lllllllllllllllll“Illlllmlllllllllljl\llHllUlHllHll 3 1293 10533 M‘glub‘lkl a»: " "£95 This is to certify that the thesis entitled STUDIES ON THE EFFECT OF DIET ON NITROGEN PASSAGE TO THE LOWER GASTROINTESTINAL TRACT IN STEERS presented by Stephen Scott Sachtleben has been accepted towards fulfillment of the requirements for Ph. 13- degree in _Anima.l_Hns.bandrY {bé {Mtg 16: 71;.VL, Major professor Date April 28, 1980 0-7639 .-_1 OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to remove chm: fro. circulation records STUDIES ON THE EFFECT OF DIET ON NITROGEN PASSAGE TO THE LOWER GASTROINTESTINAL TRACT IN STEERS By Stephen Scott Sachtleben M.S. A Dissertation Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Animal Husbandry 1980 ABSTRACT STUDIES ON THE EFFECT OF DIET 0N NITROGEN PASSAGE TO THE LOWER GASTROINTESTINAL TRACT IN STEERS By Stephen Scott Sachtleben, M.S. Nitrogen Passage Studies Three experiments were conducted to evaluate the effect of diet on nitrogen passage to the lower gastrointestinal tract in steers. In the first two experiments a total of four concentrate rations were utilized. These diets were corn based and were formulated so that the low protein diet {7.0% crude protein (CP)} served as the basal ration and the subsequent two rations were supplemented with soybean meal to final levels of 9.9% and 12.6% CF, respectively. The fourth ration was the 12.6% CP diet supplemented with urea to a final CP level of 15.6%. Lignin and chromic oxide were used as markers to determine nitrogen (N) flow rates. Total nitrogen passage per day was 60.9, 106.8 and l45.8 grams for rations 1-3, respectively. These means were significantly different (P $.05) across all treatments. Non-ammonia nitrogen (NAN) passage per day for treatments 1-3 were 53.7, 94.3 and 127.3 grams, respectively. Again, all treatments were significantly different (P <.05) across treat- ments. NAN as a percent of total N intake was not significantly differ- ent (P <.05) for rations l, 2 and 3 (87.3, 77.8 and 77.4%, respectively). Data from steers fed ration 4 could not be analyzed statistically since Stephen Scott Sachtleben different animals were utilized. Their total N and NAN passage/day were l12.7 and 98.5 grams, respectively. NAN as a percent of total N intake was 54.0%. In experiment three, four Hereford steers fitted with abomasal cannulae were fed a total of six corn silage rations differing in the amount of anhydrous ammonia applied { control, 7.8 g Anhydrous-NH3 per kilogram corn silage dry matter (AN/KGCSDM) and l5.6 g AN/KGCSDM} and the absence or presence of monensin in the supplement. Chromic oxide, polyethylene glycol and lignin were used as markers to estimate N passage to the lower gut. However, due to marker system difficulties, the estimated flow data obtained was not realistic. A correlation study between individual abomasal samples and a com- posite made from samples representing a 24 hour period showed no rela- tionship between any one or two times of the day when contrasted to a 24 hour composited sample. Thus, collections over an extended period must be made to ensure a representative sample. Nitrogen Balance A nitrogen balance was conducted to evaluate the N status of eight Hereford steers fed the six silage treatments from the previous experiment. Rumensin had no significant effect on nitrogen retention (P > .10), but the treatment of silage with anhydrous ammonia at either level resulted in significantly higher (P < .05) N retention in steers when compared to control animals. Nitrogen retained as a percent of total N intake was affected by the level of anhydrous ammonia (P < .10) and monensin addition (P <.lO). The nitrogen retained as a percent of Stephen Scott Sachtleben N intake increased with increasing dietary protein, however, with the addition of monensin, N retained as a percent of N intake decreased with the highest protein level. MEMORIAL This dissertation is dedicated to and written in the memory of my brother, Peter, whose short time on God's earth will always be cherished by all those he touched. As close as we were in life, he'll always be closer in my heart and memory. ii ACKNOWLEDGMENTS This manuscript is the end result of many years of class and research work and there are many people whose guidance and patience have been invaluable to this author. First, 0r. Werner G. Bergen has my deepest gratitude for the understanding and critical guidance he afforded to me during my graduate program. His knowledge and the intensity at which he pursued research in general was a constant reminder as to what to strive for in ruminant nutrition. Thanks are given to the members of my committee, Drs. M. T. Yokoyama, J. W. Thomas and D. R. Hawkins for their support and critical appraisal of my dissertation. Special thanks are expressed to Dr. E. R. Miller for all his guidance, extra help and personal friendship. Gratitude is extended to Dr. R. H. Nelson and the Animal Husbandry Department for financing me and making available the cattle research facilities. Without Dr. w. T. Magee, the statistical analysis of my data would have become much more laborious and my thanks are extended for his help. I would also like to thank Liz Rimpau for her aid in general laboratory analyses. Jetsy Young has my deepest appre— ciation for her diligent work in preparing the final copy of this manu- script. I would especially like to thank my parents for their continual support during my graduate program for without their love and understanding this work would not have been possible. TABLE OF CONTENTS Page Introduction ........................... 1 Review of Literature ....................... 3 Ruminal Protein Degradation and Microbial NH3-N Utilization. . . . 3 Factors Which Affect Microbial Synthesis in the Rumen ....... l2 Monensin Effects on Performance .................. l9 Marker Systems Used in Digesta Passage Studies .......... 26 Objectives ............................ 32 Materials and Methods ....................... 34 Experiment One .......................... 34 Design of Experiment ..................... 34 Cannula Design and Insertion ................. 34 Sample Collection and Preparation .............. 36 Nitrogen Determinations ................... 38 Chromium Analysis ...................... 38 Lignin Determinations .................... 39 Determination of Daily Flow Rates From the Rumen ....... 39 Statistical Analysis ..................... 39 Experiment Two .......................... 40 General Design ........................ 40 Determination of Daily Flow Rates From the Rumen ....... 40 iv Page Experiment Three ......................... 42 Design of Experiment ..................... 42 Sample Collection and Preparation .......... . . . . 43 Nitrogen Determinations .b. ................. 47 Chromium Determinations ................... 50 Lignin Determinations .................... 50 Polyethylene Glycol Determination .......... '. . . . 50 Determination of Daily Flow Rates From the Rumen ....... 51 I Statistical Analysis ..................... 52 Experiment Four .......................... 53 General Design ....................... . . 53 Sample Collection and Preparation .............. 53 Nitrogen and Dry Matter Determinations ............ 54 Statistical Analysis ..................... 54 Results and Discussion ...................... 55 Experiments One and Two ................... 55 Experiment Three ....................... 59 Experiment Four ....................... 67 Conclusions ............................ 70 Appendix ............................. 72 Literature Cited ......................... 90 \I Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table #WN 10 A1 A2 A3 A4 A5 A6 LIST OF TABLES Page Rations Fed to Steers in Experiment One ........ 35 Rations Fed to Steers in Experiment Two ........ 41 Vitamin and Mineral Premix - MSU 376 .......... 44 Vitamin and Mineral Premix - MSU 377 .......... 45 Effect of Ration Crude Protein Level on N Passage to the Abomasum of Steers ................. 56 Liquid Flow Rates From the Rumen of Steers Fed Varying Levels of Crude Protein ................ 60 Correlations for Individual Samples vs Composite (10# Anhydrous Treatment) ............... 61 Marker Concentrations in Feed and Abomasal Samples for Steers Fed Experiment Three Rations ........ 64 Marker: Marker Ratios in Feed and Abomasal Samples. . . 65 Effect of Monensin and Anhydrous Ammonia Treatment on N Metabolism in Steers ............... 68 Individual Steer Data: Intake Components - Control - Experiment 3 ...................... 72 Individual Steer Data: Intake Components - 7.80 g AN/KGCSDM - Experiment 3 ................ 73 Individual Steer Data: Intake Components - 15.60 g AN/KGCSDM - Experiment 3 ................ 74 Individual Steer Data: Abomasal Components - Control - Experiment 3 ...................... 75 Individual Steer Data: Abomasal Components - 7.80 g AN/KGCSDM - Experiment 3 ................ 76 Individual Steer Data: Abomasal Components - 15.60 g AN/KGCSDM - Experiment 3 ................ 77 vi Table Table Table Table Table Table Table Table Table Table Table Table A8 A9 A10 A11 A12 A13 A14 A15 A16 A17 A18 Individual Steer Data: Estimate of N Passage - Control - Experiment 3 ................ Individual Steer Data: Estimate of N Passage - 7.80 g AN/KGCSDM - Experiment 3 ........... Individual Steer Data: Estimate of N Passage - 15.60 g AN/KGCSDM - Experiment 3 ........... Individual Steer Data: Particulate and Liquid Flow Rates - Control - Experiment 3 ............ Individual Steer Data: Particulate and Liquid Flow Rates - 7.80 g AN/KGCSDM - Experiment 3 ....... Individual Steer Data: Particulate and Liquid Flow Rates - 15.60 g AN/KGCSDM - Experiment 3 ....... Individual Steer Data: NAN Passage Based_on Dual Marker System - Control - Experiment 3 ........ Individual Steer Data: NAN Passage Based on Dual Marker System - 7.80 g AN/KGCSDM - Experiment 3 . . . Individual Steer Data: NAN Passage Based on Dual Marker System - 15.60 g AN/KGCSDM - Experiment 3. . . Effect of Anhydrous Ammonia Treatment Level on N Passage to the Abomasum of Steers .......... Effect of Monensin on Abomasal Nitrogen Passage Parameters ...................... Effect of Monensin on Daily Ruminal Flows to the Abomasum ....................... vii Page 78 79 80 81 82 83 84 85 86 87 88 89 Figure 1 Figure 2 Figure 3 Figure 4 LIST OF FIGURES Flow Chart for Abomasal Sampling ........... Flow Chart of Sampling Protocol - Experiment 3 . . . . Scheme of Laboratory Analysis For Individual Abomasal Samples - 10# Anhydrous-NHB/Ton Treatment . . Scheme of Laboratory Analysis for Composited Abomasal Samples ................... viii Page 37 46 48 49 INTRODUCTION The optimal levels of non-protein nitrogen (NPN) addition to rations fed to high producing cattle in respect to the efficiency of microbial cell yield has been debated for a number of years. Several researchers have attempted to pinpoint at what level further increases in rumen ammonia concentration will not further enhance microbial cell protein (MCP) yields thus making further NPN additions to the diet unnecessary. ’ Drskov (1972) showed that in lambs fed barley-based rations one could supplement the diet with urea up to 12% crude protein equiva- lent (CPE) and still enhance live weight gains. Satter and Slyter (1974), in an in_yitrg_chemostat that simulated rumen activity, determined that 5.0 mg % ammonia nitrogen (NH3-N) was sufficient for maximal microbial growth. This is equivalent to approxi- mately 11-14% CPE. Diets producing rumen NH3 concentration above this point would not increase MCP synthesis. Various factors affect microbial cell yields such as energy availability, growth conditions, cofactor levels and nitrogen availability for ge_ngyg_synthesis. Research is needed to evaluate the addition of NPN above the ll-l4% CPE level and to determine the amounts of N flow to the lower gut of animals fed high concentrate rations. Silage treatment with anhydrous ammonia (AN) at ensiling time is another method of adding NPN to low protein feed sources. There is limit- ed research concerning nitrogen availability (i.e. N passage) to animals 1 fed such AN treated silage. Research is needed to evaluate the avail- ability of this NPN (at various application levels at ensilement) to the animal in regard to microbial cell yield and actual non-ammonia nitrogen passage to the lower gastrointestinal tract and subsequent absorption. REVIEW OF LITERATURE RUMINAL PROTEIN DEGRADATION AND MICROBIAL NH3-N UTILIZATION Ruminants fed diets with varying protein sources produce microbial protein of similar amino acid profiles and of similar nutritional quality (biological value = 70-80%; Bergen et_gl,, 1968; Ibraham and Ingalls, 1972). Since the amino acid composition of microbial protein synthe- sized from differing preformed protein sources is essentially the same, researchers should direct their emphasis to manipulating rumen fermenta- tion so that proteins of poorer quality are degraded in the rumen and resynthesized into microbial protein and the higher quality proteins escape rumen degradation and pass to the lower gut for direct utilization by the host. In reviewing the available literature dealing with protein metabolism in the rumen it would be beneficial to discuss the aspects of nitrogen (N) metabolism i.e., protein degradation to amino acids and ammonia (NH3), available NH3-N sources for the rumen NH3-N pool and the synthesis of microbial crude protein (MCP) and its dependency on avail- able N and carbohydrate fennentation. Two major considerations that influence protein degradation in the runen are the protein solubility in the rumen fluid (Hendrick and Marten, 1963; El Shazly, 1958 and Chalupa, 1975) and digesta retention time in the rumen (Chalupa, 1975 and Orskov and Fraser, 1973). Most highly soluble proteins are degraded extensively and rapidly regardless of the type of ration, but the less soluble proteins are not broken down as extensively with higher dietary intakes and higher dilution rates (Orskov and Fraser, 1973; Chalupa, 1975). Goering and Waldo (1974), through their work with feed processing and its effects on protein utilization, implied that protein utilization was a curvilinear function of solubility with optimal nitrogen retention occurring in ruminants which had been fed a diet with protein that was intennediary in regards to protein solubility. El-Shazly (1958) con- cluded that protein solubility was the major factor in determining ruminal ammonia production and that highly soluble protein should be considered lower in nutritive value since ruminants retain less nitrogen while on highly soluble protein diets. Bergen, Cash and Henderson (1974) reported that the water soluble nitrogen (WSN) portion of chopped whole corn plant could not be utilized or converted to NH3-N quickly enough to support rapid cellulose diges- tion. This information when coupled with an jg_yi£rg_ammonia production study (Bergen, 1974) which showed extremely slow ammonia release by rumen microbes with WSN as a N source when compared to a urea source, suggests why overall runen fennentation is suppressed when ruminants are fed an all silage ration that is low in protein and high in corn silage WSN. Aitchison and coworkers (1976) conducted a metabolic trial with dairy cattle fed corn silage and grain diets, supplemented with varying amounts of urea, that ranged from 32 to 49% WSN. Using coefficients of utilization derived from a linear regression model, data clearly showed that the insoluble nitrogen was used to a greater extent than the soluble N. The utilization of the soluble N was relatively constant, but total dietary nitrogen utilization decreased with increasing levels of soluble N. This suggests that the WSN could not be utilized fast enough by rumen microbes to support cellulose breakdown equivalent to urea hydroly- sis and ammonia formation, thus the decrease in the efficiency of total N utilization as soluble N increased. The better coefficient of utili- zation for the insoluble N could have been the result of a rumen fermen- tation in which the available energy released from the fermentation more closely matched the release of NH -N from the hydrolysis of urea or the 3 portion of the insoluble N which escaped rumen degradation was degraded and utilized by the host of the lower gut. Pichard and Van Soest (1977) proposed that feed proteins are degraded in a series of steps. First, that portion which is degraded very rapidly represents the soluble dietary protein. This rapid hydroly- sis of the soluble fraction is followed by a slower breakdown of less soluble nitrogen components and finally the rumen microbial degradation of a portion of the insoluble protein to NH3-N and amino acids. That part of the insoluble protein which escapes the rumen relatively unaltered may be degraded postruminally. Bull §t_al, (1977) stated that although a portion of the protein is insoluble and undegraded in the rumen this does not mean that the protein is undigestable in the small intestine. A review of the current literature concerning the effect of the level of dry matter intake has shown that depending on that level of intake certain physical and biochemical changes occur. An increase in dry matter intake will cause increased outflow (dilution) rate (Castle, 1956; Grovum and Hecker, 1973; Goshtasbpour-Parsi gt g1,, 1977 and Tammenga et_a1,, 1978), rumen fill (Rumsey and coworkers, 1969), rumina- tion (Welch and Smith, 1969) and volatile fatty acid concentrations (Rumsey e§_g1,, 1970). Restricted feed intake will increase rumen pH with corresponding increases in microbial populations (Slyter e§_a1,, 1970). The level of intake did not affect the degradation of highly soluble proteins, but increased intake decreased the degradation of intermediary soluble proteins due to a higher digesta outflow rate from the rumen thus decreasing the rumen retention time (Miller, 1973). Goshtasbpour-Parsi 93.91: (1977) fed lambs two dietary intake levels (500 or 1000 grams/head daily) which contained equal amounts of dietary nitrogen. Total N recovered in the omasum was higher than the dietary N intake with both diets. They found that significantly (P <.05) more total nitrogen and non-protein nitrogen (NPN) reached the omasum in sheep fed the higher level of intake. More NPN (amino acids liber- ated from proteins) was recovered at the abomasum when lambs ingested 1000 grams/day (P <.05). In other investigations greater amounts of NPN‘ reached the abomasum than the omasum, probably as a result of abomasal' hydrolysis of some preformed protein that passed from the omasum (Amos 15.31., 1970). I Tammenga, Van Der Koelen and Van Vuuren (1978) utilized dairy cows fitted with re-entrant cannulae in the small intestine to deter- mine the quantity of protein entering the small intestine when the animals were fed mixed diets which varied in N content. As dry matter intake increased, the level of N reaching the small intestine as a percentage of dietary N intake also increased. The authors felt that this was a direct consequence of a decrease in digestion of dietary N with decreased rumen turnover time. Increasing amounts of N also reached the lower gut with increasing dietary protein levels particu- larly with higher dry matter intakes. These results support the data obtained by Miller g§_al, (1973), Orskov §§_a1, (1974) and Crickenberger g§_a1, (1979). Tammenga (1978) found that when the lambs ingested more feed, that greater amounts of nonvammonia nitrogen (NAN) entered the duodenum. The author suggests that the difference may be due to an increase in microbial N flow, an increase of undegraded dietary N or a combination of both. Using a regression equation developed by Hvelplund gt _1. (1976), Tammenga determined that increased NAN passage to the lower gut in lambs that consumed more feed was mainly due to decreased dietary protein breakdown in the rumen. This observation was in agreement with the findings of other researchers who evaluated the effect of increased intake on intestinal N parameters (Zinn, 1978; Goshtasbpour-Parsi e§_a1,, 1977; Orskov and Fraser, 1973). Contradictory results were obtained by Hogan and Weston (1967a) when they fed sheep diets which ranged from 7.8 to 19.9% crude protein. The NAN recovered at the abomasum was similar regardless of the protein content of the diet