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Relation of Dietary Acetate and Lactates to Dry Matter Intake and Volatile Fatty Acid Metabolism1
Relation of Dietary Acetate and Lactates to Dry Matter Intake and Volatile Fatty Acid Metabolism 1 S. H. SENEL 2 and F. G. O W E N
Department of Dairy Science, University of Nebraska, Lincoln factor(s) other than the acetate and lactate contribution of silages.
Abstract
Effects of feeding lactates and acetate supplemental to a basal ration of two-thirds sorghum silage and one-third mixture of beet pulp and soybean oil meal were determined, using five heifers in a 5 × 5 Latinsquare design. Rations were fed ad libitum. Lactates were added to give 9.0% lactic acid equivalent in the ration dry matter (HL) and acetate at 2.8% acetic acid equivalent (HA) significantly (P < .01) increased dry- matter intake; at lower levels, 5.9% lactic (LL) and 1.5% acetic (LA), intake was not significantly different from the basal ration. Gains in body weight and efficiency of dry matter utilization were higher, but not significantly, when supplements were fed. The H A supplement increased the molar per cent of acetic acid; whereas, the t I L supplement decreased the proportion of acetic acid and increased propionic acid. Blood levels of acetic, propionic, and butyric acids were highest for the ]4L treatment. Blood glucose and ketones were not apparently affected by these supplements. The results suggest that the depression in dry matter consumption and lower rates of gain which often result from feeding silage rations compared to hay are due to Received for publication May 3, 1966. 1Published with the approval of the Director as paper no. 1746, Journal Series, Nebraska Agricultural Experiment Station, Lincoln. ~Present address: Faculty of ~reterinary Science, University of Ankara, Ankara, Turkey.
Among the theories on the regulation of consumption rate by ruminants is a chemoregulating idea which holds that blood levels of acetic and propionic acid stimulate receptors that limit appetite (5). I f this theory is correct, the relatively lower intake of fermented forages (silages) than comparable nonfermented forages, such as hay and green crop, may he related to their higher acid content. Previous studies (6, 11, 12) have indicated that lactic acid in the diet has a depressing effect, but that lactate salts have little or no effect on intake. Acetic acid administered intravenously reduced appetite (5), whereas dietary acetate had no effect on intake (1). This experiment was conducted to evaluate the effects on consumption rate of dietary acetate and lactate at levels equivalent to those of the corresponding acids in moderately and highly fermented silages. The effects of supplemental acetate and lactate on tureen and blood volatile fatty acids ( V F A ) , blood levels of glucose, and ketones also were determined. Experlmentai Procedure
Five Holstein heifers, 10-12 months of age, were used in a 5 X5 Latin-square design. A preliminary period of seven days preceded the experimental periods: Experimental periods (21 days) consisted of 11 days for transition and ten days for comparison of feed consumption. The basal ration consisted of two-thirds for-
TABLE 1 Composition of the concentrate portion of the rations Basal (B)
3.0% Acetate (LA)
7.0% Acetate (t~A)
7.5% Lactate (LL)
]7.5% Lactate (HL)
(#g)
Beet pulp, molasses 193.9 187.1 178.0 176.9 154.3 Soybean oil meal (44%) 32.6 32.6 32.6 32.6 32.6 Sodium acetate ........ 6.8 15.9 Lactate saltsa . . . . . . . . . . . . . . . . . . . . . . . 17.0 39.6 * Supplied by Sheffield Chemical, Norwich, New Jersey, as Soluble Lactate Salts®, which contalus sodium and calcium lactates, in mixture of D and L forms. 1075
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A N D F. G. O W E N
TABLE 2 Dry matter (DM) and organic acid content of the rations~ Ration
DM
Acetic
Basal 3.0% Acetate 7.0% Acetate 7.5% Lactate 17.5% Lactate Dry basis.
47.42 48.04 48.21 46.08 48.06
0.49 1.52 2.81 0.73 0.88
age sorghum silage and one-third concentrate mixture composed of beet pulp and soybean oil meal. The four treatments were 3.0 and 7.0% sodium acetate and 7.5 and 17.5% of lactates. These additives replaced beet pulp on a weight basis. Table I lists the ingredients of the concentrate mixture and Table 2 gives results of dry matter and organic acid analyses. The animals were fed these rations individually twice daily, allowing 10% in excess of the previous day's intake. I n cases of feed refusal the maximum reduction in feed per day was 10% of the previous day's allowance. Bonemeal and tracemineralized salt were provided free-choice, l%ed samples were taken daily during the comparison period and kept frozen nntil chemical determinations were nmde. Ration dry matter determinations were made on the ten-day composite samples according to recommended procedures (15). Rumen fluids were drawn by stonmeh tube with an attached stainless steel strainer at 4 and 6 hr after feeding on the last two days of every comparison period. Blood samples were taken from the jugular vein at 10 and 12 hr post-feeding. Previous studies by Bensadoun and Reid (3), Bensadoun et al. (2), and Little and Hawkins (9) showed that V F A were maximal in the rumen fluid between 2 and 6 hr and in blood 10 and 12 hr post-feeding. Rumen fluid p H was checked immediately after sampling. Preservative (1 ml of saturated mercuric chloride per 100 nil) was added to another portion of the rumen fluid which was then frozen until Y F A determinations were made. During the same two days, 50-ml blood samples were drawn into plastic bags containing heparin solution as anticoagulant. These samples also were kept frozen until analyzed. The rumen and blood samples were prepared for V F A analysis according to a modification (M. J. Marco, personal communication~) of the Erwin method (7). The modified method is as follows: 80 ml of 0.1 ~" H.~SO, was slowly added to 10 ml of heparinized blood, and mixed vigorously. After standing 5 rain, 10 ml of 10% sodium tungstate was added slowly, with vigorous mixing. The mixture was filtered after standing for 5 rain.
