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The effect of the level of roughage, diethylstilbestrol, and iron on certain blood components in growing… Ranta, James Lawrence 1967

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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 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I ag ree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y pu rposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n -t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department The U n i v e r s i t y o f B r i t i s h Co lumb ia Vancouve r 8, Canada 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 10.40 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 barley-alfalfa 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. In the second study, using an a l l barley ration and a protein supplement containing a high iron concentration, there was a stimulation i n growth rate and feed efficiency due to D.E.S . The animals were started on D.E.S . at 718 l b . (cf. 465 l b . for Study I ) . The increase i n the three blood parameters was again observed on the low iron rations but, on the high iron ration there was an apparent but insignif icant decrease i n these parameters due to D.E .S . The animals fed the control high iron rat ion produced an haematocrit and red c e l l count that was s ignif icantly higher than that of the control low iron ra t ion , indicating a possible deficiency i n the or ig inal supplements. On the low iron rat ion there was a significant increase i n to ta l blood V . F . A . from 0.88 meq. / l . to 1.19 meq. / l . i n response to D.E.S . supplementation but, on the high iron ration the increase was ins ignif icant . The difference between the two control rations (0 D . E . S . , high and low iron) t o t a l blood V . F . A . was shown to be s ignif icant , 0.88 vs. 1.08 meq. / l . at P < .05. A method of preparation of feed and l i v e r samples for analysis of the ir mineral content by atomic absorption spectrophotometry was developed. There was shown to be a significant increase i n l i v e r copper storage on the low iron rat ion with increased levels of D.E.S . The feeding of a high iron ration caused a significant decrease (P** .05) i n the leve l of copper i n the l i v e r from 84- ppm. to 37 ppm. A s l ight but insignif icant increase i n l i v e r iron levels on the high iron ration and through the treatment with D.E.S . was observed. TABLE OF CONTENTS Page A. Introduction 1 B. Literature Review 3 I. VOLATILE FATTY ACIDS 3 1. Volatile Fatty Acid Metabolism 3 a) Production of V.F.A. i n 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. i n 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. i n the Ruminant 23 2. 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 i n V.F.A. Production with Age 33 b) Species Variation i n V.F.A. Production 34 Page 3. Blood and Rumen Levels of Volatile Fatty Acids 35 4. 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 42 1. The Effect of Diethylstilbestrol on Efficiency 42 a) Rate of Gain and Feed Efficiency 42 b) Nitrogen Retention and Digestibility 44 2. Changes i n Body Composition Caused by D.E.S. 45 a) Changes i n Gross Body Composition 45 b) Changes i n Organ Size and Mineral Content 46 c) Tissue Residues of D.E.S. 47 3. Oral vs. Implantation Administration of D.E.S. 47 4. The Effect of Level of D.E.S. on Response 48 5. The Influence of Ration on D.E.S. Response 49 6. Effect of D.E.S. on the Volatile Fatty Acids 51 7. Relationship Between D.E.S. and Blood Components 52 8. Theories of D.E.S. Response 53 I I I . BLOOD 55 1. The Variation i n Normal Blood Parameters 55 a) Normal Values 55 b) The Effect of Age on Normal Values 56 2. Changes i n Blood Parameters Caused by: 57 a) Supplemental Iron and Copper 57 b) Miscellaneous Ration Additions 58 3. Relationships Between Iron and Copper and Anemia 59 4. Blood Parameters and Growth Rate 59 IV. LIVER IRON AND COPPER STORAGE 61 1. Levels of Iron and Copper Storage 61 Materials 64 I. ANIMALS 64 1. Experiment One (Barley and Barley-Alfalfa) 64 2. Experiment Two (Barley and High and Low Levels of Iron) 64 I I . HOUSING 64 I I I . RATIONS 65 1. Study I 65 Page 2. Study II 66 IV. ANALYTICAL REAGENTS 67 D. Methods 68 I. ANIMAL HANDLING PROCEDURES 68 1. Preliminary Procedures 68 2. Weighing 68 3. Feeding 68 4. Blood Collection 68 5. Tissue Sampling 69 II . VOLATILE FATTY ACID ANALYSIS 69 1. Total Blood Volatile Fatty Acid Analysis 69 2. Volatile Fatty Acid Ratio Analysis 70 II I . ANALYSIS OF BLOOD PARAMETERS 71 IV. ANALYSIS OF TISSUE SAMPLES 72 1. Thyroid Gland 72 2. Liver Mineral Storage 72 Page E. Experimental Results 74 I. STUDY I 74 1. 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 80 2. Summary of Study I 82 I I . STUDY II 83 1. 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 88 2. Summary of Study I I 91 I I I . CONCLUSIONS BASED ON STUDIES I AND I I 93 F. Bibliography 95 G. Appendices 111 LIST OF TABLES Page Table I Gross Composition of Supplements 55, 56, and 57 65 Table II Gross Composition of Supplements 60, 61, and 62 66 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 I I 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 LIST OF APPENDICES Page Appendix I. I l l 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 ACKNOWLEDGEMENTS The writer wishes to express his thanks to Dr. W.D. K i t t s , 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 i n the animal handling and tissue sampling procedures and, I thank them. I would be remiss though, i f I d id not give special mention to Messrs. W. Freding, L . Holmes, G. Huffman, and J . Ross. A. INTRODUCTION Ever since i t 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. Up to this time no definitely proven pathway has been established but, i t 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 i t 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 l i t t l e 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 l i t t l e 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 2'. blood as well as to examine changes i n the three main blood parameters on different iron intakes. An examination was also made of the effect of D.E.S. on the l i v e r storage of iron and copper at various levels of D.E.S. treatment and iron intakes. It i s 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 i n growth rate and feed efficiency due to D.E.S. This may then demonstrate a possible mode of action for D.E.S. 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 i n cattle i t has been shown that 6,000-12,000 Cal. become available from the V.F.A. produced by fermentation i n the rumen (Carroll and Hungate, 1954). The total energy turnover of fasting sheep and cattle i s about 1,100 and 6,500 Cal. respectively per day, indicating that V.F.A. make a major contribution to the energy requirements since i t i s recognized that these acids are u t i l i z e d by body tissues." (8) 1) Volatile Fatty Acid Metabolism a) Production of Volatile Fatty Acids i n the Gastrointestinal  Tract The basis for the production of volatile fatty acids, (V.F.A.) l i e s i n the nonsecretory character of the rumen epithelium. Under the conditions that prevail i n the monogastric digestive system the greater part of the breakdown of feedstuff's i s the result of endogenous enzymes secreted by the epithelium of the stomach and the organs of the upper digestive tract. The role of bacteria i n digestion of the feeds i s relatively minor from the standpoint of efficiency of u t i l i z a t i o n of the ration or i n governing the availability of the nutrients. They are, however, of some limited importance when one considers their a b i l i t y to synthesize the B complex vitamins and Vit. K i n the lower intestinal tract. In the ruminant or polygastric animal there i s essentially no secretory capability i n the largest part of the stomach. Under this condition, i t must exist i n a symbiotic relationship with the bacteria and protozoa that inhabit the largest part of i t s digestive system, the rumen. 4 The complete absence of an enzyme secretory capability i s not altogether true as i t does possess enzymes which are secreted into the abomasum and are proteolytic i n nature. They are responsible for the digestion of microbial protein. Ruminants do not have endogenous enzymes capable of digesting cellulose and must i n con-sequence, depend wholly on their rumen microflora for this function. As early as 1884 i t was theorized by Tappeiner (20) that cellulose digestion i n the ruminant animal was the result of bacterial as well as protozoan action resulting i n 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 i n the f i e l d who f e l t that the contribution of these acids to the energy requirement of the animal was relatively minor. I t was not u n t i l the 1930's that i t 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 u t i l i z a t i o n , their effects on the per-formance 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 i s more than one area i n the animal body capable of producing V.F.A. The major site of production i s the rumen where 70% of cellulose digestion takes place, (173) the second and a relatively minor s i t e , i s the caecum where 17% of cellulose 5 digestion takes place, and the third site i s the large intestine where the remaining 10% of digestible cellulose digestion occurs. As i t has been stated by Shaw (155) that 70% of ruminant energy i s derived from absorption anterior to the abomasum, the greatest part of this review w i l l deal with factors effecting V.F.A. production within the rununo-reticulum. The total level of production has been estimated i n Barnett and Reid's review (20) to be from 286-371 gm./day in the rumen of the ox. The relative level of V.F.A. concentration i n the rumen has been shown to be of major importance i n governing the productive efficiency of any particular ration (20, 155). The ratios of the three major acids, i n 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. i n the rumen when animals are fed identical rations. One of these i s the influence of time after feeding on the level of total V.F.A. i n the rumen (44, 78). Barnett and Reid i n 1961 (20) and, ljuther i n 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 6 to this drop i n propionic production there was a rise i n acetic production. Basic agreement with these observations may be found in the work of Grey et a l (70). They state that when the rate of production increases the acetic-propionic ratio gets smaller and, when i t i s decreasing, the acetic-propionic ratio gets larger. In the more recent work of Leng et a l (97, 98) and Weller (178), i t 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 i t i s 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 i t s energy contribu-tion i s 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 i n the rumen are governed by the rations fed to the animals and they a l l contribute i n 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 i n the whole 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) i t was shown that, through a series of condensa-tion 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 i t 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, i t must be. realized .that over 50% of protein digestion is the result of microbial attack in the rumen. (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 1.6-3.2% Propionic' 16 - 27% Caproic .5 - 1.0% n Butyric 6 - 11% Heptoic .04- .05% From this, i t 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 9 noted a high production of propionic ac id from simple polysaccharides and hexose sugars. With respect to prote in d igest ion and poss ib le V .F .A . produc-t i o n , i t has been shown that an increased l e v e l of starch i n the ra t i on i s capable o f reducing ammonia product ion. Since the production o f V .F .A . from an amino ac id would require i t s deamination, i t i s reasonable t o suggest that s tarch depresses V .F .A . production from prote ins i n a s i m i l a r way to the glucose e f fec t on ce l l u lose d iges t ion . There i s a lso a qua l i t a t i ve aspect to the e f fec t of prote in on V .F .A . production due to the di f ferences i n t h e i r s o l u b i l i t i e s . The second major group o f inf luences on the production of V .F .A . are changes brought about i n the microb ia l populat ion by some external in f luences. I t has been stated by Walker (173) that there ex i s t s "a f a i r l y prec ise re la t ionsh ip between the amount o f b a c t e r i a l c e l l mater ia l synthesized and the A . T . P . made ava i lab le by the fermenta-t i o n of an energy source." From t h i s i t i s postulated that the greater the amount o f c e l l ma te r ia l , the higher w i l l be the energy y i e l d from any foodstuf f and the more e f f i c i e n t w i l l be the gains produced. The importance o f the quant i ta t ive aspect o f pro te in metabolism must then be considered as i t i s known that about 50% of the bac te r i a l c e l l i s pro te in (20). I t i s therefore not unreasonable to observe an increase i n bac te r i a l numbers wi th an increase i n prote in l e v e l and a consequent increase i n the energy o r V .F .A . y i e l d from any feed under these condit ions (52). There are numerous other external inf luences on the bac te r i a l 10 population that help towards changing the production of V.F.A. concentra-tions. 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 10-20 ppm, estrogen, diethyl-stilbestrol, 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 11 by 45%. From what has previously been discussed concerning cellulose digestion, i t 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, i t 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 micro-population 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. 12 In subsequent work (44) he stressed the importance of the symbiotic relationship that exists between bacteria and protozoa i n their mutual metabolism of each other's end products. In work with protozoa-free lambs he noted an increase i n the acetic-propionic ratio and i n the acetic-butyric ratio. He theorized that this effect was due to an increase i n the level of propionic acid production and hypothesized that protozoa produce l a c t i c 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 i n the feed efficiency by 34%. The changes brought about by protozoa were greatest when the animals were on a high con-centrate diet. Luther (102) i s i n 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 i n the acetic-propionic ratio on low concentrate rations. He has also discovered that protozoa effect an increase i n 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. 13 d) The D is t r i bu t ion of V .F .A . i n the Digest ive Tract The leve ls o f t o t a l V .F .A . and the l eve ls o f each V .F .A . have been shown to change as a feedstuf f passes through the d igest ive t r ac t (89, 125, 175). The main causes o f these changes have been enumerated, f i r s t by Elsden i n 194-5 (57), and then l a t e r by Annison and Lewis i n 1959 (8). In summary, they have compiled a l i s t o f s i x fac to rs : 1. production i n the rumen 2. absorption from the rumen, e t c . 3. passage from the rumen to omasum 4. d i l u t i o n wi th s a l i v a 5. u t i l i z a t i o n by microorganisms 6. conversion to other metaboli tes In E lsden 's work i t was shown that 88% of the t o t a l V .F .A . i n the whole d igest ive system was located i n the rumino-reticulum of sheep. In steers t h i s value was 86%. These observations have been confirmed by the work o f Packett i n 1966 (125) where he was able to show that 87% of the t o t a l V .F .A . content of concentrate fed animals was found i n the rumino-reticulum. In the case o f roughage fed animals, however, t h i s percentage was 92%. This e f fec t could poss ib ly be due to a more rap id rate o f passage i n the concentrate fed animals, causing more undigested carbohydrate to pass in to the res t of the d igest ive system. I t must be remembered that the synthesis of V .F .A . a lso occurs i n the caecum and colon (125) and t h i s would tend to lower the ca lcu lab le percentage of V .F .A . w i th in the rumen. The absorptive capab i l i t y of the rumen was examined by Johnston i n 1961 (89). He showed that the decrease i n the V .F .A . concentrat ion 14 between the rumen and the ret iculum was 18%, between the ret iculum and the omasum was 51%, and between the omasum and abomasum was 83% of the amount i n the preceding sect ion o f the stomach. In an ana lys is of the changes i n t o t a l V .F .A . concentrat ion i n each sect ion o f the gas t ro in tes t ina l t r ac t one must a lso be aware of changes that occur i n the r e l a t i v e proport ions o f each V .F .A . due to the d i f fe ren t absorptive preferences or d i f fe rent condit ions governing t h e i r metabolism (89, 125, 175). The proport ions o f the three main V .F .A . are qui te var iab le throughout the d igest ive t r a c t . A breakdown of t h i s d i s t r i bu t i on has been presented by Ward i n 1961 (175) from work wi th Hereford c a t t l e , as fo l lows: Acet ic Propionic Butyr ic Formic Lac t i c Tota l molar % mM/% Rumen 56.8 26.3 16.8 11.9 Abomasum 34.9 15.7 19.7 11.7 18.0 1.3 Small In test ine 17.9 11.8 33.8 10.8 25.8 1.9 Caecum 41.6 17.3 10.0 5.4 25.7 12.1 Colon 41.6 18.4 11.1 4.0 25.0 11.2 e) Absorption o f V o l a t i l e Fatty Acids In p r i o r parts of t h i s d iscuss ion i t has been shown that the disappearance o f V .F .A . from the rumen i s brought about mainly by absorp-t i o n through the rumen epi thel ium. This absorption of V .F .A . i s cont ro l led i n both quanti ty and type by condit ions that ex i s t w i th in the rumen (32, 51, 70, 106, 135). The i n i t i a l work done on t h i s subject was that of Barcrof t i n 1943 (19) when he found, working wi th sheep, that the s i t e s of absorption 15 o f V .F .A . were the rumen, re t icu lum, omasum, and caecum. In an attempt to determine i f there were any di f ferences i n the rates of absorption from the rumen he 'infused: the sodium s a l t s of the V .F .A . and analyzed the di f ferences i n concentration between the rumen and the por ta l blood stream. He postulated that the rates o f absorption were: ace t i c the greatest , fol lowed by prop ion ic , and buty r ic the l eas t . This order i s exact ly that of the concentration of the V .F .A . i n the blood. This assumption o f po r ta l blood concentrat ion r e f l e c t i n g the rate o f absorption i s not a l together t rue as i t became apparent that the rumen epi thel ium i s capable of metabol iz ing ce r ta in o f the V .F .A . In 1945 at tent ion was s ta r t i ng to be d i rected towards an understanding o f t h i s phenomeronby D a n i e l l i (51). At a rumen pH of 7.5 he found that there was l i t t l e f ree fa t t y ac id present and that the permeabi l i ty of the rumen epi thel ium was reduced. However, at a pH of 5.8 the largest por t ion o f the V .F .A . were found i n the free form and the ra te o f t h e i r absorption was increased. The t ransportat ion o f these f ree V .F .A . was p a r t i a l l y through the water f i l l e d pores but la rge ly through the l i p o i d membranes o f the e p i t h e l i a l c e l l s . The order o f the rates of absorption that D a n i e l l i proposed under these various pH leve ls i s as fo l lows: pH 7.5 rate of absorpt ion: a c e t i c , p rop ion ic , bu ty r ic pH 5.8 rate o f absorpt ion: bu t y r i c , p rop ion ic , ace t i c Support f o r the idea that absorption i s poss ib le under both a c i d i c and basic condit ions comes from the work of Masson i n 1951 (106). His data ind icates that the e f fec t i ve rates of absorption o f V .F .A . are : 16 a c e t i c , p rop ion ic , and butyr ic due to the metabolic e f fec ts o f the rumen epithel ium i n lowering por ta l blood concentrations of propionic and buty r ic ac ids . He a lso pointed out that the rate o f absorption of ace t i c ac id i s cont ro l led by the concentrat ion i n the blood. This cont ro l d id not appear to ex i s t f o r propionic o r butyr ic ac ids . In fur ther work on the e f fec t o f pH on the rates o f absorpt ion, Gray (70) postulated a more rap id rate of absorption f o r propionic ac id when the rumen pH was between 6 and 6.5. These di f ferences between Gray, Masson, and D a n i e l l i may a l l be the resu l t of t h e i r use o f d i f fe ren t ra t ions as Gray (70) has pointed out that ra t ions do cont ro l the r e l a t i v e ra tes . Further contro ls on the r e l a t i v e rates o f absorption were proposed i n 1953 by the work of Pfander and P h i l l i p s o n (135) when they found that the rate o f disappearance o f the acids from the rumen was determined by the length of the carbon chain and by the concentrat ion of the a c i d . They a lso re la ted the t o t a l rate o f absorption o f a l l ac ids to production when they infused a mixture of the three main V .F .A . at a rate o f 78 mequivs./hr. They found that absorption was almost the same as the rate o f production at 68.5 mequivs./hr. Pfander and P h i l l i p s o n observed that when there was an increase i n ac i d i t y o f the rumen the ove ra l l rate o f absorption was increased. The work of Brown i n 1961 (32) a lso shows that absorption of the V .F .A . i s i n proport ion to t h e i r concentrations i n the rumen. In these studies the absolute rates o f absorption were 2.61 gm/hr. f o r ace t i c a c i d , 1.16 gm/hr. f o r p rop ion ic , and 0.89 gm/hr. f o r butyr ic a c i d . 