The ration of ruminants should ensure the continuous consumption of animal nitrogen in order to allow the animal to grow and regenerate properly and completely organs and tissues. There are sources of dietary nitrogen in protein and non-protein forms. Plant protein nitrogen is 60-80% of the total nitrogen present in plants. These proteins can be divided into two groups: leaf and stem proteins and storage proteins found in seeds. Proteins of leaves are basically found in chloroplast and cytosol and to a lesser extent in mitochondria, nucleus and membrane. Protein nitrogen is present in seeds in the form of albumin, globulin, prolamin and glutelin. The difference in the ratio of these proteins has an effect on the digestibility of dietary protein in the stomach. Generally, the amount of protein in the ration is calculated indirectly and from the total nitrogen, in this way, the determined nitrogen is multiplied by a factor of 6.25. This factor is used because the amount of nitrogen in most proteins is considered to be 16%.
Analysis of nitrogen compounds in rumen
An important part of the digestion process in adult ruminants is based on the action of rumen microorganisms. The key phenomenon of nitrogen nutrition of these animals is based on the capacity of the microbial population to use ammonia. In the presence of sufficient amount of energy, microorganisms can meet their protein needs by direct use of nitrogen compounds and by endogenous synthesis of new amino acids.
Endogenous protein synthesis, in the form of protein, in the form of microbial proteins, is very important in ruminants, because this synthesis allows such animals to meet their protein needs from a non-protein nitrogen source. More than 80% of rumen bacteria are able to use ammonia as the only source of nitrogen. Of these bacteria, 25% absolutely need ammonia, while 55% can use amino acids just as well as they use ammonia. Few species can use peptides directly.
Breakdown of proteins in the rumen
The rate of protein breakdown in the rumen is considerably variable. A part of leguminous nitrogen, which is between 30 and 50% of total nitrogen, is decomposed into ammonia in the rumen. Another part in the form of peptides, amino acids and nucleic acid bases are used by microorganisms, while the last part passes directly into the milk. Food protein can be decomposed after being released from plant cells, this process is done when the membrane breaks during chewing or in the rumen under the effect of fiber-decomposing bacteria. These microbial proteases and peptidases are responsible for protein breakdown in the stomach. Protein-degrading bacteria comprise between 12 and 38% of the rumen bacteria population, but their exact ratio varies according to the components used in the diet. An important part of protein-degrading enzymes is attached to the cell wall of bacteria, which are suspended in the rumen fluid or attached to food residue. Another part of protein-degrading (proteolytic) enzymes was found in free form. which are probably obtained from the decomposition of bacteria. Proteozoans, which exist exclusively in the solid phase of the rumen, participate in controlling the proliferation of bacteria and preferably have less proteolytic activity. In relation to fungi, they have no known activity on the proteolytic part. The analysis of proteins is done in two stages. First, the hydrolysis of proteins is done to release peptides and amino acids, in the second step, deamination and decomposition of amino acids takes place. It seems that the hydrolysis of peptides into amino acids is a stepwise process with limitations because the rate of deamination of amino acids is generally higher than the rate of proteolysis (breakdown of protein). Actually, the concentration of free amino acids in the rumen is low. rumen flora to be used for microbial protein synthesis or deamination. On the one hand, deamination leads to the formation of ammonia, and on the other hand, it leads to the formation of various products, the type of which depends on the type of amino acid to be decomposed. Other reactions that lead to the formation of fatty acids, carbon dioxide, methane and heat. The decomposition rate seems to be variable for different amino acids according to its free form. As a result, arginine and threonine are broken down quickly. Lysine, phenylalanine, leucine and iso-leucine show intermediate degradation rates; Valine and methionine are slowly decomposed. According to the amino acids that are found in the protein part, the order of its amino acids decomposition is as follows: leucine > valine > histidine > isoleucine > lysine. The use of dietary nitrogen depends on providing The presence of energy is inside the stomach. In fact, the excess of nitrogen relative to the available energy increases the concentration of ammonia inside the rumen, which leads to wastage of nitrogen in food and also reduces the consumption of animal feed. In the opposite case, if the amount of nitrogen is insufficient, microbial growth will be limited, which in turn causes a decrease in fiber digestion and a decrease in feed consumption, even if the energy rate is sufficient. The main factors that affect the breakdown of proteins in the rumen are: their molecular structure, their retention time in the rumen, their solubility and food processing.
