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Comparison of in Vivo and in Vitro Techniques in Ruminology Studies1
C O M P A R I S O N OF I N V I V O A N D I N V I T R O T E C H N I Q U E S IN RUMINOLOGY STUDIES 1 F. G. ttUETER, R. J. GIBBONS, J. C. SHAW, _~D R. N. DOETSCH
Depart~ents of Dairy Husbandry and Microbiology, University of Maryland, College Park SUMMARY
This paper is concerned with the comparison of in vivo and in vitro rumen bacterial dissimilations of some carbohydrates, anfino acids, and organic acids of metabolic importance. It was observed that sueeinate, I)L-aspartate, glucose, and maltose were readily fermented by rumen microorganisms, and that volatile fatty acids (VFA) were produced. The mnino acids DL-lysine, I)L-alanine, and glycine were not degraded to an appreciable extent to YFA by rmnen bacteria. In general, qualitative agreement between in vivo and in vitro washed cell suspension (WCS) experiments was obtained with DL-lysine, DL-alanine, and glycine, while the in vivo studies using D~-aspartate and suceinate agreed with the WCS in vitro studies of Sirotnak et al. (17j 18). With the substrates glucose and maltose, in vitro WCS experiments were both variable and in disagreement with the in vivo studies. Thus, the WCS technique appears most useful for studying short oneor two-step reactions presumed to occur in the rumen. This technique loses significance when studying nmltistep metabolic reactions and, in such cases, the results should be interpreted with caution until they are verified by in vivo experiments, or until further studies concerning the limitations of the WCS tec]mique are available.
Ill vitro investigations of the rumen flora require the use of one, or a combination, of the following methods : Microscopic examinations, pure culture studies of bacteria, studies on cell-free enzymes from bacterial or protozoan suspensions, and various types of the so-called artificial rumen (3). Because the determination of volatile f a t t y acids ( V F A ) in the rnmen is, at best, only a rough estimate of production and can not be used for precise studies on the metabolism of rumen bacteria, Johns et al. (9), Sijpesteijn and Elsden (16), and later, Doetsch et al. (5) have used buffered washed cell suspensions ( W C S ) of rumen bacteria in an attempt to add percision to such studies. Elsden (16) stated the following advantages for this technique: (a) The population is studied as an entity under controlled conditions; (b) the enzymatic action of the population in the absence of an added substrate is negligible; (c) since the experimental period is short and growth is not permitted, the chance of significant alteration in the composition of the population is nil, and (d) small-scale work is possible which permits the investigation of tile degradation of expensive or toxic materials. This technique and its modifications have been described by Doetsch
et at. (5). An extensive study was conducted by Doctsch et al. (4), in which the washed cell suspension technique was used to study catabolic reactions of rumen bacteria. The dissimilation of numerous substrates was studied in regard to gas and V F A production. Similar studies on amino acids, polysaecharide synthesis, and lactic Received for publication August 6, 1957. 1Scientific Article No. A641, Contribution No. 2824 of the Maryland Agricultural Experiment Station. 651
652
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acid formation were reported by Sirotnak et al. (17), Gibbons et al. (8), and J u r t s h u k and H u e t e r (10). A later study was conducted by Sirotnak et al. (18) on aspartate dissimilation reactions of rumen bacteria. The results confirmed previous findings and, in addition, indicated the probable pathways of dissimilation of aspartic acid. The data reported in this p a p e r are concerned with the comparison of in vivo and in vitro r u m e n bacterial dissimilations of some carbohydrates, amino acids, and organic acids of metabolic importance. The purpose was to determine the extent to which in vitro dissimilations are representative of in vivo occurrences. Our intention was to make a simultaneous and more direct comparison between in vivo and in vitro results than has been done heretofore. EXPERI:SIENTAL
PROCEDURE
Animal feeding. Two p e r m a n e n t l y fistulated, non]aetating and n o n p r e g n a n t cows (Jersey and Holstein) were used as experimental animals. They were fed slightly in excess of Morrison's recommended standards (14) on a ration of 8 lb. of alfalfa h a y and 6 lb. of 16% protein concentrate mixture. The concentrate ration was fed twice daily except on (lays of experimental trials, when the mornlug feeding was omitted. H a y was fed only in the evening, and in controlled amounts to insure complete consumption by 12 midnight preceding a trial. On trial days, water was withheld f r o m the animals starting at 12 midnight and ending at 4:30 ~.~I., at which time they were allowed access to water for approximately 0.5 hr. The source of water was then removed, and to afford sufficient time for the ingested water to reach equilibrium within the animal, an interval of a p p r o x i m a t e l y 3.5 hr. was allowed before the s t a r t of the trial. A f t e r the morning watering, the animals did not receive water again until the end of the trial period. The cows remained at a p p r o x i m a t e l y constant weight and were in good health throughout the entire study. Sampling. R u m e n liquor samples (590 ml.) were obtained via the fistula with the aid of a metal eannula and rubber hose connected to an aspirator pump. Samples were taken prior to administration of substrate and at intervals after substrate addition. I m m e d i a t e l y after removal, the rumen liquor was strained through two layers of cheesecloth, then eentrifuged at 1,000 × G for 15 rain. to remove the residual feed particles. Aliquot samples of the resultant s u p e r n a t a n t liquid were preserved for lactic acid and volatile f a t t y acid analyses. Samples for lactic acid determination were preserved by adding 0.1 ml. of 100% ( w / v ) trichloroacetic acid to 1 ml. of r u m e n liquor. Samples for V F A determinations were preserved by adding 1 ml. of a saturated mecuric chloride solution to 9 nd. of rumen liquor. The p H of r u m e n samples, when determined, was measured within 2 to 3 min. after removal. Although these measurements probably did not represent the precise i n t r a r u m i n a l p H , it appears reasonable to assume that a n y large changes in r m n e n liquor p H would be detected by this procedure. F o r in ¥itro dissimilation studies, cell suspensions were p r e p a r e d as described b y Doetsch et al. (5). These cell suspensions were made from the rumen liquor
C O M P A R I S O N OF T E C H N I Q U E S IN R U M I N O L O G ¥
653
of an experimental animal, on which an in vivo study of the same substrate was being made simultaneously. Two different cell suspension dissimilations were p e r f o r m e d for each in vitro study. One cell suspension, referred to hereafter as the 0-hr. suspension, was p r e p a r e d immediately before adding the substrate to the rumen. The other cell suspension, referred to hereafter as the 5-hr. suspension, was p r e p a r e d f r o m the rumen liquor of the same cow 5 hr. a f t e r the administration to the r u m e n of the substrate being studied. The bacteria in the 0-hr. suspension had no previous contact with the substrate, whereas those of the 5-hr. suspension were exposed for 5 hr. to the p a r t i c u l a r substrate being investigated. The fermentation liquor of in vitro dissimilation studies was saved for V F A determinations. The samples were preserved as just described. R u m e n liquor lactic acid was determined by the method of B a r k e r and Summerson (1) and the V F A by the method of Keeney (11). The Beckman p H Meter, Model H-2, using s t a n d a r d glass electrodes, was used for measuring the p H of rumeu liquor. All substrates used in the in vivo studies were diluted with water to a volume of 1.5 to 2.0 liters, then administered via the r u m e n fistula. The following substrates were used for in vivo studies: sodium sueeinate, DL-aspartie acid, DL-aspartie acid neutralized to p H 6.9 with Na2COs, D-glucose, nialtose, DL-lysine, and a m i x t u r e of glycine plus DL-alanine. I n order to compare in vivo with in vitro results, in vitro dissimilations were made on the same substrates, with the exception of sueeinate and aspartate. The dissimilation of these latter substrates by W C S has been stndied in detail by Sirotnak et al. (17, 18); hence, was not included. RESULTS
Sodium succinate and Db-aspartic acid. Results of in vivo studies with sodium succinate and nL-aspartie acid are summarized (Table 1). As a control, the rumen liquor analyses for a cow f r o m which feed had been withheld for 18 hr. preceding the sampling period are shown. I t will be noted t h a t the concentration of propionie acid in the r u m e n increased a p p r o x i m a t e l y 17 and 9 molar per cent, respectively, following additions of sodium suceinate and neutralized DL-aspartic acid. Decreases occurred in the tureen liquor concentrations of acetic acid and butyric plus higher acid fractions following the administration of sodium suceinate. A decrease also occurred in the acetic acid concentration of the tureen liquor of the cow which received neutralized tm-aspartic acid, with a concurrent decrease in butyric acid t h a t a p p e a r e d almost equal to the increases in valeric and higher acids. This increase in the valeric and higher acid fraction m a y have been owing to an increase in branched-chain f a t t y acids resulting f r o m the Stickland-type reaction, as suggested by E1Shazly (7). The DL-aspartie acid which was not neutralized was not dissimilated to V F A . Indeed, the concentration of total V F A decreased more r a p i d l y than in the fasting cow, p r o b a b l y owing to the low p H of the r u m e n liquor. No consistent or marked changes occurred in the relative concentrations of the various f a t t y
TABLE 1
Effect of in vivo dissimilation of sodium succinate and DL-aspartic acid on tureen liquor composition and pH Trial No.
