Determination of N metabolism parameters in an N increase trial in the rat using 15N tracers

3 groups of 8 Wistar rats each of a live weight of ca. 215 g received graded N supplements. A number of N metabolism parameters were ascertained and discussed. Among other things, the protein synthesis and degradation quotas were ascertained by means of the 15N tracer technique. For the assessment a 3-pool model was used, the content of which was in so far extended as growth and metabolic faecal nitrogen are concretely taken into consideration. A quasi-continuous method of supplementing the tracer was chosen; it was discontinued in approximately the middle of the experiment. Thus the labelling and abatement phases in urine could at the same time be included in the calculation. The protein synthesis quota increases with N intake. It can be concluded from the course of the graph that it gravitates towards a saturation value.     

15N库稀释法和15N示踪法在草地生态系统氮转化过程研究中的应用——方法与进展

氮素(N)是陆地生态系统尤其是草地生态系统第一生产力的重要限制性养分之一.陆地生态系统N素的可利用性由土壤N素的转化速率决定,其中包括氨化和硝化2个重要过程.准确测定N素在各转化过程中的量,对于估算陆地生态系统N转化非常重要.稳定性同位素¹⁵N由于其安全、准确且不干扰自然生态系统等特点,近年来在生态系统N循环研究方面得到广泛应用,常用方法包括¹⁵N自然丰度法、¹⁵N还原法、¹⁵N库稀释法和¹⁵N示踪法.在查阅大量文献的基础上,搜集整理了¹⁵N库稀释法和¹⁵N示踪法的详细操作流程并综述了其在草地生态系统应用的最新进展,分别从不同草地管理方式(增施氮肥、放牧、火烧和刈割等)和全球气候变化(增温、增雨、大气氮沉降和CO₂浓度升高等)对草地生态系统N转化过程的影响进行论述.同位素¹⁵N在草地生态系统应用的方法同样适用于森林、农田以及其他陆地生态系统.     

The role of glucose infusion on the metabolism of nitrogen in ruminants

This work determined the time necessary to stabilize the decrease in urinary N excretion after initiating continuous i.v. glucose infusion and the quantity of glucose required to maximize N balance in growing wether lambs fed a high-protein diet (21.1% CP, DM basis). In the first experiment, six wethers (30 kg) were used in a 10-d crossover design comparing jugular infusion of glucose (600 kcal gross energy/d) plus saline with saline alone. The second infusion experiment was carried out with six wethers (31 kg) assigned to two 3 x 3 plus extra period latin squares, with glucose infusion rates of 0, 300 and 600, and 0, 450 and 900 kcal/d, respectively. Urinary N decreased (P less than .02) by d 2 of glucose infusion, remained stable to the end of the 10-d infusion period, and returned to the preinfusion level within 2 d after glucose infusion was discontinued. Urinary N decreased (P less than .01) and N balance increased (P less than .005) with an increasing level of glucose infusion through 600 kcal/d. Plasma glucose and insulin were elevated (P less than .05) only by infusion of 900 kcal/d of glucose. Glucose was present in the urine of wethers infused with 900 kcal/d of glucose. Glucose infusion had no effect on diet digestibility, hematocrit or plasma urea N. The level of glucose infused into growing wether lambs that maximized reduction of urinary N and was fully utilized for protein deposition without increasing plasma glucose and insulin was about 12 g.Wk-.75.d-1.     

Methodologic aspects of the determination of N-exchange parameters from 15N-tracer studies in swine on the basis of models of N-metabolism. 3. Calculation of protein synthesis and decomposition rate on the basis of lysine flux, ascertained from the amount of 15N in the urine

The final product method introduced here is based on the use of 15N L-Lysine as tracer amino acid for the determination of the protein synthesis and decomposition quotas of the total body with the flux of lysine. For this purpose it is necessary to complete the N flow and N compartments of the 3-pool model by the values of lysine content. The ascertained values of protein synthesis--8.4 g/d/kg--and protein decomposition--6.9 g/d/kg--agree very well with those determined with 15N glycine as tracer amino acid.     