consumed by the sheep. Such a low NAN recovery in the abomasum of sheep fed the 19.9% crude protein ration was probably the result of insufficient energy available to the rumen microbes for the assimilation of NH3-N for microbial protein synthesis. This con- clusion was supported by Orskov gt gl. (1973) who showed that NAN passage to the lower gut increased with increasing amounts of dietary nitrogen consumed up to about 20% crude protein when adequate energy was made available to rumen microbes. The quality of feed protein becomes much more important when ruminants are fed at high protein intake levels since more dietary protein bypass is observed. McDonald (1948) and Mangan (1972) have shown that dietary proteins are degraded by rumen microbes to essentially amino acids and ammonia. AlthOugh NH -N is the predominant end product of ruminal protein break- 3 down, small polypeptides and nucleic acid bases are also present in much smaller concentrations (Smith, 1975). Preformed protein is not the only precursor of ammonia. Non protein nitrogen (NPN) from ingested feeds, recycled urea from saliva (McDonald, 1948; Somers, 1961) and urea diffusing through the rumen epithelium (Houpt, 1959; Dobson, 1961) may be utilized. According to Pearson and Smith (1943) in an jn_yjtrg system, rumen bacteria hydrolyzed urea to ammonia by using a microbial enzyme, urease. To substantiate this observation, the authors added toluene, a microbial inactivator to the media, and found that no hydrolysis of urea occurred, thus confirming the participation of rumen microbes in urea hydrolysis. The amount of nitrogen recycled as urea is dependent on the circulating levels of urea in the blood and the nitrogen content of the feed (Phillipson, 1962). Ammonia not assimilated by bacteria for protein synthesis will move into the vascular bed of the rumen, then to the portal vein and finally to the liver where it is synthesized to urea (McDonald, 1948; and Cocimano and Leng, 1967) recycled or excreted. Such NH 7N movement into the rumen wall is against a concentration gradient 3 and dependent on the ruminal NH -N concentration and the pH of the rumen 3 digesta (Chalmers and White, 1969). A lower pH would increase NH3-N passage to the lower gut where most NH -N absorption occurs (McDonald, 3 1948). Hogan (1967) and Smith (1971) have shown that extensive ammonia absorption occurs in the omasUm with little or no absorption further down the gastrointestional tract. The major source of nitrogen for microbial protein synthesis is ammonia. Bryant and Robinson (1962) concluded fran their work that 82% of rumen bacteria were capable of growing with ammonia nitrogen (NH3-N) N15 as their principal N source. Researchers using labelled ammonia or N15 ammonium nitrate showed that rumen bacteria employed labelled N for their own microbial protein synthesis (Warner, 1965; Phillipson, 1962) and was further found as milk protein in dairy cows (Land and Virtanen, 1959). Although NH3-N is the predominant source of N for microbial protein, amino acids and small polypeptide are assimilated by various bacterial strains but the mechanism is not known (Bryant and Robinson, 1963; Pibnan and Bryant, 1964; Hungate, 1966; Wright, 1967 and Chalupa, 1974). Nolan and Leng (1972) concluded that in sheep fed lucerne (alfalfa) chaff, 29% of the nitrogen digested was incorporated into microbial protein as amino acid nitrogen. Similar data from Pilgram ££.§l- (1970) and Mathison gt_al, (1971) have been published. Salter g§_al, (1979) have shown in rumen fistulated steers fed diets which consisted mainly of straw and tapioca and supplemented with decorticated groundnut meal (DSGM), DCGM plus urea or entirely urea, that when 15N urea was infused into the rumen, the extent at which the labelled N was assimilated into microbial amino acids was dependent on the preformed protein content of the diet. When sufficient prefonned units (amino acids and peptides) were available, proline (pro), arginine (arg), histidine (his), methionine (met) and phenylalanine (phe) were used directly by the bacteria. However, in a diet that was inadequate in preformed units such as the diet supplemented solely with urea, ‘ synthesis of pro, arg and his increased; met and phe synthesis rates remained constant. Salter e§_a1, (1979) suggest that methionine and 10 phenylalanine supplies from preformed units may limit bacterial growth on low preformed protein diets that is high in NPN. Rumen ciliate protozoa utilize bacterial amino acids and possibly other growth factors through bacterial engulfment and digestion (Allison, 1969 and Coleman, 1975). The Value of this protozoal protein to the host is not certain, but Oxford (1955) believed that such microbial protein would provide protein similar to natural animal protein. Accord- ing to Bergen g; 91' (1968a, b) the protein quality, protein digestibil- ity and lysine concentration of protozoa were superior to that of rumen bacteria. A review of current literature has shown that little is known about the quantitative contribution of protozoal protein to the host animal. However, Hungate e;_g1, (1971) has shown that sheep fed alfalfa pellets produced rumen microbes of which more than 50% were protozoa. Of this large ciliate protozoa population only a small portion was passed to the duodenum, however, Hungate (1966) indicated that perhaps the protozoa were retained in the rumen or omasum thus implying that the microfauna had a slower dilution rate than rumen liquid and small particle pool. Weller and Pilgrim (1974) compared actual appearance of protozoa to that population expected from dilution rate calculations and found only 6-29% of the theoretical yield actually appeared in the omasum. These findings confirmed what Hungate (1966) had speculated in regard to protozoal retardation within the rumen and they moved independently of the liquid pool. The data of Weller and Pilgrim (1974) and Hungate e3_al, (1971) could imply that a recycling of protozoa occurred with relatively little passage of rumen ciliate protozoa to the lower gastrointestional tract. If these protozoa are indeed retained within the rumen, they 11 would actually limit total ruminal protein production and decrease the nitrogen available to the host for its own tissue synthesis (Bergen and Yokoyama, 1977). Ration type (natural versus purified diets) will influence the quantity of total microbial protein which reaches the duodenum. Accord- ing to Bergen, Purser and Cline (1968), protozoa will represent a higher percentage of total microbial protein in animals fed natural diets when compared to those fed a purified or semi-purified diet. Increasing con- centrations of protozoa within the rumen with any given ration will decrease the amount of microbial protein available to the host. Utiliz- ing roughage rations, Hungate gt g1, (1971),Pilgrim §§_gl, (1970) and Bucholtz and Bergen (1973) found that as a ratio of bacterial: protozoal protein synthesized, considerable variation could have been the result of the experimental approach used to quantitate microbial protein. FACTORS WHICH AFFECT MICROBIAL SYNTHESIS IN THE RUMEN The amount of microbial cells synthesized in the rumen is depen- dent on two major factors: first, the presence of an energy supply adenosine triphosphate (ATP) 1derived from the fermentation of a sub— strate and second, the precursors (intermediates from rumen fermentation or its end products) of microbial cell synthesis must be present in adequate concentrations and in a form utilizable by the microbes (Hungate, 1966). Under ideal conditions, rumen fermentation is coupled to cell growth) due to carbohydrate degradation, VFA production and ATP generation and their interrelationships with the process of microbial cell synthesis (Walker and Forrest, 1965) . The major end products of this coupled rumen fermentation process are VFA, gas and microbial cells (Hungate, 1966). Anaerobic microorganisms utilize various limiting metabolites as electron acceptors in the place of oxygen, thus having a lower poten- tial of ATP generation from a particular substrate (Gunsalus and Shuster, 1961). Under such a system, the first limiting factor for microbial growth is energy availability (Gunsalus and Shuster, 1961). According to Hungate (1966) a molecule of hexose fermented in the rumen yields only 10-12% of its aerobic ATP potential. During ruminal CHO degradation by microbes, the amount of ATP generated is dependent on the type of CHO, the fermentation pathways and final end products (eg. e' acceptors). The end products of the ruminal fermentation are primarily acetic, propionic and butyric acids and methane (CH Their associated ATP yields are (as moles of ATP/mole VFA or CH4 4)' formed): acetate, 2; propionate, 3; butyrate, 3; CH4, 1 (Isaacson g;_a1,, 12 13 1975). Portions of the substrate are only broken down to intermediary compounds and these are used as precursors of microbial cell synthesis. Little, if any, ATP is generated by such partial degradation (Allison, 1969), but the energy loss is minute compared to the potential energy cost of d§_ngyg_microbial macromolecular synthesis from fermentation end products (Stouthamer, 1973). Although the basic rumen fermentation pathways have been eluci- dated (Hungate, 1966; Baldwin, 1965) there remains an uncertainty in the theoretical ATP yields associated with the production of propionate and CH4. According to Baldwin e§_al, (1963), propionate is produced via the dicarboxylic acid pathway or the direct reductive pathway depending on the CHO source (eg. diet). The pathway utilized to synthesize pro- pionate is dependent on the concentration of dietary carbohydrate ingested. The dicarboxylic pathway is the predominant route, but with increasing amounts of readily fermentable CHO in the diet, the role of the direct reductive pathway increases (Baldwin e§_a1,, 1963). The theoretical ATP yield of the dicarboxylic pathway and the direct reduction pathway differ by a single ATP, the former producing 3 ATP (Hobson and Summers, 1972) and the latter 2 ATP (de Vries e§_al,, 1973). According to Wolin (1975), most, if not all, of the propionate in the rumen is produced via succinate (eg. dicarboxylic pathway). Hydrogen is rapidly utilized in the rumen through its reaction with CO2 to form CH4 or acetate (Hungate, 1966). ln_yj§rg_work conducted by Carroll and Hungate (1955) has shown that CH4 is the predominant end product in regard to H2 utilization in an anaerobic system. Methanogenesis has been evaluated in regard to its possible effect on energy yields 14 within the rumen (Scheifinger et_§1,, 1975; Hungate, 1966). Methane production represents a loss of H2 that could be transmitted to pro- pionate in the absence of methanogenic bacteria (Scheifinger g§_§1,, 1975). There is no net change in ATP synthesis when shifting from CH4 production to propionate synthesis, yet metabolic H is conserved and propionate 2 production is enhanced (Demeyer e§_a1:, 1975). Nevertheless, in curbing CH4 synthesis rumen function and feed utilization are disrupted (Garton et_a1,, 1972; Demeyer and Van Nevel, 1975). According to Demeyer and Van Nevel (1975) inhibiting methano- genesis may result in depressed cellulolysis and fermentation rates, pro- tozoa production may be decreased, proteolysis is depressed and H2 easily accumulates. As a result of the above, microbial growth may decrease and alter the site and extent of protein and CHO degradation. Even though the propionic levels will increase with the prevention of methane production, the use of methanogenic bacteria inhibitors may supress rumen function and microbial synthesis to a point where the beneficial energy derived from increased propionate levels will not be realized to the fullest extent. Bauchop and Elsden (1960) concluded that the amount of microbial growth was directly proportional to the amount of ATP that could be ob- tained from the fermentation of the energy source present in the medium. This relationship between cell growth and ATP was defined by Bauchop and Elsden (1960) as YATP (grams dry weight synthesized/mole ATP). Initially, the YATP was thought to be a constant factor of 10.5 for all microbes (Forrest and Walker, 1971 and Stouthamer, 1969). Based on a YATP of 10.5 and assumed stoichimetry of ATP/mole VFA, Hungate (1966) calculated that 15 the rumen fermentation process can produce approximately 10.0 grams of microbial protein/100 grams of organic matter (0M) digested in the rumen. This 10 grams/100 grams OM fermented would represent the "upper limit" of microbial protein synthesis due to the available energy in 100 grams of fermented carbohydrate. Studies by Stouthamer and Bettenhaussen (1973) indicated, however, that YATP was not constant for all microbes and that the efficiency depended on the microbes' growth rate and maintenance requirements. These researchers studied the growth rate of_A. aerogenes in an energy limiting chemostat. Stouthamer and Bettenhaussen (1973) showed that as growth rate increased, a con- comitant increase in YATP was evident. The Y2$é (molar growth yield for ATP corrected for energy of maintenance) was detennined to be about 25. They showed that slower growing organisms had a much higher maintenance requirement (biosynthetic reactions). Therefore, it was suggested that YATP was a function of the dilution rate (growth rate) and maintenance requirements of that microorganism (Stouthamer and Bettenhaussen, 1973; Bergen and Yokoyama, 1977). Maintenance energy for bacteria is necessary for the turnover of cell constituents, motility, the preservation of right ionic composition and intracellular pH of the cell and replacement of lysed cells (Marr .et_gl., 1963; Stouthamer, 1977 and Owens and Isaacson, 1977). The amount of energy used for the maintenance is termed me or maintenance coefficient and the yield in grams dry cells/mole ATP above maintenance, YX$5 (Stouthamer and Bettenhaussen, 1973). Isaacson §§_g1,, (1975) and Van Nevel and Demeyer (1979) supported the observations of Stouthamer and Bettenhaussen (1973) with mixed rumen 16 cultures grown at various dilution rates in a continuous culture system with glucose as substrate. They found that YATP in rumen bacteria was not constant but varied with dilution rate. At 2% turnover per hour ATP energy used for maintenance as a percent of the total available ATP was 60%, but when the dilution rate increased to 12% per hour, only 15-20% of the total energy was utilized for maintenance. In a continu- ous culture system maintained at a constant dilution rate, Isaacson et_al, (1975) showed bacterial growth efficiency did not change with varied sub- strate levels. However, when dilution rate was increased, a correspond- ing increase in microbial growth efficiency was observed. This increase in cellular growth rate efficiency was due to a decreased microbial popu- lation and residence time within the fermentor. Cole e}; _a_l_. (1976a), Kropp e_t__a_l_. (1977) and Harrison and co— workers (1975) observed with ruminants fed various rations of roughages, grains and protein supplements that as dilution rate increased (.02 to .06 h“), microbial synthesis also increased. The above jg_ijg_data exemplify the conclusions made by Stouthamer and Bettenhaussen (1973) and Isaacson e§_§1, (1975) that the efficiency of microbial growth is dependent on maintenance expenditures which, in turn, is a function of rumen turnover. Rumen turnover will effect the molar proportion of propionic acid (Hodgson e£_a1,, 1975). With every 1% increase in rumen turnover, a decrease of 1.5 to 8% propionate is observed concomitant with increases in acetate and butyrate. Isaacson e§_a1, (1975) concluded that fermen- tation balance equations indicated that such a shift in VFA was accom- panied by increased levels of CH4 and heat losses and decreased ATP synthesis. 17 .Contrary results to those of Isaacson et_a1, (1975) and Hodgson et 21, (1975) regarding the molar proportions of VFA produced during the fermentation were reported by Van Nevel and Demeyer (1979). In a chemostat with glucose as the growth limiting energy and carbon source, mixed rumen bacteria were maintained at four dilution rates. Increased dilution rates shifted the proportions of the end products: methane decreased and propionate increased. The reasons why propionate increased with increased dilution rates are unclear. However, Van Nevel and Demeyer (1979) suggest several possibilities for such a shift. First, with increased dilution rate, more glucose can be fermented per unit of time resulting in a shift of metabolism toward lactate production and ultimately propionate synthesis. A change in microbial populations (eg. decreased methanogenic bacteria) due to faster rumen turnover may also stimulate alternate electron sink acceptors (eg. propionate). From fermentation balance equations, metabolic H2 recoveries were calculated and found to be similar to those of batch cultures (Demeyer and Van Nevel, 1975). Such data contradict the metabolic H2 recoveries calculated by Isaacson §t_a1, (1975). The difference was suggested by Isaacson and coworkers (1975) to be a result of a bacterium that could utilize acetate or longer chained fatty acids for methanogenesis. If this was the case, their Y values were erroneous since a portion of ATP their maintenance requirements were met by energy obtained from a source other than glucose. Van Nevel and Demeyer (1979) found no such bacterium within their culture system and attributed the presence of the microbe to a difference in inoculum donors and/or the rations they were consuming. The capacity for microbial cell synthesis is dependent on the ATP availability mainly derived from rapidly fermented carbohydrates and the 18 efficiency at which the ATP is utilized. From a general viewpoint, higher cereal grain diets encourage lower dilution rates (at least less than full ag_1jb, intakes) with higher ATP production but lower Y P yields. Conversely, those diets that encourage high dilution AT rates will produce less total ATP but a higher Y From this example ATP' one can see how under two different flow rates the amount of microbial cells produced could be similar. In order to obtain maximal microbial synthesis, energy avail- ability and CH0 source are not the only factors to be considered. Ade- quate supplies of NH3-N, carbon skeletons, sulfur, free amino acids and other cofactors are necessary. The whole scheme of VFA production from energy sources (eg. carbohydrates) and microbial synthesis is a coupled process and a deficiency of any one of the metabolites will create limita- tions on catabolic and anabolic processes which will ultimately lead to decreased VFA and microbial production (Bergen and Yokoyama, 1977). MONENSIN EFFECTS ON PERFORMANCE Monensin, a biologically active compound (Haney and Hoehn, 1967) synthesized by Streptomyces cinnamonensis has improved cattle perfor- mance by altering rumen fermentation patterns thereby enhancing feed efficiency (Potter g§_gl,, 1976a; Utley et_a1,, 1976; Mowat e;_§1,, 1977; Perry §£.El-: 1979; Hanson and Klopfenstein, 1979). Data from many research stations have consistently shown that feeding monensin to sheep and cattle has resulted in a decrease in rumen acetate production with a concommitant increase in propionate synthesis of 35-40%; total VFA production changed little, if any (Richardson et 11., 1976; Prange .et_g1., 1978). It has been suggested that since the propionic acid is used more efficiently in terms of a metabolizable energy source than acetate (Raun et_g1,, 1976; Van Nevel gt g1., 1969; Hungate, 1966; Wolin, 1960), cattle fed rations plus monensin should gain more effi- ciently than those fed rations without the benefit of the additive. However, Drskov e§_gl, (1979) has recently shown that in sheep infused with various volatile fatty acid (VFA) mixtures (ie. acetic, propionic and butyric) there were no significant differences in the efficiency of utilization of VFA mixtures for maintenance or energy retention. This agrees with the observations of Armstrong and Blaxter (1957), Bull et_g1, (1970) and Orskov and Allen (1966). Goodrich and coworkers at Minnesota (1976) summarized data from 28 trials with steers and heifers which were fed various types of rations with and without monensin. A range of monensin concentrations (5.5, 11.0, 22.0, 27.5, 33.0 and 44.0 ppm of