THE EFFECT OF THE LEVEL OF ROUGHAGE, DIETHYLSTILBESTROL, AND IRON ON CERTAIN BLOOD COMPONENTS IN GROWING BEEF CATTLE JAMES LAWRENCE RANTA

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1 THE EFFECT OF THE LEVEL OF ROUGHAGE, DIETHYLSTILBESTROL, AND IRON ON CERTAIN BLOOD COMPONENTS IN GROWING BEEF CATTLE by JAMES LAWRENCE RANTA B.S.A., University of British Cblumbia, 1965 A THESIS SUBMITTED IN PARTIAL FUlJrTLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRIOJLTURE in the Division of Animal Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1967

2 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver 8, Canada

3 ABSTRACT In the first study 30 Hereford steers were fed a ration of either steam rolled barley or a 50:50 mixture of barley and alfalfa leaf meal pellets. In addition to these basal rations groups of animals on each, were fed a protein supplement with Diethylstilbestrol (D.E.S.) at levels such that each animal received either 0, 10, or 18 mgm. of D.E.S./head/day. The hormone treatment of animals fed the barley ration did not affect the growth rate or feed efficiency but a significant increase in hemoglobin levels from 8.96 to gm. % Hb. and in the red cell 3 3 counts from 8.25 million/mm to 9.50 million/mm resulted. A similar 3 3 increase in red cell count from 8.7 million/mm to 9.6 million/mm resulted from hormone treatment of the animals fed the 50:50 barleyalfalfa ration. There was also an apparent, but insignificant, increase in haematocrits on both rations due to hormone treatment. This treatment resulted in a significant increase in the ratio of blood acetic to propionic acids on the barley ration from 82.3:1 to 195.3:1 but, did not cause a change in total blood volatile fatty acids (V.F.A.). There was an increase in the blood acetic-propionic ratio on the barley-alfalfa ration due to D.E.S. addition. This was from 97:1 to 159:1 at 10 mgm. D.E.S./head/day, to 233:1 at 18 mgm. of D.E.S./head/day but, was insignificant due to high within-group variability. There was an apparent difference between the three blood parameters (Haematocrit, Hemoglobin, Red Cell Counts) on the two control rations due to the higher iron content of the barley-alfalfa ration. This, and an apparent decrease in thyroid weights were shown to be insignificant.

4 In the second study, using an all barley ration and a protein supplement containing a high iron concentration, there was a stimulation in growth rate and feed efficiency due to D.E.S. The animals were started on D.E.S. at 718 lb. (cf. 465 lb. for Study I). The increase in the three blood parameters was again observed on the low iron rations but, on the high iron ration there was an apparent but insignificant decrease in these parameters due to D.E.S. The animals fed the control high iron ration produced an haematocrit and red cell count that was significantly higher than that of the control low iron ration, indicating a possible deficiency in the original supplements. On the low iron ration there was a significant increase in total blood V.F.A. from 0.88 meq./l. to 1.19 meq./l. in response to D.E.S. supplementation but, on the high iron ration the increase was insignificant. The difference between the two control rations (0 D.E.S., high and low iron) total blood V.F.A. was shown to be significant, 0.88 vs meq./l. at P <.05. A method of preparation of feed and liver samples for analysis of their mineral content by atomic absorption spectrophotometry was developed. There was shown to be a significant increase in liver copper storage on the low iron ration with increased levels of D.E.S. The feeding of a high iron ration caused a significant decrease (P**.05) in the level of copper in the liver from 84- ppm. to 37 ppm. A slight but insignificant increase in liver iron levels on the high iron ration and through the treatment with D.E.S. was observed.

5 TABLE OF CONTENTS A. Introduction 1 Page B. Literature Review 3 I. VOLATILE FATTY ACIDS 3 1. Volatile Fatty Acid Metabolism 3 a) Production of V.F.A. in the Gastrointestinal Tract 3 b) Conditions Effecting V.F.A. Production 8 c) The Influence of Protozoa on V.F.A. Production 11 d) Distribution of V.F.A. in the Digestive Tract 13 e) Absorption of V.F.A. 14 f) Epithelial Metabolism of V.F.A. 17 g) Liver Metabolism of V.F.A. 19 h) The Utilization of V.F.A. in the Ruminant Total and V.F.A. Ratio Variability as Caused by: 24 A. Ration 24 a) The Concentrate-Roughage Ratio and Forage Maturity 25. b) The Effect of Various Methods of Feed Processing 27 c) The Effect of Protein Level and Feed Additives 29 d) Animal Feeding Practices and V.F.A. Ratios 31. B. Animal 33 a) Changes in V.F.A. Production with Age 33 b) Species Variation in V.F.A. Production 34

6 Page 3. Blood and Rumen Levels of Volatile Fatty Acids Effect of Total and Volatile Fatty Acid Ratios on Growth 37 a) V.F.A. and Energetic Efficiency 37 b) The Effect of V.F.A. Ratios on the Rate and Efficiency of Gain 39 c) The Effect of V.F.A. Ratios on Body Composition 40 II. DIETHYLSTILBESTROL The Effect of Diethylstilbestrol on Efficiency 42 a) Rate of Gain and Feed Efficiency 42 b) Nitrogen Retention and Digestibility Changes in Body Composition Caused by D.E.S. 45 a) Changes in Gross Body Composition 45 b) Changes in Organ Size and Mineral Content 46 c) Tissue Residues of D.E.S Oral vs. Implantation Administration of D.E.S The Effect of Level of D.E.S. on Response The Influence of Ration on D.E.S. Response Effect of D.E.S. on the Volatile Fatty Acids Relationship Between D.E.S. and Blood Components Theories of D.E.S. Response 53

