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Utilization of Methionine and Methionine Hydroxy Analog by Rumen Microorganisms in Vitro
Utilization of Methionine and Methionine Hydroxy Analog by Rumen Microorganisms in Vitro R. L. SALSBURY, D. K. MARVIL,1 C. W. WOODMANSEE, and G. F. W. i~AENLEIN
Department of Animal Science and Agricultural Biochemistry University of Delaware, Newark 19711
Materials and Methods
Abstract
When incubated with rumen fluid under suitable conditions, methionine hydroxy analog, calcium acts like methionine in preventing inhibition of cellulose digestion by ethionine and in forming methanethiol and dimethyl sulfide. When inocula were from an animal receiving only cellulose, corn starch, urea, and water, cellulose digestion in vitro was depressed but less in the presence of methionine than in its absence. I n all of these reactions, methionine appeared to function more effectively than did the methionine hydroxy analog. When methionine-14C was incubated with rumen fluid, radioactivity was detected in methionine, methionine sulfoxide, carbon dioxide, mercaptan (presumably methanethiol), and protein.
Introduction
Recent evidence has suggested that supplementation of the diet of the dairy cow with methionine or methionine hydroxy analog, calcium (MttA) may be helpful in preventing ketosis (3) or increasing milk yield (2). The resulting heightened interest in the use of these two compounds as dietary supplements increases the need for an understanding of their utilization and dissimilation by tureen microorganisms. The experiments reported here indicate some of the reactions that methionine or its hydroxy analog undergo when exposed to the action of tureen microorganisms. Wherever it has been possible, an attempt has been made to compare the effectiveness of these two compounds as snbstrates for the same reaction(s). Received for publication August 14, 1970. Published with the approval of the Director of the Delaware Agricultural Experiment Station as Miscellaneous Paper 626, Contribution 6, of the Department of Animal Science and Agricultural Biochemistry, University of Delaware, Newark. 1 Present address: The North Carolina State University, Raleigh 27607.
Experiments with methio~dne-~"C. Inocula for these experiments were from three fistulated Holstein cows (Numbers 154, 191 and 240) receiving normal rations and maintained within the University dairy herd. The feeding program was centered around corn silage ad libitum, hay, supplementary grain as needed, and access to pasture during most o£ the year. Samples of rumen ingesta were taken in the morning before grain feeding, but experimental animals always had access to feed other than grain. Inocula were incubated in 60 ml serum bottles at 39 C with either methionine-methyl-14C to label products of transmethylation or transthiomethylation, or methionine-carboxyl-Z4C to determine whether a large proportion of the added methionine was deearboxylated. Known amounts of unlabeled methionine were added to the fermentations to have methionine ranging from physiological concentrations (0 mg added) to amounts that could be expected to saturate enzyme systems and permit detection of intermediates (Table 1). A stream of nitrogen was passed slowly over the fermenting rumen fluid, then through a series of traps designed to retain mercaptans (4% mercuric cyanide solution), sulfides (3 % ttgC12 solution), and carbon dioxide (saturated Ba(OtI)2 solution). Samples of rumen fluid of known volume were withdrawn from the reaction vessel at intervals during the fernlentation and immediately added to equal volumes of 10% trichloroacetic acid solution in 50 ml polycarbonate screw-cap centrifuge tubes. These were stored at --10 C for further analysis. After thawing, all samples were centrifuged at 10,000 X g for 30 min at l 0 C. A known volume of each supernatant was drawn off and passed through a Rexyn 101 2 cation exchange column (0.9 by 15 cm) which had been conditioned previously with 0.1 ~¢ citric acid (pH 2.2). After the supernatant had passed through, the column was washed with approximately 10 ml 0.1 ~¢ citric acid. The amino acids were then 2 Obtained from ~'isher Scientific Co., King of Prussia, Pennsylvania.
390
METHIONINE UTILIZATION
eluted from the column with 10 ml N ammonium hydroxide. The eluate was evaporated to dryness at 75 C and redissolved in 0.5 ml deionized distilled water. A 10-~liter aliquot of the resulting amino acid solution was spotted on a thin-layer chromatographic plate that had been coated with 250 tz of Avicel.3 The thin-layer chromatogram was developed in the two-phase system described by Redfield (10). The first phase consisted of water methanol and pyridine in the proportion 8 0 : 2 0 : 4 (v/v) and the second phase consisted of tertiary butanol, methylethyl-ketone, diethylamine and water in the proportion 4 0 : 4 0 : 2 0 : 4 (v/v). A f t e r developmerit of the chromatogram the amino acids were visualized by spraying the plates with a 0.3% ninhydrin-acetone solution and placing them in an oven at 100 C for 5 rain. Chromatograms showing radioactivity in an amino acid other than the substrate amino acid were exposed to 20 by 20 cm sheets of x-ray film,4 usually for 24 hr. The identity of the active amino acid was then determined by comparing the autoradiograph with a thin layer chromatogram of known amino acids. The amounts of activity in mercaptans (methanethiol) and carbon dioxide were determined by counting weighed portions of the precipitates formed in the traps. Aliquots of the anfino acid solutions for thin-layer chromatography were placed on planchets, dried in an oven, and counted similarly. Amounts of activity in the microbial protein formed in the fermentations were estimated by counting the suspended material after it had been dried on a small filter paper. All counts were with a Nuclear-Chicago Model D181 Decade Scaler. Purified ration experiment. This experiment was performed as described by Salsbury et al. (11) with an ovariectomized Holstein cow (Number 154) with a permanent rumcn eannula. F o r the first three days of the experimental period the animal received two feedings of cellulose5 daily (2 kg at each feeding) introduced directly into the rumen and as much as it would cat of the regular ration (corn silage ad lib. -b hay). F o r the remainder of the experimental period the animal received only the purified ration of cellulose 2 kg~ corn starch 200 g, and urea 30 g fed twice daily by way of the cannula. Samples of rumen fluid s Obtained from Analtech, Inc., Wilmington, Delaware. 4 Cronex II-DC X-ray film manufactured by E. I. duPont de Nemours & Co., Wilmington, Delaware. 5 Solka-Floe BW 40. A wood cellulose flour from Brown and Co., ]~erlin, New Hampshire.
