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2 UTILIZATION OP NON - PROTEIN NITROGEN BY TEE RUMINANT
2.1 Non - Protein Nitrogen in ilmn.mn.t Rationa
The concept that micro-organisms play a '.useful role in protein metabolism was put forward by Zuntz (1991) who expressed the view that rumen bacteria use by preference amides, amino acids and ammonium
salts instead of protein, and that the protein supplied by a given ration was augmented as a result of the formation of protein in the bodies of bacteria and protozoa which were later digested. These early observations showed that the protein requirement of animals especially Eerbivora
could be met in part by such non-protein nitrogenous (NPN) compounds as asparagine, urea and ammonium salts. Loosli _et_ al_ (1949) obtained
specific evidence that microbial action in the rumen can synthesize from urea all of the ten amino acids which are essential .for rat growth. In
so far as the microbial protein arises from NPN compounds such as urea, a distinct gain in amino acids available to the body results. The microbial protein is of high biological value (BY) as measured by rat growth. McNaught et al. (1954-) got the values of 81 and 80/C for the biological values of bacteria and protozoa respectively. This means that through the rumen microbial activities, rations of poor quality are enhanced in quality. Amino acids deficient in the ration are supplied by microbial synthesis. This explains why the protein
quality of tho rations as fed is much less important in the case of the ruminant than in non-ruminant animals. Hoever, the microbial action results
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in some losses also. Some of the ammonia produced, in the rumen by protein degradation or from KPN compounds such as urea is absorbed into the blood stream and converted to urea in the liver. Most of
the urea is lost in the urine and the rest is recycled to the rumen via the saliva and the walls of the rumen.
Virtanen (1967) had shown that milk production could be maintained in cows given purified, protein-free feed using urea and ammonium salts as the sole sources of nitrogen provided energy and minerals are adequate.
Deif, El - Shazly and Abou Akkada (1968) fed urea, casein and gluten in the diet of the sheep at levels which supplied 1.33
g,
3*33g, 5»33g» 7.33g, 11,33g and 14.33g nitrogen per day to each animal. A nitrogen - balanceexperiment was carried out for each nitrogen level with each of the three sources of the nitrogen supplements. They found that the faecal nitrogen was lowest when area or casein was given whereas it was highest
■with gluten at levels of 11.33 and 14-.33g/day. This is to be expected because urea and casein are rapidly degraded in the rumen with the
formation of ammonia, some of which is used for the synthesis of microbial protein while the rest is absorbed through the rumen wall into the blood stream a n d converted to urea in the liver. Thus, a large portion of the
urea and casein nitrogen is lost in the rumen, hence, the low faecal nitrogen on these two diets. Gluten is not degraded in the rumen to a
great extent and this accounts for greater faecal nitrogen on this diet than on diets of urea and casein.
The fact that casein and urea are highly degraded in the rumen,
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and gluten is not, also explains why the urinary nitrogen was higher on casein and urea than on gluten. It also explains why absorbed nitrogen is greater on casein and urea than on gluten,
k
linear relationshipexisted between nitrogen intake and nitrogen retention up to nigrogen
xntakes of 5.33, 11.33 and 7.33g/day for urea, casein and gluten respectively .i. linear relationship was also found to occur between absorbed nitrogen
and nitrogen retention up to levels of 1.33, 5.33 and 7.33g/day for gluten, area and casein respectively.
Leibholz and Naylor (1971) using early weaned calves found that the replacement of 20.1 and 39.2/6 of the meat meal protein nitrogen by urea was associated with a significantly greater weight gain of calves between 5 and 11 weeks of age. The inclusion of urea in the ration to 5 5 . °f
the total nitrogen depressed both weight gain and the intake of the
concentrate mixture. The source of carbohydrate was sorghum and at levels of 62.3 to 77.8^ of the ration. Also the faecal nitrogen was lowered, and the urinary nitrogen greater than in other urea rations. The concentration of branched chain amino acids in plasma was low on urea rations, so also was the concentration of free essential amino acids.
Limiting factors in the experiment might have been carbohydrate to provide energy and carbon skeleton.
.2.2.2 Metabolism of Ammonia Nitrogen by Rumen micro-organisms.
The manner in which the liberated ammonia from protein and non-protein is utilized in the synthesis of amino acids ia poorly understood but available evidence suggests that ammonia is a starting
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material for the synthesis of amino acids which are subsequently incorporated into microbial protein (Loosli et a l . 1949).
On the basis of available information on the synthesis of amino acids by animal tissues and bacteria, it seems probable that in the
prresence of ammonia and a keto acid such as , X ~ ketoglutaric acid, rumen micro-organisms synthesize glutamic acid through reductive aminaiion.
