N I T R O G E N F I X A T I O N IN R U M I N A N T S 1 Hector H. Li Pun and L. D. Satter
SUMMARY
The amount of nitrogen gas (N2) available for microbial protein synthesis via nitrogen fixation in the rumen was estimated. Measurements were made of nitrogen fixation by ruminal ingesta from goats fed low protein rations for a prolonged period of time, and by ingesta from sheep fed high protein rations. Nitrogen fixation by rumen microorganisms incubated for 30 or 180 min in vitro was measured with the acetylene-reduction technique. Results suggest that goats fixed approximately .2 to .3 mg of N2 daily. Results from determinations made with rumen ingesta obtained from two sheep fed a high protein diet (17% crude protein) suggested that up to 2.5 mg N2 could be fixed daily. Three mature castrated goats were fed a 5.4% crude protein diet for 16 weeks. A nitrogen balance trial conducted during the eighth week of the experiment indicated that the goats were each losing about 1 g of nitrogen daily. This amount of nitrogen loss was about what would have been expected if there was no nitrogen fixation occurring in the gastrointestinal tract. Based upon results from the acetylene-reduction test and the nitrogen balance trial, it was concluded that nitrogen fixation by rumen microorganisms is of negligible quantity. (Key Words: Nitrogen Fixation, Nitrogen Balance, Rumen Microbes, Nitrogenase.)
spheric gas could contribute to the available supply of N2. Measurements of nitrogen flux in the reticulo-rumen have suggested that N2 fixation could be operative (Mathison and Milligan, 1971 ). The amount of N2 fixed in the rumen, though, is considered small (Granhall and Ciszuk, 1971 ; Hardy et al., 1968; Hobson et al., 1973). Under normal feeding conditions, however, N2 fixation may be very low because of the ready availability of ammonia which may supplant N2 fixation. It is known that nitrogenase, the enzyme for N2 fixation, is repressed by ammonia (Postgate, 1971; Kleiner, 1974). Since many anaerobic bacteria can fix N2, it is conceivable that with low ruminal ammonia concentrations, N2 fixation could be more important than when rations adequate in protein are fed. The objective of the present experiment was to estimate the amount of N2 fixation occurring in goats f e d low protein diets for a sustained period of time. MATERIALS AND METHODS
Four adult castrate male goats, two of which had rumen fistulas, were used in this experiment. The goats were kept in individual metal pens and were fed ad libitum a pelleted ration containing 5.4% crude protein (table 1). Feed consumption was recorded daily and the goats were weighed every 10 days. Two months after the start of the experiment, one of the goats INTRODUCTION refused to eat and was retired from the experiThere is significant potential for fixation of ment. A conventional nitrogen balance was gaseous nitrogen (N2) in the rumen. Kleiber carried out during the eighth week of the (1956) has estimated that up to 56 liters (69 g) 16-week experiment. of N2 could diffuse daily from the blood to the Nitrogen fixation by incubated ruminal inbovine rumen. In addition, ingested atmo- gesta was measured by the acetylene-reduction technique (Burris, 1972). This test is based on the reduction of acetylene to ethylene and the measurement of ethylene production by gas 1Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, chromatography. It is a very sensitive test, and ethylene concentrations as low as 10-i2 molar and by Hatch Project 1891. 2 Department of Dairy Science. can be determined. Fifty milliliters of rumen 1161 JOURNAL OF ANIMAL SCIENCE, Vol. 41, No. 4, 1975
Downloaded from https://academic.oup.com/jas/article-abstract/41/4/1161/4668223 by Iowa State University user on 12 January 2019
University o f Wisconsin 2 , Madison 53 706
1162
PUN AND SATTER TABLE 1. RATION COMPOSITION
Ingredient
Dry matter Crude protein (N • 6.25, DM basis)
% composition
59 29 10 .9 1 .1 94.9 5.4
awood pulp consisting primarily of short cellulose fibers and containing approximately 2% ash and 20% partially degraded lignin. bcontains 97% NaCI, .2% Mn, .3% Fe, .033% Cu, .01% Zn, .007% I and .005% Co. Ccontains 1,000,000 IU each of vitamins A and D per kilogram and 1,000 IU of E per kilogram.
