Effect of Propionate and Citrate on Depressed Feed Intake After Intraruminal Infusions of Acetate in Dairy Cattle

TECHNICAL

NOTES

Effect of Propionate and Citrate on Depressed Feed Intake After Intraruminal Infusions of Acetate in Dairy Cattle Abstract

Intraruminal infusions of acetic acid (neutralized to p H 6.5) reduced hay consumption in fistulated dairy heifers by 39%. Substitution of a portion of the acetate with an equicalorie amount of propionate prevented the depression. Addition of citrate to the acetate partially alleviated the depression. Temperature of the tympanum of the ear increased following infusion of acetate, but was maintained lower by the acetate-propionate mixture. The data suggest that a balance of the volatile fatty acids in the tureen may be a significant factor in controlling their voluntary feed intake. The effect of increased ruminal levels of steam volatile fatty acids on feed intake behavior of sheep and cattle has been studied by several workers (7, 8, 15, 16). Some (18, 19, 21) have observed a depressing effect on feed intake, from an intraruminal infusion of free acetic acid. I n addition to its function as a metabolite, it may be that this effect was in part due to physical discomfort (t4, 18), inhibition of muscular contractions of the ruminoreticulum (4, 6), or other effects resulting from a lowered rumen p H (10, 14, 19). Propionic acid showed no effect with heifers over long term (19) or short term (18) infusions. I n sheep, however, propionic acid infused daily for 14 days depressed intake significantly (14). Results of intraruminal infusion of neutralized acetic acid have been variable. Cows fed hay showed no effect following acetate infusion (18, 20). However, intake of cows receiving pellets was depressed (20). Intraruminal propionate administration, on the other hand, reduced feed consumption in cows fed either hay or pellets (20). Preliminary experiments in this laboratory indicated that propionate could modify the depressing effect of acetate infusions upon voluntary feed intake. The purpose of this experiment was to determine more definitely the effect of intraruminal infusion of acetate on feed intake in dairy heifers and to examine the influence of propionate or citrate in modifying this effect when infused with acetate. Experimental Procedures

had a crude protein content of 14% on a dry basis. The animals were kept indoors in individual stanchions equipped with rubber mats and no bedding. Water and trace mineralized salt blocks were continuously available. The heifers were fed the hay for a period of four weeks to establish a uniform intake prior to starting the experiment. The experimental design consisted of a 5 X 5 Latin square. The intraruminal infusions were 1) water, 2) acetate, 3) acetate ~- propionate, 4) acetate + citrate, 5) citrate. Propionate and citrate were chosen to supply a source of three-carbon intermediates which might aid in the efficiency of acetate utilization. Each trial consisted of a six-day preliminary period followed by a single day of infusion. The acids used were glacial acetic, propionie acid (100%), and granulated citric acid (100%). These were dissolved in six to seven liters of water, neutralized to a p i t of 6.5 with sodium hydroxide solution, and diluted to a final volume of eight liters. This solution was infused once, one hour before the 9 AM feeding. Control animals received a tap water infusion of the same volume and at the same time as the experimental animals. The amount of free acid that was neutralized and infused was related to body weight and the dosages per kilogram live weight are shown in Table 1. A representative sample of rumen fluid was collected through the fistula, one hour after infusion, and p H determined immediately with a glass electrode. After the six-hour feeding period, jugular blood was collected for analysis of glucose, urea, and steam volatile fatty acids. TABLE 1. Infusion of several metabolites on voluntary feed intake in heifers. Hay intake

Treatment infusion

Level of infusion 1~ ca]

g

Control period

(per kg wt) a Water (control) Acetate Acetate -F propionatee Acetate ~citrate Citrate

Experimental period (kg)

............ 3.49 1.00

11.9 12.3

12.2~ 7.4~

3.49

0.91

12.0

11.5"

5.73 2.26

1.99 0.99

12.1 12.5

8.1b~ 11.2~

Trials were conducted with five rumen-fistu.. b. ~ Means on the same coIumrLhaving different lated Holstein heifers weighing an average of superscripts are significantly different (P ~ 0.01; 600 kg. They were fed a chopped hay ration ~P < 0.05). ad libitum for six hours daffy from 9 A~ to 3 aExpressed as the free acid. e4 to 1 molar ratio. P~. The mixed hay was cut in early July and 1091

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The blood and rumen data are shown in Table 2. The depression of intake could not be attributed to a fall in rumen p H (10), as little variation was observed. Total blood V F A ' s were significantly increased while urea was affected little by the i n f u s i o n s , although the trend was towards a lower value. Blood glucose was increased by all treatments. The temperature recordings of two of the heifers infused are shown in F i g u r e 1. The acetate infused group showed a distinctly higher t y m p a n i c temperature throughout

