[Glutamate dehydrogenase and glutamine synthetase activity in the digestive tract in lambs in relation to age]. / Aktivita glutamátdehydrogenázy a glutamínsyntetázy v tráviacom aparáte jehniat vo vztahu k veku.

Developmental dynamics was investigated in the activity of glutamate dehydrogenase (GDH, E.C. 1.4.1.2.-4) and glutamine synthetase (GS, E.C. 6.3.1.2) in different parts of the digestive tract of lambs, in dependence on the age from 10 to 90 days; the goal of these investigations was to elucidate in greater detail the role of the above enzymes in nitrogen metabolism. The activity of GDH, and of the coenzymes NADH and NADPH, was followed in the digesta because simple organisms (bacteria, fungi, plants) have two glutamate dehydrogenases: they differ from each other by coenzyme specificity, unlike GDH from animal sources which can utilize both NADH coenzyme and NADPH coenzyme (Fahien et al., 1965; Frieden, 1964). The following activities of GDH and GS were found out in trials with lambs at the age of 10, 20, 30, 40 and 90 days, as to the different parts of digestive tract: in the tissues of rumen, omasum, reticulum, spleen, duodenum, jejunum, ileum, int. caecum and colon the activity of GDH (NADH) varied from 0.031 to 0.305 nkat/mg dry matter, in the digesta from 0 to 2.92 nkat/mg dry matter. An investigation of GDH (NADH, NADPH) dynamics in the digesta of lambs showed the relatively high activity of GDH (NADH) in the digesta of colon at the age of 10 days and that of GDH (NADPH) in the digesta of int. caecum. The activity of GDH (NADH) was also found to be high in the digesta of int. caecum at the age of 20 days. In that period the activity of GDH (NADH, NADPH) in the digesta of rumen, omasum and reticulum was zero.(ABSTRACT TRUNCATED AT 250 WORDS)

Urease activity of adherent bacteria and rumen fluid bacteria.

In experiments on six sheep fed on a low nitrogen diet (3.7 g N/day), urease (EC 3.5.1.5) activity (nkat X mg-1 bacterial dry weight) 3 h after feeding was found to be highest in the bacteria adhering to the rumen wall (13.25 +/- 2.10), lower in the rumen fluid bacteria (8.96 +/- 1.35) and lowest in the bacteria adhering to feed particles in the rumen (5.69 +/- 2.13). The urease activity of bacteria adhering to the rumen wall and of the rumen fluid bacteria of six sheep fed on a high nitrogen diet (21 g N/day) was significantly lower than in sheep with a low N intake and in both cases was roughly the same (3.81 +/- 1.37 and 3.76 +/- 1.02 respectively); it was lowest in bacteria adhering to feed particles in the rumen (1.92 +/- 0.90). It is concluded from the results that the urease activity of rumen fluid bacteria and of bacteria adhering to the rumen wall and to feed particles in the rumen is different and that it falls significantly in the presence of a high nitrogen intake. From the relatively high ureolytic activity of bacteria adhering to the rumen wall in the presence of a low nitrogen intake it is assumed that this is one of the partial mechanisms of the hydrolysis of blood urea entering the rumen across the rumen wall and of its reutilization in the rumen-liver nitrogen cycle in ruminants.

Glutamate dehydrogenase and glutamine synthetase activity of the bacteria of the sheep's rumen after different nitrogen intake.

In experiments on 18 sheep with a differentiated nitrogen intake (3.7, 6.2 and 21 g N/day), it was found that different enzyme activities--glutamate dehydrogenase (GDH) (NADH- and NADPH-dependent) and glutamine synthetase (GS)--of bacteria adhering to the rumen wall and to food particles and the rumen fluid bacteria altered in correlation to the nitrogen intake. With a nitrogen intake of 3.7-6.2 g/day there was a significant increase, and of 6.2-21 g/day a decrease, in NADH- and NADPH-dependent GDH activity in the three given bacterial fractions, with the exception of NADPH-dependent GDH activity of the rumen fluid bacteria of sheep given 3.7-6.2 g N/day, in which the difference was nonsignificant. GS activity was significantly higher only in adherent rumen wall bacteria in the presence of a nitrogen intake of 3.7-6.2-21 g/day. The results show that the effect of the nitrogen intake on the given enzyme activities is strongest in the case of bacteria adhering to the rumen wall. The high GS activity and low GDH activities in these bacteria during lower nitrogen intakes (3.7 g/day) as well as lower rumen ammonia concentration (2.39 +/- 0.98 mmol.l-1) indicate that bacteria adhering to the rumen wall utilize ammonia at an increased rate by means of CS catalyzed reactions. Reduced GDH activity in the presence of a high nitrogen intake (21 g/day) and the relatively high rumen ammonia concentration (36.63 +/- 5.28 mmol.l-1) indicate that ammonia inhibits this enzyme in the rumen bacteria in question.