Methodologic studies for the measurement of kinetic parameters of the metabolism of whole body protein in association with measurements of energy metabolism in rats

In 3 successive experiments with growing rats the suitability of pulse labelling with [15N]glycine, linked with a 14C labelling by means of [14C]lysine (experiment 3), was tested for the determination of kinetic parameters of the protein metabolism of the whole body by the application of the compartment model in comparison with pulse labelling with a 15N amino acid mixture (experiment 2) and long-time labelling with 15N with 15N labelled wheat in the feed (experiment 1) under standardized experiment conditions. In simultaneously carried out measurings of energy metabolism with parallel groups of animals the comparability of the metabolic development was studied. The ascertained values of protein synthesis rate, protein catabolism rate and re-utilization rate showed insignificant differences only between the 3 15N tracer variants (with certain limitations for the 'protein turnover' (P)-group of experiment 2) in comparison with errors of the applied methods, from which conclusions can be drawn for the suitability of [15N] glycine as tracer, at least under the experiment conditions tested. The protein synthesis and degradation rates ascertained from 14CO2 excretion in experiment 3 were clearly below those average values ascertained with 15N. The differences in the average heat production between the main periods of the 3 experiments were statistically insignificant.     

15N transamination in the administration of various tracer substances. 1. Whole body studies in rats

4 groups of 3 growing Wistar rats each were orally given either 15N methionine, 15N lysine, 15N glycine or 15N ammonia sulphate over 10 days. By means of measuring 15N, the 15N accumulation in the amino acids (AA) of the body protein, statements were to be made on the transamination of the individual 15N substances and thus their suitability as tracer substances for studies of N metabolism. None of the tested 15N AA achieved a proportionate labelling of all AA of the body protein. The AA used as tracer in each case showed the highest 15N labelling of all AA in the body. Of the amino 15N detected in the animal body, ca. 19% were found in Met after a 15N Met application, ca. 88% in Lys after a 15N Lys application and ca. 50% in Gly after a 15N Gly application. After the application of 15N ammonia sulphate ca. 42% of the body amino 15N are apportioned to the essential and ca. 58% to the non-essential AA. Thus this substance produces a more proportional labelling of the essential and non-essential AA of the body protein than 15N Gly. The following quotas of the 15N amounts applied were found in the AA of the animal bodies: tracer substance lysine 52%, glycine 32%, ammonia sulphate 24%, methionine 21%. After summing up the amino acid 15N amounts in the animal body, eliminating in each case the tracer AA and taking into account the molecular weight of the AA, there was a good agreement of the intensity of the accumulation of 15N in the individual AA, irrespective of the applied tracer substance: arginine, glutamic acid, cysteine and aspartic acid highest, threonine, phenylalanine and lysine lowest accumulation.     

Endogenous nitrogen metabolism in 15N-labeled swine. 1. Course of 15N-labeling and 15N excretion in urine and feces under 4 different diets

Four male castrated pigs (55-65 kg) either received a wheat--fish meal diet (1 and 2) or a wheat--horse bean diet (3 and 4) without straw meal supplement (1 and 3) or with a supplement of 20% DM partly hydrolysed straw meal to the DM of the ration (2 and 4). In order to investigate whether a 15N-labelling of the pigs is also possible with a protein excess in the ration, the animals 1 and 2 received 24.8 g and the animals 3 and 4 = 11.6 g crude protein/kg0,75 live weight. During a 10-day 15N-labelling 385 mg 15N-excess (15N') per kg0,75 were applied in a mixture of ammonia acetate and ammonia chloride in the feed. During the period of 15N-labelling the following quotas of the applied 15N-amount were incorporated: 1 = 10.2%, 2 = 7.2%, 3 = 18.7%, 4 = 14.4%. 15N-excretion in both TCA fractions of faeces showed a highly significant positive correlation to the increasing content of crude fibre in the 4 diets. The immediate 15N-incorporation into the TCA-precipitable fraction of faeces (from the 2nd of the beginning of the 15N-application onwards) proves that 15N enters the large intestine endogenously (probably as 15N-urea) and serves bacterial protein synthesis. Three days after the last 15N-application the pigs were killed. The following values of atom-% 15N' could be determined in the TCA-precipitable blood plasma and in the TCA-precipitable fraction of the liver: 1 = 0.18 and 0.19 resp., 2 = 0.22 and 0.27 resp., 3 = 0.22 and 0.23 resp. and 4 = 0.24 and 0.26 resp. The other examined organs and tissues showed smaller differences between the test animals. The following atom-% 15N' were measured in the TCA-precipitable fractions on an average of the 4 test pigs: kidney = 0.20, pancreas = 0.18, intestinal wall tissue, duodenum = 0.18, jejunum (beginning) = 0.17, jejunum (end) = 0.15, ileum = 0.15, caecum = 0.16, colon (beginning) = 0.15, colon (middle) = 0.14, colon (end) = 0.13, stomach (cardia) = 0.11, stomach (fundus) = 0.12, spleen = 0.13, heart = 0.12, skin = 0.07 and skeleton muscles = 0.06. The results show that the 15N-labelling of tissues and organs of pigs is also possible at a high level of protein supply by means of an oral application of 15N ammonia salts.     