Treatment
Sampling time
Total YFA
1
P r e v i o u s l y f a s t e d 18 hr.
(hr.)
(~M/ml)
.....
0 0.5 2 5 8
91.4 87.8 78.2 75.2 54.8
68.7 68.7 68.5 68.0 68.5
16.1 ]6.0 16.1 15.9 15.3
13.2 13.4 ]3.3 13.3 13.6
2
1 kg. sodium s u c c i n a t e
O 0.5 1.5 3.5 5.5 8
21.3 49.6 55.1 56.4 66.6 47.1
69.5 66.5 65.3 61.5 53.9 57.4
18.9 19.7 22.3 27.6 35.7 32.4
11.6 '' 13.8 12.4 10.9 ]0.4 10.2
3
1 kg. DL-aspartic acid ( u n n e u t r a l i z e d )
0 0.5 2 5
47.9 35.8 24.1 18.1
68.2 69.7 67.3 66.2
15.0 ]5.1 15.2 ]7.9
12.8 9.0 8.1 9.4
4.0 6.2 9.4 6.5
7.5 4.5 5.4 6.5
4
1 kg. DL a s p a r t i c acid ( u n n e u t r a | i z e d )
0 0.5 2 5
66.5 50.6 39.2 43.5
66.0 68.2 67.6 65.0
15.7 15.8 17.2 19.8
13.0 11.7 ]3.4 11.6
4.4 4.3 1.8 3.6
5.7 6.5 7.0
0 0.5 2 5 8
70.7 60.3 58.4 64.2 69.4
66.8 67.0 65.1 62.7 62.4
15.3 15.3 18.3 22.5 24.4
15.1 14.2 13.5 10.9 9.7
2.8 3.5 3.1 3.9 3.7
6.5 7.1 7.2 7.2 7.3
5
1 kg. DL-aspartic acid ( n e u t r a l i z e d to p H 6.9 w i t h Na2CO~)
a I n c l u d e s v a l e r i c a n d other h i g h e r acids.
Acetic
Propionic
Butyric
V a l e r i c p lu s higher acids
(Molar % total V F A ) - -
Rumen liquor
(pH)
1.9 1.8 2.1 2.8 2.6
6.9 7.0 6.9 6.8 7.0
. . . . . . . ........ ........ ........ ........ ........
7.6
COMPARISON OF TECHNIQUES IN RUMINOLOGY
655
acids. However, deamination did occur. This is in agreement with the findings of Sirotnak et al. (18). Lactic acid was not observed in these rumen liquor samples. Glucose. The D-glucose dissimilation data are shown (Table 2). The principal in vivo changes were increases in r u m e n propionic, butyric, and lactic acid concentrations, and decreases in rumen liquor p H and free ammonia concentration. Valeric acid concentration did not undergo any appreciable changes. A large decrease in free ammonia concentration of the rumen liquor adds confirmation to the view that ammonia utilization is enhanced by the presence of readily available carbohydrate. Increases in propionic acid concentration also were observed during the dissimilation of glucose by WCS. The presence of large amounts of glucose in the rumen appeared to affect the activity of the bacteria, because the WCS prepared 5 hr. after administering glucose attacked this substrate more vigorously and with a greater production of propionic acid than did the WCS without previous exposure to this substrate. Maltose. In vivo maltose dissimilation (Table 3) did not result in appreciable changes in the relative proportions of V F A . Total V F A concentration increased rather markedly in most cases. This is made evident by comparing the maltose data with those of the control cow (shown in Table 1). During these trials, a relatively large increase in lactic acid occurred which, when combined with the increase in total VFA, resulted in a marked decrease in rumen pH. I t will be noted that the lactic acid concentration following the administration of maltose was much greater than that from glucose in Trial 6 (Table 2). This observation agrees with the results of Robinson et al. (15). The results of the in vitro dissimilations of maltose by cell suspensions were similar to the ill vivo results. The proportion of acetic acid was lower, and that of propionic and butyric acids higher, than in the case of the in vivo trials. F o r proper evah~ation, the butyric plus higher acid fraction in the dissimilations by cell suspensions should be compared with the combined butyric and valeric acid fractions of the in vivo trials. The one major exception noted was the large increase in the propionic acid concentration of the 0-hr. WCS dissimilation with added maltose (Trial 12). DL-lysine. The in vivo and in vitro data (Table 4) indicate that little or no V F A were produced from DL-lysine. Sirotnak et al. (17), using L-lysine, obtained similar results. It will be noted that there was a decrease in total V F A in the rumen (absorption) and an increase in rumen pH. Only slight increases were noted in rumen lactic acid concentrations. Glycine plus DL-alanine. The in vivo data provided little or no evidence of V F A production from the mixture of glycine and DL-alanine, although there was a large production of ammonia (deamination) and some production of lactic acid. The in vivo and in vitro dissimilations of the glycine and DL-alanine mixture were in close agreement, in that both indicated that the mixture, if fermented at all by the rumen microorganisms, produced negligible amounts of VFA. The uniform p H of the rumen liquor during these trials served as supporting evidenec for the preceding statement. These observations v e r i f y the results of Sirotnak et al. (17).
TABLE 2
In vivo and in vitro dissimilations of glucose by tureen microorganisms
Trial No.
Treatment 1.5 kg. glucose a d d e d in vivo
1.5 kg. glucose a d d e d ill vivo
8•
I11 vitro WCS f r o m Trial 6
S~,mpling time
Total VFA
(hr.)
(uM/ml)
0 0.5 2 5
57.2 50.0 73.5 69.5
68.5 63.1 57.6 57.9
13.0 16.0 21.5 21.5
14.3 18.1 ]8.9 18.5
4.2 2.8 2.0 2.1
7.1 6.6 6.3 6.9
0.0 2.5 3.2 0.0
0 0.5 2 5
]01.5 89.5 105.0 93.0
67.8 61.8 57.6 56.3
16.6 19.3 19.4 ]9.3
] 1.9 16.1 21.2 21.8
3.7 2.8 ].8 2.6
6.6 6.0 6.0 6.5
"v .... .... ....
0-hr. 0-hr. 5-hr. 5-hr.