7 III. BLOOD The Variation in Normal Blood Parameters 55 a) Normal Values 55 b) The Effect of Age on Normal Values Changes in Blood Parameters Caused by: 57 a) Supplemental Iron and Copper 57 b) Miscellaneous Ration Additions Relationships Between Iron and Copper and Anemia Blood Parameters and Growth Rate 59 IV. LIVER IRON AND COPPER STORAGE Levels of Iron and Copper Storage 61 Materials 64 I. ANIMALS Experiment One (Barley and Barley-Alfalfa) Experiment Two (Barley and High and Low Levels of Iron) 64 II. HOUSING 64 III. RATIONS Study I 65

8 Page 2. Study II 66 IV. ANALYTICAL REAGENTS 67 D. Methods 68 I. ANIMAL HANDLING PROCEDURES Preliminary Procedures Weighing Feeding Blood Collection Tissue Sampling 69 II. VOLATILE FATTY ACID ANALYSIS Total Blood Volatile Fatty Acid Analysis Volatile Fatty Acid Ratio Analysis 70 III. ANALYSIS OF BLOOD PARAMETERS 71 IV. ANALYSIS OF TISSUE SAMPLES Thyroid Gland Liver Mineral Storage 72

9 E. Experimental Results 74 Page I. STUDY I Results and Discussion 74 a) Growth Rate and Feed Efficiency 74 b) Blood Parameters 75 c) Total and Volatile Fatty Acid Ratios 78 d) Thyroid Weights and Dressing Percentage Summary of Study I 82 II. STUDY II Results and Discussion 83 a) Growth Rate and Feed Efficiency 83 b) Blood Parameters 85 c) Total Blood Volatile Fatty Acids 87 d) Liver Iron and Copper Levels Summary of Study II 91 III. CONCLUSIONS BASED ON STUDIES I AND II 93 F. Bibliography 95 G. Appendices 111

10 LIST OF TABLES Page Table I Gross Composition of Supplements 55, 56, and Table II Gross Composition of Supplements 60, 61, and Table III Rate of Gain and Feed Efficiency, Study I 74 Table IV Summary of Blood Parameters, Study I 76 Table V Total and Volatile Fatty Acid Ratios, Study I 78 Table VI Average Thyroid Weights, Study I 80 Table VII Average Dressing Percentage, Study I 81 Table VIII Rate of Gain and Feed Efficiency, Study II 83 Table IX Summary of Blood Parameters, Study II 86 Table X Total Volatile Fatty Acids, Study II 87 Table XI Iron and Copper Contents of Supplements 89 Table XII Liver Iron and Copper Levels 90

11 LIST OF APPENDICES Page Appendix I. Ill Appendix II. 114 Appendix III. 115 Appendix IV. 116 Appendix V. 117 Appendix VI. 118 Appendix VII. 119 Appendix VIII. 120 Appendix IX. 121 Appendix X. 122 Appendix XI. 123 Appendix XII. 124 Appendix XIII. 125 Appendix XIV. 126 Appendix XV. 127

12 ACKNOWLEDGEMENTS The writer wishes to express his thanks to Dr. W.D. Kitts, Professor of Animal Science and Chairman of the Division of Animal Science for the encouragement and guidance received throughout the course of this study. A number of students and technicians have helped in the animal handling and tissue sampling procedures and, I thank them. I would be remiss though, if I did not give special mention to Messrs. W. Freding, L. Holmes, G. Huffman, and J. Ross.

13 A. INTRODUCTION Ever since it was observed that diethylstilbestrol (D.E.S.) caused a significant increase in the rate and economy of gain in the bovine, a considerable amount of work has dealt with elucidating the pathways of its action. has been established but, Up to this time no definitely proven pathway it is known to have a stimulatory effect on certain endocrine glands (pituitary, adrenal) and a depressant effect on the thyroid gland. D.E.S. has also been shown to affect certain of the main blood parameters, hemoglobin, haematogrit, and red cell counts, which are known to cause an effect on growth rate. It has been proposed that it causes a change in the length of certain periods of physiological growth as well as an effect on enzyme systems in vitro. One criteria that has received very little attention is the effect of D.E.S. on the energy yielding substrates of bovine blood. These components, volatile fatty acids, were considered by early workers to be of little significance in the animal. But, in more recent years their importance is becoming more apparent with estimates of their energy supplying capability ranging as high as 81% of the total requirement of the animal. The three main acids, acetic, propionic, and butyric have also been shown to have different relative efficiencies in their ability to promote certain types of gain. This experiment was designed to examine the effect of orally administered D.E.S. on the total and volatile fatty acid ratios in

14 2'. blood as well as to examine changes in the three main blood parameters on different iron intakes. An examination was also made of the effect of D.E.S. on the liver storage of iron and copper at various levels of D.E.S. treatment and iron intakes. It is hoped that through these studies a parallel can be made between blood levels of these volatile fatty acids or the rates of mineral absorption and the observed increase in growth rate and feed efficiency due to D.E.S. This may then demonstrate a possible mode of action for D.E.S.

