15N-labelled lysine in colostomized laying hens. 4. Incorporation of 15N-lysine into various amino acids of egg yolk and white

Each of three colostomized laying hens received per os 0.2% L-Lysine with 48 atom-% 15N-excess (15N') labelled in alpha-position in addition to a pelleted laying hen ration of 120 g over a period of 4 days. On the following 4 days they received equal amounts of unlabelled lysine. The eggs laid during the 8 days of the experiment were separated into the white of egg, the yolk and the eggshell, and the to and heavy nitrogen in the individual fractions were determined. Above that, 17 amino acids and their atom-%15N' were determined in the 19 samples of the white and yolk of egg. Of the total 15N' from the lysine fed in the 4 days, 10.1% were found in the yolk, 10.5% in the white of egg and 1.1% in the eggshells of the eggs laid during the 8 days of the experiment. 85% of the total amino acid-15N' of the yolk and 86% of the white of egg detected to be lysine-15N'. The 15N'-amount of the other 16 amino acids was mainly concentrated in the two acid and basic amino acids. Approximately 50% of the non-lysine 15N' in the egg are contained in aspartic acid, glutamic acid, histidine and arginine. A very low incorporation of the labelled lysine only could be detected in the aromatic and sulphur-containing amino acids from both the yolk and the white of egg. 43% of the 15N' was detected in the 10 essential and semi-essential (except lysine) and 57% in the 6 non-essential amino acids of the yolk and 52% and 48% resp. of the white of egg. One can summarize that the incorporation of 15N' into the egg shows the same development as that of the labelled amino acids of the wheat protein and that 15% of the lysine-15N' could be detected in the 16 other amino acids.     

Dynamics of amino acid and protein metabolism of laying hens after the administration of 15N-labeled wheat protein. 3. Incorporation of 15N into egg shell, egg white and egg yolk

12 colostomized laying hybrids received a ration meeting their requirement of 15N labelled wheat with a 15N excess (15N') of 14.37 atom-% over 4 days. The 15N' of the total ration amounted to 4.47 atom-%. Each hen consumed 135 mg 15N' per day. On another 4 days the same rations with non labelled wheat were fed. The 12 hens laid 56 eggs during the 8 days of the experiment. They were divided into egg shell, white and yolk of egg. In addition, the protein of the white and yolk of egg was precipitated with trichloric acetic acid (TCA) and the nitrogen in these fractions was determined. On average of the 56 eggs, the N quota in the egg shell was 5.3%, in the white of egg 49.1% and in the yolk 45.6%. The atom-% 15N' in the shells of the eggs laid on the first day of the experiment was on average 0.21, whereas only 0.03 and 0.02 atom-% 15N' resp. could be detected in the white and yolks of the eggs. On the first day after the last 15N application the atom-% 15N' in the egg shell and the white of egg was highest and amounted to 2.33 and 2.43 atom-% resp. The highest value of 1.83 atom-% 15N' in the yolk was ascertained 3 days after the last 15N intake. The mean quota of TCA-precipitable N in the white of egg is 97.6% and in the yolk 94.4% of the respective total N. The atom-% 15N' in the non-protein N-compounds was higher than in the protein fractions.     

Metabolism of colostomized laying hens with 15N-labeled wheat. 2. Incorporation of the labeled nitrogen into fractions of the egg]

The colostomised hens received 15N-labelled wheat. For a period of 8 days the incorporation of the 15N excess (15N') into the albumen, the yolk and the egg-shell was measured. In addition to that, the values for atom-percent of 15N' in the amino acids lysine, histidien and arginine of the albumen and the yolk were ascertained. The egg-shell and the albumen were labelled shortly after the beginning of the experiment. In both fractions the atom-per cent of 15N' rises more quickly than in the yolk. After the withdrawal of the labelled feed the decrease in the yolk was slower than in the egg-shell and the albumen. Concerning the atom-per cent of 15N' there is hardly a difference between the total N and the amino acids. Despite the different supply with amino acids, the relation of histidine 15N' : lysine 15N' : arginine-15N' in the yolk and the albumen remains largely constant. It is 1 : 2 : 3 and corresponds to the quantitative relation of the N content of the three amino acids in the egg protein. The utilisation of 14N and 15N' of the wheat lysin for the egg synthesis amounts to approximately 50%. There are considerable differences between the utilisation of 15N' of the histidine and 15N' of the arginine and the 14N of the two dietary amino acids.     

