Department of Animal Science FEEDING & METABOLISM OF DIETARY PROTEIN Dairy Cattle Production 342-450A Page 1 of 11
Protein Fractions of Ruminant Feeds These are some of the important protein fractions and definitions that are commonly used in ruminant nutrition and particularly in dairy ration formulation Crude protein (CP) Crude protein measures total nitrogen rather than true protein. The method is based on the assumption that all proteins contain 16% protein. The most common method used to measure CP is known as Kjeldahl nitrogen. The nitrogen value obtained by this method is multiplied by 6.25 (100/16) to obtain CP content. Soluble protein (SCP) Soluble protein is the CP fraction, which is soluble in a buffer solution, water or a rumen fluid. In the new ration evaluation models, SCP is measured as protein soluble in a borate-phosphate buffer (ph 6.9). Considerable amounts of CP in young forages, silages, legume seeds, and oil seeds are in the form of SCP. Soluble protein is rapidly degraded by rumen microbes. The SCP fraction contains all the non-protein nitrogen and some true protein Non-protein nitrogen (NPN) Includes all nitrogenous compounds, which do not have the complex structure of a true protein. Non-protein nitrogen includes ammonia, small peptides, free amino acids, amines, amides. Most of soluble protein in silages and agricultural byproducts (straws and stovers) is in the form of NPN. As with SCP, NPN is rapidly converted to ammonia in the rumen. Neutral detergent insoluble protein (NDICP or NDIN) The amount of CP associated with neutral detergent fiber (cell wall). Another definition of NDICP is the amount of protein insoluble in neutral detergent solution. It is measured by analyzing the NDF residues for CP. Neutral detergent insoluble protein is slowly degraded in the rumen due to its association with the cell wall. Thus large percentage of NDICP escapes ruminal microbial fermentation and can be digested in the small intestine. Mature forages, distillers' byproducts and heat-treated feeds contain considerable amounts of NDICP. Page 2 of 11
Acid detergent insoluble protein (ADICP or ADIN) The amount of protein associated with acid detergent fiber or the amount of protein that is insoluble in acid detergent solution. This protein fraction is not degraded by ruminal microbes and can not be digested by the proteolytic enzymes in the small intestine. For these reasons, this protein fraction is also known as unavailable protein. Acid detergent insoluble protein is determined by subjecting the ADF residues to CP analysis. A high level of ADICP in feedstuffs is an indication of a poor quality protein. Feeds, which have been exposed to excessive heat treatment, contain large amounts of ADICP (heatdamaged protein). Ruminal undegraded protein (RUP) Refers to the portion of dietary protein, which is not degraded by the rumen microbes. In other words, dietary protein that is resistant to microbial attack in the rumen. Several in vivo, in vitro and in situ methods have been used to estimate RUP. The most commonly used method is the nylon bag bags (in situ) technique. The method involves incubating small nylon bags containing feed samples inside the rumen of a cow or steer for a given period of time (12-16 hours). Ruminal undegraded is then estimated as the amount of protein remaining in the bag. Ruminal bypass protein The term "bypass protein" is some times mistakenly used to refer to RUP. The term bypass protein refers to the proportion of dietary protein that evades microbial attack and bypasses the rumen without thoroughly mixing with ruminal content. Liquid feeds that pass through the esophageal groove fall into that category. Ruminal microbial protein Protein fraction synthesized by microbes in the rumen. Rumen microbes utilize ammonia, amino acids and peptides to synthesize microbial protein. About 60 to 80% of the dairy cow protein requirements are provided by rumen microbes. Microbial protein is highly digestible by the animal (80% digestible). Metabolic fecal nitrogen (MFN) The amount of protein that does not result directly from undigested feed protein or microbial protein. Sources of MFN include enzymes and intestinal epithelial cells. Metabolic fecal nitrogen can be estimated from fecal nitrogen of animals fed a nitrogenfree diet. Page 3 of 11