15 3 I VOLATILE FATTY ACIDS "Phillipson and Cuthbertson (1956) have calculated from the data of Schambye (1951, 1955) that at least 600-1, 200 Cal. of energy are absorbed as V.F.A. from the sheep rumen every 24 hours. Similarly in cattle it has been shown that 6,000-12,000 Cal. become available from the V.F.A. produced by fermentation in the rumen (Carroll and Hungate, 1954). The total energy turnover of fasting sheep and cattle is about 1,100 and 6,500 Cal. respectively per day, indicating that V.F.A. make a major contribution to the energy requirements since it is recognized that these acids are utilized by body tissues." (8) 1) Volatile Fatty Acid Metabolism a) Production of Volatile Fatty Acids in the Gastrointestinal Tract The basis for the production of volatile fatty acids, (V.F.A.) lies in the nonsecretory character of the rumen epithelium. Under the conditions that prevail in the monogastric digestive system the greater part of the breakdown of feedstuff's is the result of endogenous enzymes secreted by the epithelium of the stomach and the organs of the upper digestive tract. The role of bacteria in digestion of the feeds is relatively minor from the standpoint of efficiency of utilization of the ration or in governing the availability of the nutrients. They are, however, of some limited importance when one considers their ability to synthesize the B complex vitamins and Vit. K in the lower intestinal tract. In the ruminant or polygastric animal there is essentially no secretory capability in the largest part of the stomach. Under this condition, it must exist in a symbiotic relationship with the bacteria and protozoa that inhabit the largest part of its digestive system, the rumen.

16 4 The complete absence of an enzyme secretory capability is not altogether true as it does possess enzymes which are secreted into the abomasum and are proteolytic in nature. They are responsible for the digestion of microbial protein. Ruminants do not have endogenous enzymes capable of digesting cellulose and must in consequence, depend wholly on their rumen microflora for this function. As early as 1884 it was theorized by Tappeiner (20) that cellulose digestion in the ruminant animal was the result of bacterial as well as protozoan action resulting in the production of organic acids, which were of some nutritive value. He was able to classify these organic acids as acetic, propionic, and butyric acid and identify the gaseous products of ruminantion as carbon dioxide, methane, and hydrogen. This work was largely ignored by other workers in the field who felt that the contribution of these acids to the energy requirement of the animal was relatively minor. It was not until the 1930's that it became generally accepted that they made an important contribution. Since that time a number of major strides have been made in elucidating their pathways of utilization, their effects on the performance of the animal, and the factors which control their production. In a review of the production of V.F.A., Barnett and Reid, in 1961 (20), stated that there is more than one area in the animal body capable of producing V.F.A. The major site of production is the rumen where 70% of cellulose digestion takes place, (173) the second and a relatively minor site, is the caecum where 17% of cellulose

17 5 digestion takes place, and the third site is the large intestine where the remaining 10% of digestible cellulose digestion occurs. As it has been stated by Shaw (155) that 70% of ruminant energy is derived from absorption anterior to the abomasum, the greatest part of this review will deal with factors effecting V.F.A. production within the rununo-reticulum. The total level of production has been estimated in Barnett and Reid's review (20) to be from gm./day in the rumen of the ox. The relative level of V.F.A. concentration in the rumen has been shown to be of major importance in governing the productive efficiency of any particular ration (20, 155). The ratios of the three major acids, in the light of work by Blaxter and Armstrong (11, 12, 13) and Shaw (156), has been shown to influence productive efficiency of rations to a similar degree. There has been shown to be a number of complicating factors which govern statements made concerning the level of any particular V.F.A. or the concentration of total V.F.A. in the rumen when animals are fed identical rations. One of these is the influence of time after feeding on the level of total V.F.A. in the rumen (44, 78). Barnett and Reid in 1961 (20) and, ljuther in 1966 (102) have stated that the highest rate of V.F.A. production occurs approximately two hours after feeding. At the two hour mark the level of propionic production was at a maximum and dropped from this point on. Consequent

18 6 to this drop in propionic production there was a rise in acetic production. Basic agreement with these observations may be found in the work of Grey et al (70). They state that when the rate of production increases the acetic-propionic ratio gets smaller and, when it is decreasing, the acetic-propionic ratio gets larger. In the more recent work of Leng et al (97, 98) and Weller (178), it has been show/),through radioisotope infusion studies, that the effective rate of production of these acids was approximately 3.85 m moles/min. acetic, 1.01 m moles/jriin. propionic and, 0.64 m moles/min. butyric acid.. Leng (97) also shed light on the relative importance of these V.F.A. by theorizing that 79% of the apparent digestible energy of the ration could be accounted for by these compounds. Of this 79% apparent digestible energy, Shaw (155), states that 40% of it is made up of acetic acid, 24% made up of propionic acid, 16% made up of butyric acid, and 10% made up of valeric acid. The apparent discrepancy between the volume of acetic acid and its energy contribution is due to the higher S.D.A. (11, 12, 13, 156) and the lower caloric value, (31) than either of the two other V.F.A. The substrates present in the rumen are governed by the rations fed to the animals and they all contribute in varying degrees to the level of any particular V.F.A. present. The more readily fermentable carbohydrates; glucose, lactose, maltose, and galactose are broken down so that the distribution of carbon atoms in the whole

19 7 digestive tract is as follows (20): Volatile Fatty Acids 34.7% Lactic Acid 8.0% Carbon Dioxide 8.0% Methane 3.1% Bacterial Protein 11.8% Bacterial Polysaccharide 28.1% Undetermined 6.3% 100.0% Glucose is the only carbohydrate used to produce lactic acid, the latter being present in the rumen only when the animal is on high grain rations (88). In work completed by Grey in 1952 (71) and confirmed by VanCampen in 1960 (179) it was shown that, through a series of condensation reactions higher fatty acids may be produced from the acetic and propionic acids normally resulting from cellulose digestion. They have also shown that the isomers of these acids may result from amino acid digestion. This observation has been confirmed by Annison (8), Bath (23), and Walker (173). In a more complete analysis, Sirotnak in 1953 (157), used a mixture of rumen microorganisms to show that glutamic acid, serine, arginine, cystine, and cysteine can be metabolized to propionic acid. Although it was initially stated that the rumen microflora were wholly responsible for cellulose digestion and, that the animal's endogenous enzymes were only capable of proteolytic action, it must be. realized.that over 50% of protein digestion is the result of microbial attack in the rumen. (20)