Non-protein nitrogen (NPN)
Decomposition of non-protein compounds (amides, peptides and amino acids) that are released in the rumen fluid is generally very fast. Most of the non-protein nitrogen of food materials is hydrolyzed into ammonia by microorganisms. It is possible that the ammonia resulting from the deamination of amino acids, from the hydrolysis of urea by enzymes or from other nitrogen compounds in the diet. Ammonia formed in this way may be used by rumen bacteria for microbial protein synthesis, or absorbed and entered into the blood. Ammonia is the main form of nitrogen. It is used by rumen microbes. Bacteria can also use free amino acids and peptides. Ammonia concentration changes mainly based on the time after consuming food and depending on the source of dietary nitrogen. For this reason, if the nitrogen supplied in the form If it is urea, the ammonia peak is obtained 1 to 2 hours after the meal. At the same time, if the source of nitrogen is plant, the ammonia peak is obtained 3 to 5 hours after the meal, the same time as the peak of volatile fatty acids appears. Density Ammonia in a certain time depends only on the release rate of ammonia It does not consume nitrogen, but it also has the consumption rate of rumen microbes and the rate of absorption from the rumen wall. The use of non-protein nitrogen is reduced when the amount of glucose or energy is not sufficient or when glucose is fermented slowly. This is the result of a diet that uses non-protein nitrogen, for example urea And its glucoside is released slowly. In such conditions, the rate of ammonia increases rapidly and a significant amount of it is absorbed by the wall of the stomach and as a result is removed from the reach of the animal. Attempts are made to reduce non-protein nitrogen by inhibiting urease or by reducing its solubility, but the observed results are not completely satisfactory.
The main factors that affect the concentration of ammonia in the rumen at a specific time are:
A- Solubility and speed of protein decomposition: the peak concentration of ammonia is achieved very quickly when the proteins are easily decomposed.
B- Contribution and availability of fermentable glycosides in the diet: increasing the availability of fermentable glycosides allows more effective use of ammonia by rumen bacteria, and as a result, reduces the concentration of free ammonia in the rumen.
C- Time of food consumption: If the animals consume fodder, it has been observed that its density in the rumen increases up to 4 hours after consuming food and then decreases.
D- Frequency of feeding: the small amount of food, which is given to the animal at short intervals, produces stable conditions in the rumen, which results in fewer changes in the concentration of ammonia.
E- Dilution rate in the rumen: increasing the dilution rate will reduce the proteolysis in the rumen, and the ammonia level will also decrease.
F- Ammonia absorption rate: the degree of acidity in the stomach affects the ammonia absorption rate; At acidic pH, absorption decreases.
G- Microbial activity: the activity of proteolytic bacteria may be affected by the components used in the diet. Also, adding some food additives such as ionophores are also effective.
H- Animal health status: In case of lactic acidosis, a decrease in microbial activity has been observed, and as a result, the use of nitrogen is reduced.
Generally, the amount of nitrogen that can be used by rumen bacteria depends on two factors:
A- The availability of materials needed by bacteria.
B- Energy conversion efficiency to microbial mass that is synthesized per mole of ATP composed of glycosides.
Ammonia does not penetrate quickly through the rumen wall at a pH lower than 7.3. In fact, in acidic pH, the predominant form of NH4+ in the rumen cannot pass through the rumen wall quickly. At a pH greater than 7.3, about 50% of ammonia is found as ammonia, a form that can diffuse quickly. For this reason, in the case of urea poisoning, it is recommended to reduce the pH of rumen fluid by giving vinegar (acetic acid) to the animal. This action reduces the absorption rate of ammonia.
The normal plasma urea concentration varies between 20 and 30 mg per 100 ml (that is, 4-5 mmol). Urea can be toxic at levels of 0.3 to 0.8 grams per kilogram of live weight. If the animals have not passed an adaptation period before, the amounts of 0.27 to 0.50 grams per kilogram of live weight may be lethal. Acceptable amounts are estimated to be about 1% of the dry matter of the ration. Regarding ammonia, its normal level in blood is between 0.5 and 0.8 milligrams per 100 liters of blood and the critical limit is 1 milligram of N-NH4+ per 100 milliliters of blood. At the level of the stomach, poisoning appears when the concentration of ammonia in the stomach exceeds one gram per liter of liquid. If the liver cannot use nitrogen at the proper speed, its concentration in the blood increases, which can cause poisoning symptoms. In the state of poisoning, the level of concentration of N-NH4+ in 100 milliliters of blood has been observed to be 2 to 4 mg. pH. Above blood and blood will not be able to carry out gas exchange. In normal conditions (pH between 5.5 and 6.5), a large part of ammonia, which is not used by microbes, will be absorbed on the surface of milk. It has a stomach wall. The return rate of plasma urea fluctuates between 23 and 92%.
