THE INFLUENCE OF RATIONS CONTAINING SODIUM ACETATE AND SODIUM PROPIONATE uN THE COMPOSITION OF TISSUES FROM FEEDER LAMBS

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Gladys Williams Royal, B.A., M.S. The Ohio State University 1954

Approved by! i

TitBLE OF CONTi^TS Page

I. ACKNOWLiDXihENTS iii

II. INTRODUCTION 1

III. REVIEW OF TH^ LITrjRATURu 4

IV. EXPtli.IMENTa L METhODS

Plan of Study 16

Maintenance of TestAnimals 17

Collection of Tissue oamnles IB

Preservation of Blood Samples 19

Preservation of Rumen 3amoles 19

Preservation of Liver Samples 20

Preoaration of Meat SamDles 20

Preservation of Meat Samples 21

Extraction of Metabolic Acids for Chromatographic Analyses 21

Chromatographic Analysis of Tissue Extracts 24

determination of pH of Meat 26

Determination of Moisture in Meat 26

Determination of Fat in Meat 27

Preparation of Lamb Boasts 27

Taste Panel Evaluation of Lamb Boasts 28

V. RESULTS AND DISCUSSION

Growth Performance and Feeding Efficiency 30

Distribution of Acetic* Propionic and Butyric Acids in Lamb Tissues 38 ii

Distribution of Other Metabolic Acids in Lamb Tissues 52

Moisture, Fat, dH and Shrinkage Analyses 70

Flavor Studies Using Taste Panels 75

VI. SUMMARY 83

VII. COWULUSIONS 87

VIII. BIBLIOGRAPHY 88

IX. AUTOBIOGRAPHY % ill

Acknowledgements

It is with deepest gratitude that the author acknowledges the many instances of assistance received in the organization of material presented here. Foremost among these are the faculty and facilities of the Department of Agricultural Biochemistry, at The Ohio State

University, extended through a graduate assistantship, which made this work possible.

Special thantcs are also extended!

To ny adviser, Dr. F.E. Deatherage, for his very patient guidance and constructive criticisms throughout, and to Dr. J.E. Varner for valuable suggestions and technical assistance in the chromatographic analyses;

To Professors A.L. Moxon and O.G. Bentley of The Ohio Agricultural

£4xperiment Station, for their supervision of the feeding study and permission to use samples and data from the animals;

To Professor L.L. Kunkle and other personnel of the Meats Laboratory, and to Hr. T. Hershberger and Kir. L. Kamstra of the Department of

Agricultural Biochemistry of The Ohio State University, for assist­ ance in collecting and preserving tissue samples and for assisting in some of the analytical work;

To those persons who participated in the taste panels, for their wholehearted cooperation;

And to the many friends whose words of encouragement contributed in a very large way to the completion of this work. -1-

INTRODUCTION

It is often said by many people in the meat industry that the flavor of meat from grass-fed animals differs from that of com-fed animals. If so, this may be a reflection of differences in quantities and types of products resulting from the digestion of cellulose and starch. Starch digestion is evident throughout the animal kingdom; however cellulose digestion is more restricted.

Although some animals are adapted to cellulose digestion by means of an enlarged caecum and colon, as in the case of the horse, others, such as ruminants, also possess an enlarged gastric system in *tich cellulose digestion occurs.

Microorganisms living symbiotically in the rumen are able to digest cellulose as well as other carbohydrates present in ruminant feeds. At least two symbiotic relationships exist, one among the several groups of microorganisms in the rumen and the other between the host and the r m e n microflora* The rumen provides conditions favorable to the growth of certain microorganisms. One such group of microorganisms may alter existing environmental conditions in such a manner as to promote the growth of a second group of organ­ isms also present in the rumen. The second group may, in turn, produce substances which are utilized by the first group of micro­ organisms . The principle may be extended to include many groups of microorganisms. Feed in the rumen serves as a source of nutrients for the rumen sdcroflora* ihergy and carbon are derived -2- from fermentable carbohydrates, and ammonia or other simple nitrogen compounds, such as urea, may be utilized as a source of nitrogen for protein synthesis* The animal host utilizes some of the fermentation products for energy and some of the microbial protein as a source of amino acids.

The products of fermentation are influenced by the type of carbohydrate digested. Changes in rumen flora of sheep accompany changes in ration but the kind of flora appears to be relatively constant on any given ration ( 13, 51 )* tfhen starch or cellulose is fermented, there is a difference in the proportion of volatile acids produced from the two sources, although acetic, propionic and butyric acids are always formed ( 12, 13 )* A starch containing ration yields principally butyric acid whereas cellulose fermentation gives rise to more acetic acid ( 49 )•

Although volatile fatty acids are also produced in and ab­ sorbed from the caecum, the major portion of cellulose digestion occurs in the rumen. Once elaborated in the rumen, the volatile acids are rapidly absorbed; some through the abomasum, more through the omasum but the greatest amounts are absorbed directly into the blood draining the rumen. This absorption is equally effective for volatile acids added directly to the empty rumen ( 2, 17 )• After absorption, the volatile acids are used as anabolic intermediates.

Propionic acid is oonverted into carbohydrate ( 53 ), butyric acid is degraded to form acetic acid ( 11, 68 ), and acetic acid serves as an intermediate in carbohydrate metabolism, and in the synthesis -3- of auch compounds as fatty acids and cholaatarol ( 6, 9* 55, 56 ).

If one adds to an otherwise adequate ruminant ration, the sodium salts of volatile acids which normally result during starch or cellulose digestion, one would expect the formation of meta­ bolic products by the animal in direct response to the acid or acids added. This response would be expected to produce products similar to those formed by grass-fed and corn-fed ruminants and to provide some index as to differences in meat flavor and tissue composition, if differences do exist.

It was the purpose of this investigation to study the effects of including sodium acetate and sodium propionate in an adequate ration for feeder lambs. Comparisons were made of the volatile acids present in the rumen, their distribution as well as that of other metabolic acids in the blood, liver, and muscle tissue; and these findings were studied in relation to the flavor and composition of the meat. HiSVIS# OF THS LITERATURE

An increased interest in ruminant nutrition and particularly in the special problem of the digestive processes of these animals has been shown since iVorld Aar II. Emphasis has been placed on the study of these animals because of their ability to successfully use cellulose for food. This is of great economic importance, since the ruminant utilizes feeds, roughages, which the human cannot use and converts them into high quality human food such as milk and meat.

Compared to the carnivorous and omnivorous animals, the basic adapta­ tion of the herbivorous animal is an alteration of a portion or portions of the alimentary canal. Enlargement of the caecum and colon is the usual form of adaptation as exemplified in the rabbit or horse; however, ruminants have primarily an enlarged gastric

system. This system ie suitable for the maintenance of a large and active population of microorganisms which are capable of utilizing

various plant materials. For this reason, ruminants as a group are better adapted anatomically than other mammals to the herbivorous mode of life; they digest a greater proportion of the crude fiber

of the diet.

The three ruminants considered to be the most important to man are the ox, sheep, and goat. Little work has been done with the

goat as an experimental animal. In the earlier days it was easier to

gain access to the rumen fluid in the sheep than in the cow; hence

rumen studies with cows are relatively recent. Pearson and Smith ( 43 ) emphasized the difficulty involved in obtaining represent­ ative samples of rumen contents from the ox for kinetic studies of urea utilization. Colin ( 15 ) however, described a method for following changes in the rumen during digestion which makes cattle easier than sheep to study at present* This method consists of creating large rumen fistulas in animals to be studied. Although sheep are too small to be easily adapted to this technique, the idea has been carried over in the use of ebonite cannula. The first technique using a cannula was developed by Quin ( 50 ); Phillipson and other ( 45, 49 ) have devissd useful modifications.

The apparent digestion of cellulose by the ruminant is in reality the result of fermentation carried on by microorganisms liv­ ing symbiotically in the rumen. Fhillipson and UcAnally ( 37* 49 ) introduced individual carbohydrates, including cellulose, into the runen and in each instance the changes noticed were typical of active fermentation observed with normal rations. Two types of symbiotic relationships have been recognized, one involving the microorganisms and the ruminant host and a second involving several classes of organisms within a single host.

Gray ( 17 ) showed that 70^ of digestible cellulose was fermented in the rumen of sheep. The extent of cellulose digestion can be increased, within limits, by inducing microbial multiplication through controlled nutrition. Harris and Uitchell ( 20 ) showed that urea is a useful source of nitrogen for lambs. Its rapid hydrolysis -6- in the rumen was demonstrated by Pearson and Smith ( 45 ); the ammonia formed from this hydrolytic reaction is utilised by the organisms for growth if a suitable carbohydrate is present ( 32, 45#

46# 65 ). In their search for a definition of "suitable carbohydrate",

Pearson and Smith found that maltose and starch are the most satisfactory sources. Theoretically# in bacterial synthesis of protein, rumen organisms use ammonia irrespective of the source of nitrogen, such as urea or other protein supplements. It has been observed, however, in at least one series of experiments ( 15 ) that microbial protein synthesized from the hydrolytic products of urea was deficient in levels of methionine. This amino acid is an important factor in sheep nutrition because of the quantity of sulfur-contain­ ing amino acids needed for wool production.

The rumen microflora is capable of lending beneficial effects to its host other than that of cellulose fermentation. Numerous reports of separate and collective synthesis of vitamins of the B- complex in the rumen are in the literature ( 28# 39# 66 ). UcElroy and Goss ( 39 ) observed the biosynthesis of riboflavin, pyridoxins, thiamine and vitamin K in the rumen of animals maintained on a vitamin deficient diet. In return for these benefits the ruminant host supplies conditions and nutrients favorable to the multiplication of certain classes of microorganisms.

Changes in the type of rumen flora of a given set of sheep during the period of rumen digestion may be interpreted as a reflec­ tion of the second type of symbiotic relationship, that is, symbiosis among groups of microorganisms. nlsden ( 12 ), Uuin ( 51 )> and

McNaught ( 40 ) in independent investigations, observed that

changes in type of rumen flora also accompany changes in the diet and suggested a correlation between these changes and elaboration

of metabolic products. Many investigations have been undertaken to classify the rumen microflora. The presence of both lactic acid bacteria and lactic acid decomposing organisms was suggested by the formation of lactic acid as an intermediate in glucose de­

composition by microorganisms ( 15 )• Two types of lactic acid de­ composing bacteria have been found in the rumen of sheep, propionic acid bacteria ( 15 ) and a gram positive micrococcus which produces hydrogen, carbon dioxide and propionic acid ( 62 ). Few physio­

logical studies and bacteriological examinations have been applied simultaneously to the microorganisms considered. This deficiency is due to difficulty in duplicating the symbiotic existence necessary

for the studies. Johnson et. al. ( 32 ) counted rumen organisms after plating them on a simple medium and incubating them aerobic­ ally. This was in complete agreement with standard bacteriological procedures but no significance can be attached to the results because neither the medium nor the method resembled the conditions in the rumen. Sheep fed on a starch-rich ration contained numerous iodo- philic cocci in the rumen as reported by Van der uath ( 1, 62 ); these cocci congregated around disintegrating starch grains but neither total nor viable counts were possible.

Organisms capable of digesting cellulose have been isolated from the termite gut and from cultures of rumen protozoa, however, none of these seem to be significant in the rumen. The iodophilic bacteria which Baker ( 1 ) and Van der tfath ( 62 ) demonstrated in large numbers attached to the plant debris of the rumen contents, and a gram negative rod reported by Hungate ( 26 ) seem significant.

The gram negative rod seems to require bicarbonate for growth and accounts for less than one third of the total rumen population.

Phillipson ( 48 ) and Uyburgh ( 44 ) in separate Investigations showed that the pH of the rumen liquor of sheep is seldom above neutrality. Phillipson ( 48 ) observed that after feeding, the pH of the rumen fluid falls and this change coincides with the production of volatile fatty acids. This fall in pH of rumen fluid is more ab­ rupt with a ration containing starch than it is with a hay-type ration, otarch fermentation yields principally butyric acid where­ as cellulose fermentation gives rise to more acetic acid. It was not until the work of Slsden ( 13 ), vfcich introduced a suitable partition chromatographic procedure, that the acids present in the rumen fluid were identified. His studies indicated the constant presence of acetic, propionic and butyric acids, however, acetic acid was present in the greatest amount under his conditions. During the fermentation of glucose, the ratio of propionic acid to acetic acid increased.

The volatile acids normally found in the rumen are absorbed into the blood stream as evidenced by the higher concentration of volatile fatty acids in the blood draining the rumen than in the arterial or venous blood from any other part of the alimentary tract.

This fact was established through the investigation of kiddle and -9- co-workers ( 33 )• In a subsequent study Bareroft ( 2 ) introduced equimolar solutions of sodium acetate, sodium propionate and sodium butyrate into the empty, washed-out rumen. The levels of these salts in the blood draining the rumen were such as to indicate that acetate is most rapidly absorbed and the size of the molecule is perhaps a rate governing factor.

Gray ( 17 ) studied the change in ratio of acetic to propionic acid in solutions introduced into the empty rumen of sheep. No difference in concentration was observed when sodium salts alone were present but at pH 6.3, propionic acid disappeared more rapidly than acetic acid.

Through studies of intermediary metabolisdi acetic, propionic, and butyric acids have been designated as important anabolic inter­ mediates. The literature is particularly rich with studies involv­ ing acetic acid. Although many different pathways have been proposed, it seems to be firmly established that acetic acid is involved in fat and cholesterol metabolism. Zelmen et. al. ( 70 ) found that acetic acid increased the rate of synthesis of milk fat in the dairy cow.