20 8 A rather complete table of volatile fatty acids present in the rumen has been devised by Gray (71) and shows the following distribution: Formic 0-5% iso Butyric.3 -.6% Acetic 62-70% Valeric % Propionic' 16-27% Caproic.5-1.0% n Butyric 6-11% Heptoic % From this, it is readily apparent that one need only be concerned with acetic, propionic, and butyric acids when dealing with the significance of changes in the V.F.A. ratios. b) Conditions Favoring the Production of V.F.A.'s The digestibility of cellulose, the main substrate of V.F.A. production, is influenced to a large degree by conditions within the rumen which act to change the effectiveness of microbial attack. The first major influence could be the provision of other substrates, such as glucose, which are more readily attacked by rumen micro-organisms than are cellulose or starch (20). It has been shown by Barnett and Reid (20) that the inclusion of fat in the ration has a similar depressant effect on cellulose digestion. But, Shaw (155), states that the fatty acids oleic and. linoleic have a stimulatory effect on total V.F.A. production which, is an apparent contradiction of the above statement, although he doesn't mention any specific effect on cellulose digestion. Shaw is in agreement when he was able to show that these two fatty acids resulted in an increase in propionic acid production and a decrease in acetic production. Barnett and Reid also

21 9 noted a high production of propionic acid from simple polysaccharides and hexose sugars. With respect to protein digestion and possible V.F.A. production, it has been shown that an increased level of starch in the ration is capable of reducing ammonia production. Since the production of V.F.A. from an amino acid would require its deamination, it is reasonable to suggest that starch depresses V.F.A. production from proteins in a similar way to the glucose effect on cellulose digestion. There is also a qualitative aspect to the effect of protein on V.F.A. production due to the differences in their solubilities. The second major group of influences on the production of V.F.A. are changes brought about in the microbial population by some external influences. It has been stated by Walker (173) that there exists "a fairly precise relationship between the amount of bacterial cell material synthesized and the A.T.P. made available by the fermentation of an energy source." From this it is postulated that the greater the amount of cell material, the higher will be the energy yield from any foodstuff and the more efficient will be the gains produced. The importance of the quantitative aspect of protein metabolism must then be considered as it is known that about 50% of the bacterial cell is protein (20). It is therefore not unreasonable to observe an increase in bacterial numbers with an increase in protein level and a consequent increase in the energy or V.F.A. yield from any feed under these conditions (52). There are numerous other external influences on the bacterial

22 10 population that help towards changing the production of V.F.A. concentrations. One of the most common of these is the effect of the essential buffering ability of the saliva (Na Bicarbonate). In 1959 Matrone (108) demonstrated the essentiality of these buffers in maintaining growth in sheep. Shaw (155) has also stated that a change in the ph of the rumen can result in a shift in the balance of rumen microorganisms and a consequent shift in the level of V.F.A. production or in the relative level of production of any one of the acids. An explanation for this shift in the relative proportions of each of the three main acids has been put forward by VanCampen in 1960 (179). He showed that the previously mentioned interconversions of butyric acid to propionic and acetic acids within the rumen is enhanced by the presence of Na and K bicarbonate. Bath (23) is in basic agreement with this underlying idea of a particular set of nonbiologic conditions governing shifts in the relative proportions of the three V.F.A. produced. He feels that the conditions necessary for the production of butyric acid are somewhat intermediate between those that produce acetic and those enhancing the production of propionic acid. The results of other workers in this field of microbial stimulation (20), indicate that at levels of ppm, estrogen, diethylstilbestrol, and cholesterol increase cellulose digestion and in so doing may alter or increase the levels of V.F.A. produced. The addition of ordinary salt to the ration at a level of 1.4% has been shown by Walker (173) to reduce cellulose or more specifically cellobiose fermentation

23 11 by 45%. From what has previously been discussed concerning cellulose digestion, it is obvious that this reduction will effect the total V.F.A. production and possibly their ratios. One final relationship has been brought out in the work of Weldy (177) where he has been able to show a positive relationship between body temperature and V.F.A. concentration. In work on cattle, it was demonstrated that a decrease in rumen V.F.A. concentration, mainly due to a decrease in acetic acid levels, resulted from an increased temperature. This effect, however, may just be the result of a decrease in feed intake due to the hot conditions and, not due to any change in the physiologic conditions within the rumen. c) The Influence of Protozoa on V.F.A. Production In the preceding two sections of this review the greatest stress has been placed on the influence of the more numerous bacteria on the production of V.F.A. in the gastrointestinal tract of the rununant. This, however, does not represent a true picture of the total micropopulation influencing V.F.A. production. The protozoan component of the rumen has been show/5,mainly through the work of Christiansen (41), to be of major importance. In 1962, he presented an estimate that 20-25% of the total energy of the animal is derived from the ciliate protozoa. The relative effectiveness of their production was influenced to a large degree by the ph of the rumen contents. As the ph was raised the viability of the smaller protozoa was increased with the greatest production occurring at ph 7.