17 f ) E p i t h e l i a l Metabolism of V o l a t i l e Fatty Acids Ever since research has s tar ted concerning i t s e l f wi th the usefulness o f V . F . A . , a considerable deal of a t tent ion has been d i rec ted towards determining the causes o f the di f ferences between rumen and blood leve ls o f V .F .A . In 1952, Pennington (128) determined that the main s i t e s o f V .F .A . metabolism were the rumen epithel ium and the l i v e r . The metabolism of ace t i c and propionic acids by the rumen epithel ium was found to be much less than butyr ic a c i d . In 1960, Brown (32) analyzed the po r ta l blood f o r end products of e p i t h e l i a l metabolism and confirmed the foregoing observation that butyrate was metabolized to the greatest extent. The main products of butyrate metabolism were ketones. In t h i s same year Shaw (155) demonstrated that butyrate metabolism resu l ted i n lac ta te as we l l as ketones. He a lso found that e p i t h e l i a l t i ssue removed glucose from the blood as a source o f energy. Ramsey (146), i n 1964, worked on the i d e n t i f i c a t i o n of the ketones produced by e p i t h e l i a l metabolism of butyr ic ac id and, through the use o f - l abe l l ed ac ids , demonstrated that the greatest part of the l abe l n was found i n £3 hydroxybutyric a c i d . This observation was confirmed by Spahr i n 1965 (1960). Spahr has a lso shown that small amounts o f ace t i c t ac id were metabolized to ^ hydroxybutyric acid-. In 1952, Pennington (128) stated that poss ib l y , propionic ac id was the main source o f carbohydrate f o r the ruminant animal and demonstrated 18 that t h i s conversion could occur. In 1956 (130) he showed that the epi thel ium would metabolize propionic ac id in to lac ta te and carbon d iox ide. Subsequent to t h i s i t was shown that propionic ac id and carbon dioxide would combine to form succinate, an intermediate i n the T.C.A. cyc le . The r e l a t i v e degree to which metabolism of propionic and buty r ic acids takes place i n the rumen epithel ium has been shown by Kiddle (95). By r a t i o ana l ys i s , he was able to show that propionic metabolism was much less than butyrate metabolism i n passing through the rumen epi thel ium. Prop ion ic :Acet ic Rumen Blood (Porta l ) Bu ty r i c :Ace t i c Rumen Blood (Por ta l ) 1. 2. 3. .26 .30 .28 .26 .33 .21 post. .32 ant. .24 .21 .21 .10 .11 .07 post. .10 ant. Ace t i c Propionic Butyr ic Rumen 63.2% 16.7% 15.4% Por ta l Blood 76.7% 13.4% 5.2% In 1952, Pennington (128) incubated sheep rumen e p i t h e l i a l s l i c e s wi th a mixture o f V .F .A . and noted the fo l lowing average u t i l i z a t i o n from a sample o f 100 moles: Ace t i c Propionic Butyr ic 14.1 moles 10.3 moles 40.0 moles These observations have been confirmed by Bensadoun i n 1962 (26), 19 In previous sect ions of t h i s report i t was stated that absorption occurred from the rumen and caecum. Packett i n 1966 (125), attempted to determned i f any di f ferences ex is ted i n the metabolic charac te r i s t i cs o f these two areas. Working wi th sheep t i ssues from animals fed on roughage and concentrate ra t i ons , he was able to postulate the p o s s i b i l i t y of d i f ferences i n the pathways o f ox idat ion of V .F .A . and i n the preference o f each t i ssue f o r a pa r t i cu l a r a c i d . He found that caecal t i ssue u t i l i z e d 75% of the amount of V .F .A . that r\m_nal t i ssue would wi th an oxygen uptake of 35%. I t was a lso graph ica l ly i l l u s t r a t e d that the rumen epithel ium had a great preference f o r bu ty r ic ac id and that the caecal t i ssue had a preference f o r ace t i c a c i d . These resu l t s give strong evidence to the theory o f d i f fe ren t metabolic pathways when one considers that the caecal t i ssue u t i l i z e d so much more V . F . A . / u l . o f oxygen consumed. Evidence a lso ex is ted to support the p o s s i b i l i t y of changes i n these absorptive t i ssues to accommodate changes i n ra t i ons . I t has been shown (9, 181) that con-centrate ra t ions produced a higher proport ion of propionic ac id and, would therefore require a greater rate o f metabolism by the absorptive t i s sues . This hypothesis appears to be borne out by the data from the rumen t i ssue but, i s most s t r i k i n g l y shown by caecal t i ssue where the absorption o f propionate from concentrate ra t ions was 2.5 times that on the roughage r a t i o n . g) L ive r Metabolism of V o l a t i l e Fatty Acids In the preceding sect ion of t h i s l i t e ra tu re review i t was shown that the proport ions of V .F .A . reaching the l i v e r were approximately 20 t 76-86% a c e t i c , 11-13% prop ion ic , and 1-5% butyr ic (26, 95) depending on the ra t i on used. Upon enter ing the l i v e r , var iab le metabolism of these acids occurs to produce the ra t i os normally found i n blood. The ox idat ion of acetate i s qui te minimal i n comparison to that o f other ac ids . The main metabolic products are ketone bodies and carbon dioxide (96, 128, 139). These ketone bodies and the ace t i c sub-s t ra te can enter the T.C.A. cyc le as ace ty l CoA wi th no net synthesis o f carbohydrates (9, 14, 96). The oxidat ion o f ace t i c ac id i s inf luenced to a very major extent by the presence o f other metabol i tes. When t h i s occurs, greater amounts of ace t i c ac id enter the blood stream and cause an e levat ion of t o t a l blood V .F .A . One of the most ac t ive i nh ib i t o r s o f acetate metabolism i s propionic ac id (14, 96, 129, 131, 139). In 1956, Perinington (129) incubated sheep l i v e r s l i c e s with l abe l l ed ace t i c ac id and equimolar amounts of propionic ac id and found a 50% drop i n l abe l l ed carbon dioxide product ion. In tes ts wi th other metabol i tes, i t was found that butyrate, (96) i sova le ra te , v a l i n e , i so leuc ine , and methionine caused a s im i l a r reduct ion but , not to such an extent. Pennington reasoned that the propionic i n h i b i t i o n acted by masking Coenzyme A and therefore prevented the formation of Ace ty l CoA. In order to examine t h i s hypothesis a s i m i l a r experiment was run i n 1958 (131). The i nh ib i t o ry e f fec t was el iminated i n the presence of added Coenzyme A. This resu l t has been confirmed by the work of Pr i tchard i n 1960 (139) and Annison i n 1963 (9). 21 The metabolism of propionic ac id resu l t s mainly i n glucose (128) or i n i t s being ox id ized to carbon dioxide and water (14, 139) (v ia T.C.A. cyc le wi th entry through succinate or pyruvate) (50). Because the primary carbohydrate product of l i v e r metabolism of propionate i s g lucose, i t makes a considerat ion o f propionic metabolism espec ia l l y important consider ing that only 10% of the animals requirement i s absorbed each day (14). In 1958, McCarthy (111), working wi th goat l i v e r s , infused propionic ac id and found that i t was almost completely removed from the blood. This l i v e r metabolism resu l ted i n a s l i g h t increase i n blood acetate (9) but more important, there was a f i ve fo ld increase i n blood glucose and a three f o l d increase i n l a c t i c a c i d . Lac t i c ac id can then enter the T.C.A. cyc le through pyruvate and malate (14). In a more complete analys is o f the fate o f l abe l l ed propionic a c i d , Pr i tchard i n 1960 (139) demonstrated that the l a b e l occurred on not only carbon dioxide and glucose but a lso on succ inate, malate, and fumarate, a l l intermediates of the T.C.A. cyc le . Pr i tchard a lso found that the metabolism of propionic ac id would be st imulated by the presence o f most normally occurr ing l i v e r metabolites such as aceta te , butyrate (96), g lucose, and carbon dioxide (128). The greatest part o f t h i s work was confirmed by Annison i n 1963 (9). In 1957, Leng et a l (98) attempted to determine the s ign i f i cance o f l i v e r conversion o f propionate to glucose. They found that i f a l l the propionate was converted to glucose i t would account f o r 100% of i t . 22 However, t h e i r experimental resu l t s ind icate that only 32% of the absorbed propionate i s metabolized to glucose, making up 54% of that present. The l a s t and smaller por t ion (1-5%) o f por ta l V .F .A . enter ing the l i v e r has been found to be metabolized almost completely (9, 111). In 1958, i t was proposed by McCarthy (111) that the greatest amount of bu ty r ic ac id was used i n the formation o f l i v e r glycogen. However, s ince that time the general consensus of opinion i s that butyrate i s metabolized to Q hydroxybutyric ac id (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. cyc le v i a ace ty l CoA although, no net synthesis, o f carbohydrate has taken place (9, 14, 96). In a study which involved the sources of ^ hydroxybutyrate Annison, i n 1963, (9) was able to show that of a mixture o f l abe l l ed a c e t i c , prop ion ic , and butyr ic ac i ds , 50% of the butyr ic l a b e l , 7-15% of the ace t i c l a b e l , and 2% of the propionic ac id l a b e l turned up on t h i s product". The metabolian of l abe l l ed butyr ic ac id was a lso examined by Ho l te r (85), i n 1963, when he found that 76% of the l abe l turned up on ^ hydroxybutyric a c i d , 9.9% on carbon d iox ide , and 4.0% on ace t i c a c i d . Ho l ter (85) a lso presented a complete summary o f the changes that occur i n l i v e r metabolism during the in fus ion o f a 9:3:1 mixture of a c e t i c , p rop ion ic , and butyr ic acids as fo l lows: 15 0 mgm./lOO ml. 60 Change Butyrate 8.2 2.6 1.9 down Propionate 17.3 3.8 1.3 down Acetate 51.3 64.4 65.1 up Formate 16.5 47.5 64.2 up Lactate 47.7 132.5 173.0 up Glucose 92.0 376.0 615.0 up Ketones 5.2 10.6 18.0 up 23 h) The U t i l i z a t i o n o f V .F .A . i n the Ruminant At the present stage i n the development o f the energy supply o f the ruminant, from V . F . A . , the main sources are i n the form of ace t i c a c i d , ketones,(mainly£ hydroxybutyrate) smal l amounts o f absorbed glucose, and intermediates of the gluconeogenesis from propionate and other nu t r ien ts . The r e l a t i v e importance o f each o f these catagories i s the subject of much speculat ion wi th estimates of the importance o f V .F .A . der iva t ives ranging as high as 80% of the t o t a l energy requirement (120). Of t h i s 80%, Kiddle i n 1951 (95) estimates that 58% was due to ace t i c a c i d , 24% due to metabolites of propionic ac id and 18% due to the metabolites of butyr ic a c i d . The knowledge that the u t i l i z a t i o n o f any o f these compounds f o r energy resu l t s i n the production o f carbon d iox ide , has led many workers to an at tack o f measuring the amount o f l abe l present on the exhaled gas from animals fed l abe l l ed V .F .A . In 1965, Leng (97), using t h i s technique, found that 17.6% of exhaled carbon dioxide came from a c e t i c , 12.9% from propionic (glucose and intermediates), and 14.3% from butyr ic ac id (^ hydroxybutyric a c i d ) . Reid i n 1950 (148) has stated that propionic ac id i s present i n blood only when the rate of absorption i s greater than the rate of i t s use i n gluconeogenesis i n the l i v e r . The metabolism of ace t i c a c i d , and the butyr ic ac id de r i va t i ve , ^ hydroxybutyric a c i d , occurs i n the mitochondria i n the same way as the g l yco l ys i s products o f monogastric metabolism. In 1958, Annison (7) has shown that t h i s u t i l i z a t i o n ranges 24 from 1.4 to 2.1 m moles/hr/kg. body weight which, on a da i l y b a s i s , f o r a mature sheep, i s approximately 144 gm. In order f o r t h i s pathway o f V .F .A . metabolism to operate i n a most e f f i c i e n t manner, a source of three carbon intermediates, i n the form of oxaloacet ic a c i d , i s necessary. This requirement leads to a t y ing together o f the metabolism of ace t i c and butyr ic acids with that o f propionic ac id which can form t h i s intermediary v i a succ iny l CoA (14). I t i s poss ib le that t h i s supply o f cyc le intermediar ies can a lso be met by amino ac id breakdown (8). Even though the absorption of glucose forms a r e l a t i v e l y minor part o f the t o t a l energy absorption (26), i t s u t i l i z a t i o n by the ruminant body i s quan t i t a t i ve l y , per un i t metabolic s i z e , very s i m i l a r to that o f monogastric animals. This has been shown i n a summary by Armstrong (14) 75 to be 2.5 to 3.8 mgm./kg.' body weight/mm. f o r the dog and 1.5 mgm./ .75 kg.* body weight/min. f o r fasted sheep. The metabolism of glucose occurs i n the same way as i t does i n nonruminants v i a the g l yco l ys i s -T .C .A . pathway or the hexosemonophosphate shunt. 2) Tota l and V .F .A . Rat io V a r i a b i l i t y as Caused by: A. Ration The importance of changes i n the V .F .A . r a t i o i n the ruminant has been stressed by many workers because o f the inf luence i t has on the e f f i c i ency o f u t i l i z a t i o n o f nut r ients (20). The reasons fo r these di f ferences i n e f f i c i ency form the basis fo r a large part o f the work of B laxter and Armstrong (11, 12, 13) and w i l l be covered i n a l a t e r part o f 25 t h i s ana lys i s . Pfander i n 1961 (136) presented a l i s t o f fac tors which have been shown by past research to inf luence the V .F .A . r a t i o and i t i s apparent from t h i s that the ra t i on forms the major source o f t h i s va r i a t i on . 1. Proport ion of roughage to concentrate 2. P e l l e t i n g 3. P a r t i c l e Size 4. Heat Increment 5. Various O i l s 6. Prote in Level 7. Environment 8. Frequency of feeding 9. Mineral adequacy of the d ie t a) The Concentrate-Roughage Rat io and Forage Matur i ty The e f fec t o f any increase i n the l e v e l of concentrates i n the ra t i on i s no longer a subject of conjecture. I t has been shown by Balch (17) and Luther i n 1966 (102) that the response i n rumen micrcorganisms i s one which culminates i n an increase i n t o t a l V .F .A . (18) and an increase i n the r e l a t i v e l eve ls of propionic (48) and butyr ic acids at the expense o f ace t i c ac id (169). Along wi th t h i s change there i s an e f fec t o f lowering pH. Further observations concerning the cha rac te r i s t i c V .F .A . response to an increased proport ion o f concentrate i n the ra t i on came from the work o f Balch and Rowland (16). They noted the occurrence o f large f luc tuat ions i n the t o t a l V .F .A . l eve ls that d id not occur when a higher proport ion of roughage was fed . I t was a lso proposed that rumen 26 ace t i c ac id percentages ranged from 40.6 to 73.7, propionic ac id percentages ranged from 16.5 to 39.1, and buty r ic ac id percentages ranged from 6.6 to 13.9 depending on the types o f concentrates and roughages and the r a t i os i n which they were used. Emery, i n 1956 (6) , and Maki i n 1958 (105) examined the microb ia l pop-u la t ions o f concentrate fed animals and noted apparently c o n f l i c t i n g r e s u l t s . Emery, using the a r t i f i c i a l rumen technique and inocu la from concentrate fed animals, found a higher propionic ac id product ion. Maki , however, t r i e d to do an ana lys is o f the types of bac ter ia found i n the rumen and observed that gra in fed animals had few buty r ic ac id producers and no propionic ac id producers. A poss ib le explanation f o r t h i s contrast may be due to the fac t that Emery used a complete rumen sample and Maki , used bac ter ia without the protozoan populat ion. In add i t i on , Maki noted a two to three f o l d increase i n bac te r i a l numbers on concentrate-fed cows. roughage and the r e l a t i v e l e v e l of maturity of the forage on these V .F .A . r a t i os (23), as r e f l e c t i n g changed microb ia l populat ions. Card i n 1953 (39) showed that the l a t e r a forage i s cu t , the higher w i l l be the resul tant ace t i c ac id production and the lower the buty r ic and propionic ac id production as fo l lows: In an attempt to exp la in the causes of these r a t i o changes I t i s a lso o f some s ign i f i cance to note the e f fec t o f type of Ear ly Cut Si lage Late Cut Si lage Barn Dried Hay (ear ly) F i e l d Cured Hay Acet ic % 52.5 54.8 56.9 60.3 Propionic % 23.8 23.1 19.9 19.5 23.7 22.1 23.2 20.2 27 These resu l t s were subsequently confirmed by Parks, i n 1964 (127) and suggested that these changes be due to a decrease i n d i g e s t i b i l i t y , soluble sugar l e v e l , pro te in l e v e l , and an increase i n l i g n i n content. Barnett and Reid (20) have stated that the t o t a l V .F .A . production i s at a maximum when forages are at the stage o f greatest lea fy growth. In 1965, Bath (23) has a lso shown that the addi t ion of supplements such as sugar beet pulp and brewers grains depress ace t i c product ion. b) The Ef fec t of Various Methods o f Feed Processing The feeds usual ly fed to rundnant animals under in tensive care pract ices have almost always been subjected to some form of processing such as gr inding or p e l l e t i n g . I t i s therefore o f some importance to look at any di f ferences that occur i n the V .F .A . r a t i os o r t o t a l production caused by such handl ing. A great deal of work has been done i n determining the e f fec t o f gr inding o f hay on the V .F .A . r a t i os as produced. Ba lch , i n 1958, (17) fed l ac ta t i ng Shorthorn cows long or ground hay and noted a marked drop i n rumen ace t i c l eve l s from 57% to 46%, and a r i s e i n rumen propionic l eve ls from 22% to 33%. There was no change i n butyr ic ac id percentage. These resu l t s have been confirmed by Ensor, i n 1959, (62) and Thompson, i n 1965, (169) who a lso found that gr inding increased the t o t a l V .F .A . l e v e l i n the rumen. This e f fec t i s probably jus t the resu l t o f increased d i g e s t i b i l i t y on the ra t i on of smal l p a r t i c l e s i ze rather than a change i n the species of bac te r ia present (182). 