fluid were o b t a i n e d f r o m each of the three goats fed the low protein ration and f r o m t w o rumen fistulated sheep fed a pelleted alfalfa meal diet (17% crude protein). Ruminal ingesta was obtained f r o m the goats after they had been on the low protein ration for 9 to 11 weeks. The sheep served as controls. R u m e n fluid samples were obtained via r u m e n fistula or stomach tube and immediatety strained through a single layer of cheesecloth. Ten milliliters of the filtrate were injected through the rubber
RESULTS A N D DISCUSSION
Results of the nitrogen balance study are shown in table 2. The goats lost weight during the experiment, probably because of protein deficiency. All of the goats were in negative nitrogen balance, losing a b o u t 1 g of nitrogen per day as measured during the eighth week. This a m o u n t of nitrogen loss was a b o u t what w o u l d have been expected, based u p o n overall b o d y weight change, if there were no nitrogen fixation occurring in the gastrointestinal tract. If there was a significant a m o u n t of N2 fixation, total nitrogen excretion should have exceeded nitrogen intake by m o r e than approximately 1 g daily in these goats which were losing only m o d e s t a m o u n t s of weight. The length of this e x p e r i m e n t was similar to that of a dry season in the tropics. If N2
TABLE 2. AVERAGE BODY WEIGHT, FEED CONSUMPTION, AND NITROGEN BALANCE OF GOATS FED A LOW PROTEIN RATION
Item
246
Goat no. 269
278
Initial weighta Eight-week weight Final weighta Average dry matter consumption (kg/day for 16-week period) Dry matter consumption during nitrogen balance trial (kg/day) Nitrogen intake (g/day) Nitrogen excreted (g/day) In feces In urine Nitrogen retained (g/day)
52.0 48.6 51.8
55.2 50.4 49.9
46.1 40.9 40.4
a
9
Sixteen-week experiment. bSEM.
1.13 + .09 b
1.16 + .O7
.67 +- .04
1.17 10.1 11.1 + .8b 7.40 + .35 b 3.67 + .67 b -1.01
1.21 10.4 11.5 +_.80 7.61 +- .47 3.86 + .49 -1.07
.81 6.95 8.01 + .31 5.05 -+ .19 2.96 +- .25 -1.06
Downloaded from https://academic.oup.com/jas/article-abstract/41/4/1161/4668223 by Iowa State University user on 12 January 2019
Oats, straw (1), chopped, 1-O3-283 Wood pulp fines (4)a Sugarcane, molasses, dehy. (4), 4-04-695 Salt (6) Trace mineral salt (6) b Vitamin ADE premix (7) c
stopper of a serum vacuum tube that was previously flushed with nitrogen gas. In addition, 1 ml of acetylene was injected into the tube. Care was taken to maintain anaerobiosis in the tube. The sealed tubes were incubated in a shaker water bath at 38 C for either 30 or 180 minutes. A f t e r the incubation, fermentation was stopped by injecting .5 ml of a 50% v/v H2SO4 solution through the rubber stopper into the tubes. A sample of gas f r o m the tubes was taken up in a syringe and i m m e d i a t e l y analyzed in a gas c h r o m a t o g r a p h for e t h y l e n e p r o d u c t i o n according to the procedure described by Burris (1972).