15.0 19.5 12.6 35.5
68.0 65.0 58.5 60.9
13.5 ]6.8 20.1 28.0
9.1 10.4 14.1 8.2
9.4 7.3 7.3 2.9
Control, no s u b s t r a t e 400 ~M g lu co s e ~dded Control, no s u b s t r a t e 400 ~M glucose a d d e d
Acetic
Propionic
--(Molar
Butyric
V a l e r i c pl us h i g h e r a c i ds
Rumen liquor R u m e n liq u o r free lactic acid ammonia
% totalVFA)--
Rumen liq u o r
(pH)
---(~M/ml)---
a D i s s i m i l a t i o n s by W C S p r e p a r e d f r o m r u m e n l i quor o b t a i n e d b e f o r e (0-hr,) a n d a f t e r (5-hr.) a d d i n g glucose in T r i a l 6 above.
7.2 4.3 4.5 1.0
TABLE 3
In vivo and in vitro dissimilations of maltose by rumen microorganisms Trial No.
Treatment
Sampling time
Total VFA
Acetic
. . . .
Proplonie
Butyric
Valeric plus higher acids
(Molar % total V F A ) - - - -
Rumen liquor
Rumen liquor lactic acid
(hr.)
(t~M/ml)
(pH)
(~M/ml)
9
1.5 kg. maltose added in vivo
0 0.25 0.5 2 5
10].3 85.0 91.5 140.2 125.8
64.2 61.8 63.1 62.0 62.7
17.5 ]8.4 19.3 18.1 ]6.9
]1.3 14.1 13.2 16.1 ].6.4
7.0 5.7 4.4 3.8 4.0
6.6 6.1 6.0 5.6 5.9
0.0 11.0 13.0 4.5 0.45
10
1.5 kg. maltose added in vlvo
0 0.25 0.5 2 5
114.4 100.8 111.9 129.4 134.8
62.4 60.6 62.t 59.3 59.4
16.3 19.0 17.6 18.4 15.5
15.2 14.8 14.8 17.6 18.8
6.1 5.6 5.5 4.7 6.3
6.7 6.2 6.0 5.8 6.0
O.0 8.7 ]1.6 5.1 0.11
11
1.5 kg. maltose added in vivo
0 0.25 0.5 2 5
122.1 121.5 127.4 ].20.6 97.3
59.9 6].4 58.5 59.1 60.7
20.8 20.7 19.8 20.4 18.9
14.2 13.6 13.2 15.7 15.3
5.1 4.3 8.5 4.8 5.1
6.0 5.6 5.5 5.2 4.9
...... ...... ...... ...... ......
12 a
I n vitro WCS f r o m Trial 9
0-hr. 0-hr.
29.1 46.2
55.7 52.9
18.7 28.2
25.6 e 18.9
Control, no s u b s t r a t e 200 t~M maltose added
5-hr.
34.1
52.0
24.9
23.1
200 ~M maltose added
13 b
I n vitro WCS f r o m Tria 10
0-hr. 0-hr.
21.1 30.5
53.5 55.2
23.0 22.6
23.5 ~ 22.2
Control, no s u b s t r a t e 200 ~M maltose added
5-hr.
34.0
53.5
23.0
23.5
200 #M maltose added
Dissimilations by WCS p r e p a r e d f r o m tureen llquor obtained before (0-hr.) and a f t e r (5-hr.) a d d i n g maltose in Trial 9 above. b Dissimilations by WCS p r e p a r e d f r o m r u m e n liquor obtained before (0-hr.) and a f t e r (5-hr.) adding maltose in Trial 10 above. e Includes valerie ~md other higher acids.
TABLE 4
I n vivo and in vitro dissimilations of DL-lysinc by rumen microorganis'ms Trial No.
Treatment
Sampling time
Total VFA
Acetic
Propionic
--(Molar
Butyric
Valeric plus higher acids
% total V F A ) - -
Rumen liquor
Rumen liquor lactic acid
(hr.)
(~M/ml)
(pH)
(tzM/ml)
14
1.5 kg. 4 -lysine added in vivo
0 0.5 2 5
50.4 54.2 49.9 42.5
69.6 71.1 73.4 78.6
21.4 18.1 17.8 18.9
8.5 10.5 8.5 1.7
0.5 0.3 0.3 0.8
7.4 7.0 7.2 7.4
0.0 0.18 0.0 0.0
15
1.5 kg. DL-lysine added in vivo
0 0.5 2 5
38.0 35.5 33.8 33.5
64.2 65.4 66.0 65.0
19.5 18.3 18.1 17.8
13.0 11.6 12.6 14.7
3.3 4.7 3.3 2.5
6.9 6.8 7.1 7.0
O.0 0.33 0.0 0.0
16 a
I n vitro WCS f r o m Trial 17
0-hr. 0-hr.
17.0 18.0
68.3 69.3
22.3
16.6
9.4 ~ 14.1
Control, no s u b s t r a t e 200 #M DL-]ysine added
5-hr. 5-hr.
17.0 17.0
66.8 73.6
17.6 16.3
15.6 10.1
Control, no s u b s t r a t e 200 ~M I)L-lysine added
Dissimilations by WCS p r e p a r e d f r o m r u m e n liquor obtained b e f o r e (0-hr.) and a f t e r (5-hr.) a d d i n g DL-lysine in Trial 14 above. b Includes valerie and other higher acids.
COMPARISON OF T E C H N I Q U E S IN I~UMINOLOGY
659
DISCUSSION
In three trials, the in vivo and in vitro results were in close agreement qualitatively. Quantitative differences existing between in vivo and in vitro results could be owing to intraeonversions among the V F A and to absorption occurring during the in vivo trials. The results of the in vivo fermentation of suecinate was similar to the in vitro results reported by Doetsch et al. (4). The failure to obtain any apparent fermentation of aspartie acid in vivo is attributed to the abnormal p H of the rumen contents caused by the DL-aspartic acid. The extremely high acidity (approximately p H 4.5) may have adversely affected rumen bacterial metabolism and interfered with normal dissimilation. When aspartic acid was partially neutralized to p H 6.9 with Na2CO~, it was attacked by rumen microorganisms and resulted in a marked increase of VFA, mainly propionic acid. The in vivo results of partially neutralized aspartie acid are similar to in vitro results of WCS reported by Sirotnak et al. (18). It is of interest to note that in vivo trials with glucose indicated that the presence of fermentable carbohydrates enhances ammonia utilization by rumen microorganisms. I t was noted also that the in vivo fermentation of maltose and glucose resulted in the production of lactic acid, as well as VFA. The lactic acid reached a maximum concentration within 2 hr. after the administrations of the substrates and in 5 hr. had almost completely disappeared from the rumen. This high concentration of lactic acid may be explained by assuming that it is a normal intermediate in carbohydrate metabolism in the rumen, and that its formation is occurring at a faster rate than its dissimilation a n d / o r absorption. Similar results concerning lactic acid production have been reported by Waldo and Sehultz (19). DL-lysine or a combination of glyeine and DL-alanine was not fermented to V F A by rumen microorganisms. These results agree with in vitro WCS results of Sirotnak et al. (17). However, glycine plus DL-alanine was deaminated in vivo by rumen microorganisms. The identity of the compound (s) resulting is as yet unknown. Loosli et al. (12), Black et al. (2), and Duncan et al. (6) have provided evidence demonstrating the synthesis of essential amino acids by rumen microorganisms. Perhaps reactions in the rumen involving essential amino acids are entirely assimilatory in nature. Deamination processes would aid in supplying ammonia required for bacterial (McDonald, 13), and possibly host, protein synthesis. Observations made on rumen liquor p H values during in vivo trials indicated that the p H of the rumen liquor is directly related to the total V F A concentration of the ruminal contents. It is thought that these measurements, although not representing the precise intraruminal pH, do reflect any large changes in rumen liquor p H values. During the in vitro trial with glucose (Trial 8, Table 2), differences were observed between the fermentation of the added glucose by the 0-hr., as compared to the 5-hr., WCS. I t should be noted that increases in butyric acid occurred during both the in vivo and in vitro trials. However, with added substrate, the in vitro concentration of butyric acid by the 5-hr. WCS did not keep pace with
