De stikstofhuishouding van het melkrund en de betekenis van de gluconeogenese uit aminozuren

The nitrogen metabolism of the ruminant has received close attention during the last decades. At the beginning of this century, the use of various protein sources in the rations of cattle and sheep was investigated. In addition to the biochemical and physiological processes of N metabolism, the literature paid much attention to microbiological aspects of ruminal fermentation. Literature on interpretation of qualitative results in a quantitative approach to protein requirement is scarcer.In monogastric animals, the quantity and composition of protein reaching the stomach is determined by the quality and quantity of the protein in the ration. Ruminants behave in a different way. Through activity of bacteria and protozoa in the forestomachs, there is less similarity between the protein of the ration and the protein reaching the abomasum. Hence, in ruminants, the amino acid supply to the blood is less determined by the amino acid pattern of the ration than in monogastric animals.There are several ways of determining the protein requirement. One can determine the quantity of protein in the ration just sufficient for maintenance or for a certain production. The requirement can also be determined factorially, the various parts (factors) in the total requirement being analysed and summed.Chapter 2 gives a survey of the literature on general aspects of N metabolism. Figure 1 shows the processes in the N metabolism of the ruminant. Part of the protein in the ration passes the rumen undegraded. An average of about 60 % of the protein is degraded to peptides and amino acids or even ammonia. Section 2.2 deals with methods of measuring the protein degradation in the reticulo-rumen. The extent of break-down of protein is largely determined by the solubility of the protein.Ammonia can be formed and absorbed in the rumen. The greater part of the absorbed ammonia is converted into urea. Part of the urea is returned to the rumen by saliva. This recirculation is known as the 'urea cycle.'Amino acids can also be absorbed from the rumen and probably from the reticulum and omasum too. The concentration of free amino acids, however, is usually so small that this absorption can be ignored.The micro-organisms in the rumen can synthesize amino acids from simple N compounds. They can use these as well as other amino acids present in the rumen for synthesis of proteins. Carbohydrates in the rumen form an important substrate for bacteria. They provide energy and are the important material for the synthesis of cells.Results from the literature on degradation and synthesis of protein in the rumen assist in estimating the real amount of protein that reaches the small intestine and can be utilized by the ruminant. This amount can be smaller than the amount ingested as feed, or larger as a result of urea recycling. The microbial activity in the rumen has a levelling effect on the amount of protein reaching the duodenum.It is generally accepted that the digestion of the protein in the small intestine is about the same in ruminants and monogastric animals. Several published trials with cows and sheep showed that the net absorption of non-ammonia N in the small intestine was about 70 %.The activity of the microbes can also be detected in the caecum and colon. Usually, however, the amount of N in faeces equals or is somewhat smaller than the amount reaching the large intestine. The amount of organic matter in the large intestine influences the extent of N excretion with the faeces.Section 2.9 gives a model calculation to indicate the quantitative influence of degradation and synthesis of protein in the rumen on the protein supply in the small intestine.Chapter 3 deals with literature on the estimation of the protein requirement of lactating cows by the factorial method.Metabolic faecal N (MFN) consists of the residues of gastric and intestinal juice, bile and pancreatic juice, epithelial cells and mucus from the intestinal wall and residues from microbial fermentation in the gastro-intestinal tract. Only part of the MFN is of endogenous origin. The MFN is mainly determined by the amount of food passing through the gastro-intestinal tract. It is a common usage to relate MFN to dry matter intake. In monogastric animals, one can observe the influence of crude fibre, whereas in ruminants this influence is of no importance.Section 3.2.1 reviews many correct and less correct methods for determining the MFN. Table 3 surveys original data from the literature on the excretion of MFN by cattle and sheep. Published data from analysis by linear regression gave an average of 4.9 g MFN per kg dry matter intake for both cattle and sheep.Animals given protein-free rations also excrete N in urine. The fraction of N excreted in urine that is independent of the ingested N is called endogenous urinary N (EUN, Section 3.3). EUN seems proportional to metabolic weight. Section 3.3.1 gives some methods for the estimation of EUN and discusses excretion of urinary N on the basis of a simple model. Table 4 shows original data for the estimation of EUN. A graph of collected data (fig. 6) shows the EUN is not quite proportional to metabolic weight. At a lower weight, the excretion is larger. For a weight of more than 200 kg EUN in g equals 0.10 * W 3/4, where W is live weight in kg. The loss of N in hair and scurf is estimated to be 0.02 * W 3/4g.About 15-20% of live weight gain during growth and pregnancy is due to gain in protein. According to the literature, the maternal fraction of the growth during pregnancy can easily be influenced, whereas foetal growth can be influenced less easily. Retention of N during the last 2 months of pregnancy increases from about 20 to 50 g N per day. After parturition it is easy to mobilize the N laid down in maternal tissues, resulting in a negative N balance. This negative N balance is not the result of an insufficient protein supply.Excretion of N in milk can be estimated in a simple way. The protein content of milk varies less than the fat content and is about 3.3 %. An ample energy supply can influence total production and protein content of milk. A larger energy supply is often coupled with a larger portion of concentrates in the ration. Rations causing an increase in protein