391
were taken daily before the morning feeding and used as inocula for laboratory fermentations to which cellulose -k methionine, cellulose + methionine hydroxy analog, or cellulose alone had been added. Experiments on the inhibition of cellulose digestion by ethionine. Laboratory fermentations were similar to those described by Salsbury et al. (12). Inocula were from two fistulated Holstein cows (Numbers 154, 191) receiving normal rations as described. I n the laboratory triplicate fermentations were run for each of the following treatments: cellulose, cellulose -~ ethionine, cellulose ~- ethionine -t- methionine, cellulose + ethionine -t- methionine analog. Gas chromatography experiments. Inocula for the experiments were frmn the fistulated Holstein cows (Numbers 154, 191 and 240) used as a source of inocula for the experiments with methionine-14C. Conditions for incubation of the inocula with the substrates and chromatographic determination of volatile products were as described by Zikakis and Salsbury (17). Portions of rumen fluid (15 ml) were incubated in 60 ml serum bottles at 39 C with 25 mg methionine or 28 mg methionine hydroxy analog. Methanethiol and dimethyl sulfide were determined by gas chromatography on 250 fairer aliquots of the headspace gas of each serum bottle. A series of determinations was made for each serum bottle so that curves showing the rates of production of methanethiol and dimethyl sulfide could be constructed. Results and Discussion
Experiments with methionine-l~C. The results of some of the experiments with labeled methionine are in Table 1. When :10 ~Ci (0.14 mg) of methionine-methyl-14C were added to 20 ml of rumen fluid, there was no activity in the amino acids after three hours of incubation. When the same amount of labeled compound was used with 10 mg of unlabeled methionine, radioactive methionine and methionine sulfoxide were detected. I n each subsequent experiment both labeled and unlabeled methionine were used, and labeled methionine sulfoxide was invariably detected after incubation. Visual examination of autoradiograms indicated that, in general, activity of the methionine sulfoxide increased as fermentation progressed. The exception to this generalization occurred in an experiment (not in Table 1) which had the least concentration of unlabeled methionine (2.10 rag/50 ml) of those in which sequential autoradiograms were made. I n this experiment activity of the methionine sulfoxide appeared to increase up to three hours, then to decrease. JOUR2/AL OF DAIRY SCIENCE ~GL. 54, NO. 3
392
S A L S B U R Y E T AL.
Carboxyl-labeled methionine was used in this experiment. The consistent finding of methionine sulfoxide is of interest because it apparently can serve as a source of dimethyl sulfide (1) or methanethiol and it also partially prevents inhibition of cellulose digestion by ethionine (14). Our results suggest that sulfoxide is formed in the presence of relatively high concentrations of methionine; the reports cited and the aforementioned experiment imply that sulfoxide is probably reduced to methionine and utilized as the methionine concentration decreases. Shoekman and Toennies (15) have shown that methionine sulfoxide formation can be due to surface phenomena during chromatography and can be caused by catalytic reaction in the sterile medium as well as by the activity of microorganisms. The apparent increase in methionine sulfoxide with increased time of incubation in our experiments suggests that it arose, at least in part, from catalytic or microbial action in the rumen fluid. Approximately 6% of the activity added as methionine carboxylA4C was recovered as carbon dioxide and more than 10% of the activity added as methionine methylA4C was recovered as methanethiol (Table 1). The value of these estimates is diminished by the counting method
which would tend to make them low. That they represent the total amounts trapped over 9 and 10 hr, respectively, tends to conceal differences in rates of evolution at different times during the fermentation period. To this should be added the possibility that some of the activity determined as methanethiol could be in the form of dimethyl sulfide for, although we have found that a 4% solution of mercuric cyanide would remove essentially all of the methanethiol from a headspace gas mixture of methanethiol and dimethyl sulfide, there seemed to be a slight reduction in the amount of dimethyl sulfide remaining. I n Experiment 4 (Table 1) a measurable portion of activity added as methionine-methyl14C appeared to be incorporated into microbial protein, rapidly at first then at a slower rate. The rate of incorporation in Experiment 5 was much slower and more uniform over the short fermentation period. This may have been due to competitive inhibition by the s-ethyl cysteine and s-methyl cysteine added to Experiment 5 although this possibility seems unlikely because of the quantity of unlabeled methionine present along with the substituted cysteines. However, the possibility of another explanation arises from procedural differences in the two expert-
TABLE 1. Experiments with labeled methionine. Labeled compounds found Rumen fluid Meth. Expert- vol14C ment ume added (ml) 20 20 30 50
(cps × --10 -'4) 37a 37a 93b 185 a
Unlabeled cornpounds added Meth.
Other
--(rag)10 0 22.25 35.0
-----
5A
20
46 a
20
20 c
5B
20
46 a
20
20 d
Sampling time (hr) 3 3 9 1 2.5 5 7.5 10 1.5 3 1.5 3
MicrobiaI Free proamino rein acids
Meth.
Meth. sulfoxide
CO2
MeSH
+ 0 + + + -? + -k+ + + +
+ 0 + + + + + + -4+ + +
--(cps ND ND 5.7 ND ND ND ND ND ND ND ND ND
× --10-4) ND ND ND ND ND ND ND 15.3 lqD 17.2 ND 19.1 :ND 19.6 20.1 21.9 ND .40 21.4 .84 ND .77 2.7 1.39
ND ND ND 69.2 57.6 30.1 16.7 6.9 16.0 7.7 15.0 12.9
a Labeled in methyl group. b Labeled in earboxyl group. c S-Ethyl eysteine. a S-Methyl eysteine. Abbreviations: + , present; --, not added; 0, not detected; l~D, not determined; cps, count~ per second. JOVR~AL OV DA.~Y SCY~NOE VOL. 54, NO. 3
METHIONINE
80'
~o~
",k
~60 (::} b.I
~ 50 W
" 40 W 0
~ 3o .J .J
__
L-METHIONINE
N zo
__
MHA
....