Blood glucose was determined, using the glucose oxidase method (22). Blood urea was determined according to the method of Cou]ombe and F a v r e a u (12). Total steam volatile f a t t y acids were determined by steam distillation in a Markham still (17), according to the procedure of Annison (1). Data f r o m the trial were subjected to an analysis of variance. The multiple range test (13) was used to test f o r significance among treatment groups. I n a subsequent preliminary trial, two heifers were used to observe rectal temperature and the temperature of the tympanic membrane of the ear following daily infusions of water (control), acetate, or acetate plus propionate at the levels described in Table 1. The infusion schedule was identical to the previous experiment. The method used for temperature measurement was according to Bhattaeharya and W a r n e r (11).

TABLE 2. Blood and rmnen data as influenced by intraruminal infusion of acetate, acetate-tpropionate, acetate Jr citrate, and citrate in heifers. Blood values Treatment infusions

Rumen pH

Results

Daily air-dried hay consumption is shown in Table 1. I n f u s i o n of acetate significantly depressed intake, while an equicaloric mixture of acetate plus propionate showed no such depression. The level of citrate used here did not depress intake and, in a p r e l i m i n a r y experiment (9), even twice this level appeared not to influence intake. W h e n citrate was added to the basal level of acetate, intake was depressed significantly less than for acetate alone.

Glucose

Urea

YFA (meq/ liter)

--(mg/100 ml)-Water (control) Acetate Acetate -~propiona.te Acetate + citrate Citrate

6.80 6.87

42.6" 51.3

39.5 32.5

0.58 ~ 1.01

6.80

47.6

33.0

1.21

6.90 6.94

47.5 47.3

29.0 * 35.0

0.97 0.72

" Significantly less (P < 0.01) than the other values in the same column.

40.0

TREATMENTS: CONTROL 39.5 A

u

ACETATE

-.

ACETATE + PROPIONATE

-.

TIME OF I N F U S I O N

o v

t

uJ

ID

LLI

I"

38"0 I 37.5

i -1

t 0

i

I 1

I

I 2 HOURS

i

I 3 OF

I

I 4

I

I 5

I

I 6

I

I

FEEDING

FIG. 1. Effect of intraruminal infusion of water, acetate, or acetate plus propionate solutions on tympanic (ear drum) temperature pattern in heifers. "Vertical lines are standard errors and apply to all treatment means at any given time. J . DAIRY SCIENCE VOL. 51, NO. 7

TECHNICAL NOTES

the feeding period, as compared to the control. A t times the difference narrowed to zero. The acetate + propionate group was somewhat lower. Discussinn

The depressing" effect of intraruminal acetate infusion on hay intake in heifers has not been reported before. Simkins et al. (20) did not find any reduction in hay intake when cows were receiving infusions of acetate at the rate of 1.3 or 1.7 g of the acid per kilogram of body weight over a five-hour ~)eriod during the morning feeding. However, they (20) found a significant depression in the consumption of pellets containing 60% alfalfa meal and 40% ground corn under similar conditions while infusing the neutralized acid at the rate of 1.3 or 2.1 g / k g body weight. In sheep, however, Egan (14) depressed hay intake in two out of four when infusing neutralize~t acid (0.6 g / k g body wt) over a four-hour period daily for five days. The decline in feed consumption was observed only after the first day. The present studies add a new parameter to the problem, in that a single, substantial load of acetate (1.0 g / k g live weight) infused prior to a meal markedly depressed the intake of hay. This depression was not evident when an eqniealoric amount of 4 to 1 molar ratio of neutralized acetic and propionic acid was used. While the possibility exists that the failure to reduce the intake was due to a reduction in acetate infused, the data also suggest that a synergism between tureen levels of acetate and propionate may have a profound effect on feed intake patterns. Propionic acid or propionate per se has been shown to reduce intake (14, 20) and a combination of acetate and propionate (molar ratio of about 3 to 1) has also reduced intake (14) when administered over one day. In another experiment, however, Egan (14) infused free acetic acid at a level of 0.3 g / k g live weight per day for 14 days and caused a decline in feed intake. Propionic acid at a level of 0.12 g / k g live weight did not ~ffect intake when administered in the same manner. When an additive combination of these metabolites was used, intake was not depressed, strongly suggesting that a synergism between these two metabolites exists. Our observations tend to confirm those of Egan (14) and suggest that a fine balance of intraruminal metabolites may be critical in affecting voluntary food intake patterns. As citrate~ while not depressing intake of itself, was able to partially alleviate the depressing effect of acetate, it appears that the balance is not a simple one. The fact that Simkins et al. (20) depressed pellet intake but not hay intake when a mixture of acetic, propionic, and butyric acids was infused supports the contention that the relationship is complex.