Lactic (%) L43 1.33 1.30 5.90 9.03
Butyric
Propionic
0.27 0.21 0.27 0.24 0.31
3.12 4.07 4.40 5.08 4.80
The salts of V F A were prepared by addition of 1 ~ K O H to 25 ml of filtrate until alkaline to Alkacid test paper. The alkaline filtrate was reduced to dryness on steam bath. The fatty acid salts were dissolved in 2 ml of 4% metaphosphoric acid and injected into the gas chromatograph. V F A in the rumen fluid and venous blood were measured, using a Jarrell-Ash gas chromatograph. A two-meter Teflon chromatographic co/man 1/8 in. in diameter was used. It was packed with 20% Tween 80 and 2% phosphoric acid (85%) on Anakrom mesh size 90/100. Blood was also analyzed for total ketone bodies (13) and glucose (14). The organic acid content of the rations was determined by column chromatography, as described by Wiseman and Irvin (17). The heifers were weighed on two successive days at the beginning of each experimental period. Statistical analyses were made for the 5 × 5 Latin-square design and comparisons among treatment means by applying Duncan's new multiple-range test, as described by Steel and Torrie (14). Results and Discussion
Addition of 3.0% acetate to the beet pulp and soybean oil meal mixture yielded 1.5% acetic acid in the ration, which corresponds to a level of acetic acid often found in hay-crop silages. The 7.0% acetate addition yielded 2.8% acetic acid in the ration. This level corresponds to the upper levels sometimes found in highmoisture legume silage. The inclusion of 7.5 and 17.5% lactate in the beet pulp and soybean oil meal mixture resulted in 5.9 and 9.0% lactic acid, respectively, in the total rations. The lower level of lactic acid is similar to that in common corn silage and the higher level is approximately the concentration reported for corn silage preserved with added lime. The considerable amount of propionic acid, ranging from 3.1-5.1% in the rations, probably came mainly from the beet pulp. A variable amount Monsanto Missouri.
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FATTY ACID ~[ETABOLISI~
TABLE 3 Dry matter consumption, body weight gain, and efficiency~
Ration
Average daily DM intake
Efficiency Average DM intake dMly per kg gain wt gain
(~g) Basal 8.52 ~'2 0.861 3.0% Acetate 9.33 ~,8 1.00 ~ 7.0% Acetate 9.65 a'4 1.16 ~ 7.5% Lactate 8.02 ~ 1.061 17.5% Lactate 9.96 ~'4 1.191 CV (%) 7.3 22.2 " Values with common superscripts are nificantly different (P ~ .05).
9.90 ~ 9.331 8.32 ~ 7.57 ~ 8.371 9.1 not sig-
of fermentation probably occurred following harvest and during processing, before the pulp was dehydrated. Dry matter intake. Table 3 shows the effect of acetate and lactate supplements on dry matter consumption, body weight gains, and feed efficiency. Compared to the basal ration, addition of the higher levels of acetate and lactate significantly (P ( . 0 1 ) increased consumption of dry matter by 13.3 and 17.0%, respectively. The slightly lower level of dIT matter intake resulting from an average intake of 0.725 kg of supplemental lactate daily confirms results of Radloff and Schultz (12), ~[ontgomery et al. (11), and Emery et al. (6). They fed or infused lactate or lactic acid into the tureen in amounts ranging from 0.234-0.950 kg per day. The significant (P ( . 0 5 ) increase in total dry matter intake with a high level of lactate (averaging 1.45 kg/day) in the present study appears to be a new finding. No explanation for this effect is apparent. Acetate feeding also increased dry matter consumption. Montgomery et al. (11) infused acetic acid into the tureen and found a significant decrease in daily hay consumption, but when the acetic acid was supplied as the sodium salt the reduction in hay consumption was small. Balch et al. (1) found that feeding 0.340 kg
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sodium acetate daily produced no significant depression in feed intake. Higher dry matter consumption was associated with higher concentrations of acetic acid in the blood, i.e., rations which produced increases in blood acetic acid also increased dry matter intake. Of all rations, the high level of lactate produced the highest concentration of acetic acid (Table 7) in the blood. It was also consumed in the highest amount. This tends to detract from the hypothesis of Dowden and Jacobson (5), whose findings suggested that blood acetic acid level has a depressing effect on appetite. On the other hand, this result is in agreement with the result of Little and Itawkins (9), who found that total feed intake was positively correlated with blood content of acetic and propionic acids. Body weight gai~ and e~iciency. Rations containing the high level of acetate and lactate resulted in 34 and 38% higher weight gains, respectively, and required 82 and 80% as much dry matter per pound of gain as the basal ration (Table 3). Although these responses were not statistically significant, their magnitude suggests possible important effects. Similar increases in efi%iency from supplemental lactic acid or lactate salts have been reported by Montgomery et al. (11) and Emery et al. (6). Blood glucose and ketone bodies. Blood glucose levels were slightly higher for the acetate than for the basal or lactate rations (Table 4). Blood glucose values were somewhat higher at 12 hr following feeding than at 10 hr. However, none of the differences in blood glucose was statistically significant at P ~ .05. The lactate salts were probably degraded by fermentation in the tureen to nonglucogenic substances as well as propionic acid. Although the lactates tended to increase blood propionate, the net effect was insufficient to elevate blood glucose. These results are in general agreement with those of Radloff and Schultz (12), who used a similar lactate product. Hueter (8)
TABLE 4 Ration effect on blood glucose and ketones at 10 and 12 hr after feeding s Ration
Blood glucose 10 hr 12 hr
Blood ketones 10 hr 12 hr
(mg/lO0 ml) Basal 3.0% 7.0% 7.5% 17.5% " Differences P ~ .05.