28 I t has been shown by Rhodes (150) i n 1962, that the p e l l e t i n g o f a roughage ra t i on does not e f f e c t the V .F .A . r a t i o but he does lend support to the above hypothesis when he found that t h i s process d id increase d i g e s t i b i l i t y (182) and consequently feed e f f i c i e n c y . Further support f o r t h i s idea i s gained through the work of Shaw (150) when he found that the gr inding and p e l l e t i n g o f hay resu l ted i n a two f o l d increase i n t o t a l V .F .A . These two sets o f resu l t s taken together ind icate that there w i l l be an increase i n t o t a l V .F .A . wi th an increase i n d i g e s t i b i l i t y as proposed e a r l i e r . Ol t jen has shown, i n 1965, (120, 121) that p e l l e t i n g an a l l concentrate ra t i on does not e f fec t the molar percentage o f V .F .A . o r the t o t a l ruminal l e v e l s . These observations suggest that the pe l l e t i ng o f a concentrate ra t i on w i l l have no e f fec t on the V .F .A . r a t i os but , the p e l l e t i n g o f a roughage-concentrate o r a s t ra igh t roughage ra t i on w i l l cause a decrease i n ace t i c ac id l e v e l and an increase i n propionic ac id l e v e l when compared to the resu l t s obtained on a long hay r a t i o n . In t h i s regard, Woods i n 1962 (181) was able to demonstrate that the p e l l e t i n g o f a complete ra t i on ( i . e . containing concentrates and roughage) resu l t s i n an increase i n propionic production and a decrease i n ace t i c product ion. But, the pe l l e t i ng o f an a l l concentrate ra t i on had no e f fec t on V .F .A . r a t i os or t o t a l s . Woods a lso observed that the f ineness o f gr ind of hay has an e f fec t o f increasing the propionate percentage thereby causing a decrease i n the acet ic -prop ion ic r a t i o . This work was confirmed i n 1963 by the work o f Wright (182) when he found the acet ic -prop ion ic r a t i o resu l t i ng from long hay was 2.62, from coarsely ground hay was 2.12, and 1.67 from f i n e l y ground hay. 29 Another process designed to accomplish the same resu l t as gr inding i s that o f r o l l i n g but, i t i s usua l ly appl ied to the processing of gra ins. In an experiment i n 1961, Hayes (80) compared the e f fec ts o f dry r o l l e d bar ley and steam r o l l e d bar ley on the ga ins, e f f i c i e n c y , and V .F .A . r a t i os i n beef c a t t l e . In t h i s ana l ys i s , he found no di f ferences i n ra te o f ga in , 2 l b . / d a y , o r i n feed e f f i c i e n c y , 8.69 l b . / pound of ga in , between the two methods o f r o l l i n g . The V .F .A . data from the experimental animals showed no di f ferences up to 70 days on feed but, at 110 days there was s i gn i f i can t d i f ferences i n t o t a l V . F . A . , and molar proport ions of V .F .A . as fo l lows: Dry Rol led Steam Rol led Tota l V .F .A . 1230 mgm./lOO ml . 811 mgm./lOO ml . Acet ic 23.9% 32.6% Propionic 59.5% 49.1% Butyr ic 8.7% 10.1% Higher 7.8% 8.2^ These data serve to i l l u s t r a t e the high leve ls o f propionic ac id production on concentrate feeding which Hayer states i s the main reason f o r the greater feed e f f i c i ency on t h i s type o f r a t i o n . c) The Ef fec t o f Prote in Level and Feed Addi t ives In previous discussions on the factors in f luenc ing bac te r i a l numbers i t has been pointed out that an increase i n ra t i on pro te in l e v e l resu l t s i n an increase i n the t o t a l V .F .A . concentrat ion. I t has been shown by the work o f Davis i n 1957 (52) that such an increase i s accompanied by a change i n the molar proport ions of the three main V .F .A . 30 This change consists of a decrease i n the molar percentage of acetic acid and an increase i n the molar percentage of propionic and butyric acids with increasing levels of protein. Putnam, working i n 1958 (140), related these changes i n protein level to concurrent changes in the energy level. He found that the high energy rations produced a higher percentage of butyric acid and a lower percentage of acetic acid., The highest concentration of a l l acids was obtained on the high energy ration but, at the medium level of protein intake. In an attempt to isolate the cause of these changes (i.e. whether i t was protein or energy causing the change) Putnam, i n 1966 (143), conducted a similar experiment with beef heifers. He confirmed the previous observations and proposed an optimum level of protein for maximum V.F.A. production. There was also an increase i n total V.F.A. with an increase in energy intake. His results are as follows: Acetic Propionic Butyric Total m moles/1. Energy Intake H. 8.0 therms 64.0 25.1 21.3 110.4 M. 6.1 therms 56.3 18.8 15.6 90.7 L. 5.3 therms 53.9 18.8 15.1 87.8 Protein Intake H. .22 kg. 55.0 19.1 16.3 90.4 M. .12 kg. 66.3 23.5 19.9 109.7 L. .03 kg. 52.9 20.1 15.7 88.7 There are numerous other additives which are commonly used in cattle feeds which could influence the V.F.A. of the rumen. One of the 31 most common of these are a n t i b i o t i c s , which have been shown by Re id , i n 1954 (149) to cause a decrease i n the concentration o f propionic and buty r ic acids but no change i n the concentration of ace t i c ac id i n ca lves . Thompson i n 1965 (169) has shown that such a change does not occur i n s teers , poss ib ly because i t acts to prevent the establishment of a populat ion rather than a l t e r an ex i s t i ng one. The act ion o f supplemental urea i s the same as that o f supplemental pro te in i n causing an increase i n t o t a l V .F .A . production (162) at low l e v e l s . Stewart (162) showed that the common p e l l e t b inder, molasses, causes a decrease i n the ace t i c ac id l e v e l . Rhodes and Woods (150), i n 1962, reported that the addi t ion o f cobalt to a ra t i on causes a decrease i n butyr ic ac id percentage and an increase i n propionic a c i d . A poss ib le explanation of t h i s phenomena i s found i n the work of Barnett and Reid (20) when they found the cobalt would reduce c e l l u l o l y t i c a c t i v i t y . As ace t i c ac id i s the main V .F .A . produced from c e l l u l o s e , such a reduct ion i n ce l l u lose d igest ion would resu l t i n a decrease i n i t s product ion. There would a lso be a consequent reduct ion i n bu ty r ic ac id production because i t i s formed from the condensation of two molecules o f ace t i c a c i d . This could lead to a r e l a t i v e increase i n the propionic percentage as i t has been shown to respond to prote in l e v e l and protozoan numbers, factors not d i r e c t l y re la ted to c e l l u l o l y t i c a c t i v i t y . The act ion of z inc has a lso been shown by the work o f Ol t jen i n 1965 (120) of e f fec t a widening of the a c e t i c -propionic r a t i o . d) Animal Feeding Pract ices and V .F .A . Ratios 32 The plane o f nu t r i t i on inf luences the t o t a l l e v e l o f V .F .A . i n the rumen and a lso the V .F .A . r a t i o s . The only remaining var iab le i n animal feeding pract ices i s that o f the frequency of feeding. In 1961, Putnam (141) fed Angus he i f e r calves e i the r two or ten times da i l y i n amounts so that each group received the same d a i l y feed in take. He observed an apparent increase i n e f f iehcy on the more frequent feeding regime as we l l as an increase i n the t o t a l V .F .A . concentrat ion i n the rumen. The more frequent ly fed animals had a s l i g h t l y lower percentage of ace t i c and propionic ac id and an increased bu ty r i c ac id l e v e l . These ra t i on changes were not s i g n i f i c a n t . A poss ib le explanation f o r the increase i n t o t a l V .F .A . concentration i s protozoan numbers which have been shown i n previous sect ions to cause such an e f f ec t : 2X 10X molar % Acet ic 67.7 67.1 Propionic 17.5 17.2 Butyr ic 14.8 15.7 Tota l m moles/1. 62.6 70.1 . Protozoan Counts 2.04 2.60 x 10 /m l . Chr is t iansen , in 1964, (43) re la ted the changes i n the population of protozoa to changes i n the rate o f passage as caused by d i f fe ren t feeding p rac t i ces . He found that when an animal was f u l l fed a pe l le ted ra t i on a resu l tant increase i n the rate o f passage occurs along wi th complete e l iminat ion o f the protozoan populat ion. When the feed intake l e v e l was cut to 2/3 o f f u l l feed there was a drop i n the rate o f passage and a re-establishment o f the rumen protozoal populat ions. 33 Animal a) Changes i n V .F .A . Production wi th Age The production o f V .F .A . has been shown to be pr imar i l y the resu l t o f ce l l u lose d igest ion i n the rumen. In order f o r t h i s breakdown to occur i t i s necessary fo r the animal to have an establ ished microbia l population and, i t i s therefore poss ib le to t i e the r e l a t i v e l eve l s of V .F .A . to the age of the animal. In the young ruminant the major energy source i s blood glucose, as i s the case i n non-ruminants. As the animal ages, t h i s blood glucose l e v e l drops and the V .F .A . l e v e l r i s e s . In an experiment using goats, C ra i re in 1952, ("48) showed a drop i n blood glucose from the non-ruminant l e v e l of 115 mgm./lOO ml . to that i n ruminants, 40-60 mgm./ml. As the animals matured t h i s drop i n glucose concentrat ion occurred as rumen development progressed, as measured by e i t he r an increase i n volume o r i n t i ssue weight. Craine a lso noted that the greatest percentage change i n blood V .F .A . occurred i n butyr ic and propionic ac ids . In"1956, McCarthy (110) worked on a s im i l a r ana lys is with Hols te in calves and noted the same t rend. For the f i r s t f i v e weeks there was a steady decl ine which slowed down i n the s i x t h week and remained at t h i s l e v e l . The l e v e l of blood V .F .A . on the other hand, increased s tead i l y f o r a per iod of 15 weeks to a l e v e l of 6.53 mg./lOO ml . Rumen V .F .A . increased during t h i s same per iod but at a much fas te r ra te . McCarthy has been able to cor re la te t h i s increased blood V .F .A . l e v e l to an increase i n ce l l u lose d iges t ion . 34 The work o f Liang i n 1967 (99) ind icates that young calves have the a b i l i t y to absorb and metabolize V .F .A . i n the rumen or fo re -stomach and large in tes t ine at a few days of age. There was no di f ference i n the animals a b i l i t y to handle any o f these three ac ids . b) Species Var ia t ion i n V .F .A . Production The greatest va r ia t ion i n t o t a l V .F .A . production and V .F .A . ra t i os occurs between ruminants and non-ruminants due to t h e i r d i f fe ren t d igest ive processes. In a summary o f these d i f fe rences, McClymont (112), presented the fo l lowing r e s u l t s : Tota l Acet ic Propionic Butyr ic Higher mg.% molar % Sheep 8.0 93.0 2.3 1.1 3.6 Guinea P ig 4.0 92.0 .5 2.0 5.5 Rabbit 4.2 78.3 1.9 15.8 4.0 Horse 3.2 81.4 7.0 5.3 6.3 P ig 4.6 75.5 9.6 3.7 11.2 Human 6.7 88.0 4.2 1.1 6.7 These data i l l u s t r a t e the wide v a r i a b i l i t y i n t o t a l s and ra t i os o f V .F .A . I t was suggested by McClymont that there would be the same ra t i os and t o t a l s i n any two true rundnants i f they were fed the same ra t i on and were ra i sed under the same condi t ions. The importance o f t h i s l a s t condi t ion has been brought out by Wright, i n 1963, (182) when he stated that the di f ferences i n V .F .A . production were due to the species or a c t i v i t i e s o f those bac ter ia present rather than to the t o t a l numbers. I t i s therefore a reasonable assumption that the true ruminants being compared should be ra ised under the same condit ions so that they had equal opportuni t ies o f contamination. 35 The establishment o f i d e n t i c a l populations i n the rumens o f two animals i s f a i r l y impossible (23) due to t h e i r d i f fe ren t hab i t s . Therefore, one would expect minor d i f ferences i n response from animal to animal on the same r a t i o n . The major r espons ib i l i t y f o r a l t e r i ng t o t a l o r V .F .A . r a t i os on the same ra t i on s t i l l remains wi th processing (150). 3) Blood and Rumen Levels of V o l a t i l e Fatty Acids The ra t i os of V .F .A . i n the rumen and blood stream o f the bovine have prev ious ly been shown to be qui te d i f fe ren t due to the metabolic e f fec ts o f the rumen epi thel ium and the l i v e r . One of the e a r l i e r workers i n t h i s f i e l d was P h i l l i p s o n , who wi th Schambye i n 1949 (154) looked at the changes i n rumen and blood (por ta l and carot id) l eve ls o f V .F .A . wi th time a f t e r feeding. They observed that the peak rumen l e v e l was achieved at about four hours a f t e r feeding at 124 m moles/1. The peak por ta l l e v e l was reached at approximately the same time at 3.69 m moles/1, but, the peak caro t id blood l e v e l was at two hours a f te r feeding at 1.14 m mo les / l . These data on po r ta l blood were confirmed by Annison, i n 1957, (6) when he found that the peak per iphera l blood V .F .A . o f 0.92 m moles/1, occurred at one and a h a l f hours a f te r feeding. In 1951, McClymont (112) presented a breakdown of the various acids i n the per iphera l blood system and the corresponding rumen values on a molecular bas i s . 36 Rumen Blood (A r te r i a l ) Acet ic 60.0% Propionic 21.8 2.4 Butyr ic 14.4 2.5 Higher 3.8 1.8 Cra ine , in 1952,(48) performed essen t i a l l y the same experiment on goats and obtained a t o t a l blood l e v e l o f 5.4 mgm./lOO ml . wi th a d i s t r i bu t i on of 63.6 micro M/100 ml . a c e t i c , 21.0 micro M/100 ml . propionic , and essen t i a l l y no buty r ic a c i d . This t o t a l V .F .A . value compares favorably wi th the value o f McCarthy (110) of 6.53 mgm./lOO ml . i n work i n growing ca lves . In 1954 Annison (5) examined the jugular blood o f c a t t l e and sheep and found that ca t t l e per iphera l blood contained no propionic ac id but, sheep blood d id along wi th minimal amounts of bu ty r ic ac id (1%). The observed peak blood leve ls ranged from 0.55 to 1.32 m moles/1. Hol ter (84) stated that bu ty r ic ac id i s completely removed from bovine por ta l blood by the l i v e r . Bensadoun (26) using the new and more accurate gas chromatographic technique, proposed a s i m i l a r V .F .A . pat tern i n the rumen but , a lso examined the ra t i os present i n the por ta l blood: Rumen Blood (Porta l ) Ace t i c 53.0% 86.3% Propionic 24.5 11.4 Butyr ic 16.5 1.3 In a comparison of the values obtained here f o r po r ta l bu ty r ic ac id wi th that o f McClymont f o r a r t e r i a l blood i t i s apparent that the gas chromatographic technique would resu l t i n a much lower a r t e r i a l blood 37 l e v e l . Perhaps, as has been suggested by Craine (48), there would be no per iphera l butyr ic a c i d . The length of time required f o r the V .F .A . to enter the blood stream i s a funct ion of the rate of d igest ion and i s not cont ro l led by the length o f time required f o r e p i t h e l i a l metabolism. Aaf jes , i n 1964, (1) reported that a ruminal ly infused sample o f V .F .A . i s detectable i n the blood stream i n f i ve minutes. 4) The Ef fec t o f Tota l and V o l a t i l e Fatty Ac id Ratios on Growth a) V .F .A . and Energetic E f f i c i ency The var ia t ions i n the net energy y i e l d from a feedstuf f are cont ro l led i n large degree by the ra t i os o f the V .F .A . produced therefrom. An estimate o f the importance of each ind iv idua l a c i d ' s cont r ibut ion to the t o t a l energy derived from V .F .A . has been prepared by Kiddle i n 1951, (95) and by Emery i n 1956, (60). Of the t o t a l V .F .A . energy, 58% i s from a c e t i c , 25% from propionic,and 18% from bu ty r i c . The reason fo r the large contr ibut ion made by the long chain ac ids , i n such low concentrat ions, i s that they have a higher c a l o r i c value than ace t i c a c i d . Accorxiing to Brody, (31) these values are : ace t i c 3.49 Ca l . / gm. , propionic 4.96 Ca l . / gm. , and buty r ic 5.95 Cal . /gm. An even more dramatic d i f ference between normal rumen l eve l s o f these acids and the energy contr ibut ion of each to the t o t a l i s found i n the work o f Pfander i n 1953 (135). In sheep, he showed that V .F .A . absorption occurs such that ace t i c : 32 mequivs. /hr . , propionic : 18 mequivs. /hr . , and butyr ic : 23 mequivs./hr. f o r a t o t a l energy intake o f 38 25.3 C a l . / h r . A breakdown of the energy contr ibut ions resu l t s i n ace t i c wi th 6.7 C a l . , propionic with 6.6 C a l . , and butyr ic with 12.0 C a l . due to the great d i f ference i n c a l o r i c equivalents. Another fac to r contr ibut ing to the net energy d i f ference between these four: acids i s that of S.D.A. The greatest part of the work on t h i s subject i s that o f Armstrong and Blaxter (11, 12, 13). In 1956, (11) they infused i nd i v idua l samples o f the three fa t t y acids in to the rumen o f fasted sheep and noted large di f ferences i n S.D.A. Ace t i c Ac id had an S.D.A. o f 4-0-41%, propionic was 13%, and buty r ic was 16%. They of fered the explanation that the high S.D.A. o f ace t i c ac id was due to a shortage of metabolic intermediates, poss ib ly three carbon oxaloacet ic a c i d . To tes t t h i s hypothesis, a mixture of 5:3:1 a c e t i c , p rop ion ic , and buty r ic acids was infused i n the rumen o f sheep and they obtained an S.D.A. o f only 17%, f a r short o f what one would expect i f the acids d id not act symbio t ica l l y . In the same year , they (12) examined the inf luence of l e v e l o f ace t i c ac id on the S.D.A. of a mixture and found that there was no s i gn i f i can t d i f ference at l eve ls of 25, 50, 75, and 90% ace t i c a c i d . However, at l eve l s above 90% ace t i c a c i d , the S.D.A. rose qu ick ly to reach a l e v e l o f 40%. They have a lso noted that a l l mixtures o f acids have a ni t rogen sparing ac t ion wi th the greatest e f fec t caused by propionic . This observation tends to re in force the i n i t i a l hypothesis that propionic ac id lowered ace t i c S.D.A. by supplying a three carbon intermediate. 