Metabolizable protein (MP) Is defined as the net quantity of true protein or amino acids (feed and microbial true protein) absorbed in the small intestine. It is the summation of digested feed and microbial protein minus nucleic acid. Page 4 of 11
Background Protein Metabolism in Ruminants Due to the synthesis of microbial protein in the rumen, the cow has the ability to survive and produce some milk without a source of a dietary protein. Rumen microbes are capable of utilizing non-protein nitrogen (primarily ammonia) to synthesize microbial protein. The microbes are subsequently digested by the animal and the amino acids produced will be used to supply the animal's amino acid requirements for various production purposes. The presence of rumen microbes makes it possible for ruminant animals to utilize non-protein sources such as urea to produce a high quality microbial protein. Dietary protein consumed by the dairy cow has three fates: 1- Fermentation in the reticulo-rumen by ruminal microbes 2- Enzymatic hydrolysis in the small intestine 3- Excretion of indigestible protein in feces Ruminal protein degradation Rumen microbes, especially bacteria, degrade most of dietary protein entering the rumen. However, some of dietary protein will escape ruminal degradation (RUP). Some of the RUP will be digested in the small intestine by proteolytic enzymes produced in the pancreas protein and some of it will be excreted in feces. The end product of dietary protein degradation in the rumen is ammonia and microbial protein. The end product of digestion of RUP and microbial protein in the small intestine is amino acids. Page 5 of 11
Ration crude protein True protein Non protein N Small intestine MP absorbed Microbial protein Microorganisms RUMEN Energy Excess NH 3 Liver Excreted in urine NPN in saliva Metabolizable Protein Ammonia A B Degradable protein Undigested protein Undegradable protein ADI-protein (C) Crude protein In the diet Two major steps are involves in protein degradation in the rumen: 1- Hydrolysis of peptide bonds to produce peptides and amino acids 2- Deamination and degradation of amino acids Hydrolysis Protein hydrolysis is a multi-step process. Insoluble dietary protein is solubilized and then hydrolyzed by a variety of endo- and exo-peptidases, which cleave the peptide bonds. The hydrolysis of peptides occurs extracellularly by proteolytic enzymes associated with the bacterial cell wall. Many of the protease enzymes produced by rumen microbes are "trypsin-like" in nature suggesting that proteolytic activities in the rumen can be reduced by trypsin inhibitors. Free peptides and amino acids are absorbed rapidly by rumen microbes and used as such or deaminated. Non-structural carbohydrate bacteria utilize peptides and amino acids as N sources. Deamination Metabolism of amino acids is the next step in protein degradation by rumen microbes. The main end product of amino acid deamination is ammonia. An important byproduct of amino acid deamination is branched chain VFA, which enhance the growth of cellulolytic bacteria. Ammonia produced from deamination of amino acids is used by structural carbohydrate bacteria use as a nitrogen source. Page 6 of 11
Metabolism of Urea Urea is broken down rapidly in the rumen to ammonia by urease enzyme. This activity is combined with microbial synthesis from ammonia, enables ruminants to utilize urea entering the rumen either with the feed or in salivary secretion. Recycling of blood urea to the rumen allows ruminant animals to survive on diets very low in nitrogen. The amount of blood urea recycled to the rumen depends on the ammonia concentration in the rumen and plasma urea concentration. Plasma urea enters the rumen with the saliva or by diffusion through the ruminal wall. Microbes adhering to the ruminal epithelium have the ability to produce urease. The enzyme hydrolyzes urea as it passes through the rumen to ammonia and CO 2. High ruminal ammonia levels reduce recycling by inhibiting urease activity. Recycled nitrogen (urea) is useful only if it is incorporated into microbial protein. Incorporation of recycled nitrogen into microbial protein causes duodenal nitrogen flow to exceed nitrogen intake when the level of protein in the diet is low. Ammonia assimilation Ammonia is the most important source of nitrogen for microbial protein synthesis in the rumen. The