NO ADDITION
'~/'---- ~r"" m
I0
0
I
I
I
I
I
I
I
I
2
3
4
5
6
7
DAYS
ON
393
UTILIZATION
EXPERIMENT
FIG. 1. Effect of L-methionine and methionine hydroxy analog on cellulose digestion in vitro. Inocula from a cow receiving corn silage + hay (Day 0), corn silage + hay + cellulose (Days 1, 2 and 3) or cellulose + starch + urea (Days 4, 5, 6 and 7). ments. Although the fraction examined in both experiments was the trichloroacetic acid-precipitable material, the triehloroaeetic acid-treated aliquots in Experiment 5 were sonicated whereas those in Experiment 4 were not. Hence the apparent incorporation of methionine in Experiment 4 may actually represent uptake by the microorganisms, rapid until a limiting concentration was reached, then at a slower rate that balanced utilization. Purified ration experiment. When laboratory
fermentations were inoculated with rumen fluid from the cow receiving the purified ration, L-methionine stimulated cellulose digestion more than the analog (Fig. 1). I n this experiment, Student's "t" test (16) showed that cellulose digestion was greater (P < .05) for the cellulose .5 L-methionine treatment than for cellulose alone on Days 3, 4, 6, and 7; greater for the cellulose + the analog treatment than for cellulose alone on Day 6; and greater for the cellulose .5 L-methionine treatment than for cellulose .5 the analog on Days 3, 4, and 6. The particularly low values on Day 5 followed by higher ones on Days 6 and 7 may indicate a major shift in the nficrobial population of the rumen. I n a previous experiment, both L-methionine and the analog showed a stimulation (P < .05) of cellulose digestion but the difference between the cellulose + L-methionine and cellulose + methionine hydroxy analog treatments was not significant. Experiments on the inhibition of cellulose digestion by ethionine. To compare effects of methionine and the analog with inocula from animals receiving normal rations, it was necessary to use the methionine antagonist, ethionine. This proved to be an effective inhibitor of cellulose digestion in our laboratory fermentations. Usually addition of methionine along with ethionine prevented most of the inhibition with ethionine alone (Table 2). With rumen fluid from Cow 154, the analog was only slightly less effective than methionine but the difference in effectiveness of methionine and the analog appeared to be greater with tureen fluid from Cow 191. This suggests that the results may have been affected by differences in the characteristic microbial populations of the animals. I n Experiment 7 (Table 2) the difference be-
TABLE 2. Effectiveness of methionine and methionine hydroxy analog in preventing ethionine inhibition of cellulose digestion. Cellulose digestion (%) and SE
Experiment 1 2 3 4 5 6 7
Cow
Rumen fluid
Rumen fluid -5 ethionine
Rumen fluid + ethionine + methionine
154 154 154 191 154 191 154
44.6 51.7 47.7 68.7 65.8 58.8 65.8
18.2 16.1 23.4 36.0 43.7 28.3 19.4
41.4 48.3 29.0 65.7 71.2 50.5 58.9
± 4.8 ± 1.5 ±13.1 ± 2.8 ± 4.7 ± 4.5 ± 4.0
± ± ± ± ± ± ±
2.3 0.2 3.4 0.5 0.6 5.7 1.4
± 3.2 ± 5.4 ± 3.3 ± 2.8 ± 4.6 ± 2.7 ±19.6
Rumen fluid + ethionine + MHA 37.2 41.5 27.6 49.9 66.9 36.9 43.7
± ± ± ± ± ± ±
1.7 2.5 4.9 3.3 8.6 7.7 8.2
JOUI~NAL OF DAIRY 801EIqCE V(>L, 54, NO. 3
394
SALSBURY
tween methionine and the analog was greater than in other experiments with Cow 154. This may have been an artifact (note the large standard error for the methionine treatment) or may reflect the lapse of time (more than a year) between this experiment and the previous experiments with Cow 154. The latter possibility is considered more likely because Cow 154 had received a rumen transfusion from another animal (not Cow 191) during this period and this transfusion had brought about a visible change in the protozoal population of the rumen and possibly, therefore, a change in its bacterial population. The results shown by methionine and methionine hydroxy analog in Table 2 need not be interpreted as indicating a difference in the utilization of these compounds by rumen microorganisms. They could be interpreted as indicating that methionine competes effectively for the site(s) occupied by ethionine in functioning as an antimetabolite and that it does so at a lower concentration than does the analog. Under these circumstances the possibility would still exist that rumen microorganisms could utilize the analog as well as methionine if ethionine were not present and that the difference between the effects of methionine and methionine hydroxy analog could be narrowed by increasing the metabolite: antimetabolite ratio. When this possibility was tested by holding ethionine at 0.1 meq per liter (the same concentration as in experiments shown in Table 2) and varying methionine and the analog from 0.27 to 5.37 meq per liter, the results in Figure 2 were obtained. At this ethionine concentration the effectiveness of methionine and the analog in preventing inhibition appeared to differ (P < .05) only at the lowest concentration (0.27 meq per liter) of these two compounds. When ethionine was increased to 3.9 meq per liter, 2.6 to 26 meq per liter of the analog were not effective, but 67 and 124 meq per liter of methionine partially prevented inhibition (Fig. 3). Methionine hydroxy analog was more sensitive to increases in ethionine, and this was verified by holding methionine and the analog constant at 1.3 and 2.6 meq per liter, respectively, and varying ethionine from 0.02 to 2.0 meq per liter (Fig. 4). The effectiveness of fixed concentrations of methionine and its analog decreased with inereasing ethionine but much more precipitously with the analog than with methionine. These results support the view that methionine competes more effectively with ethionine than does the analog. The greater sensitivity of the analog to increasing ethionine, therefore, may be simply a manifestation of a requirement for a higher ~OURI~AL OF D A I R Y SCI EI~CE ~rOIJ. 54, N O .
E T AL.
90
80 70 6C --
~_ 50 c~ ~ nO o
DL-ETHIONINE + DL-METHIONINE
- - -
DL-ETHIONINE +MHA
D
~ 30
20 IO I
0.27
1.34
I
2.68
I
5.37
CONCENTRATION OF METHIONINEOR MHA(MEG/L) FIG. 2. Effectiveness of different levels of DLmethionine and methionine hydroxy analog in preventing inhibition by DL-ethionine of ce]lulose digestion by rumen microorganisms. Ethionine 0.1 meq per liter. metabolite:antimetabolite ratio to prevent inhibition. This would imply that the analog has a lower affinity for the enzyme(s) of the reaction(s) being inhibited and that probably it is not as well utilized by rumcn microorganisms as is methionine under normal conditions. Alternatively, if ethionine inhibition is specific for methionine, the greater sensitivity of the analog may indicate an inhibition of methionine
50
DL-ETHIONINE + L-METHIONINE
~4o
_~3c a uJ ~ ~c ~o
DL-ETHIONINE+ MHA G
CONCENTRATION OF METHIONINE OR MHA (IMEO/L)
FIG. 3. Relative effectiveness of L-methionine and methionine hydroxy analog in preventing inhibition by higher concentration of DL-ethionine. Ethionine 3.9 meq per liter.