660
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G. H U E T E R
ET AL
t h e in v i v o c o n c e n t r a t i o n increases, a l t h o u g h a c o n s i d e r a b l e i n c r e a s e d i d occur. These r e s u l t s i n d i c a t e t h a t th e in v i v o a d m i n i s t r a t i o n of r e l a t i v e l y l a r g e a m o u n t s of c e r t a i n c a r b o h y d r a t e s c a n a l t e r t h e a c t i v i t y of r u m e n m i c r o o r g a n i s m s . Th e effects of c e r t a i n c a r b o h y d r a t e s on lactic acid a n d p o l y s a c c h a r i d e p r o d u c t i o n b y b o v i n e r u m e n b a c t e r i a h a v e been r e p o r t e d by R o b i n s o n et al. ( 1 5 ) a n d Gibbons et al. ( 8 ) . A l t e r a t i o n s m a y o c c ur f o r one or m o r e of t h e f o l l o w i n g r e a s o n s : ( a ) a b a c t e r i a l p o p u l a t i o n in w h i c h one or m o r e species h a v e i n c r e a s e d in n u m b e r s ; ( b ) an i n c r e a s e d m e t a b o l i c a c t i v i t y of one or m o r e species of microo r g a n i s m s ; or ( c ) th e e s t a b l i s h m e n t of a m e t a b o l i c s h u n t p a t h w a y in the p r e s e n c e of excess s u b s t r a t e b y one or m o r e species of r u m e n m i c r o o r g a n i s m s . A l t h o u g h q u a l i t a t i v e in v i v o a n d in v i t r o r esu l t s w e r e s i m i l a r , q u a n t i t a t i v e differences w e r e noted. V a r i a t i o n s m a y be the r e s u l t of d i f f e r e n t i a l a b s o r p t i o n of e n d - p r o d u c t s , c h a n g e s in p H p l u s o t h e r in v i v o factors, a n d a l t e r e d b a c t e r i a l m e t a b o l i s m . These differences also c o u ld be o w i n g to e n z y m e i n a c t i v a t i o n in p r o c e s s i n g cell suspensions, e x c l u s i o n of protozoa, omission of i m p o r t a n t b a c t e r i a because of a d h e r e n c e to v a r i o u s m a t e r i a l , lack of c o f a c t o r s f o r c e r t a i n reactions, assimilations, a n d / o r u t i l i z a t i o n of some V F A , o w i n g to t h e absence of a b s o r p t i o n . Th e in v i t r o cell s u s p e n s i o n m e t h o d a p p e a r s to be a u s e f u l t e c h n i q u e f o r s t u d y i n g toxic a n d / o r e x p e n s i v e s u b s t r a t e s ; f o r a r t i f i c i a l l y a l t e r i n g the c h e m i c a l e n v i r o n m e n t in a p r e d i c t a b l e m a n n e r a n d d i r e c t i o n ; f o r p h a r m a c o l o g i c a l investigations, and for pilot studies for subsequent animal experiments. REFERENCES (1) BARK~, S. B., A~D SUM~E~RS0N,W. H. The Colorimetric Determination of Lactic Acid in Biological Material. J. Biol. Chem., 138: 535. 194'1. (2) BL_~CK, A., KI~IBE~, M., ANI) S_~ITH, A. H. Carbonate and Fatty Acids as Precursors of Amino Acids in Casein. J. Biol. Chem., 197: 365. 1952. (3) DOETSCtt, R. N., AND ROBINSON, R. Q. The Bacteriology of the Bovine Rumen: A Review. J. Dairy Sci., 36: 115. 1953. (4) DOETSCH,:[~. N., ROBINSON, R. Q., BICOWN, R. E., AND SHA\¥, J. C. Catabolic Reactions of Mixed Suspensions of Bovine Rumen Bacteria. J. Dairy Svi., 36: 825. 1953. (5) DOETSCH, R. N., SHAW, J. C., McNE~I~L, J. J., ANI) JUaTSKUK, P., Jm Observations on the Use of Mixed Suspensions of Bovine Rumen Bacteria as a Tedlnique of the Rumen Microbiologist. Maryland Agr. Expt. Sta., Misc. Publ. 238: 1. 1955. (6) DUNCAN,C. W., AGRAWALA,I. P., ]:[UFFMAN,C. F., AND,LUECKE, R. W. A Quantitative Study of Rumen Synthesis in the Bovine on Natural and Purified Diets. II. Ami~:o Acid Content of Mixed Rumen Proteins. J. Nutrition, 49: 41. 1953. (7) EL-SHAZLY, K. Degradation of Protein in the Rumen of Sheep. II. The Action of Runlen Micro-organisms on Amino Acids. Biochem. J., 51 : 647. 1952. (8) GIBBONS, ]~. J., D0]gTSCH, R. N., AND SHAW, J. C. Further Studies on Polysaccharide Production by Bovine Rumen Bacteria. J. Dairy Sci., 38: 1147. 1955. (9) JOHNS, A. T. Isolation of a Bacterium, Producing Propionic Acid, from the Rumen of Sheep. J. Gen. Microbiol., 5: 317. 1951. (10) JUI~TSHUK,P., JR., AND HUETEE, F. G. In Vitro Studies on the Dissimilation of Purines and Pyrimidines by Bovine Rumen Bacteria. Maryland Agr. Expt. Sta., Misc. Publ. 238: 4. 1955. (11) KEBSrE¥, M. Direct Chromatographic Determination of C~ to C~ Fatty Acids in Rumen Fluid. Maryland Agr. Expt. Sta., Misc. Publ. 238: 23. 1955.
COMPARISON
OF TECHNIQUES IN RUMINOLOGY
661
(12) LOOSLI, J. K., WILLIAMS, H. ~I., THOMAS, W. E., FE,RUlS, F. tt., AN1) MAYNAt~I), L. A. Synthesis of Amino Acids in the Rumen. Science, 110: 144. 1949. (13) McDONALD, I. W. The Role of Ammonia in Ruminal Digestion of Protein. Bioche~t. J., 51: 86. 1952. (14) MO~alSO~, F. B. Feeds and Feeding. 1949,
21st ed. The Morrison Publ. Co., Ithaca, 1~. Y.
(15) ~OBINSON, R. Q., DOE.TSCH, i~.. N., SIROTNAK, F. M., .~-ND SHAW, J. C. Production of Lactic Acid and an Iodine Staining Substance by Bovine Rumen Baeteria. J. Dairy Sci., 38: 13. 1955.
(16) SIJPESTEIIJN, A. K., AND ELSI)E,N, S. R. The Metabolism of Succinic Acid in the Rumen of the Sheep. Biochem. J., 52: 41. 1952. (17) SII~0TNAK, F. M., DOETSCH, R. 1~., BROWN, R. E., AND SHA~V, J. C. Amino Acid Metabolisnl of Bovine Rumen Bacteria. J. Dairy Sci., 35: 1117. 1953. (18) SIgOTNAK, F. M., DOETSCH, l~. N., ROBINSON, R. Q., AND SHAW, J. C. Aspartate Dissimilation Reactions of Rumen Bacteria. J. Dairy Sci., 37: 531. ]954. (19) WALDO, D. ]:~., AIqD SCHULTZ, L. H. Lactic Acid Production in the Rumen. J. Dairy Sci., 38: 605. 1955.