Rittenberg ( 55 ) demonstrated the utilization of the carboxyl carbon of acetic acid for fatty acid synthesis in experiments with rata.

Bucher ( 9 ) obtained evidence for the formation of cholesterol as 14 well as fatty acids from C labeled acetate in rat liver homogenates.

Bloch ( 6 ) summarized the known facts concerning the utilization of this volatile acid. The fact that the amount of acetic acid in the systemic circulation is small while relatively large amounts are found in the blood draining the rumen originally suggested to Deuel ( 11 ) that sheep too were capable of metabolizing acetic acid. This has since been experimentally confirmed by UcClymont ( 3^ ) in a study involving both sheep and cattle.

Propionic and butyric acids are involved in carbohydrate and fat metabolism, respectively. Held ( 52, 53 ) indicated that sheep are able to metabolize both of these acids, however, their roles in metabolism have been explained through the use of microorganisms.

Lorber ( 35 ) using microorganisms, demonstrated the conversion of propionic acid to glycogen and proposed a mechanism involving the initial formation of succinate, then pyruvate and finally glycogen*

Larsen ( 34 ) experimentally confirmed the feasibility of such a mechanism by actually forming succinate from a three-carbon inter­ mediate. Evidence gained from butyrate oxidation by Clostridium kluyveri ( 15 ) indicated that aceto-acetate is a direct product of butyrate oxidation in this organism. Zabin ( 69 ) considered butyric acid as a precursor for the synthesis of cholesterol. In experiments with doubly labeled butyrate he showed that the ck- carbon was more frequently incorporated into the cholesterol molecule than was the carboxyl carbon atom.

Uany investigations have undertaken to define the "normal11 microbial population of the rumen and the factors which affect this population. Other research has been centered on the metabolic pro­ ducts arising in the rumen with different microbial populations; and the role of these products in intermediary metabolism. There has been - u -

no att«npt however, to relate these studies to flavor in the meat

of ruminants. This is suprising, in view of the fact that flavor

includes odor as well as taste and there is a decided difference

in the odor of different metabolic acids.

Iamb has a flavor distinctive in itself, however, virtually

no experimental reports exist which attempt to assign this flavor

to one or more chemical entities. Howe and Barbella ( 25 ) sought

to define the flavor of meat, without any differentiation for types

of animals, as a composite of salts, acids and a group of products

resulting from cooking. Possibly, disintegration products of proteins

and lipids, typical of the animal, become more evident on heating the meat* Flavor effects are observed with the aging of meat.

In spite of the lack of concrete supporting evidence, the

contribution of acids and salts to flavor has been explained in terms

of their stimulation on taste buds or other nerve receptors. Various

authors have attempted to explain acid taste, intensity and character

from a chemical standpoint with particular emphasis on the effects

of molecular weight or substituent chemical groups. Berlatzky ( 5 )

concluded in determining the "threshold of gustatory sensation for

acids", that both anion and cation of an Inorganic salt influenced

taste by affecting intensity and character, respectively, rfithin a given series of compounds, taste variations result from chemical and

physical differences. According to Henqvist ( 54 ), increasing the molecular weight results in a decreased taste stimulation effect, although isomers yield irregular results. Taylor ( 59 ) speculated -12-

that organic acids were absorbed by a tissue in the taste organs which was fatty in nature rather than proteinaceous and that lengthening the

acid chain by CH^ makes the penetration of this tissue easier.

The existing literature is proportionately as rich in proximate

analyses defining the gross composition of lamb carcasses as i t is

poor in lamb flavor studies. The effects of breed and dietary constit­

uents, fermentable carbohydrate, fat and protein on this gross compo­

sition constitute the general trends of study. As early as 1916,

Wright (66) had tabulated and reported data for the proximate analysis of leg and loin cuts of lamb carcasses and simultaneously noted the inverse relationship between fat and moisture contents, that is, the

fattest animals contain the least moisture. In the years to follow, various groups compiled data on the composition and biological value of specific cuts; however, Hoagland ana co-workers ( 23) demonstrated equal biological value for the leg, shoulder and entir* carcass.

Recent advances in techniques for studying metabolic acids make it feasible to study the possible relation between these acids and meat flavor. A literature search of methods for the determination of organic acids and specifically those of the tricarboxylic acid cycle in animal tissues, revealed many reports of individually determined acids but only one which investigated the complete tricarboxylic acid cycle on a single tissue. Vladimirov ( 63 ) determined organic acids in whole blood of dogs and in plasma of dogs and humans. The data for dogs indicated that more than one-half of the organic acids on a milliequiv­ alent basis, is in the plasma. Licarboxylic acids were demonstrated as -13- intermediate products in the biological breakdown of normal saturated

fatty a d d s and their derivatives*

Trautaan ( 60), among others, made chemical and physical measure

■ants on blood of healthy sheep* These analyses include pH, density, viscosity, blood sugar, non-protein nitrogen, and alkali reservej how­ ever, no determination of the metabolic acids other than volatile

fatty acids were aade*

Bulan, Varner and Burrell ( 10 ), and Isherwood ( >0 ), inde­

pendently employed partition chromatography on silica gel to separate and determine several organic acids, sasie of which belong to the tri­ carboxylic a d d cycle, in plant tissue* Marshall, Orten and Smith

( 42 ) applied this technique to several animal tissues for the de­ termination of fuaaric acid; coincidentally, other members of the tri­ carboxylic acid cycle were separated. In a later publication Marshall et. al. ( 43 )» followed by Frohaan ( 16 ), reported an extension of the procedure to include the simultaneous determination of all members of the cycle in a single sample of pooled rat tissues* In general,

the metabolic acid studies have been carried out with animals whose tissues are not used for huaan concumption. In the volatile acid

studies with ruainants, no atteapt has been made to relate the find­

ings to meat flavor.

Froa the existing literature several basic facts stand out and may be suemarised as follows: It is evident that the emphasis of sci­

entific research on the problems involved in ruainant nutrition is -Ir­ relatively recent. The increased interest in ruminants stems from an economic consideration, for they effectively utilize feeds and roughages which other mammals cannot use.

Cattle and sheep have been studied more extensively than other ruminants. Earlier difficulties encountered in physiological studies with cattle have been eliminated by the basic technique of creating large rumen fistulas* Modifications of this technique are also used in studying sheep.

Cellulose digestion by the ruminant is accompanied by two symbiotic relationships; one between the microorganisms and the ruminant host, the second between the several types of microorganisms existing at various stages of the digestion process. Although many organisms have been found which are capable of digesting cellulose, at least two seem to be definitely associated with its digestion in the rumen, the iodophilic cocci which aggregate around starch and a gram negative bacilli which requires bicarbonate for growth.

The digestive products found in the rumen include volatile acids, vitamins, and ammonia; however, concentrations are dependent upon the types of ration and microorganisms present in the ruaen.

Irrespective of the type of carbohydrate present, acetic acid as well as propionio and butyric acids are always found. Urea, as well as natural protein, may serve as a precursor for ammonia. The volatile acids are used as important anabolic intermediates in carbohydrate and fat metabolism whereas ammonia is used in the synthesis of -15- microbial protein. The synthesis of vitamins in the rumen is of microbial origin; however, specific food pracursoro ara not clearly da fined•

Heat flavor has bean described as a composite of salts, acids, and a group of products resulting from cooking* Consideration has bean given to the effects of taste bud stimulation and chemical group­ ings on flavor. No experimental data have conclusively related chemi­ cal groupings to the flavor typical of lamb meat.

A relatively recent technique, which allows the chromatographic separation of metabolic acids on a single tissue, has made it feasible to study the possible relation between these acids and meat flavor*

The technique has been applied most extensively to plant studies and to animals not commonly used for human food. No attempts have been made up to this time to relate volatile acids found in the rumen by this technique to swat flavor. The purpose of this study is to de­ termine if some relation may exist between meat flavor and the meta­ bolic acids produced in the rumen when different rations are fed. -16-

EIPERIMENTAL METHODS

The lambs in this study wars from a project on the use of organ­

ic acid salts in the ration of fssdsr lambs. The feeding study of that

project was an outgrowth of fundamental studies involving rumen function

in cellulose and staroh digestion. In general, the investigation re­

ported in this paper was divided into four parts; (l) maintenance of

test animals; (2) collection of tissue samples; (3) preparation of

tissue samples; (4) analysis of tissue samples.

The twenty-four experimental lambs were maintained under con­

trolled conditions at The Ohio Agricultural Experiment Station by the

personnel of the Station. A basal ration adequate for normal growth was employed. Representative numbers of these animals received, in add­

ition to the basal ration, one or more organic acid salts. Soditmt acet­ ate and sodium propionate were the acid salts used for the supplements.

After reaching the average weight of marketable laabs which cor­ responded to a feeding period of 109 days, the animals were slaughtered at the Meats Laboratory of The Ohio State University. Samples of blood, rumen Juice, liver were collected Aron each animal at the time of slaught­ er. Meat samples were refrigerated and later collected when needed for the taste panels.

All samples were quick-fro sen, lyophilised and then stored in air­ tight containers to preserve the sample composition present at the time of slaughter. Blood, liver, r men-Juice and meat samples were chromatograph-

ically analyzed for volatile and other metabolic acids. In addition,

the meat from these lambs were studied with respect to pH, fat and

moisture content, and flavor. The meat samples used in the flavor tests

and pH, fat, moisture and metabolic acid analyses were the same.

Maintenance of test ajri a

Twenty-four feeder lambs of the Shropshire breed and Columbia-

Shropshire crosses were randomly assigned in such a manner as to give

five crosses and one pure bred animal in each of four lots. This

method of distribution also provided three wethers and three ewes in

each lot except the control. The control lot contained five wethers

and one ewe. All lambs were born in the Spring of 1952 and, after

shearing, weighed from 63*3 to 64*0 pounds at the beginning of the

experiment. During the growth period the animals were housed in the

Animal Nutrition Building at The Ohio Agricultural Kxperiment Station,

Aooster, Ohio.

A basal ration consisting of urea - glucose (cerelose) mixture,

yellow corn (cracked), mixed clover-timothy^alfalfa hay, and a mineral mixture was fed to each of the four lots. Lot 1 received no supplement

and served as the control; Lot 2 received a supplement of sodium acet­

ate; Lot 3 received a supplement of sodium propionate; and the sup­

plement of Lot 4 consisted of sodium acetate and sodium propionate. -18-

The quantities of basal ration and supplements fed are listed below.

Basal Ration (pounds/day/lamb)

Urea 0.0652 Cerelose 0.25 Yellow corn 0.5 (cracked) Hay (mixture) 3.1 clover timothy alfalfa Mineral mixture free choice

3WylOT1HlVy ( grams/day/lamb )

Lot l...No supplement Lot 2...Sodium acetate 46.7 Lot 3.. .Sodium propionate 46.6 Lot 4.•.Sodium acetate 23.4 Sodium propionate 23.4

Urea and cerelose were mixed together and fed at the same rate to each lot. The hay mixture was full fed to each lot and all animals had free choice of the mineral mixture, hiach lamb was weighed weekly; all feed was weighed in; the amount of hay rejected was estimated.

Collection of Tissue Samples

After 109 days, one half of each lot was slaughtered at The Ohio

State University Meats Laboratory, Columbus, Ohio. The remaining three animals in each lot were slaughtered after 123 days. Blood, liver and rumen juice were obtained from each animal for analysis, at the time of slaughter. Meat samples were kept in cold storage until required for the taste panels. -19-

Preservation of Blood Samples

Arterial blood was collected fro* the firet set of twelve ani­ mals and iraediately deflbrlnated. The defibrinated blood was centri­ fuged and the serum from each sample was decanted Into a deep culture dish.

Arterial blood wae collected from the second set of twelve ani­ mals and 25 ml. of blood from each animal were plpeted Into a deep culture dish.

To each 25 ml. of blood or serum, 1.5 ml. of 4 N. NaOH was added.

This quantity of base had been determined previously as necessary to adjust the pH to 10. Precautions were taken to add the base to the whole blood samples before clotting occurred. Immediately after the addition of sodium hydroxide all samples were frozen In an ether-dry Ice mixture, and then lyophilised. Approximately 15 hours were required to dry the samples. After lyophilization the samples were stored In air­ tight containers.

Preservation of Rumen Samples

The rumen was removed tram the carcass and ligated at both ends.

After Its transfer to the analytical laboratory the rumen was opened and the entire contents were weighed. The contents were then pressed through two layers of cheese-cloth and the weight of the filtrate,

"rumen Juice", wae determined; 100 ml. of the filtrate were taken and -20- and 3 ml. of 4 N. NaOH m n added to adjust the pH to approximately

10. One half of this mixture was placed in a deep culture dish, frozen and then lyophilized for 15 hours, aftei^rhich the sample was stored in an air-tight container.

Preservation of Liver Samples

From each carcass of the first set of twelve animals, sixty grams of liver were homogenized in a Waring blender along with 25 ml. of water and 10 ml. of 2 N. NaOH. This quantity of alkali was found necessary to adjust the homogenate to pH 10 - 11. The samples were transferred to deep culture dishes, frozen immediately in an ether-dry- ice mixture and lyophilized for 15 hours. The resulting powder was stored in air-tight containers.

For the second set of twelve animals the amount of liver, water and base were altered because the original quantities provided a mix­ ture somewhat thick for the blender. After adding 50 ml. of water and

40 ml. of 2 N. NaOH to 45 grams of liver, the samples were froamn, lyo­ philised for 24 hours and then stored in air-tight containers.