24 12 In subsequent work (44) he stressed the importance of the symbiotic relationship that exists between bacteria and protozoa in their mutual metabolism of each other's end products. In work with protozoa-free lambs he noted an increase in the acetic-propionic ratio and in the acetic-butyric ratio. He theorized that this effect was due to an increase in the level of propionic acid production and hypothesized that protozoa produce lactic acid which was then converted to propionic acid by the rumen bacteria. The overall effect of a lack of protozoa on the growth of the animals was quite dramatic as he has shown a decrease in the rate of gain by 28% and in the feed efficiency by 34%. The changes brought about by protozoa were greatest when the animals were on a high concentrate diet. Luther (102) is in basic agreement with these observations but noted that protozoa have a marked stimulatory effect upon butyric acid production and a depressant effect on propionic production. This contradiction of a part of Christiansen's work may be due to differences in the species of protozoa present even though both sets of data show a decrease in the acetic-propionic ratio on low concentrate rations. He has also discovered that protozoa effect an increase in the ammonia production by 40%. This could be expected, as a result of the previous discussion, to cause an increase in the total V.F.A. production. This reasoning has been confirmed by the results of his experiment showing a 50% increase on the low concentrate rations and a 38% increase on the high concentrate rations.

25 13 d) The Distribution of V.F.A. in the Digestive Tract The levels of total V.F.A. and the levels of each V.F.A. have been shown to change as a feedstuff passes through the digestive tract (89, 125, 175). The main causes of these changes have been enumerated, first by Elsden in (57), and then later by Annison and Lewis in 1959 (8). In summary, they have compiled a list of six factors: 1. production in the rumen 2. absorption from the rumen, etc. 3. passage from the rumen to omasum 4. dilution with saliva 5. utilization by microorganisms 6. conversion to other metabolites In Elsden's work it was shown that 88% of the total V.F.A. in the whole digestive system was located in the rumino-reticulum of sheep. In steers this value was 86%. These observations have been confirmed by the work of Packett in 1966 (125) where he was able to show that 87% of the total V.F.A. content of concentrate fed animals was found in the rumino-reticulum. percentage was 92%. In the case of roughage fed animals, however, this This effect could possibly be due to a more rapid rate of passage in the concentrate fed animals, causing more undigested carbohydrate to pass into the rest of the digestive system. It must be remembered that the synthesis of V.F.A. also occurs in the caecum and colon (125) and this would tend to lower the calculable percentage of V.F.A. within the rumen. The absorptive capability of the rumen was examined by Johnston in 1961 (89). He showed that the decrease in the V.F.A. concentration

26 14 between the rumen and the reticulum was 18%, between the reticulum and the omasum was 51%, and between the omasum and abomasum was 83% of the amount in the preceding section of the stomach. In an analysis of the changes in total V.F.A. concentration in each section of the gastrointestinal tract one must also be aware of changes that occur in the relative proportions of each V.F.A. due to the different absorptive preferences or different conditions governing their metabolism (89, 125, 175). The proportions of the three main V.F.A. are quite variable throughout the digestive tract. A breakdown of this distribution has been presented by Ward in 1961 (175) from work with Hereford cattle, as follows: Acetic Propionic Butyric Formic Lactic Total molar % mm/% Rumen Abomasum Small Intestine Caecum Colon e) Absorption of Volatile Fatty Acids In prior parts of this discussion it has been shown that the disappearance of V.F.A. from the rumen is brought about mainly by absorption through the rumen epithelium. This absorption of V.F.A. is controlled in both quantity and type by conditions that exist within the rumen (32, 51, 70, 106, 135). The initial work done on this subject was that of Barcroft in 1943 (19) when he found, working with sheep, that the sites of absorption

27 15 of V.F.A. were the rumen, reticulum, omasum, and caecum. In an attempt to determine if there were any differences in the rates of absorption from the rumen he 'infused: the sodium salts of the V.F.A. and analyzed the differences in concentration between the rumen and the portal blood stream. He postulated that the rates of absorption were: acetic the greatest, followed by propionic, and butyric the least. This order is exactly that of the concentration of the V.F.A. in the blood. This assumption of portal blood concentration reflecting the rate of absorption is not altogether true as it became apparent that the rumen epithelium is capable of metabolizing certain of the V.F.A. In 1945 attention was starting to be directed towards an understanding of this phenomeronby Danielli (51). At a rumen ph of 7.5 he found that there was little free fatty acid present and that the permeability of the rumen epithelium was reduced. However, at a ph of 5.8 the largest portion of the V.F.A. were found in the free form and the rate of their absorption was increased. The transportation of these free V.F.A. was partially through the water filled pores but largely through the lipoid membranes of the epithelial cells. The order of the rates of absorption that Danielli proposed under these various ph levels is as follows: ph 7.5 rate of absorption: acetic, propionic, butyric ph 5.8 rate of absorption: butyric, propionic, acetic Support for the idea that absorption is possible under both acidic and basic conditions comes from the work of Masson in 1951 (106). His data indicates that the effective rates of absorption of V.F.A. are:

28 16 acetic, propionic, and butyric due to the metabolic effects of the rumen epithelium in lowering portal blood concentrations of propionic and butyric acids. He also pointed out that the rate of absorption of acetic acid is controlled by the concentration in the blood. This control did not appear to exist for propionic or butyric acids. In further work on the effect of ph on the rates of absorption, Gray (70) postulated a more rapid rate of absorption for propionic acid when the rumen ph was between 6 and 6.5. These differences between Gray, Masson, and Danielli may all be the result of their use of different rations as Gray (70) has pointed out that rations do control the relative rates. Further controls on the relative rates of absorption were proposed in 1953 by the work of Pfander and Phillipson (135) when they found that the rate of disappearance of the acids from the rumen was determined by the length of the carbon chain and by the concentration of the acid. They also related the total rate of absorption of all acids to production when they infused a mixture of the three main V.F.A. at a rate of 78 mequivs./hr. They found that absorption was almost the same as the rate of production at 68.5 mequivs./hr. Pfander and Phillipson observed that when there was an increase in acidity of the rumen the overall rate of absorption was increased. The work of Brown in 1961 (32) also shows that absorption of the V.F.A. is in proportion to their concentrations in the rumen. In these studies the absolute rates of absorption were 2.61 gm/hr. for acetic acid, 1.16 gm/hr. for propionic, and 0.89 gm/hr. for butyric acid.