Kinetic parameters of protein metabolism in rats on protein-free diets

16 male rats of 100 g live weight were given 50 mg of a mixture containing 15N labelled amino acids as a single dose within a protein-free feeding period. Following this the 15N excretion in faeces and urine as well as the development of the 15N excess in different organs and tissues were estimated over 3 days by slaughtering the animals within given 7 time intervals. Using a 3 pool model and the computer program for the interpretation of 15N tracer experiments by T?we et al. (1984), kinetic parameters such as the rate of protein synthesis, protein breakdown and the rate of reutilization were calculated. Despite a negative N balance (-41.8 mg N/d) under protein-free conditions the protein metabolism of the rat shows high dynamics characterized by a high flux rate (225 mg N/d) and a high rate of body protein synthesis (181 mg/d). The reutilization was 85%. Depending on time the 15N excess in the tested organs and tissues showed significant differences and seems to demonstrate that under these conditions protein synthesis mainly takes place in the most important organs (e.g. intestinal tract, liver). Under protein-free feeding conditions protein synthesis and protein breakdown of the whole body seems to be slightly increased in comparison to N balanced feeding conditions.     

Utilization of 15N marked urea by the laying hen. 2. Incorporation and metabolism of 15N for the synthesis of egg protein

In an N-metabolism experiment 3 colostomized laying hybrids received 2870 mg 15N-excess (15N') per animal in 6 days in the form of urea with their conventional feed rations. During the 8-day experiment the 21 eggs laid were separated into eggshell, white of egg and yolk. Weight, N-content and 15N' were determined of the individual fractions of the eggs. On an average of the 21 eggs 4.6% of the heavy nitrogen was in the egg-shells, 50% in the white of egg and 45.5% in the yolk. 2.8%, 4.5% and 5.5% (hens 1...3) of the 15N' consumed were detected in the eggs. The maximum 15N'-output in the white of egg was reached on the 6th day, whereas 15N'-output in the yolk showed a nearly linear increase in the time of the experiment. The results show that labelled nitrogen from urea is incorporated into the egg to a lower degree than after the feeding of 15N-labelled proteins and that the development of its incorporation into the white of egg and the yolk differ from that after the feeding of 15N-labelled native proteins.     

The effect of stanozolol on 15nitrogen retention in the dog.

The objective of the study was to determine the influence of either oral or intramuscular administration of stanozolol on nitrogen retention in dogs by using a non-invasive 15N-amino acid tracer technique. Ten healthy, intact, adult male sled dogs received either stanozolol tablets, 2 mg/dog PO, q12h, for 25 days (Group 1, n = 5) or an intramuscular injection of 25 mg of stanozolol on Days 7, 14, 21, and 28 (Group 2, n = 5). A 15N amino acid (5.27 mmol) was infused intravenously into each dog on Day 0 (before stanozolol treatment) and on Day 31 (after stanozolol treatment). Urine was collected by catheterization from each animal 3 times daily for 3 consecutive days. The 15N-urea enrichment in urine was determined by high-resolution mass spectrometry and the total amount of urea in the urine was determined. Both oral and injectable stanozolol resulted in significant (P < 0.05) increases in amino acid nitrogen retention compared to pretreatment values. Oral stanozolol increased nitrogen retention from 29.2 +/- 8.2% to 50.3 +/- 9.2%, while stanozolol injection increased nitrogen retention from 26.6 +/- 9.9% to 67.0 +/- 7.5%. The response to intramuscular administration was significantly greater than the response to the oral dosing regime. Stanozolol increases amino acid nitrogen retention in dogs, as has been previously observed in rats. This action of stanozolol may be beneficial in dogs under stress of surgical trauma and chronic disease.     