METHIONINE
4O ~
DL-ETHIONINE
~ 3o •,9 {3 2 0
DL-ETHIONINE + MHA
3 m _1 w
o
DL-ETHIONINE i
i
0.10 0.49 C O N C E N T R A T I O N OF E T H I O N I N E
i
1.96 (MEQ/L)
~IG. 4. Inhibition of cellulose digestion by different concentrations of ethionine at fixed methionine and methionine hydroxy analog concentrations. Methionine 1.3 meq per liter; analog 2.6 meq per liter. formation from the analog at higher ethionine levels. The failure of the analog to prevent inhibition of cellulose digestion by higher concentrations of ethionine may explain the negative results with the analog in earlier work (13). Gas chromatography experiments. Figure 5 shows the results of a typical experiment with inoculum from Cow 240. Inocula from Cows :[54 and 19:[ gave similar results. I n previous work (:[7) addition of methionine to rumen fluid resulted in the formation of measurable quantities of methanethiol. I n the present study methanethiol was also produced from the analog but at a much lower level (Fig. 5). A relatively small quantity of dimethyl sulfide was formed from both substrates. Dunham et al. (1) reported production of dimethyl sulfide from methionine by rumen microorganisms. Because they did not mention methanethiol or other related compounds, it can be assumed that dimethyl sulfide was the main, if not the only, volatile sulfur compound detected. I n our experiments, methanethiol has been consistently the main volatile sulfur compound even though there has been considerable variation in amounts produced by inocula from the same animals at different times or from different animals at the same time. Dunham et al. (1) used alfalfa-fed animals and added alfalfa to their fermentations. We found that addition of alfalfa alone to our fermentations resulted in the formation of measurable quantities of dimethyl sulfide. Reddy (9) reported that the dimethyl sulfide concentrations in milk from cows fed chopped alfalfa were greater than in milk from cows on rye or bromegrass pasture. Moreover, previous work (17) has shown that when a laboratory fermentation was inoculated with rumen fluid from a pasture-fed animal, methanethiol
395
UTILIZATION
was evolved, then rapidly disappeared. This phenomenon requires further investigation but does suggest that pasture feeding favored development of a flora capable of utilizing methanethiol. I n the reactions examined, it appears that methionine and its analog behave similarly but that the analog is less effective as a substrate. I n part at least, this may be due to lower solubility of the analog. I n the cellulose digestion experiments with normal rnmen fluid, it was necessary to use a methionine antagonist to show a significant methionine response. This suggests that there is sufficient methionine available in normal rumen fluid to meet the requirements for cellulose digestion under our conditions. However, recent work with dairy cattle and sheep indicates that methionine and methionine hydroxy analog affects lipid metabolism, both in milk fat synthesis (3, 8) and in the rumen itself (4, 5, 6) and that feeding the analog increases protozoal numbers (7). The inference from these reports is that feeding methionine or the analog may be beneficial. There is little doubt that the question is complicated by the multiplicity of reactions which utilize methi-
~ 45 40 = ~ 35 ,,< z o 30
/M __ H A .... NOADDITION / O METHANETHIOL / PRODUCTION / • DIMETHYL_SULFIDE /
~ 25
PRODUCTION
.~ 20 ~ 15 ,. o ~.10
-
,0
A
.
.o---~
L.¢~---.---',
I00
.... ....
200
300
•
. ....
400
7. . . . 500
INCUBATIONTIME(MIN.)
: ....
:
600
FIe. 5. Production by rumen microorganisms of methanethiol and dimethyl sulfide from equivalent concentrations of L-methionine, and methionine hydroxy analog, and in a control fermentation to which no substrate was added. Inoculum from Cow 240. JOURNAL OF DAIRY SOIEI~0E VOL, 54, NO. 3
396
SALSBURY ET AL.
o n i n e in t h e r u m e n ; o u r w o r k here h a s i n d i c a t e d f o u r : m e t h i o n i n e sulfoxide f o r m a t i o n , decarboxylation, d e t h i o m e t h y l a t i o n , a n d p r o t e i n s y n t h e sis. I t is p r o b a b l e t h a t these a n d o t h e r r e a c t i o n s would d e m a n d different p r o p o r t i o n s of the met h i o n i n e p o o l as conditions varied. N e v e r t h e less, it seems logical t h a t the circumstance most likely to benefit f r o m m e t h i o n i n e s u p p l e m e n t a tion would be one in which the i n t a k e of p r e f o r m e d m e t h i o n i n e f r o m o t h e r sources was limited b y a relatively h i g h p r o p o r t i o n of t h e crude p r o t e i n r e q u i r e m e n t coming f r o m n o n p r o t e i n n i t r o g e n , a n d t h e loss o f m e t h i o n i n e f r o m the b o d y was h i g h because o f h i g h milk production.
(7)
(8)
(9)
(10)
References
(1) Dunham, J. R., G. Ward, R. Bassette, and M. C. Reddy. 1968. Methionine as a precursor of methyl sulfide in cow's milk. J. Dairy Sci., 51: 199. (2) Grie], L. C., Jr., R. A. Patton, R. D. McCarthy, and P. T. Chandler. 1968. Milk production response to feeding methionine hydroxy analog to lactating dairy cows. J. Dairy Sci., 51: 1866. (3) McCarthy, R. D., G. A. Porter, and L. C. Griel, fir. 1968. Bovine ketosis and depressed f a t test in milk; a problem of methionine metabolism and serum lipoprotein aberration. J . Dairy Sci., 51:459. (4) Patton, i~. A., R. D. McCarthy, and L. C. Griel, Jr. 1968. Lipid synthesis by tureen microorganisms I. Stimulation by methionine in vitro. J. Dairy Sci., 51: 1310. (5) Patton, R. A., R. D. McCarthy, and L. C. Griel, Jr. 1970. Lipid synthesis by rumen microorganisms II. F u r t h e r characterization of the effects of mcthionine. J. Dairy Sci., 53 : 460. (6) Patton, R. A., R. D. McCarthy, and L. C. Grie], Jr. 1970. Observations on rumen fluid, blood serum and milk ]ipids of cows
JOURNAL OF DAIRY SOI]~NCE VOL. 54, NO. 3
(11)
(12)
(13) (14)
(15)
(16)
(17)
fed methionlne hydroxy analog. J . Dairy Sci., 53: 776. P a t t o n , R. A., R. D. McCarthy, L. G. Keske. L. C. Griel, Jr., and B. R. Baumgardt. 1970. Effect of feeding methlonine hydroxy analog on the concentration of protozoa in the rumen of sheep, ft. Dairy Sci., 53:933. Polan, C. B., P. T. Chandler, and C. N. Miller. 1970. Methlonlne hydroxy analog: varying levels for lactating cows. ft. Dairy Sci., 53 : 607. Reddy, M. C. 1966. Effect of surface active agent poloxalene on milk flavor when fed to cows. M.S. thesis, Kansas State University, Manhattan. Redfield, R. R. ]953. Two-dimensional paper chromatographic systems with high resolving power for amino acids. Biochim. Biophys. Acta, 10: 344. Salsbury, R. L., J. A. Hoefer, and R. W. Lueeke. 1961. Effect of feeding certain defined nutrients on cellulose digestion and volatile f a t t y acid concentrations of the rumen. J. Dairy Sci., 44: 1122. Salsbury, R. L., C. K. Smith, and C. F. Huffman. 1956. The effect of high levels of cobalt on the in vitro digestion of cellulose by rumen microorganisms, ft. Animal Sci., 15: 863. Salsbury, R. L., and ft. P. Zikakis. 1965. Stimulation of cellulose digestion by methionine. J. Animal Sci., 24: 902. Salsbury, R. L., and J. P. Zikakis. I967. Sulfur metabolism of rumen microorganisms. J. Animal Sci., 26: 929. Shockman, G. D., and G. Toennies. 1954. Formation of D-methionine from L- by Streptococcus faeealis. Arch. Biochem. and Biophys., 50: 9. Steel, R. G. D., and J. It. Torrie. 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York. Zikakis, J. P., and R. L. Salsbury. 1969. Metabolism of sulfur amino acids by rnmen microorganisms. J. Dairy Sci., 52: 2014.