Preliminary Preparation of Meat Samples

One complete shoulder of eacn lamb was boned and each such meat sample was ground in the Meats Laboratory through a l/4 inch plate, three times, with mixing. Prior to its use for flavor tests or fat, moisture, pH and metabolic acid analyses, each meat sample was sdxed in the Foods Laboratory in a Kitehen-Aid adxer to a homogenous oon- -21- sis tency. The neat samples used for flavor tests and pH, fat, moisture and metabolic acid analyses sere the same. Meat samples from those animals in Lot 4 which received sodium acetate and sodium propionate supplements, sere not included in these studies.

Preservation of Meat Samples

Meat samples shich sere used for chromatographic analyses were preserved in the following manner: Portions of the meat pre­ pared according to the preceding directions were homogenized with sodium hydroxide and water in a Waring blender. For each 100 gram sample of meat, 2.5 ml. of 4 N. NaOH and 250 ml. of distilled water were added. Limited quantities of available sample required nearer

20 gram samples from the second set of nine animals; however, the proportions of water, base and meat were vnaltered. Homogenates were placed in deep culture dishes, frozen inmediately in an alcohol- dry ice mixture and lyophilized for 36 hours. At the end of this time the samples were stored in air-tight containers.

Bxtraction of Metabolic Acids for Chromatographic Analyses of Iamb Tissues

Rumen

Preliminary extractions of acidified, lyophilized rumen Juice indicated that extraction with ethyl ether did not increase the re­ covery of the short chain fatty acids, acetic, propionic and butyric acids, in chromatographic analyses. Therefore, the following pro­ cedure was used! 0 . 1 0 0 0 gram of lyophilized rumen Juice was put into -22-

* small evaporating dish and one ml. of 4 N. sulfuric acid was added to acidify the sample to pH 1.0. This sample was mixed thoroughly with two grams of silica gel and applied to the top of the chromato­ graphic column of siliea gel.

Blood. Liver. Meat

The extraction procedure applied to these samples was in keep­ ing with that outlined by Isherwood ( 30 ). An acidified ether ex­ tract was employed. The size of sample used in any one extraction was necessarily small. Helatively small quantities were originally prepared for lyophilization and some portions of blood, liver, and meat samples were lost in an accident involving the lyophilization equipment.

Serum samples ( 1.0 - 1.5 )» blood samples ( 0.5 - 1.0 grams), liver ( 2.5 - 3.0 grama ) and meat ( 5 . 0 - 1 0 grams ) were moistened with 4 N. sulfuric acid prior to extraction. Serum, blood and liver samples were pulverized in a mortar and pestle; meat samples were cut into fragments about the size of a pea since due to their high fat content, grinding was not practical. Desired amounts of each sample were accurately weighed into extraction thimbles. After pipeting the sulfuric acid onto the samples and mixing the samples and acid thoroughly, the top of each thimble was plugged with glass wool. The quantities of 4 N. sulfuric acid used for moistening were as follows: 1.5 ml. for serum and blood samples, 3.5 ml. for the second set of liver samples, 2.5 ml. for the first set of liver samples, and 2.0 ml. for meat samples. These values represent -23- amounts used for each sample. detraction thimblaa containing the prepared samples were placed in cylindrical tubes equipped with cold finger condensers and extracted.

Diethyl ether was used as the extraction solvent. Prior to its use the ether was treated with potassium permanganate

( 0.5 - 1.056 in 0. 1 N. NaOH ) until no additional green color was evident in the permanganate solution. The excess permanganate was removed by repeated washing with distilled water. After filtering through filter paper the ether was stored over anhydrous sodium sulfate until needed.

Ether extraction was allowed to proceed until the extract showed no additional acid reaction to the phenol red indicator for a two hour period following neutralisation of the extract with sodium hydroxide. At the end of approximately six hours the extraction was stopped, two drops of 156 aqueous phenol red were added to the extract and the extract was titrated with I N . NaOH to the end point of this indicator. Extraction was resumed and checked at intervals for the additional formation of acid accord­ ing to the preceding directions. A period of approximately eight hours was adequate for extraction of the blood and serum samples} twelve hours was necessary for the liver and twenty-four hours was satisfactory for the meat.

At the end of the extraction period the neutralised extracts were evaporated to dryness on a steam bath; however, it was necessary to treat the meat samples further because of their high -24- fat content. The neutralized ether extracts from the meat samples were washed with distilled water until the ether layer gave no evidence of the basic form of the phenol red indicator. The combined water extracts were then evaporated to dryness on a steam bath; the ether layer was discarded.

Chromatographic Analysis of Tissue Extracts

The procedure used for the determination of the volatile organic acids and other metabolic acids was essentially that of

Bulen, et. al, ( 10 ). A solvent system composed of l£ butanol- chloroform ( v/v ) was used before the 5% butanol-chloroform ( v/v ) of the Bulen method because the solvent system described by Bulen does not separate butyric acid from propionic acid. It should be pointed out that the Bulen system was not designed for this purpose.

The chromatographic column was prepared by acidifying 8.0 grams of silica gel ( Uallinckrodt ) with 5.5 ml* of 0.5 N. sulfuric acid. The free-flowing powder which resulted, after mixing acid and gel thoroughly, was slurried onto the 12 mm. column with a total of

40 ml. of chloroform. A slight pressure was applied to the top of the column to hasten the removal of excess solvent. When the chloro­ form level dropped to within a few centimeters of the top of the

1 4 . 6 cm. column, the dried tissue extract was acidified with 4 N. sulfuric acid, taken up in 2 grama of silica gel and the slurry was transferred to the column with a minimum of chloroform. Excess solvent was drained from the column and a glass wool plug was pressed -25- fir ml y on top of the sample. Solvents in the n-butanol-chloroform system were then added according to the following schedule.

n-butanol-chloroform Volume used percent ( v/v ) ml.

Solvent 1 1 % 50 Solvent 2 5 % 100 Solvent 3 15 % 135 Solvent 4 25 % 100 Solvent 5 35 % 100

A multiple pressure system was incorporated which allowed

four such columns to be run simultaneously under approximately equal pressure conditions. As far as possible one sample from each

lot of animals was included in each chromatographic run. The effluent was collected in fractions of 2.9 - 3*1 ml* with the aid of an Autonomos fraction collector. Each fraction was titrated with 0.0137 or 0.0122 N. NaOH. The latter concentration was used

for the rumen samples. Phenol red, 1 % aqueous solution, was used as the indicator throughout the titrations.

All fractions were saved from the 15 % ( v/v ) n-butanol- chloroform solvent and samples from similarly treated animals which had been slaughtered at the same time were pooled. The fractions were separated into their respective layersj chloroform layers were washed with distilled water and the combined water fractions were evaporated to dryness on a steam bath. After dilution of the residue to 50 ml. with distilled water, 25 ml. aliquots were em­

ployed in the isolation of the members designated by Bulen and co­ workers as Groups I and II. Group 1 includes formic, glutaric and -26-

fumaric acids while Group II includes trans-aconitic, succinic, lactic and -keto glutaric acids.

The aliquots to be rechromatographed were first evaporated to dryness on a steam bath, acidified drop by drop with sufficient 4 N.

sulfuric acid to release the acids from their sodium salts, and then

chromatographed. The chromatographic technique followed that

previously described on pages 24 and 25* however, the elution sol­

vent was 100 ml. of 25 M v/v ) n-butanol-benzene as specified by

the Bulen method.

Determination of pH of Meat

Approximately one gram of sample from each ground shoulder

was allowed to come to room temperature while standing on a watch

glass, unrefrigerated, for one hour with the maximum surface exposed.

Enough carbon dioxide-free water was added to make a slurry. The

pH of the slurry was detersiined on a battery operated pH meter.

Determination of Moisture in Meat

Duplicate samples from each ground shoulder, varying in size

from 2.0 - 2.5 grams were weighed into small moisture pans. The

samples were dried in air at 105° C for six hours, cooled in a

dessicator and re-weighed. Loss in weight represents moisture con­

tent. This method was reported by Husani ( 29 ) to give optimum

results with beef samples. -27-

Determination of Fat in Meat

The method employed was essentially one outlined by Hoover

{ 24 ), 1951* A mixture of 440 ml. of a 24 % Rochelle salts solution, 264 ml. of 90 % ethanol ( v/v ), 210 ml. of 20 % sodium hydroxide and 1?6 ml. of distilled water was used as the extraction solvent. Samples weighing 5*0 - 5.5 grams of the meat slurry were pipeted directly from the homogenate into 20 % Palsy cream flasks.

This weight of sample correspond to 1.4 - 1*5 grams of meat.

Thirty ml. of the extraction mixture were added to each flask and the flasks were maintained at 75° - 80°C on a steam bath with frequent agitation until the meat was entirely digested, mough extraction mixture was added to bring the volume of the flask con­ tents to the base of the flask neck. The flasks and contents were centrifuged for three minutes in a centrifuge preheated to approxi­ mately 7 0° C to remove suspended particles from the fat layer.

Sufficient water at 70°C was added to bring the volume of the flask contents within a few millimeters of the top of the flask. The samples were cnetrifuged again for two minutes, maintained in a water bath at 70°C for an additional two minutes and the fat volume was then read directly. Each determination was carried out in duplicate.

Preparation of Lamb Roasts for Flavor Tests

Roasts for shrinkage and flavor tests were prepared from Lots

1, 2, and 3; those animals which received no supplement, sodium acetate, sodium propionate, respectively. Three and one-half pounds -28- of each ground lamb shoulder which had been well mixed were packed into a loaf pan lined with wax paper. In this manner all loaves were molded to nearly uniform size and shape. The loaves were re­ moved from the mold and all three loaves from animals receiving the same ration were placed on one aluminum tray. Ueat thermo­ meters were inserted into the middle loaf and one of the outer loaves of one tray to indicate differences in internal temperature of the roasts due to position in the oven. Ueat loaves from all nine animals were roasted simultaneously in a rotary hearth oven operating at 32 $°F until the internal temperature of the indicator loaves registered 180°C. The roasts were drained of fat when necessary, to prevent spilling in the oven and each roast was weighed as soon as practical upon its removal from the oven. After roasting was complete, the loaves were maintained, uncovered, in a warming oven and used for a taste panel within one hour after cook­ ing. This procedure of preparing the roasts was employed for each of the two taste panels.

Taste Panel Evaluation of Lamb Roasts

Twelve persons, six of whom had previous experience on taste

panels for flavor in meat or dairy products, took part in each of

the two panels. The participants were not the same twelve in each

panel* Triangular tests were conducted in which each member of the

panel was given a plate containing three pieces of meat; each

portion of meat was appropriately designated by number. All

samples from a given animal had the same number, however, numbers -29- were different for samples from different animals. In all teste

the odd piece of meat placed on the plate was from the control

lot of animals. The other two pieces of meat were from the same animal, an animal which had received a supplement of sodium

acetate or sodium propionate in its ration. In this manner each

of the three control roasts were compared with roasts from sodium

acetate and sodium propionate supplemented animals. During the

course of each panel this procedure allowed 3 comparisons of each

treatment with the control. The tabulated results were based on

the ability of each panel member to successfully identify which

two samples were from the same animal. These results were then

studied statistically to determine possible differences in flavor

and to establish the degree of reproducibility of the individual

scores as well as the scores of the entire panel. -30-

RESULTS AND DISCUSSION

Growth Performance and Feeding Efficiency

Management of the feeding experiment was carried out at The

Ohio Agricultural iixperiment Station by the personnel there. The feeding experiment was part of a project on the use of sodium acetate and sodium propionate in the ration of feeder lambs. This was an outgrowth of fundamental studies involving rumen function in cellulose and starch digestion.

A summary of the feeding management data appears in Table 1 which is found on the following page. During the experiment it was observed that the salts, sodium acetate and sodium propionate, are fairly palatable. Data included in Table 1 substantiate this observation. The lambs consumed as high as 83 grams per day of sodium acetate or sodium propionate per lamb and as much as 83 grams per day of a mixture of equal weights of acetate and propionate per lamb.

From the economic standpoint it is not feasible to feed either sodium acetate or sodium propionate as a ration supplement for lambs.

For example, approximately 45.00 worth of feed was saved in obtain­ ing desired gains by adding approximately 410.00 worth of sodium acetate. Hence, the saving in feed is offset by the present cost of sodium acetate. It is significant however, that feeding end pro­ ducts of carbohydrate metabolism resulted in replacement of other feeds and did not interfere with the appetite of the lambs. The TABLE 1

Growth Performance and feed Efficiency

N\aber of Sodiimi salt Average Average Average Feed required / 100 pound gain Lot No. aninale of fatty starting final daily " acid fed weight weight gain Corn Hay Cerelose Urea Fatty Ibe lbs lbs lbs lbs lbs lbs acid salt lbs

1 6 - 63.5 67.0 .22 230.3 1405 114.5 30.2 —

2 6 sodiw acetate 64*0 91.0 .25 200.9 1293 100.3 26.3 41.35

3 6 sodium propion­ 63.6 90.5 .25 202.5 1228 101.1 26.5 41.71 ate

4 6 sodium acetate 63.3 91.2 .25 195.2 1190 97.4 25.6 20.12 and sodiua propionate 20.12 -32- incorporation of the salt of either organic acid in the ration resulted in a decrease in the pounds of corn, hay, cerelose and urea required per 100 pounds of gain in body weight. This seems to be a better criterion for feeding efficiency than average final weight of animals in each lot because of variations in average starting weight between the four lots. Nevertheless, average starting weight, average final weight, and average daily gain are included in Table 1 for each of the four lots. Lot 2, which received the sodium acetate supplement had the highest average starting weight; 0.7 pounds higher than Lot 4 which received the mixture of sodium acetate and sodium propionate. Lots 1, 3 and 4 had approximately equal starting weights and yet the differences in their average final weights ranged from

2.5 - 4*2 pounds. Although the difference in average starting weight between Lots 2 and 4 was 0.7 pounds, their difference in average final weight was o n l y 0 . 2 pounds. This suggested that the gains in body weight should be given more attention. Average daily gains were approximately equal for all lots which received either of the supple­ ments; however, these were o n l y .03 povnds per day higher than the average daily gain of the control lot. Attention was than focused upon the feed required per 100 poisids of gain in body weight. These data are tabulated in Table 1.