The occurence of many keto - acids including pyruvic acid and
ketoglutaric acid in the rumen liquor may be offered in support of this view. Synthesis of other amino acids would be expected to occur through transamination reactions involving the appropriate keto acids and glutamate Evidence of transaminase activity has boen presented b y Otogald., Black, Goss and Kleiber (1955)•
Investigations by Allison and Bryant (1963) have shown that
cellulolytic rumen bacteria, Ruminococcus flavefaciens required either isovalerate or isobutyrate for growth but that neither 2 - ketoisovalerate, 2 - ketoisocaproate nor leucine supported the growth of these organisms.
The organism failed to incorporate labelled leucine into protein but labelled isovalerate or isobutyrate is required because of inability of the organism to synthesize isopropyl group,
Suphur - containing amino acids (SAA) are structural units of rumen micro-organisms as well as of ruminant tissue protein. It has been
shown that these amino acids can be synthesized by the rumen micro-organism
• t
utilizing inorganic sulphur to synthesize cysteine, cystine and methionine.
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Lambs fed rations containing urea and inorganic sulphur as th® sole source of sulphur were found to produce normal wool growth. Orally administered labelled sulphur has been found to appear in the cystine
35
of wool. Block, Stekol and Loosli (1957) reported that S fed as
Sodium sulphate to a lactating goat was detected in cystine and methionine of mi11c protein. These investigators also showed that was incorporated into rumen micro-organisms of the sheep. Emery, Smith and Huffman
(1957) found that S"^inorganic sulphate was synthesized more rapidly into cystine than into methionine. Lewis (1954) reported that reduction of sulphate to sulphide was brought about by rumen micro-organism, and the sulphide was believed to be ’used in sulphur amino acid synthesis.
Although the ability of rumen microbial population to synthesize amino acids from am m onia nitrogen has been shown (Loosli e_t a l . 1949), evidence exists to indicate that some supplementary organic nitrogen is required for maximum nitrogen utilization. The nitrogen requirement of most bacteria can be met by ammonia but some bacteria also require amino acids and evidence has been presented to show that growth stimulation of some species may be brought about by peptide (Bryant' and Robinson, 196l).
Supplementation of high urea ration with organic nitrogen may result in the development of a broader spectrum of rumen bacteria by providing
nutrients required by some of the most fastidious species. The
possibility also exists that a general improvement in rumen microbial metabolism might occur by virtue of the supplementary organic nitrogen
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supplying a irate-limiting nutrient. Ammonia is an essential nutrient for the growth of Bacteroid.es succinogenes, Ruminococcus flavefacicn
3
,JAminococcus alh u s . Bacteriodes amylophylus, Nethanobacterium .rumiiiantium and Bubacterium ruminantium (Bryant and Robinson, 1963; Hungate, 1966).
Addition o f nitrogenous sources yielding ammonia stimulated in vitro digestion of cellulose and starch. Both cellulo^tic and amylolytic activities in vitro of mixed rumen micro-organisms were increased when urea replaced soybean meal as the sole crude protein supplement, showing that ammonia is important in the nutrition of both cellulolytic and amylolytic rumen bacteria.
Synthesis of amino acids from ammonia by rumen m i c r o - o r g a m s n
3
requires the presence of ammonia, carbon skeleton and energy.
Utilization of carbon from carbohydrate, (Hoover, Kesler and Flipse, 1963), carbon dioxide (Huhtanen, Carleton and Roberts 1954; Otogaki, at al 1965), Isovaleric acid, acetate and other volatile fatty acids
(Hoover et al. 1963) indicates that carbon from a wide variety of sources could be used for synthesis of amino acids. However, synthesis of leucine from isovalerate (Allison, Bucklin and Robinson, 1966), isoleucine from 2 - methylbutyrate (Hungate, 1966), valine from isobutyrate (Allison and Bryant, 1963), phenylalanine from
phenylacetate (Allison, 1965) and tryptophan from Indole -
3
- acetate (Allison and Robinson, 196?), indicates a requirement for certain specific carbon skeleton in the synthesis of certain amino acids.UNIVERSITY OF IBADAN LIBRARY
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Energy for amino acid synthesis is provided by carbohydrates and other organic compounds in the form of ATP. Hungate (1966) estimated that 1.1g microbial nitrogen is utilized for synthetic purposes for
©ach 100g of carbohydrate fermented.
•2.3 Factors Affecting the Utilization of Ammonia in the R u m e n .
Recent studies have been concentrated on factors which will promote the maximum bacterial synthesis of protein in the rumen to provide for the more effective use of rations of poor quality protein and parti
cularly non - protein sources of nitrogen such as urea. A readily available source of energy i3 necessary for the efficient utilization of the end-products of protein fermentation. Pure starch or starch feeds such as cereals, cassava and potatoes are usually most satisfactory.