Ammonia-utilizing enzymes of adherent bacteria in the sheep's rumen.

In experiments on 6 sheep the authors found the following enzyme activities in bacteria in the rumen fluid, bacteria adhering to the epithelium of the rumen wall and bacteria adhering to food particles in the rumen (given in nkat X g-1 bacterial dry weight): GDH (NADH): 725 +/- 165, 558 +/- 127, 661 +/- 153; GDH (NADPH): 558 +/- 338, 255 +/- 88, 565 +/- 139; GOAT (NADH): 46 +/- 23, 67 +/- 31, 66 +/- 14; GOGAT/NADPH: 58 +/- 27, 56 +/- 15, 65 +/- 29; GS: 153 +/- 65, 69 +/- 35, 71 +/- 32; ALT: 71 +/- 25, 43 +/- 20, 52 +/- 11; AST: 52 +/- 12, 33 +/- 16, 28 +/- 15. The results show that, except for GDH (NADPH), there were no significant differences between the given enzyme activities in the rumen fluid and in bacteria adhering to the rumen wall and to food. Adherent rumen bacteria have the same potential possibilities as the rumen fluid bacteria for the utilization of ammonia, particularly for the synthesis of glutamic acid, glutamine, alanine and aspartic acid, with the above enzymes as catalysts. By means of the GS/GOGAT system, adherent rumen bacteria can probably synthesize glutamic acid in the presence of a limited NH3 concentration in the rumen.

[The effect of zeolite (clinoptilolite) on the post-feeding dynamics of N metabolism in the portal vein, jugular vein and the rumen fluid of bulls]. / Stúdium vplyvu zeolitu (klinoptilolitu) na dynamiku niektorých ukazovatel'ov N-metabolizmu po nakrmení vo v. port ae, v. jugularis a v bachorovej tekutine u býkov.

If easily digestible saccharides are deficient in the feed ration of bulls with the live weight of 300 kg and at simultaneous single application of urea at a rate of 0.2 g per 1 kg live weight, zeolite (with 50.6% clinoptilolite content) administered at a rate of 2.5% per 1 kg dry matter influenced significantly (P less than 0.05) the ammonia concentration in rumen, v. portae and v. jugularis. The rumen contents and blood were sampled at the intervals of 0, 15, 30, 60, 90, 120, 180 and 360 minutes after feeding. Basal feed ration consisted of 1 kg feed mixture and 3 kg meadow hay. After urea administration, zeolite reduced the ammonia concentration in rumen by 20-40% in comparison with the control group and in v. portae by 60-70%. In v. jugularis in the 90th minute after feeding significant hyperammonemia was observed in bulls with no zeolite supplement. Zeolite administration did not influence urea concentration in plasma.

Urease activity of adherent bacteria in the sheep rumen.

In experiments on six sheep fed on a low protein diet (6.2 g N/day), it was found that the urease activity of the rumen fluid did not change significantly in the first 6 hours after feeding and that it ranged from 45 to 75 nkat.ml-1. The major portion was bound to the bacterial fraction and formed about 70% of total rumen fluid activity. Urease activity determined in food particles with adherent bacteria removed from the rumen before and 3 and 6 hours after feeding ranged from 20 to 26 nkat.g-1 food (wet weight), and on rumen wall samples with adherent bacteria from 30 to 800 nkat per 2.5 cm2 tissue. Again, no significant changes correlated to the time after feeding were found. The results show that urease activity in the sheep rumen is localized on food particles and on rumen wall epithelium with adherent bacteria, as well as in the rumen fluid.

Nucleic acid synthesis from blood urea 15N in the sheep.