Depart~ents of Dairy Husbandry and Microbiology, University of Maryland, College Park SUMMARY
This paper is concerned with the comparison of in vivo and in vitro rumen bacterial dissimilations of some carbohydrates, anfino acids, and organic acids of metabolic importance. It was observed that sueeinate, I)L-aspartate, glucose, and maltose were readily fermented by rumen microorganisms, and that volatile fatty acids (VFA) were produced. The mnino acids DL-lysine, I)L-alanine, and glycine were not degraded to an appreciable extent to YFA by rmnen bacteria. In general, qualitative agreement between in vivo and in vitro washed cell suspension (WCS) experiments was obtained with DL-lysine, DL-alanine, and glycine, while the in vivo studies using D~-aspartate and suceinate agreed with the WCS in vitro studies of Sirotnak et al. (17j 18). With the substrates glucose and maltose, in vitro WCS experiments were both variable and in disagreement with the in vivo studies. Thus, the WCS technique appears most useful for studying short oneor two-step reactions presumed to occur in the rumen. This technique loses significance when studying nmltistep metabolic reactions and, in such cases, the results should be interpreted with caution until they are verified by in vivo experiments, or until further studies concerning the limitations of the WCS tec]mique are available.
Ill vitro investigations of the rumen flora require the use of one, or a combination, of the following methods : Microscopic examinations, pure culture studies of bacteria, studies on cell-free enzymes from bacterial or protozoan suspensions, and various types of the so-called artificial rumen (3). Because the determination of volatile f a t t y acids ( V F A ) in the rnmen is, at best, only a rough estimate of production and can not be used for precise studies on the metabolism of rumen bacteria, Johns et al. (9), Sijpesteijn and Elsden (16), and later, Doetsch et al. (5) have used buffered washed cell suspensions ( W C S ) of rumen bacteria in an attempt to add percision to such studies. Elsden (16) stated the following advantages for this technique: (a) The population is studied as an entity under controlled conditions; (b) the enzymatic action of the population in the absence of an added substrate is negligible; (c) since the experimental period is short and growth is not permitted, the chance of significant alteration in the composition of the population is nil, and (d) small-scale work is possible which permits the investigation of tile degradation of expensive or toxic materials. This technique and its modifications have been described by Doetsch
et at. (5). An extensive study was conducted by Doctsch et al. (4), in which the washed cell suspension technique was used to study catabolic reactions of rumen bacteria. The dissimilation of numerous substrates was studied in regard to gas and V F A production. Similar studies on amino acids, polysaecharide synthesis, and lactic Received for publication August 6, 1957. 1Scientific Article No. A641, Contribution No. 2824 of the Maryland Agricultural Experiment Station. 651
652
~. G. H U E T E R
E T .A.L
acid formation were reported by Sirotnak et al. (17), Gibbons et al. (8), and J u r t s h u k and H u e t e r (10). A later study was conducted by Sirotnak et al. (18) on aspartate dissimilation reactions of rumen bacteria. The results confirmed previous findings and, in addition, indicated the probable pathways of dissimilation of aspartic acid. The data reported in this p a p e r are concerned with the comparison of in vivo and in vitro r u m e n bacterial dissimilations of some carbohydrates, amino acids, and organic acids of metabolic importance. The purpose was to determine the extent to which in vitro dissimilations are representative of in vivo occurrences. Our intention was to make a simultaneous and more direct comparison between in vivo and in vitro results than has been done heretofore. EXPERI:SIENTAL
PROCEDURE
Animal feeding. Two p e r m a n e n t l y fistulated, non]aetating and n o n p r e g n a n t cows (Jersey and Holstein) were used as experimental animals. They were fed slightly in excess of Morrison's recommended standards (14) on a ration of 8 lb. of alfalfa h a y and 6 lb. of 16% protein concentrate mixture. The concentrate ration was fed twice daily except on (lays of experimental trials, when the mornlug feeding was omitted. H a y was fed only in the evening, and in controlled amounts to insure complete consumption by 12 midnight preceding a trial. On trial days, water was withheld f r o m the animals starting at 12 midnight and ending at 4:30 ~.~I., at which time they were allowed access to water for approximately 0.5 hr. The source of water was then removed, and to afford sufficient time for the ingested water to reach equilibrium within the animal, an interval of a p p r o x i m a t e l y 3.5 hr. was allowed before the s t a r t of the trial. A f t e r the morning watering, the animals did not receive water again until the end of the trial period. The cows remained at a p p r o x i m a t e l y constant weight and were in good health throughout the entire study. Sampling. R u m e n liquor samples (590 ml.) were obtained via the fistula with the aid of a metal eannula and rubber hose connected to an aspirator pump. Samples were taken prior to administration of substrate and at intervals after substrate addition. I m m e d i a t e l y after removal, the rumen liquor was strained through two layers of cheesecloth, then eentrifuged at 1,000 × G for 15 rain. to remove the residual feed particles. Aliquot samples of the resultant s u p e r n a t a n t liquid were preserved for lactic acid and volatile f a t t y acid analyses. Samples for lactic acid determination were preserved by adding 0.1 ml. of 100% ( w / v ) trichloroacetic acid to 1 ml. of r u m e n liquor. Samples for V F A determinations were preserved by adding 1 ml. of a saturated mecuric chloride solution to 9 nd. of rumen liquor. The p H of r u m e n samples, when determined, was measured within 2 to 3 min. after removal. Although these measurements probably did not represent the precise i n t r a r u m i n a l p H , it appears reasonable to assume that a n y large changes in r m n e n liquor p H would be detected by this procedure. F o r in ¥itro dissimilation studies, cell suspensions were p r e p a r e d as described b y Doetsch et al. (5). These cell suspensions were made from the rumen liquor
C O M P A R I S O N OF T E C H N I Q U E S IN R U M I N O L O G ¥
653
of an experimental animal, on which an in vivo study of the same substrate was being made simultaneously. Two different cell suspension dissimilations were p e r f o r m e d for each in vitro study. One cell suspension, referred to hereafter as the 0-hr. suspension, was p r e p a r e d immediately before adding the substrate to the rumen. The other cell suspension, referred to hereafter as the 5-hr. suspension, was p r e p a r e d f r o m the rumen liquor of the same cow 5 hr. a f t e r the administration to the r u m e n of the substrate being studied. The bacteria in the 0-hr. suspension had no previous contact with the substrate, whereas those of the 5-hr. suspension were exposed for 5 hr. to the p a r t i c u l a r substrate being investigated. The fermentation liquor of in vitro dissimilation studies was saved for V F A determinations. The samples were preserved as just described. R u m e n liquor lactic acid was determined by the method of B a r k e r and Summerson (1) and the V F A by the method of Keeney (11). The Beckman p H Meter, Model H-2, using s t a n d a r d glass electrodes, was used for measuring the p H of rumeu liquor. All substrates used in the in vivo studies were diluted with water to a volume of 1.5 to 2.0 liters, then administered via the r u m e n fistula. The following substrates were used for in vivo studies: sodium sueeinate, DL-aspartie acid, DL-aspartie acid neutralized to p H 6.9 with Na2COs, D-glucose, nialtose, DL-lysine, and a m i x t u r e of glycine plus DL-alanine. I n order to compare in vivo with in vitro results, in vitro dissimilations were made on the same substrates, with the exception of sueeinate and aspartate. The dissimilation of these latter substrates by W C S has been stndied in detail by Sirotnak et al. (17, 18); hence, was not included. RESULTS
Sodium succinate and Db-aspartic acid. Results of in vivo studies with sodium succinate and nL-aspartie acid are summarized (Table 1). As a control, the rumen liquor analyses for a cow f r o m which feed had been withheld for 18 hr. preceding the sampling period are shown. I t will be noted t h a t the concentration of propionie acid in the r u m e n increased a p p r o x i m a t e l y 17 and 9 molar per cent, respectively, following additions of sodium suceinate and neutralized DL-aspartic acid. Decreases occurred in the tureen liquor concentrations of acetic acid and butyric plus higher acid fractions following the administration of sodium suceinate. A decrease also occurred in the acetic acid concentration of the tureen liquor of the cow which received neutralized tm-aspartic acid, with a concurrent decrease in butyric acid t h a t a p p e a r e d almost equal to the increases in valeric and higher acids. This increase in the valeric and higher acid fraction m a y have been owing to an increase in branched-chain f a t t y acids resulting f r o m the Stickland-type reaction, as suggested by E1Shazly (7). The DL-aspartie acid which was not neutralized was not dissimilated to V F A . Indeed, the concentration of total V F A decreased more r a p i d l y than in the fasting cow, p r o b a b l y owing to the low p H of the r u m e n liquor. No consistent or marked changes occurred in the relative concentrations of the various f a t t y
TABLE 1
Effect of in vivo dissimilation of sodium succinate and DL-aspartic acid on tureen liquor composition and pH Trial No.