42.1 44.3 Acetate 44.6 46.2 Acetate 46.4 47.4 Lactate 40.0 42.0 Lactate 43.0 44.9 among treatments and sampling times were not
4.91 4.53 3.58 4.50 3.23 3.97 4.37 4.85 3.49 3.45 statistically significant at
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S E N E L AND F. G. O W E N
TABLE 5 Period effect on blood glucose and ketones ~ Period
Glucose -
Sept. 16-Oct. 6 Oct. Oct. ~ov. Dec.
7-Oct. 27 28-Nov. 18 19-Dec. 9 10-Dec. 30
-
Ketones
(mg/lO0
38.8 a 41.2 *'2 45.9 ~'z'a 47.32'3'~ 50.8 ~'~
m l ) - -
3.65 ~ 6.852 4.151 3.45* 3.20*
Values with common superscripts are not significantly different ( P < .05). r e p o r t e d a rise i n blood glucose w i t h sodium lactate but n o t w i t h calcimu lactate. The k e t o n e bodies in the blood were lower f o r acetate- a n d l a c t a t e - s u p p l e m e n t e d r a t i o n s t h a n f o r control rations. S i m i l a r results were o b t a i n e d f r o m i n t r a r u m i n a l i n f u s i o n o f acetic acid (11) a n d f o r feeding lactates (12). Acetic acid is generally classified as ketogenie; but, the basal r a t i o n c o n t a i n e d a p p r e c i a b l e a m o u n t s of p r o p i o n i c acid, which m a y h a v e s t i m u l a t e d r a p i d metabolism of ketone p r e c u r s o r s a n d p r e v e n t e d elevation of blood ketones. The p e r i o d effect o f blood glucose a n d ketones, as shown in Table 5, is p r o b a b l y seasonally influenced. Blood glucose values increased p r o gressively ( P < .05) with e x p e r i m e n t a l periods. Total ketone bodies were also different a m o n g periods. K e t o n e s increased m a r k e d l y f r o m the first to the second period, t h e n decreased p r o gressively t h r o u g h the r e m a i n i n g periods. Seasonal effects of this n a t u r e h a v e been r e p o r t e d previously (16).
Rumen volatile f a t t y acids. The r a t i o n effects on r u m e n V F A ratios a n d p H are shown in Table 6. The addition o f acetate to t h e r a t i o n increased the m o l a r p e r cent of acetic acid significantly ( P < .01) at b o t h levels a n d decreased p r o p i o n i c a n d b u t y r i c acids only a t the low level ( P < .05). Isovalerie a n d valeric acids were n o t significantly affected. L a c t a t e a d d i t i o n decreased acetic acid a n d increased p r o p i o n i c acid, as m o l a r p e r cent, significantly ( P < .01) a t b o t h levels. R u m i n a l i n f u s i o n of lactic acid p r o d u c e d similar results (10). Effects of lactate on the o t h e r acids, ineluding butyric, were n o t a p p a r e n t . This is in c o n t r a s t to results o b t a i n e d b y M o n t g o m e r y a n d B a u m g a r d t (10), who f o u n d a s h a r p increase in b u t y r i c acid f o l l o w i n g i n f u s i o n of lactates. The m o l a r ratio of acetic-to-propionic acid in the r u m e n fluid was 4.4 : 1 f o r t h e basal ration. This r a t i o was 4 . 7 : 1 with the acetate supplem e n t s a n d 3 . 6 : 1 w i t h lactate additions. The h i g h e r total V F A f o r t h e 7 % - a c e t a t e r a t i o n is m a n i f e s t m a i n l y in a g r e a t e r concentration of acetic acid. The absence of a n effect o f these s u p p l e m e n t s on r u m e n p H s u b s t a n t i ates the findings of 5 [ o n t g o m e r y a n d B a u m g a r d t (10). Blood volatile fatty acids. The d a t a p r e s e n t e d in Table 7 show t h a t a d d i t i o n of acetate a t b o t h levels a n d lactate a t t h e h i g h e r level increased acetic acid significantly ( P < .01). Blood levels of p r o p i o n i c acid were h i g h e r f o r the acetate a n d lactate s u p p l e m e n t s , b u t t h e differences were n o t significant. B u t y r i c acid was increased
TABLE 6 Ration effect oi1 rumeu V F A and pH a Ration
Acetic
Propionie
Butyric
Isovalerie
Valeric Total V F A b
OH
(mole % ) - Basal 72.4 a 16.41 3.0% Acetate 75.02 15.6 ~ 7.0% Acetate 73.93 16.01,2 7.5% Lactate 69.6 ~ 19.0 ~ 17.5% Lactate 69.5 ~ 19.63 a Values With common superscripts are b Micromoles per milliliter.