Department of Animal Science and Agricultural Biochemistry University of Delaware, Newark 19711
Materials and Methods
Abstract
When incubated with rumen fluid under suitable conditions, methionine hydroxy analog, calcium acts like methionine in preventing inhibition of cellulose digestion by ethionine and in forming methanethiol and dimethyl sulfide. When inocula were from an animal receiving only cellulose, corn starch, urea, and water, cellulose digestion in vitro was depressed but less in the presence of methionine than in its absence. I n all of these reactions, methionine appeared to function more effectively than did the methionine hydroxy analog. When methionine-14C was incubated with rumen fluid, radioactivity was detected in methionine, methionine sulfoxide, carbon dioxide, mercaptan (presumably methanethiol), and protein.
Introduction
Recent evidence has suggested that supplementation of the diet of the dairy cow with methionine or methionine hydroxy analog, calcium (MttA) may be helpful in preventing ketosis (3) or increasing milk yield (2). The resulting heightened interest in the use of these two compounds as dietary supplements increases the need for an understanding of their utilization and dissimilation by tureen microorganisms. The experiments reported here indicate some of the reactions that methionine or its hydroxy analog undergo when exposed to the action of tureen microorganisms. Wherever it has been possible, an attempt has been made to compare the effectiveness of these two compounds as snbstrates for the same reaction(s). Received for publication August 14, 1970. Published with the approval of the Director of the Delaware Agricultural Experiment Station as Miscellaneous Paper 626, Contribution 6, of the Department of Animal Science and Agricultural Biochemistry, University of Delaware, Newark. 1 Present address: The North Carolina State University, Raleigh 27607.
Experiments with methio~dne-~"C. Inocula for these experiments were from three fistulated Holstein cows (Numbers 154, 191 and 240) receiving normal rations and maintained within the University dairy herd. The feeding program was centered around corn silage ad libitum, hay, supplementary grain as needed, and access to pasture during most o£ the year. Samples of rumen ingesta were taken in the morning before grain feeding, but experimental animals always had access to feed other than grain. Inocula were incubated in 60 ml serum bottles at 39 C with either methionine-methyl-14C to label products of transmethylation or transthiomethylation, or methionine-carboxyl-Z4C to determine whether a large proportion of the added methionine was deearboxylated. Known amounts of unlabeled methionine were added to the fermentations to have methionine ranging from physiological concentrations (0 mg added) to amounts that could be expected to saturate enzyme systems and permit detection of intermediates (Table 1). A stream of nitrogen was passed slowly over the fermenting rumen fluid, then through a series of traps designed to retain mercaptans (4% mercuric cyanide solution), sulfides (3 % ttgC12 solution), and carbon dioxide (saturated Ba(OtI)2 solution). Samples of rumen fluid of known volume were withdrawn from the reaction vessel at intervals during the fernlentation and immediately added to equal volumes of 10% trichloroacetic acid solution in 50 ml polycarbonate screw-cap centrifuge tubes. These were stored at --10 C for further analysis. After thawing, all samples were centrifuged at 10,000 X g for 30 min at l 0 C. A known volume of each supernatant was drawn off and passed through a Rexyn 101 2 cation exchange column (0.9 by 15 cm) which had been conditioned previously with 0.1 ~¢ citric acid (pH 2.2). After the supernatant had passed through, the column was washed with approximately 10 ml 0.1 ~¢ citric acid. The amino acids were then 2 Obtained from ~'isher Scientific Co., King of Prussia, Pennsylvania.
390
METHIONINE UTILIZATION
eluted from the column with 10 ml N ammonium hydroxide. The eluate was evaporated to dryness at 75 C and redissolved in 0.5 ml deionized distilled water. A 10-~liter aliquot of the resulting amino acid solution was spotted on a thin-layer chromatographic plate that had been coated with 250 tz of Avicel.3 The thin-layer chromatogram was developed in the two-phase system described by Redfield (10). The first phase consisted of water methanol and pyridine in the proportion 8 0 : 2 0 : 4 (v/v) and the second phase consisted of tertiary butanol, methylethyl-ketone, diethylamine and water in the proportion 4 0 : 4 0 : 2 0 : 4 (v/v). A f t e r developmerit of the chromatogram the amino acids were visualized by spraying the plates with a 0.3% ninhydrin-acetone solution and placing them in an oven at 100 C for 5 rain. Chromatograms showing radioactivity in an amino acid other than the substrate amino acid were exposed to 20 by 20 cm sheets of x-ray film,4 usually for 24 hr. The identity of the active amino acid was then determined by comparing the autoradiograph with a thin layer chromatogram of known amino acids. The amounts of activity in mercaptans (methanethiol) and carbon dioxide were determined by counting weighed portions of the precipitates formed in the traps. Aliquots of the anfino acid solutions for thin-layer chromatography were placed on planchets, dried in an oven, and counted similarly. Amounts of activity in the microbial protein formed in the fermentations were estimated by counting the suspended material after it had been dried on a small filter paper. All counts were with a Nuclear-Chicago Model D181 Decade Scaler. Purified ration experiment. This experiment was performed as described by Salsbury et al. (11) with an ovariectomized Holstein cow (Number 154) with a permanent rumcn eannula. F o r the first three days of the experimental period the animal received two feedings of cellulose5 daily (2 kg at each feeding) introduced directly into the rumen and as much as it would cat of the regular ration (corn silage ad lib. -b hay). F o r the remainder of the experimental period the animal received only the purified ration of cellulose 2 kg~ corn starch 200 g, and urea 30 g fed twice daily by way of the cannula. Samples of rumen fluid s Obtained from Analtech, Inc., Wilmington, Delaware. 4 Cronex II-DC X-ray film manufactured by E. I. duPont de Nemours & Co., Wilmington, Delaware. 5 Solka-Floe BW 40. A wood cellulose flour from Brown and Co., ]~erlin, New Hampshire.