Treatment
Sampling time
Total YFA
1
P r e v i o u s l y f a s t e d 18 hr.
(hr.)
(~M/ml)
.....
0 0.5 2 5 8
91.4 87.8 78.2 75.2 54.8
68.7 68.7 68.5 68.0 68.5
16.1 ]6.0 16.1 15.9 15.3
13.2 13.4 ]3.3 13.3 13.6
2
1 kg. sodium s u c c i n a t e
O 0.5 1.5 3.5 5.5 8
21.3 49.6 55.1 56.4 66.6 47.1
69.5 66.5 65.3 61.5 53.9 57.4
18.9 19.7 22.3 27.6 35.7 32.4
11.6 '' 13.8 12.4 10.9 ]0.4 10.2
3
1 kg. DL-aspartic acid ( u n n e u t r a l i z e d )
0 0.5 2 5
47.9 35.8 24.1 18.1
68.2 69.7 67.3 66.2
15.0 ]5.1 15.2 ]7.9
12.8 9.0 8.1 9.4
4.0 6.2 9.4 6.5
7.5 4.5 5.4 6.5
4
1 kg. DL a s p a r t i c acid ( u n n e u t r a | i z e d )
0 0.5 2 5
66.5 50.6 39.2 43.5
66.0 68.2 67.6 65.0
15.7 15.8 17.2 19.8
13.0 11.7 ]3.4 11.6
4.4 4.3 1.8 3.6
5.7 6.5 7.0
0 0.5 2 5 8
70.7 60.3 58.4 64.2 69.4
66.8 67.0 65.1 62.7 62.4
15.3 15.3 18.3 22.5 24.4
15.1 14.2 13.5 10.9 9.7
2.8 3.5 3.1 3.9 3.7
6.5 7.1 7.2 7.2 7.3
5
1 kg. DL-aspartic acid ( n e u t r a l i z e d to p H 6.9 w i t h Na2CO~)
a I n c l u d e s v a l e r i c a n d other h i g h e r acids.
Acetic
Propionic
Butyric
V a l e r i c p lu s higher acids
(Molar % total V F A ) - -
Rumen liquor
(pH)
1.9 1.8 2.1 2.8 2.6
6.9 7.0 6.9 6.8 7.0
. . . . . . . ........ ........ ........ ........ ........
7.6
COMPARISON OF TECHNIQUES IN RUMINOLOGY
655
acids. However, deamination did occur. This is in agreement with the findings of Sirotnak et al. (18). Lactic acid was not observed in these rumen liquor samples. Glucose. The D-glucose dissimilation data are shown (Table 2). The principal in vivo changes were increases in r u m e n propionic, butyric, and lactic acid concentrations, and decreases in rumen liquor p H and free ammonia concentration. Valeric acid concentration did not undergo any appreciable changes. A large decrease in free ammonia concentration of the rumen liquor adds confirmation to the view that ammonia utilization is enhanced by the presence of readily available carbohydrate. Increases in propionic acid concentration also were observed during the dissimilation of glucose by WCS. The presence of large amounts of glucose in the rumen appeared to affect the activity of the bacteria, because the WCS prepared 5 hr. after administering glucose attacked this substrate more vigorously and with a greater production of propionic acid than did the WCS without previous exposure to this substrate. Maltose. In vivo maltose dissimilation (Table 3) did not result in appreciable changes in the relative proportions of V F A . Total V F A concentration increased rather markedly in most cases. This is made evident by comparing the maltose data with those of the control cow (shown in Table 1). During these trials, a relatively large increase in lactic acid occurred which, when combined with the increase in total VFA, resulted in a marked decrease in rumen pH. I t will be noted that the lactic acid concentration following the administration of maltose was much greater than that from glucose in Trial 6 (Table 2). This observation agrees with the results of Robinson et al. (15). The results of the in vitro dissimilations of maltose by cell suspensions were similar to the ill vivo results. The proportion of acetic acid was lower, and that of propionic and butyric acids higher, than in the case of the in vivo trials. F o r proper evah~ation, the butyric plus higher acid fraction in the dissimilations by cell suspensions should be compared with the combined butyric and valeric acid fractions of the in vivo trials. The one major exception noted was the large increase in the propionic acid concentration of the 0-hr. WCS dissimilation with added maltose (Trial 12). DL-lysine. The in vivo and in vitro data (Table 4) indicate that little or no V F A were produced from DL-lysine. Sirotnak et al. (17), using L-lysine, obtained similar results. It will be noted that there was a decrease in total V F A in the rumen (absorption) and an increase in rumen pH. Only slight increases were noted in rumen lactic acid concentrations. Glycine plus DL-alanine. The in vivo data provided little or no evidence of V F A production from the mixture of glycine and DL-alanine, although there was a large production of ammonia (deamination) and some production of lactic acid. The in vivo and in vitro dissimilations of the glycine and DL-alanine mixture were in close agreement, in that both indicated that the mixture, if fermented at all by the rumen microorganisms, produced negligible amounts of VFA. The uniform p H of the rumen liquor during these trials served as supporting evidenec for the preceding statement. These observations v e r i f y the results of Sirotnak et al. (17).
TABLE 2
In vivo and in vitro dissimilations of glucose by tureen microorganisms
Trial No.
Treatment 1.5 kg. glucose a d d e d in vivo
1.5 kg. glucose a d d e d ill vivo
8•
I11 vitro WCS f r o m Trial 6
S~,mpling time
Total VFA
(hr.)
(uM/ml)
0 0.5 2 5
57.2 50.0 73.5 69.5
68.5 63.1 57.6 57.9
13.0 16.0 21.5 21.5
14.3 18.1 ]8.9 18.5
4.2 2.8 2.0 2.1
7.1 6.6 6.3 6.9
0.0 2.5 3.2 0.0
0 0.5 2 5
]01.5 89.5 105.0 93.0
67.8 61.8 57.6 56.3
16.6 19.3 19.4 ]9.3
] 1.9 16.1 21.2 21.8
3.7 2.8 ].8 2.6
6.6 6.0 6.0 6.5
"v .... .... ....
0-hr. 0-hr. 5-hr. 5-hr.