NITROGEN F IXATION IN RUMINANTS
1163
TABLE 3. ACETYLENE REDUCTION BY RUMINAL INGESTA Time of incubation C: H4 formation (nmoles/10 ml tureen fluid)
30 min
No. 246 No. 269 No. 278 Sheep 1 Sheep 2
1.35a (1.24 - 1.46) b 1.31 (1.19 -- 1.43) 1.27 (1.21 -- 1.33) 5.88 (3.43 -- 8.32) 15.8 (9.21 - 22.4)
180 rain 7.36 (7.07 -- 7.65) 6.81 (4.46 -- 9.15) 8.88 (5.20 --!2.6) 27.6 (23.1 -- 32.1) 68.4 (64.5 - 72.4)
aAverage of two incubations. b Range.
fixation in the reticulo-rumen were of any biological significance, it should reduce nitrogen loss by a measurable amount. The nitrogen balance data suggest that N2 fixation was not important. Results of the acetylene-reduction test (table 3) suggested that the a m o u n t of N2 fixed by rumen contents in vitro was very low. Assuming that: (1) the ratio of ethylene production to fixed N2 is 3:1 (Postgate, 1971); ( 2 ) t h e rumen volume of goats used in this experiment was 5 liters; and (3) the production of ethylene followed a linear fashion, then, on the basis of ethylene production during the 180-rain incubation ( ~ 7 nmoles/10 ml of rumen fluid), it can be calculated that about .26 mg of N2 was being fixed daily by the goats and 1.1 and 2.5 nag in the case of the sheep. These values are similar to those reported by Granhall and Ciszuk (1971), who estimated that 1.28 mg of Nz/day were fixed by sheep eating dried clover. They .also obtained estimates of N2 fixation by goats consuming a diet containing 2 to 3% crude protein and determined that the a m o u n t of N2 fixed was approximately .6 mg/day, assuming 5 kg of rumcn contents. The goats were consuming this tow protein ration for as short a period as 2 weeks, however. Hobson et al. (1973) recently verified that estimates of N2 fixation obtained from in vitro studies agreed well with estimates obtained in vivo, and that the average rate of N2 fixation was about .4 mg/sheep/day. The concerltration of ammonia which will inhibit nitrogenase activity under anaerobic conditions is not known. Kleiner (1974) has determined that 25/aM concentration of ammonia will completely repress the nitrogenase system of the aerobe, A z o t o b a c t e r vinelandii.
From the few studies reported, it is suggested that the amount of N2 fixed in the rumen is very small and of milligram quantity (Granhall and Ciszuk, 1971 ; Hardy et al., 1968; Hobson et al., 1973). The results obtained in the present experiment support these observations. This experiment further suggests that even with prolonged protein deficiency, a condition which would favor N2 fixation, N2 fixation by rumen microorganisms is of negligible quantity. It has to be recognized, however, that the approaches used in this study have been indirect ones, and that the number of animals involved was small. There is agreement among reported studies and the present research.
LITERATURE CITED
Burris, R. H. 1972. Nitrogen fixation-assay methods and techniques. In A. san Pietro (Ed.) Methods in Enzymology. Vol. 24, p. 415. Granhall, V. and P. Ciszuk. 1971. Nitrogen fLxationin tureen contents indicated by the acetylene reduction test. J. Gen. MicrobioL 65:9t. Hardy, R. W. F., R. D. Holsten, E. K. Jackson and R.C. Burns. 1968. The acetylene-ethylene assay for nitrogen fixation: Laboratory and field evaluation. Plant Physiol. 43:1185. Hobson, P. N., R. Summers, J. R. Postgate and D. A. Ware. 1973. Nitrogen fixation in the rumen of a living sheep. J. Gen. Microbiol. 77:225. Kleiber, M. 1956. Sources of tureen gases. In A Review of Bloat in Ruminants. Pub. 380:10. National Academy of Science-National Research Council, Washington, D. C. Kleiner, D. 1974. Quantitative relations for the repression of nitrogenase synthesis in Azotobacter vinelandii by ammonia. Arch. Microbiol. 101:153. Mathison, G. W. and L. P. Milligan. 1971. Nitrogen metabolism in sheep. Brit. J. Nutr. 25:351. Postgate, J. R. 1971. The chemistry and biochemistry of nitrogen fixation. Plenum Press, New York.