In experiments on 4 sheep fed on a low protein diet [6.2 g N/day] and given a single i.v. dose of 15N-labelled urea [15 mg 15N/kg body mass], the authors found that, from 0.5 to 6 h, mean 15N incorporation rose progressively in the total rumen fluid nitrogen from 0.23 to 0.44 at. % 15N and in the rumen bacterial nitrogen from 0.11 to 0.51 at. % 15N. Up to 3 h, total nitrogen enrichment was greater (0.5 at. % 15N) than enrichment of bacterial nitrogen (0.28 at. % 15N), but from 3 to 6 h there was little difference between them. The mean 15N values in the nucleic acids isolated from rumen fluid bacteria in samples collected 3 and 6 hours after injecting labelled urea into the blood were 0.15 and 0.19 at. % 15N respectively, in nucleic acids isolated from the liver 0.042 and 0.04 at. % 15N, in the total rumen bacterial nitrogen 0.28 and 0.51 at. % 15N and in the total liver nitrogen 0.11 and 0.11 at. % 15N. It is concluded from the results that blood urea nitrogen is utilized for synthesis of the total nitrogenous substances of the sheep's rumen bacteria and liver far more intensively than for synthesis of the nucleic acids isolated from them. At the same time, it is utilized more intensively for nucleic acid synthesis in the rumen bacteria than in the liver.

Incorporation of 15N-urea into individual amino acids of rumen bacteria and blood plasma protein in sheep.

Two sheep weighing about 25 kg were fed on rations with similar nitrogen content (about 9 g N/day including 15N from 5 g labelled urea) but with different content of readily digestible carbohydrate. Concentrate ration consisted of maize meal (whole plant) 300 g + barley meal 100 g + mineral mixture 20 g + urea 5 g; fibrous ration--meadow hay 250 g + oat straw 170 g + mineral mixture 20 g + urea 5 g. Retention of 15N was 53.8 and 43.6% of intake in sheep on concentrate or fibrous diet, respectively. Incorporation of 15N into individual amino acids of bacterial protein isolated from the rumen and blood plasma proteins was different, depending on the ratio and amino acid. Enrichment of 15N of the bacterial matter and plasma proteins in both sheep exceeded that of individual amino acids, indicating that urea nitrogen was utilized to a large extent for the synthesis of nitrogen compounds other than amino acids.

Utilization of urea 15N for the synthesis of the rumen microorganisms and blood plasma protein in sheep.

Two sheep were fed a diet containing urea and high level of easy fermentable carbohydrate or crude fibre. Incorporation of urea 15N into the total and bacterial N in rumen and NPN of blood plasma was higher, but lower into plasma protein in the sheep fed the diet with easy fermentable carbohydrate. The analysis of data of the rate of enrichment with 15N in protein and NPN of plasma and rumen liquid suggests that urea nitrogen was fixed transiently into plasma protein and after releasing and recirculation throughout the rumen incorporated permanently into plasma protein.

[Effect of fasting on the concentration of amide nitrogen in the blood plasma of sheep]. / Vplyv hladovania na koncentráciu amidického dusíka v krvnej plazme oviec.

The experiments with sheep starving 24, 48, 72 and 96 hrs after the last feeding showed that on the second day of starving the concentration of the total amide nitrogen in plasma increased significantly (P less than 0.01) both at a lower (7.6 g of nitrogen per day) and at a higher (24 g of nitrogen per day) nitrogen uptake before the beginning of starvation. The concentration of free amide nitrogen, unbound to plasma proteins, increased only at the lower nitrogen intake before starving. At the studied stages of fasting no significant differences in the levels of total and free amide nitrogen in plasma having a relation to nitrogen intake before starvation were found. With the increased urea level in plasma a parallel increase of the total amide nitrogen was observed only in the plasma of sheep fed before starvation the diet with a higher nitrogen content. The results confirm that a short-term starvation is one of the factors which can influence the relatively stable concentration of amide nitrogen in plasma.

[Amide nitrogen in blood plasma of sheep]. / Amidický dusík v krvnej plazme oviec.

The physiological concentrations of amide nitrogen in blood plasma of sheep taken before feeding, 60, 180, and 360 min, after feeding were determined. The concentration of the total amide nitrogen in plasma at intake of 7.6 g of nitrogen per day (45.55 +/- 3.85 mmol.1-1) was significantly higher (P less than 0.001) than at intake of 24 g of nitrogen per day (42.34 +/- 4.07 mmol.1-1). Between the concentrations of amide nitrogen unbound to plasma proteins (1.5 +/- 1.36 mmol.1-1) no significant differences in dependence on the nitrogen intake were found. The results prove that a substantial part of amide nitrogen in plasma is bound to proteins and that its level is relatively stable. A significant change was observed only in the concentration of the total amide nitrogen after a long-term feeding of sheep with some other diet. When the concentration of the total amide nitrogen in plasma was lower in relation to nitrogen intake, the urea concentration was higher.