With a low rate of nitrogen consumption, an important part of urea is recycled and a small amount appears in the urine. The amount of recycled nitrogen to the rumen may be so high that it reaches 15 grams per day in sheep and 60 grams per day in cattle. With a suitable ration, 10 to 15% of nitrogen in food will be recycled. The amount of nitrogen that reaches the duodenal surface may be more than the amount that has been eaten, especially when the amount of protein in the diet is low. This is explained by the recycling of nitrogen by bacteria. When there is a lot of non-protein nitrogen in the feed of ruminants, special attention should be paid to it in terms of proper supplementation of sulfur, potassium and phosphorus. These elements generally do not exist in most non-protein nitrogen sources, but they may exist in proteins. Sulfur can be a limiting factor in microbial protein synthesis, although an important part of nitrogen in the diet is of non-protein origin. The ratio between nitrogen and sulfur should be kept at about 14 to 1 (1:14) in order to meet the sulfur needs of microorganisms. Nucleic acids comprise between5/2% and 9.5% of the total nitrogen content of the forage, they are rapidly decomposed inside the rumen. As a result, the dietary nucleic acids have a small share in relation to the nucleic acids that enter the duodenum. Dietary RNA is broken down by bacterial nucleosidase and produces nucleotides, sugars (ribose or deoxyribose), phosphate, purines and pyrimidines. A small amount of rumen nucleic acids can originate from mucus secretions and the destruction of rumen mucous cells. Purines and pyrimidines are resistant to decomposition in the rumen and can be absorbed from the rumen wall. Catabolism of pyrimidines produces ammonia, carbon dioxide, beta-alanine and beta-amino-isobutyric acid.
Microbial protein synthesis
The share of microbes in the protein that is absorbed on the surface of the small intestine is 60 to 80 percent. The synthesis of microbial proteins in the rumen is limited by the amount of ATP energy (or digestible organic matter) available to the bacteria and by the energy efficiency of the bacteria from the materials used. It is difficult to estimate protein synthesis inside the rumen because the available energy and The energy efficiency of diets is different.Also, the estimation of protein synthesis in vivo is limited due to the accuracy of existing methods, so it is necessary to limit the results obtained with in vitro techniques.
Post-ruminal digestion of nitrogenous compounds
Intestinal digestion and absorption of proteins in ruminants is similar to digestion and absorption in non-ruminants. Along with the digestive contents, proteins and microbial nucleic acids, the indigestible part of the digestible protein of the food material that was spared from the attack of microbes and as a result of microbial digestion, as well as non-protein nitrogenous substances, leave the rumen. It is estimated that 40 to 80% of the nitrogen that reaches the duodenum is in non-protein form. Another source of nitrogen in the intestine is the internal secretions of the stomach, milk and pancreas. This part is made up of enzymes, mucus and dead epithelial cells.
Digestion in the small intestine
The small intestine is the second important organ that is related to protein digestion. Chymus quickly moves into the duodenum and jejunum. But on the contrary, the stopping time in the ileum is longer. It is at this level that protease is more active. Because the pH becomes less acidic. Protease activity is not affected by the processing that feed materials undergo or by the source of nitrogen consumed by cows. If the rate of protein passing into the intestine increases, the secretion of pancreatic protease can be increased, but the percentage of protein absorption remains constant. Between 13 and 19% of microbial nitrogen is found in the form of DNA and RNA. High secretion of pancreatic ribonuclease is a characteristic of ruminants. Is. This phenomenon explains the high digestion (80%) of the nucleic acids of microbes in the small intestine. RNA digestion is done in the initial part of the duodenum. A small part of pyrimidines are absorbed and used by animal tissues, while an important percentage of purines are excreted by urine and RNA digestion helps to protect nitrogen. This nitrogen will be recycled later in the liver. In addition, the duty of ribonucleases is to protect and recycle phosphorus.
Important proteolytic enzymes of the small intestine: endopeptidases-exopeptidases
Endopeptidases include: trypsin, chymotrypsin and pancreatopeptidase.
Exopeptidases include: carboxypeptidase A – carboxypeptidase B.
Digestion at the level of the cecum
The caecum receives protein residues of food, undigested micro-organisms in the small intestine, endogenous substances (mucus) and blood-derived urea. Its main role is based on slowing down the passage rate and continuing anaerobic fermentation. The pH of the caecum changes between 6.6 and 7.8, which is favorable for the growth of a bacterial flora that has the same enzyme activity as the enzyme activity of rumen bacteria. The existing enzymes provide the basis for the production of volatile fatty acids, peptides, amino acids and ammonia, which are finally absorbed by the wall of the cecum. In sheep, the concentration of ammonia in the cecum is the same as in the rumen, the digestible organic matter in the cecum is mainly composed of hemicellulose particles and bags belonging to rumen bacteria. Microbial proteins are used. The entry of starch or other glycosides that are not digested in the small intestine can stimulate the synthesis of more microbial proteins in the cecum. Because this protein is not digested in the large intestine and appears in the feces, and this causes an increase in nitrogen excretion, which causes a decrease in the apparent digestibility of food protein.
Digestion in the large intestine
The structure and activity of the large intestine allows digestive substances to pass. The large intestine receives the nitrogenous residues that are not digested in the small intestine. In addition, the urea that reaches the large intestine has two sources: one is obtained from the blood and the other is obtained from the fermentation of microorganisms in the cecum. Proteolytic activity in the cecum and large intestine is more than in the stomach, but absorption of amino acids is less in these two parts. Ammonia absorbed in the blood circulation turns into urea, which may return to the stomach. Microorganisms in this part are finally eliminated with feces and form an important part of nitrogen in feces.
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