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The mechanism of action of the infused metabolites is not clear. Rumen p H is not the answer (9). The somewhat lower tympanic temperature following infusion of acetate and propionate compared to acetate alone may offer a clue. While the infusion of acetate at the rate of 1.0 g of acetic acid equivalent per kilogram live weight increased tympanic temperature in our studies, Baile et al. (5) showed a decline in medial hypothalamic temperature when approximately 0.4 m g / k g of acetic acid were injected abruptly in the rumen. Clearly, differences between the experimental techniques may account for these differences in results. The reduced intake in the acetate group could be interpreted as an over-all reflection of its high heat increment (2, 3). The synthesis of long chain fatty acids, the unavailability of glycerol, and the deamination due to a consequent gluconeogenesis are factors contributing to this thermal imbalance. The observed effect due to inclusion of propionate or citrate could be explained by their being glucogenic. Additional data must be available before a rational hypothesis integrating the relative impact of the chemostatic and thermostatic mechanisms on voluntary intake control can be formulated for ruminants. Acknowledgments

This study was supported in part by a grant from the United States Department of Agriculture CSRS, no 427-15-17. The authors gratefully acknowledge the assistm~ce of l%bert Brokaw and Vance Eakins for their expert care of the experimental animals and the assistance of Marlin Hiller with the chemical analyses. ASOK NATH BHATTAGHARYA and R. G. WARNER Department of Animal Science Comell University, Ithaca, New York References

(1) Annison, E. P. 1954. Studies on the volatile fatty acids of sheep blood with special reference to formic acid. Biochem. J., 58: 670. (2) Armstrong, D. G., and K. L. Blaxter. 1957. The heat increment of steam volatile fatty acids in fasting sheep. Brit. J. Nutrition, 11 : 247. (3) Armstrong, D. G., and K. L. Blaxter. 1957. The utilization of acetic, propionic and butyric acids by fattening sheep. Brit. J. Nutrition, 11: 413. (4) Ash, 1% W. 1956. Inhibition of reticulorumen contraction by acid. J. Physiol., 133 : 75. (5) BMle, C. A., A. W. Mahoncy, and J. Mayer. 1967. Feeding behavior and hypothalamlc temperature of goats. J. Dairy Sci., 50: 1854. (6) Baile, C. A., and W. It. Pfander. 1966. A possible chemosensitive regulatory mech,~. DAIRY SCIENOE ~V-C~5,51, NO. 7

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(7)

(8)

(9) (10)

(11)

(12) (13) (14)

J O U R N A L OF D A I R Y SCIENCE

anism of ovine feed intake. Amer. J. Physiol., 210: 1234. Bentley, O. G., R. R. Johnson, E. W. Klosterman, J. If. Cline, and A. L. Moxon. 1960. Feed rich in growth stimulating factors for rumen organisms and certain volatile f a t t y acids as supplements for rations for cattle and sheep. Ohio Agr. Exp. Sta., Res. Bull. 863. Bentley, O. G., R. :R. Johnson, G. W. Royal, F. Deatherage, L. E. Kunkle, W. J. Tyznik, and D. S. Bell. 1956. Studies on the feed~ ing value of acetic, propionic and lactic acids with growing fattening lanlbs. Ohio Agr. Exp. Sta., Res. Bull. 774. Bhattacharya, A. N., and R. G. Warner. 1967. Unpublished data. Bhattacharya, A. N., and R. G. Warner. 1967. Rumen pIf as a factor for controlling feed intake in ruminants. J. Dairy Sci., 50 : 1116. Bhattacharya, A. N., and R. G. Wa.rner. 1967. Influence of varying rumen temperature on central cooling or warming and on regulation of voluntary feed intake in dairy cattle. J. Dairy Sei., 50 : 968. (Abstr.) Coulombe, J. J., and L. Favre'm. 1963. A new semi-micro method for colorimetrie determination of urea. Clin. Chem., 9: 102. Duncan, D. B. 1955. Multiple Range and Multiple ]~ Tests. Biometrics, 11: 1. Egan, A. R. 1966. Nutritional status and intake regulation in sheep. V. Effects of intra-ruminal infusions of volatile f a t t y

(15)

(16)

(17) (18)

(19)

(20)