39 When working with growing sheep, Armstrong and Blaxter, (13) noted that there was a large increase in the V . F . A . , S.D.A. to levels such that acetic was 67%, propionic was 44%, and butyric was 38%. At this plane of nutri t ion the S.D.A. sparing action of propionic acid was s t i l l shown to be operative. When ratios of 7.5:1.5:1.0 of the V . F . A . were fed the resultant S.D.A. was 68%. However, when i t was dropped to 0.83:1.5:1.0 the S.D.A. dropped to 42%. This observation leads one to the conclusion that the rations producing the highest proportion of propionic acid should be the most e f f ic ient ly used. b) The Effect of V . F . A . Ratios on the Rates and Efficiency of Gain The ultimate test of whether or not these changes in V . F . A . are of any rea l importance i s in whether or not they are capable of altering the rates of gain or feed efficiency of those animals commonly raised for meat. A number of workers have shown that this i s possible. In 1959, Ensor (62) reported that ground, pelleted rations produce faster gains than those fed i n the unground form. From the previous discussion on feed processing and V . F . A . ra t ios , i t i s known that grinding a ration produces a higher proportion of propionic acid at the expense of acetic ac id , for example (62): Ave. D. Gain Feed D.M./lb. Gain Low Propionic 2.05 3.99 High Propionic 2.55 3.29 Shaw,in 1960, (156) in work with steers, produced a higher propionic ration by again grinding and pel let ing a hay ration and 40 observed a 22% increase i n average da i l y gain and a 15% increase i n feed e f f i c i ency . The e f fec t of t o t a l rumen concentration of V .F .A . has been shown by the work o f Rae i n 1963, (144) to be p o s i t i v e l y re la ted to the rate of gain i n lambs. He a lso noted that those animals achieving the most rap id gains had a higher proport ion o f propionic and butyr ic ac ids . This decrease i n the acet ic -prop ion ic r a t i o causing a st imulat ion of the rates of gain and feed e f f i c i ency has a lso been shown by Olt jen i n 1965, (120) i n work on a l l concentrate fed ca t t l e and Weiss i n 1967 (176) i n roughage-con-centrate fed s teers . In 1966, Orskov (124) demonstrated that the add i t ion of sa l t s of V .F .A . on an i s o c a l o r i c basis can cause an increase i n gains. He has a lso shown that each o f these acids are not only used equal ly as e f f i c i e n t l y i n promoting t h i s increased gain but a lso as e f f i c i e n t l y as concentrate ra t i on energy. c) The E f fec t of V .F .A . Rat ios on Body Composition The most complete analys is of the e f fec ts of changes i n the V .F .A . r a t i o on body composition i s that of Weiss i n 1967 (176). He proposed the p o s s i b i l i t y that the r a t i o of fa t to lean i n an animal could be cont ro l led by changes i n the acet ic -prop ion ic r a t i o . The r a t i o of fa t to lean i s independent of the rate o f gain (82). Weiss found that the major i ty of the variance i n body composition could be accounted fo r by changes i n t h i s r a t i o . F i f t y - s i x per cent of the variance i n fa t and 58% of the variance 41 i n prote in was associated wi th changes i n the acet ic -prop ion ic r a t i o . He has a lso confirmed the fac t that propionic ac id i s the most e f f i c i e n t V .F .A . f o r l ipogenes is . Raun i n 1962, (147) had shown that 63% of the va r ia t i on i n carcass grade was associated wi th rumen buty r ic ac id l e v e l s . Orskov (124) found, through the supplementation of a ra t i on wi th the s a l t s o f V .F .A . that ace t i c ac id was used the leas t e f f i c i e n t l y f o r l ipogenes is . He a lso noted an increase i n dressing percentage from such treatment. Shaw (156) and Ol t jen (120) have both shown that the desi rable increase i n the acet ic -prop ion ic r a t i o ( i . e . due to increased propionic production) a lso resu l t s i n an increase i n the degree of unsaturation of body f a t . 42 I I DIETHYLSTILBESTROL 1) The Ef fec t of D i e t h y l s t i l b e s t r o l on Ef f iency a) Rate o f Gain and Feed E f f i c i ency One of the e a r l i e s t workers i n the f i e l d of animal growth st imulat ion was Andrews who, wi th Dinusson, i n 1950, (55) implanted 42 mgm. p e l l e t s o f d i e t h y l s t i l b e s t r o l (D.E.S.) i n beef he i fe rs and noted an increased rate o f body weight ,gain, feed e f f i c i e n c y , and feed con-sumption. Since that time a large amount of work has been done i n determining the e f fec ts of ra t ions and leve ls o f D.E.S. on the response of many types o f animals (45, 151, 168). In 1954, Burroughs (35) used several d i f fe rent types of ra t ions ranging from high roughage to high concentrate and noted an average st imulat ion i n gains of 20% and i n feed e f f i c i ency of 11% when D.E.S. was administered o r a l l y . He a lso ind icated (36) that there was no inf luence on ove ra l l response caused by feeding D.E.S. at the beginning o f the feeding per iod rather than at the end. A poss ib le refinement of t h i s l a s t observation of Burroughs comes from the work o f Per ry , (132) when he showed that the greatest e f fec t o f feeding D.E.S. at a l e v e l of 10 mgm./head/day was achieved i n the f i r s t 28 days. He a lso found a st imulat ion i n feed e f f i c i ency of 11% but d id not observe an increased feed consumption and, postulated that D.E.S. increased ce l l u lose d igest ion to e f fec t the increased feed e f f i c i ency . In 1957, Clegg (47) fed high gra in ra t ions o f ground bar ley 143 and r o l l e d oats and observed a 15% increase i n feed e f f i c i ency . The estimates o f the o v e r a l l ef fect iveness of D.E.S. i n increasing rates o f gain and feed e f f i c i ency i n ca t t l e have been reviewed by Riggs (151). The increase i n rates o f gain ranged from 4 to 37% with an average o f 17% and the increased feed e f f i c i ency from 4 to 24% with an average o f 12%. This i s a summary o f 21 t r i a l s i n steers rece iv ing 10 mgm./head/day at a weight greater than 600 pounds. The response of steers to various leve ls o f D . E . S . , shown by O'Mary, (123) appears to be somewhat dependent on ra t i on add i t ions. In an experiment where he fed a ra t i on containing 33% molasses he found that o ra l adrdnis t ra t ion or implantat ion of D.E.S. d id not resu l t i n any increase i n the rate o f gain or feed e f f i c i ency . A l s o , i n ra t ions of corn and hay he d id not observe any s ign i f i can t response. P r i o r to t h i s work, however, he had obtained s i gn i f i can t response which ind icates an inconsistency o f D.E.S. ac t ion . A s i m i l a r observation was made by Wallentine i n 1961 (174) when he fed an a l l -concent ra te ra t i on of corn and soybean meal. He obtained an i ns i gn i f i can t increase i n gains but , a s i gn i f i can t increase i n feed e f f i c i ency . The use o f D.E.S. i n animals that are pasture fed p r i o r to being brought to the feedlot has been examined by McCtormick (113). This type o f s i t ua t i on i s probably the most common i n the r e a l sense as most beef animals are ra ised on the range fo r about s i x months before being brought to the feedlot to be f i n i shed . In t h i s experiment, he implanted D.E.S. i n animals e i the r at the beginning of the pasture feeding t r i a l o r as they entered the feedlot o r at both t imes. Those animals implanted 44 as they went in to the feedlot had a 14-20% bet ter feed e f f i c i ency than those that were reimplanted, and a 11-14% bet ter feed e f f i c i ency than the cont ro ls . However, the ove ra l l rates of gain fo r those animals implanted twice was greater than those implanted only once. His resu l t s are as fo l lows: Implanted on Feedlot Only Pasture and Feedlot Gain/Head/Day Control Pasture 2.94 2.49 2.39 Feedlot 2.13 2.38 1.96 Tota l Per iod 2.54 2.44 2.18 These data ind icate that i t i s more economically e f f i c i e n t to implant only as the animal enters the feedlot as the more expensive feedlot gains are made 11-20% more e f f i c i e n t l y and 12% more qu ick ly under these condi t ions. b) Nitrogen Retention and D i g e s t i b i l i t y In attempting to e luc idate the act ion o f D.E.S. i n promoting these increased gains and feed e f f i c i e n c i e s , Whitehair i n 1953 (179) implanted two 12 mgm. pe l l e t s of D.E.S. i n lambs. He observed a s l i gh t increase i n crude f i b e r d i g e s t i b i l i t y and an 83% increase i n ni trogen re tent ion. Calcium and phosphorus retent ions were a lso increased by 60% but , no change i n serum Ca leve ls (55) was observed. In 1954, Clegg (45) reported that there was a .024 gm./kgm. feed, increase i n ni t rogen retent ion i n s teers . This l e v e l was twice that observed i n the cont ro l animals. Perry (132) postulated an increased prote in d i g e s t i b i l i t y as a 45 resu l t o f D.E.S. feeding but , t h i s i s probably jus t an increased ni trogen retent ion as there was no change i n faeca l ni t rogen excret ion wi th D.E.S. (10). Urinary excret ion i s decreased. 2) Changes i n Body Composition Caused by D.E.S. a) Changes i n Gross Body Composition From the point o f view of the producer the only importance of a change i n body composition due to D.E.S. i s whether o r not i t resu l t s i n a lower carcass grade or dressing percentage. From the volume of l i t e ra tu re that ex i s t s on t h i s subject i t has been d e f i n i t e l y shown (34, 69, 93, 123, 174) that there i s no change i n e i t he r o f these para-meters. The percentage shr ink , however, i s the subject o f a l i t t l e more controversy as i t has been shown to be unaffected (113, 69) o r to decrease (45). In e i t he r case i t i s not a disadvantage to the use o f D.E.S. In a more de ta i led ana lys is workers have shown (10, 45, 174, 180) D.E.S. reduces marbling or the amount of intramuscular f a t . Clegg (46) stated that the body o f t reated steers contained less separable f a t , more l ean , and was not d i f fe ren t from the contro ls with respect to the percentage bone o r moisture (113). He d id observe, however, that D.E.S. produced an increase i n the moisture content of h e i f e r s . This same analys is has been conducted by other workers (119, 174, 180) who agree b a s i c a l l y wi th Clegg but they a l l observed an increase i n carcass moisture. A sample o f the resu l t s obtained from t h i s sort o f study would be those o f Og i lv ie (119) 46 as fo l lows: Prote in % Moisture % Fat % 23.66 23.46 21.88 0 mgm. D.E.S. 10 mgm. D.E.S. 30 mgm. D.E.S. 16.80 17.12 17.39 58.96 59.00 60.22 b) The Ef fec t of D.E.S. on Organ Size and Mineral Content In fur ther attempts to show the s i t e s of act ion of D . E . S . , Clegg and Cole , i n 1954, (45) have examined cer ta in of the endocrine glands f o r increased s i ze or a c t i v i t y . They found that D.E.S. t reated steers had la rger p i t u i t a r y and adrenal glands. An increased p i t u i t a r y weight has a lso been observed by Preston (138) i n lambs. Clegg has a lso shown a s l i g h t but none the less i ns i gn i f i can t increase i n thyro id weights i n steers from 21.1-23.5 gm. to 22.5-26.1 gm. f o r the 60 mgm. implanted animals but Thompson (168), showed a 21% decrease i n 12 mgm. implanted lambs. I t has been shown by other workers (10) that there was a two f o l d increase i n the rates o f release o f growth st imulat ing hormone from the anter io r p i t u i t a r y along wi th an increase i n adrenal c o r t i c a l a c t i v i t y . In l a t e r work, Clegg and C a r r o l l (46) observed no change i n blood prote in bound iodine and, therefore, concluded that D.E.S. had no r e a l e f fec t on the thy ro id . weight was due p r imar i l y to an increase i n ra t i on prote in l e v e l . He a lso observed an increase i n kidney and l i v e r weights. In the same year , Bohman (30) examined the l i v e r f o r changes i n gross composition wi th In 1958, Preston (138) theor ized that an increase i n thyro id 47 D.E.S. feeding and found no change i n the dry matter, f a t , p ro te in , ash, o r carbohydrate percentage. However, Magee i n 1963 (104) examined the ash component of the spleen and l i v e r o f D.E.S. fed ra ts and found an increase i n the t i ssue leve ls o f i ron and z inc but, no change i n the leve ls o f copper. c) Tissue Residues o f D.E.S. In 1954, Perry (132) fed D.E.S. to steers at a l e v e l of 10 mgm./head/day and was unable to detect res idua l estrogen i n the t i s sues . Preston i n 1956 (137) used a bioassay technique sens i t i ve enought to detect D.E.S. at l eve ls of two p a r t s / b i l l i o n but was unable to f i nd any D.E.S. i n the lean meat, f a t , l i v e r , hear t , kidney, o r i n t e s t i n a l t i s sues . Preston's work was a lso done on animals fed D.E.S. However, Stob (163) (10) has been able to detect s l i g h t res idua l l eve ls i n D.E.S. implanted animals, i n the carcass and l i v e r . These leve ls were at approximately .01 u gm./gm. 3) Oral vs Implantation Administrat ion of D.E.S. In the i n i t i a l work on D.E.S. the primary method of administrat ion was by implantat ion. However, t h i s method resu l ted i n ce r ta in undesirable charac te r i s t i cs such as depressed l o i n s , elevated t a i l heads, pawing and bel lowing, and mammary development (113). I t has been suggested by Burroughs (34) that the implantat ion o f D.E.S. presents a po ten t ia l heal th hazard as we l l as the p o s s i b i l i t y o f some animals showing tox i c react ions 48 to i t . A l s o , he has shown (34, 36) that the o r a l adrniri istration o f D.E.S. produces the same desirable e f fec ts as implantat ion but without the un-desi rable side e f fec ts . Oral administrat ion al lows a more cont ro l led supply o f D.E.S. as implants tend to be released at varying ra tes . Add i t iona l advantages are presented by the ease with which D.E.S. i s administered and the p o s s i b i l i t y of making correct ions to the dosage. There i s , however, one disadvantage to t h i s method. The amount o f D.E.S. used per animal i s from 20 to 100 times (36) that used i n implantation but, the increased gains and feed e f f i c i ency are not propor-t i o n a l to t h i s increased usage. I t has been stated by Clegg and C a r r o l l , i n 1957, (47) that the feeding of 10 mgm./head/day i s equivalent i n response to one 15 mgm. implant. His resu l t s are as fo l lows: Gain/Head/Day 0 mgm. D.E.S. 2.12 10 mgm. D.E.S. fed 2.35 15 mgm. D.E.S. implant 2.40 O'Mary, (123) states that a 24 mgm. implant i s superior fo r feeding periods of 120-150 days than e i the r a 12 mgm. implant o r the feeding of 10 mgm./head/day. In 1958, Perry (133) has shown a 36 mgm. implant to be s l i g h t l y more e f fec t i ve than 10 mgm./head/day. ( i . e . 2.61 vs . 2.58 lb. /head/day) 4) The Ef fec t of the Level of D.E.S. on Response Dinusson, i n 1950 (55) f i r s t proposed that D.E.S. response, as rate o f ga in , was proport ional to the amount o f estrogen present. In t h i s 49 same year , Andrews (3) implanted Hereford steers with 60 and 120 mgm. o f D.E.S. and observed the fo l lowing resu l t s on 580 l b . animals: Gain/Head/Day Feed E f f i c i ency 0 mgm. D.E.S. 2.24 7.07 60 mgm. D.E.S. 2.47 6.76 120 mgm. D.E.S. 2.68 6.24 These resu l t s were confirmed by work on 680 l b . steers i n 1954 (4). The use of o r a l D.E.S. at l eve ls o f 0 ,2 .5 ,5 .0 , and 10.0 mgm./ head/day has shown essen t i a l l y the same pattern o f response. Burroughs, i n 1954, (34) fed a high concentrate ra t ion and obtained the fo l lowing resu l t s i n beef s teers . Gain/Head/Day Feed E f f i c i ency 0 mgm. D.E.S. 2.50 11.57 2.5 mgm. D.E.S. 2.71 10.76 5.0 mgm. D.E.S. 3.15 9.97 10.0 mgm. D.E.S. 3.41 9.15 These resu l ts have been confirmed by Kas te l i c (93) and Og i lv ie (119) at leve ls as high as 30 mgm./head/day. Og i l v ie a lso noted that there were consistent changes i n the p ro te in , moisture, and fa t contents of the body with increasing l e v e l s . 5) The Influence of Ration o f D.E.S. Response 0 The response o f beef steers to treatment wi th D.E.S. has been shown by Burroughs (35) and Eve r i t t (65) to be greatest on high concentrate ra t i ons . But, Hartman (78) has demonstrated a greater increase i n growth 50 o f lambs on high roughage rat ions than on high concentrate ra t i ons . I t i s probably of more importance, however, to consider the e f fec t of ra t i on nutr ient balance on D.E.S. response considering the advances that have been made i n the understanding of the nutr ient requirements o f ruminant animals. In 1958, Preston and Burroughs (138) fed ra t ions containing 9, 13, and 17% prote in at two energy l eve ls o f 530 and 665 k c a l / l b . to lambs fed 0, . 3 , and .6 mgm. D . E . S . / l b . r a t i o n . They observed a s t imula-t i o n i n the rate of gain on a l l ra t ions except the high p ro te in , low energy one. On the low energy ra t i on the increasing leve ls o f pro te in produced no increase i n gains i n response to D.E.S. treatment but , on the high energy r a t i o n , t h e i r was a 25% increase i n gains on the 13% prote in ra t i on and a 44% increase on the 17% prote in r a t i o n . On any one o f the low energy ra t ions the response to increasing leve ls o f D.E.S. decreased whereas on the high energy ra t ions the response increased wi th the l e v e l o f D.E.S. The feed e f f i c i ency fol lowed the expected trend and improved when gains increased. This same experiment has been run by Jones (90) at prote in l eve ls of 8.4% and 11.2% and at unstated energy l eve ls (LE and HE). He observed that the greatest s t imulat ion of gains occurred on the LE-LP r a t i o n , fol lowed by LE-HP, and HE-LP. The leas t response was i n the HE-HP r a t i o n . These two sets of data tend to point out the necessi ty of the aforementioned considerat ion of the nu t r i t i ona l balance, o r i n t h i s case p ro te i n : ca l o r i e , o f a ra t i on before examining D.E.S. response. Clegg and C a r r o l l (46) examined the e f fec t o f changing roughage concentrate ra t i os on the response o f ca t t l e to D.E.S. They fed ra t i os 51 o f 55:45, 60:40, and 65:35 and observed the greatest response on the 55:45 ra t i on fol lowed by the 65:35 r a t i o n , wi th almost no response on the intermediate 60:40 r a t i o n . Although they d idn ' t go in to the changes i n nut r ient balances caused by these s h i f t s t h e i r data ind icates the p o s s i b i l i t y of ra t ions whose nutr ient balances respond more to D.E.S. than other energe t ica l l y i d e n t i c a l ra t ions . This p o s s i b i l i t y has been explored by Goodwin (69) when he fed i s o c a l o r i c ra t ions at three prote in l eve l s of D.E.S. implanted s teers . There was an increase i n the growth rate of the cont ro l animals i n response to increasing leve ls of p ro te in , i nd ica t ing a more n u t r i t i o n a l l y balanced ra t ion being fed . However, the growth rate s t imulat ion o f the D.E.S. t reated animals got smal ler as the prote in l e v e l increased, as fo l lows: Prote in Level Control D.E.S. St imulat ion L 1.34 lb . /day 61% M 1.79 lb . /day 19% H 1.84 lb . /day 9% These data along wi th that of Jones (90) and Hartman (78) ind icate that the response to D.E.S. decreases as the ra t i on becomes more per fec t l y balanced wi th respect to energy and p ro te in . I t i s , there fore , not impossible that t h i s same re la t ionsh ip holds true f o r other nu t r ien ts . 