The total feed requirement per 100 pounds of gain was reduced for all lots which received a supplement; however, the role of the supplesmnt in this econony can be better demonstrated in an itemised consideration of reductions in each of the major feed components. -33-

The corn requirement waa lowered for Lot 2 which received

sodium acetate, by 30 pounds per 100 pounds of gain. The re­

duction in corn requirement for Lot 3 which received sodium pro­

pionate was not quite as pronounced as that for Lot 2. The de­

crease of 28 pounds of corn required per 100 pounds of gain found

in Lot 3, however, was substantial. The combined supplement which

contained both sodium acetate and sodium propionate was more

efficient than either of these organic acid salts in reducing the

corn requirement for weight gains. The corn requirement was re­

duced by 35 pounds per 100 pounds of gain in Lot 4 which received

the mixed supplement.

Hay consumption was decreased progressively from Lot 1 to

Lot 2 to Lot 3 to Lot 4* Here again the effects in Lot 4 which

received the mixed supplement, were superior to those found with

either of the separate supplements. Sodium acetate supplemented rations reduced the hay required for each ICO pounds of weight gain by 112 pounds; sodium propionate supplemented rations reduced the

requirement by 177 pounds and the mixed supplement in the rations

reduced the hay requirement by 215 pounds per ICO pounds of gain in body weight of the lambs. All of these reductions are based on re­ quirements of the control animals, Lot 1*

Cerelose and urea requirements followed the same pattern of reduction previously described for corn, that is, animals receiving acetate supplement required an average of 1 4 . 2 pounds less cerelose and 3*9 pounds less urea than did the control per 100 pounds of gain. -34-

Animals receiving the propionate supplement required 13*4 pounds less

cerelose and 3*7 pounds less urea than did the control per 100 pounds

of gain in body weight. Animals receiving both acetate and propion­

ate required 17*1 pounds less cerelose anl 4.6 pounds less urea than

did the control per 100 pounds of gain in body weight.

The reductions in c o m , hay,cerelose and urea required by

animals which received either sodium acetate or sodium propionate for each 100 pounds of gain in body weight suggest that, when present in

the ration, these organic acid salts are used preferentially to the more complex feed components.

Prom the nutritional viewpoint it is significant that a reduc­

tion in quantity of feed required for weight gains is accompanied by

a reduction in caloric requirement. Table 2 contains the caloric dis­

tribution of the four major components of the basal ration. The values presented here are based on the assumptions of complete utiliz­ ation of ration consumed and net energy values per 100 pounds of com­ ponent as follows: c o m , 8 0 , 0 0 0 Calories, hay, 41,000 Calories and

cerelose, 1 6 1 , 6 0 0 Calories. These values may not allow the computa­ tion of absolute caloric units; however, they do serve as an adequate

tasis for comparing caloric requirements of the four lots of animals.

There is, in Table 2, a reduction in the number of calories re­ quired from the basal ration for each pound gained by those lots which received one or both of the organic salts. Sodiuo propionate seemed to be more effective in reducing the caloric requirement than sodium TABLE 2

Caloric Values of Ration Components

Caloric Requirement Per 100 Pound Gain in height

Sodium salt of Total calories Calories Total fatty acid used Corn Hay Cerelose from basal from sup- Calories ration plement in ration

Hone 184,200 576,000 207,900 968,000 0 968,000 -SC- Sodium acetate 160,700 530,100 182,100 873,000 47,600 920,600

Sodium propionate 162,000 503,500 183,600 849,100 70,000 919,100

Sodium acetate plus Sodium propionate 156,200 487,900 176,900 821,000 56,900 877,900 -J*6 acetate. This is to be expected. From the caloric values listed in Table 2 for sodium acetate alone and sodium propionate alone, it can be seen that propionate liberates more calories per mole than does acetate during oxidation. These data are based on the heat of combustion for propionic acid, 363 Kilogram calories per mole of acid and for acetic acid, 20£ Kilogram calories per mole of acid. A com­ bination of the two salts in equal weight quantities in the ration of Lot U caused the greatest reduction in caloric requirement below that of the control, quantitatively, this caloric differential corresponds to an 8 & reduction for the s-jlium acetate supj lamented animals, 12 jL reduction for the sodium propionate supplemented ani­ mals, and a 15 4> reduction for the nixed supplemented animals be­ low the caloric requirement of the control, for corresponding weight gains.

The conversion of available energy in the rumen into weight

Is by no means complete. From Table 2, it is apparent that a larger amount of energy was available to those animals which received corn or cerelose in the ration than was available to animals which re­ ceived sodium acetate or sodium propionate. Hence there was a less efficient conversion of calories from c o m or cerelose than from i acetate or propionate. It is estimated that the rumen conversion is only about 50 % efficient since a lot of energy is used up in digest­ ing feeds and in synthesis, itesults here suggest that ener&r such as available in corn or cerelose may stimulate synthesis or other mechanisms of dissipation. -37-

From & consideration of growth performance of the lambs and

the corresponding feed requirements it seems that the incorporation

of sodium acetate or sodium propionat*- in the ration does produce a

somewhat more efficient ration. Smaller quantities of hay, corn,

urea and cerelose are requirei for unit gains in body weight when

either of these salts are included in the ration. Larger weight

gains observed in those animals receiving either supplement are

accompanied by lower caloric requirements supplied from the basal

ration. The acids resulting from the supplements are oxidized and

thus supply additional energy to the animals. Sodium acetate seems

somewhat more efficient than sodium propionate in reducing feed re­

quirements. On the other hand, due to a higher heat of combustion,

sodium propionate seems somewhat more efficient than sodium acetate

in reducing caloric requirements, per unit gain in weight. A com­

bination of sodium acetate and sodium propionate is superior to

either salt alone, in reducing feed requirements.

In spite of the large amounts of available energy, there is

a less effective conversion of calories to gains in oody weight in

animals which received only c o m or cerelose than in animals which

received sodium acetate or sodium propionate.

In general, there appears to be a substitution by sodium acetate or sodiua propionate for com, urea, hay and cerelose when these organic salts are Included in the ration of feeder laobs. -38.

Distribution of Acetic. Propionic

end Butyric Acide in Lamb Tissues

Results from the analyses of acetic, propionic and butyric

acids are tabulated together and discussed apart from the other metabolic acids* This method of presentation has been chosen be­ cause (l) the sodiua salts of two of these acids were included in the maintenance rations and hence cosmand a special consideration of their distribution in the several tissues investigated here;

(2) earlier experiments in ruminant nutrition have definitely established the consistent presence of these three acids in the rumen fluid during controlled feeding; (3) these three acids may be absorbed directly from the rumen into the blood and subsequent­ ly carried to various tissues and organs of the body; (A) it has been shown experimentally that these three acids are involved in fat, carbohydrate and protein metabolism*

Data from the chromatographic analyses of tissue extracts from the several lots are tabulated in Tables 3, k, 5* 6, and 7.

Data fTom all animals investigated are presented in the tables*

Those animals receiving identical rations are grouped together and designated as Lot 1, the control ration; Lot 2, the acetate supple­ mented ration; Lot 3* the propionate supplemented ration; and Lot 4, the mixed acetate and propionate supplemented ration* £ach table contains data from tissues of a single type* -39-

TABLE 3

Distribution of Volatile Organic Acids in ths Livsr of Lambs

Butjric Propionic Acetic Treatment Animal acid aoid acid milliequivalents of acid per ten grams of lyophilised tissue 1 .075 .061 .317 2 .069 .058 •440 Lot 1 ^ .031 .041 .125 LO L 4 * .052 .023 .447 5* .043 .056 .324 6* .058 .039 .111 Average .061 ± .017 .046 * .016 .294 * . 1 4 4

7 .058 .082 .350 8 .026 .013 .384 .024 •044 .419 L o t 2 ^ o o 10 * .088 • .390 11* .080 — .109 12* .160 .029 .438 Average .072 ± .050 .039 *. 0 2 6 .348 *. 0 6 8

13 .071 .078 .294 14 .080 .148 .317 Lot 3 ^ .030 .073 .105 16* .150 - .336 17* .394 .188 .545 18* .080 - .096 Average .134**132 .121 *.056 .282 ± .166

19 .050 .055 .301 2 0 .079 .132 .210 Lot 4 21 .046 .041 .334 2 2 * .011 .013 •122 23 * .058 .140 .622 2 4 * .149 .058 .334 Average .065 *. 0 4 6 .073 * *051 .320 *. 1 6 8

Lot 1 - Control Lot 3 - Propionate treatment Lot 2 - Acetate treatment Lot 4 - Acetate and Propionate treatment * Thsss animals wars slaughtered two weeks after the others -40-

TABLE 4

Distribution of Volatile Organic Acids in ths Blood of Lambs

Treatment Animal Butyric Propionic Acatic acid acid acid milliequivalents of acid par tan grams of lyophilised tissua 1 .033 .045 .171 2 .163 .029 .051 LUvT X 1 3 .079 .035 .190 4* .034 .034 .215 5* .062 .059 .089 6* .041 .039 .062 Average .052 * .019 .060 * .024 ,129 * .071

7 — .027 8 .022 •040 .363 Lot* 2 9 •012 .062 .386 10* .028 .015 .227 11* .049 .035 .259 12* .030 .017 .187 Average .028 *.014 .034 * .019 .260 * .060

13 .017 .073 .393 14 .013 .085 .464 .022 — .480 1LOv nf J'l 15 16* .053 .034 .055 17* -- .147 18* - - .190 Average .026 * .018 .064 * .02? .286 *.172

19 .034 .022 .277 20 .071 .116 .124 Lot 4 21 .028 - .199 22* —— — 23* - .019 .264 24* .079 .106 .246 Average .053 *.026 .066 * .052 .222 *.062

Lot 1 - Control Lot 3 ~ Propionata traatmant Lot 2 - Acatata treatment Lot 4 - Acatata and Propionata traatmant * These anismls vara slaughtered two weeks after the others* -41-

TABLE 5

Distribution of Volatile Organic Acida in the Muscle of Luba

Butyric Propionic Aoatio Traataant Aniaal acid acid acid

ailliequivalente of acid par tan grua of ljophilisad tiaaua 1 .017 .015 .051 4 Lot 1 3 .098 .023 .038 4* .006 .018 .076 5* .009 .011 .055 6* .008 .006 .066 Avaraga .027 A .038 . 0 U * .006 .057 * .013

7 .017 •021 .078 8 .010 .036 •142 Lot 2 ^ .009 . o n .188 10* .026 •010 •036 11* .031 .019 .039 12* .028 •012 .030 Avaraga .020 * .008 .018 *.009 .085 * .051

13 14 .036 .016 .060 x * , 1 5 .109 .047 .177 Lot 3 16* .026 .035 .076 17* .005 • o n .066 18* .013 .018 .059 Avaraga .037 ±.042 .025 *.015 .087 ±.050

Lot 1 - Control Lot 3 - Propionata traataant Lot 2 - Acatata traataant Lot 4 - Acatata and Propion­ ata traataant

* Thaaa anInala wars slaugbtarad two waaks aftar tba othara. -42-

TABLE 6

Distribution of Volatile Organic Acida in tha Ruasn Julca of Laabe

Traataant Aniaal Butyric Propionic Acetic acid acid acid milliequivalents of acid par tan grams of lyophilissd tissue 1 3.30 2.79 12.66 2 3.38 2.09 11.11 Lot 1 3 3.54 3.09 15.08 4* 2.93 1.45 8.65 5* 2.40 1.07 7.35 6* 3.17 2.31 10.35 Avaraga 3.12 ± .401 2.13 ± .79 10.86± 2.54

7 2.58 3.71 16.3i 8 3.00 4.24 17.06 9 3.40 3.75 14.63 Lot 2 10* 2.51 3.02 13.93 11* 3.57 4.94 18.06 12* 1.58 0.69 4.78 Avaraga 2.77 * .73 3*38* 1.26 16.12*5.28

13 3.22 2.67 13.43 14 3.89 2.81 13.28 15 4.10 3.35 16.53 Lot 3 16* 2.81 2.44 13.95 17* 2.01 1.48 9.50 18* 3.07 2.82 11.18 Avaraga 3.18 * 1.03 2.59 * .62 12.97*1.83

19 3.20 3.48 16.28 20 3.40 4.25 18.45 21 3.38 4.16 18.13 Lot 4 22* 2.44 2.98 13.65 23* 3.39 3.16 15.31 24* 3.31 3.24 15.08 Avaraga 3.18 * .35 3.54 *.45 16.10* 1.90

Lot 1 - Control Lot 3 - Propionata traataant Lot 2 - Acatata traataant Lot 4 - Acatata and Propionata traataant * Thaaa animals wars slaugbtarad two weeks aftar tha others -43-

Table 3 shows results from liver extracts; Table 4 shows results from blood extracts; Table 5 shows results from muscle extracts; Table 6 contains results from rumen Juice; and Table

7 summarizes the results presented in Tables 3-6. Average levels of butyric, propionic and acetic acids are included for each lot with the standard deviation of these averages. Identical numbers for the designation of an animal in different tables re­ present data from the same animal. The plan of the experiment required slaughter of half the animals in each lot at two weeks time interval. Those animals which were slaughtered in the latter interval are designated by an asterisk.

