K. Adam

Lysinbedarfsbestimmung bei wachsenden Ratten anhand der Katabolisierungsrate von 14C- und 15N-markiertem Lysin

Male Wistar rats (weighing some 80 g at the start of the experiment) were fed diets containing maize gluten as protein carrier and which was supplemented with amino acids (except lysine) in such way that their concentrations came up to the requirement norms. Lysine was gradually supplemented this resulting in 10 diets of different lysine content (1.6-10.6 g lysine/16 g N). On the 7th experimental day, 4 animals of each group were labelled with 14C-lysine and subjected to 2-hour measuring of 14CO2-excretion. On the following day, the animals were injected i.p. 15N-lysine, the urine being collected over 24 hours to determine 15N-frequency in urine. Both 14CO2-excretion and 15N-frequency in urine were found to remain constant at a lysine content of the diet up to 4.5 g/16 g N and rose steeply from 5.8 g lysine/16 N on. Under the experimental conditions chosen the lysine requirement is deduced to be 5 g/16 g N. This method of lysine requirement determination is highly sensitive and exact because it covers the catabolization of the amino acids under study and not so parameters that are known to be influenced by other factors such as growth, N-balance, total N-conversion or CO2-formation. The method can also be applied to metabolic situations not connected with productive performances.

Stoffwechselorientierte Aminosäurenbedarfsbestimmung anhand der Katabolisierungsrate von 14C- und 15N-markiertem Lysin im Erhaltungszustand

Male Wistar rats (of 60 g live weight) allotted in 10 groups were fed diets with gradually increasing lysine levels ranging from 1.4 to 7.4 g lysine/16 g N. Feed intake was restricted so much that the experimental animals did not change their live weights during the last 3 days of the 8-day experiment period. On the 7th experimental day, 4 animals of each group were injected i.p. 14-C-L-lysine, the 14CO2-excretion being subsequently measured over a period of 2 hours. On the next day, 6 animals of each group were applied an i.p. injected of 15N-L-lysine, the urine being collected over the following 24-hour period to measure the 15N-frequency. Applying both labelling methods, an increased catabolisation of the amino acid was observed after the metabolically necessary lysine requirement had been covered. The methods are very sensitive and revealed, under the experimental conditions chosed, a lysine requirement coverage of about 3 g lysine/16 g N. The possibility of using also 15N-labelled compounds in the metabolism-oriented amino acid requirement determination is likely to facilitate the transfer of the methodology to farm animals and would thus allow to study the amino acid requirement of man. The metabolism-oriented amino acid requirement determination will likewise allow to estimate exact amino acid requirement data under conditions that cannot be rated on the basis of productive yields.

Dynamik von 15N-Isobutylidendiharnstoff (IBDH) bei Schafen

Four Merino Landrace wethers averaging 47.6 kg body weight were adapted to a semi-synthetic diet containing as the only N-source 60 g of IBDU per day. After the adaptation phase, on the 1 st experimental day the IBDU of the morning feed was given in 15N-labelled form (701 mg 15N-excess). After 2 1/2, 7 1/4, 12 and 24 hours the experimental animals were killed without having been fed again. The comparison of the IBDU-concentrations in the content of the rumen bottom with the residual rumen content did not allow to draw conclusions regarding IBDU-sedimentation at the bottom of the rumen. For the 15N-decline in the rumen content, a relationship was established following y = 76.3 - 2.62 (r = 0.96) (see fig. 2). In the order of killing times the following 15N-IBDU amounts were retrieved (% of intake): I = 15.6%, II = 24.1%, III = 3.3% and IV = 3.6%. 7 1/4 hours after starting the experiment, 40% of the 15N-labelled material were found in the rumen in the form IBDU; after 12 hours it came to 10%. Except for sheep I, 15N-urea was not found but in small amounts. Only sheep I and III revealed IBDU-traces in the abomasum, but in the small intestine of all sheep 2 to 6% of the amount taken in. This fact is explained with the endogenous influx of IBDU from the blood. An additional experimental sheep provided with a ligature at the abomasum entry, revealed that IBDU is absorbed from the rumen and allowed to enter the individual segments of the intestine in small amounts.