6) The Ef fec t of D.E.S. on V o l a t i l e Fatty Acids There has been very l i t t l e work done on examining the r e l a t i o n -ship between D.E.S. and V .F .A . and o f tha t , most i s c o n f l i c t i n g . Browning, 52 (33) treated one animal in each of two pairs of lactating dairy cows with D.E.S. and noted, in one case, a decrease in total rumen V.F.A. concentration by 1.29 mM/100 ml. and in the other case, an increase of .26 mM/100 ml. There was also an apparent decrease, in propionic and butyric acids molar proportions. These results are, however, not significant due to their size and the small sample size. Christiansen, in 1964 (43) has also shown that D.E.S. stimulates protozoan numbers which, from the previous discussions should indicate an increase in total V.F.A. and butyric acid but a decrease in propionic acid. The previous results of Browning do not show this but, Christiansen (42) has shown increased acid production and protozoan numbers on D.E.S. work in vitro. 7) The Relationship Between D.E.S. and Blood Components Dinusson, 1950, (55) implanted 42 mgm. pellets of D.E.S. in beef heifers and noted a reduction in the erythrocyte counts from 8.53 3 to 7.86 million/mm. . However, Erain (63) in feeding D.E.S. at a level of 0.45 mgm./lb. of ration observed no change in hemoglobin or haematocrit in beef steers. As would be expected from such a neutral reaction Bohman (30) has shown no change in erythrocyte counts in animals fed 10 mgm./head/ 3 day. (8.06 million/mm. ). He also confirmed the lack of response in blood hemoglobin values to D.E.S. treatment. In additional work Clegg and Carroll (46) have shown no change in most other blood constituents attributable to D.E.S. treatment. They examined blood glucose, NPN, protein bound iodine, and potassium and sodium levels. 53 8) Theories o f D.E.S. Response There are many theor ies or var ia t ions o f theor ies aimed at deciphering the mode o f ac t ion o f D.E.S. I t has been shown (100) that D.E.S. w i l l enhance cer ta in ox idat ive react ions i n i n v i t r o breakdown of amino ac ids . A l so , D.E.S. response has been shown to have no e f fec t (119) o r a s l i g h t l y depressant e f fec t (33, 168) on ra t i on d i g e s t i b i l i t y so i t does not act by making exogenous nutr ients more ava i l ab le . One of the poss ib le explanations of i t s act ion has been presented by Og i lv ie (119) and Wilson (180) who state that D.E.S. prolongs the growth phase, o r the deposi t ion o f p ro te in . Because pro te in i s o f lower c a l o r i c value than f a t , i t r esu l t s i n more rap id gains and bet ter feed e f f i c i ency from the same c a l o r i c in take. In l a t t e r stages of fa t ten ing , these more e f f i c i e n t prote in gains lead to a lower maintenance cost per un i t o f feed and therefore contr ibute to the o v e r a l l more e f f i c i e n t gains at t h i s stage of growth. This hypothesis receives some support from Clegg and C a r r o l l (46) who found that the added weight was not due to add i t iona l bone growth or water re tent ion. In 1954, Clegg and Cole (45) proposed that D.E.S. act ion was mediated through the anter io r p i t u i t a r y (37) when they observed an increase i n i t s weight and i n adrenal weight. Support f o r t h i s hypothesis has been shown as a two f o l d increase i n the rates o f release o f an ter io r p i t u i t a r y , growth st imulat ing hormone (10). Preston (138) bel ieves D.E.S. has a growth hormone-like ac t ion and has shown that i t increases anter io r p i t u i t a r y weight. An increase i n adrenal c o r t i c a l a c t i v i t y has a lso been 54 shown. Burroughs (37) has postulated that D.E.S. causes the release of A .C .T .H . which acts on the adrenal cortex to st imulate androgen release o r , causes the release o f T .S .H . which acts on the thyro id to re lease thyrox in . Both o f these compounds are capable o f st:Lmulating growth ra tes . Two fur ther theor ies have been proposed by M e l l i n , i n 1965 (115), and are act ions s i m i l a r to that proposed f o r hormones. He theor ized that D.E.S. st imulated the synthesis or a c t i v i t y o f enzyme systems. Jensen (88) postulated that D.E.S. enhances the mobi l i ty of metabolites in to o r w i th in the c e l l by augmenting the permeabi l i ty o f c e l l u l a r membranes. I t may a lso e f fec t prote in synthesis by <_hanging the permeabi l i ty of nuclear membranes to increase the passage o f messenger R.N.A. 55 I I I BLOOD 1) The Var ia t ion i n Blood Parameters a) Normal Values The blood parameters which are usual ly considered as being necessary f o r descr ib ing i t s oxygen carry ing capacity o r i r on status are hemoglobin (Hb), haematocrit ( P . C . V . ) , and red c e l l counts ( R . C . C . ) . I t i s d i f f i c u l t , however, to state any normal values because o f the inf luence o f age, m ine ra l - l eve ls , and balance on these parameters. I t i s of in te res t to note that Byers (38) sampled many hundreds o f Jersey and Hols te in ca t t l e and found that 87% o f them had hemoglobin values between 9.0 and 13.0 gm./lOO ml . wi th an o v e r a l l average of 11.1 gm. %. He a lso observed a s i gn i f i can t d i f ference between the hemoglobin values o f Jersey ca t t l e (11.3 gm. %) and Ho ls te in ca t t l e (10.6 gm. %). This average value o f 11.1 gm. % has a lso been shown i n Hereford ca t t l e by Dent (54). Bhannasir i (27) has presented a good review o f a l l these parameters i n beef ca t t l e and concludes that the hemoglobin ranges between 11.8 and 12.4 gm. %, that the haematocrit ranges between 37.6 and 33.3%, 3 and that the red c e l l count ranges between 8.1 and 7.4 mil l ion/mm. depending on the weight of the animal. There was no change i n the hemoglobin concentration w i th in the R.B.C. The i n i t i a l values are f o r 500 pound animals and the second ones, f o r 800 pound animals. Greatorex, (72) showed that there i s very l i t t l e change i n the s i ze of the erythrocytes over t h i s weight range. 56 b) The Ef fec t of Age on Normal Blood Values Holman, i n 1956 (83) found that there was a change i n the erythrocyte s i ze i n the ear ly stages o f l i f e . Over the f i r s t two months 3 the volume dropped from 52 to 40 micron , and rose again i n two years 3 to an adult l e v e l of 57 microns . Byers (38) has stated that there was no change i n blood hemoglobin values when measured every h a l f year f o r a per iod of eight years. These observations of Byers (38), Holman (83), and Greatorex (72) ind icate that any changes occurr ing i n blood parameters must occur i n the f i r s t s i x months. This p o s s i b i l i t y has been explored by a number o f workers (83, 166) and Greatorex (72) who produced a tab le o f the weekly values of P . C . V . , Hb. , and R.C.C. Thomas (166) found that blood Hb. was highest during the f i r s t 15 days and then decreased u n t i l the 30th to 70th days. Within t h i s time per iod Greatorex (72) showed that the R.C.C. increased from b i r t h to the 50th to 80th days and then gradual ly decreased. He has a lso shown that the P.C.V. bears a c lose re la t ionsh ip wi th t h i s R.C.C. I t has a lso been shown that there i s an increase i n the hemoglobin concentration wi th in these red blood c e l l s (27). Holman (83) presented the fo l lowing table showing s h i f t s i n these three parameters: P.C.V. Hb. R.C.C. , % gm./lOO ml. mil l ions/mm 2 months 29.1 10.3 9.60 4 11 37.1 11.7 10.62 6 " 34.8 10.6 8.04 8 11 33.6 10.8 8.43 10 11 35.7 10.3 8.14 12 " 35.9 10.4 7.65 57 2) Changes i n Blood Parameters Caused by: a) Supplemental Iron and Copper The inf luence of supplemental i ron and copper on the three blood parameters previously mentioned has been examined by Matrone (107). He supplemented calves wi th 0, 30 and 60 mgm. o f i ron/day, and copper at l eve ls o f 0, 3 and 6 mgm./day and found that the minimal i ron requirement was 30 mgm./day. There was no observed di f ference i n blood parameters between animals on the 30 mgm. and 60 mgm. l eve l s but the 0 mgm. l e v e l produced anemia. Copper supplementation had no e f fec t on these values. Swenson (164-) observed a s i m i l a r occurrence i n a milk ra t i on supplemented with Fe, Cu, Mn,-. Co, Zn, I, Ca, P, Mg, and Vitamins A , D, E , C, and K and a lso found a depression i n adrenal weights as the ra t ions became more n u t r i t i o n a l l y per fec t . He pointed out the wide v a r i a b i l i t y i n the l i t e r a t u r e resu l t s from t h i s type o f experiment due to the d i f fe ren t 3 c l i n i c a l condi t ions. The R.C.C. ranged from 4.9 to 9.0 mil l ion/mm. , Hb. ranged from 7.6 to 16.5 gm./lOO m l . , and P . C . V . ' s ranged from 29 to 42%. The quant i t ive aspect o f mineral nu t r i t i on has been brought out by Cunningham (49) when he observed that Mn caused a decrease i n hemoglobin values at l eve ls between 820 and 2460 ppm. Magee (103) has shown a s im i l a r e f fec t wi th high l eve ls o f z i n c . The e f fec t o f l e v e l of i ron supplementation on the blood parameters has been examined by Hibbs (81) i n ca lves , and he found that as higher and higher doses of i ron dextran were in jec ted these blood values increased. 58 His results are as follows: Hb. P.C.V. % R.C.C. millions/mm. •3 Iron Level gm./lOO ml. H M L 12.7 9.9 7.4 39.2 31.9 26.4 9.1 8.0 6.1 This work has been confirmed Natz (118) and Hansen (76) in veal calves and by Perry (134) i n beef cattle. b) Miscellaneous Ration Additions A number of apparently unrelated ration additives have been shown to influence certain of these three blood measurements. Of these normal ration components; fat has tended to increase blood hemoglobin (38) but insignificantly so, and crude protein at levels less than 5.10% has produced a drop i n hemoglobin and haematocrit in beef bulls (114). Meacham (114) has also shown an increase i n thyroid weight per unit body weight with this decrease i n protein level. of 20 to 80 gm. of D.E.S./100 gm. body weight resulted i n a several fold increase in plasma iron levels. This increase was shown to be proportional to the amount of estrogen injected and i t was theorized that i t stimulated the synthesis of iron binding pho^hoproteins. shown the P.C.V. i s positively related to body temperature. Therefore, i t explains some of the c l i n i c a l v a r i a b i l i t y that i s observed from one piece of research to the next. In work on cockerals, Greengard (73) showed that the injection Of the numerous other miscellaneous factors, Weldy (177) has 59 3) Relat ionships Between Iron and Copper and Anemia Anemic animals have been shown to have a low P . C . V . , Hb. , and R.C.C. as we l l as a decreased volume of the erythrocytes (29). In 1957 Blaxter (29) stated that 20 to 35 mgm. of i ron i s not s u f f i c i e n t to meet the da i l y requirements o f ca lves . He f e l t that 40 mgm./day i s required i f an animal i s gaining 1 kg. /day. This requirement ra ises to 100 mgm./ day i f the animal i s expected to store 15-20 mgm./day. I t has a lso been shown (166) that the add i t ion of i ron to the ra t i on o f anemic calves resu l t s i n an increase i n the R.C.C. and Hb. values i f the other minerals are i n proper supply (166). This inf luence o f other minerals on the e f fec t o f i r on addi t ions on blood parameters has been examined by Elvehjem (59). This ana lys is t i e s together the importance of l i v e r i r on and copper storage with the blood: P . C . V . , Hb . , and R.C.C. He found that when an animal was copper d e f i c i e n t , add i t iona l feed i ron would only be stored i n the l i v e r wi th no change i n blood hemoglobin. When copper i s again fed there i s a decrease i n l i v e r i r on storage and an increase i n blood hemoglobin values. This deplet ion o f l i v e r i ron stores w i l l continue u n t i l the l e v e l reaches 0.1 mgm./gm. o f dry t i s s u e . When both copper and i ron are fed there i s an increase i n Hb. formation proport ional to the l e v e l o f i ron fed. 4) Blood Parameters and the Growth Rate I t has been shown by numerous workers that animals fed supplemental l eve ls o f i ron high enough to prevent anemia w i l l gain fas te r than those 60 allowed to develop t h i s condi t ion (29, 91, 107, 164). Natz (118) showed an increase i n feed e f f i c i ency and a greater res is tance to resp i ra tory disease from such treatment. However, Hibbs (81) reported that changes i n the l e v e l of i ron suppl ied to an animal by i n j ec t i on resu l ted i n changes i n blood parameters but not i n the growth ra te . Perry (134) stated that the i n j ec t i on of i ron had a depressant e f fec t on the growth rate o f a l l animals other than suck l ing ca lves . He d id recognize that lowered hemoglobin was associated with a lowered growth ra te . This co r re la t ion has been examined by Thomas (166) and Arthand (15) i n presumably non-anemic animal's. Arthan found that 33% of the va r ia t i on i n the rate o f gain was associated with var ia t ions i n s i x blood const i tuents , age, and i n i t i a l weight. Of the blood const i tuents measured, 19% of the va r ia t i on i n gain was associated wi th va r ia t ion i n blood glucose l e v e l s . Prom these examples i t i s apparent that var ia t ions i n the three main blood parameters o f non-anemic animals are not very important i n determining the rate o f ga in . However, the di f ference between anemic and non-anemic animals i s qui te s i g n i f i c a n t . 61 IV LIVER IRON AND COPPER STORAGE 1) Levels of Iron and Copper Storage The s i t e s o f i r on storage were f i r s t examined by Elvehjem i n 1927 (58) when he found the fo l lowing i ron concentrations i n sof t t i s s u e s : From t h i s , the importance of l i v e r storage of i ron i s read i l y apparent. The in te r re la t ionsh ips betwen i ron and copper i n the l i v e r have been discussed (59) i n the previous sect ion and showed that copper was necessary fo r the metabolism of l i v e r i r on in to hemoglobin with a consequent decrease i n l i v e r storage. The f luc tuat ions i n l i v e r i ron storage have been shown by Hibbs (81), on copper su f f i c i en t r a t i ons , to r e f l e c t the l e v e l o f i ron supple-mentation. In work on new born calves in jec ted wi th i ron dextran, he has shown the fo l lowing r e s u l t s : Ha i r Lung Spleen L i ve r Pancreas Kidney .0811% .0122% .0089% .0083% .0060% .0055% Hide Muscle Intest ine Brain Bone Marrow .0047% .0041% .0034% .0023% .0009% Iron Level L ive r Iron Levels H M L 125.2 ppm. 78.7 ppm. 47.4 ppm. At an intake o f .25 gm./day/100 l b . body weight, Thacker (165) showed l i v e r i ron storage to be 184 ppm. 62 Dent (54) reported essen t i a l l y the same re la t ionsh ip f o r calves supplemented with copper at an average l e v e l of 17.3 mgm./animal/ day. His resu l t s are as fo l lows: L ive r Copper Level L i ve r Iron Level Control 18.7 ppm. 278 ppm. Experimental 40.5 ppm. 262 ppm. I t i s s i gn i f i can t to note, however, that as the animals aged and increased t h e i r i r on intake there was a consistent drop i n l i v e r storage from 478 ppm. to 178 ppm. i n s i x months. Hardison (77) confirmed the changes i n l i v e r copper l eve l s associated wi th d i f fe rent copper intakes and found that the presence of Ca, P, Mn, Co, Zn, and Fe inh ib i ted t h i s storage on low copper in takes. VanCampen (171) proposed that these minerals act at the s i t e of absorp-t i o n , the i n t e s t i n a l mucosa. The absorption o f i r on was examined by Tompsett (167) who found that i t was hindered by the presence o f calcium and phosphoproteins but st imulated by ra t ions causing an increase i n rumen pH. These observations have been confirmed by Magee (103) i n respect to z inc l e v e l s . Gubler (74) examined the quant i ta t ive e f fec ts of i ron and copper de f i c ienc ies on the storage of both of these minerals i n l i v e r . He found that there was an increase i n l i v e r , k idney, and a 200% increase i n heart weights per un i t body weight on copper de f i c ien t ra t i ons . On the i ron de f i c ien t ra t ions there was a 50% increase i n heart weight per un i t body weight. The di f ferences i n the concentration of these two minerals at pa r t i cu l a r s i t e s i n the l i v e r have been examined by Chapman (40). The concentration of copper was found to vary depending on the 63 area from which the sample was taken but, the l i v e r was found to be completely homogeneous with respect to iron content. Kennedy (94) found that the variation i n duplicate l i v e r iron determinations was no more than 4%. 64 C. MATERIALS I. Animals 1. Study I (Barley and Bar ley -A l fa l fa ) Th i r ty grade Hereford feeder calves were obtained on November 8th, 1965 at an average weight o f 415 l b . They were maintained on decreasing leve ls o f hay and increasing leve ls of bar ley and a l f a l f a l ea f meal p e l l e t s u n t i l December 4 th , 1966 when they were placed on the experimental ra t i ons . At t h i s time the animals had an average weight o f 465 l b . They were f u l l fed on these rat ions fo r approximately 200 days to an average weight of 1010 l b . by Ju l y 1966. 2. Study I I (Barley and High and Low Levels of Iron) Th i r ty grade Hereford steers were obtained on Ju l y 28th, 1966 at an average weight of 688 l b . and were gradual ly condit ioned to an a l l bar ley ra t i on u n t i l September 2nd. At t h i s time t h e i r average body weight was 718 l b . The experiment terminated at the end of November at which time the average body weight o f the steers was 984 l b . I I . Housing The animals were placed i n covered pens, 24 feet square, g iv ing each ardmal an area o f 115 sq . f t . The f ront of each pen was f i t t e d with a feed bunk 11 feet long, a two foot constant ly f lowing watering system, and 12 foot metal gate. The pens were bedded wi th coarse sawdust. 65 I I I . Rations 1. Study I The 30 animals were d iv ided up in to s i x groups so that each group had as s i m i l a r a s ta r t i ng average weight as poss ib le . Three groups o f these received a basal ra t i on o f bar ley and the other three groups received a 50:50 mixture of bar ley and a l f a l f a l ea f meal p e l l e t s . Each o f these s i x groups o f animals a lso received two pounds of a prote in supplement containing su f f i c i en t D.E.S. to give each animal an intake of e i t he r 0, 10, o r 18 mgm. per day. The gross composition o f these supple-ments i s presented i n tab le I. Table I Gross Composition o f Supplements 55, 56, and 57 Constituent 55 56 57 lb . /Ton A l f a l f a Leaf Meal 1450 1450 1450 F ish Meal 72% 50 50 50 Soybean Meal 44% 50 50 50 Iodized Sa l t 150 150 150 Dicalciumphosphate 100 100 100 Molasses 195 195 195 Vitamin A 10,000 I.U./gm. 5 5 5 Aurofac 10 (aureomycin) 2.8 2.8 2 D.E.S. 1 gm./ lb . 10 18 On the basis of 20 l b . o f basal ra t i on supplemented with two pounds o f pro te in supplement the bar ley ra t i on contained 7.9% d iges t ib le prote in and the b a r l e y - a l f a l f a ra t ion contained 12.0% d iges t ib le p ro te in . 