ration DM) were provided in the various rations. The results showed that all cattle with the 19 20 exception of those fed 44.0 ppm monensin, had gains equal to or greater than the contrdls. All cattle fed the additive decreased their feed intakes with increasing monensin levels. Feed efficiency was improved across all treatment levels with the maximum improvement (eg. feed/gain decreased but improved) noted With cattle fed 27.5 ppm monensin. Two separate trials were conducted by Boling g3 g1, (1977) to study: (1) the influence of monensin level on gain and VFA production in Angus steers grazing on a Kentucky Bluegrass - clover mixture supple- mented with either 0, 25, 50 or 100 milligrams monensin per head per day and; (2) the influence of monensin (0, 100, 200 or 300 mg + 4.54 kg corn gain/head/day) on finishing steers fed corn silage gg_libitum. In the first trial, average daily gains were greatest for those Angus fed the 50 or 100 mg monensin per day (P <.01) when contrasted to steers fed either 0 or 25 mg monensin. Ruminal propionate increased (P <.01) in , all monensin fed groups. Carcass data were similar although those cattle fed 300 mg monensin per day tended to have lower marbling scores, smaller ribeye areas and less fat over the rib. Data from Brown e§_al, (1974), Potter gt 1. (1976) and Thonmey (1977) support the findings of Boling in that monensin had no consistent effect on carcass quality or cut- ability. Heifers fed monensin (33 ppm) and implanted with Synovex-H gained 11.5% faster and 6.5% more efficiently than heifers fed monensin without the benefit of a hormonal implant (Woody and Fox, 1977). Similar results were obtained by Burroughs e§_al, (1976) in feedlot steers fed monensin. 21 According to Nissen and Trenkle (1976), the addition of monensin to feedlot rations initially reduced dry matter intake by as much as 15-30%. However, intake returns to 90% of the intake recorded for steers fed the control ration by the end of 30 days. The adaptation of both the animal and rumen ecosystem to the monensin may have made an adapta- tion period a necessity (Poos et_a1,, 1979). In vitro studies by Simpson 23.21: (1976) and Simpson (1978) found monensin to inhibit cellulolytic activity in an inoculum obtained from an animal not previ- ously exposed to the additive. However, if the animals are adapted to monensin for three weeks prior to inoculum removal from the rumen, no inhibition of cellulolytic activity could be observed (Dinius et_a1,, 1976). In cattle that had been adapted to monensin and then fed a diet supplemented with the additive (33 ppm),Dinius e;_§1, (1976) reported that the additive had no major effect on dry matter, protein, hemicellu— lose or cellulose digestibilities nor did it effect the total microbial population. This is in conflict with the report by Simpson et 11. (1976). Simpson (1978) and P005 e;_a1, (1979) found that monensin was a potent inhibitor of protozoa and cellulolytic bacteria. The relationship among fermentation rate, dilution rate and the extent of digestion in the rumen is paramount in the determination of protein and AA nitrogen (NAN) reaching the duodenum (Bergen and Yokoyama, 1977). The conversion of dietary N to microbial protein is less effi- cient in terms of energy utilized with slower rumen turnover rates. When rumen turnover increased, efficiency is significantly increased since microbial populations and residence time in the rumen decline (Owens and Isaacson, 1977). 22 Lemenager, Owens and coworkers (1978) in Oklahoma conducted a series of in yjy9_studies to evaluate the effect of monensin on rumen turnover, cellulose disappearance and nitrogen components of rumen fluid. Rumen cannulated steers fed low quality winter grass, supplemented with soybean meal and 200 mg monensin per day had a 15.6% (P <.02) lower daily feed intake than steers fed grass and soy without monensin. The authors suggested that in steers fed monensin supplemented roughage rations, the lower feed intake was due to a decreased rumen digestion rate resulting in a 44% slower solid and 31% slower liquid turnover rate. Hence the limit of feed intake in steers fed monensin roughage ration is rumen capacity. In the second trial, steers fed a high concentrate ration with or without monensin maintained constant intake levels, however, rumen turnover rate decreased. Lemenager gt_a1, (1978) suggests several reasons why there seems to be a relationship between forage intake and rumen dilution rate. With roughage rations, rumen turnover may simply be depressed due to a decrease in intake (Balch and Campling, 1965). However, with high con- centrate rations, intake usually remains constant, yet turnover decreases thus indicating the monensin's effect of depressed dilution rate is inde- pendent of intake. Therefore, decreased rumen turnover in ruminants fed monensin may cause a decrease in intake dependent on the type of ration fed, ie. concentrate or roughage. The authors further speculated that a decreased rate of digestion possibly was responsible for reduced intake. If the digestion of feed particles is retarded, the small size needed to pass into the omasum will not have been reached for a longer period of time resulting in a prolonged rumen retention time. The decrease in dry matter intake 23 (energy) does not necessarily indicate a detriment to animal performance since increased propionate synthesis, decreased methane (CH4) production, heat loss and gross fecal energy compensate for that decrease in intake (Lemenager and coworkers, 1978). With the shift of molar percentages of VFA‘s towards more pro- pionic and less acetic and butyric acids, the question arose as to how this increase in propionic acid caused improved efficiencies within the ruminant's metabolic system. Hungate (1966) concludes that propionic fermentations are more efficient due to the reduction in CH4 losses which occur with the production of acetate and butyric. Using the equations for the calculation of theoretical fermentation balances of Wolin (1960), it could be seen that the reduction in CH4 production accounted for some increase in propionate efficiency. Demeyer and Van Nevel (1975) stated in a review on methanogenesis and its control, that a decline in CH4 production as a result of high rates of rumen fermentation or turnover would increase the molar percentage of propionate and decrease that of acetate. A shift from a 60% acetate: 30% propionate: 10% butyrate to a 52:40:8 ratio would reflect a 5.6% increase in gross energy savings to the animal (Hungate, 1966). Eskeland et_a1, (1974) reported that propionic acid infusion increased N retention in ruminants when contrasted to infusions of acetic and butyric acids. The increase in retention may have been mediated through a protein sparing effect. Since both propionate and amino acids can serve a precursors of gluconeogenesis (Leng et_al,, 1977), a greater efficiency of propionate utilization may allow more amino acids to be used for anabolism rather than gluconeogenesis (Reilly and Ford, 1971). 24 Potter gt 11. (1976) showed increased blood glucose concentrations in cattle pastured on orchard grass, alfalfa, brome and ladino clover mixtures and supplemented with soybean meal and monensin when compared to those fed only the forage. , Blaxter (1962) suggested that the efficiency of propionate utili- zation in ruminant tissue is greater than for either acetate or butyrate. Smith (1971), in a general review of the subject has found that researchers are not in agreement as to propionate's efficiency at the tissue level. However, if the propionate is used more efficiently at this point, the benefit would be additive to that obtained during rumen fermentation by ~1owering CH4 losses. Poos 23.21: (1979) conducted a metabolic study with steers fitted with abomasal cannulae to determine any differences in digestibility, ruminal protein degradation (bypass) and microbial protein synthesis of ground corn-milo based ration supplemented with Brewer's dried grain or urea; with and without monensin. Monensin decreased bacterial nitrogen flow (P <.05) while increasing plant protein passage to the abomasum regardless of protein supplement. As previously discussed, monensin tends to reduce the cellulolytic bacteria population. These bacteria are required to have NH -N as an N source irregardless of the presence of 3 preformed amino acids (Blackburn, 1964). A decrease in the total N and NAN flow might be indicative of this decrease in cellulolytic bacteria in ruminants fed diets supplemented with urea and monensin. Further work at Nebraska by Hanson and Klopfenstein (1979) with similar rations indicated that when a preformed protein source was used as the supplement, that the addition of monensin did not result in an 25 increased dietary protein need as dry matter intake was depressed. An increase in dietary levels of preformed protein (10.5% to 12.5%) caused significant (P<=.05) increases in gain. Such an increase in protein equivalent from urea did not improve performance, however. Hanson and Klopfenstein (1979) suggested that the protein reaching the duodenum was the limiting factor for improved animal performance. Gain differences were not apparent at different urea levels thus indicating that ammonia nitrogen was not the limiting factor but rather microbial protein syn- thesis. If monensin caused increased microbial protein synthesis, one would expect increased average daily gain in those steers fed the higher urea levels, however, the opposite occurred. The work of P005 et_g1, (1979), Richardson e§_g1, (1978), and Hanson and Klopfenstein (1979) support the idea that microbial protein synthesis is inhibited (or at least not changed) with the addition of monensin, but that total ruminal degradation of preformed protein is markedly lower. Producers would benefit to the greatest extent if they utilized monensin when feeding a natural protein supplement. Although monensin depresses protein degradation at any dietary protein level, once the animal's requirement has been exceeded with a large supply of protein escaping rumen degrada- tion any further increase in ruminal bypass of protein would not stimu- late