Urease activity in the contents and tissues of the sheep, pig and chicken gastrointestinal apparatus.

Urease activity, expressed as mg N-NH3/g dry weight per 30 min at 25 degrees C, was determined in the various parts of the sheep, chicken and pig digestive apparatus. The results were as follows. Sheep: contents--rumen 1.25"/-0.09, reticulum 0.78+/-0.02, omasum 0.44+/-0.02, abomasum 0.002+/-0.001, duodenum 0.003+/-0.001, jejunum 0.18+/-0.03, ileum 0.42+/-0.03, caecum 1.34+/-0.11, colon 0.76+/-0.08, walls-rumen 0.88+/-0.16, reticulum 0.38+/-0.04, omasum 0.11+/-0.02, abomasum 0.01+/-0.002, ileum 0.092+/-0.01, caecum 0.14+/-0.03, colon 0.16+/-0.02. Chicken: contents--jejunum 0.028+/-0.009, ileum 0.043+/-0.013, caecum 0.17+/-0.03, colon and cloaca 0.04+/-0.013. Pigs: contents--jejunum 0.02+/-0.01, ileum 0.14+/-0.08, caecum 0.62+-0.12, colon 0.43+/-0.06. No urease activity was found in the walls of the digestive apparatus or the contents of the duodenum in chickens, or in the walls of the stomach and intestine and the contents of the duodenum in pigs. The results show that urease activity in the digestive apparatus of pigs and poultry is lower than in sheep. Inadequate urease activity in the digestive apparatus explains why chickens and pigs are significantly less capable than ruminants of utilizing urea nitrogen as a substitute for some of the protein in the diet.

Distribution of glutamate dehydrogenase and glutamine synthetase activity in the sheep and chicken digestive tract.

Glutamate dehydrogenase (GLDH, EC 1.4.1.3) and glutamine synthetase (GS, EC 6.3.1.2) activity were determined in the contents and tissues of the various parts of the sheep and chicken digestive tract, GLDH activity in the tissues of the sheep omasum, duodenum, rumen, reticulum, colon, caecum, jejunum and ileum ranged from 3.25+/-0.7 U (mumol/g dry weight . min) to 5.94+/-2.28 U; in the abomasum it was 9.67+/-1.27 U. GLDH activity in the contents of the ileum, abomasum, jejunum and duodenum varied from 0.85+/-0.19 U to 3.29+/-0.53 U and in the colon, caecum, reticulum, omasum and rumen from 6.34+/-2.64 U to 16.96+/-3.83 U. GS activity in the tissues of these parts of the digestive tract varied from 2.8+/-0.59 U to 8.6+/-1.4 U and their contents from 2.49+/-0.85 U to 10.76+/-2 U. GS activity in the contents of the colon was very low (0.26+/-0.07 U). In the tissues of the chicken duodenum, caecum, jejunum and ileum we found GLDH activity of 4.68+/-1.64 U to 7.96+/-1.73 U; in their contents it was 3.31+/-1.06 U to 3.8+/-0.73, but in the caecum it attained up to 66.7+/-24.3 U. GS activity was high from 57.6+/-2.0 U to 231+/-84 U in the tissues and 357+/-53 U to 383+/-76 U in the contents (in the caecum up to 2,500+/-233 U). The results show that conditions for the utilization of ammonia are present in the tissues and the contents in the whole of the sheep and chicken digestive apparatus. The hypothesis is confirmed that the different ability of ruminants and fowls to utilize ammonia formed from urea added to their feed, including ammonia formed by hydrolysis of blood urea, is due to the different GLDH and GS activity in their digestive tract as well as in their liver.

[Changes in the distribution of urea in body fluids during growth]. / Zmeny distribúcie mocoviny v telových tekutinách krýs pocas rastu.

Urea concentration in plasma, muscle, and liver fluids of Wistar rats at the age of 3, 6, and 12 weeks was determined. In rats at the age of 3 weeks urea was cumulated in liver and muscle tissue fluids, in older rats (6 and 12 weeks) in plasma fluid. According to the stated urea distribution in individual age categories, a higher urea transport from tissues to blood is considered to be in rats at the age of 6 and 12 weeks. The urea transport proved to be one of the mechanisms of facilitating better utilization of endogenous urea nitrogen in bio-synthetic processes of the nitrogen metabolism in ruminants as well as in monogastric animals.