9.51' 0.71 x 1.071 104 7.942 0.52* 0.82 x 103 8.69* 0.62* 0.801 115 9.53* 0.57 ~ 1.31 ~ 99 9.56* 0.37 ~ 0.99* 105 not significantly different ( P < .05).
6.71 ~ 6.81 a 6.68* 6.88 ~ 6.85 ~
TABLE 7 :Effect of lactates and acetate supplements on blood V F A Ration
Acetic a
Propionic ~
Butyric ~
Isovalerie b
Valeric b
(~M/ml) Basal 1.267' 3.0% Acetate ].6132'3 7.0% Acetate 1.6392'3 7.5% Lactate 1.457" 2 17.5% Lactate 1.6982 Values with common superscripts are b Not analyzed statistically.
.0301 .0151 .003 .081' .0381'2 .008 .045* .047 *'2'3 .006 .056* .0441, 2 .005 .1141 .0753 .011 not significantly different ( P < .05).
.001 .007 .010 .010 .037
FATTY ACID 5~ETABOLISM at all levels of acetate a n d l a c t a t e feeding. However, the difference was significant ( P < .01) only f o r the h i g h level of lactate salts. 0 n l y a b o u t o n e - h a l f of the blood samples c o n t a i n e d detectable a m o u n t s of valeric a n d isovaleric acids. R e l a t i o n of blood V F A to intake was j u s t discussed u n d e r D r y 5 I a t t e r I n t a k e . References
(1) Baleh, C. C., Taylor, J., and Thomson, I. T. 1961. The Short-Term Effects of the Level of Concentrates Given to Ayrshire Cows and Adding 0.75 lb. Sodium Acetate to the Daily Diet. J . Dairy Sci., 28:5. (2) Bensadoun, A., Paladines, O. L., and Reid, J. T. ]962. Effect of Level of I n t a k e and Physical Form of the Diet on Plasma Glucose Concentration and Volatile F a t t y Acid Absorption in Ruminants. J. Dairy Sci., 45 : 1203. (3) Bensadoun, A., and l~eid, J. T. 1962. Estimation of Rate of P a r t i a l Blood Flow in ]¢uminants: Effect of Feeding, Fasting, and Anesthesia. J. Dairy Sci., 45:540. (4) Campbell, L. A., and Kronfcld, D. S. 1961. The Estimation of Low Concentrations of Plasma Glucose ~ s i n g Glucose Oxidase. Am. J. Vet. Research, 22:587. (5) Dowden, D. R., and Jacobson, D. R. 1959. Normal Intake Variation and Inhibition of Appetite in the Bovine by Certain Intermediary Metabolites. Ph.D. thesis, University of Kentucky, Lexington. (6) Emery, ~. S., Brown, L. D., ttuffman, C. ~., Lewis, T. R., Everett, J. P., and Lassiter, C. A. 1961. Comparative Feeding Value of Lactic Acid and Grain for Dairy Cattle. J. Animal Sci., 20:159.
1079
(7) Erwin, E. S., Marco, G. J., and Emery, E. ~ . 1961. Volatile F a t t y Acid Analyses of Blood and l~umen Fluid by Gas Chromatography. J. Dairy Sci., 44:1768. (8) tIueter, F. G., Shaw, J. C., and Doetsch, R . N . 1956. Absorption and Dissimilation of Lactates Added to the Bovine ]¢umen and the Resulting Effects on Blood Glucose. J. Dairy Sci., 39:1430. (9) Little, J. A., and Hawkins, G. E. 1963. Relationship Between Acetic and Propionic Acid Content of Blood and Feed Intake in Young Dairy Animals. J. Dairy Sci., 46 : 634. (10) Montgomery, M. J., and Baumgardt, B. R. 1963. Effect of Lactic Acid Infusion on RuminaI Volatile F a t t y Acids. J. Dairy Sci., 46 : 639. (11) Montgomery, M. J., Schu]tz, L. /-L, and Baumgardt, B. R. 1963. Effect of Intraruminal Infusion of Volatile F a t t y Acids and Lactic Acid on Voluntary Hay Intake. J. Dairy Sci., 46:1380. (12) Radloff, H. D., and Schultz, L~ B:. 1963. Some Effects of Feeding Lactates to Dairy Cows. J. Dairy Sei., 46:517. (13) Reid, R. L. 1960. The Determination of Ketone Bodies in Blood. The Analyst, 85:265. (14) Steel, R. G. D., and Torrie, J. It. 1960. Principles and Procedures of Statistics. McGraw-~Iill Book Company, Inc., New York. (15) Sumnlary of Second Silage Conference. 1959. P a r t I. ]~eltsville, Maryland. (16) Van Soest, P. J. 1963. Ruminant :Fat Metabolism with Particular Reference to Factors Affecting Low Milk F a t and Feed Efficiency. J. Dairy Sci., 46:204. (17) Wiseman, H. G., and Irvin, H. M. 1957. Chromatographic Separation of Silage Acids. J. Food Chem., 5:213.