production any further. MARKERS SYSTEMS USED IN DIGESTA PASSAGE STUDIES Various types and combinations of marker systems have been em- ployed to assess digesta passage to the abomasum or duodenum of rumin- ants in an effort to estimate rumen liquid and dry matter turnover rates, non-ammonia nitrogen passage, extent of ruminal organic matter digestion, microbial protein yields and protein which escapes ruminal degradation. An ideal marker must satisfy the following criteria in order to be considered for use in passage studies (Engelhardt, 1974 and Kotb e;_al,, 1972): (1) the marker must be non-absorbable, (2) it must not affect or be affected by the animal's gastrointestinal tract, its environment or the microbes, (3) the substance should be physically similar to or closely associated with the fraction of digesta under study, and (4) the method of analysis for the marker must be sensitive and specific for that marker and not interfere with any other chemical analyses. As researchers can attest, there is not one marker available for animal research today that will qualify as an ideal marker and satisfy all the criteria previously described. It next becomes the main objective of the researcher to obtain a marker that will satisfy as many criteria as possible. Any great deviation from the criteria aforementioned will result in serious miscal- culations in regards to the extent of digestion, time of rumen retention (i.e. dilution rate) and digesta passage. Markers may be administered by many techniques such as continuous infusion with time-sequenced sampling, continuous infusion with total collection or single dosage with time sequence sampling. Liquid passage may be measured through employing polyethylene glycol (PEG) (Sperber gt g1., 1953), the 5Ichromium complex of ethylenediaminetetra-acetic acid 26 27 (5]Cr-EDTA) (Downes and McDonald, 1964), Cr-EDTA (Downes and McDonald, 1964) or phenol red (Hecker gt_g1,, 1964) as inert markers either singu- larly, in combination with each other and/or in a mixture with particu- late markers chromic oxide (Purser and Moir, 1966), ruthenuim phenathro- line (Tan et_g1,, 1971), other rare-earth elements (Hartnell and Salter, 1979) and the internal marker already present in the ration, lignin (this by no means is the total number of markers available to researchers). Since this author dealt only with Cr203, PEG and lignin as indigestible markers, the review of literature will pertain only to these entities. Faichney (1975), in a review article on the use of markers in digesta studies, pointed out that Cr203 moves independently of the liquid and particulate phases of the digesta thus making any accurate estimate of flow rates rather dubious. Drennan, Holmes and Garrett (1970) con- cluded that in trials with sheep and cattle fed high concentrate diets, that chromic oxide, fed as a powder mixed into the ration, flowed much more rapidly out of the rumen than did the digesta. Their calculations of ruminal digestion using Cr203 as the marker gave digestion coeffi- cients which were not possible, i.e. too high. I The amount of the starch digested in the rumen of these sheep when lignin was used as a marker was less than the organic matter (OM) digested, but when chromic oxide was used as the marker, more starch was digested than total ON by about 500 grams. Likewise, Cr203 data depicted that twice as much protein left the abomasum than was ingested, indi- cating a rather absurd value for endogenous nitrogen secretion. Lignin based data for nitrogen passage was credible. The data of Drennan gingl. (1970) was in agreement with that pro- vided by other workers that used chromic oxide powder and similar ‘28 sampling techniques. The general conclusion was that passage data based on Cr203 powder was unusable. The values recorded by using lignin as the marker were reasonable and consistent when contrasted to investigators (Topps g3_§1,, 1968; Nicholson and Sutton, 1969; Orskov and Fraser, 1968) using PEG or Cr203 impregnated paper as their reference sources. It would seem from this study that lignin represented a better estimate of digestion and passage than Cr203 powder. Chromic oxide, whether administered as a powder, impregnated into paper or baked with wheat flour and ground into a powder, shows a pattern of diurnal variation (Faichney, 1975; Wilkinson, 1970, Kane §§_a1,, 1952 and Davis e§_al,, 1958) which may correspond to cyclic changes in gastrointestinal physiology with the lowest point in the cycle occuring at night. It would therefore become apparent that when Cr203 is utilized, regardless of form, that long sampling periods be employed at various times, day or night. These individual samples in order to be representa- tive should be pooled and subsampled for analysis since the concentration ofCr203 will vary (MacRae and Armstrong, 1969; Harris and Phillipson, 1962). Incomplete marker recoveries are quite common in the majority of experiments reviewed. According to Sutton e;_a1, (1976), incomplete recoveries could be the result of a depression in flow due to sampling interference with the animal. Sutton e§_al, (1976) claimed marker collection for recovery purposes beyond day three is useless and an ad- justment to 100% recovery should be made at this point. Sutton g§_§1, (1976) further pointed out that adjusted recoveries are often undesir- able due to higher standard errors associated with such an adjustment. 29 Pitzen (1974) obtained recoveries of Cr203 as low as 59% and when ad- justed to 100% recovery, the flow data was much more variable and incon- sistent. Some of the adjusted microbial crude protein values were higher than theoretically possible under normal ruminal conditions (Walker, 1975). Pitzen concluded that Cr203 paper was unsuitable for determining digesta flow rates in the ruminant. A decrease in digesta flow is not the only possible theory for incomplete marker recoveries. Curran, Leaver and Weston (1967) suggested that losses due to regurgitation, fecal loss, loss while grinding samples, analytical losses and the absorption of soluble chromates will decrease marker recovery. Deinium et_a1, (1962) found traces of chromate in the liver, lymph, kidneys and lungs of cows fed chromic oxide impregnated paper. The data of Deinium e§_g1, (1962) clearly shows a violation of one of the paramount criteria of a marker; that it be non-absorbable. In more recent data, Poo; et a1. (1979) using a dual marker system (Cr203 and PEG) in steers, calculated that total N reaching the abomasum exceeded the N intake. The researchers suggest that an under- estimation of ruminal degradation and an over-estimation of particulate matter passage to the abomasdm was the reason for the increased total N reaching the abomasum when compared to the N intake. Such problems with Cr203 may be found without difficulty throughout current literature. Polyethylene glycol was first introduced as a soluble marker to study the movement of water in the rumen by Sperber, Hyden and Ekman (1953). Their method of administration (injection into the rumen) resulted in a "hit and miss" type of approach which ultimately led to misconceptions and misleading data (Termouth, 1967). Under some conditions 30 approximately 5% of the PEG (molecular weight = 4000) was found to be associated with the particulate phase of the digesta, a violation of Engelhardt's (1974) and Kobt et al.‘s (1972) criteria for a reference marker. Another serious drawback to use of PEG as a marker is the lack of a specific, sensitive and accurate method of analysis (Downes et_a1., 1964). All known methods are based on the precipitation of PEG from an aqueous solution. With this type of procedure, incomplete recoveries of the marker were obtained due to analytical losses (Smith, 1955; Corbett, 1958). This underestimation of PEG concentration will ulti- mately result in an overestimate of total liquid flow from the gastroin- testinal site (Bergen, 1979; Lemenager e§_a1,, 1978). I Lignin is often employed as an internal marker to check the validity of the other liquid and particulate markers calculated digesta flows. Lignin, although there is no question as to its association with the particulate phase of the digesta, does have problems associated with its usage as a reference marker. In high grain rations the concentration of lignin is rather small, thus sampling and analytical procedures become limiting. Acceptable methods for lignin analysis (Van Soest, 1963; Collings, 1979; Johnson e; 31,, 1961) are highly variable among labora- tories thus limiting the usefulness of lignin as a marker since compari- sons of flow data would become futile. According to Drennan e;_a1, (1970), data based on lignin calculations may not be correct, but in comparison to data based on Cr203 as a marker, it is at least feasible. The section of the gastrointestinal (GI) tract choosen for cannulation, the location of cannulation within the choice of organ and the type of cannual system utilized (eg. "T" type or re-entrant) may influence passage data. Sampling digesta through an abomasal "T" type 31 cannula will not result in an increase of digesta flow from the upper GI tract to compensate for that lost during sample collection. However, obtaining a representative sample of abomasal digesta is difficult because of the stratification of feed within the organ resulting in channels for liquid flow. Thus, a dual marker system (one each for the liquid and particulate phase) should be used since the liquid and solid portions do not necessarily move together. The cannula itself should be placed in the abomasum just anterior to the pyloric junction in order to obtain the best possible representative sample of digesta that would be available for absorption in the duodenum. A duodenual "T" type cannual has been acceptable for spot sampling of digesta flow from the abomasum. A cannula inserted anterior to the common bile duct represents an excellent site for the collection of samples that are adequately mixed and highly representative of chyme available for absorption. However, the surgical procedure proves more difficult compared to the abomasal insertion with more complications occurring post-operatively. A