391
were taken daily before the morning feeding and used as inocula for laboratory fermentations to which cellulose -k methionine, cellulose + methionine hydroxy analog, or cellulose alone had been added. Experiments on the inhibition of cellulose digestion by ethionine. Laboratory fermentations were similar to those described by Salsbury et al. (12). Inocula were from two fistulated Holstein cows (Numbers 154, 191) receiving normal rations as described. I n the laboratory triplicate fermentations were run for each of the following treatments: cellulose, cellulose -~ ethionine, cellulose ~- ethionine -t- methionine, cellulose + ethionine -t- methionine analog. Gas chromatography experiments. Inocula for the experiments were frmn the fistulated Holstein cows (Numbers 154, 191 and 240) used as a source of inocula for the experiments with methionine-14C. Conditions for incubation of the inocula with the substrates and chromatographic determination of volatile products were as described by Zikakis and Salsbury (17). Portions of rumen fluid (15 ml) were incubated in 60 ml serum bottles at 39 C with 25 mg methionine or 28 mg methionine hydroxy analog. Methanethiol and dimethyl sulfide were determined by gas chromatography on 250 fairer aliquots of the headspace gas of each serum bottle. A series of determinations was made for each serum bottle so that curves showing the rates of production of methanethiol and dimethyl sulfide could be constructed. Results and Discussion
Experiments with methionine-l~C. The results of some of the experiments with labeled methionine are in Table 1. When :10 ~Ci (0.14 mg) of methionine-methyl-14C were added to 20 ml of rumen fluid, there was no activity in the amino acids after three hours of incubation. When the same amount of labeled compound was used with 10 mg of unlabeled methionine, radioactive methionine and methionine sulfoxide were detected. I n each subsequent experiment both labeled and unlabeled methionine were used, and labeled methionine sulfoxide was invariably detected after incubation. Visual examination of autoradiograms indicated that, in general, activity of the methionine sulfoxide increased as fermentation progressed. The exception to this generalization occurred in an experiment (not in Table 1) which had the least concentration of unlabeled methionine (2.10 rag/50 ml) of those in which sequential autoradiograms were made. I n this experiment activity of the methionine sulfoxide appeared to increase up to three hours, then to decrease. JOUR2/AL OF DAIRY SCIENCE ~GL. 54, NO. 3
392
S A L S B U R Y E T AL.
Carboxyl-labeled methionine was used in this experiment. The consistent finding of methionine sulfoxide is of interest because it apparently can serve as a source of dimethyl sulfide (1) or methanethiol and it also partially prevents inhibition of cellulose digestion by ethionine (14). Our results suggest that sulfoxide is formed in the presence of relatively high concentrations of methionine; the reports cited and the aforementioned experiment imply that sulfoxide is probably reduced to methionine and utilized as the methionine concentration decreases. Shoekman and Toennies (15) have shown that methionine sulfoxide formation can be due to surface phenomena during chromatography and can be caused by catalytic reaction in the sterile medium as well as by the activity of microorganisms. The apparent increase in methionine sulfoxide with increased time of incubation in our experiments suggests that it arose, at least in part, from catalytic or microbial action in the rumen fluid. Approximately 6% of the activity added as methionine carboxylA4C was recovered as carbon dioxide and more than 10% of the activity added as methionine methylA4C was recovered as methanethiol (Table 1). The value of these estimates is diminished by the counting method
which would tend to make them low. That they represent the total amounts trapped over 9 and 10 hr, respectively, tends to conceal differences in rates of evolution at different times during the fermentation period. To this should be added the possibility that some of the activity determined as methanethiol could be in the form of dimethyl sulfide for, although we have found that a 4% solution of mercuric cyanide would remove essentially all of the methanethiol from a headspace gas mixture of methanethiol and dimethyl sulfide, there seemed to be a slight reduction in the amount of dimethyl sulfide remaining. I n Experiment 4 (Table 1) a measurable portion of activity added as methionine-methyl14C appeared to be incorporated into microbial protein, rapidly at first then at a slower rate. The rate of incorporation in Experiment 5 was much slower and more uniform over the short fermentation period. This may have been due to competitive inhibition by the s-ethyl cysteine and s-methyl cysteine added to Experiment 5 although this possibility seems unlikely because of the quantity of unlabeled methionine present along with the substituted cysteines. However, the possibility of another explanation arises from procedural differences in the two expert-
TABLE 1. Experiments with labeled methionine. Labeled compounds found Rumen fluid Meth. Expert- vol14C ment ume added (ml) 20 20 30 50
(cps × --10 -'4) 37a 37a 93b 185 a
Unlabeled cornpounds added Meth.
Other
--(rag)10 0 22.25 35.0
-----
5A
20
46 a
20
20 c
5B
20
46 a
20
20 d
Sampling time (hr) 3 3 9 1 2.5 5 7.5 10 1.5 3 1.5 3
MicrobiaI Free proamino rein acids
Meth.
Meth. sulfoxide
CO2
MeSH
+ 0 + + + -? + -k+ + + +
+ 0 + + + + + + -4+ + +
--(cps ND ND 5.7 ND ND ND ND ND ND ND ND ND
× --10-4) ND ND ND ND ND ND ND 15.3 lqD 17.2 ND 19.1 :ND 19.6 20.1 21.9 ND .40 21.4 .84 ND .77 2.7 1.39
ND ND ND 69.2 57.6 30.1 16.7 6.9 16.0 7.7 15.0 12.9
a Labeled in methyl group. b Labeled in earboxyl group. c S-Ethyl eysteine. a S-Methyl eysteine. Abbreviations: + , present; --, not added; 0, not detected; l~D, not determined; cps, count~ per second. JOVR~AL OV DA.~Y SCY~NOE VOL. 54, NO. 3
METHIONINE
80'
~o~
",k
~60 (::} b.I
~ 50 W
" 40 W 0
~ 3o .J .J
__
L-METHIONINE
N zo
__
MHA
....