Department of Dairy Science, University of Nebraska, Lincoln factor(s) other than the acetate and lactate contribution of silages.
Abstract
Effects of feeding lactates and acetate supplemental to a basal ration of two-thirds sorghum silage and one-third mixture of beet pulp and soybean oil meal were determined, using five heifers in a 5 × 5 Latinsquare design. Rations were fed ad libitum. Lactates were added to give 9.0% lactic acid equivalent in the ration dry matter (HL) and acetate at 2.8% acetic acid equivalent (HA) significantly (P < .01) increased dry- matter intake; at lower levels, 5.9% lactic (LL) and 1.5% acetic (LA), intake was not significantly different from the basal ration. Gains in body weight and efficiency of dry matter utilization were higher, but not significantly, when supplements were fed. The H A supplement increased the molar per cent of acetic acid; whereas, the t I L supplement decreased the proportion of acetic acid and increased propionic acid. Blood levels of acetic, propionic, and butyric acids were highest for the ]4L treatment. Blood glucose and ketones were not apparently affected by these supplements. The results suggest that the depression in dry matter consumption and lower rates of gain which often result from feeding silage rations compared to hay are due to Received for publication May 3, 1966. 1Published with the approval of the Director as paper no. 1746, Journal Series, Nebraska Agricultural Experiment Station, Lincoln. ~Present address: Faculty of ~reterinary Science, University of Ankara, Ankara, Turkey.
Among the theories on the regulation of consumption rate by ruminants is a chemoregulating idea which holds that blood levels of acetic and propionic acid stimulate receptors that limit appetite (5). I f this theory is correct, the relatively lower intake of fermented forages (silages) than comparable nonfermented forages, such as hay and green crop, may he related to their higher acid content. Previous studies (6, 11, 12) have indicated that lactic acid in the diet has a depressing effect, but that lactate salts have little or no effect on intake. Acetic acid administered intravenously reduced appetite (5), whereas dietary acetate had no effect on intake (1). This experiment was conducted to evaluate the effects on consumption rate of dietary acetate and lactate at levels equivalent to those of the corresponding acids in moderately and highly fermented silages. The effects of supplemental acetate and lactate on tureen and blood volatile fatty acids ( V F A ) , blood levels of glucose, and ketones also were determined. Experlmentai Procedure
Five Holstein heifers, 10-12 months of age, were used in a 5 X5 Latin-square design. A preliminary period of seven days preceded the experimental periods: Experimental periods (21 days) consisted of 11 days for transition and ten days for comparison of feed consumption. The basal ration consisted of two-thirds for-
TABLE 1 Composition of the concentrate portion of the rations Basal (B)
3.0% Acetate (LA)
7.0% Acetate (t~A)
7.5% Lactate (LL)
]7.5% Lactate (HL)
(#g)
Beet pulp, molasses 193.9 187.1 178.0 176.9 154.3 Soybean oil meal (44%) 32.6 32.6 32.6 32.6 32.6 Sodium acetate ........ 6.8 15.9 Lactate saltsa . . . . . . . . . . . . . . . . . . . . . . . 17.0 39.6 * Supplied by Sheffield Chemical, Norwich, New Jersey, as Soluble Lactate Salts®, which contalus sodium and calcium lactates, in mixture of D and L forms. 1075
S. }/. S E N E L
1076
A N D F. G. O W E N
TABLE 2 Dry matter (DM) and organic acid content of the rations~ Ration
DM
Acetic
Basal 3.0% Acetate 7.0% Acetate 7.5% Lactate 17.5% Lactate Dry basis.
47.42 48.04 48.21 46.08 48.06
0.49 1.52 2.81 0.73 0.88
age sorghum silage and one-third concentrate mixture composed of beet pulp and soybean oil meal. The four treatments were 3.0 and 7.0% sodium acetate and 7.5 and 17.5% of lactates. These additives replaced beet pulp on a weight basis. Table I lists the ingredients of the concentrate mixture and Table 2 gives results of dry matter and organic acid analyses. The animals were fed these rations individually twice daily, allowing 10% in excess of the previous day's intake. I n cases of feed refusal the maximum reduction in feed per day was 10% of the previous day's allowance. Bonemeal and tracemineralized salt were provided free-choice, l%ed samples were taken daily during the comparison period and kept frozen nntil chemical determinations were nmde. Ration dry matter determinations were made on the ten-day composite samples according to recommended procedures (15). Rumen fluids were drawn by stonmeh tube with an attached stainless steel strainer at 4 and 6 hr after feeding on the last two days of every comparison period. Blood samples were taken from the jugular vein at 10 and 12 hr post-feeding. Previous studies by Bensadoun and Reid (3), Bensadoun et al. (2), and Little and Hawkins (9) showed that V F A were maximal in the rumen fluid between 2 and 6 hr and in blood 10 and 12 hr post-feeding. Rumen fluid p H was checked immediately after sampling. Preservative (1 ml of saturated mercuric chloride per 100 nil) was added to another portion of the rumen fluid which was then frozen until Y F A determinations were made. During the same two days, 50-ml blood samples were drawn into plastic bags containing heparin solution as anticoagulant. These samples also were kept frozen until analyzed. The rumen and blood samples were prepared for V F A analysis according to a modification (M. J. Marco, personal communication~) of the Erwin method (7). The modified method is as follows: 80 ml of 0.1 ~" H.~SO, was slowly added to 10 ml of heparinized blood, and mixed vigorously. After standing 5 rain, 10 ml of 10% sodium tungstate was added slowly, with vigorous mixing. The mixture was filtered after standing for 5 rain.