Der Umsatz von 15N-14C-Azetylharnstoff beim Schaf

: 3 male sheep (phi 48.3 kg) were fed a semisynthetic diet containing acetyl urea as sole protein source and 15N-14C labelled acetyl urea (urea-C labelled) by intraruminal tube. A half life period of 4 hrs was established for the removal of labelled acetyl urea from the TCE-soluble portion of the ruminal fluid. The degree of 14C labelling in ruminal proteins was very low whereas the extent of 15N labelled protein synthesis was quite marked reaching a maximum between the 18th and 24th hour of experiment. The steepest rise of 15N incorporation into ruminal proteins was found to occur between 8 to 12 hrs after start of the experiment, i.e. at the time of peak level of 15N returned from 15N urea via the rumino-hepatic circulation. 23.3% of the amount of 14C activity administered (mean of all 3 experimental animals) was excreted through respiration. The curve patterns of both isotopes in the TCE soluble portion of the ruminal fluid were similar to that of the degasified TCE soluble portion of the blood blasma. At the peak time (8 hrs) a concentration of the nitrogen isotope of about 4 atom% excess of 15N was observed. The level of 14C labeling in blood plasma proteins was insignificant when compared with that of 15N labelling. The ratio at the peak time was 1:10; the same ratio was found for ruminal proteins. From this it can be concluded that the process of labelling of blood plasma proteins proceeds mainly through microbial protein synthesis. Sheep I and III excreted an average of 60.6% of 14C activity and 57.0% of the administered excess of 15N in the urine. 6 hrs after the beginning of the experiment 81% of the amount of urinary 14C activity was found to occur as acetyl urea; after 48 hrs this amount had decreased to 50%. All experimental sheep excreted a urinary sediment consisting mainly of acetyl urea. The level of faecal 14C excretion (1.4%-2.9% of the amount administered) was considerably lower than that of 15N excretion (9.1%--15.6% of the administered dose). The TCE soluble fraction of the faeces contained up to 2% of the 14C dose and 3% of the 15N dose. The true digestibility data of 15N from 15N acetyl urea varied between 96.4% and 98.2%. An average of 40.9% was obtained for the 15N balance over the 7-day trial period.

Untersuchungen zur Messung der ilealen Proteinverdaulichkeit an 15N-markierten Versuchsratten

After 15N-labelling over 7 days male albino rats (92-95 g live weight) received either a wheat or whole egg diet (10 animals each) for 4 days. On the following day of the experiment 5 animals each continued to receive their diets as their morning meal (group 1 whole egg, group 3 wheat) and 5 animals each after the previous feeding of a wheat diet received a 2.9 g whole egg diet (group 2) and after the previous feeding of a whole egg diet a 2.85 g wheat diet (group 4) resp. This morning meal was supplemented with chromium(III)oxide. The rats consumed their meals within 20 minutes. The animals were killed 3.5 hours after the beginning of feed intake. At that time the following relative amounts (in % of the intake) could be detected in the stomach in the sequence of groups 1 to 4: Cr2O3 = 22.5; 26.5; 57.5 and 64.2; dry matter = 25.4; 22.1; 43.2 and 38.5. The better agreement between the whole egg diet and Cr2O3 can be explained with the hydrophobic qualities of Cr2O3 and the small disposition of the Cr2O3 to decompose in combination with the whole egg diet. In the first third of the small intestines less than 1% of the intake of Cr2O3 and a maximum of 3.5% of the DM could be detected. Between 20 and 36% of the Cr2O3 and between 15 and 20% of the dry matter intake were ascertained in the small intestines as a whole; in the large intestines the values were 12-20% of the Cr2O3 and 16-23% of the DM. Endogenous 15N-secretion could be ascertained in all parts of the digestive tract. According to the method suggested by U. Bergner and H. Bergner (1982), protein digestibility in the last third of the small intestines was calculated as follows: (formula; see text) The following ileal digestibility values were calculated for crude protein: whole egg = 95.6%; whole egg (wheat previously) = 95.5%; wheat = 94.1%; wheat (whole egg previously) = 85.1%. It is a precondition for the application of this method that at the time of killing representative quotas of the diet sample to be tested can be detected both in the stomach and the large intestine so that the decrease of 15N-labelling in the ileum is actually caused by the test protein.

Isobutylidendiharnstoff als neue NPN-Quelle für Wiederkäuer

2 experimental cows received isobutylidenedi urea added to a natural diet in amounts of 175 g (I) and 730 g (II) per day for a period of several weeks before the trial was started. On the 1st day of experiment the morning dose was labelled with 5.05 g of excess 15N. 8 hrs after the beginning of the trial of 15N level in the TCE soluble portion of blood plasma (TCE=trichloroacetic acid) increased and remained at an elevated level until the 36th hour of experiment. Similarly, the values for maximum urinary 15N concentrations were maintained for a prolonged period of time. Isobutylidenedi urea was excreted with the urine in rates related to its solubility. Only small percentages of the 15N intake were excreted in the TCE soluble portion of the milk (cow I: 0.03%; cow II: 0.05%). The 15N-labelling of milk protein provides evidence for the fact that nitrogen from IBDU is utilized for the synthesis of milk in the cows. The amount of urea in milk averaged 400 mg per litre. None of the milk samples tested contained IBDU.2 experimental cows received isobutylidenedi urea added to a natural diet in amounts of 175 g (I) and 730 g (II) per day for a period of several weeks before the trial was started. On the 1st day of experiment the morning dose was labelled with 5.05 g of excess 15N. 8 hrs after the beginning of the trial of 15N level in the TCE soluble portion of blood plasma (TCE=trichloroacetic acid) increased and remained at an elevated level until the 36th hour of experiment. Similarly, the values for maximum urinary 15N concentrations were maintained for a prolonged period of time. Isobutylidenedi urea was excreted with the urine in rates related to its solubility. Only small percentages of the 15N intake were excreted in the TCE soluble portion of the milk (cow I: 0.03%; cow II: 0.05%). The 15N-labelling of milk protein provides evidence for the fact that nitrogen from IBDU is utilized for the synthesis of milk in the cows. The amount of urea in milk averaged 400 mg per litre. None of the milk samples tested contained IBDU.