Molasses or sugars are less satisfactory because they pass out of the rumen too rapidly. On the other hand, cellulose is made available too slowly. Rations low in protein and high in readily available carbohydrate are most favourable to protein synthesis in the rumen. In ruminants,
t is generally considered that soluble carbohydrates exert a positive influence on protein metabolism. Addition of readily available
carbohydrate to protein - rich rations fed to ruminants, wa3 followed by a depression in the concentration of ammonia in the rumen (Chahers and Synge, 1954)* The yield of protein produced by incubating ammonium salts with rumen liquor can be markedly increased by the addition of readily available carbohydrate. Nitrogen retention also increases consistently by supplement of readily available carbohydrate. Lower
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concentrations of blood urea are also observed in ruminant receiving a supplement of readily available carbohydrate. The observed inhibition of ammonia accumulation in the rumen has been explained by the fact that unionised ammonia molecules pass through rumen epithelia much quicker
than the ionised form (Lewis, Hill and Annison 1957). At high pH, the ammonia molecules are mostly present in the unionised form. The presence of glucose or its derivative, lactic acid, lowers the pH and the ammonia molecules are mostly present in the ionized form and their passage through the rumen epithelia i3 much delayed, giving time for the rumen micro-organism to incorporate ammonia for microbial protein.
The pH of the rumen liquor also affe^cts utilization of ammonia.
The pH affects the production of ammonia and also the absorption of ammonia. Reis and Reid (1959) found that high pH favours ammonia production in the rumen. The optimum pH for ammonia production in the rumen varied between 6.0 and 7.0. The observed effect of pH is on deamination as well as on proteolysis. The enzymes concerned with the deamination of amino acids are affected by the pH of rumen liquor.
Warner (1955) Las shown that pH affects the rate of proteolysis and that optimum pH range is 6.5 to 7. The pH also affects the rate of growth of bacteria in the rumen.
Annison (1956) showed that the formation of ammonia in the rumen varies with the type of protein - rich supplement. He compared casein,
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groundnut meal, herring meal and flaked maize gluten. He found that the groundnut meal can be deaminated to an extent equal to or greater than casein.* This is because groundnut meal is also very soluble in rumen liquor.
Maize gluten yielded very low level of ammonia. Herring meal is intermediate between groundnut meal and maize gluten. Protein supplements such as
casein and groundnut meal are easily degraded giving high levels of ruminal ammonia. Urea is easily hydrolysed by the urease of the rumen giving high levels of ammonia. Therefore, the more soluble the protein supplement in the rumen liquor the more ammonia is produced. The method
of processing of the protein supplement also affects the rate of degradation of the protein supplements in the rumen. Formaldehyde - treated casein has been shown to be less soluble than untreated casein and therefore gives lower levels of ammonia in the rumen that* untrsated casein.
1.2,2.4 Absorption of Ammonia Through the Rumen Wal l .
The absorption of ammonia across the rumen irall was reported by McDonald (1948). It is influenced by both the concentration gradient
(Lewis, Hill and Annison, 1957) and pH of the rumen liquor. Ammonia is a weak base with a pKa of 8.80 to 9«15. A n increase in pH causes the ammonium ion (NH
4
+ ) to be converted to ammonia (NH^), and this is rapidly absorbed. Absorbed ammonia is carried via the portal circulation to theliver where it is converted to urea, Hogan (1961) has estimated that
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if efferent blood contains 1.5 mg NHj - N per 100 ml and the rate of flow is 200 ml/minute, then ammonia absorption is 4.3 g/day. The rate of saliva urea secretion has also been estimated at 0.5g/day (Hogan, 1961).
The deamination of protein and the hydrolysis of (NPN) substance such as urea in the rumen forms large amounts of ammonia which if allowed to accumulate would be highly toxic to the animal. Ruminant blood contains about 1.5 mg - N/100 ml blood in the ruminal vein but only traces about 0.1 m - mole/litre in the peripheral circulation (Chalmers, 1954).
It has been shown that a concentration of 0.4 to 0.5 m - mole per litp©
is toxic to the sheep. It is therefore, important that the animal
detoxifies this ammonia in the liver before releasing it into the systemic circulation. This is done by its conversion into urea.
Krebs and Henseleit (1932), working with liver slices, established the general chemical mechanism by which ammonia is converted to urea.
They discovered that the rate of urea production in"liver slices incubated with ammonium salts, bicarbonate and lactate, was increased by addition
of ornithione or citrulline, and that arginine was an intermediate product of the reaction. It was also observed that the quantity of ammonia disappearing was equivalent to the urea formed. On the basi3
of these observations, Krebs and Henseleit (1932) proposed a cyclic mechanism for urea synthesis, involving ornithine, citrulline, arginine, ammonia and carbon dioxide. It was found that 2 molecules of ammonia and
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1 molecule of carbon dioxide are converted to a molecule of urea for each turn of the cycle and the ornithine is regenerated. Therefore, synthesis of urea involves the primary fixation of carbon dioxide and ammonia. It must be known that of the two nitrogen atoms present in a molecule of urea, an atom comes from ammonia, and the other from aspartic acid and could be shown as follows:
KHj +
hco3"
COOH COOH\
\
CH RH0
C H 2 ______ > |
i
— NHp f •‘ 2 H 2 ° *
~—0 ii 0
' COOH
I
COOH
ASPARTIC ACID
FUMARIC ACID