Lactic (%) L43 1.33 1.30 5.90 9.03
Butyric
Propionic
0.27 0.21 0.27 0.24 0.31
3.12 4.07 4.40 5.08 4.80
The salts of V F A were prepared by addition of 1 ~ K O H to 25 ml of filtrate until alkaline to Alkacid test paper. The alkaline filtrate was reduced to dryness on steam bath. The fatty acid salts were dissolved in 2 ml of 4% metaphosphoric acid and injected into the gas chromatograph. V F A in the rumen fluid and venous blood were measured, using a Jarrell-Ash gas chromatograph. A two-meter Teflon chromatographic co/man 1/8 in. in diameter was used. It was packed with 20% Tween 80 and 2% phosphoric acid (85%) on Anakrom mesh size 90/100. Blood was also analyzed for total ketone bodies (13) and glucose (14). The organic acid content of the rations was determined by column chromatography, as described by Wiseman and Irvin (17). The heifers were weighed on two successive days at the beginning of each experimental period. Statistical analyses were made for the 5 × 5 Latin-square design and comparisons among treatment means by applying Duncan's new multiple-range test, as described by Steel and Torrie (14). Results and Discussion
Addition of 3.0% acetate to the beet pulp and soybean oil meal mixture yielded 1.5% acetic acid in the ration, which corresponds to a level of acetic acid often found in hay-crop silages. The 7.0% acetate addition yielded 2.8% acetic acid in the ration. This level corresponds to the upper levels sometimes found in highmoisture legume silage. The inclusion of 7.5 and 17.5% lactate in the beet pulp and soybean oil meal mixture resulted in 5.9 and 9.0% lactic acid, respectively, in the total rations. The lower level of lactic acid is similar to that in common corn silage and the higher level is approximately the concentration reported for corn silage preserved with added lime. The considerable amount of propionic acid, ranging from 3.1-5.1% in the rations, probably came mainly from the beet pulp. A variable amount Monsanto Missouri.
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Company,
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Louis,
FATTY ACID ~[ETABOLISI~
TABLE 3 Dry matter consumption, body weight gain, and efficiency~
Ration
Average daily DM intake
Efficiency Average DM intake dMly per kg gain wt gain
(~g) Basal 8.52 ~'2 0.861 3.0% Acetate 9.33 ~,8 1.00 ~ 7.0% Acetate 9.65 a'4 1.16 ~ 7.5% Lactate 8.02 ~ 1.061 17.5% Lactate 9.96 ~'4 1.191 CV (%) 7.3 22.2 " Values with common superscripts are nificantly different (P ~ .05).
9.90 ~ 9.331 8.32 ~ 7.57 ~ 8.371 9.1 not sig-
of fermentation probably occurred following harvest and during processing, before the pulp was dehydrated. Dry matter intake. Table 3 shows the effect of acetate and lactate supplements on dry matter consumption, body weight gains, and feed efficiency. Compared to the basal ration, addition of the higher levels of acetate and lactate significantly (P ( . 0 1 ) increased consumption of dry matter by 13.3 and 17.0%, respectively. The slightly lower level of dIT matter intake resulting from an average intake of 0.725 kg of supplemental lactate daily confirms results of Radloff and Schultz (12), ~[ontgomery et al. (11), and Emery et al. (6). They fed or infused lactate or lactic acid into the tureen in amounts ranging from 0.234-0.950 kg per day. The significant (P ( . 0 5 ) increase in total dry matter intake with a high level of lactate (averaging 1.45 kg/day) in the present study appears to be a new finding. No explanation for this effect is apparent. Acetate feeding also increased dry matter consumption. Montgomery et al. (11) infused acetic acid into the tureen and found a significant decrease in daily hay consumption, but when the acetic acid was supplied as the sodium salt the reduction in hay consumption was small. Balch et al. (1) found that feeding 0.340 kg
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sodium acetate daily produced no significant depression in feed intake. Higher dry matter consumption was associated with higher concentrations of acetic acid in the blood, i.e., rations which produced increases in blood acetic acid also increased dry matter intake. Of all rations, the high level of lactate produced the highest concentration of acetic acid (Table 7) in the blood. It was also consumed in the highest amount. This tends to detract from the hypothesis of Dowden and Jacobson (5), whose findings suggested that blood acetic acid level has a depressing effect on appetite. On the other hand, this result is in agreement with the result of Little and Itawkins (9), who found that total feed intake was positively correlated with blood content of acetic and propionic acids. Body weight gai~ and e~iciency. Rations containing the high level of acetate and lactate resulted in 34 and 38% higher weight gains, respectively, and required 82 and 80% as much dry matter per pound of gain as the basal ration (Table 3). Although these responses were not statistically significant, their magnitude suggests possible important effects. Similar increases in efi%iency from supplemental lactic acid or lactate salts have been reported by Montgomery et al. (11) and Emery et al. (6). Blood glucose and ketone bodies. Blood glucose levels were slightly higher for the acetate than for the basal or lactate rations (Table 4). Blood glucose values were somewhat higher at 12 hr following feeding than at 10 hr. However, none of the differences in blood glucose was statistically significant at P ~ .05. The lactate salts were probably degraded by fermentation in the tureen to nonglucogenic substances as well as propionic acid. Although the lactates tended to increase blood propionate, the net effect was insufficient to elevate blood glucose. These results are in general agreement with those of Radloff and Schultz (12), who used a similar lactate product. Hueter (8)
TABLE 4 Ration effect on blood glucose and ketones at 10 and 12 hr after feeding s Ration
Blood glucose 10 hr 12 hr
Blood ketones 10 hr 12 hr
(mg/lO0 ml) Basal 3.0% 7.0% 7.5% 17.5% " Differences P ~ .05.