duodenal re-entrant cannula system is capable of collecting a good representative sample and excellent flow data can be obtained, but one major drawback is the necessity to replace the volume of digesta sampled with donor digesta. If the digesta is not replaced, digesta flow rates from the upper GI tract will increase to compensate for the losses to the jejenum (MacRae, 1975) thus giving erroneous rumen outflow data. Other drawbacks which cause some researchers to choose "T" type cannulae in preference to the re-entrant type are the increased labor involved with sampling, more complicated surgical procedures with increased post- operative problems and a shorter life expectancy of the animal after sur- gery. OBJECTIVES The optimal level of non-protein nitrogen addition to rations fed to high producing cattle in respect to the efficiency of microbial cell yield has been debated for a number of years. Several researchers have attempted to pinpoint at what level further increases in rumen NH3-N concentrations will not enhance microbial cell protein yield thus making NPN additions to the diet unnecessary. Satter and Slyter (1974) in an jn_vjt§o chemostat experiment determined that 5.0 mg % NH3-N was suffi- cient for maximal microbial growth. This was equivalent to approxi- mately ll-l4% crude protein equivalent (CPE) in the ration. Diets that produce rumen NH3-N concentrations above this level should not increase microbial cell protein synthesis and the added CPE would be wasted. Therefore, it was the objective of Experiments One and Two to: (1) determine the effect of protein level on total nitrogen and NAN passage to the abomasum in steers. (2) determine if supplemental NPN above 13% CP will result in increased non-ammonia nitrogen passage to the lower gut in steers. Corn silage may be supplemented with either preformed protein or NPN in order to provide adequate nitrogen to the rumen microbes for microbial protein synthesis. Corn silage is well suited for NPN supple- mentation due to its high energy and relatively low protein content. The addition of NPN at ensiling has decreased the amount of feed steers con- sumed per unit gained when compared to untreated silage supplemented with NPN at feeding (Ely, 1967). Data concerning N passage to the abomasum is limited in steers fed anhydrous ammonia treated silage supplemented 32 33 with monensin. Therefore, it was the objective of Experiment Three to: (1) determine the effect of silage treatment (protein level) on nitrogen parameters that pass to the abomasum in steers, (2) determine if monensin affects nitrogen flow to the abomasum in steers, (3) determine if there is an anhydrous ammonia-monensin inter- action on nitrogen passage to the abomasum in steers, (4) determine the validity of using a two phase marker system in determining N passage from the rumen and ruminal liquid and particu- late outflow rates, (5) determine if monensin effects dilution rates in the ruminant and (6) compare two systems of abomasal sample analyses (individual time analysis vs composite analysis) and to see if there is a correlation between any one or two times and the composite. There has been little work done on the subject of nitrogen reten- tion in steers fed corn silage that had been treated with various levels of anhydrous ammonia and supplemented with monensin. It was the purpose of Experiment Four to: (1) determine the effect of silage treatment (CP level) on nitrogen retention and (2) determine if monensin had any effect on nitrogen retention in steers fed the various anhydrous ammonia treated rations. MATERIALS AND METHODS A. EXPERIMENT ONE 1. Design of the Experiment A total of five Holstein steers with a body weight of approxi- mately 300 kg were fitted with an abomasal "T" type cannulae and housed in 91 x 244 cm metabolic stalls. The animals were fed one of three ex- perimental rations twice daily (Table l) at 90 percent voluntary intake and had free access to water. Each steer was fed at least two of the three rations in this completely randomized trial. The concentrate diets fed were corn based with relatively constant percentages of oats and corn cobs present across treatments (see Table 1). They were formulated in order that the low protein diet{ 7% crude protein (CP)} served as the basal ration and the subsequent two rations were formulated by adding soybean meal to final levels of 9.9 and 12.6% CP respectively. A chromic oxide-wheat flour mixture (1:4) was added at one percent of the ration dry matter (Orskov eg_a1,, 1971). The Cr203 was mixed with the flour to form a paste, oven dried and then ground to pass through a 1 mm wire mesh screen in a Wiley mill. All diets were similar in digestible energy values (meal/kg). 2. Cannula Design and Insertion The abomasal "T" type cannulae were manufactured from viscous plastisol (Norton's Plastic and Synthetics Div., Akron, Ohio) and Mystaflex (M & R Plastics and Coatings, Maryland Heights, Missouri). The liquid was placed under a strong vacuum for at least one hour to alleviate 34 35 .>~m>?uumammc .A.:.HV muwcz chowpmccmucH oom.mm Ucm ooo.mNF m000.000... mm tmvum mew: m .o .< mcwEmuw> a w.m_ m.m o.“ Am_m»_ccmv mm.ovxz Pm.m mm.m wo.m Aumpmfisoqu .GX\_muzv mm o._ 0., o._ L:o_c “was; -momtu o.N o.m c.m mpmcmcwe munch o._ o.F o._ ome-_c-m momeqwoga xuoe empmceczo_cmo - - o.m mmm-mo-q nucmpm :Lou o.m. o.m o.m mm©-qo-e mammmfioe memo 0.0? o.o_ 0.0? mom-mo-e muse o.oN 0.0N o.mm NmN-No-_ mnou ctou o.o_ o.m - eoo-¢o-m me-»om o._m o.wm o._¢ Nmm-mo-m ceou & m N _ , .o: .cmm .ch IpcmwumtmcH mco ucmecqum c? mcmmum ou vow mcowpmm .P m4madaptation for 17 days 1 Day I collection at 0 + 6 hours postfeeding Day‘2 collection at 2 + 8 hours postfeeding 1 Day 3 collection at 4 + 10 hours postfeeding l adjust to new ration for 4 days 1 adapt for 17 days 1 collect as above for new ration 38 stored. Daily feed samples were also retained and composited for sub- sequent analysis. Prior to analyses for total nitrogen, ammonia nitrogen, chromium and acid lignin, the abomasal composites were thawed, acidified with 6N hydrochloric acid to a pH of 2 and then oven dried at 55°C. Subsequent to drying, the samples were ground through a .5 mm mesh screen in a Wiley mill. 4. Nitrogen Determinations Total nitrogen was determined on all feed and abomasal composites with a Technicon Auto Kjeldahl system. Non-ammonia nitrogen levels were calculated by subtracting ammonia nitrogen values obtained by steam distillation over alkali from corresponding total nitrogen figures for that sample. 5. Chromium Analysis Chromium concentrations in feed and abomasal samples were deter- mined by a nitric-perchloric acid digestion followed by atomic emission spectrophotometry utilizing an I.L. 453 Atomic Absorption-Emmission Spectrophotometer. Approximately 200-300 mg of sample was placed in a 250 ml Phillips beaker followed by the addition of 25 ml of nitric and 4 ml of perchloric acid. The samples were digested under a hood at a slow boil until only 1-2 ml of liquid remained. At this point the sample should have been yellow to orange in color. After cooling the sample was diluted to 100 ml (grams). The oxidized samples were then read on the Atomic Emmission spectophotometer at a wavelength of 424.8 nm, scale of 2.5, slit width 39 of 80 and using the nitric oxide burner head. A chromium standard curve was obtained by preparing solutions with 0, 2, 5, 10, 15 and 20 ppm chromium and subjecting them to the nitric-perchloric digestion and dilution. 6. Lignin Determinations Dried feed and abomasal samples were subject to the permanganate lignin procedure of Van Soest (1963). 7. Determination of Daily Flow Rates From the Rumen Daily N flow was determined by a marker ratio technique (Fenderson and Bergen, 1975) utilizing the following relationship: Total N flow = N: marker ratio in abomasal digesta x g N consumed (g/day) N: marker ratio in feed ingested per day Non-ammonia nitrogen passage was calculated by subtracting the NH3-N from the total N flow. The NAN data were not adjusted for endo- genous protein secretions. The above nitrogen: marker relationship may be used when chromium or lignin are used as reference markers. 8. Statistical Analysis Data were statistically analyzed by analyses of variance on a Hewelett Packard 9825 A computer. The difference among means were deter- mined by the Studentized Range Test utilizing the table of Rohlf and Sokal (1969). 40 B. EXPERIMENT TWO 1. General Design Three Hereford steers averaging 325 kg and housed under the iden- tical environmental conditions as in Experiment One were fed the 12.6% CP ration used in the first trial but supplemented with urea to 15.7% CP (Table 2). The feeding regime, abomasal collection schedule, and labora- tory analyses performed were similar to that described for Experiment One. The Holstein steers from Experiment One could not be used in this trial because they developed severe feet and leg problems prior to the collection period of abomasal samples for the urea supplemental ration. Due to this problem, a switch in steers to Herefords was necess- itated and, hence, the data obtained from the Hereford steers could not be statistically compared with data accumulated from the abomasal samples obtained from steers in the first trial. 2. Determination of Daily Flow Rates from the Rumen Daily N flow was determined by a marker ratio technique (Fenderson and Bergen, 1975) utilizing the following relationship: Total N flow = N: marker ratio in abomasal digesta - (g/day) N: marker ratio in feed ingested X g N intake/day Non-ammonia nitrogen passage was calculated by subtracting the NH3-N from the total N flow. The NAN data were not adjusted for endogen- ous protein secretions. The above nitrogen: marker relationship may be used when chromium or lignin are used as reference markers. 41 .x—m>wuomammc ..:.H oom.mm new oco.mNF mooo.ooo.o_. mm vmuum mew: m “a .