All three acids were present in each lot considered, however, not all animals within the lots contained the three acids in sufficient quantities to allow detection in each tissue. On page

39, Table 3, Animals 11, 16, and 18 did not show the presence of propionic acid in the liver. No butyric or propionic acid was found in blood of animals 7, 17, and 16 ( Table 4, page 40 ). In the same table, animal 21 did not show the presence of butyric acid and no propionic acid was detected in animal 23* Repeated extract­ ions and chromatographic analyses did not alter these results. The absence of either of these volatile acids in detectable amounts under the oonditions of the experiment is interpreted as differences due to the individual animal rather than losses in the experimental procedure. Due to lack of sufficient sample, the test was not re­ peated for animal 18 with blood. All meat samples analyzed did -DA­

TABLE 7

The Effect of Ration* containing Sodium Acetate and Sodium Propionata on tha Diatribution of Volatile Organic Acida in tha Tiaauaa of Lamba

Tiaaua Butyric acid Propionic acid Acetic acid mi lli equivalent a of a d d par tan grama of tiaaua - dry matter RUMEN JUICE Lot 1 3.12 ±.A1 2.13 1 .79 10.8612.54 Lot 2 2.77 ♦ .73 3.38 11.26 16.12 ±5.28 Lot 3 3.1811.03 2.59 ± .62 12.97 ±1.83 Lot A 3.18 1.35 3.54 1 .45 16.10 2 1.90

LIVER - Lot 1 .061± .017 .046 2.016 .294 1.144 Lot 2 .072 ± .050 .039 2.026 .348 2.068 Lot 3 •13A ± .132 .121 t .056 .282 2.166 Lot A .065 i .GA6 .073 1 .051 .320 t .168

BLOOD Lot 1 .052 ± .019 .060 ± .024 .129 ±.171 Lot 2 .028 ± .014 .034 t .019 .260 ±.060 Lot 3 .026 2 .018 .064 1.027 .288 * .172 Lot A .053 1.026 .066 1.052 •222 ± .062

MUSCLE Lot 1 .027 ± .038 .014 t .006 .0571.013 Lot 2 .020 ± .008 .018 ± .009 .850 ± .051 Lot 3 .037 1.042 .025 1 .015 .087 ± .050

Lot 1 - Control Lot 2 - Acetate treatment Lot 3 - Propionata traatmant Lot A - Acatata and propionata traatmant -45-

Figure 1

The Influence of Rations Containing Sodium Acetate and Sodium Prooionate on the Distribution of Volatile Organic Acids in the Tissues of Lambs

1 - Control 3 - Propionate 2 -r Acetate 4 - Acetate plus propionate

CO CO RUMEN JUICE

4 r~i

.3 - LIVER

no

BLOOD

MUSCLE

BUTYRIC PROPIONIC ACETIC ACID ACID ACID -46- did contain detectable quantities of all three acids, it may be seen in Table 6 that samples from animals 2 and 13 were not analyzed. This was due to uncontrollable losses in lyophilization chamber. All animals showed each of the three acids in their rumen juice, Table 6.

Table 7 summarizes the data from Tables 3-6, page 44* The data presented in Table 7 are graphed in Figure lf page 45. Eleden

( 12 ) pointed out originally that acetic, propionic and butyric acids could be detected consistently in the rumen fluid of sheep.

This is confirmed by the graphs in Figure 1* He also reported that acetic acid was present in the greatest amounts, under his conditions.

This too was confirmed in this investigation, however, the extent to which acetic acid was present in greater quantities than butyric and propionic acids varied for each lot and for each tissue. Ranges based on all lots studied in each tissue shown in Table 7 indicate that acetic acid represents 67-70 % ( mole percent ) of the total volatile acids in the rumen juice, 50-88 % ( mole percent ) of the total volatile acida in the liver, 45-87 % ( mole percent ) of the total volatile acids in blood and 45-71 % ( mole percent ) of the total volatile acids in muscle.

Rations containing sodium acetate produced animals with a higher content of acetic acid in rumen juice, liver, blood and muscle than did the control ration, as shown in Figure 1. Rations contain­ ing sodium propionate produced animals with more acetate in rumen juice, blood and liver than did the control ration, however, the level -47-

of acetic acid found in the liver of control animals was unaltered

in animals which received the sodium propionate ration. The sodium

propionate ration was less effective in increasing the acetic acid

level in rumen juice than the sodium acetate ration. In blood and

muscle however, there was no difference in acetic acid level pro­

duced with either supplemented ration.

Figure 1 shows no appreciable difference in rations of

butyric and propionic acids in either of the four tissues, however,

the rations did produce animals with different absolute concent­

rations of these acids. The ration containing sodium propionate

gave significantly higher butyric and propionic acid levels in liver

and muscle, than did the control ration. The ration containing

sodium acetate gave a slightly higher propionic acid level in rumen

juice and a somewhat lower butyric acid level in the muscle than did

the control ration. These results emphasize the role of acetic acid

in volatile acid metabolism and point to it as a key acid in the

rumen.

Cienerally, the presence of both sodium acetate and sodium

propionate in the ration seemed to produce animals with volatile acid

levels based on the combined effects of the two supplements. Two

notable exceptions are found in the blood and liver. The presence

of sodium propionate and acetate showed a level of butyric acid in

liver more typical of the sodium acetate alone. In blood, a decrease was observed in acetic acid level below that produced with rations

containing either acetate or propionate and yet greater than the level - r e ­

produced with the control ration. These results suggest that acetic acid has a more dominating effect on the level of butyric acid in

liver than does propionic acid when both are present in the ration,

it may be further suggested that sodium acetate anu sodium propion­ ate, when present simultaneously in the ration, stimulate a compet­

ing reaction which decreases the level of acetic acid in blood. The magnitude of this decrease does not give an acetic acid level as low as that exhibited with the control ration.

Other trends observed in combined effects of supplements can­

not be used as criteria for making definite conclusions. Large

variations within the lots, indicated by standard deviations in Table

7, make their significance questionable. While some of the variations

may be attributed to a collective analysis of data from animals

slaughtered at two different times, it is more likely that length of

feeding, ration and age of animals are variables of equal magnitude.

Apparent differences in data within lots at the two week intervals

may be regarded as insignificant.

The levels of all three volatile acids decreased from rumen

juice, to liver, to blood and were lowest in muscle. For purposes

of greater clarity this distribution is schematically diagramed in

Figure 2, on page 49# Both sodium acetate and sodium propionate pro­

duced animals whose acetic, propionic and butyric acid levels were

different from those in the control animals. Sodium acetate lowered

the level of propionic acid in blood and lowered the levels of

butyric acid in both blood and muscle. This suggests a possible -49-

Figure 2 ochematic Distribution of Volatile Acids Among Tissues of Lambs

Acetate - bupolemunt for Lot 2

Pronionate - bupplement for Lot 3

ACETATE PROPIONATE tiff ACETIC A C E T IC +++ PROPION PROPION +++ - I C - I C 0 0 B U TYR IC BUTYRIC 0

A C ETA TEl PROPION A T ACETIC o ACETIC ♦+ + A ACETIC PROPION PROPION BLOOD )PROPION -IC ++ -IC o - IC BUTYRIC ♦♦+ BUTYRIC - ^ BUTYRIC

I aCC'TATE I *------IPROPIONATEl + ACETIC / \ ACETIC + 0 PROPION I MUSCLE I PROPION + -ic V J -'c - BUTYRIC \ y BUTYRIC +

Concentration: + greater than control o same as control - less than control -50-

utilization of t h e s e acids, or a decreased absorption in the presence of sodium acetate a d d e d to the ration. lJropionic acid was higher in rumen juice when s o d i u m acetate was included in the ration. This may be explained i n terms of lowered absorption of propionic acid

from the rumen due t o pH changes, or absorption selectivity induced b y the acetate ion.

flhen sodium propionate was included in the ration, the level

of acetic acid was increased in the rumen juice. Again, pH changes and selective absorption may account for lowered absorption from

the rumen or acetic acid may be increased by a stimulated rumen microflora. Both a c e t i c acid and butyric acid are increased in

muscle, butyric a n d propionic acids are increased in liver, acetic acid is increased i n blood, a n d butyric acid is decreased in blood when sodium propionaite is included in the ration.

From these observations it is suggested that both sodium acetate and sodium propionate, when incorporated in a nutritionally

complete ration, a r e capable of altering metabolic pathways. As a

result of such alterations, if they do exist, the quantities of the

volatile organic a c i d s in certain tissues are varied, iihere the

levels of acetic, propionic a n d butyric acids are decreased

significantly below those found in the control tissues the meta­

bolic alteration m a y be increased utilization or absorption of the

acid in question. I n instances where volatile acid levels are higher

than those found i n control tissues, the metabolic alteration may be

stimulated production or decreased absorption. The possibility -51 exists that the formation of other metabolic products in various tissues is affected by each of the supplements used, in a manner specific for sodium acetate and sodium propionate, Differences ob­ served in volatile acid levels of meat samples may in part account for flavor differences as indicated by taste tests. Results from taste tests referred to here may be seen on pages 76 and 77* -52-

Bistribution of Other metabolic ncids in Lamb Tissues acids from chromatographic column peaks designated as Groups 1.11.Ill

According to the Bolen procedure, the initial chromatographic analysis of a given extract with the n-butanol-chloroform solvent system does not provide a complete separation of all the metabolic acids which may be present, beveral of these acids are eluted from the initial ( survey j column in one elution peak and the acids in each such peakj have been designated as Group 1, 11 or ill, depend­ ing on their order of elution from the survey column. iroup 1 in­ cludes glutaric, formic ana furaaric acids; Iroup it includes lactic, succinic and -ketoglutaric acids; ana those acids in

"roup ill are glycolic, oxalic and cis-aconitic acids.

Bata resulting from the initial chromatographic analysis of each extract for acids in Group i, il ana iii are presented in

Tables 8, 9 and 10. ^ach Table is restricted to results from extracts of a single type of tiaaue. Table d contains data from blood extracts; Table 9 contains data from meat extracts and Table

10 contains data from liver extracts. ;iuraen juice did not contain sufficient quantities of any of the acids considered here to permit detection in the size of samples analyzed by this method.

^ach extract is designated in Tables 8, 9 ana lu by a number indicating tha animal from which the original sample was taken, nil animals which received similar treatment are listed together ana collectively represented as Lot 1, for the control animals; Lot 2 -53-

TAHLE 6

Distribution of Metabolic Aoids in ths Blood of T.anba

Olutaric Lactic Oljcolie Treatment Animal Formic Suoeinie Oxalic Fumarie Alpha-keto- cie-Aoonltic acids glutaric aoids acids

^ilUequivalents of aoid psr ton grama of lyophllisod tissuo

1 .186 •115 .241 2 .030 .170 .055 Lot 1 3 .121 .381 .183 4* .244 .511 .094 5* .176 .288 .263 6* .227 •167 .157 Average ,164*.079 .272±.154 .165 ±.081

7 .261 .777 .358 3 •096 .694 .244 9 .166 •611 .218 Lot 2 10* .056 .112 .098 11* -— - 12* .090 .135 .077 Average . 1 34* .082 •466 ±.284 •199 *.115

13 .295 .195 .111 14 .147 .655 .101 Lot 3 15 .005 .298 .062 16* .300 .520 .266 17* .167 .556 .191 id* .170 .363 .308 Average .181 ±.109 .431 *.175 .173 ±.098

19 .622 .225 .243 20 .074 .348 •283 XL •085 .112 .177 Lot 4 22* --- 23* .273 • 502 •088 24* .757 .370 .137 Average .363 *. 3 1 2 .311 *.148 ,186 ±.078

Lot 1 - Control Lot 3 - Propionsts treatment Lot 2 - Acetate treatment Lot 4 - Acetate snd propionate treatment * Thsso animals were slaughtered two weeks sftsr tho othors -54-

TABLE 9

Distribution of Metabolic Aoids in the Muscle of Lsmbs

Olutaric Lactic Glycolic Treatment Animal Formic Succinic Oxalic Fumarie

7 .106 .201 .049 8 .216 .347 .050 Lot 2 9 .144 .465 .061 10* .076 .027 .113 11* .074 .024 .038 12* .071 .039 .123 Average •114 *.057 .183 ±.059 .072 ±.036

13 — _— 14 .055 .050 .019 15 .131 .142 .018 Lot, 3 16* .060 .181 .162 17* .023 .081 .058 18* •024 .244 .104 Average .058 ±.044 .139 ±.078 .072 ±.061

Lot 1 - Control Lot 3 - Propionsts treat Lot 2 - Acststs treatment ment Lot 4 - Not included in these teste

* These animals were slaughtered two weeks after the others• -55-

TABLE 10

Distribution of Uetabolic Acids in the liver of Lambs

Crlutaric Lactic G l y c o l i c Treatment Animal Formic Succinic O x a l i c Fusmric el -keto ci s-Acor* ±.t,ic acids glutaric acids a c i d s mllliequivalents of acid in ten grams o f lyophilised tissue

1 .237 .561 . 2 9 3 2 .352 .703 . 0 8 5 Lot 1 3 .182 .194 . 1 5 0 4* .110 .592 . 4 5 6 5* .074 .799 .262* 6* .111 .222 . 4 0 7 Average .178 ± .094 .511 ± .251 . 2 7 5 ^ 1 4 3 7 .419 1.170 • 44 3 6 .568 .640 . 4 0 7 9 .420 .485 . 1 9 4 Lot 2 10# .245 1.010 . 2 5 9 11# .167 .798 . 4 8 3 12# .117 .758 . 4 0 0 Average .322 ±.175 .810 ±.239 . 36 4 ^±.112

13 .334 .810 . 1 3 3 14 .518 .624 . 2 0 0 Lot 3 15 .320 .563 . 1 8 9 16# .353 .842 . 2 5 3 17# .363 .466 . 2 0 4 18# .201 .482 . 3 0 0 Average .356±.107 .631 ±.151 . 2 X 3 ^.057

19 .353 .608 . 2 2 1 20 .217 .586 . 1 2 7 Lot 4 21 .469 .845 . 3 5 2 22# .136 .235 . 2 0 6 23# .493 .731 • 4 4 6 24# .188 .626 . 2 7 4 Average .310 ±.151 .605 ±.205 . 2 7 0 ±\ 1 1 4

Lot 1 - Control Lot 3 - Propionate treatment Lot 2 - Ace tats treatment Lot 4 - Acetate and proplonm.'t,* treatment # These animals were slaughtered two weeks after the o t h e r s . -56- for the aodium acetate supplemented animals; Lot 3* for the sodium propionate supplemented animals; Lot U for the sodium acetate and propionate supplemented animals. Animals of a given lot which were slaughtered at the same time are listed together and asterisks are placed beside the numbers of those animals which were slaughtered two weeks after the others. Milliequivalents of acids designated as Groups I, II and III per ten grams of lyophiliaed tissue are re­ ported separately for each extract. The average milliequivalents of acid and standard deviations of these averages are recorded for each lot of animals. The same number is used to indicate samples from a given animal in all tissues; for instance, milliequivalents of acid in the blood of animal # 1 and in the liver of animal § 1 are concentrations of acid in the blood and liver extracts from the same animal.

