Relationship Between Ruminal A m m o n i a and Nonprotein Nitrogen Utilization by Ruminants. II1. Influence of Intraruminal Urea Infusion on Ruminal A m m o n i a Concentration 1 R. E. ROFFLER, C. G. SCHWAB2, and L. D. SATTER Department of Dairy Science University of Wisconsin Madison 53706 ABSTRACT
concentration, therefore, would not be expected to influence ammonia uptake by the microbes, nor to increase microbial protein production. This study was to examine this question under in vivo conditions, and to develop an independent line of supportive evidence that 5 mg NH3-N/100 ml are sufficient to support maximal growth rates of rumen microorganisms. This experiment postulates that a plot of ruminal ammonia concentration as a function of urea intake would be linear beyond the point of ammonia accumulation (5 mg NH3-N/100 ml) if total ammonia uptake by the microbes was unaffected by ammonia in excess of this concentration, or if the extraction rate (fractional amount of ruminal ammonia incorporated into the microbes) remained unchanged. This assumes that ammonia absorption and passage out of the rumen are directly proportional to concentration of ammonia (4). A curvilinear plot would suggest a changing extraction rate by the microbes and would constitute indirect evidence that ammonia nitrogen concentrations in excess of 5 mg/100 ml were influencing total ammonia uptake by the microbes. A linear ammonia accumulation response, therefore, would be consistent with the in vitro observations (13) while a curvilinear accumulation response would be inconsistent with the hypothesis that 5 mg NH3-N/100 ml is a saturating concentration of ammonia for microbial growth.
In three trials, we studied the effect of incremental amounts of intraruminally infused urea on mean ruminal ammonia concentration of steers fed at 2-h intervals. Basal rations contained these percentages of crude protein and total digestible nutrients (dry matter basis): Trial I, 11.1 and 81; Trial II, 6.0 and 54; Trial III, 6.5 and 58. Mean ruminal ammonia concentration reached 5 mg ammonia nitrogen/100 ml rumen fluid at crude protein equivalents of 12.0, 9.3, and 9.496 in I, II, and III. Once ruminal ammonia began to accumulate, there was a linear relationship between intake of urea and mean concentration of ruminal ammonia. The concentration of amino acids of plasma, serving as an indirect measure of amino acid absorption from the intestine, was not increased by increased intake of urea in III. Results of this experiment support the concept from in vitro data that microbial protein synthesis is unaffected by ruminal ammonia concentration in excess of 5 mg ammonia nitrogen/100 ml rumen fluid.
INTRODUCTION Studies of in vitro continuous culture fermentation with ruminal ingesta have indicated that maintenance of ruminal ammonia at 5 mg NH3-N/100 ml rumen fluid is adequate to support maximal growth rates of rumen microorganisms (13). Ammonia in excess of this
EXPERIMENTAL PROCEDURE In three trials, we studied the influence of urea infusion on mean concentration of ruminal ammonia. Each trial consisted of a series of 7-day periods during which increasing amounts of urea were infused intraruminally. In each trial, four rumen-fistulated steers were fed a basal ration at 2-h intervals by mechanical feeders controlled by time clocks. Steers were
Received April 28, 1975. Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, and by the Soybean Research Council of the National Soybean Processors Association, Washington, DC. 2Departrnent of Animal Science, University- of Kentucky, Lexington 40506. 80
RUMINAL AMMONIA AND UREA INTAKE different in Trial II1 f r o m I and 11. Body weights averaged a p p r o x i m a t e l y 450, 3 50, and 240 kg in I, II, and III. Dry m a t t e r intake was held c o n s t a n t across infusion periods within a trial. Daily feed intake was limited to a b o u t 1.5% o f b o d y weight in I and II and 1.8% of body weight in IIl. C o m p o s i t i o n of the basal rations is in Table 1. Crude protein (CP) and estimated total digestible nutrient (TDN) content (dry m a t t e r basis) of the basal rations are in Fig. 1, 2, and 3. Urea solutions were infused continuously into the tureen of each steer with infusion v o l u m e at 2 liters/24 h. The a m o u n t of urea infused was controlled by varying the concentration of urea in the infusates. Ruminal a m m o n i a concentrations were m o n i t o r e d during the last 2 days of each 7-day period. R u m e n contents were sampled via rumen fistula at 0, 20, 40, 60, 80, and 100 min following feeding. Thus, six samples for each of f o u r steers, or a total of 24 samples, were collected each period• Sampling times were selected r a n d o m l y with not more than two samples per steer during a single 2-h feeding interval. In III, jugular blood samples were obtained on the 6th and 7th days of each period. The t w o samples f r o m each steer were p o o l e d for a m i n o acids analysis of plasma• Crude protein (N x 6.25) in the basal rations was determined by Kjeldahl procedure. Ration
81
TDN was calculated from National Research Council (NRC) (6) tabular values. A m m o n i a was d e t e r m i n e d in the supernatant of centrifuged l u m e n fluid by steam distiliation over magnesium o x i d e (1). Blood plasma for a m i n o acid analysis was o b t a i n e d by centrifuging 25 ml of heparinized blood at 8000 x g for 15 rain at 5 C. The plasma was d e p r o t e i n i z e d immediately by mixing 4 volumes of plasma with 1 v o l u m e of 15% sulfosalicylic acid and centrifuging at 28,700 × g for 30 rain at 5 C. The supernate was stored at - 2 0 C until analyzed by a J E O L - 6 A H amino acid analyzer. Since the primary objective was to test linearity of the response of ruminal a m m o n i a c o n c e n t r a t i o n to incremental a m o u n t s of intraruminally-infused urea, analyses were regression. H o m o g e n e i t y of regressions was tested according to Steel and Torrie (14).