Studies on nitrogen metabolism in the large intestine of ruminants. 5. Metabolism of intra-cecally infused 15N-urea without and with fermentable material in heifers

Six heifers with a live weight of 215, 227 and 238 kg (experiment 1) and 220, 227 and 233 kg, resp. (experiment 2), were supplied with ileocaecal re-entrance cannulae, jugular venous catheters and bladder catheters. The ration consisted of 4 kg maize silage and 4 kg wheat straw pellets per animal per day. Up to 3.5 kg of the straw pellets, consisting of 73% wheat straw, 10% barley, 12% molasses, NPN salts and a mineral mixture, were consumed per animal per day. In a preliminary period 50% of the digesta flow was collected over 12 h/d on 5 consecutive days and stored in a deep-freeze. During the main trial the re-entrance cannula was disrupted and the flowing digesta were quantitatively collected at the end of the ileum; previously collected digesta were supplemented with 15N urea and every hour over 24 h infused into the caecal part of the re-entrance cannula. Between the 24th and 30th hours the digesta were infused without 15N urea supplement. In trial 2 the digesta were also supplemented with partly hydrolysed straw meal between the 1st and 30th hours (approximately 10% straw meal DM related to digesta DM). There were no differences between trials 1 and 2 with regard to the increase of atom-% 15N excess (15N') in the plasma urea. The 15N labelling decrease of the plasma urea N shows that the half-life is 7.9 h in trial 1 and 7.0 h in trial 2. The NH3 nitrogen in faeces was distinctly higher labelled in trial 2 after the supplement of straw meal than in trial 1. The total N in faeces was also twice as highly labelled as in trial 1. Atom-% 15N' in urine was significantly higher in trial 2 than in trial 1 between the 6th and 16th hours after the beginning of 15N urea supplementation. In the decrease curve of atom-% 15N' (after the 26th hour of trial) the values in trial 1 were generally higher than in trial 2. The higher bacterial protein synthesis in the large intestine in trial 2 (after the supplement of partly hydrolysed straw meal) had the effect that 13.6% of the supplemented 15N' was excreted in faeces by the 30th hour of trial, in contrast to this only 4.7% in group 1. Up to the 4th day after the 15N urea infusion these values increased to 16.2 and 6.1%, resp., only.     

Nitrogen metabolism in the large intestine of ruminants. 6. Metabolism of intracecally administered 14C and 15N urea in sheep with simultaneous intracecal doses of heat-damaged hay

Two wethers (28 kg and 33 kg) were supplied with ileocaecal re-entrance cannulae and received a straw pellet ration rich in crude fibre (70.5% straw, 12% chopped sugar beet, 10% cereals, 2% urea, 3% NH4HCO3 and 2.5% of a mineral mixture). In a preliminary period 50% of the digesta flow was collected on 6 successive days for 18 h each. An amount of digesta sufficient for 24 h was apportioned for hourly application and stored at a temperature of -20 degrees C for the main trial. In the main trial the two animals received intracaecally the collected digesta with a supplement of ca. 6 g hay damaged by heat/kg LW(0.75) in hourly portions over 24 h (hay made up ca. 15 and 20% resp. of the DM amount). In addition, each digesta sample was supplemented with 14C and 15N labelled urea (19.7.10(6) Bq 14C urea and 364 mg 15N excess from 15N urea). About 9% of the applied 15N amount was microbially utilized; the utilization quota was thus lower than after the application of partly hydrolyzed straw meal (16% in a previous trial). The 14C activity from 14C urea was quickly eliminated in the form of CO2 in the respiratory gases (at the 18th hour after the end of the infusion 70% excreted as CO2). The half-lives for the urea resulting from the semi-logarithmic decrease of the atom-% 15N excess in the blood plasma were 7.9 and 7.7 resp. 23% and 34% resp. of the applied 15N excess were excreted in urine. The excretion of radioactive carbon in urine, however, was at 2.8% and 4.3% resp. of the applied amount very low 120 h after the beginning of the trial (96 h after the end of the infusion). On the whole one can conclude from this trial that hay damaged by heat has only a low stimulating effect on microbial activity in the large intestine.     