Utilization of 15N-labeled urea in laying hens. 7. 15N-incorporation into the amino acids of different muscle types

3 colostomized laying hybrids received 1% 15N labelled urea with 96.06 atom-% 15N excess (15N') with a commercial ration over a period of 6 days. After the application of the same ration with unlabelled urea on the following 2 days the animals were butchered. In the muscles of the breast, the leg and the heart, the labelling of total nitrogen and the incorporation of urea 15N' into 15 amino acids of the 3 different kinds of muscles were ascertained. On average, significant differences could be ascertained between the atom-% 15N of the muscles of the skeleton and those of the heart. The 15N' of the breast and leg muscles was 0.25 and 0.34 atom-% resp.; that of the cardial proteins 0.71 atom-% 15N'. The incorporation of urea 15N into the basic amino acids is low and varies both between the kinds of muscles and between the amino acids. On average the highest level of labelling was found among the essential amino acids valine, isoleucine and leucine; the average atom-% 15N' for the muscles of the breast is 0.13, of the leg 0.17, and of the heart 0.27; the 15N' quota of branched chain amino acids in the total 15N' of the respective muscle is accordingly 6.0%, 5.0% and 4.5%. The non-essential amino acids, particularly glutamic acid, are more highly labelled in the muscles than the essential ones. A 15N' for glutamic acid of 0.24 atom-% in the breast muscles, of 0.27 atom-% in those of the legs and of 0.64 atom-% in the heart muscle could be detected. The average quota of the 15N' of these acid amino acids in the 15N' for breast, leg and heart muscles is 7.4, 6.2 and 6.7 resp. The quota of the 15N' in the 6 non-essential amino acids in the total 15N' in all 3 kinds of muscles is approximately two thirds and in the 9 essential ones one third of the total 15N'. Although the results show that there is a certain incorporation of 15N' from urea into the amino acids of the muscle proteins, their contribution to meeting the demands is to be considered irrelevant.     

Utilization of 15N-labeled urea in laying hens. 8. 15N-incorporation in the amino acids of the oviduct

3 colostomized laying hybrids received orally with a conventional ration 1% urea with 96.06 atom-% 15N excess (15N'). over a period of 6 days. In the period of the experiment every hen consumed 2.87 g 15N'. After another 2 days, on which they received conventional feed urea, the animals were butchered. 15N' was determined in the total N and in 15 amino acids of the oviduct. Of the 15 amino acids the labelling of glutamic acid, glycine and serine was highest and on average amounted to 0.80, 0.66 and 0.67 atom-% 15N'. In lysine and arginine only 0.10 and 0.11 atom-% 15N' could be detected. The amino acid N with natural isotopic frequency amounted to a quarter for the basic amino acids, a tenth for the branched chain ones and for the non-essential ones (glutamic acid, aspartic acid, serine, glycine, alanine, proline) a third of the total oviduct 14N, The average quota of 15N' is only 3.6%, that of the branched chain amino acids 4.5 and that of the non-essential ones 21.1%. Consequently, the 15N' of the urea is mainly used for the synthesis of the non-essential amino acids of the oviduct.     

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.     

Dynamics of amino acid and protein metabolism of laying hens after administration of 15N-labeled wheat protein. 5. Incorporation of 15N into the blood fraction and its amino acids

12 colostomized laying hens which received 15N labelled wheat over 4 days were butchered 12 h, 36 h, 60 h and 108 h (3 animals each) after the last 15N application. The intake of 15N excess (15N') from the wheat amounted to 540 mg 15N' during the application period. The 15N' in the blood plasma decreased after the last 15N' application from 0.76 atom-% to 0.55 atom-% after 108 h, the labelling of the corpuscular components at the same measuring points increased from 0.28 to 0.50 atom-% 15N'. 96.6% of the plasma 15N' and 93.8% of that in the corpuscles is precipitable in trichloric acetic acid. The atom-% 15N' of histidine in the total blood remained unchanged in dependence on the butchering time. The 15N amount in lysine and arginine and that in the non-basic amino acids decreased inconsiderably in the period between 12 h and 108 h after the last 15N' wheat feeding.     