Glutamate dehydrogenase and glutamine synthetase activity in some organs of ruminants and monogastric animals.

A comparative study of glutamate dehydrogenase (GLDH 1.4.1.2) and glutamine synthetase (GS 6.3.1.2.) activity in liver, kidney and spleen homogenates from cattle, sheep, pigs and chickens showed that chicken liver contained on an average 3.5%, pig liver 8.3% and bovine liver 45.6% of the glutamate dehydrogenase activity present in sheep liver. Relatively low trace activity was found in the spleen and kidneys, except for the renal cortex of cattle (32% of activity in the liver). GS activity was the highest in chicken liver; in pigs it amounted to 33.40%, in cattle to 24.2% and in sheep to 19.7% of this activity. No marked interspecies differences were found in the values in the kidneys and spleen. It can be concluded from the results that the relatively high GLDH activity in the liver of ruminants compared with pigs and chicken is associated with the greater ability of ruminants to utilize ammonia. The higher GS activity and lower GLDH activity in chicken liver can be attributed to higher uric acid synthesis from ammonia via glutamine and purine bases and the lower ability of birds to utilize ammonia for protein synthesis. The presence of alanine dehydrogenase was not demonstrated in chicken liver, where the maximum oxidation of NADH after the addition to pyruvate and ammonia substrate was found.

Incorporation of intravenously administered urea-15N into sheep plasma proteins and their amide groups.

Two sheep with a low and high nitrogen intake (7.6 and 24 g N/day respectively) were given a single intravenous dose of 15N-labelled urea (15.3 mg 15N/kg b.w.) The findings were as follows. The greater part of non-retained 15N from the administered dose was excreted during the first day after the intravenous administration of 15N-urea. Daily excretion in the faeces amounted to 1.35-2.37% of the 15N in the given dose. With a low N intake, more 15N from the given dose (59.4%) was retained in the N pool than with a high N intake (50.5%). The net passage of 15N into the rumen and 15N incorporation into the amide-N of the plasma proteins was likewise greater. 15N incorporation into the amide-N of the plasma proteins rose steadily for 3 days. The porportion of amidic 15N in the plasma proteins rose steadily for 3 days. The proportion of amidic 15N in the plasma protein total 15N changed on the second and third day after administering 15N-urea from 8% to 16%, with the maximum at the beginning of the second day. The amount of 15N incorporated into the proteins in 1 litre plasma attained up to 3% of the given dose. It is concluded from the results that the synthesis of amino acids and their amide groups is both a quantitatively and a qualitatively important metabolic route for the reutilization of blood urea nitrogen for protein synthesis in ruminants.

Passage of the intravenously administered 15N urea into the digestive tract and its excretion in the sheep.

The experiments performed on two wethers provided with simple rumen cannulas and reentrant cannulas, inserted into the proximal duodenum and ileum, showed a passage of 15N from labelled urea, injected intravenously, from the blood to the digestive tract. The amount of the 15N in the digesta was the highest in duodenum, slightly lower in the rumen and slightly lower in ileum. Approximately 50% of the injected 15N was excreted in urine. The amount of the 15N eliminated with feces was very small; 0.6 to 2.8% of the dose injected per day. About 73--84% of the 15N which passed the duodenum was absorbed in further parts of the digestive tract. It can be concluded that all parts of the digestive tract take part in utilization of the endogenous urea.

Incorporation of N from intravenously administered 15N labelled urea into the bacterial protein in the sheep.

The experiment carried out on two wethers demonstrated that nitrogen of intravenously injected urea, labelled with 15N was incorporated into total and bacterial nitrogen fraction of the digesta flowing through the rumen and duodenum. The amount of 15N in the bacterial fraction flowing throught the rumen and duodenum was relatively low in comparison with the amount of 15N in the total nitrogen (14,8% and 8,1% in the rumen and 6,6% and 7,9% in the duodenum. The ratio of the amount of bacterial-N to total-N in the rumen content (12,7 and 7,5%) was only slightly lower than the ratio of bacterial 15N to total 15N. In the duodenum this ratio was a little higher (8,7 and 10,0%). Blood urea nitrogen was utilized only partly in biosynthesis of bacterial protein. The results showed that only a small amount of blood urea nitrogen retained in the organism was utilized for microbial protein synthesis and the majority in some different way.