15.0 19.5 12.6 35.5
68.0 65.0 58.5 60.9
13.5 ]6.8 20.1 28.0
9.1 10.4 14.1 8.2
9.4 7.3 7.3 2.9
Control, no s u b s t r a t e 400 ~M g lu co s e ~dded Control, no s u b s t r a t e 400 ~M glucose a d d e d
Acetic
Propionic
--(Molar
Butyric
V a l e r i c pl us h i g h e r a c i ds
Rumen liquor R u m e n liq u o r free lactic acid ammonia
% totalVFA)--
Rumen liq u o r
(pH)
---(~M/ml)---
a D i s s i m i l a t i o n s by W C S p r e p a r e d f r o m r u m e n l i quor o b t a i n e d b e f o r e (0-hr,) a n d a f t e r (5-hr.) a d d i n g glucose in T r i a l 6 above.
7.2 4.3 4.5 1.0
TABLE 3
In vivo and in vitro dissimilations of maltose by rumen microorganisms Trial No.
Treatment
Sampling time
Total VFA
Acetic
. . . .
Proplonie
Butyric
Valeric plus higher acids
(Molar % total V F A ) - - - -
Rumen liquor
Rumen liquor lactic acid
(hr.)
(t~M/ml)
(pH)
(~M/ml)
9
1.5 kg. maltose added in vivo
0 0.25 0.5 2 5
10].3 85.0 91.5 140.2 125.8
64.2 61.8 63.1 62.0 62.7
17.5 ]8.4 19.3 18.1 ]6.9
]1.3 14.1 13.2 16.1 ].6.4
7.0 5.7 4.4 3.8 4.0
6.6 6.1 6.0 5.6 5.9
0.0 11.0 13.0 4.5 0.45
10
1.5 kg. maltose added in vlvo
0 0.25 0.5 2 5
114.4 100.8 111.9 129.4 134.8
62.4 60.6 62.t 59.3 59.4
16.3 19.0 17.6 18.4 15.5
15.2 14.8 14.8 17.6 18.8
6.1 5.6 5.5 4.7 6.3
6.7 6.2 6.0 5.8 6.0
O.0 8.7 ]1.6 5.1 0.11
11
1.5 kg. maltose added in vivo
0 0.25 0.5 2 5
122.1 121.5 127.4 ].20.6 97.3
59.9 6].4 58.5 59.1 60.7
20.8 20.7 19.8 20.4 18.9
14.2 13.6 13.2 15.7 15.3
5.1 4.3 8.5 4.8 5.1
6.0 5.6 5.5 5.2 4.9
...... ...... ...... ...... ......
12 a
I n vitro WCS f r o m Trial 9
0-hr. 0-hr.
29.1 46.2
55.7 52.9
18.7 28.2
25.6 e 18.9
Control, no s u b s t r a t e 200 t~M maltose added
5-hr.
34.1
52.0
24.9
23.1
200 ~M maltose added
13 b
I n vitro WCS f r o m Tria 10
0-hr. 0-hr.
21.1 30.5
53.5 55.2
23.0 22.6
23.5 ~ 22.2
Control, no s u b s t r a t e 200 ~M maltose added
5-hr.
34.0
53.5
23.0
23.5
200 #M maltose added
Dissimilations by WCS p r e p a r e d f r o m tureen llquor obtained before (0-hr.) and a f t e r (5-hr.) a d d i n g maltose in Trial 9 above. b Dissimilations by WCS p r e p a r e d f r o m r u m e n liquor obtained before (0-hr.) and a f t e r (5-hr.) adding maltose in Trial 10 above. e Includes valerie ~md other higher acids.
TABLE 4
I n vivo and in vitro dissimilations of DL-lysinc by rumen microorganis'ms Trial No.
Treatment
Sampling time
Total VFA
Acetic
Propionic
--(Molar
Butyric
Valeric plus higher acids
% total V F A ) - -
Rumen liquor
Rumen liquor lactic acid
(hr.)
(~M/ml)
(pH)
(tzM/ml)
14
1.5 kg. 4 -lysine added in vivo
0 0.5 2 5
50.4 54.2 49.9 42.5
69.6 71.1 73.4 78.6
21.4 18.1 17.8 18.9
8.5 10.5 8.5 1.7
0.5 0.3 0.3 0.8
7.4 7.0 7.2 7.4
0.0 0.18 0.0 0.0
15
1.5 kg. DL-lysine added in vivo
0 0.5 2 5
38.0 35.5 33.8 33.5
64.2 65.4 66.0 65.0
19.5 18.3 18.1 17.8
13.0 11.6 12.6 14.7
3.3 4.7 3.3 2.5
6.9 6.8 7.1 7.0
O.0 0.33 0.0 0.0
16 a
I n vitro WCS f r o m Trial 17
0-hr. 0-hr.
17.0 18.0
68.3 69.3
22.3
16.6
9.4 ~ 14.1
Control, no s u b s t r a t e 200 #M DL-]ysine added
5-hr. 5-hr.
17.0 17.0
66.8 73.6
17.6 16.3
15.6 10.1
Control, no s u b s t r a t e 200 ~M I)L-lysine added
Dissimilations by WCS p r e p a r e d f r o m r u m e n liquor obtained b e f o r e (0-hr.) and a f t e r (5-hr.) a d d i n g DL-lysine in Trial 14 above. b Includes valerie and other higher acids.
COMPARISON OF T E C H N I Q U E S IN I~UMINOLOGY
659
DISCUSSION
In three trials, the in vivo and in vitro results were in close agreement qualitatively. Quantitative differences existing between in vivo and in vitro results could be owing to intraeonversions among the V F A and to absorption occurring during the in vivo trials. The results of the in vivo fermentation of suecinate was similar to the in vitro results reported by Doetsch et al. (4). The failure to obtain any apparent fermentation of aspartie acid in vivo is attributed to the abnormal p H of the rumen contents caused by the DL-aspartic acid. The extremely high acidity (approximately p H 4.5) may have adversely affected rumen bacterial metabolism and interfered with normal dissimilation. When aspartic acid was partially neutralized to p H 6.9 with Na2CO~, it was attacked by rumen microorganisms and resulted in a marked increase of VFA, mainly propionic acid. The in vivo results of partially neutralized aspartie acid are similar to in vitro results of WCS reported by Sirotnak et al. (18). It is of interest to note that in vivo trials with glucose indicated that the presence of fermentable carbohydrates enhances ammonia utilization by rumen microorganisms. I t was noted also that the in vivo fermentation of maltose and glucose resulted in the production of lactic acid, as well as VFA. The lactic acid reached a maximum concentration within 2 hr. after the administrations of the substrates and in 5 hr. had almost completely disappeared from the rumen. This high concentration of lactic acid may be explained by assuming that it is a normal intermediate in carbohydrate metabolism in the rumen, and that its formation is occurring at a faster rate than its dissimilation a n d / o r absorption. Similar results concerning lactic acid production have been reported by Waldo and Sehultz (19). DL-lysine or a combination of glyeine and DL-alanine was not fermented to V F A by rumen microorganisms. These results agree with in vitro WCS results of Sirotnak et al. (17). However, glycine plus DL-alanine was deaminated in vivo by rumen microorganisms. The identity of the compound (s) resulting is as yet unknown. Loosli et al. (12), Black et al. (2), and Duncan et al. (6) have provided evidence demonstrating the synthesis of essential amino acids by rumen microorganisms. Perhaps reactions in the rumen involving essential amino acids are entirely assimilatory in nature. Deamination processes would aid in supplying ammonia required for bacterial (McDonald, 13), and possibly host, protein synthesis. Observations made on rumen liquor p H values during in vivo trials indicated that the p H of the rumen liquor is directly related to the total V F A concentration of the ruminal contents. It is thought that these measurements, although not representing the precise intraruminal pH, do reflect any large changes in rumen liquor p H values. During the in vitro trial with glucose (Trial 8, Table 2), differences were observed between the fermentation of the added glucose by the 0-hr., as compared to the 5-hr., WCS. I t should be noted that increases in butyric acid occurred during both the in vivo and in vitro trials. However, with added substrate, the in vitro concentration of butyric acid by the 5-hr. WCS did not keep pace with