29 17 f) Epithelial Metabolism of Volatile Fatty Acids Ever since research has started concerning itself with the usefulness of V.F.A., a considerable deal of attention has been directed towards determining the causes of the differences between rumen and blood levels of V.F.A. In 1952, Pennington (128) determined that the main sites of V.F.A. metabolism were the rumen epithelium and the liver. The metabolism of acetic and propionic acids by the rumen epithelium was found to be much less than butyric acid. In 1960, Brown (32) analyzed the portal blood for end products of epithelial metabolism and confirmed the foregoing observation that butyrate was metabolized to the greatest extent. The main products of butyrate metabolism were ketones. In this same year Shaw (155) demonstrated that butyrate metabolism resulted in lactate as well as ketones. He also found that epithelial tissue removed glucose from the blood as a source of energy. Ramsey (146), in 1964, worked on the identification of the ketones produced by epithelial metabolism of butyric acid and, through the use of - labelled acids, demonstrated that the greatest part of the label n was found in 3 hydroxybutyric acid. This observation was confirmed by Spahr in 1965 (1960). Spahr has also shown that small amounts of acetic t acid were metabolized to ^ hydroxybutyric acid-. In 1952, Pennington (128) stated that possibly, propionic acid was the main source of carbohydrate for the ruminant animal and demonstrated

30 18 that this conversion could occur. In 1956 (130) he showed that the epithelium would metabolize propionic acid into lactate and carbon dioxide. Subsequent to this it was shown that propionic acid and carbon dioxide would combine to form succinate, an intermediate in the T.C.A. cycle. The relative degree to which metabolism of propionic and butyric acids takes place in the rumen epithelium has been shown by Kiddle (95). By ratio analysis, he was able to show that propionic metabolism was much less than butyrate metabolism in passing through the rumen epithelium. Propionic:Acetic Rumen Blood (Portal) Butyric:Acetic Rumen Blood (Portal) post..32 ant post..10 ant. Rumen Portal Blood Acetic Propionic Butyric 63.2% 16.7% 15.4% 76.7% 13.4% 5.2% In 1952, Pennington (128) incubated sheep rumen epithelial slices with a mixture of V.F.A. and noted the following average utilization from a sample of 100 moles: Acetic Propionic Butyric 14.1 moles 10.3 moles 40.0 moles These observations have been confirmed by Bensadoun in 1962 (26),

31 19 In previous sections of this report it was stated that absorption occurred from the rumen and caecum. Packett in 1966 (125), attempted to determned if any differences existed in the metabolic characteristics of these two areas. Working with sheep tissues from animals fed on roughage and concentrate rations, he was able to postulate the possibility of differences in the pathways of oxidation of V.F.A. and in the preference of each tissue for a particular acid. He found that caecal tissue utilized 75% of the amount of V.F.A. that r\m_nal tissue would with an oxygen uptake of 35%. It was also graphically illustrated that the rumen epithelium had a great preference for butyric acid and that the caecal tissue had a preference for acetic acid. These results give strong evidence to the theory of different metabolic pathways when one considers that the caecal tissue utilized so much more V.F.A./ul. of oxygen consumed. Evidence also existed to support the possibility of changes in these absorptive tissues to accommodate changes in rations. It has been shown (9, 181) that concentrate rations produced a higher proportion of propionic acid and, would therefore require a greater rate of metabolism by the absorptive tissues. This hypothesis appears to be borne out by the data from the rumen tissue but, is most strikingly shown by caecal tissue where the absorption of propionate from concentrate rations was 2.5 times that on the roughage ration. g) Liver Metabolism of Volatile Fatty Acids In the preceding section of this literature review it was shown that the proportions of V.F.A. reaching the liver were approximately

32 20 t 76-86% acetic, 11-13% propionic, and 1-5% butyric (26, 95) depending on the ration used. Upon entering the liver, variable metabolism of these acids occurs to produce the ratios normally found in blood. The oxidation of acetate is quite minimal in comparison to that of other acids. The main metabolic products are ketone bodies and carbon dioxide (96, 128, 139). These ketone bodies and the acetic substrate can enter the T.C.A. cycle as acetyl CoA with no net synthesis of carbohydrates (9, 14, 96). The oxidation of acetic acid is influenced to a very major extent by the presence of other metabolites. When this occurs, greater amounts of acetic acid enter the blood stream and cause an elevation of total blood V.F.A. One of the most active inhibitors of acetate metabolism is propionic acid (14, 96, 129, 131, 139). In 1956, Perinington (129) incubated sheep liver slices with labelled acetic acid and equimolar amounts of propionic acid and found a 50% drop in labelled carbon dioxide production. In tests with other metabolites, it was found that butyrate, (96) isovalerate, valine, isoleucine, and methionine caused a similar reduction but, not to such an extent. Pennington reasoned that the propionic inhibition acted by masking Coenzyme A and therefore prevented the formation of Acetyl CoA. In order to examine this hypothesis a similar experiment was run in 1958 (131). The inhibitory effect was eliminated in the presence of added Coenzyme A. This result has been confirmed by the work of Pritchard in 1960 (139) and Annison in 1963 (9).