Studies using labeled urea on lactating ruminants. 4. Incorporation of 15N-urea into the basic amino acids of goat milk]

Goats were used in a trial to investigate the rate of 15N incorporation from labelled urea into the basic amino acids of the milk (excess of 15N = 24.9 atom%). As early as 20 minutes after administration of the first dose of 15N urea nitrogen labelling in arginine and lysine was observed. The highest level of 15N labelling was noticed for lysine and arginine 24 hrs after start of the trial. The peak values of labelling for histidine were essentially lower. Reference is made to peculiarities of the ruminal metabolism of histidine.     

Absorption and utilization of amino acids infused into the cecum of growing pigs. 2. 15N-labeled lysine

Over a period of 4 days 15N-labelled lysine was infused into two growing female pigs (live weight approximately 50 kg) through a caecal cannula. The feeding was restrictive (1,400 g dry matter/day) and, with regard to lysine, it didn't meet the requirement. In a 7-day experiment the N- and 15N-content was measured periodically in the excretions (feces and urine), in various fractions of the blood and in selected slaughtering samples. From the infused 15N 3-5% are excreted as lysine in feces, another 5% are in other amino acids of the bacteria protein. The disappearance rate of 15N' from the large intestine makes greater than or equal to 90%. The biggest part of this 15N (78-88%) is excreted with the urine in form of 15N-urea. Obviously the infused amino acid is decomposed to NH3 in the large intestine and then absorbed. The absorbed ammonia is changed into urea in the ornithine cycle and excreted in urine. The recovery rate of the 15N infused as 15N-lysine is 93 and 84% resp. Incorporation of 15N in to serum protein or other body protein could not be detected so that the remaining difference of 7-16% cannot necessarily be interpreted as incorporation rate of 15N into the body protein. Under practical conditions the maximal utilisation of lysine from the feed in the large intestine is 1.6% and should thus be without importance.     

Utilization of N15-labelled urea in the laying hen. 6. Incorporation in the liver and kidneys

In an experiment 3 colostomized laying hybrids received a normal ration containing 1% 15N labelled urea with 96.06% atom-% 15N excess (15N') over six days. Subsequently the same ration with unlabelled urea was given over 2 days, after which the animals were butchered. In the kidneys the 15N' amounted to 1.1 atom-% and 1.8 atom-% in the liver. The TCA soluble N fraction and the ammonia were more highly labelled than the total N. Lysine, histidine and arginine were lowly labelled in the kidneys. This also applies to the liver with the exception of histidine. In the branch-chained and aromatic amino acids of the liver the 15N' was between 0.2 and 0.3 atom-%. The highest labelling of non-essential amino acids was found in glutamic acid with 0.9 atom-% 15N' and aspartic acid with 1.1 atom-% 15N'. The evaluation of the amino acid in the liver showed that the 6 non-essential amino acids account for two thirds of the total amino acid 15N' whereas the 9 essential ones account for one third of the amino acid 15N' only.     

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.     

Endogenous nitrogen metabolism processes in 15N-labeled swine. 2. Fecal excretion of amino acids and 15N-labeled amino acids with diets differing in crude fiber content