66 The d iges t ib le energy content of bar ley was approximately 1520 k c a l . / l b . and that o f the 50:50 b a r l e y - a l f a l f a ra t ion i s approximately 1320 k c a l . / l b . (117). These two values in terac t to give the bar ley ra t i on a p ro te in : ca lo r i e of 23.5 mgm./kcal . , and the b a r l e y - a l f a l f a ra t i on 4-1 mgm./kcal. A l l animals received one pound o f a l f a l f a hay per day. 2. Study I I The basal ra t i on was steamed r o l l e d bar ley wi th h a l f o f the animals being fed the supplements numbered 55, 56, and 57 and the other h a l f received supplements designated 60, 61, and 62. The gross composition o f these supplements i s presented i n table I I . Table I I Gross Composition of Supplements 60, 61, and 62 Constituent 60 61 62 lb . /Ton A l f a l f a Leaf Meal 1191 1191 1191 F ish Meal 72% 150 150 150 Soybean Meal 44% 250 250 250 Iodized Sa l t 80 80 80 Dicalcium phosphate 100 100 100 Molasses 195 195 195 Vitamin A 10,000 I.U./gm. 5 5 5 Zinc Oxide 2 2 2 Mineral Supplement 24. 5 24.5 24.5 Aurofac 10 (aureomycin) 2. 8 2.8 2.8 D.E.S. 1 gm./ lb . 10 18 The composition o f the mineral supplement was as fo l lows: Iron Fumarate 16.08 l b . Potassium Sul f ide 8.32 l b . Manganese Dioxide 23.70 gm. 67 The pro te in to ca lo r i e r a t i o obtained on t h i s ra t i on (20 l b . bar ley and 2 l b . supplement) was 23.8 mgm./kcal . , i n s i g n i f i c a n t l y d i f fe ren t from that i n study I These supplements, 60, 61, and 62, were designed to contain i ron at a l e v e l of 1290 mgm./lb. and supplements 55, 56, and 57 have been ca lcu la ted to contain 104 mgm./lb. A comparison of the proximate ana lys is of these supplements has been prepared by Huffman (86) as fo l lows: Supplements 55, 56, 57 60, 61, 62 Barley Dry Matter 89.60% 92.90% 83.20% Crude F iber 16.14% 22.35% 9.52% Fat 5.14% 5.60% 5.10% Ash 19.67% 17.58% 2.61% F iber 19.28% 20.20% 4.17% IV. Ana l y t i ca l Reagents A l l chemicals used met the American Chemical Society spec i f i ca t i ons . 68 D. METHODS I. Animal Handling Procedures 1. Prel iminary Procedures Upon enter ing the feedlot a l l animals were given an i n t e r -muscular i n j ec t i on of 5 cc . of I_quamycin (Oxytetracycl ine 100 mgm. /cc ) . The second group a lso received 2 c c . ' s of Pro-V i te (V i t . A 500,000 I .U . , V i t . D 75,000 I .U . , and V i t . E 50 I .U . / cc . ) and a second i n jec t i on of Liquamycin two days l a t e r . At times throughout both of these studies a few animals developed d igest ive upsets which were t reated by spr ink l ing 5 gm. o f Aurofac 10 (aureomycin) on the feed o f that group. This treatment proved to be very successfu l . 2. Weighing A l l animals were weighed weekly, p r i o r to t h e i r morning feeding. 3. Feeding Da i ly feed intake records were maintained f o r each o f the groups. The animals o f study I were fed twice a day up u n t i l March 3rd at which time they were only fed i n the morning. Feeding i n study I I was only done i n the morning. 4. Blood Co l lec t ion To c o l l e c t blood samples the animal was placed i n a stanchion which had attached to i t , at r i gh t angles, a f i ve foot gate. With t h i s * Source: Charles P f i z e r Co. L td . * * Source: Ayerst Laboratories L td . 69 apparatus the animals body could be he ld per fec t l y s t i l l and yet the head had complete freedom of movement. A rope ha l t e r was used to elevate the head so that the r i gh t and l e f t jugular veins were more access ib le . The blood from each animal was co l l ec ted in to vacutainer tubes; 2 x 15 ml. i n tubes wi th potassium oxalate as the anticoagulant f o r V .F .A . r a t i o , haematocrit, hemoglobin, and red c e l l count ana lys i s . 5. Tissue Sampling In the f i r s t study the only t i ssue sample co l l ec ted was the thyro id gland. The gland was obtained at the abat to i r by removing a sect ion o f the trachea i n the region o f the e p i g l o t i s . The thyro id was then excised from i t s res t ra in ing t issues i n the laboratory and weighed. In the second study, l i v e r samples were taken fo r i ron and copper storage ana lys i s . An approximate 100 gm. sample was removed from the t i p o f the caudate lobe and frozen u n t i l ana lys i s . I I . V o l a t i l e Fatty Ac id Analys is 1. Tota l Blood V o l a t i l e Fatty Ac id Analys is Tota l blood V .F .A . were analyzed by the method o f Bensadoun (25) wi th s l i g h t modi f icat ions. I t was found by Ross (152) that a s ing le 100 ml . sample o f d i s t i l l a t e contained essen t i a l l y 100% o f any added V .F .A . sample. A l so , he showed that i t i s not necessary to bubble ni trogen gas through the sample before t i t ra t i on . , provided that a blank i s run with each sample. 70 In t h i s case, a fur ther modi f icat ion was made i n the choice o f an ind ica to r . A sharper end point was achieved through the use of an ind ica to r made up o f two parts 0.2% methylene blue i n ethanol and one part 0.1% methyl red i n ethanol d i l u ted to 250 ml . 2. V o l a t i l e Fatty Ac id Ratio Analys is Samples o f blood used i n the analys is o f V .F .A . r a t i os were prepared by the method of Baumgardt (24) wi th a few modi f icat ions. In t h i s case blood serum was used. Sodium heparin was used as the a n t i -coagulant because i t was the only anticoagulant tested that d id not cause hemolysis under the condit ions the samples were taken. The samples were deproteinized and stored i n a frozen state without deleter ious e f f ec t s . In order to prevent contamination during steam d i s t i l l a t i o n of the samples, the system was completely washed wi th d i s t i l l e d water and the holding and c o l l e c t i o n f l asks were washed i n soap and water. P r i o r to a d i s t i l l a t i o n , the system was closed and steam was allowed to pass through i t f o r ten minutes. The recovery o f a mixture o f V .F .A . under these condit ions was as fo l lows: Ac id Input Ac id Output Recovery meq. 1. .0385 .0367 95.4% 2. .0410 .0407 99.3% 3. .0410 .0412 100.5% 4. .0410 .0409 99.9% The d i s t i l l a t e s were then made b a s i c , wi th 3N NaOH, and f rozen. They were l yoph i l i zed to dryness and placed i n long pointed tubes f o r storage. 71 The ana lys is o f V .F .A . ra t i os was made through the use o f a MicroTec (Model 2000 MF) gas chromatograph f i t t e d wi th a hydrogen flame ion iza t i on detector. A neopentylglycol succinate (N.P.G.S. ) s i x foot columnwas used as recommended by Baumgardt (24). The instrument was found to operate best , f o r the purposes at hand, under the fo l lowing condi t ions: Detector Temperature 190° C In le t Temperature 180° C Column Temperature 130° C Hydrogen Gas 60 cc . /m in . Nitrogen Car r ie r Gas 60 cc . /m in . A i r 1.2 C.F .H. A l l regulators were set at 20 p s i . The lypho l ized frozen basic samples were made a c i d i c wi th three or four drops of 60% ortho phosphoric ac id and 0 . 7 / / 1 . o f t h i s so lu t ion was in jec ted by the use of a l .Oy /1 . Hamilton syr inge. Each sample was run at l eas t four t imes. The use o f acetone f o r c leaning these syringes proved detr imental to the columns as ind icated by a r i s e i n the base l i n e a f t e r a sample had passed through them. Chloroform was l a t e r used as i t d id not cause t h i s problem. The analys is o f the peaks obtained on the recorder (Westronics S t r i p Chart Recorder, Model LS11B) i s presented i n Appendix I. I I I . Analys is o f Blood Parameters In these studies the blood parameters measured were haematocrit, 72 hemoglobin, and red c e l l counts. The haematocrits were prepared by centr i fug ing f o r four minutes i n an Internat ional Micro Cap i l l a ry Centrifuge (Model MB). The hemoglobins were determined d i r e c t l y i n a Photovolt Hemoglobinometer (Model 10) and the red c e l l counts were made i n a Neubauer Hemacytometer (Clay Adams Co . ) . IV. Methods o f Analys is of Tissue Samples 1. Thyroid Gland The thyro id gland was excised from the e p i g l o t y l region of the trachea and weighed. 2* L i ve r Mineral Storage The analys is o f l i v e r copper and i ron l eve ls was performed using a Perkin-Elmer atomic absorption spectrophotometer (Model 303). Small samples of l i v e r , averaging about 2 gm., and feed samples averaging about 1 gm, were oven dr ied at 110° C to constant weight. This required about two days f o r the l i v e r samples due to t h e i r high moisture content (approximately 70%). L i ve r samples were always taken from the same pos i t ion on the caudate lobe as i t has been shown (40) that the concentration of copper w i l l vary from s i t e to s i t e . Iron does not show t h i s v a r i a b i l i t y . The samples were then wet ashed using 10 ml . of concentrated n i t r i c ac i d . This d igest ion was allowed to continue u n t i l the samples 73 were completely d isso lved. At frequent in te rva ls during t h i s time the so lu t ion was heated to the point of fuming and held at t h i s temperature f o r about one hour. A f te r the second day of d iges t ion , i t was found necessary to add an add i t iona l 5 ml . o f n i t r i c a c i d . When the samples were completely d i sso lved , they were again heated and d i lu ted wi th 20 ml . o f t r i p l e d i s t i l l e d water from an a l l g lass s t i l l . They were a l l f i l t e r e d when hot and brought to a constant volume o f 250 ml . This whole preparat ional procedure was ca r r i ed out i n Coors c ruc ib les (Size 000). The samples were stored at t h i s point under 0.5 ml . o f toluene at 5° C. The ana lys is was ca r r ied out d i r e c t l y i n the atomic absorption spectrophotometer f o r both copper and i r o n . The standard curves f o r both o f these minerals are presented i n Appendix I I . 74 E. EXPERIMENTAL RESULTS I. Study I This study was designed to show the e f fec ts o r o r a l administ ra-t i o n o f 0, 10, and 18 mgm./day o f D.E.S. on the t o t a l and ra t i os o f v o l a t i l e f a t t y acids i n the blood. These animals were fed e i t he r an a l l bar ley ra t i on or a 50:50 mixture o f bar ley and a l f a l f a l ea f meal p e l l e t s . Both groups received two pounds o f prote in supplement per day. Thyroid weights and the three main blood parameters were a lso measured and re la ted to the l e v e l o f D.E.S. fed . 1. Results and Discussion a) Growth Rate and Feed E f f i c i ency The growth rate data f o r each animal fo r the en t i re experimental per iod i s presented i n Appendix I I I and IV. A summary i s given i n table I I I . t Table I I I Rate of Gain and Feed E f f i c i e n c y , Study I D.E.S. Gain/Head/Day Feed E f f i c i ency l b . l b . f eed / lb . gain Bar ley: 0 mgm. 3.00 5.76 10 mgm. 2.84 5.86 18 mgm. 2.87 5.88 B a r l e y - A l f a l f a : 0 mgm. 2.71 7.72 10 mgm. 2.60 7.35 18 mgm. 2.63 7.40 75 These data ind icate that the animals o f t h i s study d id not produce the expected increasing gains/day and feed e f f i c i e n c i e s wi th increasing l eve ls o f D.E.S. as reported by Dinusson (55), Burroughs (35), and as summarized by Riggs (151). A poss ib le reason fo r t h i s lack o f response i n e f f i c i e n c i e s may be due to the weight at which these animals were s tar ted on D.E.S. In the review of the e f fec ts of D . E . S . , Riggs (151) tabulated data from animals which were s tar ted on D.E.S. at weights greater than 600 pounds. A l s o , the use o f molasses, i n t h i s case 10% of the r a t i o n , has been shown by O'Mary (123), at l eve ls o f 33%, to negate the b e n e f i c i a l e f fec ts o f D.E.S. He has a lso observed t h i s inconsistent response o f D.E.S. on the same ra t ions at d i f fe rent t imes. The only growth data obtained i n t h i s study which fol lowed the expected trends was the increase i n feed e f f i c i ency due to the use o f high energy ra t i ons . The feed e f f i c i ency on the bar ley ra t i on was 5.76 l b . f eed / lb . of gain whereas that i n the b a r l e y - a l f a l f a ra t i on group was 7.72 l b . / l b . o f ga in . A l s o , there was an apparent increase i n the rate o f ga in , 3.00 lb . /day vs . 2.71 l b . / day . There i s good cor re la t ion between the feed e f f i c i ency and rate of gain on the bar ley ra t ion which doesn't ex i s t on the b a r l e y - a l f a l f a r a t i o n . The decrease i n rates of gain that apparently resu l ted from the higher l eve ls o f D.E.S. was shown to be i ns i gn i f i can t at P > . 0 5 . b) Blood Parameters The measurement o f the normally considered blood parameters, 76 hemoglobin, haematocrit, and red c e l l counts resu l ted i n a more consistent pat tern of response. A summary o f these data i s given i n Table IV. A complete set of data used to obtain t h i s summary i s presented i n Appendix V. Table IV Summary o f Blood Parameters, Study I D.E.S. Hemoglobin gm. "o Haematocrit % Red C e l l Counts mil l ions/mm Bar ley: 0 mgm. 8.96 32.02 8.25 10 mgm. 10.36 37.75 9.11 18 mgm. 10.40 37.35 9.50 B a r l e y - A l f a l f a : 0 mgm. 9.84 36.35 8.73 10 mgm. 9.93 36.54 8.67 18 mgm. 10.20 38.25 9.63 When examining these data i t i s important to remember that there are inate di f ferences i n the i ron l eve ls o f these basal r a t i ons , therefore, one would expect that the blood parameters o f the cont ro l animals should r e f l e c t t h i s . According to Morrison (117) the l e v e l o f i ron i n a l f a l f a l ea f meal i s about 4.5 times that o f bar ley which, i n a 50:50 ra t i on would give an i ron concentrat ion o f 2.25 times that of the a l l bar ley r a t i o n . This erythropoiet ic s t imulat ion by the b a r l e y - a l f a l f a ra t i on i s apparent when the 0 l e v e l of D.E.S. groups are compared. There i s an increase i n hemoglobin from 8.96 to 9 .8 , i n haematocrit from 32.0 to 36.3, and i n red c e l l count from 8.25 to 8.70. However, due to the v a r i a b i l i t y w i th in each o f these groups, these increases were shown to be i ns i gn i f i can t (P > .05). 77 This observation of the di f ferences i n the i ron content and hence cont ro l blood parameters i s important when one analyzes the. inf luence o f D.E.S. on both the bar ley and the b a r l e y - a l f a l f a ra t i ons . In the f i r s t case there was shown to be a s i gn i f i can t increase (P < .05) i n blood hemoglobin from 8.96 to 10.40 wi th the use o f D.E.S. However, i n the second s i t u a t i o n , b a r l e y - a l f a l f a , there was an i ns i gn i f i can t increase (P >-.05) due to the higher cont ro l l e v e l on t h i s r a t i on . I t i s i n te res t ing to note the c lose s i m i l a r i t y between t h i s blood parameter at 10 and 18 mgm. o f D.E.S./day on both the bar ley and b a r l e y - a l f a l f a ra t i ons . This tends to support the above hypothesis as we l l as to ind icate that D.E.S. compensates f o r ra t i on i nsu f f i c i enc ies up to a ce r ta in po in t . An examination o f the data f o r the red c e l l counts o f animals on the various ra t ions presents an even more convincing p ic ture of the e f fec t of D.E.S. on these parameters. In the case o f the bar ley ra t ion there was shown to be a s i gn i f i can t increase i n c e l l counts wi th each subsequent increase i n the l e v e l o f D.E.S. In the b a r l e y - a l f a l f a ra t i on the response was shown to be s i gn i f i can t between the 0 and 18 mgm. D . E . S . / head/day l e v e l s . From these two observations one would expect an increase i n the haematocrits i n animals fed increasing leve ls o f D.E.S. The trends establ ished i n the. bar ley r a t i o n , an increase from 32% to 37% and i n the b a r l e y - a l f a l f a r a t i o n , 36% to 38%, were shown to be i n s i g n i f i c a n t , however (P > .05). This same observation has been made by Erwin (63) feeding D.E.S. at 0.45 mgm./lb. of feed. He observed no change i n hemoglobin 78 values on t h i s same r a t i o n . Bohman (30) has a lso shown that there was no change i n erythrocyte counts when feeding D.E.S. at 10 mgm./head/day. A poss ib le explanation f o r t h i s apparent cont rad ic t ion between these two workers and the resu l t s obtained here could be the resu l t o f d i f ferences i n ra t i on mineral content. As w i l l be shown l a t e r , there was a decrease or no change i n these blood parameters on high i ron ra t ions wi th increasing leve ls of D.E.S. I t i s , therefore, possib le that Erwin (63) and Bohman (30) fed ra t ions o f a higher i r on content than those used i n t h i s study and as a consequence, made d i f f e r i n g observations concerning the response o f blood parameters to supplemental D.E.S. c) Tota l and V o l a t i l e Fat ty Ac id Ratios The complete set of data f o r the analys is o f changes i n blood t o t a l V .F .A . and i n blood acet ic -prop ion ic ra t i os i s presented i n Appendices VI and VII respec t ive ly . A summary of these data i s given i n table V. Table V Tota l and V o l a t i l e Fat ty Ac id Ra t ios , Study I D.E.S. Tota l V .F .A . Ace t i c :Prop ion ic meq . / l .  Bar ley: 0 mgm. 1.84 82.3 10 mgm. 1.23 76.8 18 mgm. 1.