(21) (22)

acids upon voluntary intake of roughage of sheep. Australian J. Agr. Res., 17: 741. Essig, If. W., V. S. Garrigus, and B. C. Johnson. 1962. Studies on the levels of volatile f a t t y acids for growing-fattening lambs. J. Anim. Sci., 21: 37. Essig, If. W., U. S. Ifatficld, and B. C. Johnson. 1959. Volatile f a t t y acid rations for growing lambs. J. Nutrition, 69: 135. Markham, R. 1942. A steam distillation apparatus suitable for micro-Kjeldahl analysis. Biochem. J., 36: 790. Montgomery, M. J., L. If. Schultz, and B. ~. Baumgardt. 1963. Effect on intra-ruminal infusion of volatile f a t t y acids. J. Dairy Sci., 46: 1380. Rook, J. A. F., C. C. Balch, R. C. Campling, and L. J. Fisher. 1963. The utilization of acetic, propionic and butyric acids by the growing heifer. Brit. J. Nutrition, 17 : 399. Simkins, K. L., Jr., J. W. Suttie, aa~d B. R. Baumgardt. 1965. Regulation of food intake in ruminants. 4. Effect of acetate, propionate, butyr~te, and glucose on voluntary food intake in dairy cattle. J. Dairy Sci., 48: 1635. Ulyatt, M. g. 1964. Studies on some factors influencing food intake in sheep. Proc. New Zealand Soe. Anim. Prod., 24: 43. Worthington Biochemical Corporation. 1963. Glucostat, a prepared enzymatic glucose reagent. Freehold, New Jersey.

Effect of Carbon Chain Length of Fatty Acids on Ketogenesis in the Ruminant Abstract E v e n - c a r b o n f a t t y acids with c h a i n lengths f r o m 2 to 18 were a d m i n i s t e r e d to goats in v a r i o u s metabolic states. T h e r e was a r a p i d k e t o n e m i a of decreasing magnitude a f t e r a d m i n i s t e r i n g the C, t h r o u g h C,o acids, s u g g e s t i n g direct a b s o r p t i o n f r o m the rumen, w i t h ketone b o d y f o r m a t i o n in the r u m e n wall or liver. I n fed animals, the C~ t h r o u g h C~8 acids h a d no effect on b l o e d ketones or glucose. I n f a s t e d a n d phlorizinized a n i m a l s e x h i b i t i n g h y p o g l y cemia, C8 caused increases b o t h in blood ketones a n d in blood glucose, while C16 h a d no effect on ketones, b u t did increase glucose. Results suggest e n o u g h early abs o r p t i o n in the r u m e n of the long-chain acids to s t i m u l a t e existing gluconeogenesis, b u t m a j o r a b s o r p t i o n in the lower tract, p r o b a b l y as triglycerides, w i t h o u t r e s u l t a n t ketogenesis. 1 Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. 2 Supported by N I H Grant AM08546-03 from the National Institute of Arthritis and Metabolic Diseases. ~. DAIRY SCIENCE VOL. 51, No. 7

The ketogenic n a t u r e of b u t y r i c acid, the lack of ketogenesis f r o m acetic acid, a n d the antiketogenic a c t i v i t y of p r o p i o n i c acid in the r u m i n a n t are well-known p h e n o m e n a (6). The i m p o r t a n c e of o t h e r f a t t y acids as ketogenic agents has not been v e r y clear. R e c e n t studies in m a n (1, 2) h a v e s h o w n t h a t the i n g e s t i o n of m e d i u m - c h a i n triglycerides caused a significant increase in blood ketones. The longchain triglycerides, however, h a d a negligible effect. I n monogastrics, it a p p e a r s t h a t the m e d i u m - c h a i n triglycerides are t r a n s p o r t e d almost e n t i r e l y via the p o r t a l s y s t e m to t h e liver, whereas a b o u t 8 5 % of the l o n g - c h a i n triglycerides are t r a n s p o r t e d v i a the l y m p h a t i c system w i t h o u t o b l i g a t o r y p a s s a g e t h r o u g h the liver ( 3 ) . The p u r p o s e of this s t u d y was to determine the effect of c a r b o n chain l e n g t h of f a t t y acids on the p r o d u c t i o n of k e t o n e bodies in the r u m i n a n t . These d a t a were r e p o r t e d previously in a b s t r a c t f o r m (7).

Experimental Procedure A p r e l i n l i n a r y t r i a l was conducted b y u s i n g one fistulated g o a t f o r each t r e a t m e n t . T h i r t y g r a m s of each of the e v e n - n u m b e r e d f a t t y acids