Action of proteinase inhibitors. 4. Effect of short-term application of chymostatin on nitrogen metabolism in the digestive tract and protein metabolism in tissue

Chymostatin is an effective inhibitor of intracellular proteinases in vitro. In the present experiment male rats were injected intraperitonealy during a 3 days period twice daily with a solution containing 0,9 mg Chymostatin per 100 g live weight. Reference animals received a control injection containing the same solvents but no chymostatin. During this period a daily nitrogen balance was made and metabolic faecal nitrogen and true digestibility of nitrogen were estimated using 15N-labelled animals. Furthermore, apparent biological half lives of proteins in liver and intestinal tissues were determined following the decay curves for radioactivity in proteins 48 hours after injection of L-[5-3H]-arginine und L-[guanido-14C]-arginine. The fractional rate of protein synthesis in tissues was measured by a 6 hours continuous infusion technique with L-[U-14C]-tyrosine and L-[U-14C]-leucine. Among the parameters estimated only the apparent biological half lives of proteins in liver and intestinal tissues were influenced by chymostatin. However, the prolonged half lives seemed to be rather an effect of an increased reutilisation of amino acids resulting from the intracellular protein breakdown than a decreased rate of protein degradation. The in vivo effect of the proteinase inhibitor was by far inferior compared with the action in vitro. Factors like distribution, degradation and excretion of the inhibitor could be responsible for the moderate in vivo action of chymostatin.     

Dynamics of amino acid and protein metabolism in laying hens after the administration of 15N-labeled wheat protein. 2. 15N excretion with nitrogen and basic amino acids of the feces

Over 4 days 12 colostomized laying hens received together with a commercial ration labelled wheat with a 15N excess (15N') of 14.37 atom-%. The labelling of the basic amino acids amounted to 13.58 atom-% for lysine, to 14.38 atom-% for histidine and to 13.63 atom-% 15N' for arginine. 3 animals each were butchered 12 h, 36 h, 60 h and 108 h resp. after the last application of 15N. The heavy nitrogen in the total N and in the N fraction of non-protein origin as well as in the basic amino acids in faeces was daily determined for the individual hens in the total experimental period. On average the crude protein of faeces contained 5.45 % lysine, 2.32% histidine histidine and 3.68% arginine: the protein of faeces correspondingly contained 5.43% lysine, 2.32% histidine and 4.07% arginine. The quota of TCA soluble N in the total N of faeces amounts to one third on the 3rd und 4th days of the experiment and that of 15N' to 28%. The average atom-% 15N' of the protein fraction is 3.48 atom-% 15N' and that of the non-protein N fraction of faeces 2.93 atom-% 15N'. The apparent digestibility and that of the non-protein N fraction of faeces 2.93 atom-% 15N'. The apparent digestibility of the 14N of the ration on average amounts to 82.8% and that of the wheat 15N' to 87.5%. The average quota of the basic amino acids in the protein compounds of faeces amounts to 70.9% for lysine 15N', 73.7% for histidine 15N' and 70.3% for arginine 15N'. The digestibility of the 15N labelled amino acids amounts to 80.4% for lysine, 90.8% for histidine and 90.2% for arginine.     

Dynamics of amino acid and protein metabolism of laying hens after the administration of 15N-labeled wheat protein. 8. 15N-labeling of nitrogen and 15N incorporation into the amino acids of the liver

Over 4 days 12 colostomized laying hens received, together with the ration, 36 g wheat with 14.37 atom-% 15N excess (15N'), The basic amino acids were nearly equally labelled. Three animals each were butchered after 12 h, 36 h, 60 h, and 108 h after the last 15N' application. Emission spectrometric determination of 15N' in the liver and in the amino acids was carried out. In addition, atom-% 15N' was determined in the free amino acids and the peptides. The labelling in the liver 12 h after the last 15N' application amounted to 1.75 atom-% 15N' and decreased after 108 h to 0.81 atom-% 15N'. The average TCA precipitable 15N' quota in the total 15N' amounted to 81.4% and was nearly identical at all measuring times. The arginine 15N' amount in the liver was twice as high as that of lysine 15N'. In dependence on the period of time after the last 15N' application the decrease in the labelling of the free arginine is considerable in comparison to free lysine. At the first measuring time (12 h) it was 1.69 atom-% 15N' and at the last one (108 h) 0.57 atom-% 15N'. Based on the results of 15N' labelling of the peptides in the liver further, more detailed series of experiments for studies of the peptide metabolism in the liver should be carried out.     

