fer-
mentation patterns were altered. Consistent increases in butyrate,
with decreased acetate, were observed for both d-limonene and citrus
oil at the 1.0 mg/ml level.

These studies suggest that the decreased hay intake due to citrus
oil observed in the previous trial may have been due to decreased cel-
lulose digestion rates. Of particular interest is the apparent differ-
ence in the inhibitory action of citrus oil on cellulose fermentation in
comparison to non-cellulose organic nutrients. The inhibitory action o-
citrus oils seems to be primarily, if not completely, due to d-limonene.
Dried citrus pulp would not be expected to contain d-limonene since this
compound is volatile.

GENERAL SUMMARY

Citrus oils, which are by-products obtained from fruit peel waste
and from molasses production, are effective limiters of intake of con-
centrate feed mixtures: supplements) offered ad libitum to mature rumi-
nating cattle. At a concentration of 5%, midseason change oil and
citrus stripper oil composed of more than 95% d-limonene, s owed the
rate of consumption and limited daily consumption of a concentrate
mixture to about 5 to 6 lb. (2200-2700 gm.) without decreasing consump-
tion of grass hay. A large variation in response (voluntary daily intake
of supplement) existed among individual steers; consequently, citrus oils
are not precise, uniform regulators of supplemental feed intake for
cattle. The effect of citrus oils on forestomach (ruminoreticulum) fer-
mentation is not comet ely understood; however, a 5% concentration in
supplement apparently did not inhibit fermentation of suppler.ent or hay
in yearling steers. An artificial rumen (in vitro) study indicated th:-.
cellulose and bermudagrass digestion was inhibited by a low level of
citrus oil (1 mg/ml) in a synthetic rumen flui.d




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