Four pigs were labelled with 15N-ammonium salt over a period of 10 days in the feeding of a fishmeal diet, a fishmeal diet + partly hydrolysed straw meal, a field bean diet and a field bean diet + partly hydrolysed straw meal. The 14N-amino acids and the 15N-amino acids excreted in faeces showed highly significant correlation coefficients with the increasing content of crude fibre in the diets, which amounted to 3.0, 5.3, 10.0 and 12.1% in the DM. The following sequence was established for the growth angle (tan alpha) of the essential 14N-amino acids: Leu, Lys, Arg, Thr, Phe, Ile, Val, His and of the 15N-amino acids: Lys, Arg, Val, Leu, Ile, Thr, Phe and His. As Lys, His and Thr cannot incorporate 15N in transamination reactions in the intermediate metabolism, their level of labelling was considerable in case of diet 4. Nevertheless, tan alpha is highest for 15N-Lys and lowest for 15N-His. This means that His in contrast to Lys, parallel to increased synthesis, is also increasingly decomposed in the large intestine. In contrast to this, proline was not labelled with 15N even with the highest content of crude fibre in the diet. Despite this, 14N-proline excretion, next to glutamic acid, increased most with the growing content of crude fibre in the diet. Due to the hydrophilic character of glutamic acid and the increased water influx in the large intestine and the increased content of crude fibre in the diet, a growing proline transport parallel to the increased influx of crude fibre and water must be assumed. If the growth angle tan alpha for the excretion of 14N-amino acids is ascertained regressively for a crude fibre content of diet of 10%, one can prove from the proportion of the amino acids and a comparison from literature for faecal bacteria and ileum digesta that the amino acid composition for this measuring point largely corresponds to that of bacteria protein.     

The utilization of crude proteins from 15N-labeled straw by broiler hen breeds

In an experiment with 10 colostomized broiler breeding hens the digestibility of wheat straw meal labelled with 15N and the incorporation of heavy nitrogen into individual body fractions were studied. The straw meal contained a 15N excess (15N') of 14.88 atom-%. Before the experiment part of the straw meal was treated with gamma-rays (2.0 MGy). 5 animals each received in addition to the basic ration 30 g untreated (group I) and irradiated 15N labelled straw meal (group II). The apparent 15N' digestibility amounted for untreated straw meal to 49% and for irradiated straw meal to 46% (p less than 0.05). The labelling of uric acid amounted to 0.25 atom-% 15N', urine with 0.30 atom-% 15N' was more highly labelled (p less than 0.05). On an average of both groups the same labelling of 0.18 atom-% could be detected in the follicles and the liver, whereas 0.17 were ascertained in the blood plasma and 0.16 atom-% 15N' in the oviduct. 18% of the digested 15N' were incorporated in the muscles. There were only insignificant differences between the two groups with regard to the incorporation of 15N'. In conclusion one can say that the apparent digestibility of straw protein is 47.5% and that the utilization of the absorbed N is about the same as that of wheat protein.     

Dynamics of amino acid and protein metabolism of laying hens after administration of 15N-labeled wheat protein. 7. Incorporation into the amino acids and peptides of the gastrointestinal tract and the pancreas

In a 15N labelling experiment 12 colostomized laying hens received 15N labelled wheat with 14.37 atom-% 15N excess (15N') over 4 days. 3 hens each were butchered after 12 h, 36 h, 60 h and 108 h after the last 15N' application. The gastro-intestinal tract was divided into 3 parts (oesophagus with crop and gizzard as well as glandular stomach, small intestine, large intestine). These parts and the pancreas were hydrolysed with 6 N HCl and the individual basic as well as the sum of acid and neutral amino acids were determined in the hydrolysed fractions. In addition, the amino acids and peptides were determined in the TCA soluble N fraction. The atom-% 15N' was determined in the individual amino acid and peptide fractions. The labelling of the basic amino acids in the individual tract segments was lower than in the acid and neutral amino acids. In comparison to the peptides, a higher atom-% 15N' could be determined in the free amino acids.     

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.     

Utilization of 15N-labeled urea in laying hens. 9. Bones, feathers and 15N-incorporation in the rest of the body

For studying the incorporation of the 15N labelled urea into individual organs and tissues 3 colostomized laying hens were butchered after they had received 1% urea (96.06 atom-% 15N excess) with a high quality ration over a period of six days and after receiving conventional urea for another two days. Nitrogen and atom-% 15N excess (15N') were determined in the bones, the feathers and the remaining body (skin, lungs and windpipe, head with comb and wattle, lower leg without bones and with skin, pancreas and fatty tissue). In the remaining body the atom-% 15N' was determined in 15 amino acids. The labelling in the remaining body and the bones was approximately the same and averaged 0.37 atom-% 15N'. A significantly lower relative frequency could be detected in the feathers. The lysine of the remaining body contained only 0.04 atom-% 15N', tyrosine 0.06, histidine and arginine 0.07. The phenylalanine and proline molecules were labelled with 0.11 atom-% 15N'. Most 15N' was incorporated in serine and glutamic acid with over 0.30 atom-%. In the six non-essential amino acids out of the 15 amino acids studied, 48.6 of the non isotopic nitrogen of the total N of the remaining body and 70.7% of the isotopic nitrogen of total 15N' could be detected. Consequently the urea-N is mainly used for the synthesis of the non-essential amino acids, with its utilization being very low.     