42.1 44.3 Acetate 44.6 46.2 Acetate 46.4 47.4 Lactate 40.0 42.0 Lactate 43.0 44.9 among treatments and sampling times were not
4.91 4.53 3.58 4.50 3.23 3.97 4.37 4.85 3.49 3.45 statistically significant at
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S.H.
S E N E L AND F. G. O W E N
TABLE 5 Period effect on blood glucose and ketones ~ Period
Glucose -
Sept. 16-Oct. 6 Oct. Oct. ~ov. Dec.
7-Oct. 27 28-Nov. 18 19-Dec. 9 10-Dec. 30
-
Ketones
(mg/lO0
38.8 a 41.2 *'2 45.9 ~'z'a 47.32'3'~ 50.8 ~'~
m l ) - -
3.65 ~ 6.852 4.151 3.45* 3.20*
Values with common superscripts are not significantly different ( P < .05). r e p o r t e d a rise i n blood glucose w i t h sodium lactate but n o t w i t h calcimu lactate. The k e t o n e bodies in the blood were lower f o r acetate- a n d l a c t a t e - s u p p l e m e n t e d r a t i o n s t h a n f o r control rations. S i m i l a r results were o b t a i n e d f r o m i n t r a r u m i n a l i n f u s i o n o f acetic acid (11) a n d f o r feeding lactates (12). Acetic acid is generally classified as ketogenie; but, the basal r a t i o n c o n t a i n e d a p p r e c i a b l e a m o u n t s of p r o p i o n i c acid, which m a y h a v e s t i m u l a t e d r a p i d metabolism of ketone p r e c u r s o r s a n d p r e v e n t e d elevation of blood ketones. The p e r i o d effect o f blood glucose a n d ketones, as shown in Table 5, is p r o b a b l y seasonally influenced. Blood glucose values increased p r o gressively ( P < .05) with e x p e r i m e n t a l periods. Total ketone bodies were also different a m o n g periods. K e t o n e s increased m a r k e d l y f r o m the first to the second period, t h e n decreased p r o gressively t h r o u g h the r e m a i n i n g periods. Seasonal effects of this n a t u r e h a v e been r e p o r t e d previously (16).
Rumen volatile f a t t y acids. The r a t i o n effects on r u m e n V F A ratios a n d p H are shown in Table 6. The addition o f acetate to t h e r a t i o n increased the m o l a r p e r cent of acetic acid significantly ( P < .01) at b o t h levels a n d decreased p r o p i o n i c a n d b u t y r i c acids only a t the low level ( P < .05). Isovalerie a n d valeric acids were n o t significantly affected. L a c t a t e a d d i t i o n decreased acetic acid a n d increased p r o p i o n i c acid, as m o l a r p e r cent, significantly ( P < .01) a t b o t h levels. R u m i n a l i n f u s i o n of lactic acid p r o d u c e d similar results (10). Effects of lactate on the o t h e r acids, ineluding butyric, were n o t a p p a r e n t . This is in c o n t r a s t to results o b t a i n e d b y M o n t g o m e r y a n d B a u m g a r d t (10), who f o u n d a s h a r p increase in b u t y r i c acid f o l l o w i n g i n f u s i o n of lactates. The m o l a r ratio of acetic-to-propionic acid in the r u m e n fluid was 4.4 : 1 f o r t h e basal ration. This r a t i o was 4 . 7 : 1 with the acetate supplem e n t s a n d 3 . 6 : 1 w i t h lactate additions. The h i g h e r total V F A f o r t h e 7 % - a c e t a t e r a t i o n is m a n i f e s t m a i n l y in a g r e a t e r concentration of acetic acid. The absence of a n effect o f these s u p p l e m e n t s on r u m e n p H s u b s t a n t i ates the findings of 5 [ o n t g o m e r y a n d B a u m g a r d t (10). Blood volatile fatty acids. The d a t a p r e s e n t e d in Table 7 show t h a t a d d i t i o n of acetate a t b o t h levels a n d lactate a t t h e h i g h e r level increased acetic acid significantly ( P < .01). Blood levels of p r o p i o n i c acid were h i g h e r f o r the acetate a n d lactate s u p p l e m e n t s , b u t t h e differences were n o t significant. B u t y r i c acid was increased
TABLE 6 Ration effect oi1 rumeu V F A and pH a Ration
Acetic
Propionie
Butyric
Isovalerie
Valeric Total V F A b
OH
(mole % ) - Basal 72.4 a 16.41 3.0% Acetate 75.02 15.6 ~ 7.0% Acetate 73.93 16.01,2 7.5% Lactate 69.6 ~ 19.0 ~ 17.5% Lactate 69.5 ~ 19.63 a Values With common superscripts are b Micromoles per milliliter.