-58 195.3 B a r l e y - A l f a l f a : 0 mgm. 1.58 97.0 10 mgm. 1.50 159.0 18 mgm. 1.48 233.0 79 The r e l a t i v e leve ls of t o t a l V .F .A . i n blood are f a i r l y consistent with those presented i n the l i t e r a t u r e . Schanbye (154-) observed ca ro t id blood leve ls of 1.14 m e q . / l . , and Annison (5) has shown ca t t l e jugular blood leve ls to range from 0.55 to 1.32 meq . / l . A l s o , these data agree with the l i t e r a t u r e reports showing essen t i a l l y no buty r ic ac id (5 , 48, 84) and minimal amounts o f propionic ac id (5, 112). This i s ind icated by the high acet ic -prop ion ic r a t i o s . These resu l t s obtained i n t h i s f i r s t study ind icate no s i gn i f i can t e f fec t on t o t a l blood V .F .A . as a resu l t o f feeding D.E.S. This e f fec t was observed i n both ra t i ons . A l s o , one would expect that there would be a higher l e v e l o f blood V .F .A . i n animals fed on the high concentrate ra t i on and t h i s was observed except at l eve ls o f 10 mgm. o f D.E.S. /day. This discrepancy may be the resu l t of the large f l uc tua -t ions i n t o t a l rumen V .F .A . on the high concentrate ra t i on as noted by Balch and Rowland (2) . These f luc tuat ions would then be re f lec ted i n changes i n the blood V .F .A . (6, 154). The acet ic -prop ion ic ra t i os i n blood have been shown by t h i s study to fo l low the pattern o f rumen ra t i os resu l t i ng from the addi t ion of concentrates to a r a t i o n . I t has been shown that there w i l l be an increase i n the r e l a t i v e l eve ls of propionic and butyr ic acids i n the rumen at the expense o f ace t i c ac id (48, 147, 169, 181). This means that there would be a decrease i n rumen acet ic -prop ion ic r a t i o i n animals fed a ra t ion containing more concentrates. This r a t i o change i s apparently re f l ec ted i n the blood as the bar ley ra t i on fed animals had an a c e t i c -propionic r a t i o o f 82.3:1 whereas the b a r l e y - a l f a l f a ra t i on resul ted i n 80 a r a t i o of 97.0 :1 . A l s o , i t was shown that there was a s i gn i f i can t (P •<.05) increase i n t h i s blood r a t i o due to the addi t ion o f D.E.S. to the bar ley r a t i o n . S ign i f icance was only obtained on the bar ley ra t i on where, the r a t i o increased from 82.3:1 at 0 mgm. D.E.S./head/day to a r a t i o o f 195.3:1 at 18 mgm. D.E.S./head/day. This trend appears even stronger i n the blood of animals fed the b a r l e y - a l f a l f a ra t ion where they increase from 97:1 to 159:1 at 10 mgm./head/day, and to 233:1 at 18 mgm./head/day. But, because o f the large v a r i a b i l i t y i n the values w i th in each group these averages were shown to be i ns i gn i f i can t (P > .05) . This observation i s i n agreement wi th the work o f Browning (33) who observed an apparent decrease i n the rumen molar proport ions o f propionic and buty r ic acids wi th the addi t ion o f D.E.S. to the ra t ions o f da i ry cows. His observations were not s i gn i f i can t because o f the smal l sample s i z e , only two. d) Thyroid Weights and Dressing Percentage The data f o r the thyro id weights of each animal are presented i n Appendix VII I wi th a summary i n Table VI . Table VI Average Thyroid Weights, Study I Treatment 0 mgm. D.E.S. 10 mgm. D.E.S. 18 mgm. D.E.S. gm. Bar ley: 22.9 14.6 16.2 B a r l e y - A l f a l f a : 17.3 14.4 14.8 81 There i s an apparent decrease i n the weight o f t h i s gland wi th increased leve ls of D.E.S. but , again t h i s has been shown to be an i n -s ign i f i can t (P > .05) decrease on both ra t i ons . This decrease i s i n c o n f l i c t with the apparent increase observed by Clegg (45) i n steers implanted with 60 mgm. o f D.E.S. I t i s poss ib le , however, that there could be a di f ference i n response due to o r a l vs . implantat ion administ ra-t i on o f D.E.S. There has been shown to be a decrease i n thyro id weights i n sheep when implanted with 12 mgm. o f D.E.S. (55). The dressing percentage data i s summarized i n tab le VII from the data i n Appendix IX. Table VII Average Dressing Percentage, Study I Treatment 0 mgm. D.E.S. 10 mgm. D.E.S. 18 mgm. D.E.S. % Barley: 57.8 58.1 57.2 Ba r l ey -A l f a l f a : 57.5 57.2 57.8 These data confirm the previous observations (34, 69, 93) that D.E.S. has no e f fec t on dressing percentage o r carcass grade. A l s o , there i s no s ign i f i can t e f fec t o f ra t i on on dressing percentage. 82 2. Summary o f Study I a) When animals were fed D.E.S. at the l eve ls o f 0, 10, and 18 mgm./ head/day no increase i n rate o f gain or feed e f f i c i ency was observed. b) There i s an apparent increase i n hemoglobin, haematocrit , and red c e l l counts through the feeding o f a b a r l e y - a l f a l f a ra t i on (50:50) over those obtained on a s t ra igh t bar ley r a t i o n . c) There was a s i gn i f i can t increase i n blood hemoglobin and red c e l l count on the bar ley ra t ion through the addi t ion o f D.E.S. and an apparent increase i n haematocrit. d) There was a s i gn i f i can t increase i n red c e l l count on the bar ley-a l f a l f a ra t i on caused by D.E.S. supplementation. A l s o , an apparent increase i n hemoglobin and haematocrit e) There was no s i gn i f i can t increase i n t o t a l blood V .F .A . as a resu l t o f D.E.S. treatment on e i the r o f these ra t i ons . f ) There i s an apparently higher acet ic -prop ion ic r a t i o i n blood o f roughage fed animals when compared to that of concentrate fed animals. g) There i s a s i gn i f i can t increase i n blood acet ic -prop ion ic ra t i os on the bar ley ra t i on due to the addi t ion of D.E.S. and an apparent but i n s i gn i f i can t response on the b a r l e y - a l f a l f a r a t i o n . h) An apparent decrease i n thyro id weights was observed on both ra t ions as a resu l t o f D.E.S. treatment. 83 I I . Study I I The second study was designed to show the e f fec t o f D.E.S. on t o t a l blood V .F .A . and to examine l i v e r mineral storage, p r imar i l y i ron and copper. The animals were fed a basal ra t ion o f bar ley wi th an add i -t i o n a l two pounds o f prote in supplement. This supplement contained D.E.S. at l eve ls such that selected groups would receive 0, 10, o r 18 mgm./head/ day as i n the f i r s t study. Three groups o f animals were fed on a high i ron ra t i on and the other three on the 55, 56, and 57 supplements used i n Study I. The blood parameters were a lso measured at the beginning o f the study and at s laughter. 1. Results and Discussion a) Growth Rate and Feed E f f i c i ency A complete set o f growth rate data i s presented i n Appendix X and XI f o r the i nd i v idua l growth rates and f o r the body weights respec t ive ly . A Summary o f these e f f i c i e n c i e s i s as fo l lows: Table VII I Rate o f Gain and Feed E f f i c i e n c y , Study I I Gain/head/day Feed E f f i c i e n c i e s l b . l b s . / l b . gain Low Iron 0 mgm. D.E.S. 3.00 6.9 10 mgm. D.E.S. 3.58 5.7 18 mgm. D.E.S. 3.89 5.2 High Iron 0 mgm. D.E.S. 3.25 5.9 10 mgm. D.E.S. 3.47 5.5 18 mgm. D.E.S. 3.34 5.7 84 An examination o f the growth rate data f o r animals on the low i ron r a t i o n , supplements 55, 56, and 57, reveals an apparent trend towards increasing growth rate with each subsequent increase i n the l e v e l of D.E.S. used. This i s what one should observe according to the l i t e r a t u r e (34, 93, 119). But, these increases with each increase i n D.E.S. o r , the di f ference between the cont ro l l e v e l of 3.00 lb. /head/day and the 18 mgm. D.E.S./head/day l e v e l of 3.89 lb. /head/day were shown to be i ns i gn i f i can t due to the small sample s i ze and the range of growth rates w i th in each group. The high i ron group d id not produce any d iscern ib le trend resu l t i ng from the addi t ion of D.E.S. I t i s of in te res t to note, however, that there was an apparent increase i n the cont ro l growth rate i n animals on t h i s ra t i on i n comparison to those on the low i ron r a t i o n . This observation has been made previously concerning anemic and non-anemic animals (29, 91, 107) ind ica t ing the p o s s i b i l i t y that supplemented ra t ions 55, 56, and 57 were i n s u f f i c i e n t i n i r o n . The r e l a t i v e growth rates on these two rat ions seems to give support to an idea developed i n the l i t e r a t u r e review that D.E.S. compensates f o r ra t i on i nsu f f i c i enc ies by producing the greatest response on the more imperfect ly balanced ra t i ons . In t h i s case i t i s f a i r to suppose that the h igh- i ron ra t i on met, more exac t l y , the nutr ient requirements of the animals due to i t s higher cont ro l growth r a t e , i . e . 3.24 l b . vs . 3.00 lb. /head/day. The response to increasing leve ls of D.E.S. was more consistent and pronounced on the low i ron ra t i on than on the high i ron ra t i on . There was a lso good cor re la t ion between the rates of gain and the feed e f f i c i e n c i e s o f these animals on both ra t ions (high and low i r on ) . A comparison of the resu l t s 85 obtained i n the growth ana lys is of t h i s study with those obtained on the bar ley ra t i on i n Study I show them to be qui te incompatible. In actua l f a c t , both o f these rat ions are exact ly the same, a basal ra t i on o f bar ley and supplements 55, 56, and 57. However, the growth rate s t imulat ion i n Study I was apparently negative whi le that of Study I I was qui te apparently pos i t i ve . The only d i f ference i n the treatment o f these s i x groups of animals was that those i n Study I were star ted on D.E.S. at a weight o f 465 l b . and those o f Study I I at 718 l b . This tends to ind icate that there may be a c r i t i c a l weight below which animals should not be fed D.E.S. i f optimum response i s to be obtained. b) Blood Parameters The complete set o f data f o r the analys is o f the blood para-meters i s presented i n Appendices XII and XI I I f o r the i n i t i a l and f i n a l values respec t ive ly . The ove ra l l average haematocrit f o r animals at the s ta r t o f t h i s study per iod was 37.5% and f o r hemoglobin i t was 9.5 gm. % Hb. This was at an average weight of 718 l b . The d i v i s i o n of these animals in to groups i n Appendix XII i s of no s ign i f i cance as these blood samples were co l lec ted p r i o r to the actual d i v i s i o n f o r experimental purposes. A summary of the data obtained upon analys is o f these blood parameters at slaughter i s as fo l lows: 86 Table IX Summary of Blood Parameters, Study I I Hemoglobin Haematocrit Red C e l l Count gm. % % mill ions/mm^ Low Iron 0 mgm. D.E.S. 9.66 33.35 7.57 10 mgm. D.E.S. 10.02 34.47 7.91 18 mgm. D.E.S. 10.50 35.30 7.85 High Iron 0 mgm. D.E.S. 10.70 36.20 8.10 10 mgm. D.E.S. 10.00 34.50 7.84 18 mgm. D.E.S. 10.30 34.15 7.25 I t should be noted that animal 73 (Appendix XI I I ) was not used i n the ca lcu la t i on of average hemoglobin, haematocrit , or red c e l l counts f o r the cont ro l h igh i ron r a t i o n . This animal produced exceedingly high values f o r these parameters and was obviously exc i ted at the time of sampling. On the low i ron rat ions there i s an apparent, but none the less i n s i g n i f i c a n t , increase i n the quant i ta t ive values f o r these three para-meters wi th increasing leve ls o f D.E.S. This trend gives support to the observations made i n the f i r s t part of t h i s experiment where a s i gn i f i can t increase was observed i n the hemoglobin and red c e l l counts. But, i n the animals fed the high i ron ra t ions (60, 61, and 62) there i s an apparent decrease i n the values of these parameters wi th increasing leve ls o f D.E.S. As mentioned i n the d iscussion of Study I o f t h i s experiment, these two divergent trends seem to ind icate that D.E.S. causes an increase i n these 87 blood parameters on an i ron de f i c ien t ra t ion and a decrease on rat ions containing su f f i c i en t o f excess i r o n . When comparing the cont ro l l eve ls o f these three blood parameters i t was shown that there was a s ign i f i can t increase i n red c e l l counts from 3 3 7.57 mill ion/mm on the low i ron rat ions to 8.1 mill ion/mm on the high i ron ra t i ons . A l s o , there was shown to be a s i gn i f i can t increase i n haematocrit from 33.3% on the low i ron ra t i on to 36.3% on the high i ron r a t i o n . There was an increase i n hemoglobin from 9.66 to 10.70 but , t h i s proved to be i ns i gn i f i can t as a resu l t o f h igh , w i th in group v a r i a b i l i t y . A s i m i l a r observation to t h i s has been made by Thomas (166) who noted an increase i n red c e l l counts and hemoglobins when i ron addi t ion was made to the ra t ion o f anemic ca lves . I f i t i s accepted that the hemoglobin of normal beef ca t t l e ranges between 11.8 and 12.4 gm. % Hb. , haematocrit ranges between 3 33.3% and 37.6%, and red c e l l counts range between 7.4 and 8.1 mill ion/mm (27) then the low i ron ra t i on produced a border l i n e case o f anemia. c) Tota l Blood V o l a t i l e Fatty Acids The blood v o l a t i l e fa t t y acids are presented i n Appendix XIV wi th a summary i n Table X. Table X Tota l V o l a t i l e Fatty Ac ids , Study I I Treatment 0 mgm. D.E.S. 10 mgm. D.E.S. 18 rngm. D.E.S. meq . / l . Low Iron 0. 88 1.15 1.19 High Iron 1. 08 1.15 1.19 88 The average value f o r the high i ron cont ro l ra t i on was again ca lcu la ted without the values from animal No. 73 because of the reasons previously mentioned. I t had a t o t a l V .F .A . value o f 2.04 meq . / l . i nd ica t ing that the blood l eve l s o f V .F .A . respond to s t ress i n much the same way that blood glucose does. The r e l a t i v e leve ls o f t o t a l V .F .A . obtained i n t h i s study produced a more uniform trend i n response to subsequent l eve ls o f D.E.S. On the low i ron ra t ions there was a s i gn i f i can t increase i n the leve ls o f t o t a l V .F .A . as a resu l t o f treatment wi th D.E.S. The cont ro l l e v e l was 0.88 meq . / l . r a i s i n g to 1.19 meq . / l . i n animals fed D.E.S. at l eve l s o f 18 mgm./head/day. There was no s i gn i f i can t d i f fe rence, however, between the two leve ls of D.E.S. used. On the high i ron ra t i on there was a s im i l a r trend to that establ ished on the low i ron ra t ion but , i n t h i s case, the change was i ns i gn i f i can t due to the high l e v e l of t o t a l V .F .A . i n the blood o f the cont ro l animals on t h i s r a t i o n . This di f ference between the low i ron cont ro l ra t i on (0.88 meq. / l . ) and the high i ron cont ro l r a t i on (1.08 meq./ 1.) was shown to be s i gn i f i can t at P < .05. The s i m i l a r i t y between the t o t a l blood V .F .A . i n the 10 and 18 mgm./head/day groups on both ra t ions ind icates again the compensatory e f fec ts o f D.E.S. on ra t i on i n s u f f i c -ienc ies . d) L i ve r Iron and Copper Levels In the sect ion on mater ia ls i t was stated that ra t i on supplements 50, 61, and 62 were designed to provide i ron at a l e v e l o f 1290 mgm./lb. 89 These supplements as we l l as 55, 56, and 57 have been analyzed and the actua l i r on and copper contents are presented i n Table X I . A complete set o f data concerning l i v e r i r on and copper leve ls i s shown i n Appendix XV with the summary i n Table XII (next page). These data show an apparent increase i n l i v e r i r on storage wi th increased i ron in take. In t h i s case t h i s d i f ference was found to be i ns i gn i f i can t but, previous reports have shown i t to be s i gn i f i can t (81). The l eve l s o f i ron are wi th in acceptable l i m i t s as they have been shown to range from 47-125 ppm. (81) to 184 ppm. (165) to as high as 278 ppm. (54) depending on the mineral balance o f the ra t i ons . Table XI Iron and Copper Contents of Supplements Iron Copper mgm./lb. Low Iron 55 259 15.4 56 264 14.8 57 250 15.6 Mean 258 15.2 High Iron 60 1340 14.5 61 1297 13.0 62 1235 12.4 Mean 1290 13.3 In both o f these ra t i ons , high and low i r o n , there i s a s l i gh t but i n s i g n i f i c a n t trend towards increased l i v e r i ron storage with subsequent addi t ions o f D.E.S. The copper storage on the low i ron 90 Table XII L i ve r Iron and Copper Levels Iron Copper PJ__ Low Iron 0 mgm. D.E.S. 145 84 10 mgm. D.E.S. 142 116 18 mgm. D.E.S. 160 131 High Iron 0 mgm. D.E.S. 158 37 10 mgm. D.E.S. 167 61 18 mgm. D.E.S. 170 45 ra t i on has been shown to be s i g n i f i c a n t l y increased through the addi t ion o f D.E.S. In the ca lcu la t i on o f the copper averages and s t a t i s t i c a l ana l ys i s , the data o f animal 86 has been el iminated because i t was obviously out of l i n e , ind ica t ing fore ign contamination. In a comparison of the r e l a t i v e l eve ls of copper i n both the cont ro l groups of these ra t ions a s i g n i f i c a n t l y lowered l e v e l was obtained i n those l i v e r s from high i ron fed animals. There was a drop from 84 ppm. to 37 ppm. upon i ron add i t ion . Con f l i c t i ng reasons f o r t h i s drop can be found, i n the work of Hardison (77), who stated that i ron i nh ib i t ed l i v e r copper storage by act ing at the s i t e o f absorpt ion, and i n the work of Elvejem (59) who proposed the idea o f the in te r re la t ionsh ip between copper and the use o f i ron i n blood hemoglobin. In Study I I there was an apparent increase i n hemoglobin leve ls ind ica t ing that poss ib ly the second reason i s more v a l i d . 91 2. Summary o f Study I I a) There was an apparent, but i ns i gn i f i can t increase i n the growth rate o f animals on the low i ron ra t ion caused by increasing leve ls o f D.E.S. No trends were observed on the high i ron ra t i on . b) The high i ron ra t i on produced a higher contro l growth r a te , ind ica t ing that supplements 55, 56, and 57 were i nsu f f i c i en t i n i r o n . c) The low i ron ra t ions produced a trend o f increasing blood parameters with increasing leve ls of D.E.S. d) The high i ron ra t i on produced a trend o f decreasing values o f these blood parameters with increasing D.E.S. e) There was a s i gn i f i can t d i f ference between the red c e l l counts and haematocrits i n the blood o f animals fed the high and low i ron ra t ions . f ) There was a s i gn i f i can t d i f ference between the t o t a l blood V .F .A . i n the cont ro l groups of both ra t i ons . The high i ron ra t ion had the highest cont ro l t o t a l blood V .F .A . g) A s i gn i f i can t increase i n t o t a l blood V .F .A . was observed on the low i ron ra t i on with the addi t ion o f D.E.S. but , there was only an apparent increase i n t o t a l blood V .F .A . on the high i ron r a t i o n . h) Barley contains i ron at a l e v e l o f 62 mgm./lb. and copper at 10.1 mgm./lb. 92 i ) Feeding high i ron rat ions resu l ted i n an i ns i gn i f i can t increase i n l i v e r i r on storage. j ) An apparent increase i n l i v e r i ron storage with increasing leve ls o f D.E.S. was observed. k) There was a s i gn i f i can t increase i n l i v e r copper storage with increased leve ls o f D.E.S. on the low i ron r a t i o n . 1) The average l e v e l o f copper i n the l i v e r s o f animals fed the high i ron ra t i on was s i g n i f i c a n t l y lower than i n those fed the low i ron r a t i o n . 93 I I I . Conclusions Based on Studies J_ and I I In the f i r s t study there was a s i gn i f i can t increase i n hemoglobin and red c e l l counts wi th increasing leve ls o f D.E.S. This same trend was observed i n the second study on i d e n t i c a l ra t ions but , on ra t ions o f a higher i ron content there was an apparent decrease i n the three main blood parameters. This f ind ing agrees wi th the l i t e ra tu re reports and suggests that D.E.S. can act to cause e i t he r an increase or a decrease depending on the ra t i on mineral l e v e l . In assoc ia t ion with t h i s , the growth data o f Study I I suggests that D.E.S. acts to cause the greatest s t imulat ion on ra t ions that are the leas t per fec t l y balanced ind ica t ing that D.E.S. tends to compensate f o r ra t i on i n s u f f i c i e n c i e s . There was a s i gn i f i can t increase i n the acet ic -prop ion ic r a t i o i n blood as a resu l t o f treatment wi th D.E.S. This change was not accompanied by a s i gn i f i can t change i n t o t a l blood V .F .A . except i n the case where growth rates respond to D.E.S. i n the proven manner. There was a lso a s i gn i f i can t d i f ference i n t o t a l blood V .F .A . between the cont ro l ra t ions on high and low i r o n . The high i ron ra t ion produced the highest l e v e l . In both studies there was an apparent co r re la t ion between the r e l a t i ve blood t o t a l v o l a t i l e f a t t y ac id l eve ls and the r e l a t i ve growth ra tes . Because of the inconsistent response o f animals to s i m i l a r treatments, d i f f e r i n g only i n the weight at which the animals were 94 s tar ted on D . E . S . , there appears to be a c r i t i c a l weight below which animals should not be t reated wi th t h i s hormone i f optimum response i s to be achieved. A s i gn i f i can t drop i n the l e v e l o f l i v e r copper storage was observed when high leve ls of i ron were fed . 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S c i . 17: 164-170, 1958 134. Perry , T.W., Smith, W.H., Beeson, W.M., Peterson, R . C . , Heath, M .E . , Webb, D. , and N i c h e l , C H . In jectable i ron f o r beef c a t t l e . Jour . An. S c i . 