Downloaded from https://academic.oup.com/jas/article-abstract/41/4/1161/4668223 by Iowa State University user on 12 January 2019
Animal
SUMMARY
The amount of nitrogen gas (N2) available for microbial protein synthesis via nitrogen fixation in the rumen was estimated. Measurements were made of nitrogen fixation by ruminal ingesta from goats fed low protein rations for a prolonged period of time, and by ingesta from sheep fed high protein rations. Nitrogen fixation by rumen microorganisms incubated for 30 or 180 min in vitro was measured with the acetylene-reduction technique. Results suggest that goats fixed approximately .2 to .3 mg of N2 daily. Results from determinations made with rumen ingesta obtained from two sheep fed a high protein diet (17% crude protein) suggested that up to 2.5 mg N2 could be fixed daily. Three mature castrated goats were fed a 5.4% crude protein diet for 16 weeks. A nitrogen balance trial conducted during the eighth week of the experiment indicated that the goats were each losing about 1 g of nitrogen daily. This amount of nitrogen loss was about what would have been expected if there was no nitrogen fixation occurring in the gastrointestinal tract. Based upon results from the acetylene-reduction test and the nitrogen balance trial, it was concluded that nitrogen fixation by rumen microorganisms is of negligible quantity. (Key Words: Nitrogen Fixation, Nitrogen Balance, Rumen Microbes, Nitrogenase.)
spheric gas could contribute to the available supply of N2. Measurements of nitrogen flux in the reticulo-rumen have suggested that N2 fixation could be operative (Mathison and Milligan, 1971 ). The amount of N2 fixed in the rumen, though, is considered small (Granhall and Ciszuk, 1971 ; Hardy et al., 1968; Hobson et al., 1973). Under normal feeding conditions, however, N2 fixation may be very low because of the ready availability of ammonia which may supplant N2 fixation. It is known that nitrogenase, the enzyme for N2 fixation, is repressed by ammonia (Postgate, 1971; Kleiner, 1974). Since many anaerobic bacteria can fix N2, it is conceivable that with low ruminal ammonia concentrations, N2 fixation could be more important than when rations adequate in protein are fed. The objective of the present experiment was to estimate the amount of N2 fixation occurring in goats f e d low protein diets for a sustained period of time. MATERIALS AND METHODS
Four adult castrate male goats, two of which had rumen fistulas, were used in this experiment. The goats were kept in individual metal pens and were fed ad libitum a pelleted ration containing 5.4% crude protein (table 1). Feed consumption was recorded daily and the goats were weighed every 10 days. Two months after the start of the experiment, one of the goats INTRODUCTION refused to eat and was retired from the experiThere is significant potential for fixation of ment. A conventional nitrogen balance was gaseous nitrogen (N2) in the rumen. Kleiber carried out during the eighth week of the (1956) has estimated that up to 56 liters (69 g) 16-week experiment. of N2 could diffuse daily from the blood to the Nitrogen fixation by incubated ruminal inbovine rumen. In addition, ingested atmo- gesta was measured by the acetylene-reduction technique (Burris, 1972). This test is based on the reduction of acetylene to ethylene and the measurement of ethylene production by gas 1Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, chromatography. It is a very sensitive test, and ethylene concentrations as low as 10-i2 molar and by Hatch Project 1891. 2 Department of Dairy Science. can be determined. Fifty milliliters of rumen 1161 JOURNAL OF ANIMAL SCIENCE, Vol. 41, No. 4, 1975
Downloaded from https://academic.oup.com/jas/article-abstract/41/4/1161/4668223 by Iowa State University user on 12 January 2019
University o f Wisconsin 2 , Madison 53 706
1162
PUN AND SATTER TABLE 1. RATION COMPOSITION
Ingredient
Dry matter Crude protein (N • 6.25, DM basis)
% composition
59 29 10 .9 1 .1 94.9 5.4
awood pulp consisting primarily of short cellulose fibers and containing approximately 2% ash and 20% partially degraded lignin. bcontains 97% NaCI, .2% Mn, .3% Fe, .033% Cu, .01% Zn, .007% I and .005% Co. Ccontains 1,000,000 IU each of vitamins A and D per kilogram and 1,000 IU of E per kilogram.