Nitrogen metabolism in the large intestine of ruminants. 1. Metabolism of i.v. infused 15N-urea without additional carbohydrate supply to the large intestine

The experiments were carried out on 3 bulls (body weight: 172, 229 and 193 kg), equipped with ileo-caecal cannulas and with catheters in the jugular veins on both sides. The offered pelleted ration consisted of straw 73%, molasses 12%, cereals 10%, ammonium hydrogen carbonate 3% and urea 2%. Feed intake amounted to about 3 kg per animal and day. During a preliminary period of 5 days 50% of ileal digesta were collected for 12 hours daily, deep-freezed and stored. In the main experiment 15N-urea was infused intravenously for 24 hours. During this period and during the following 6 hours ileal digesta were collected and replaced by precollected, unlabelled digesta. The urea metabolism was estimated by the 15N-labelling of blood urea, by the 15N-excretion via faeces and urea, by the appearance of 15N in ileal digesta and by the 15N-labelling of faecal NAN, NH3 and bacterial fraction. The time course of the 15N-labelling of plasma urea during infusion can be described by an exponential function. The urea flux rate was estimated from the calculated plateau value. The flux rate for the 3 animals was 28.8, 30.7 and 34.8 mumol per minute per kg0.75, respectively. During the infusion of 15N-urea 1.0-2.4% of the infused amount of 15N' appeared in ileal digesta, half of it in the TCA precipitable fraction. At the same time the 15N-labelling of faecal NH3 increased sharply, however, the 15N-labelling of the faecal bacterial fraction was smaller by one order of magnitude. Deficiency of fermentable substrates and problems of inhomogenity of the NH3 pool are supposed as reasons for this result. 30 to 50% of the urea flux entered the digestive tract, the direct entry of urea into the large intestine seems to be only very low.     

Endogenous nitrogen metabolism in 15N-labeled swine. 3. Endogenous excretion of 15N- and 14C-labeled secretions following a 14C-leucine injection

Four pigs (59-65 kg live weight) were labelled over a period of 10 days with 15N in the feeding of a fishmeal diet (1), a fishmeal diet + partly hydrolysed straw meal (2), a horse bean diet (3) and a horse bean diet + partly hydrolysed straw meal (4). After a 24-hour fasting the animals were provided with simple cannulae in the upper part of the small intestines. After a fasting period of 24 h all four pigs received a 14C-leucine injection and the cannula secretion was collected in the subsequent 24 h. After the feeding of the diets without straw meal supplement (1 and 3) there were distinct differences in the secretion in comparison with the feeding with straw meal supplements (2 and 4) despite the long fasting period (48-72 h). 14C-activity could already be detected in the TCA-precipitable fraction of the secretion after 3-6 min of the injection in 1 and 3 but only 20 to 25 min after the 14C-leucine injection in 2 and 4. The specific 14C-leucine activity of the TCA-soluble fraction of the secretion was, after the straw meal supplementation to the fish meal diet, 15 times higher 25 min after the 14C-leu-injection, 25 times higher after 70 min, 36 times after 2 h and 1.8 times after 4 h than without straw meal supplementation. For all four diets a specific correlation (r = 0.96) could be ascertained between the increase of 14C-activity/mg N in the TCA-soluble fraction and the increasing crude fibre content in the diet between 25 and 180 min after the injection. Furthermore, a distinctly decreased N-secretion/h could be ascertained (correlation coefficient r = 0.84) with the increasing crude fibre content in the diet. The influence of the crude fibre on the parameters mentioned is seen in the changed osmotic conditions in the secretion, which may be caused by the changed regulation by hormones of the gastro-intestinal tract. The atom-% 15N' in both TCA-fractions of the secretion underwent big rhythmic variations, which is explained by different ratios of the components pancreatic juice, bile, and intestinal juice.     