33 21 The metabolism of propionic acid results mainly in glucose (128) or in its being oxidized to carbon dioxide and water (14, 139) (via T.C.A. cycle with entry through succinate or pyruvate) (50). Because the primary carbohydrate product of liver metabolism of propionate is glucose, it makes a consideration of propionic metabolism especially important considering that only 10% of the animals requirement is absorbed each day (14). In 1958, McCarthy (111), working with goat livers, infused propionic acid and found that it was almost completely removed from the blood. This liver metabolism resulted in a slight increase in blood acetate (9) but more important, there was a five fold increase in blood glucose and a three fold increase in lactic acid. Lactic acid can then enter the T.C.A. cycle through pyruvate and malate (14). In a more complete analysis of the fate of labelled propionic acid, Pritchard in 1960 (139) demonstrated that the label occurred on not only carbon dioxide and glucose but also on succinate, malate, and fumarate, all intermediates of the T.C.A. cycle. Pritchard also found that the metabolism of propionic acid would be stimulated by the presence of most normally occurring liver metabolites such as acetate, butyrate (96), glucose, and carbon dioxide (128). The greatest part of this work was confirmed by Annison in 1963 (9). In 1957, Leng et al (98) attempted to determine the significance of liver conversion of propionate to glucose. They found that if all the propionate was converted to glucose it would account for 100% of it.

34 22 However, their experimental results indicate that only 32% of the absorbed propionate is metabolized to glucose, making up 54% of that present. The last and smaller portion (1-5%) of portal V.F.A. entering the liver has been found to be metabolized almost completely (9, 111). In 1958, it was proposed by McCarthy (111) that the greatest amount of butyric acid was used in the formation of liver glycogen. However, since that time the general consensus of opinion is that butyrate is metabolized to Q hydroxybutyric acid (9, 85, 96) and carbon dioxide (96, 139). g hydroxybutyrate can then be used as a source of energy through entry to the T.C.A. cycle via acetyl CoA although, no net synthesis, of carbohydrate has taken place (9, 14, 96). In a study which involved the sources of ^ hydroxybutyrate Annison, in 1963, (9) was able to show that of a mixture of labelled acetic, propionic, and butyric acids, 50% of the butyric label, 7-15% of the acetic label, and 2% of the propionic acid label turned up on this product". The metabolian of labelled butyric acid was also examined by Holter (85), in 1963, when he found that 76% of the label turned up on ^ hydroxybutyric acid, 9.9% on carbon dioxide, and 4.0% on acetic acid. Holter (85) also presented a complete summary of the changes that occur in liver metabolism during the infusion of a 9:3:1 mixture of acetic, propionic, and butyric acids as follows: 15 0 mgm./loo ml. 60 Change Butyrate down Propionate down Acetate up Formate up Lactate up Glucose up Ketones up

35 23 h) The Utilization of V.F.A. in the Ruminant At the present stage in the development of the energy supply of the ruminant, from V.F.A., the main sources are in the form of acetic acid, ketones,(mainly hydroxybutyrate) small amounts of absorbed glucose, and intermediates of the gluconeogenesis from propionate and other nutrients. The relative importance of each of these catagories is the subject of much speculation with estimates of the importance of V.F.A. derivatives ranging as high as 80% of the total energy requirement (120). Of this 80%, Kiddle in 1951 (95) estimates that 58% was due to acetic acid, 24% due to metabolites of propionic acid and 18% due to the metabolites of butyric acid. The knowledge that the utilization of any of these compounds for energy results in the production of carbon dioxide, has led many workers to an attack of measuring the amount of label present on the exhaled gas from animals fed labelled V.F.A. In 1965, Leng (97), using this technique, found that 17.6% of exhaled carbon dioxide came from acetic, 12.9% from propionic (glucose and intermediates), and 14.3% from butyric acid (^ hydroxybutyric acid). Reid in 1950 (148) has stated that propionic acid is present in blood only when the rate of absorption is greater than the rate of its use in gluconeogenesis in the liver. The metabolism of acetic acid, and the butyric acid derivative, ^ hydroxybutyric acid, occurs in the mitochondria in the same way as the glycolysis products of monogastric metabolism. In 1958, Annison (7) has shown that this utilization ranges

36 24 from 1.4 to 2.1 m moles/hr/kg. body weight which, on a daily basis, for a mature sheep, is approximately 144 gm. In order for this pathway of V.F.A. metabolism to operate in a most efficient manner, a source of three carbon intermediates, in the form of oxaloacetic acid, is necessary. This requirement leads to a tying together of the metabolism of acetic and butyric acids with that of propionic acid which can form this intermediary via succinyl CoA (14). It is possible that this supply of cycle intermediaries can also be met by amino acid breakdown (8). Even though the absorption of glucose forms a relatively minor part of the total energy absorption (26), its utilization by the ruminant body is quantitatively, per unit metabolic size, very similar to that of monogastric animals. This has been shown in a summary by Armstrong (14) 75 to be 2.5 to 3.8 mgm./kg.' body weight/mm. for the dog and 1.5 mgm./.75 kg.* body weight/min. for fasted sheep. The metabolism of glucose occurs in the same way as it does in nonruminants via the glycolysis-t.c.a. pathway or the hexosemonophosphate shunt. 2) Total and V.F.A. Ratio Variability as Caused by: A. Ration The importance of changes in the V.F.A. ratio in the ruminant has been stressed by many workers because of the influence it has on the efficiency of utilization of nutrients (20). The reasons for these differences in efficiency form the basis for a large part of the work of Blaxter and Armstrong (11, 12, 13) and will be covered in a later part of