fluid were o b t a i n e d f r o m each of the three goats fed the low protein ration and f r o m t w o rumen fistulated sheep fed a pelleted alfalfa meal diet (17% crude protein). Ruminal ingesta was obtained f r o m the goats after they had been on the low protein ration for 9 to 11 weeks. The sheep served as controls. R u m e n fluid samples were obtained via r u m e n fistula or stomach tube and immediatety strained through a single layer of cheesecloth. Ten milliliters of the filtrate were injected through the rubber
RESULTS A N D DISCUSSION
Results of the nitrogen balance study are shown in table 2. The goats lost weight during the experiment, probably because of protein deficiency. All of the goats were in negative nitrogen balance, losing a b o u t 1 g of nitrogen per day as measured during the eighth week. This a m o u n t of nitrogen loss was a b o u t what w o u l d have been expected, based u p o n overall b o d y weight change, if there were no nitrogen fixation occurring in the gastrointestinal tract. If there was a significant a m o u n t of N2 fixation, total nitrogen excretion should have exceeded nitrogen intake by m o r e than approximately 1 g daily in these goats which were losing only m o d e s t a m o u n t s of weight. The length of this e x p e r i m e n t was similar to that of a dry season in the tropics. If N2
TABLE 2. AVERAGE BODY WEIGHT, FEED CONSUMPTION, AND NITROGEN BALANCE OF GOATS FED A LOW PROTEIN RATION
Item
246
Goat no. 269
278
Initial weighta Eight-week weight Final weighta Average dry matter consumption (kg/day for 16-week period) Dry matter consumption during nitrogen balance trial (kg/day) Nitrogen intake (g/day) Nitrogen excreted (g/day) In feces In urine Nitrogen retained (g/day)
52.0 48.6 51.8
55.2 50.4 49.9
46.1 40.9 40.4
a
9
Sixteen-week experiment. bSEM.
1.13 + .09 b
1.16 + .O7
.67 +- .04
1.17 10.1 11.1 + .8b 7.40 + .35 b 3.67 + .67 b -1.01
1.21 10.4 11.5 +_.80 7.61 +- .47 3.86 + .49 -1.07
.81 6.95 8.01 + .31 5.05 -+ .19 2.96 +- .25 -1.06
Downloaded from https://academic.oup.com/jas/article-abstract/41/4/1161/4668223 by Iowa State University user on 12 January 2019
Oats, straw (1), chopped, 1-O3-283 Wood pulp fines (4)a Sugarcane, molasses, dehy. (4), 4-04-695 Salt (6) Trace mineral salt (6) b Vitamin ADE premix (7) c
stopper of a serum vacuum tube that was previously flushed with nitrogen gas. In addition, 1 ml of acetylene was injected into the tube. Care was taken to maintain anaerobiosis in the tube. The sealed tubes were incubated in a shaker water bath at 38 C for either 30 or 180 minutes. A f t e r the incubation, fermentation was stopped by injecting .5 ml of a 50% v/v H2SO4 solution through the rubber stopper into the tubes. A sample of gas f r o m the tubes was taken up in a syringe and i m m e d i a t e l y analyzed in a gas c h r o m a t o g r a p h for e t h y l e n e p r o d u c t i o n according to the procedure described by Burris (1972).
