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.     

Methodic recommendations for the determination of whole body protein synthesis rates in tracer studies

Methodical recommendations are suggested--predominantly for laboratory and small animals (rats and young chickens)--for the determination of parameters of the protein metabolism of the whole body after single doses of a mixture of 15N labelled amino acids by means of the determination of the temporal course of cumulative 15N excretion in urine and the assessment of the tracer kinetic data in a compartment model. These recommendations are to make it possible to carry out purposefully such experiments under comparable conditions. The advantages of this method are: the non-invasive character of the method; the possibility of repeating the experiment with the same animal; the adaptability to other methods of investigation (e.g. measuring energy metabolism); the relatively low expenditure of labour and requirement of test animals; the relatively good reproducibility of the method. Thus this method is a good supplement to the flooding and permanent infusion methods and should be used wherever the determination of parameters of the protein metabolism of the total body is sufficient.     

Studies with 15N-labeled lysine in colostomized hens. 1. 15N excretion in urine

0.2% L-lysine with an atom-% 15N-excess (15N') of 48% were given per day through a throat probe in an experiment to three colostomised laying hybrids in addition to a pelleted ration of 120 g per animal and day. In the following 4 days unlabelled L-lysine was given. As the labelled lysine was given three times a day, the development of 15N'-excretion could be pursued. 80 minutes after the 15N'-lysine dose a distinct atom-% 15N' could be detected in urine. 6 hours after the 15N'-application we rediscovered 2.9% (hen 1), 4.2% (hen 2) and 2.7% (hen 3) of the applied 15N'-amount in urine. 8 days after the beginning of the experiment the excretion of 15N' in urine averaged 17.5% of the consumed 15N'-amount. 44% of the nitrogen in the ration, however, was excreted in urine. The results showed that the lysine-N is excreted to a considerably lower extent in urine than the nitrogen in the remaining ration.     

Whole body protein turnover of growing rats in response to different dietary proteins--soy protein or casein.

Whole body protein turnover was studied in growing rats fed restrictively on isoenergetic (GE 17.6 MJ/kg DM) and isonitrogenous (104 g CP/kg DM) diets based on soy protein isolate or casein supplemented with D,L-methionine. During each of the three separate experiments six male Fischer rats per group were housed individually in metabolic cages at 24 degrees C. Prefeeding of both dietary groups up to similar body weights at the start of the main experimental periods (105-134 g) lasted up to 16 d for casein-fed rats and up to 30 d for the soy protein-fed rats. Following the energy and nitrogen balance periods whole-body protein synthesis was estimated by the end-product method using a single tracer dose of a mixture of 15N-labelled amino acids. Fractional protein accretion rate [% of the protein pool accreted per day] was significantly lower in soy protein-rats than in casein-fed rats in all three experiments whereas fractional synthesis rate was not significantly lower. Therefore, protein breakdown subsequently calculated as the difference between synthesis and accretion showed a tendency towards higher values in this group. In soy protein-fed rats also a tendency towards higher excretion of 3-methylhistidine as a marker of myofibrillar protein breakdown was observed. It is concluded that increase in lean tissue growth resulting from improved protein quality is brought about by changes of both rates, by small increase of protein synthesis and by reduced rate of body protein breakdown.     

Infection studies in laboratory mice with erysipelas and coli bacteria after the administration of biotechnical substances

Chlormadinone-acetate, human chorionic gonadotrophin, oestrophan, and zinc-metallibur, all of them substances used for biotechnological indications, were tested in generation experiments for their bearings on the immune status. The tests were applied to untreated descendants of biotechnologically treated mothers. Though the model animal experiments were continued through 6 generations of laboratory mice, no clue was obtained as to immunosuppressive action of any of the substances involved.     

Relation between 15N excretion in feces following oral 15N urea administration and blood urea concentration in relation to raw fiber intake in swine

9 pigs of a live weight at the beginning of the experiment of 33 kg received in 3 consecutive series of experiments (3 animals/group) a basic barley ration of 1.0-1.2 kg per animal and day. In groups 1 to 9 the following supplements were given: 1 = without N-supplement, 2 = 10.5 g urea, 3 = 79 g dried skim milk, 4 = 11 g urea, 5 = without N-supplement, 6 = 110 g horse bean coarse meal, 7 = without N-supplement, 8 = 95 g dried skim milk, 9 = 120 g horse bean coarse meal. In groups 1-6 the ration was supplemented with 150-165 g DM partly hydrolysed straw meal per animal and day. After 20 days the animals received a single dosis of 0.5 g/kg0.75 15N-urea (72.1 atom-% 15N-excess) with the morning meal of the first day of the experiment. During the four days of the experiment groups 1-6, due to the straw meal supplement, excreted significantly higher N-amounts than the corresponding groups 7-9. In comparison with the first day of the experiment (1 h after the morning meal) urea concentration in the blood decreased to the following percentage in the sequence 1-9: 64; 65; 77; 54; 64; 73; 82; 88; 84 on the second day of the experiment (1 h before the evening meal. Between the excretion of 15N-excess in faeces (y = mg) during the four days of the experiment and the concentration of urea in the blood (x = mmol/l) there was the following significant negative correlation: y = -40.1 X +340.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Resorption and incorporation of radioactive labeled amino acids during administration of various protein carriers in rats. 1. Resorption of 14C leucine and 3H glycine after intragastric administration]