Extracts analysed from each animal showed elution peaks for the Group I, II and III acids in all blood, liver and meat samples.

The extracts from the blood of animals 11 and 22 as shown in Table, and from the swat of animals 2 and 13 as shown in Table 9* were not chromatographed, due to insufficient amounts of sample.

The average milliequivalents of Group II acids ( lactic, atxcclnie and e\ -ketoglutaric acids ) were, in general, higher than the average milliequivalents found in the Group I or Group III eluates. This was evident in liver and meat extracts of all lots and in the blood of Lots 1, 2 and 3. Greater variation in the milliequivalents of acid found within lots was also apparent for the -57-

Group II acids, ( lactic, succinic, and eA-ketoglutaric ) than for those in Groups I and III. The average milliequivalents and variation from this average by animals within the lot were found to be approximately equal for Groups I and III in a given lot. For example, in muscle of Lot 3» there was an average of 0.058 milli­ equivalents of Group I with a standard deviation of £ .OLA; there was an average of 0.072 milliequivalents of Group III with a standard deviation of £ .061. Other examples may be cited to substantiate this, especially blood and muscle of Lots 1, and 3# blood and liver of Lot 2, and liver of Lot A*

Deviations in the number of milliequivalents of Group 1, II and III acids within lots were such as to make deviations between lots insignificant. Nevertheless, it was felt that a consideration of the levels of individual acids in these chroaiatogrephic groups may be significantly different. Hence, eluates containing Group 1 acids were re-chromatographed according to the procedure previously outlined* Differences in intervals between feeding time as well as differences in the activity of individual animals may account for the levels of the Group II acids found in the tissues. If this were true, it would be partially reflected in the lactic acid con­ tent of these extracts. Eluates of the Group II acids were also re-chromatographed according to the procedure previously outlined with particular interest in the lactic acid content in this group.

Chromatographic Group III consists of glycolic, oxalic, and cis- aconitic acids. It is believed, however, that the group does not -58- in elude any quantitlea of cia-aconitic acid under theae conditions, as employed in the sample extraction; this acid would have been converted to the trans-isomer which is separated completely on the survey column.

The data in Tables 8-10 do suggest the possibility that differences in content of one or more of the acids in Group XI deserve closer consideration. Mo effects are immediately evident from the presence of sodium acetate or sodium propionate in the rations of feeder lambs. -59-

Indivldual acid a a a para tad completely on the survey column.

I^ruvic acid and three acids of the tricarboxylic acid cycle were completely separated on the initial (survey) column. The three acids of the tricarboxylic acid cycle are trans-aconitic, malic and citric acids. Data from these analyses are presented in Tables 11,

12 and 13 in a manner consistent with that described on page 52 for

Chromatographic Groups I, II and III. £ach table contains data from extracts of a single type of tissue; animals receiving a similar ration are listed together and designated as Lots 1, 2, 3 and 4; ani­ mals slaughtered two weeks after the others are indicated by an aster­ isk; levels of each acid in each extract are reported as milliequival­ ents of acid per ten grams of lyophilised tissue; average levels of each acid and standard deviation of each average are included for all lots considered. Table 11 contains acid levels found in blood; Table

12 contains acid levels found in meat and Table 13 contains acid levels found in liver.

All extracts analysed contained detectable amounts of pyruvic, trans-aconitic, malic and citric acids in the blood extracts, Table 11; meat extracts, Table 12; and in liver extracts, Table 13* with three exceptions. Animals 9, 15# and 21 in Table 11 did not contain quanti­ ties of pyruvic acid in their blood sufficiently high for detection by this method. No significance is attached to the absence of detectable

pyruvic acid levels in the blood of animals 9# 15 and 21 since all

other animals In Lots 2, 3 and 4 did show measurable amounts of pyruvic • 6 0 -

TABLE LI Distribution of Metabolic Aoids in ths Blood of Laabs

Malic Treatment Animal Pyruvic Trans- Citric acid aconitic acid acid acid milliequivalents of acid psr tsn grams of lyophilissd tissue 1 .0 2 7 .286 .1 8 0 .2 6 2 2 .02 0 .113 .298 .307 Lot 1 3 .103 .163 .153 .18 2 4* .026 .075 .083 .193 .06 2 .13 2 .1 1 2 .1 4 1 6* .14 1 .095 .0 9 9 .099 Avsrags .063 * . 0 4 9 . 1 4 4 * . 0 7 6 .154 ±.079 .197*076

7 .057 .3 9 4 . 24 4 .210 8 .06 9 .078 .1 1 4 .0 4 0 L o t 2 9 - .05 6 •22 2 .111 1 0 * .031 . 0 4 2 .136 .079 1 1 * -- - 1 2 * .035 . 0 3 2 .10 7 .193 Average .048 ± . 0 1 8 . 1 2 0 ± . 1 5 5 . 1 6 4 *0 6 4 . 1 2 6 ± . 0 7 3 1 3 .0 2 4 .18 4 .3 0 7 .532 14 .0 3 4 .105 .16 8 . 2 7 4 - L o t 1 1 5 .111 .273 •162 16* .037 .0 2 8 .099 .30 7 1 7 * •0 2 6 .0 3 7 .13 8 .10 9 1 8 * .030 .0 3 7 . 0 8 4 . 1 9 8 Avsrags .030 ± . 0 0 5 •0 8 3 * . 0 6 1 . 1 7 8 *0 9 0 .263*151

19 .021 .183 .75 4 .319 20 .040 .186 .422 .42 8 2 1 - .19 0 .5 9 6 .301 L o t 4 2 2 * . • - • - 2 3 * .on .00 8 .2 8 8 .11 6 2 4 * • 1 4 0 .013 .15 8 .095 Avsrags .053 ± . 0 5 9 . 1 1 6 ± . 0 9 6 . 4 4 3 4 2 3 8 • 2 5 1 ^ 1 4 4

Lot 1 - Control Lot 3 - Propionat* treatment Lot 2 - Aostats treatment Lot 4 - Aoststs and propionate treatment * Thsss animals wars slaughtered two weeks aftsr ths othsrs. -61

TABUS 12 Distribution of Metabolic Acids in ths MuseIs of Lambs

Pyruvic Transaeonitic Malic Citric Treatswrrt Animal acid acid acid acid milliequivalents of acid per ten grams of lyo- philised tissue 1 .009 .016 .016 .028 2 L o t 1 3 . 01 2 .028 . 0 2 0 .035 4 * .013 .019 .018 .019 5* .019 .056 .043 .021 6 * .035 . 02 8 . 0 4 9 .025 Average .017 * . 0 1 0 . 0 2 9 ± . 0 0 5 . 0 2 9 ± . 0 1 6 • 0 2 1 ±.006

7 •012 .05 2 .018 .021 8 .032 .07 5 .008 .011 L o t 2 9 .047 .025 .02 0 .0 2 8 1 0 * .017 .027 .027 •0 4 0 1 1 * .022 .009 .023 .033 1 2 * .041 .0 3 2 .015 .019 Average .028 ± . 0 1 4 .036 ± . 0 2 3 .01 8 ± . 0 0 6 .025 ±0 1 0

13 — — — 14 .018 . 0 2 2 .02 0 . 014 L o t 3 15 .056 .028 . 0 0 8 . 012 1 6 * . 0 0 7 . 0 6 0 .03 6 .04 4 1 7 * .011 .018 . 0 1 0 .047 1 8 * . 0 1 0 .037 •036 .09 9 Average .02 0 ± . 0 2 0 .033 ±.017 •022 ± .014 .043 ±0 3 4

Lot 1 - Control Lot 3 - P r o p ! c o s t s trsatnsnt Lot 2 - Aoststs treatment Lot 4 - Not included in these tests* * These animals were slaughtered two weeks after the others. -62-

TABLE 13

Distribution of Metabolic Acids in ths livsr of Lambs

Treatasnt Animal Pyruvic Trane- Malic Citric Mid aoonitic acid acid acid milliequivalents of acid per ten grans of lyophllised tissue

1 .034 .11 7 .12 7 .1 4 5 2 .045 .065 - 2 4 6 .15 9 L o t 1 3 .037 .045 . 0 6 1 .062 4 * .125 .1 1 4 .1 5 2 .11 4 5* .036 .063 .0 9 6 . 0 7 6 6 * .042 .0 3 7 .130 .137 Average .053 * . 0 3 5 . 0 7 4 ± . 0 3 4 . 1 3 5 ^ 0 6 3 .115 * . 0 3 9 7 .049 .128 .175 .098 8 .058 .123 .07 1 .075 9 .061 . 1 2 0 .114 .105 Lot 2 1 0 * .083 .175 .140 .11 6 1 1 * .015 .116 .057 .055 12* .117 . 0 4 8 .095 .018 Average .063 * . 0 3 4 •118 * . 0 2 9 .108 ±0 4 4 . 0 7 8 ±0 3 7 13 .062 . 0 8 1 .070 .036 1 4 .020 .177 .051 .113 1 5 .047 .045 .079 .1 6 7 Lot 3 1 6 * •060 .19 4 .14 0 .192 1 7 * .128 .143 .133 .116 1 8 * .083 . 1 4 4 .068 .084 Average .066 * . 0 3 7 . 1 3 0 * . 0 5 8 . 0 9 0 * 1 0 3 7 .1 2 1 * . 06 0 1 9 .063 . 0 8 4 .09 6 .1 3 9 2 0 .093 . 1 5 0 .050 .0 5 9 Lot 4 2 1 .158 .352 .071 . 1 7 4 2 2 * .030 . 0 5 2 .193 .096 2 3 * .097 . 11 1 .140 .126 2 4 * .131 .055 .0 8 6 .137 Average .098 *. 0 4 5 . 1 3 4 * . 1 1 0 .10 6 *. 052 . 1 2 2 * 0 3 9

Lot 1 - Control Lot 3 - Propionats trsataant Lot 2 - Aostats trsataant lot 4 - Acs tats and Propionate trsataant * These aniaals ears slaughtered two weeks after the others. -63- acid. Blood extracts of animals 11 and 22 In Table 12, and s e a t ex­ tracts of animals 2 and 13 were not analysed due to insufficient sample.

Deviations from the average acid levels by animals within the lots were apparently unrelated to the presence of sodium acetate or sodium oropionate in the ration. The respective averages of millieq­ uivalents of acids are included in Table 14 and observed effects are discussed in the consideration of that table. -64- Pyruvic and lactic aoids and six acids of the tricarboxylic acid cycle.

Table 14 is * summary of Tablas 8-13* The avsrags milli­ equivalents of eight acids found in liver, blood, and muscle of each of the 4 lots are presented* Averages of pyruvic, trans-aconitic, malic and citric acids were taken directly from Tables 11, 12 and 13, however, lactic, fumarie, succinic and alphaketoglutarlc acid values result from the chromatographic separation of acids in Group I, and

Group II, Tables 8- 10* The results in Table 14 are graphically re­

presented in Figure 3* Individual acids in Group III were not separated because (1) after the acidic extraction of the tissues,

aconitlc acid would be completely separated as the trans isomer on

the survey column; (2) it may be presumed that Group III values re­

present levels of glycolic or oxalic acid*

The chromatograpliic analyses of Group I eluates showed peaks

characteristic of glutaric, formic and fumarie acids in all lots of

animals. Average fumarie acid levels are presented in Table 14 be­

cause of its role in the tri-carboxylie acid scheme. Henoe, differ­

ences between levels of Group II acids in Tables 8, 9 and 10 and

fumarie acid in Table 14, represent formic and glutaric acid. Formic

acid concentrations were so m u l II, however, that characteristic peaks

were detectable only in pooled eluates of all animals slaughtered at

the same time and maintained on a similar ration.

The sumsution of lactic, succinic and ^-ketoglutarlo acid con­

centrations resulting from the second set of chromatographic separations

constitute a 90-97Jf recovery of the Group I acids detected on the survey -65- column. The fact that such a recovery falls short of 1 0 0 % may in part be explained by the reversal of peaks in the n-butanol-benzene system.