NO ADDITION
'~/'---- ~r"" m
I0
0
I
I
I
I
I
I
I
I
2
3
4
5
6
7
DAYS
ON
393
UTILIZATION
EXPERIMENT
FIG. 1. Effect of L-methionine and methionine hydroxy analog on cellulose digestion in vitro. Inocula from a cow receiving corn silage + hay (Day 0), corn silage + hay + cellulose (Days 1, 2 and 3) or cellulose + starch + urea (Days 4, 5, 6 and 7). ments. Although the fraction examined in both experiments was the trichloroacetic acid-precipitable material, the triehloroaeetic acid-treated aliquots in Experiment 5 were sonicated whereas those in Experiment 4 were not. Hence the apparent incorporation of methionine in Experiment 4 may actually represent uptake by the microorganisms, rapid until a limiting concentration was reached, then at a slower rate that balanced utilization. Purified ration experiment. When laboratory
fermentations were inoculated with rumen fluid from the cow receiving the purified ration, L-methionine stimulated cellulose digestion more than the analog (Fig. 1). I n this experiment, Student's "t" test (16) showed that cellulose digestion was greater (P < .05) for the cellulose .5 L-methionine treatment than for cellulose alone on Days 3, 4, 6, and 7; greater for the cellulose + the analog treatment than for cellulose alone on Day 6; and greater for the cellulose .5 L-methionine treatment than for cellulose .5 the analog on Days 3, 4, and 6. The particularly low values on Day 5 followed by higher ones on Days 6 and 7 may indicate a major shift in the nficrobial population of the rumen. I n a previous experiment, both L-methionine and the analog showed a stimulation (P < .05) of cellulose digestion but the difference between the cellulose + L-methionine and cellulose + methionine hydroxy analog treatments was not significant. Experiments on the inhibition of cellulose digestion by ethionine. To compare effects of methionine and the analog with inocula from animals receiving normal rations, it was necessary to use the methionine antagonist, ethionine. This proved to be an effective inhibitor of cellulose digestion in our laboratory fermentations. Usually addition of methionine along with ethionine prevented most of the inhibition with ethionine alone (Table 2). With rumen fluid from Cow 154, the analog was only slightly less effective than methionine but the difference in effectiveness of methionine and the analog appeared to be greater with tureen fluid from Cow 191. This suggests that the results may have been affected by differences in the characteristic microbial populations of the animals. I n Experiment 7 (Table 2) the difference be-
TABLE 2. Effectiveness of methionine and methionine hydroxy analog in preventing ethionine inhibition of cellulose digestion. Cellulose digestion (%) and SE
Experiment 1 2 3 4 5 6 7
Cow
Rumen fluid
Rumen fluid -5 ethionine
Rumen fluid + ethionine + methionine
154 154 154 191 154 191 154
44.6 51.7 47.7 68.7 65.8 58.8 65.8
18.2 16.1 23.4 36.0 43.7 28.3 19.4
41.4 48.3 29.0 65.7 71.2 50.5 58.9
± 4.8 ± 1.5 ±13.1 ± 2.8 ± 4.7 ± 4.5 ± 4.0
± ± ± ± ± ± ±
2.3 0.2 3.4 0.5 0.6 5.7 1.4
± 3.2 ± 5.4 ± 3.3 ± 2.8 ± 4.6 ± 2.7 ±19.6
Rumen fluid + ethionine + MHA 37.2 41.5 27.6 49.9 66.9 36.9 43.7
± ± ± ± ± ± ±
1.7 2.5 4.9 3.3 8.6 7.7 8.2
JOUI~NAL OF DAIRY 801EIqCE V(>L, 54, NO. 3
394
SALSBURY
tween methionine and the analog was greater than in other experiments with Cow 154. This may have been an artifact (note the large standard error for the methionine treatment) or may reflect the lapse of time (more than a year) between this experiment and the previous experiments with Cow 154. The latter possibility is considered more likely because Cow 154 had received a rumen transfusion from another animal (not Cow 191) during this period and this transfusion had brought about a visible change in the protozoal population of the rumen and possibly, therefore, a change in its bacterial population. The results shown by methionine and methionine hydroxy analog in Table 2 need not be interpreted as indicating a difference in the utilization of these compounds by rumen microorganisms. They could be interpreted as indicating that methionine competes effectively for the site(s) occupied by ethionine in functioning as an antimetabolite and that it does so at a lower concentration than does the analog. Under these circumstances the possibility would still exist that rumen microorganisms could utilize the analog as well as methionine if ethionine were not present and that the difference between the effects of methionine and methionine hydroxy analog could be narrowed by increasing the metabolite: antimetabolite ratio. When this possibility was tested by holding ethionine at 0.1 meq per liter (the same concentration as in experiments shown in Table 2) and varying methionine and the analog from 0.27 to 5.37 meq per liter, the results in Figure 2 were obtained. At this ethionine concentration the effectiveness of methionine and the analog in preventing inhibition appeared to differ (P < .05) only at the lowest concentration (0.27 meq per liter) of these two compounds. When ethionine was increased to 3.9 meq per liter, 2.6 to 26 meq per liter of the analog were not effective, but 67 and 124 meq per liter of methionine partially prevented inhibition (Fig. 3). Methionine hydroxy analog was more sensitive to increases in ethionine, and this was verified by holding methionine and the analog constant at 1.3 and 2.6 meq per liter, respectively, and varying ethionine from 0.02 to 2.0 meq per liter (Fig. 4). The effectiveness of fixed concentrations of methionine and its analog decreased with inereasing ethionine but much more precipitously with the analog than with methionine. These results support the view that methionine competes more effectively with ethionine than does the analog. The greater sensitivity of the analog to increasing ethionine, therefore, may be simply a manifestation of a requirement for a higher ~OURI~AL OF D A I R Y SCI EI~CE ~rOIJ. 54, N O .
E T AL.
90
80 70 6C --
~_ 50 c~ ~ nO o
DL-ETHIONINE + DL-METHIONINE
- - -
DL-ETHIONINE +MHA
D
~ 30
20 IO I
0.27
1.34
I
2.68
I
5.37
CONCENTRATION OF METHIONINEOR MHA(MEG/L) FIG. 2. Effectiveness of different levels of DLmethionine and methionine hydroxy analog in preventing inhibition by DL-ethionine of ce]lulose digestion by rumen microorganisms. Ethionine 0.1 meq per liter. metabolite:antimetabolite ratio to prevent inhibition. This would imply that the analog has a lower affinity for the enzyme(s) of the reaction(s) being inhibited and that probably it is not as well utilized by rumcn microorganisms as is methionine under normal conditions. Alternatively, if ethionine inhibition is specific for methionine, the greater sensitivity of the analog may indicate an inhibition of methionine
50
DL-ETHIONINE + L-METHIONINE
~4o
_~3c a uJ ~ ~c ~o
DL-ETHIONINE+ MHA G
CONCENTRATION OF METHIONINE OR MHA (IMEO/L)
FIG. 3. Relative effectiveness of L-methionine and methionine hydroxy analog in preventing inhibition by higher concentration of DL-ethionine. Ethionine 3.9 meq per liter.