first step in ammonia uptake is transportation across the cell membrane. Glutamate is the first amino acid into which ammonia is assimilated. Once nitrogen has been fixed into an appropriate compound such as glutamatic acid, synthesis of amino acid from available energy and carbon sources must occur. Ammonia in excess of microbial protein synthesis in converted to urea in the liver. Most of the urea will be excreted in the urine. Some however, will be recycled via saliva. Protein digestion in the small intestine The nitrogen entering the duodenum is a combination of microbial protein, undegraded protein and endogenous protein. Nitrogen entering the small intestine from the stomach can range from 30 to 100% microbial protein and 0 to 70% undegraded protein. Digestion of protein in the abomasum and the small intestine in ruminants is similar to that in monogastric animals. The digestion of protein in the abomasum is carried out mainly by pepsin in a very acidic environment (ph 2). Slow neutralization of digesta in the upper part of the small intestine extends the activity of the abomasal pepsin but delay the onset of intestinal enzyme activities. Optimal activity of trypsin, chymotrypsin, and carboxypeptidase does not occur until the middle jejunum, and peak activity of exopeptidases and dipeptidases is found in the mid Page 7 of 11
ileum. A unique characteristic to ruminants is the abundant excretion of pancreatic ribonuclease. An important role of this enzyme is to release nucleic acid phosphorous for recycling in the rumen via saliva. Absorption of amino acids and peptides The most active site of amino acid and peptide absorption is the mid to lower ileum. There is a preferential absorption of essential over nonessential amino acids from digesta flowing through the small intestine. Differences in absorption also occur within the two groups of amino acids. For example, absorption of lysine and histidine is higher than the absorption of leucine and phenylalanine. Page 8 of 11
Protein Requirements of Dairy Cows The goals of dairy cow protein nutrition are 1) to provide adequate amounts of ruminal degradable protein (RDP) to maximize ruminal microbial protein synthesis and 2) the type of ruminal underrated protein (RUP) that will optimize the profile and amount of absorbed amino acids. Amino acids and not protein per se are the vital elements required by the dairy cow for maintenance, growth, reproduction and lactation. According to the NRC (1989), Protein requirements of the dairy cow are expressed as crude protein, degradable intake protein and undegraded intake protein. Several researchers have recently introduced requirements for soluble crude protein. Table 1 shows protein requirements for dairy cows compiled from the literature and NRC (1989). Table 1. Protein requirements for lactating dairy cows Stage of lactation Early Mid Late Crude protein (CP) 17-19 15-16 13-15 Ruminal degradable protein (% CP) 65-60 70-65 75 Ruminal undegraded protein (% CP) 35-40 30-35 25 Soluble protein (% CP) 25-33 25-35 25-43 The new NRC (2001) contains CP, ruminal degraded protein (RDP) and ruminal undegraded protein (RUP) requirements for small and large breeds of dairy cows at different production levels (not available in the old NRC). Requirements of crude protein: As crude protein content of the ration increases milk yield increases in quadratic fashion. Increasing CP content from 15 to 16% would be expected to increase milk yield an average of 0.75 kg/day and increasing CP from 19 to 20% would be expected to increase milk yield by 0.35 kg/day. Although milk production may be increased by feeding diets with very high levels of CP, the economic and environmental cost must be compared with lower CP diets. The average recommended level of CP in early lactation is 18%. Lower levels should be fed during mid and late lactation. Requirements of degradable protein: Degradable protein is used as a nitrogen source by rumen microbes. Degradable protein can be divided based on rate of degradation in the rumen into: 1- Rapidly degradable protein (soluble protein). 2- Intermediately degradable protein. 3- Slowly degradable protein. The rapidly degradable protein is available for microbial utilization as soon as it enters the rumen. If the amount of soluble protein is greater than the amount the bacteria can use, the excess nitrogen will be absorbed through the rumen wall and be recycled or excreted. Therefore the amount of soluble protein in rations of high producing dairy cows should be kept around 30% of the dietary CP. High levels of soluble protein should be Page 9 of 11