37 25 this analysis. Pfander in 1961 (136) presented a list of factors which have been shown by past research to influence the V.F.A. ratio and it is apparent from this that the ration forms the major source of this variation. 1. Proportion of roughage to concentrate 2. Pelleting 3. Particle Size 4. Heat Increment 5. Various Oils 6. Protein Level 7. Environment 8. Frequency of feeding 9. Mineral adequacy of the diet a) The Concentrate-Roughage Ratio and Forage Maturity The effect of any increase in the level of concentrates in the ration is no longer a subject of conjecture. It has been shown by Balch (17) and Luther in 1966 (102) that the response in rumen micrcorganisms is one which culminates in an increase in total V.F.A. (18) and an increase in the relative levels of propionic (48) and butyric acids at the expense of acetic acid (169). Along with this change there is an effect of lowering ph. Further observations concerning the characteristic V.F.A. response to an increased proportion of concentrate in the ration came from the work of Balch and Rowland (16). They noted the occurrence of large fluctuations in the total V.F.A. levels that did not occur when a higher proportion of roughage was fed. It was also proposed that rumen

38 26 acetic acid percentages ranged from 40.6 to 73.7, propionic acid percentages ranged from 16.5 to 39.1, and butyric acid percentages ranged from 6.6 to 13.9 depending on the types of concentrates and roughages and the ratios in which they were used. In an attempt to explain the causes of these ratio changes Emery, in 1956 (6), and Maki in 1958 (105) examined the microbial populations of concentrate fed animals and noted apparently conflicting results. Emery, using the artificial rumen technique and inocula from concentrate fed animals, found a higher propionic acid production. Maki, however, tried to do an analysis of the types of bacteria found in the rumen and observed that grain fed animals had few butyric acid producers and no propionic acid producers. A possible explanation for this contrast may be due to the fact that Emery used a complete rumen sample and Maki, used bacteria without the protozoan population. In addition, Maki noted a two to three fold increase in bacterial numbers on concentrate-fed cows. It is also of some significance to note the effect of type of roughage and the relative level of maturity of the forage on these V.F.A. ratios (23), as reflecting changed microbial populations. Card in 1953 (39) showed that the later a forage is cut, the higher will be the resultant acetic acid production and the lower the butyric and propionic acid production as follows: Early Cut Silage Late Cut Silage Barn Dried Hay (early) Field Cured Hay Acetic % Propionic %

39 27 These results were subsequently confirmed by Parks, in 1964 (127) and suggested that these changes be due to a decrease in digestibility, soluble sugar level, protein level, and an increase in lignin content. Barnett and Reid (20) have stated that the total V.F.A. production is at a maximum when forages are at the stage of greatest leafy growth. In 1965, Bath (23) has also shown that the addition of supplements such as sugar beet pulp and brewers grains depress acetic production. b) The Effect of Various Methods of Feed Processing The feeds usually fed to rundnant animals under intensive care practices have almost always been subjected to some form of processing such as grinding or pelleting. It is therefore of some importance to look at any differences that occur in the V.F.A. ratios or total production caused by such handling. A great deal of work has been done in determining the effect of grinding of hay on the V.F.A. ratios as produced. Balch, in 1958, (17) fed lactating Shorthorn cows long or ground hay and noted a marked drop in rumen acetic levels from 57% to 46%, and a rise in rumen propionic levels from 22% to 33%. There was no change in butyric acid percentage. These results have been confirmed by Ensor, in 1959, (62) and Thompson, in 1965, (169) who also found that grinding increased the total V.F.A. level in the rumen. This effect is probably just the result of increased digestibility on the ration of small particle size rather than a change in the species of bacteria present (182).

40 28 It has been shown by Rhodes (150) in 1962, that the pelleting of a roughage ration does not effect the V.F.A. ratio but he does lend support to the above hypothesis when he found that this process did increase digestibility (182) and consequently feed efficiency. Further support for this idea is gained through the work of Shaw (150) when he found that the grinding and pelleting of hay resulted in a two fold increase in total V.F.A. These two sets of results taken together indicate that there will be an increase in total V.F.A. with an increase in digestibility as proposed earlier. Oltjen has shown, in 1965, (120, 121) that pelleting an all concentrate ration does not effect the molar percentage of V.F.A. or the total ruminal levels. These observations suggest that the pelleting of a concentrate ration will have no effect on the V.F.A. ratios but, the pelleting of a roughage-concentrate or a straight roughage ration will cause a decrease in acetic acid level and an increase in propionic acid level when compared to the results obtained on a long hay ration. In this regard, Woods in 1962 (181) was able to demonstrate that the pelleting of a complete ration (i.e. containing concentrates and roughage) results in an increase in propionic production and a decrease in acetic production. But, the pelleting of an all concentrate ration had no effect on V.F.A. ratios or totals. Woods also observed that the fineness of grind of hay has an effect of increasing the propionate percentage thereby causing a decrease in the acetic-propionic ratio. This work was confirmed in 1963 by the work of Wright (182) when he found the acetic-propionic ratio resulting from long hay was 2.62, from coarsely ground hay was 2.12, and 1.67 from finely ground hay.