Trans-aconitic acid, if present, is eluted from the second column as a sharp peak Just before -ketoglutaric acid emerges. The titrations do not decrease completely to the base line before elution of ^ -keto­ glutaric acid begins. Under the conditions of the experiment, some trans-aconitic acid was present, hence concentrations of -keto­ glutaric acid reported in Table 14 may be admittedly low.

The concentrations of pyruvic, lactic and succinic acids were approximately the same in muscle, blood and liver. Fumarie, trans- aconitic, malic and citric acids were present in nearly equal concent­ rations in blood and liverj but were somewhat lower in the muscle.

Alpha ketoglutaric acid content increased from muscle to blood to liver for each lot tested and represented the highest concentration of ths individual acids considered. Equal molar amounts of malic, citric and trans-aconitic acids seem to be present in blood and liver, how­ ever, the absolute quantity is exceeded in the liver by 4 -ketoglutaric acid.

From the data in Table 14, it appears that the presence of sodium acetate in the ration, separately or combined with sodium pro­ pionate, produced animals with less lactic acid in the liver than did the control ration. Animals whose rations contained sodium acetate or sodium propionate had a higher average content of lactic acid in blood and muscle than did the control animals. This effect was some­ what more pronounced for the acetate ration than for the propionate TABLE 14

Distribution of Metabolic Acids in ths Tissues of feeder L s n b s

Alpha- irans- Puaric Succinic Pyruvic Lactic keto­ Aconitic Malic Citric TLssoe acid acid acid acid glutaric acid acid acid acid

Lot average in ailliequivalents of acid per ten graas of lyophilised tissue per aniasl LIVER

Lot 1 *053 .134 .013 *058 .296 .074 .135 .115 Lot 2 .063 .084 .022 .067 .593 .118 .108 .078 Lot 3 .066 .132 .031 .015 .444 •130 .090 .121 Lot 4 .098 .051 .016 .021 .470 .134 .106 .122

BLOOD Lot 1 •063 .194 .063 .028 .038 .144 .154 .197 L o t 2 .048 .304 .011 .067 .096 .120 .120 •126 L o t 3 .030 .241 .017 •084 .105 .063 .178 .263 L o t 4 .053 .215 .032 .046 .054 .116 .443 .251

MUSCLE Lot 1 .017 .134 .006 •008 .015 .029 .029 .021 L o t 2 .028 •145 .008 .019 .069 .036 .018 .025 L o t 3 .020 .200 .021 .033 .012 .033 .022 .043

Lot 1 - Control Lot 3 * Propionate treataent Lot 2 - Acetate treataent Lot 4 - Acetate and propionate treataent -67- Figure 3

The Influence of Rations Containing Sodium Acetate and Sodium Prooionate on the distribution of Other Metabolic Acids in the Tissues of Lembs MM_L(EQUIVALENTS OF AC fD PER lOg. OF DRY TISSUE

*- K> - U. L> U» J____ L-- L j______i______i 1 3 M PYRUVIC > w *

f\> LACTIC ifc»

I j— I f M 3 FUMARIC 3 ? 6 » SL □ i

SUCCINIC JOI cz t3 <* t3 o o =>U) 8 =3

3 3 r TRANS- fc 3 ACONITIC g

3 3 MALIC 3 W

3 3 CITRIC 3

1 - Control 3 - Propionate

2 - Acetate 4 - Acetate d Iu s nropionate -68- ration in blood, whereas in muscle, the effect was in the reverse

order. It is likely that differences in blood and muscle lactic acid

for the several groups are merely reflections of differences among

individual animals. On the other hand, the differences in the lactic acid content of liver are regarded as significant and suggest that acetate may be instrumental in the utilisation of lactic acid or lactic acid precursors. This would account for decreased accumulation

of lactic acid in the liver*

Both organic salt rations produced animals whose liver concent­

rations of -ketoglutaric acid were somewhat higher than those on the

control ration. Acetate ration animals showed 0.593 milliequivalents

per ten grams of dried tissue; propionate animals showed 0 . 4 4 4 mi111-

aquivalants; animals receiving both salts in the ration showed 0.470 milliequivalents per ten grama of tissue as compared with 0 . 2 9 6 milli- equivalents per ten grama of tissue with the control animals. From an evaluation of the data, there is a possibility that trans-aconitic, malic, and citric acid concentrations are typical of a normally functioning metabolic cycle, thus preventing accumulations of these acids.

Several parallelissw were observed between data on metabolic acids and data on volatile acids. In Lot 2, it was pointed out that a decrease in propionic acid content of blood below the content ob­

served with the control lot seems apparent. Succinic and -keto­ glutaric acids were found to be increased in blood samples of Lot 2.

Similarly, the elevated butyric acid content of liver shewn by Lot 2 -69- ia accompanied by a low lactic acid content. The butyric acid decrease in blood observed with animals in Lot 3 was paralleled by an increase in succinic and -ketoglutaric acids in blood for the same Lot of animals. These parallelisms do suggest an interrelationship between the volatile acids propionic and butyric acids; and succinic and keto­ glutaric acids in the blood. Fatty acid oxidation involved the de­ struction of butyric acid, one of the intermediates of fat metabolism.

An increase in oxygen consumption and carbon dioxide production was observed when surviving liver sections destroyed butyric acid ( 15 )•

This designated the liver as a site for fatty acid oxidation, manifested in oxygen consumption and carbon dioxide production. It may be expected that products from this site of formation would find their way into the blood. -70-

Moisture. Fat. p H and Shrinkage Analyses of Ls»b Shouldera

The data from pH, fat, moisture and shrinkage analyses of the lamb shoulders are presented in Table 15* Meat used for these analyses was taken from the bulk samples obtained for the taste panels and prepared according to the procedure outlined on page 21. Average percentage values from duplicate analyses are tabulated for samples of meat from each of the animals studied.

All animals which were maintained on a given ration are grouped together in Table 15 and designated as Lots 1, 2 and 3, respectively.

Those animals in a given lot which were slaughtered two weeks after the others are indicated by an asterisk. Lot 1 consists of animals that did not receive either supplement, the control group. Lot 2 con­ tains animals that received the sodium acetate supplement; and Lot 3 contains animals that received the sodium propionate supplement.

Exact amounts of each supplement are found on page 18*

Although another variation in ration was used to maintain a fourth lot of lambs, samples from these animals were not included in these analyses not in the taste tests. The original experimental plan waa to determine if any relationship existed between pH, fat, moisture, shrinkage and flavor differences in meat, if flavor differences wese found by the taste panel. Animals that received both sodium acetate and sodium propionate were not expected to give flavor or analytical differences other than those based on the combined effects of the two salts. TABLE 15

Cumulative Analjaaa of Ground Lamb Shoulder* Demonstrating the Effect* of Sodium Aeatat* and Sodium Propionate

Pareant Pareant Pareant ahrinkaga Traataant Animal PH moiatura fat during roasting

1 6.18 56.08 33.39 44.00 2 5.89 50.32 40.67 44.50 Lot 1 3 5.76 46.41 45.01 51.70 4* 5.98 49.99 37-91 46.10 5* 6.05 42.05 38.08 44.50 6* 6.10 41.84 36.30 49.50 Average 47.81*4.64 38.56 *3.43 46.70 * 3.52

7 6.37 44.12 43.05 46. 70 6 6.15 45.72 34.62 45.80 Lot 2 9 5.90 51.62 37.62 45.80 10* 6.55 44.92 33.01 42.50 11* 6.18 35.53 37.56 48.10 12* 6.17 45.56 34.20 45.50 Avaraga 44.56*4.73 40.04 *5.75 45.70*1.66

13 6.05 44.56 48*92 51.30 14 5.89 44.11 49.72 49.70 Lot 3 15 6.02 42.95 40.64 48.70 16* 6.39 37.47 46.24 45.10 17* 6.13 45.38 38.01 44.70 IS* 6.12 42.94 37.03 50.00 Avaraga 42.90*2.48 42.09 * 5.06 48.35*2.13

Lot 1 - Control Lot 3 - Propionate treatment Lot 2 - Aeotato treatment Lot 4 “ Mot lneludod in thoao teata.

* Ihoaa animala war* slaughtered two week* aftor tba othara. -72-

Differences are apparent in Table 15 between averages of moisture, fat and shrinkage values for the three lots of animals*

The standard deviations recorded for each fat, moisture and shrink­ age average indicate that variations within individuals in the lots are such as to make real differences due to the presence of sodium acetate or propionate questionable* The ranges of moisture and fat percentages obtained in these analyses agree with the values tabulat­ ed by Jacobs ( 31 ) as 51*3 ^ end 34*3 £» respectively* It is to be expected that these averages are subject to variation in individual lambs. The data imply a relationship between shrinkage while roasting and fatjmoisture contents/ however, the exact relationship is not clear from the data.

The pH values were found to be higher in the meat from animals which had received sodit» aoetate or sodium propionate than from animals in Lot 1* This observation may be interpreted as the pre­ sence of less free acid in meat due to an altered metabolism in re­ sponse to the presence of sodium acetate or sodium propionate in the ration* This effect nay be due in part to the basic ration employed in the maintenance of animals supplemented with sodium acetate or prop­ ionate .

Perhaps it is significant that the ration which contained sodium propionate produced animals with a higher percentage of edible carcass than did the control or sodium acetate supplemented rations.

Data presented in Table 16 and compiled in part by the personnel of the Heats Laboratory of The Ohio State University, Columbus, Ohio, TABLE 16

Dressing Percentages and Cut Out Values of Feeder Lambs

Ar.iaal Entrance Killed Hot carcass Cold carcass Carcass Treatment number weight weight weight weight % lbs lbs lbs lbs

1 86 84 48 44.8 57.1 Lot 1 2 83 81 45 43.8 55.5 3 87 85 51 49.7 60.0 Average 57.6

7 88 82 50 47.6 60.9 Lot 2 8 98 96 56 53.8 58.3 9 81 79 43 40.9 54.4 Average 57.9

13 103 103 59 57.4 57.3 Lot 3 14 79 77 48 46.0 62.3 15 93 91 54 52.4 59.3 Average 59.7

19 83 78 46 43-7 58.9 Lot 4 20 87 85 51 49.2 60.0 21 101 104 57 55.6 54.8 Average 57.9

Lot 1 - Control Lot 3 K Propionate treatment Lot 2 - Acetate treatment Lot 4 - Acetate and propionate treatment -74- support this observation. On the basis of killed weight and weight

of the hot earcass for three animals in each of the four lots of animals it can be seen that Lot 3 had the highest average percentage

of edible carcass. There was little or no difference in the percent­

age of edible carcass for Lots 1, 2 and 4* It is also interesting to

note from the data in Table 16 that the animals which received the

acetate supplement showed the highest amount of average shrinkage

during dressing while the animals which received the propionate supple­

ment showed the least amount of average shrinkage during dressing.

Results obtained in pH, fat, moisture -and roast shrinkage

analyses indicate that while sodium acetate and sodium propionate in

the ration may alter values somewhat, no prediction as to the extent

of this alteration is practical because of the biological variable.

Sodium acetate apparently promotes the presence of substances which are

lost in the dressing process causing a higher percentage in dressing

shrinkage than found in the absence of aodiua acetate. Sodium pro­

pionate on the other hand apparently causes a higher percentage of

edible carcass than found in animals without access to sodium pro­

pionate • -75-

&«v o r gtudjsi Using Tggt* Pgfljlft

Differences in the flavor of l u b due to organic aalt supple- dents in the ration were sufficient to distinquish these uuaples from those of the control animals in each of the two panels conducted, al­ though differences wave not detected by all members of the panels.

Due to lack of sufficient seat the procedure for the panels as pre­ sented on pages 28 and 29 includes a bias. The odd sample in every trial was frosi an animal Maintained on the control ration. Neverthe­ less, repetition of taste tests, triplicate cosqparison of flavor in each group of animals with flavor in the control group, as well as the use of experienced and inexperienced panel members, serve to minimise the significance of this bias.

The results from the two taste panels are recorded in Tables

17 and 18. Twelve persons participated in each of the panels, how­ ever, only six of the participants in each panel were regarded as experienced on the basis of previous participation in flavor tests with meat or dairy products. Results from all panel members thus designated as experienced are presented together in one table. Table

17 contains data from the experienced panel members used in both flavor tests. Those experienced panel members which participated in the second flavor test, using meat from animals slaughtered after 123 days, are further distinquished by an asterisk. In Table 17 tabulations of successful identification of meat samples from the same animal are included for each trial or comparison. These successful identifications from comparison of sodium acetate supplemented animals and control -76-

TABLE 17

Results of Experienced Taste Panel Using the Triangular Tost in the Detection of Pairs

Saeosssful Idsntifiostion Psnsl ■■■■■■ Ueaber Aostats Propionate Total

Trial No. 1 2 3 T 1 2 3 T

I x x x 3 X x X 3 6

II x x 4 x ft ft 3 5

III x ft 4 X x ft 3 5

IV x 1 X x ft 3 L

V x ft 4 X ft 2 _ 4

VI 0 X 1 1

I* X x x 3 JL ft ft 3 6

III* x x 4 X._ X x 3 5

IV* XX X 3 X X ft 3 6

V* x x x 3 . X ft ft 3 6

XV* x x 4 X 1 3

XVI* ft x x 3 X ft 2 5 Total 3 11 7 26 11 11 8 30

♦Results frost experienced parsons in ths ssoond tasts pansl. Pansl aanbers I, III, IV and V participated in each of ths two panels. Acetate X2 = (26-12) m l6#33 12 Propionate X2 = (30-12)2 s

X2 = ( .01, 2 ) = 9.210 -77-

TABLE IB

Rssuits of Insxpsrisnosd Tasts Pansl Using tbs Triangular Tsst in ths Dstsotion of Pairs

Suoossaful Idsntifioation Pansl Aostats Propionate Total Ifisabsr Trial No. 1 2 3 T 1 2 3 T

VII x X X 3 x X 4 VIII x 1 x X 2 3 IX 2 X 3

X . _ X X 3 9 2

XI X 1 X X 2

XII 9 X X 3 2 VII* x 1 9 1 VIII* x X 0 1

IX* _x _ X 2 0 2

X* x X X X 2

XIII* 0 X X 1 _ XIV* X x x 3 0 3 Total 5 4 B 17 4 2 3 9

* Rssults A tom insxpsrisnosd psrsons in ths ssoond tasts pansl. Pansl nsMbsrs VII, VIII, IX, and X partleipatsd in sach of tbs two pansls.