fed with different sources of non-fiber carbohydrates to ensure adequate available energy for bacterial growth. The remaining degradable protein will be available to rumen bacteria only after bacterial proteolytic enzymes have been secreted. The feeding of several protein sources will extend the degradation time so the availability of the degradable protein will match the growth of rumen bacteria. The new NRC (2001) suggests that maximum milk and milk protein yields occur when RDP is 12.2% of diet dry matter. Requirements of ruminal undegraded protein: High producing cows have total protein requirements that exceed the amount of microbial protein produced. Thus rations of high producing cows must include a source (s) of ruminal undegraded protein. These could be heat-treated proteins (e.g. heat-treated soybean), distillers' byproducts and animal protein sources (e.g. feather meal, blood meal). Unlike degradable protein, RUP is resistant to microbial attack in the rumen but will be available for digestion in the small intestine. Therefore the amino acid composition of RUP consumed is similar to that digested in the small intestine. The response of dairy cows to high levels of RUP is inconsistent. While some studies showed increase in total milk and protein yields, most studies found that dairy cows do not benefit from feeding ruminally undegraded protein sources such as heated oilseed meals. Protein Sources for Dairy Cows Protein is the most expensive nutrient fed to dairy cows. Protein sources can be divided based on the type of nitrogen they provide: 1- Sources of non-protein nitrogen 2- Sources of ruminally degraded protein 3- Sources of ruminally undegradable protein Sources for non-protein nitrogen: Urea is the main feed source for non-protein nitrogen. Urea contains 46% N (287% CP). Due to its very high solubility in the rumen, the level of urea that can be included in dairy cow rations is very small. Urea can be added to dairy cow rations when soluble protein content is low (such as high corn silage or low quality legume-grass forage-based rations. The amount of protein supplied by urea should not exceed 1/3 of the total dietary CP. Under practical, situations, urea should be limited to 100-150 g per day per cow. Forages particularly silages are excellent source of non-protein nitrogen for dairy cows. During ensiling process, bacteria convert large portion of protein in the silage into non-protein nitrogen. Sources of ruminally degraded protein: Oilseed meals such as soybean meal, canola meal are the main source of RDP for dairy cows. Due to their high palatability, oilseed meals can serve as the only source of supplementary protein. Soybean meal is the major protein source for dairy cows. Several types of soybean meal can be purchased including dehulled soybean meal (48% CP) and hull-containing soybean meal (45% CP). Cereal grains (other than corn) can also contribute to RDP in dairy cow rations. Page 10 of 11
Sources of ruminally undegraded protein: Ruminally undegraded protein sources are slowly degraded in the rumen by microbial proteolytic enzymes. Therefore, large portion of RUP escapes ruminal degradation and becomes available for enzymatic digestion in the small intestine. Sources of RUP can be of plant or animal origin. Meat and bone meal, fishmeal and feather meal are main animal RUP. Heated oilseeds and oilseed meals are the major plant RUP sources. Sources of RUP are more expensive than those of RDP. Another problem with RUP sources of animal origin is their low palatability. Protein fractions and ruminal degradable (RDP) and undegradable (RUP) protein content of various protein supplements CP SCP (% CP) NPN (% SCP) RDP (%CP) RUP (% CP) Alfalfa hay 19 30 96 73 28 Alfalfa silage 19 50 100 77 23 Corn silage 9 45 100 69 31 Dry corn 10 11 70 40 60 High moisture corn 10 40.0 100 20 80 Barley 13 17 29 73 27 Oats 13 53 90 83 17 Soybean meal 55 20 55 65 35 Canola meal 42 32 65 72 28 Sunflower meal 26 30 37 74 26 Fishmeal 67 12 0 40 60 Feather meal 89 4 5 29 71 Blood meal 92 5 5 18 82 Page 11 of 11