26: 106-109, 1967 135. Pfander, W.H., and P h i l l i p s o n , A.T . The rates o f absorption o f a c e t i c , p rop ion ic , and buty r ic ac ids . Jour . Phys. 122: 102-110, 1953 136. Pfander, W.H. Fat ty acids and energy i n rimdnant n u t r i t i o n . A century o f n u t r i t i o n a l progress. Midwest Feed Man. A s s n . , Kansas C i t y , Mo. 176 137. Preston, R . , Cheng, E . , Story, C D . , Homeyer, P . , Pau ls , J . , and Burroughs, W. The inf luence of o r a l administrat ion o f d ie thy-s t i l b e s t r o l upon estrogenic residues i n the t i ssues of beef c a t t l e . Jour. An. S c i . 15: 3-12, 1956 138. Preston, R . L . , and Burroughs, W. S t i l b e s t r o l responses i n lambs fed ra t ions d i f f e r i n g i n ca lo r i e to prote in r a t i o s . Jour. . An. S c i . 17: 140-151, 1958 139. P r i t cha rd , G . I . , and Tove, S.B. In ter re la t ionships between the metabolism of short chain fa t t y acids by ruminant l i v e r s l i c e s . Biochem. et Biophys. Ac ta . 41: 130-137, 1960 140. Putnam, P . A . , and Davis, R.E. The e f fec ts o f various l eve ls of d ie tary prote in and energy upon the v o l a t i l e f a t t y acids i n the rumen o f beef cows. Jour. An. S c i . 17: 1192-1193, 1958 141. Putnam, P . A . , Gut ie r rez , J . , and Davis , R.E. Ef fec ts of frequency o f feeding upon rumen v o l a t i l e ac ids , protozoa populations and weight gains i n Angus he i f e r ca lves. Jour. Dairy S c i . 44: 1364-1365, 1961 142. Putnam, P . A . , Bovard, K . , Pr iode, B .M. , and Lehmann, R. Rumen v o l a t i l e f a t t y acids and gains o f record o f performance b u l l s . Jour. An. S c i . 24: 166-167, 1965 107 143. Putnam, P . A . , Elam, C . J . , Dav is , R . E . , and Wiltbank, J . N . 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U t i l i z a t i o n o f ace t i c and propionic acids i n sheep. Nature 165: 448, 1950 149. Reid , J . T . , Warner, R . G . , and L o o s l i , J . K . An t i b i o t i c s i n the n u t r i t i o n of ruminants. Jour. Agr. Food Chem. 2: 186-192, 1954 150. Rhodes, R.W., and Woods, W. V o l a t i l e fa t t y ac id measurements on the rumen contents of lambs fed rat ions o f various phys ica l form. Jour . An. S c i . 21: 483-488, 1962 151. Riggs, J . K . F i f t y years of progress i n beef ca t t l e n u t r i t i o n . Jour. An. S c i . 17: 981-1006, 1958 152. Ross, J . P . Ration e f fec ts on blood metabolites i n pregnant ewes. M.S.A. thes is U.B.C. 1967 153. Rusoff, L .L . A n t i b i o t i c feed supplement (Aureomycin) f o r dai ry ca lves . Jour. Dairy S c i . 34: 652-655, 1951 154. Schambye, P . , and P h i l l i p s o n , A.T. V o l a t i l e ' f a t t y acids i n po r ta l blood o f sheep. Nature 164: 1094, 1949 155. Shaw, J . C . Nu t r i t i ona l Physiology o f the Rumen. Gen. Rep. I-V of the counc i l o f the European Assn. of An. Prod. 29-52 108 156. Shaw, J . C , Ensor, W.L. , Te l lechea, H . F . , and Lee,S.D. Relat ion o f d ie t to rumen v o l a t i l e fa t t y ac ids , d i g e s t i b i l i t y , e f f i c i ency o f gain and degree o f unsaturation o f body fa t i n s teers . Jour . Nutr. 71: 203-208, 1960 157. Si rotnak, F .M . , Doetsch, R . N . , Brown, R . E . , and Shaw, J . C . Amino acids producing V .F .A . Amino ac id metabolism of bovine rumen bac te r i a . Jour. Dairy S c i . 36: 1117-1123, 1953 158. S len , S . B . , and Connel l , R. Wool growth i n sheep as af fected by the administrat ion of ce r ta in sex hormones. Jour . Dairy S c i . 38: 38-47, 1955 159. Spahr, S . L . , Ho l te r , J . B . , and Kes le r , E.M. Separation of organic acids from rununant blood by the Wiseman-Irvin method. Jour . Dairy S c i . 46: 1139-1142, 1964 160. Spahr, S . L . , Kes le r , E . M . , and F l i p s e , R.U. U t i l i z a t i o n of blood acetate and butyrate by the i s o l a t e d , perfused goat rumen. Jour. Dairy S c i . 48: 228-237, 1965 161. Stanley, R.W., and Mor i t a , K. Ef fec t of feeding thyroprotein to da i ry ca t t l e i n a subt rop ica l environment on mi lk composition and product ion, rumen metabolism, and fa t t y ac id composition of mi lk f a t . Jour. Dairy S c i . 50: 1097-1100, 1967 162. Stewart, W.E. , and Schu l tz , L.H. In v i t r o V .F .A . production from various feeds by bovine rumen micro-organisms. Jour . An. S c i . 17: 737-742, 1958 163. Stob, M. , Andrews, F . N . , Zarrow, M.X. , and Beeson, W.M. Estrogenic a c t i v i t y of the meat of c a t t l e and poul t ry fo l lowing treatment with synthet ic estrogens and progesterone. Jour. An. S c i . 13: 138-151, 1954 164. Swenson, M . J . , Underbjerg, G . K . L . , Bar t ley , E . E . , and Jones, W.G. Ef fec ts o f t race minera ls , aureomycin, and other supplements on cer ta in hematologic values and organ weights o f da i ry ca lves . Jour. Dairy S c i . 40: 1525-1533, 1957 165. Thacker, E . J . , Alderman, M.L . , and Brat ton, R.W. The e f fec t of plane o f nu t r i t i on on the mineral composition o f blood serium, l i v e r , and on the growth o f bone. Jour . An. S c i . 15: 447-455, 1956 166. Thomas, J .W. , Okamoto, M . , Jacobson, W.G., and Moore, L.A. A study of hemoglobin l eve ls i n the blood of young dai ry calves and the a l l e v i a t i o n of anemia by i r on . Jour. Dairy S c i . 37: 805-812, 1954 109 167. Tompsett, S .L . Factors in f luenc ing the absorption o f i r on and copper from the alimentary t r a c t . Biochem. Jour. 34: 961-969, 1940 168. Thompson, J . R . Metabolic e f fec ts of d i e t h y l s t i l b e s t r o l on growing sheep. M.S.A. thes is U . B . C . , 1966 169. Thompson, J . T . , Bradley, N.W., and L i t t l e , C O . 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Ind iv idua l and t o t a l v o l a t i l e fa t t y ac ids . Aust. Jour. Ag. Res. 18: 107-118, 1967 110 179. Whitehair , C K . , Ga l lup , W.D., and B e l l , M.C. Ef fec t o f s t i l b e s t r o l on ra t i on d i g e s t i b i l i t y and on calc ium, phosphorus, and nitrogen re tent ion i n lambs. Jour . An. S c i . 12: 331-336, 1953 180. Wi lson, L . L . , D inke l , C . A . , Ray, D .E . , and Minyard, J . A . Beef carcass composition as inf luenced by d i e t h y l s t i l b e s t r o l . Jour . An. S c i . 22: 699-701, 1963 181. Woods, W., and Luther, R. Further observations o f the e f fec t of phys ica l preparat ion of the ra t i on on v o l a t i l e f a t t y ac id product ion. Jour . An. S c i . 21: 809-814, 1962 182. Wright, P . L . , Pope, A . L . , and P h i l l i p s , P.H. E f fec t o f phys ica l form of ra t i on upon d igest ion and v o l a t i l e fa t t y ac id production i n v ivo and i n v i t r o . Jour. An. S c i . 22: 586-591, 1963 I l l APPENDIX I. Analys is and Interpretat ion o f G.C. Response The f i r s t use o f gas chromatography fo r the i d e n t i f i c a t i o n of v o l a t i l e fa t t y acids was by James (87). This app l ica t ion was not quant i ta t ive i n nature and was only used to separate the ac ids . The t o t a l l e v e l o f each ac id was subsequently determined by t i t r a t i o n . In 1960, Bensadoun (25) developed the procedures o f preparat ion o f blood samples that were la rge ly used i n t h i s experiment. He was able to quant i fy the r e l a t i v e amounts o f each ac id present by determining the percentages o f each ac id i n the sample by using peak area as a measurement o f amount. He used the less sens i t i ve and water sens i t i ve thermal con-duc t i v i t y detector . Further modif icat ions to t h i s method were made by Erwin (64) when he used a flame ion i za t i on detector and found that peak height could be used as p rec ise ly as area i n determining the amount o f any pa r t i cu l a r V .F .A . present. I t has a lso been shown that these flame ion iza t ion detectors provide the added advantage o f not responding to water (2, 61) therefore e l iminat ing the masking of peaks. The response has been stated by Emery (61) to be i n proport ion to the carbon content o f compounds. I t i s therefore necessary to develop a cor rect ion fac to r to convert the recorder resu l t s to those that would be obtained on a s t ra igh t t i t r a t i o n bas i s . In t h i s experiment the correct ion f ac to rs , r e l a t i ve to butyr ic ac id as 1. are as fo l lows : T i t r a t i on Ratio Chromatograph Ratio Ace t i c 1.66 1.72 Propionic 1.24 1.59 Butyr ic 1.00 1.00 112 But, the t i t r a t i o n r a t i o i s based on mequivs. of ac id and the chromato-graphic response i s based on the weight o f ac id present. These two parameters are not equal wi th respect to these ac ids . The mul t ip les with respect to bu ty r i c ac id at 1. are: Acet ic .96 Propionic .78 Ace t ic ac id i s i n a much higher concentrat ion i n blood than i s propionic a c i d . In order to get a su f f i c i en t response f o r propionic ac id from the recorder, the samples must be very concentrated and the gas chromatograph (G.C.) must be run at near maximum s e n s i t i v i t y . But, t h i s can lead to an o f f sca le de f lec t ion caused by the more abundant ace t i c a c i d . Fenner i n 1963 (66) stated that the s e n s i t i v i t y o f the G.C. may be changed during a run i n order to maintain both peaks on the graph and i n a useable form. The base l i n e must then be re-es tab l ished at the beginning and end of such a peak. The l i n e a r i t y of G.C. response to increased leve ls of the three main V .F .A . has been establ ished by Baumgardt (24). This proport ional re la t ionsh ip has a lso been shown to e x i s t , i n the instrument used i n t h i s work, f o r l eve ls higher than he used. For ace t i c a c i d , response measured as peak height times at tenuat ion, was found to be l i n e a r to approximately 5000 cm. Baumgardt used t h i s same measurement of G.C. response. The storage of frozen whole blood or blood f i l t r a t e s has been examined by Fr iend i n 1964 (68). He examined the changes that occurred i n the ra t i os of a c e t i c , p rop ion ic , and buty r ic acids during storage up to 26 days and observed no e f fec t . His r e s u l t s , taken from bovine jugular 113 b lood,a lso serve to i l l u s t r a t e the very low leve ls of acids other than ace t i c : 7 days 12 days 26 days Acet ic 18.2 16.9 18.8 Propionic Butyr ic molar % 0.2 0.0 0.1 0.0 0.0 0.0 Formic Lac t i c 41.2 48.9 35.6 40.4 34.2 45.4 This same problem has been examined by Packett i n 1967 (126) f o r periods up to s i x months wi th no deleter ious e f fec ts on the proport ions o f the three main V .F .A . present. 114 APPENDIX I I . Standard Curves f o r Iron and Copper L i ve r Concentrations The i ron standards were made up from f e r r i c ammonium su l fa te d issolved i n 10 ml . o f concentrated hydrochlor ic a c i d . These standards were made up i n concentrations o f : Parts Per M i l l i o n % Absorbance 0.1 1.00 0.2 2.06 0.3 2.98 0.4 4.00 0.5 4.96 0.6 6.00 The copper standards were prepared i n a s i m i l a r manner from copper su l fa te and produced the fo l lowing data: Parts Per M i l l i o n % Absorbance 0.1 5.13 0.3 15.36 0.4 21.36 0.5 24.43 0.6 29.57 115 APPENDIX I I I Table I I I , Growth Data from Study I Barley Ration No. 0 mgm. D.E.S. No. 10 mgm. D.E.S. No. 18 mgm. D.E.S. 69 3.11 44 2.40 53 3.12 97 3.07 45 3.16 54 2.89 58 3.07 46 2.54 55 2.58 74 2.52 47 2.69 56 2.73 76 3.22 49 3.42 68 3.07 Mean 3.00 l b . 2.84 l b . 2.87 l b . B a r l e y - A l f a l f a Ration No 0 mgm. D.E.S. No. 10 mgm. D.E.S. No. 18 mgm. D.E.S. 63 2.27 48 2.61 59 2.45 64 2.69 50 2.61 60 2.93 65 2.75 51 2.72 61 2.48 66 3.37 52 2.36 62 2.48 67 2.50 57 2.69 70 2.82 Mean 2.71 l b . 2.60 l b . 2.63 l b . 116 APPENDIX IV The I n i t i a l and F i n a l Body Weights Obtained i n Study I Barley Ration 0 mgm. D.E.S. 10 mgm. D.E.S. 18 mgm. D.E.S. I n i t i a l F i n a l I n i t i a l F i n a l I n i t i a l F i n a l No. No. No. 69 510 1025 44 492 1010 53 466 1030 97 406 1005 45 448 1020 54 476 1020 58 576 1090 46 404 990 55 406 1000 74 416 996 47 446 1006 56 392 1000 76 422 1005 49 518 1090 68 508 1020 Mean 466 l b . 462 l b . 450 l b . Ba r l ey -A l f a l f a Ration No. No. No. 63 468 990 48 542 1015 59 448 995 64 518 1005 50 368 970 60 460 1010 65 448 1005 51 437 1004 61 492 975 66 442 1005 52 516 1008 62 414 984 67 460 1000 57 450 1010 70 446 1015 Mean 467 l b . 463 l b . 452 l b . 117 APPENDIX V Table IV, Blood Parameters o f Study I Barley 0 mgm. . D.E.S. 10 mgm. D.E.S. 18 mgm. D.E.S. No. C C . " P .C.V. " " AAA " Hb. No. , C C P.C.V. Hb. No. C C P.C.V. Hb. 69 8.18 34.5 9.45 44 9.05 38.50 11.1 53 8.99 33.0 9.3 97 8.15 28.9 8.10 45 8.72 35.00 9.3 54 8.80 37.5 9.9 58 8.20 34.3 9.60 46 8.49 38.25 10.2 55 9.05 37.8 10.8 74 8.23 29.5 8.20 47 8.32 39.75 10.2 56 10.18 41.0 11.1 76 8.46 33.0 9.45 98 10.98 37.25 10.8 68 10.45 37.5 10.8 X 8.25 32.02 8.96 9.11 37.75 10.36 9.50 37.3 10.4 Ba r l ey -A l f a l f a No. No. No. 63 8.79 40.75 11.4 48 8.08 34.5 9.15 59 9.56 37.7 11.1 64 8.84 38.75 9.3 50 9.01 39.2 10.20 60 8.48 33.5 9.0 65 9.31 33.50 9.3 51 9.11 35.0 10.80 61 9.45 39.2 10.5 66 8.50 31.50 8.7 99 8.21 35.7 9.90 62 9.32 39.7 9.9 67 9.24 37.25 10.5 57 8.91 38.2 9.60 70 11.32 41.0 10.5 X 8.73 36.35 9.84 8.67 36.5 9.93 9.63 38.25 10.2 3 Red C e l l Count mil l ions/mm Packed C e l l Volume (Haematocrit) % Hemoglobin gm. % 118 Barley APPENDIX VI Table V, Tota l Blood V o l a t i l e Fatty Ac ids , Study I meq . / l . No. 0 mgm. D.E.S. No. 10 mgm. D.E.S. No. 18 mgm. D.E.S 69 2.34 44 2.07 53 1.60 97 1.39 45 .74 54 .59* 58 1.45 46 .90 55 1.81 74 2.10 47 1.43 56' 1.59 76 1.94 98 1.03 68 1.30 Mean 1.84 1.23 1.58 Ba r l ey -A l f a l f a 63 1.88 48 1.83 59 1.45 64 1.27 50 1.07 60 1.29 65 1.23 51 1.66 61 2.65 66 1.83 99 .75 62 .85 67 1.68 57 2.16 70 1.15 Mean 1.58 1.50 1.48 119 APPENDIX VII Table V, Acet ic -Prop ion ic Ratios i n Blood, Study J_ Barley 0 mgm. D.E.S. 10 mgm. D.E.S. 18 mgm. D.E.S. No. Ace t i c Propionic No. Ace t i c Propionic No. Ace t i c Propionic 69 44 586 12.87 45.5 53 2125 8.12 261 97 974 12.01 81.1 45 630 9.34 67.4 54 1498 10.61 141 58 787 6.52 120.7 46 404 5.69 81.5 55 2226 9.11 244 74 439 6.49 67.6 47 923 9.67 95.4 56 1635 6.16 265 76 1119 18.70 59.8 98 603 6.40 94.2 68 834 13.06 64 Mean 82.3 76.8 195 B a r l e y - A l f a l f a 63 1012 10 .37 97.6 48 642 10.11 63.5 59 3083 6.86 449.4 64 699 8 .58 81.4 50 1830 19.42 94.2 60 1220 13.54 90.1 65 2034 18 .17 111.9 51 909 4.99 182.1 61 588 8.89 65.2 66 1199 12 .36 97.0 99 3030 16.38 184.9 62 4158 14.27 291.4 67 57 3779 13.88 272.2 70 1792 6.69 267.8 Mean 97.0 159 232.8 120 APPENDIX VII I Table V I , Thyroid Weights at Slaughter, Study I Barley gm. No. 0 mgm. D.E.S. No. 10 mgm. D.E.S. No. 18 mgm. D.E.S. 69 16.95 44 13.4 53 17.4 97 16.20 45 20.2 54 18.1 58 13.59 46 15.0 55 17.8 74 37.53 47 13.5 56 12.8 76 30.33 98 11.0 68 15.1 Mean 22.9 14.6 16.2 Ley-A l fa l fa No. No. No. 63 15.6 48 19.8 59 12.6 64 19.1 50 12.2 60 7.6 65 14.9 51 9.6 61 25.2 66 16.7 99 17.6 62 13.3 67 19.7 57 12.9 70 15.5 Mean 17.3 14.4 14.8 121 APPENDIX IX Table V I I , Dressing Percentage and Grade Barley No. 0 mgm. D.E.S. No. 10 mgm. D.E.S. No. 18 mgm. D.E.S. % Grade % Grade % Grade 69 56.6 A 44 58.9 A 53 56.6 A 97 58.3 A 45 58.8 A 54 58.4 A 58 58.3 A 46 59.5 A 55 56.3 A 74 58.6 A 47 54.9 A 56 59.4 A 76 57.4 A 98 58.5 A 68 55.1 A Mean 57.8 58.1 57.2 Ley-A l fa l fa No. No. No. 63 58.2 A 48 57.2 A 59 59.0 A 64 57.3 A 50 57.4 A 60 58.6 A 65 55.7 A 51 57.8 A 61 57.4 A 66 57.2 B 99 56.4 A 62 57.2 A 67 59.1 A 57 57.4 B 70 56.8 A Mean 57.5 57.2 57.8 122 APPENDIX X Table V I I I , Growth Data o f Study I I Low Iron High No. 0 mgm. D.E.S. No. 10 mgm. D.E.S. No. 18 mgm. D.E.S. 60 2.88 56 4.53 81 4.26 86 3.63 72 4.21 95 3.84 64 2.60 84 2.52 89 4.16 69 2.06 87 3.64 67 2.36 74 3.83 71 2.99 62 4.84 Mean 3.00 l b . 3.58 l b . 3.89 l b . i Iron No. No. No. 57 3.74 78 3.93 70 2.86 59 2.83 79 3.82 76 2.96 65 3.93 80 3.53 77 3.48 82 2.23 83 4.26 85 4.39 73 3.51 93 1.84 88 3.01 Mean 3.25 l b . X 3.47 l b . X 3.34 l b . 123 APPENDIX XI I n i t i a l and F i n a l Body Weights, Study I I Low Iron No. 0 mgm. D.E.S. No. 10 mgm. D.E.S. No. 18 mgm. D.E.S. I n i t i a l F i n a l I n i t i a l F i n a l I n i t i a l F i n a l 60 710 960 56 760 1044 81 628 990 86 612 960 72 618 970 95 826 1018 64 686 900 84 732 960 89 760 1000 69 830 996 87 757 1000 67 718 924 74 722 1018 71 732 980 62 654 998 Mean 718 l b . 719 l b . 717 l b . High Iron No. No. No. 57 722 994 78 790 1018 70 730 966 59 698 932 79 790 1004 76 745 970 65 764 1000 80 598 916 77 675 940 82 652 840 83 716 1000 85 765 1002 73 750 1012 93 702 894 88 680 932 Mean 717 l b . 719 l b . 719 l b . 124 APPENDIX XII <, I n i t i a l Blood Parameters, Study I I Low Iron High No. 0 mgm. D.E.S. No. 10 mgm. D.E.S. No. 18 mgm. D.E.S. P.C.V. Hb. P.C.V. Hb. P.C.V. Hb. 60 35.5 9.0 56 41.5 10.95 81 38.5 9.9 86 36.0 9.3 72 38.0 10.2 95 39.5 9.6 64 38.5 9.3 84 39.0 9.6 89 39.0 9.6 69 34.5 8.7 87 38.0 9.3 67 38.5 9.6 74 35.0 9.0 71 39.5 9.6 62 34.0 8.25 Iron No. No. No. 57 35.5 9.6 78 40.0 9.6 70 36.5 8.4 59 37.0 9.6 79 33.5 8.4 76 36.0 9.3 65 39.5 9.9 80 37.0 9.3 77 36.5 9.0 82 36.0 9.3 83 37.0 9.6 85 40.5 9.9 73 42.5 10.8 93 35.0 9.9 88 39.0 10.5 Overa l l Average Hemoglobin Haematocrit 9.5 37.55 125 APPENDIX XI I I Table IX, F i n a l Blood Parameters, Study I I Low Iron No. P.C.V. Hb. C C . No. P.C.V. Hb. C C No. P.C.V. Hb. C C 60 32.25 9.6 7.66 56 35.75 10.8 7.72 81 33.25 10.2 8.53 86 36.50 10.8 6.77 72 30.75 8.7 7.04 95 32.50 10.2 8.44 64 37.25 10.8 8.65 84 34.00 10.2 8.27 89 35.50 10.5 8.04 69 29.75 8.1 6.95 87 33.37 9.3 8.23 67 37.75 10.5 7.71 74 31.00 9.0 7.81 71 38.50 11.1 8.29 62 37.50 11.1 6.56 Mean 33.35 9.66 7.57 34.47 10.02 7.91 35.30 10.5 7.85 High Iron No. P.C.V. Hb. C C No. P.C.V. Hb. C C No. P.C.V. Hb. 57 37.25 10.5 7.62 78 40.00 11.9 9.66 70 33.50 9.9 59 34.50 10.2 7.77 79 31.75 9.5 7.23 76 35.50 10.5 65 35.25 10.8 8.52 80 35.00 10.1 7.31 77 34.75 10.2 82 38.00 11.4 8.50 83 31.50 9.0 8.25 85 33.50 10.4 7.3 41.00 12.3 9.51 93 34.25 9.6 6.77 88 33.50 10.5 * Haematocrit % ; " Hemoglobin gm. % Hb. 3 ; * Red C e l l Count mil l ions/mm 7.55 6.62 7.87 6.74 Mean 36.20 10.7 8.10 34.50 10.0 7.84 34.15 10.3 7.25 126 APPENDIX XIV Table X, Total Blood Volatile Fatty Acids, Study II Low Iron Higl 0 mgm. D.E.S. 10 mgm. D.E.S. 18 mgm. D.E.S meq. / I . No. No. No. 60 1.148 56 .681 81 1.476 86 .758 72 1.076 95 1.018 64 1.051 84 1.157 89 .919 69 .810 87 1.672 67 1.194 74 .651 71 1.193 62 1.363 Mean .884 1.156 1.194 n Iron Mo. No. No. 57 1.466 78 1.682 70 1.101 59 .892 79 1.078 76 1.392 65 1.029 80 .797 77 1.021 82 .927 83 .922 85 1.442 73 2.038 93 1.292 88 1.022 Mean 1.08 1.154 1.196 127 APPENDIX XV Table X I I , L i ve r Iron and Copper Storage, Study I I Low Iron Hig] 0 mgm. D.E. S. 10 mgm. D.E. ,S. 18 mgm. D.E. S. ppm. No. Fe Cu No. Fe Cu No. Fe Cu 60 137.6 97 56 170.0 92.5 81 132.2 96.3 86 181.9 148 72 145.3 116.6 95 212.4 150.9 64 116.7 94 84 121.4 118.3 89 184.4 117.7 69 109.3 55 87 140.5 126.9 67 147.5 130.3 74 177.5 91 71 134.6 127.3 62 123.3 160.0 Mean 144.6 84 142.4 116.4 159.9 131.0 i Iron No. No. No. 57 172.3 35.4 78 130.8 118.9 70 191.1 41.7 59 153.3 15.4 79 152.7 20.2 76 224.3 34.1 65 162.8 20.6 80 139.1 51.3 77 186.9 29.6 82 184.7 37.6 83 229.7 95.5 85 132.3 21.9 73 115.6 76.8 93 181.1 20.8 88 113.6 99.4 Mean 157.7 37.2 166.7 61.3 169.6 45.3 

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