Ao.t.t. I2 - ■<» - M >*. - 2.06 12 2 _ ( 9 - 12 )2 Proplonats X m — . - * 0.75 12

I2 * ( .05, 2 ) «= 5.991 -78-

animals are totaled and treated statistically at the end of the table*

A similar treatment has been used In evaluation of successful identi­

fications from the comparison of flavor in meat from sodium acetate

supplemented animals and control animals. Chi-square has been com­

puted at the bottom of Table 17 in an effort to determine if the

differences noted in flavor are due to the element of chance alone.

Table 18 contains results from those members participating in each

of the two panels which were not regarded as experienced because of

no previous participation in panels on flavor in meat or dairy pro­

ducts. Tabulations, notations and test statistics follow the pattern

just described for Table 17*

In general, rations containing sodium acetate and sodium pro­

pionate caused an alteration in meat flavor such that the meat could

be distinquished, by taste, from meat resulting from a ration lacking

in either of these salts. Certain deviations and substantiations

seem worthy of mention.

It should be noted that a consideration of results involving

experienced panel members alone, Table 17$ indicates a flavor differ­

ence due to sodium acetate and propionate feeding supplements. This difference seems somewhat more pronounced for propionate animals than

for acetate animals in the younger set of animals. There was almost no difference in the number of successful identifications of the

animals which were two weeks older whether the feeding supplement was

sodium acetate or sodium propionate. In Table 17 it is shown that

there were 10 successful identifications of the acetate pairs compared to 14 successful idsntiflections of ths propionate pairs in the first panel. In the second panel, 16 successful identifications were obtained with the acetate pairs and 15 successful identifications with the propionate pairs. Although the difference in number of successful identifications is smaller for the second panel there is no real difference in the number of successes on samples from either feeding supplcswnt by the first or second panel. Panel member number VI in

Table 17* although listed as experienced, had experience limited to dairy products. This individual was unable to detect a flavor differ­ ence between the control sample and any of the acetate samples; and observed a flavor difference between the control samples and only one of the propionate samples. All of the remaining panel members in Table

17 had served on meat flavor panels previously.

Five of the six experienced panel members in the first taste test were able to successfully pick out the odd sample and to reproduce their findings. The flavor distinction seemed to be more pronounced for the propionate supplemented animals since four out of the six experienced panel members successfully picked out ths odd sample in all three trials. Each of the six successfully identified the odd sample for at least one trial. Only one panel member obtained triplicate agreement for the samples from acetate supplemented animals and one member was unable to pick out the odd sample for any of the acetate sables.

In the second set of taste tests, the animals were two weeks

older than those represented in the first set. All the experienced -ao- panel members, indicated by an asterisk in Table 17 were able to re­ produce their distinction of the odd sample from the acetate pairs in the second panel. The same was true for the propionate samples, with one exception. Moreover, four out of these six persons were able to triplicate this distinction.

Results were not so clear cut in the teats with inexperienced panel members. The data are presented in Table 18. In the first set of tests with panel members VII through XII, all except one member detected the odd sample from the acetate sas^les for at least one trial. Only two of these persons were able to duplicate their findings, and, only one of these two made a third successful identification. With respect to the propionate samples, two persons detected a difference in flavor but neither detection was duplicated.

The divergence from a set pattern was evident among the in­ experienced panel members in the second set of taste tests, Table

18, panel members with asterisks. Three of these six persons duplicated their detection of the odd sample in the acetate trials while only two out of the six duplicated their detection of the odd sasple in the propionate trials. One inexperienced member, XIII, detected no flavor difference between the acetate and control animal

flavor and yet indicated differences between the propionate and control animal flavor. The reverse situation was true of yet another member who detected no flavor differences between the control and pro­ pionate samples and yet detected flavor differences in duplicate between the control and acetate samples. All of the persons classed —Bi­ as inexperienced tasters were students, who were admittedly affected by duration and scope of the panels.

Chi-square, which is the appropriate test statistic for these numbers, was computed for the pooled results from all experienced panel members and from the pooled results of all inexperienced panel members. These computations are presented for the experienced and inexperienced tasters at the bottom of Tables 17 and IB, respective­ ly. The results indicate that the deviations of the odd sample a n too large to be assigned to chance alone, in the case of the experienced panel members. This was true for meat from animals re­ ceiving either supplement. In the tests with the inexperienced panel members, differences in numbers of successful identifications from those expected by chance alone were not sufficiently great to be significant.

Findings of the taste panel, that is, the experienced taste panel, may reflect the differences, though small, in fat, pH and moisture presented in Table 15• Then too, the sodium acetate and sodium propionate feeding supplements may conceivably influence the flavor of meat through their alteration in volatile acid content of meat as indicated in Figure 1, page 45* The differences in volatile acid content seem to be paralleled by an alteration in the concentrations of other metabolic acids, particularly succinic and d -ketoglutaric acids; moisture, fat and roast shrinkage. - 82-

Although meat samples were not obtained from the group of lambs maintained on a ration which contained both sodium acetate and sodium propionate, it may be predicted on the basis of volatile acid and other metabolic acid analyses of rumen juice, liver, blood in Lot 4, that a flavor, different from the control samples, might be found along with differences in volatile and other metabolic acids. These differences would be expected to approach a magnitude equal to the combined effects of the two salts.

These findings in flavor differences have no bearing on a preference evaluation. Although the successful identification of the odd sample indicated a difference in flavor between the control and the sample to which it was compared, there was no information to indicate that the controls were accepted as superior or inferior to the other samples. -63-

SUMMAHX

1 Twenty-four lambs of Shropshire breed end Columbia-Shropshire crosses were randomly assigned to one of four rations. Animals maintained on one of these rations were collectively designated as

Lot 1, Lot 2, Lot 3, Lot 4. Three wethers and three ewes were in­ cluded In each lot which received a supplement, five wethers and one ewe were in the control lot. Six of the original twenty-four lambs were fed a basal ration which contained corn, urea, cerelose, hay, Lot 1; six animals were fed the basal ration and a supplement of sodium acetate, Lot 2; six animals were fed the basal ration and a supplement of sodium propionate Lot 3} *nd the remaining six ani­ mals received the basal ration and a supplement of equi-molar quan­ tities of sodium acetate and sodium propionate, Lot 4.

After 109 days, one half of the animals which received each of the rations were slaughtered; the remaining animals were slaugh­ tered after 123 days. Samples of blood, rumen juice, liver were col­ lected from each animal and preserved by lyophilisation. Iyophillsed samples of blood, rumen juice, liver and meat were individually ana­ lysed for acetic, propionic, butyric, formic, fumaric, glutaric, lac­ tic, succinic, alpha-ketoglutaric, trans-aconitic, malic and citric

1 All lambs were b o m in the Spring of 1952 and weighed from 63*3 to 64.0 pounds, after shearing, at the beginning of the experiment. "8/^ acids by ths chromatographic technique of Bulan, Varner and Burrsll

(10), Fra eh lamb shoulder samples from Lots 1, 2 and 3 were tested for roast shrinkage; pH, moisture end fat content; and for flavor in roasts by use of the taste panel.

Growth and feeding data substantiated the observations that so­ dium acetate and sodium propionate are palatable and seemed to in­ crease the efficiency of the basal ration when Included as a supple­ ment. The amount of corn, urea-cerelose mixture, and hay required to

produce a 100 pound gain in weight was reduced, A combination of the two organic acid salts in a mixture increased the efficiency above

that observed with acetate or propionate alone.

The volatile acids, acetic, propionic and butyric, were present

in each of the tissues studied. Although all lots contained these

acids, s o M animals within the lots did not contain quantities in all

tissues which could be detected, under the conditions of the experi­

ment • The concentration of all three acids increased from shoulder to

blood, to liver, to rumen juice. Acetic a d d represented from 45 - 88%

of the total volatile acids; however, there appeared to be no signifi­

cant difference in the ratios of butyric and propionic acids in any of

the tissues. Variations within the lot were such as to make any real

differences between the volatile acid levels produced with the control

ration and either supplemented ration, questionable.

All metabolic acids considered, pyruvic, lactic, fumaric, sue- -85- cinic, alpha-ketoglutaric, trans-aconltlc, malic and citric acids, were detected in liver, blood, and meat; however none of these acids were detected in rumen juice at the sample level employed, fyruvic, lactic and succinic acid were present in approximately equal molar con­

centrations in muscle, blood and liver. Fumaric, trans-aconitic, malic and citric acids were present in approximately equal siilllequivalent a- rnounta in blood and liver but decreased fay a factor of ten in Ma t .

Alpha-ketoglutaric acid levels were approximately equal in blood and

meat but were 4 to 20 times higher in the liver. Animals whose rations

contained either organic salt, showed higher levels of alpha-ketogluta­

ric acid in the liver than did the control animals. Acetate was more

effective in causing this elevated level than propionate.

Moisture, pH, fat and roast shrinkage analysis Indicated that

variations within the lots were of the same order of magnitude as the

variations between the lots. Hence, real differences between values

for the control animals and animals which received either organic salt,

are questionable. Acetate supplemented rations produced animals with

lowest average roast shrinkage and least variation within the lot.

Acetate and propionate supplemented rations each produced animals with

higher average fat and lower average moisture contents than did the

control ration. pH measurements indicated that less free acid was

present in the muscle of animals which reoeived the acid supplements•

Taste panel studies indicate that differences in flavor between

control and acetate or propionate supplemented animals were of such -86- magnitude as to allow an odd sample to ba differentiated, whan compar- lng tha control with two samples from ona an Inal in othar lots* Tha dataction in flavor diffarancas was more unifora with experienced tastars than with inaxpariancad tastars. Whan aithar sodium acatats or propionata are includad in tha ration of faadar lambs, tha flavor in aaat apparently is altarad* Thar# was no attempt to determine su­ periority or infarioritj of tha altarad flavor*

It nay ba possibla that an intarralationship doas axist batwaan tha prasanca of organic acid salt in tha ration and tha drasaing per- cantaga of faadar lambs, production of volatils acids in blood, liver and meat, alpha-ketoglutaric acid content of liver, pH, moisture and fat content, and flavor in meat* Nevertheless wide variations within lots do not allow a dear cut definition of this relationship. Tha in­ clusion of sodium acetate or sodium propionata to replace oorn or othar feed stuff in tha ration of faadar lambs is possible* -87-

CONCUUSIONS

Feeding sodium acetate or sodium propionate to lambs causes:

a. A decrease in the amounts of corn, cerelose and hay required

for a unit gain in weight;

b. An increase in acetic acid levels in the rumen juice, liver,

blood and muscle;

c. An increase in the -ketoglutaric acid levels in liver;

d. An alteration in pH, moisture, fat content and roast shrink­

age of meat although no definite pattern is evident;

e. An alteration in meat flavor to such an extent that the

sample can be distinguished by taste from the control.

Feeding sodium propionate causes an increase in butyric and propionic

acid levels in muscle and liver.

Feeding sodium propionate and sodium acetate has generally the combined

effects of the individual acids.

The sodium salts of acetic and propionic acids are capable of

replacing components of the basal ration. This replacement is accoah-

panied by alterations in the levels of metabolic acids in rumen, blood,

liver and muscle. Acetio and ^ -ketoglutaric acids seem to be key

metabolic products. Flavor differences in neat,which are detectable

by taste, and differences in pH, moisture and fat content of meat are

other effects observed when sodium acetate or sodium acetate are in­

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AUTOBIOGRAPHY

I, Gladys Williams Royal, was born in Dallas, Texas, August 29,

1926* It was in tha public schools of that city that I received dqt sacondary school education* My undergraduate training was obtained at

Dillard University, New Orleans, La., supplemented by simmer attend­ ance at Atlanta University, Atlanta, Ga. In Hay, 1945 I received the degree Bachelor of Arts from Dlllard University with special concen- tratlon in Chemistry.

Immediately after graduation I received an appointment as the first Research Assistant in Chemistry at the George Washington Carver

Foundation, Tuskegee Institute, Ala. I held this position for one and one half years while pursuing courses leading to the degree Master of

Science. During the year 1946 - 47 I attended the University of Wis­ consin where I pursued courses in the Department of Biochemistry. In

September 1947, I received an appointment as Teaching Assistant in general chemistry at Tuskegee Institute. I held this position for one year while completing requirements for the degree Master of Science in

chemistry which I received August, 1943.

In the interim between August 1946 and January, 1951, I give

birth to a son and devoted mr attentions to his care. In January 19-

51, I received an appointment as Graduate Assistant in the Ohio State

University, where I specialised in the Department of Agricultural Bio­

chemistry. I held this position for two and one half years while com­

pleting the requirements for the degree, Doctor of Philoeopgr•