Isobutylidendiharnstoff als neue NPN‐Quelle für Wiederkäuer: 2. Mitteilung: Stoffwechsel von 14C-15N-Isobutylidendiharnstoff bei Schafen

2 male sheep (weighing 45 kg and 44 kg) were fitted with a ruminal fistula and a jugular vein catheter and received isobutylidendi-urea for a 42-day period of adjustment. The diet contained 25% starch, 23.8% glucose, 29.0% cellulose, 10.0% straw, 1.7% sunflower seed oil, 4.3% isobutylidendi-urea, 5.6% minerals and vitamins. Each animal received 60 g of isobutylidendi-urea in daily amounts of 1.4 kg of the ration-4.4% of the total dietary N came from the straw. At the begin of the trial each sheep received 30 g of 14C15N isobutylidendi-urea (C1-siobutyl labelling) administered as a suspension. The animals were then placedin respiration cages. The peak of specific 14C activity in the expired air (including ruminal gas) was observed 2 hrs after the beginning of the trial. 18--30 hrs after the beginning of the trial the highest level of 15N incorporation into the TCE (trichloroacetic acid) soluble fraction of the ruminal fluid was noted resulting from the reflow of urea via the rumeno-hepatic circulatory system in the rumen. A high concentration of 15N was shown to be present, for prolonged period, in the TCE soluble fraction of the ruminal fluid (up to the 30 hr of experiment). The 15N concentration in the blood plasma (TCE soluble portion) was found to increase reaching a peak value 23 hrs after administration of the isotope. The highest level of 14C activity in this fraction appeared 1 hr after isotope administration. The 15N incorporation into the protein fraction of blood plasma reached a constant high level between the 29th and 47th hr of experiment. The highest 15N concentrations in urine were noted after 1 day. 3.5% of the administered dose of 14C activity and 23% of the supplied amount of N were excreted in the urine. 20% of the total amount of 15N excreted in the urine could be detected as 14C isobutyl residues. An excess of between 0.05 and 0.17 atom% of the isotopes were found in muscular tissue and in different organs of the sheep when these were slaughtered on the 7th day of experiment (liver: 0.17%, kidneys: 0.14%, muscle: 0.05%, heart: 0.08%). The results obtained in the present trial clearly indicate that ruminants are able to utilize nitrogen from isobutyldi-urea.

Isobutylidendiharnstoff als NPN-Quelle für Wiederkäuer

Two cows with rumen cannulae and duodenal re-entrance cannulae received in the first experiment (Ia and IIa) a conventional diet on the basis of a mixture of maize silage, hay and concentrated feed and after a three-week adaptation to isobutylidene diurea (IBDU)--276 g per animal and day--138 g IBDU with 3.865 mg 15N-excess as a single supplementation to their first meal. In the second experiment (IIa and IIb) after a 6-week break the same cows served a repeated experiment without IBDU adaptation. Irrespective of the adaptation, a re-increase of the 15N-labelling in the TCA-soluble N in the rumen could be proved between the 6th and the 8th hour after the intake of the isotopes, which resulted from the backflow of 15N to the rumen. In the duodenal digesta the maximum labelling of the TCA--soluble N-fraction appeared 12 hours after the intake of the isotopes. At that moment a labelling plateau began in the protein fraction, which lasted to the 36th hour. On an average of all 4 cows approximately 30% of the 15N taken in the TCA-precipitable fraction and 55 and 60% in the TCA-soluble fraction had passed the duodenum up to the 72nd hour after the beginning of the experiment. Up to the 72nd hour after the beginning of the experiment, approximately 15% were excreted in urine, approximately 16% in feces and approximately 7% in the milk. This shows that roughly one half of the 90% 15N-amount measured at its passage in the re-entrance cannula (related to the intake) was metabolised in the rumen at least twice, resp. after the first passage through the duodenum it originated from the intermediary metabolism (e.g. as amino acids and their incorporation as digestion secretions). Negative correlations could be ascertained between the pH-value of the rumen fluid and the 15N incorporation in the rumen proteins as well as 15N-excretion through the TCA-soluble and -precipitable quota of feces. An adaptation to IBDU is obviously not necessary.