Male Albino rats (90-100 g) were fed ad libitum (with limited periods of feeding) for 14 days. The diets were adjusted to a crude protein content of 10%. Powdered whole egg, fish meal, yeast and gelatine were used as protein sources. Additionally, one group of rats was fed a protein-free diet. On the 15th day of experiment the rats were fed a test diet at a level of 2 g per 100 g of body weight. 2 hrs after that the rats received 25 muCi of 3H glycine and 5 muCi of 14C-L-Leucine per 100 g of body weight administered by way of intragastric infusion. It was found that a large proportion of the radioactive amino acids were absorbed as early as after 0.5 hr. The highest rate of absorption was observed in animals fed dietary proteins of poor quality or a protein free diet, so that in animals receiving a gelatine diet or a protein-free diet only 68.4% or 56.4% of the administered amount of 14C activity were detected inside the gastro intestinal tract after 0.5 hr. Analogous data for the 3H activity were 52.4% and 25.3%. Maximum absorption occurred after 3-7 hrs. Following this the level of radioactivity in the intestinal contents again increased reaching a peak value after 14-24 hrs; in the case of 14C activity this peak value amounted to 25.4% of the administered dose in animals fed the gelatine diet and 32.8% in the group receiving the protein-free diet. It was established that the major proportion of the resecreted amount of 14C activity was present in leucine. Until 72 hrs after the intake of 14C activity the level of radioactivity was again found to decline, a processes which was induced by processes occurring in the large intestines. Moreover, evidence was obtained in confirmation of previous findings, indicating that the composition of faecal amino acids was constant and unaffected by dietary proteins.     

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.     

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.     

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.     

Urea utilization in growing lambs. 6. N balance with 15N urea

Lambs of an age of 2 or 4 months and of an average live weight of 14.7 and 27.4 kg resp. received rations consisting of 44% cereals, 46% dried sugar beet pulp, 6% wheat starch, 2% urea and 2% mineral-vitamin mixture. The crude protein content was 17.1 and 15.9% resp. in the dry matter, that of native crude protein 10.6 and 9.4% resp. During a 6-day N balance period 8 and 16 g 15N urea resp. with a 15N excess (15N') of 9.26 and 9.40 atom-% were fed orally instead of commercial feeding urea. There were no significant differences between the two age groups with regard to the digestibility of the organic matter and the crude nutrients. The average N balance of 372 +/- 85 mg/kg LW 0.75/day were in the intermediate range of N retention capacity and accounted for 26 +/- 5% of the consumed N. N retention in per cent. was slightly lower in younger lambs. Projections of urea utilization in a quasi stationary state resulted in an efficiency of the utilization of 33 +/- 4%. The dismembering of the lambs at the end of the main period showed between 0.02 and 0.22 atom-% 15N' in the total N, TCA precipitable N and amino acid N of the meat. At between 0.24 and 0.38 atom-% 15N' they were highest in the heart and jaw muscles. The quota of 15N' amounts found in the total N of the meat were 10.6 +/- 3% of the 15N-intake and 20.1 +/- 5.1% of the 15N' amount remaining in the body. The bones contained 7.7 +/- 1.7% and the fleece 7.9 +/- 3.1% of the 15N'-intake. Generally seen, the total N and urea utilization was slightly lower in younger lambs than in older ones.     

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.     

Influence of sex and live body mass on the true protein and amino-acid digestibility of various animal feeds in pigs. 1. Comparative studies between barrows and female pigs

Growing barrows and female pigs of 3 liveweight ranges (20-30 kg, 50-60 kg, 80-90 kg) were used to study the true digestibility of protein and essential amino acids from 4 cereal species and 4 high-protein concentrate using the fecal analysis method and regression techniques. No significant differences were found between the females and the barrows in the true digestibility of the protein and most of the amino acids from the protein carriers under study. In the 3rd liveweight range, the true digestibility of some amino acids of the cereals proved somewhat lower in tendency with female animals. In comparison with the true digestibility of protein, in the cereals under study that of lysine gave significantly lower, and that of cystine, arginine, phenylalanine and histidine significantly higher values. The relative values (protein = 100%) were 85.0, 110.6, 106.8, 106.5 and 105.5 for lysine, cystine, arginine, phenylalanine and histidine, respectively. The true digestibility of the remaining amino acids is comparable to that of protein.     

Utilization of N15-labelled urea in the laying hen. 5. N15-labelling of the content and tissue of separate parts of the gastrointestinal tract

In the series of experiments with labelled urea three colostomized laying hybrids were butchered after a six-day application of 1% urea with 96.06 atom-% 15N excess (15N') in the ration and another 2 days with a supplement of 1% unlabelled urea. Out of the individual samples from crop, gizzard, small intestine, caecum and rectum, the content of the small intestine and the caecum showed the highest labelling with greater than 1 atom-% 15N'. The TCA soluble fraction of the content of the gizzard was more highly and that of the intestines less labelled than the total nitrogen. The tissue of the gizzard is distinctly less labelled than the "omasum system" and the small intestine. The atom-% 15N' of the oesophagus with crop and glandular stomach largely showed agreement in the individual hens with that of intestinal tissue and ranged between 0.71 and 0.89 atom-%. 2% of the 15N' supplemented with the urea could be recovered in the content and the tissue of the gastro-intestinal tract.     

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.