content are usually the same as those causing a decrease in fat content.Net protein requirement is the amount of protein necessary for maintenance and production if the utilization of protein is 100%. The net protein requirement equals the sum of all factors mentioned above.The relation between protein utilized and protein absorbed is called ' biological value' of the protein (BV). For ruminants, one can hardly speak of a correct evaluation of the ration protein if the protein is subjected to microbial fermentation before it is absorbed. So it is incorrect to ascribe an equal meaning to BV in ruminants and monogastrics. As in ruminants, BV is only a measure of the utilization of protein, it is more correct to replace BV by 'protein efficiency factor' (e N ). Nevertheless, for ruminants BV (v N ) could be reserved for the indication of the quality of the protein reaching the small intestine. Data from the literature suggest that the optimum utilization of ingested truly digestible protein is about 70 % (e N = 0. 70).Table 7 surveys the way in which the protein requirement of the animal is expressed and the way in which the protein in the ration meeting this requirement is expressed. Section 3.10 deals with the use of crude protein, digestible crude protein, available protein and metabolizable protein as measures of protein supply, and discusses the estimation of the digestibility. There has been few studies on the influence of feeding level on protein digestibility. They create the impression that the percentage digestibility decreases about 3-6 units per unit increase in feeding level, that is at least 5%. Moreover, there is a difference in the digestibility of protein between cattle and sheep in favour of sheep. The data in European feeding tables are usually based on digestion with sheep, which means that cattle receive less apparently digestible protein than is calculated from the tables. When establishing the protein requirement and when discussing the results of trials with cattle one should take this into account. The differences in protein digestibility ranged from 0 to 7 units. One may assume an average difference of about 5 units, that is 8%.Table 15 estimates the digestible protein requirement for maintenance and production by the factorial method and surveys standards used in several countries.Section 3.4.10 discusses the metabolizable protein, a measure for the protein supply that allows for the degradation and synthesis of protein in the rumen. It is worthwhile to follow up the investigations on this subject as a model based on metabolizable protein could be elaborated in a few years, allowing better evaluation of feeds than from digestible protein. Especially when extreme and less conventional rations are used there is more chance of errors being made with a system based on crude protein or digestible protein.Chapter 4 discusses the literature on gluconeogenesis from amino acids. In ruminants most of the carbohydrates in the feed are broken down into volatile fatty acids. Of these acids only propionic acid can serve as a precursor for glucose. If insufficient sugars can be absorbed from the ration to meet the glucose requirement, the body synthesizes glucose from other compounds such as propionic acid and amino acids. The need to use amino acids for the synthesis of glucose may mean an increase in protein requirement.Section 4.2 deals with glucose supply from carbohydrates passing the rumen undegraded and from propionic acid. There is little agreement among the results of the investigations on the amount of starch reaching the duodenum. The amount of absorbed glucose would be in most cases equal to or somewhat more than 10% of the starch ingested. The absorbed propionic acid is only partly used to synthesize glucose.Section 4.3 attempts to estimate the glucose requirement for maintenance and production. Section 4.4 compares the glucose supply and requirement This comparison does not answer the question whether the use of other cornpounds than glucose and propionic acid is essential to meet the glucose requirement, as the estimation of the requirement and the supply is uncertain.The mechanism of gluconeogenesis is discussed in Section 4.5, in which also attention is paid to the precursors used. The literature often assumes that amino acids are an important source of glucose. Section 4.6 critically discusses the literature on this subject and considers the techniques used and the interpretation of the data. In particular the use of amino acids for glucose synthesis is distinguished from the essentiality of this use. Experiments in the literature clearly demonstrate the synthesis of glucose from amino acids. However, the the synthesis of glucose from amino acids as a result of a surplus of amino acids is not distinguished from that as result of shortage of glucose from other precursors: most experiments concern the former situation. That is why these experiments give no information whether amino acids are indeed necessary to provide in a glucose shortage.Gluconeogenesis from amino acids (Chapter 5) was studied with fullgrown lactating cows. The intake and excretion of N was measured under normal circumstances and during administration of glucose through a fistula into the duodenum or by infusion into the blood. Figure 18 schematizes the intake and excretion of N. If amino acids are used for glucose synthesis, the N of the amino acids is excreted in the urine as urea. If the synthesis of glucose from amino acids is prevented by administration of glucose into the blood and if the freed amino acids are utilized for protein synthesis, the amount of urinary N decreases. This decrease is a measure of gluconeogenesis from amino acids. All in all 8 comparisons were made of cows with a glucose infusion, ranging from 200-900 g/day, and without one. The preliminary period for the blank and infusion experiments lasted for 7-14 days, and the experimental periods were 7-10 days each. In one experiment (G 7) glucose or water was alternately infused for 4 to 5 days each.During glucose infusion no glucose was observed in urine. The glucose concentration of the bloodplasma was normal, even shortly after the infusion started. On the whole the milkproduction increased during the first 2-3 days of the infusion. Afterwards the production decreased to