9.51' 0.71 x 1.071 104 7.942 0.52* 0.82 x 103 8.69* 0.62* 0.801 115 9.53* 0.57 ~ 1.31 ~ 99 9.56* 0.37 ~ 0.99* 105 not significantly different ( P < .05).
6.71 ~ 6.81 a 6.68* 6.88 ~ 6.85 ~
TABLE 7 :Effect of lactates and acetate supplements on blood V F A Ration
Acetic a
Propionic ~
Butyric ~
Isovalerie b
Valeric b
(~M/ml) Basal 1.267' 3.0% Acetate ].6132'3 7.0% Acetate 1.6392'3 7.5% Lactate 1.457" 2 17.5% Lactate 1.6982 Values with common superscripts are b Not analyzed statistically.
.0301 .0151 .003 .081' .0381'2 .008 .045* .047 *'2'3 .006 .056* .0441, 2 .005 .1141 .0753 .011 not significantly different ( P < .05).
.001 .007 .010 .010 .037
FATTY ACID 5~ETABOLISM at all levels of acetate a n d l a c t a t e feeding. However, the difference was significant ( P < .01) only f o r the h i g h level of lactate salts. 0 n l y a b o u t o n e - h a l f of the blood samples c o n t a i n e d detectable a m o u n t s of valeric a n d isovaleric acids. R e l a t i o n of blood V F A to intake was j u s t discussed u n d e r D r y 5 I a t t e r I n t a k e . References
(1) Baleh, C. C., Taylor, J., and Thomson, I. T. 1961. The Short-Term Effects of the Level of Concentrates Given to Ayrshire Cows and Adding 0.75 lb. Sodium Acetate to the Daily Diet. J . Dairy Sci., 28:5. (2) Bensadoun, A., Paladines, O. L., and Reid, J. T. ]962. Effect of Level of I n t a k e and Physical Form of the Diet on Plasma Glucose Concentration and Volatile F a t t y Acid Absorption in Ruminants. J. Dairy Sci., 45 : 1203. (3) Bensadoun, A., and l~eid, J. T. 1962. Estimation of Rate of P a r t i a l Blood Flow in ]¢uminants: Effect of Feeding, Fasting, and Anesthesia. J. Dairy Sci., 45:540. (4) Campbell, L. A., and Kronfcld, D. S. 1961. The Estimation of Low Concentrations of Plasma Glucose ~ s i n g Glucose Oxidase. Am. J. Vet. Research, 22:587. (5) Dowden, D. R., and Jacobson, D. R. 1959. Normal Intake Variation and Inhibition of Appetite in the Bovine by Certain Intermediary Metabolites. Ph.D. thesis, University of Kentucky, Lexington. (6) Emery, ~. S., Brown, L. D., ttuffman, C. ~., Lewis, T. R., Everett, J. P., and Lassiter, C. A. 1961. Comparative Feeding Value of Lactic Acid and Grain for Dairy Cattle. J. Animal Sci., 20:159.
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(7) Erwin, E. S., Marco, G. J., and Emery, E. ~ . 1961. Volatile F a t t y Acid Analyses of Blood and l~umen Fluid by Gas Chromatography. J. Dairy Sci., 44:1768. (8) tIueter, F. G., Shaw, J. C., and Doetsch, R . N . 1956. Absorption and Dissimilation of Lactates Added to the Bovine ]¢umen and the Resulting Effects on Blood Glucose. J. Dairy Sci., 39:1430. (9) Little, J. A., and Hawkins, G. E. 1963. Relationship Between Acetic and Propionic Acid Content of Blood and Feed Intake in Young Dairy Animals. J. Dairy Sci., 46 : 634. (10) Montgomery, M. J., and Baumgardt, B. R. 1963. Effect of Lactic Acid Infusion on RuminaI Volatile F a t t y Acids. J. Dairy Sci., 46 : 639. (11) Montgomery, M. J., Schu]tz, L. /-L, and Baumgardt, B. R. 1963. Effect of Intraruminal Infusion of Volatile F a t t y Acids and Lactic Acid on Voluntary Hay Intake. J. Dairy Sci., 46:1380. (12) Radloff, H. D., and Schultz, L~ B:. 1963. Some Effects of Feeding Lactates to Dairy Cows. J. Dairy Sei., 46:517. (13) Reid, R. L. 1960. The Determination of Ketone Bodies in Blood. The Analyst, 85:265. (14) Steel, R. G. D., and Torrie, J. It. 1960. Principles and Procedures of Statistics. McGraw-~Iill Book Company, Inc., New York. (15) Sumnlary of Second Silage Conference. 1959. P a r t I. ]~eltsville, Maryland. (16) Van Soest, P. J. 1963. Ruminant :Fat Metabolism with Particular Reference to Factors Affecting Low Milk F a t and Feed Efficiency. J. Dairy Sci., 46:204. (17) Wiseman, H. G., and Irvin, H. M. 1957. Chromatographic Separation of Silage Acids. J. Food Chem., 5:213.