Evaluation of 15N marked urea in the laying hen. 4. Incorporation of 15N in blood, its fractions and follicles in various developmental stages

In order to study the utilization of urea in poultry, 3 colostomized laying hybrids were orally supplied with a traditional ration supplemented with 1% 15N'-labelled urea with a 15N excess (15N') of 96.06 atom-% over a period of 6 days. After another 2 days on which the hens received the same ration with unlabelled urea, they were butchered. The atom-% 15N' of the blood on an average of the 3 hens was 0.64, of the plasma 1.40 and of the corpuscles 0.47. The TCA-soluble fraction of the blood had an average 15N' of 1.14 atom-%; the 15N amount is 9.7% of the total amount of 15N in the blood. The amount of 15N' in the urea in the blood was 6.8 atom-%. This shows that the absorbed urea is decomposed very slowly. The quota of 15N' in the basic amino acids from the total 15N' of the blood plasma is only 0.3% and that of the corpuscles 2.2%. The average 15N' of the mature follicles is 2.39 atom-% whereas the smallest and the remaining ovary contain 1.12 atom-%. The labelling level of lysine in mature egg cells was, in contrast to this, only 0.08 atom-% 15N' and in infantile follicles 0.04 atom-% 15N'. 1% of the 15N' quota is in the follicles and the remaining ovary. Of the basic amino acids, histidine is most strongly labelled. The as a whole lower incorporation of the 15N from urea into the basic amino acids shows that the nitrogen of this compound can be used for the synthesis of the essential amino acids to a low degree only.     

Metabolism of colostomized laying hens with 15N-labeled wheat. 3. Incorporation of 15N into and its distribution over follicles of various stages of development, the restovar and the oviduct

Of colostomised hens fed with labelled wheat protein the atom-% 15N-excess (15N') was ascertained in the bigger follicles, the restovar and the oviduct from the total N, the lysine-N, histidine-N and arginine-N. The labelling of the basic amino acids decreases the smaller the follicles are. In contrast to the yolk, in which a constant relation between the three amino acids was found, the relations were inconsistent and typical of the individual hens. In the atom-% 15N' in the three amino acids in the restovar and the oviduct there were greater differences between the hens. In the lysine, histidine and arginine we found, on an average, 21.2% of the total labelled N of the follicles.     

Dynamics of amino acid and protein metabolism in laying hens after the administration of 15N-labeled wheat protein. 10. 15N-labeling of crude protein and amino acids of the oviducts

Over a period of 4 days 12 colostomized laying hens daily received 36 g coarse wheat meal containing 14.37 atom-% 15N excess (15N') together with a conventional ration. After the homogenisation of each oviduct N and 15N' were determined. After the precipitation with TCA the 15N' of the amino acids was analysed in both the precipitate and the supernatant. In addition, the free amino acids and the peptides were determined in the TCA soluble fraction. The atom-% 15N' in the total N and in the non-basic amino acid N showed a parallel decrease; it diminished from 1.75 atom-% 15N' to 0.64. Of the three basic amino acids, lysine shows the lowest labelling at all four measuring points. The quotas of non-basic amino acid 14N and 15N' in the total 14N and 15N' of the oviduct are the same and amount to 53%. In contrast to this, the quota of the 14N of the basic amino acids in the total 14N of the oviduct only amounts to 21.6% and that of 15N' only to 15.4%. The average atom-% 15N' of the free amino acids 12 h after the last 15N application is 1.54 and is considerably above that of the peptides with 1.15 atom-% 15N'. 36 h after the last 15N application the ascertained value of 1.25 is identical in both fractions. The labelling of the free amino acids decreases more quickly than that of the peptides the more time has passed after the last 15N application.