the normal level. Hence, no increase in production was observed during the experimental period. The production increase during the first couple of days was mostly accompanied with a decrease of the protein content. The administration of glucose more often than not resulted in a decrease of the fat content. The lactose content was less variable than the contents of protein and fat and tended to decrease during the glucose infusion.Both in the balance experiments with a protein poor ration (from G 7 onwards) and in the experiments with a normal protein supply no differences showing the occurrence of gluconeogenesis from amino acids could be observed between the blank and infusion periods (table 29). In the experiment with alternate infusion of glucose and water, however, one can observe a distinct influence of the glucose (fig 24). In this experiment only 75 % of the decrease in the excretion of urinary N was caused by a decreased excretion of urea and ammonia. At a production rate of less than 25 kg of milk no evidence was found for the assumption that amino acids are needed to meet the glucose requirement. At a higher production rate, however, the use of amino acids for glucose synthesis may occur. At an ample protein supply glucogenic amino acids may be used for glucose synthesis. This does not mean, however, that amino acids are needed for this process.Chapter 6 discusses the results of the research into a number of aspects of the N metabolism. The influence of processes in the caecum and colon on the utilization of protein was studied in difference trials. During one trial of each pair either starch or protein was infused via an ileum fistula. The infusion of about 500 g starch resulted in a large increase of the faecal dry matter and the faecal N excretion (table 37). The N content of the increase in dry matter amounted to about 4 % (25 % protein). The increase in the faecal N excretion was coupled with a decrease in urinary N.No influence of the infusion of about 75 g or 150 g protein on the faecal dry matter production could be observed. About 10 g of the infused N was recovered in the faeces, whereas the other N was found in the urine (table 37).The capacity of the microbes in the large intestine to fix N seems to be more restricted by the energy supply than by the N-supply.The utilization of protein at a different protein level was studied in differencetrials with lactating cows receiving a normal or a protein poor ration. The results are mentioned in figure 26 and tables 43 and 44. The tangent of the line connecting 2 points in figure 26 is a measure for the utilization of the difference in digestible protein supply between the normal and the protein poor rations. With 10 out of 13 lines this utilization lies between 10 and 30% (table 44).With the data available from 1192 energy- and/or N-balance experiments from this and other countries and with the results of digestion-trials with sheep concerning 254 feedstuffs, several calculations could be made (Section 6.4).The true digestibility of the protein in rations for cattle amounts to about 85 %. The digestibility of the crude protein in concentrates seems to be higher than the digestibility of the protein in roughage. The MFN values calculated are on the whole somewhat higher than the average of 4.9 g which was taken from the literature (4.8 to 6.6 g N).The digestible protein for cattle is based on the results of digestion trials with sheep. It should be taken into account that there is a considerable difference in the digestibility of protein between cattle and sheep. The difference in the digestibility of the protein found in processing the data can be ascribed to a difference between cattle and sheep of about 8 % and to a difference of about 2.5 % per unit of feeding level.For a further study of the utilization of the protein from the Wageningen-data those experiments were selected in which less than 65 % of the Dutch standard for digestible protein was given. Experiments with a negative energy-balance were eliminated if the N balance was smaller than -5 g N. The result is shown in figure 32. The calculated minimum line is a measure for the maximum efficiency of the utilization of the digestible protein and for the minimal protein requirement. The minimum line corresponds with about 70% of the Dutch standard and closely fits the other data (figure 28, 29, 30 and 3 1).The utilization of the digestible protein was also calculated from a collection of pairs of experiments, each of which pair consisted of two successive experiments with the same animal. Furthermore the difference of the digestible protein in such a pair had to be at least 10 % (6.4.6). Each of the 54 selected pairs corresponds with 2 points in figure 28. For each of the 54 pairs the slope and the position of the line connecting the 2 points were estimated as shown in figure 33. These provided the points in figure 33b. Out of the 54 points 39 points are situated in the rectangle of figure 33b. The mean and standard deviation of the mean is 21 ± 5.2 %. So the average percentage of the utilization of the digestible protein amounts to about 20%. This utilization is for the greater part the result of a difference in N-retention, and is only to a smaller part due to a difference in the production of milk protein.The protein content of the milk is related to the fat content, the amount of milk and the extent of the protein supply. An increase of the fat content by I % (e.g. from 3 to 4 %) results in an increase of the milk N content by 0.4 g/kg (8 % of the average content), but in a decrease of the N content in the FCM (4 % of the average content). If the milk production increases by 10 kg the N content decreases by about 8%. This decrease of the milk N content more than counterbalances the decrease of the digestibility at an increasing feeding level. The influence of the change in protein content of the milk is also shown in the relation between the milk protein produced in the experiments and the digestible protein requirement for these experiments according to the Dutch standard (Figure 34). It should be mentioned that the results of the experiments from Chapter 6 only show short term effects. If the supply of extra protein has a stimulating effect on the milk production on a long term, this effect should also be considered.