< mewEmpw> « e.m_ AnmNaemcmv mm.m x: mm.m Aumomesuqu,.mx\Pmuzv me o._. pmmzz I MONLU n. woe: o.m mchmaE munch o._ ome-_o-m mpmgamoea xuoe nmom:weso_cmo o.m mmo-¢o-e mammmpoe memo o.o_ mom-mo-s mpmc o.om NwN-No-_ _ mnou ctou m.o_ qoc-¢o-m me how o.cm Nmm-mo-e . :200 s P .o: .cmm .ch RostrumtmcH 02H ucmewcqum :? mcmmum op vow cowpmm .N meqk 42 C. EXPERIMENT THREE 1. Design of the Experiment Four Hereford steers with an average body weight of approximately 380 kg and fitted with abomasal "T" type cannulas were fed a total of six corn silage rations differing in the level of anhydrous ammonia R - Elanco applied at the blower with or without monensin (Rumensin Products Co.) in the supplenent. Every steer received each ration during the duration of this switchback designed trial. The animals were fed twice daily (8 A.M. and 8 P.M.) at 90% of voluntary intake. Voluntary intake levels were dropped to 90% at least one week prior to the collec- tion of abomasal digesta. Water was available free choice. Exogenous chromic oxide and lignin were used as insoluble markers. The former was added to the vitamin-mineral supplement as a chromic sesquoxide:wheat flour mixture (1:4). As a soluble marker, polyethylene glycol (PEG - molecular weight 4000) was also added to the vitamin mineral premix. These markers were utilized to quantitate total nitrogen and NAN passage to the abomasum as well as ruminal liquid turnover rates for the various rations studied. All corn silage used was obtained from the same source on the Michigan State University farm system and differed only in the level of anhydrous ammonia applied upon ensiling. Untreated corn silage served as the control and silage treated with either five (7.8 g anhydrous ammonia/ kilogram corn silage dry matter AN/KGCSDM) or ten (15.6 g AN/KGCSDM) pounds of anhydrous ammonia per ton of harvested silage comprised the other two protein treatments. The control silage contained 7.4% crude 43 protein on a dry matter basis and the 5 lb. anhydrous and 10 1b. anhydrous ammonia treated rations contained 9.8 and 12.4% crude protein, respectively. All three silage treatments were supplemented with a vitamin and mineral premix with or without Rumensin (Table 3 and 4), thus bringing the total number of treatments tested to six. 3. Samp1e Collection and Preparation Abomasal samples were collected three times daily (around the clock) over a four day period. The designated times for sampling (Figure 2) varied in order of sequence from day to day in an attempt to obtain a representative sample from the abomasum, thus alleviating any error due to diurnal variation of chromic oxide flow. The abomasal digesta were aerated jn_§j§g_to promote mixing as outlined in the first experiment and approximately 125 m1 of abomasal digesta were collected and frozen for analyses. It was an objective of this study to perform all analyses (i.e., dry matter, total nitrogen, ammonia nitrogen, chromium, ADF, lignin and PEG) on one silage treatment (15.6 g AN/KGCSDM with and without Rumensin) on an individual sample basis and compare that data to data obtained when abomasal samples were pooled for that particular treatment and steer. One or two time periods that abomasal samples were obtained could conceivably be highly correlated with the total sample composite thus possibly alleviating the need for around the clock or multi-collection sampling. The other silage treat- ments were analyzed on a composite basis, i.e., all samples per steer per treatment were pooled and analyzed. The individual 125 ml samples were divided into three portions; 30, 70 and 25 gm. The 30 gm fraction was centrifuged at 15,000 rpm for 44 .2o cowpmc Fmpop to &m. mm umuum ooo.¢ xmzoncmu . ammo m N .29 coves; _muou &_ mm Acne—e paws; mpcma q op o co pcma EV umuum muwxozcmmm sawEoczuo .me>Fuumamwc .Emcm can mprcn choppmccmucH 000 m ucm ooo om op Pascm o w < mcpsmpr>n so cowpmc mo Egg o.mm mo mpmc a pm umcum :wmcm23mm m. upouz—m mcmPASpmzpoa o._ ox?e muvxo umsocgu _.om mpmgqmoca xuoc cmpmcwczopmmo a. no swampw> e. n< swampw> m.~ mom . :wmcm53m N.m prom chmcwe munch F.mF mumszm Ezwopmu ¢.n¢ mmmnmo-¢ ccou uczocw coupe: xco _mDOH Longsz EmoH mo pcmocma mucmcwwmm chovpmccmch mum 2m: 1 wawca PoemCTz ncw chmpm> .m momwpomammc .Emcm emu muwc: chowpmccmucH ooo.m can ooo.om op _mzum o a < mcwEmuw>m m. opouxpm m:m_xgumzpoa o._ nxwe mvwxo oesoccu P.mm mpmcamosa xooc ummeweaoremo a. mo :PEmpw> v. m< swamuw> N.m ”Pam Pmemcwe munch P.MF oped—3m Ezwofimu m.mq Nmm-mo-¢ :Loo ecsoco cmppmz ago _mpoe Longsz EmpH mo pcmoeoa mocmcmmmm chowpmccmch mum 2m: - xwemcm pmcmcwz can swampw> .c u4mmp cwwaoca mczco covpmc mo pumwwm .m momocczp ".mmmuimmflm coasc mewfi om m mcw53mm< a _\zo some x Agoemv eeeme_ e - zee\m¥epee 2o x Aemwcv eeemwr e - meet gape e om. om.mm o~.mF «o._ mm.Fm oo.m_. em. mo.m¢ om.m mm. mm.mm oo.m nxmo\cm>occze Ame\FV mu comumm magma 30pm .cempocn mezco no mfim>mr mcmxgm> com mcmmpm mo amaze mew Eocm mmpmc 30F» weave; .m m4m

vpmpcmmmcamc mew pcmumccmazm use meme CI. .mFQmp menu cw umeemee:m mew; oo..M mcowpmpmccoo now; mosey AFco c." mm. 2a m on. z< m m mm. 24 2 $238285 z- :2 n- u- :wcmw4 mm. zq o mm. sq m mm. 2a m cue no. za 0 mm. 2e m me. 2; m Ezweoceu mm. sq m cm. 2e NF Fm. 2m m xefipcmpmccmqsmv z Fmpo» «R. 2a N_ 3. E 0 13233 2 EB om. 2a m we. sq m 2 Page» ow. E m . No. z< m coupes xgo mpwmoaeoo ou coquPmccou emewh ”EmpH Aungummcp mzocuxccm wopv mu_manoo .m> mmPQEmm Fmsnw>wucw com meowompmccou N m4mcmmno mm: cowpomcmucw cwmcwcoe x z< c< .Amo.v av mocmpmn z :o pummmm acmumewcmwm um; Fm>mP “cospmmcp maocuxccm m.~m u m2mn .pcmEpmmcp cwmcwcoe con mcmmpm v “acmEpmmep mmmpmm can mcmmpm pcmemm N.mm m.~m m.mm mm e.mm m.FN o.m_ o emxmmem z e we .ememmmmm z m.m~ n.4F m.mr mm P.mN m.e_ m.m o mame\m .mmemmmmm z o.mm m.eN _.oP mm e.em F.mm F.ee o eme\m .mmmmemxm 2 meme: “.mm. m.eN m.eF mm . m.mN m.eN N.m_ o sme\m .emmmemxm 2 memm o.em m.em m.mm mm m.em m.em F.mm o eme\m .mxmmem z om.mF om.“ o Asemv smmm Azomuu¥\z<1mv :wmcmcoe mFm>mF mmcerm mzocvxcc< mo Fm>m4 m mcmmum cw EmwFonmpmz 2 co pcmEpmmcH mmcoee< msocvzcc< use :wmcmcoz mo pommwm .oF mgmqh 69 at both levels resulted in significantly higher (P <.05) N retention when contrasted to the control silage. As the level of crude protein in the ration increased, so did the nitrogen retained per day. A slight anhy- drous ammonia x monensin interaction was evident (P = .087) pertaining to nitrogen retention. Nitrogen retained as a percent of total nitrogen intake was affected by the level of anhydrous ammonia (P <.10) and the additionof monensin (P <.lO). Monensin supplementation resulted in 20.8 and .9% increases in N retention in the O and 7.80 g AN/KGCSDM (anhydrous ammonia/ kilogram corn silage dry matter) treated rations respectively, but decreased the N retention by 5.2% in steers fed the 15.6 g AN/KGCSDM supplemented ration. A NH3-N x monensin interaction was also observed concerning the nitrogen retained as a percent of intake. This data supports earlier similar results obtained by Van Nevel and Demeyer (1977), Hanson and Klopfenstein (1977) and P005 e3_g1, (1979) which suggests that steers fed rations supplemented with monensin retained more nitrogen when the CP level is borderline to deficient in regards to their require- ment and when the rations were supplemented with preformed protein rather than NPN. The decrease in N retention/N intake in steers fed the 15.6 g AN/KGCSDM ration may be the result of less efficient NH -N use by 3 ruminal bacteria at the higher NH -N uptake from the rumen and omasum 3 thus resulting in greater N losses in the urine (Poos e§_g1,, 1979). CONCLUSIONS In the first two experiments the data indicate that the following conclusions may be made: (a) increasing CP levels in rations fed steers resulted in increased (P<<.05) total N and NAN flows to the abomasum across all treatments, and; (b) supplementation with urea above 13.0% crude protein did not result in any increase in abomasal total N or NAN flow although data from the two sets of steers could not be compared statistically. The data from the third experiment suggest that: (a) rumensin supplementation did not affect N intake. (b) the effect of increasing dietary crude protein levels on nitrogen passage to the abomasum could not be evaluated due to poor marker flows as indicated by marker: marker contrasts between feed and. abomasal samples. (c) PEG overestimated the flow of liquid pools from the rumen thus making the validity of a dual marker system in this trial rather dubious, (d) composites of abomasal samples collected over a 3-4 day period should be made since individual sample analysis correlated poorly with composited values. The last experiment regarding rumensin effects on nitrogen reten- tion showed: (a) rumensin had no effect on N retention per day, 70 71 (b) treatment of silage at both levels resulted in signifi-' cantly higher (P <.05) N retention when contrasted to the control, (c) a slight anhydrous ammonia x rumensin interaction (P = .087) was evident in regards to N retention, (d) the addition of monensin and treatment of silage with anhy- drous ammonia resulted in an increase of N retained/N intake (P <.lO), and (e) an NH3-N x rumensin interaction (P = .01). APPENDIX 72 com. . oom. NF.N mm.mm #m.— m¢.¢ mm wmm com. com. NF.N mm.m~ ¢N.F Fm.m 0 com com. com. u~.~ mm.~n em.— Pw.m mm mam oom. com. NF.N mm.mm «N.~ m¢.¢ o mom com. com. NP.N mm.mm «N.F m¢.¢ mm mmm com. com. NP.~ NF.N~ «N.F Fm.m 0 man com. com. NF.N NF.NN em._ _w.m mm mmm com. com. NF.N mm.mm em.~ m¢.¢ o mmm new; mom ummd xeu\m wood Amxv AEQaV .oz Pmewc< .oma & mo Lu & .cecmwg & mxmch z .z & oxmucH so :mmcmE3m m pcmewcmaxm . recycou . mpcmcoaeou mxmch ”mama cmmpm Fauce>wucH .F< mom<~ 73 com. com. ne.~ we.ee mm.~ mm.e mm emm com. com. em.N me.mm mm.P me.e o emm com. com. em.m ow.om mm.e _m.m mm Nam com. com. em.N me.mm mm.F me.e o Nmm .oom. com. Ne.N me.mm mm.~ me.e mm mom com. com. em.N me.mm mm._ me.e o mom com. com. em.N me.mm mm._ me.e mm mNm com. com. ee.m me.mm mm._ me.e o mmm vow... m wow“. wow“. 33m com... A9: €53 .02 35:5 .mmm m o to m .ememem e mxmmem z .z e mxmmeH so cememe:m m memeemmmxm - Zamom¥\z< m om.“ - mmemeomeOo mxmmem ”ammo mmmmm Fmsmm>meem .N< mque 74 oom. _o.m mo.wm mm.~ m¢.¢ mm com com. Fo.m mm.wm mm._ m¢.¢ o emm oom. Fm.m N¢.mm mm.r mF.m mm Nam oom. Fm.m mw.¢n mm.— mm.m o mom 00m. Po.m mm.mw mm._ m¢.¢ mm man oom. Fm.m mn.wo mm.~ m¢.m 0 man oom. Fm.m mo.wm om.~ mq.¢ mm mum oom. 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