RESULTS A N D DISCUSSION
In I, where the basal ration c o n t a i n e d 81% TDN, mean c o n c e n t r a t i o n of ruminal a m m o n i a increased linearly (P
concentration, therefore, would not be expected to influence ammonia uptake by the microbes, nor to increase microbial protein production. This study was to examine this question under in vivo conditions, and to develop an independent line of supportive evidence that 5 mg NH3-N/100 ml are sufficient to support maximal growth rates of rumen microorganisms. This experiment postulates that a plot of ruminal ammonia concentration as a function of urea intake would be linear beyond the point of ammonia accumulation (5 mg NH3-N/100 ml) if total ammonia uptake by the microbes was unaffected by ammonia in excess of this concentration, or if the extraction rate (fractional amount of ruminal ammonia incorporated into the microbes) remained unchanged. This assumes that ammonia absorption and passage out of the rumen are directly proportional to concentration of ammonia (4). A curvilinear plot would suggest a changing extraction rate by the microbes and would constitute indirect evidence that ammonia nitrogen concentrations in excess of 5 mg/100 ml were influencing total ammonia uptake by the microbes. A linear ammonia accumulation response, therefore, would be consistent with the in vitro observations (13) while a curvilinear accumulation response would be inconsistent with the hypothesis that 5 mg NH3-N/100 ml is a saturating concentration of ammonia for microbial growth.
In three trials, we studied the effect of incremental amounts of intraruminally infused urea on mean ruminal ammonia concentration of steers fed at 2-h intervals. Basal rations contained these percentages of crude protein and total digestible nutrients (dry matter basis): Trial I, 11.1 and 81; Trial II, 6.0 and 54; Trial III, 6.5 and 58. Mean ruminal ammonia concentration reached 5 mg ammonia nitrogen/100 ml rumen fluid at crude protein equivalents of 12.0, 9.3, and 9.496 in I, II, and III. Once ruminal ammonia began to accumulate, there was a linear relationship between intake of urea and mean concentration of ruminal ammonia. The concentration of amino acids of plasma, serving as an indirect measure of amino acid absorption from the intestine, was not increased by increased intake of urea in III. Results of this experiment support the concept from in vitro data that microbial protein synthesis is unaffected by ruminal ammonia concentration in excess of 5 mg ammonia nitrogen/100 ml rumen fluid.
INTRODUCTION Studies of in vitro continuous culture fermentation with ruminal ingesta have indicated that maintenance of ruminal ammonia at 5 mg NH3-N/100 ml rumen fluid is adequate to support maximal growth rates of rumen microorganisms (13). Ammonia in excess of this
EXPERIMENTAL PROCEDURE In three trials, we studied the influence of urea infusion on mean concentration of ruminal ammonia. Each trial consisted of a series of 7-day periods during which increasing amounts of urea were infused intraruminally. In each trial, four rumen-fistulated steers were fed a basal ration at 2-h intervals by mechanical feeders controlled by time clocks. Steers were
Received April 28, 1975. Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, and by the Soybean Research Council of the National Soybean Processors Association, Washington, DC. 2Departrnent of Animal Science, University- of Kentucky, Lexington 40506. 80
RUMINAL AMMONIA AND UREA INTAKE different in Trial II1 f r o m I and 11. Body weights averaged a p p r o x i m a t e l y 450, 3 50, and 240 kg in I, II, and III. Dry m a t t e r intake was held c o n s t a n t across infusion periods within a trial. Daily feed intake was limited to a b o u t 1.5% o f b o d y weight in I and II and 1.8% of body weight in IIl. C o m p o s i t i o n of the basal rations is in Table 1. Crude protein (CP) and estimated total digestible nutrient (TDN) content (dry m a t t e r basis) of the basal rations are in Fig. 1, 2, and 3. Urea solutions were infused continuously into the tureen of each steer with infusion v o l u m e at 2 liters/24 h. The a m o u n t of urea infused was controlled by varying the concentration of urea in the infusates. Ruminal a m m o n i a concentrations were m o n i t o r e d during the last 2 days of each 7-day period. R u m e n contents were sampled via rumen fistula at 0, 20, 40, 60, 80, and 100 min following feeding. Thus, six samples for each of f o u r steers, or a total of 24 samples, were collected each period• Sampling times were selected r a n d o m l y with not more than two samples per steer during a single 2-h feeding interval. In III, jugular blood samples were obtained on the 6th and 7th days of each period. The t w o samples f r o m each steer were p o o l e d for a m i n o acids analysis of plasma• Crude protein (N x 6.25) in the basal rations was determined by Kjeldahl procedure. Ration
81
TDN was calculated from National Research Council (NRC) (6) tabular values. A m m o n i a was d e t e r m i n e d in the supernatant of centrifuged l u m e n fluid by steam distiliation over magnesium o x i d e (1). Blood plasma for a m i n o acid analysis was o b t a i n e d by centrifuging 25 ml of heparinized blood at 8000 x g for 15 rain at 5 C. The plasma was d e p r o t e i n i z e d immediately by mixing 4 volumes of plasma with 1 v o l u m e of 15% sulfosalicylic acid and centrifuging at 28,700 × g for 30 rain at 5 C. The supernate was stored at - 2 0 C until analyzed by a J E O L - 6 A H amino acid analyzer. Since the primary objective was to test linearity of the response of ruminal a m m o n i a c o n c e n t r a t i o n to incremental a m o u n t s of intraruminally-infused urea, analyses were regression. H o m o g e n e i t y of regressions was tested according to Steel and Torrie (14).
RESULTS A N D DISCUSSION
In I, where the basal ration c o n t a i n e d 81% TDN, mean c o n c e n t r a t i o n of ruminal a m m o n i a increased linearly (P