660
F
G. H U E T E R
ET AL
t h e in v i v o c o n c e n t r a t i o n increases, a l t h o u g h a c o n s i d e r a b l e i n c r e a s e d i d occur. These r e s u l t s i n d i c a t e t h a t th e in v i v o a d m i n i s t r a t i o n of r e l a t i v e l y l a r g e a m o u n t s of c e r t a i n c a r b o h y d r a t e s c a n a l t e r t h e a c t i v i t y of r u m e n m i c r o o r g a n i s m s . Th e effects of c e r t a i n c a r b o h y d r a t e s on lactic acid a n d p o l y s a c c h a r i d e p r o d u c t i o n b y b o v i n e r u m e n b a c t e r i a h a v e been r e p o r t e d by R o b i n s o n et al. ( 1 5 ) a n d Gibbons et al. ( 8 ) . A l t e r a t i o n s m a y o c c ur f o r one or m o r e of t h e f o l l o w i n g r e a s o n s : ( a ) a b a c t e r i a l p o p u l a t i o n in w h i c h one or m o r e species h a v e i n c r e a s e d in n u m b e r s ; ( b ) an i n c r e a s e d m e t a b o l i c a c t i v i t y of one or m o r e species of microo r g a n i s m s ; or ( c ) th e e s t a b l i s h m e n t of a m e t a b o l i c s h u n t p a t h w a y in the p r e s e n c e of excess s u b s t r a t e b y one or m o r e species of r u m e n m i c r o o r g a n i s m s . A l t h o u g h q u a l i t a t i v e in v i v o a n d in v i t r o r esu l t s w e r e s i m i l a r , q u a n t i t a t i v e differences w e r e noted. V a r i a t i o n s m a y be the r e s u l t of d i f f e r e n t i a l a b s o r p t i o n of e n d - p r o d u c t s , c h a n g e s in p H p l u s o t h e r in v i v o factors, a n d a l t e r e d b a c t e r i a l m e t a b o l i s m . These differences also c o u ld be o w i n g to e n z y m e i n a c t i v a t i o n in p r o c e s s i n g cell suspensions, e x c l u s i o n of protozoa, omission of i m p o r t a n t b a c t e r i a because of a d h e r e n c e to v a r i o u s m a t e r i a l , lack of c o f a c t o r s f o r c e r t a i n reactions, assimilations, a n d / o r u t i l i z a t i o n of some V F A , o w i n g to t h e absence of a b s o r p t i o n . Th e in v i t r o cell s u s p e n s i o n m e t h o d a p p e a r s to be a u s e f u l t e c h n i q u e f o r s t u d y i n g toxic a n d / o r e x p e n s i v e s u b s t r a t e s ; f o r a r t i f i c i a l l y a l t e r i n g the c h e m i c a l e n v i r o n m e n t in a p r e d i c t a b l e m a n n e r a n d d i r e c t i o n ; f o r p h a r m a c o l o g i c a l investigations, and for pilot studies for subsequent animal experiments. REFERENCES (1) BARK~, S. B., A~D SUM~E~RS0N,W. H. The Colorimetric Determination of Lactic Acid in Biological Material. J. Biol. Chem., 138: 535. 194'1. (2) BL_~CK, A., KI~IBE~, M., ANI) S_~ITH, A. H. Carbonate and Fatty Acids as Precursors of Amino Acids in Casein. J. Biol. Chem., 197: 365. 1952. (3) DOETSCtt, R. N., AND ROBINSON, R. Q. The Bacteriology of the Bovine Rumen: A Review. J. Dairy Sci., 36: 115. 1953. (4) DOETSCH,:[~. N., ROBINSON, R. Q., BICOWN, R. E., AND SHA\¥, J. C. Catabolic Reactions of Mixed Suspensions of Bovine Rumen Bacteria. J. Dairy Svi., 36: 825. 1953. (5) DOETSCH, R. N., SHAW, J. C., McNE~I~L, J. J., ANI) JUaTSKUK, P., Jm Observations on the Use of Mixed Suspensions of Bovine Rumen Bacteria as a Tedlnique of the Rumen Microbiologist. Maryland Agr. Expt. Sta., Misc. Publ. 238: 1. 1955. (6) DUNCAN,C. W., AGRAWALA,I. P., ]:[UFFMAN,C. F., AND,LUECKE, R. W. A Quantitative Study of Rumen Synthesis in the Bovine on Natural and Purified Diets. II. Ami~:o Acid Content of Mixed Rumen Proteins. J. Nutrition, 49: 41. 1953. (7) EL-SHAZLY, K. Degradation of Protein in the Rumen of Sheep. II. The Action of Runlen Micro-organisms on Amino Acids. Biochem. J., 51 : 647. 1952. (8) GIBBONS, ]~. J., D0]gTSCH, R. N., AND SHAW, J. C. Further Studies on Polysaccharide Production by Bovine Rumen Bacteria. J. Dairy Sci., 38: 1147. 1955. (9) JOHNS, A. T. Isolation of a Bacterium, Producing Propionic Acid, from the Rumen of Sheep. J. Gen. Microbiol., 5: 317. 1951. (10) JUI~TSHUK,P., JR., AND HUETEE, F. G. In Vitro Studies on the Dissimilation of Purines and Pyrimidines by Bovine Rumen Bacteria. Maryland Agr. Expt. Sta., Misc. Publ. 238: 4. 1955. (11) KEBSrE¥, M. Direct Chromatographic Determination of C~ to C~ Fatty Acids in Rumen Fluid. Maryland Agr. Expt. Sta., Misc. Publ. 238: 23. 1955.
COMPARISON
OF TECHNIQUES IN RUMINOLOGY
661
(12) LOOSLI, J. K., WILLIAMS, H. ~I., THOMAS, W. E., FE,RUlS, F. tt., AN1) MAYNAt~I), L. A. Synthesis of Amino Acids in the Rumen. Science, 110: 144. 1949. (13) McDONALD, I. W. The Role of Ammonia in Ruminal Digestion of Protein. Bioche~t. J., 51: 86. 1952. (14) MO~alSO~, F. B. Feeds and Feeding. 1949,
21st ed. The Morrison Publ. Co., Ithaca, 1~. Y.
(15) ~OBINSON, R. Q., DOE.TSCH, i~.. N., SIROTNAK, F. M., .~-ND SHAW, J. C. Production of Lactic Acid and an Iodine Staining Substance by Bovine Rumen Baeteria. J. Dairy Sci., 38: 13. 1955.
(16) SIJPESTEIIJN, A. K., AND ELSI)E,N, S. R. The Metabolism of Succinic Acid in the Rumen of the Sheep. Biochem. J., 52: 41. 1952. (17) SII~0TNAK, F. M., DOETSCH, R. 1~., BROWN, R. E., AND SHA~V, J. C. Amino Acid Metabolisnl of Bovine Rumen Bacteria. J. Dairy Sci., 35: 1117. 1953. (18) SIgOTNAK, F. M., DOETSCH, l~. N., ROBINSON, R. Q., AND SHAW, J. C. Aspartate Dissimilation Reactions of Rumen Bacteria. J. Dairy Sci., 37: 531. ]954. (19) WALDO, D. ]:~., AIqD SCHULTZ, L. H. Lactic Acid Production in the Rumen. J. Dairy Sci., 38: 605. 1955.