METHIONINE
4O ~
DL-ETHIONINE
~ 3o •,9 {3 2 0
DL-ETHIONINE + MHA
3 m _1 w
o
DL-ETHIONINE i
i
0.10 0.49 C O N C E N T R A T I O N OF E T H I O N I N E
i
1.96 (MEQ/L)
~IG. 4. Inhibition of cellulose digestion by different concentrations of ethionine at fixed methionine and methionine hydroxy analog concentrations. Methionine 1.3 meq per liter; analog 2.6 meq per liter. formation from the analog at higher ethionine levels. The failure of the analog to prevent inhibition of cellulose digestion by higher concentrations of ethionine may explain the negative results with the analog in earlier work (13). Gas chromatography experiments. Figure 5 shows the results of a typical experiment with inoculum from Cow 240. Inocula from Cows :[54 and 19:[ gave similar results. I n previous work (:[7) addition of methionine to rumen fluid resulted in the formation of measurable quantities of methanethiol. I n the present study methanethiol was also produced from the analog but at a much lower level (Fig. 5). A relatively small quantity of dimethyl sulfide was formed from both substrates. Dunham et al. (1) reported production of dimethyl sulfide from methionine by rumen microorganisms. Because they did not mention methanethiol or other related compounds, it can be assumed that dimethyl sulfide was the main, if not the only, volatile sulfur compound detected. I n our experiments, methanethiol has been consistently the main volatile sulfur compound even though there has been considerable variation in amounts produced by inocula from the same animals at different times or from different animals at the same time. Dunham et al. (1) used alfalfa-fed animals and added alfalfa to their fermentations. We found that addition of alfalfa alone to our fermentations resulted in the formation of measurable quantities of dimethyl sulfide. Reddy (9) reported that the dimethyl sulfide concentrations in milk from cows fed chopped alfalfa were greater than in milk from cows on rye or bromegrass pasture. Moreover, previous work (17) has shown that when a laboratory fermentation was inoculated with rumen fluid from a pasture-fed animal, methanethiol
395
UTILIZATION
was evolved, then rapidly disappeared. This phenomenon requires further investigation but does suggest that pasture feeding favored development of a flora capable of utilizing methanethiol. I n the reactions examined, it appears that methionine and its analog behave similarly but that the analog is less effective as a substrate. I n part at least, this may be due to lower solubility of the analog. I n the cellulose digestion experiments with normal rnmen fluid, it was necessary to use a methionine antagonist to show a significant methionine response. This suggests that there is sufficient methionine available in normal rumen fluid to meet the requirements for cellulose digestion under our conditions. However, recent work with dairy cattle and sheep indicates that methionine and methionine hydroxy analog affects lipid metabolism, both in milk fat synthesis (3, 8) and in the rumen itself (4, 5, 6) and that feeding the analog increases protozoal numbers (7). The inference from these reports is that feeding methionine or the analog may be beneficial. There is little doubt that the question is complicated by the multiplicity of reactions which utilize methi-
~ 45 40 = ~ 35 ,,< z o 30
/M __ H A .... NOADDITION / O METHANETHIOL / PRODUCTION / • DIMETHYL_SULFIDE /
~ 25
PRODUCTION
.~ 20 ~ 15 ,. o ~.10
-
,0
A
.
.o---~
L.¢~---.---',
I00
.... ....
200
300
•
. ....
400
7. . . . 500
INCUBATIONTIME(MIN.)
: ....
:
600
FIe. 5. Production by rumen microorganisms of methanethiol and dimethyl sulfide from equivalent concentrations of L-methionine, and methionine hydroxy analog, and in a control fermentation to which no substrate was added. Inoculum from Cow 240. JOURNAL OF DAIRY SOIEI~0E VOL, 54, NO. 3
396
SALSBURY ET AL.
o n i n e in t h e r u m e n ; o u r w o r k here h a s i n d i c a t e d f o u r : m e t h i o n i n e sulfoxide f o r m a t i o n , decarboxylation, d e t h i o m e t h y l a t i o n , a n d p r o t e i n s y n t h e sis. I t is p r o b a b l e t h a t these a n d o t h e r r e a c t i o n s would d e m a n d different p r o p o r t i o n s of the met h i o n i n e p o o l as conditions varied. N e v e r t h e less, it seems logical t h a t the circumstance most likely to benefit f r o m m e t h i o n i n e s u p p l e m e n t a tion would be one in which the i n t a k e of p r e f o r m e d m e t h i o n i n e f r o m o t h e r sources was limited b y a relatively h i g h p r o p o r t i o n of t h e crude p r o t e i n r e q u i r e m e n t coming f r o m n o n p r o t e i n n i t r o g e n , a n d t h e loss o f m e t h i o n i n e f r o m the b o d y was h i g h because o f h i g h milk production.
(7)
(8)
(9)
(10)
References
(1) Dunham, J. R., G. Ward, R. Bassette, and M. C. Reddy. 1968. Methionine as a precursor of methyl sulfide in cow's milk. J. Dairy Sci., 51: 199. (2) Grie], L. C., Jr., R. A. Patton, R. D. McCarthy, and P. T. Chandler. 1968. Milk production response to feeding methionine hydroxy analog to lactating dairy cows. J. Dairy Sci., 51: 1866. (3) McCarthy, R. D., G. A. Porter, and L. C. Griel, fir. 1968. Bovine ketosis and depressed f a t test in milk; a problem of methionine metabolism and serum lipoprotein aberration. J . Dairy Sci., 51:459. (4) Patton, i~. A., R. D. McCarthy, and L. C. Griel, Jr. 1968. Lipid synthesis by tureen microorganisms I. Stimulation by methionine in vitro. J. Dairy Sci., 51: 1310. (5) Patton, R. A., R. D. McCarthy, and L. C. Griel, Jr. 1970. Lipid synthesis by rumen microorganisms II. F u r t h e r characterization of the effects of mcthionine. J. Dairy Sci., 53 : 460. (6) Patton, R. A., R. D. McCarthy, and L. C. Grie], Jr. 1970. Observations on rumen fluid, blood serum and milk ]ipids of cows
JOURNAL OF DAIRY SOI]~NCE VOL. 54, NO. 3
(11)
(12)
(13) (14)
(15)
(16)
(17)
fed methionlne hydroxy analog. J . Dairy Sci., 53: 776. P a t t o n , R. A., R. D. McCarthy, L. G. Keske. L. C. Griel, Jr., and B. R. Baumgardt. 1970. Effect of feeding methlonine hydroxy analog on the concentration of protozoa in the rumen of sheep, ft. Dairy Sci., 53:933. Polan, C. B., P. T. Chandler, and C. N. Miller. 1970. Methlonlne hydroxy analog: varying levels for lactating cows. ft. Dairy Sci., 53 : 607. Reddy, M. C. 1966. Effect of surface active agent poloxalene on milk flavor when fed to cows. M.S. thesis, Kansas State University, Manhattan. Redfield, R. R. ]953. Two-dimensional paper chromatographic systems with high resolving power for amino acids. Biochim. Biophys. Acta, 10: 344. Salsbury, R. L., J. A. Hoefer, and R. W. Lueeke. 1961. Effect of feeding certain defined nutrients on cellulose digestion and volatile f a t t y acid concentrations of the rumen. J. Dairy Sci., 44: 1122. Salsbury, R. L., C. K. Smith, and C. F. Huffman. 1956. The effect of high levels of cobalt on the in vitro digestion of cellulose by rumen microorganisms, ft. Animal Sci., 15: 863. Salsbury, R. L., and ft. P. Zikakis. 1965. Stimulation of cellulose digestion by methionine. J. Animal Sci., 24: 902. Salsbury, R. L., and J. P. Zikakis. I967. Sulfur metabolism of rumen microorganisms. J. Animal Sci., 26: 929. Shockman, G. D., and G. Toennies. 1954. Formation of D-methionine from L- by Streptococcus faeealis. Arch. Biochem. and Biophys., 50: 9. Steel, R. G. D., and J. It. Torrie. 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York. Zikakis, J. P., and R. L. Salsbury. 1969. Metabolism of sulfur amino acids by rnmen microorganisms. J. Dairy Sci., 52: 2014.