Studies on the 14C-15N-acetamide turnover in sheep. 2. Studies on 15N turnover and comparative studies on the 14C turnover]

Three fistula sheep with average weights of 52.2 kgs were given 37.9 g of 15N and 14C labelled acetamide (= 1.09 mg 15N' and 0,95 mCi14C) which were administered directly through the fistula. The half-life period of 15N retention in the ruminal fluid (TCE soluble portion) was found to be 4 hrs. 18 hrs after 15N administration increasing amounts of 15N were carried back to the rumen by way of the rumino-hepatic circulation. The 15N concentration in the blood (TCE soluble portion) rapidly increased up to a peak value and, from 3 hrs after isotope administration, the 15N concentration was found to decline continuously, with a slight discontinuation at about the 10th hr of experiment. The rate of 15N incorporation into the protein fraction (TEC soluble portion) of the blood was delayed by 4 hrs, relative to the rate of 15N incorporation into ruminal proteins. An average of 43.1% of the administered amount of 15N was excreted in the urine within 7 days. Up to the 4th day of experiment the half-life period of urinary 15N excretion was 19 hrs. An average of 15% of the administered total amount of 15N was excreted in the faeces. In this process, the peak values in both TCE fractions were observed to occur on the 2nd day of experiment. The proportion of isotope in the TCE soluble fraction was found to increase continuously compared with the total amount of the isotope excreted in the faeces. Isotope concentrations between 0.03 and 0.13 atom% of surplus 15N were found in organ and muscle tissues of a sheep that had been slaughtered 7 days after administration of the isotope. The results obtained are discussed on the basis of comparisons made with the analogous behaviour of 14C activity.     

Nitrogen metabolism in the large intestine of ruminants. 7. Metabolism of intracecally-infused 15N urea with supplement of starch in bulls

Three bulls with an average live weight of 228 kg were fitted with ileo-caecal reentrant cannulas for the experiment. The rations were composed of 3 kg maize silage and 3 kg wheat straw pellets per animal and day. In a previous period 50% of the digesta was collected over 12 hours and stored deep-frozen. In the main period the digesta flow was interrupted for 30 hours. The digesta flow was collected quantitatively. In the caecal part of the re-entrant cannula previously collected digesta and starch (over 30 hours) as well as 15N urea (over 24 hours) were supplemented. The amount of starch corresponded to about 10% of the DM of the digesta. Analyses of the urine, faeces, ileum digesta and blood plasma were carried out. The quota of starch clearly stimulates bacterial processing in the large intestine so that 20.5% of the supplemented 15N was excreted in faeces within 24 hours. 91.2% of the 15N in the faeces was localised in the bacteria fraction. Individual differences of the animals distinctly show the connection between the excretion of the 15N in faeces and urine. A decreased isotope excretion in faeces of 17.2% for animal 3 in contrast to the 23% for animals 1 and 2 showed an increased elimination of the 15N through the kidney with 32.7% instead of 25.2%. The largest proportion of the ileum digesta, i.e. 46%, can be localised in the 15N urea fraction; the NH3-fraction is also distinctly labelled. With time progressing, the 15N quota flowing from the rumen to the small intestine increases.     

Studies on the protein and amino-acid metabolism of laying hens using 15N-labelled casein. 15N-incorporation into N-fractions and amino acids of various parts of the body

Four colostomized Leghorn hens were fed, during 6 days, 15N-labelled casein as sole protein source. Two animals were slaughtered 48 hours, the other two 144 hours after the last 15N-application. The share of TCE-soluble N in total N averaged 16% for the body parts analysed, i.e. meat, bone, liver, kidneys, oviducts, residual viscera and other. The variation of the lysine, histidine and arginine levels in the body parts ranged from 3.6 to 7.9 g, 1.1. to 3.7 g and 6.4 to 7.4 g in 16.7 g hydrolysate N, respectively. Except for feathers, the analysed body parts contained and excess amount of heavy nitrogen. The degree of labelling was found to depend on the time of slaughtering after the tracer application. In the liver and in the oviduct being metabolically active organs, the 15N-excess in the total N fraction decreased by 45% between the 2nd and the 6th days after 15N-feeding, whilst in the meat it went down by 20%. The decline of the 15N-concentration in the TCE-soluble N compounds was faster than in the total N-fraction. Out of the body samples analysed, the lysine of the liver having 0.26 atom % 15N-excess was found to be more strongly labelled in hens 1 and 2. The amino acid arginine reached about the same level of labelling, the 15N-frequency of histidine being the lowest.