NITROGEN F IXATION IN RUMINANTS
1163
TABLE 3. ACETYLENE REDUCTION BY RUMINAL INGESTA Time of incubation C: H4 formation (nmoles/10 ml tureen fluid)
30 min
No. 246 No. 269 No. 278 Sheep 1 Sheep 2
1.35a (1.24 - 1.46) b 1.31 (1.19 -- 1.43) 1.27 (1.21 -- 1.33) 5.88 (3.43 -- 8.32) 15.8 (9.21 - 22.4)
180 rain 7.36 (7.07 -- 7.65) 6.81 (4.46 -- 9.15) 8.88 (5.20 --!2.6) 27.6 (23.1 -- 32.1) 68.4 (64.5 - 72.4)
aAverage of two incubations. b Range.
fixation in the reticulo-rumen were of any biological significance, it should reduce nitrogen loss by a measurable amount. The nitrogen balance data suggest that N2 fixation was not important. Results of the acetylene-reduction test (table 3) suggested that the a m o u n t of N2 fixed by rumen contents in vitro was very low. Assuming that: (1) the ratio of ethylene production to fixed N2 is 3:1 (Postgate, 1971); ( 2 ) t h e rumen volume of goats used in this experiment was 5 liters; and (3) the production of ethylene followed a linear fashion, then, on the basis of ethylene production during the 180-rain incubation ( ~ 7 nmoles/10 ml of rumen fluid), it can be calculated that about .26 mg of N2 was being fixed daily by the goats and 1.1 and 2.5 nag in the case of the sheep. These values are similar to those reported by Granhall and Ciszuk (1971), who estimated that 1.28 mg of Nz/day were fixed by sheep eating dried clover. They .also obtained estimates of N2 fixation by goats consuming a diet containing 2 to 3% crude protein and determined that the a m o u n t of N2 fixed was approximately .6 mg/day, assuming 5 kg of rumcn contents. The goats were consuming this tow protein ration for as short a period as 2 weeks, however. Hobson et al. (1973) recently verified that estimates of N2 fixation obtained from in vitro studies agreed well with estimates obtained in vivo, and that the average rate of N2 fixation was about .4 mg/sheep/day. The concerltration of ammonia which will inhibit nitrogenase activity under anaerobic conditions is not known. Kleiner (1974) has determined that 25/aM concentration of ammonia will completely repress the nitrogenase system of the aerobe, A z o t o b a c t e r vinelandii.
From the few studies reported, it is suggested that the amount of N2 fixed in the rumen is very small and of milligram quantity (Granhall and Ciszuk, 1971 ; Hardy et al., 1968; Hobson et al., 1973). The results obtained in the present experiment support these observations. This experiment further suggests that even with prolonged protein deficiency, a condition which would favor N2 fixation, N2 fixation by rumen microorganisms is of negligible quantity. It has to be recognized, however, that the approaches used in this study have been indirect ones, and that the number of animals involved was small. There is agreement among reported studies and the present research.
LITERATURE CITED
Burris, R. H. 1972. Nitrogen fixation-assay methods and techniques. In A. san Pietro (Ed.) Methods in Enzymology. Vol. 24, p. 415. Granhall, V. and P. Ciszuk. 1971. Nitrogen fLxationin tureen contents indicated by the acetylene reduction test. J. Gen. MicrobioL 65:9t. Hardy, R. W. F., R. D. Holsten, E. K. Jackson and R.C. Burns. 1968. The acetylene-ethylene assay for nitrogen fixation: Laboratory and field evaluation. Plant Physiol. 43:1185. Hobson, P. N., R. Summers, J. R. Postgate and D. A. Ware. 1973. Nitrogen fixation in the rumen of a living sheep. J. Gen. Microbiol. 77:225. Kleiber, M. 1956. Sources of tureen gases. In A Review of Bloat in Ruminants. Pub. 380:10. National Academy of Science-National Research Council, Washington, D. C. Kleiner, D. 1974. Quantitative relations for the repression of nitrogenase synthesis in Azotobacter vinelandii by ammonia. Arch. Microbiol. 101:153. Mathison, G. W. and L. P. Milligan. 1971. Nitrogen metabolism in sheep. Brit. J. Nutr. 25:351. Postgate, J. R. 1971. The chemistry and biochemistry of nitrogen fixation. Plenum Press, New York.
Downloaded from https://academic.oup.com/jas/article-abstract/41/4/1161/4668223 by Iowa State University user on 12 January 2019
Animal
