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V‘nr‘ I13 3.1.1 f‘L‘rl‘ “9A 1“ 1 J. .x, .. 1:313» - ...1 ‘.:1..;1f'r.f.;: 1.4de Pi; 1.23.3 :g‘gt V“ 1 I :‘ijhZ' IHESIS This is to certify that the thesis entitled FRACTIONATION OF THE COMPONENT(S) RESPONSIBLE FOR SEX ODOR IN RORK presented by Harris Bradford Craig has been accepted towards fulfillment of the requirements for Ph 0 D. degree in An. HUSb o _ 7 MaYor professor Date Dec. 21, 1960 0-169 LIBRARY Michigan State University ABSTRACT FRACTIONATION OF THE couromrmts) RESPONSIBLE FOR SEX ODOR IN PORK by Harris Bradford Craig An objectionable odor, commonly referred to as sexual or boar odor, is produced on heating the flesh from most boars and stags. It is de- scribed as onionlike, unpleasantly perspirative, or sickly sweet. In federally inspected plants about 3,000 animals are condemned in the Uni- ted States each year because of sex odor. Little information is available as to the nature of the odor. There- fore, preliminary studies were undertaken to ascertain if the component(s) responsible for sex odor was/were present in fat, lean or both and to study the solubility properties. Finally attempts were made to collect, isolate and identify the responsible agent(s). Boar tissues and organs, as well as water and ether extracts of lean and fat, were heat tested for sex odor. Lean tissue was freeze dried; the fat was extracted, and the various fractions were heat tested. Bellies from boars were cured by accepted curing methods and examined for the pres- ence of sex odor after zero, 90 and 120 days of cooler storage. Nitrogen determinations were made on solvent rendered fat from both boars and bar- rows. Various distillation and fractionation procedures were employed in an attempt to isolate the agent(s) responsible for sex odor. Barrow fat was similarly treated and served as a control. Distillation methods included: low temperature - high vacuum, high temperature - atmospheric pressure and steam distillation. The volatiles produced by the various distillation techniques were collected in traps immersed in dry ice - ethanol or in ice Harris Bradford Craig water. The contents of the traps were checked for sexual odor by the heat test and the volatiles were separated by gas chromatography. Fat from boars and barrows was saponified at room temperature using sodium ethylate. The unsaponifiable fraction was heat tested and sepa- rated by gas chromatOgraphy. In addition, separation by paper and silic- ic acid column chromatography was attempted. Sex odor could not be detected until a temperature of 100 to 108 de- grees C. was achieved, indicating that the odor component(s) were not volatile under 100 degrees C. or that their formation was heat dependent. Sex odor was not present in the fat-free lean nor in water extracts of lean and fat. The component(s) responsible for sex odor was/were sol- uble in ether and other fat solvents and appeared to be associated with the fatty tissues. Appreciable quantities of ammonia were given off by both intact boar and barrow fat, but on rendering the quantity was greatly reduced indi- cating that connective tissue was the source of this compound. The sim- ilarity in nitrogen content of rendered boar and barrow fat indicated that sex odor is probably not due to nitrogenous compounds. The agent(s) responsible for sex odor in pork was/were not obtained in recognizable form with the distillation methods used in this study. Gas chromatographic analysis of the contents of the traps failed to show any consistent and reproducible differences between volatiles obtained from.boar and barrow fat. Sexual odor was produced when the unsaponifiable fraction of boar fat was exposed to heat, but no reproducible differences were noted be- tween boar and barrow fat on gas chromatographic analysis. Identifica- tion of two components from the unsaponifiable fraction was accomplished Harris Bradford Craig by using retention times from the gas chromatograph and/or various qual— itative tests. Cholesterol and squalene were found to be present in the unsaponifiable fraction of both boar and barrow fat, but these compounds did not produce sexual odor when heated alone in the pure form. Attempts to identify the unsaponifiable component(s) responsible for sex odor by formation of their derivatives and determination of their melting points were unsuccessful. Three fractions were obtained from boar unsaponifiables when sepa- rated by silicic acid column chromatography. The fraction eluted with ethyl ether was the largest and contained most of the sex odor. Gas chromatographic analysis of this fraction indiCated that it contained essentially the same components as the unfractionated unsaponifiable matter. FRACTIONATION OF THE COMPONENT(S) RESPONSIBLE FOR SEX ODOR IN PORK by Harris Bradford Craig A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Husbandry 1961 (7 jazz 36. ///7/s1 ACKNOWLEDGMENTS The author is grateful to Dr. A. M. Pearson, Professor of Food Sci- ence, for his interest, aid, and constructive criticism during the course of this investigation. The author also wishes to express his appreciation to Dr. C. A. Hoppert, Professor of Chemistry, and to Dr. E. P. Reineke, Professor of Physiology and Pharmacology, for their critical reading of this manuscript. To Dr. J. R. Brunner, Professor of Dairy Technology, the author wishes to express his gratitude for suggestions and advice. The aid of Dr. Basil Tarladgis, and the late R. C. West, Department of Animal Hus- bandry, is also gratefully acknowledged. The writer wishes to dedicate this thesis to the memory of his Mother who passed away during the course of his graduate study. Her sac- rifices and encouragement will always be remembered. To his wife, Doris, the author is especially grateful for her en- . couragement, understanding, patience, and sacrifice and for the typing of this manuscript. Gratitude is expressed to Mr. Charlie Finkel of Crown Packing Com- pany, Detroit, Michigan, for supplying some of the research material utilized in this study. The financial assistance of Michigan State University in the form of a Graduate Research Assistantship is gratefully acknowledged. ii TABLE OF CONTENTS Page MRmUCTION'OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0... 1 REVIEW OF LITERATURE.............................................. SOME FLAVOR AND ODOR PROBLEMS IN FOODS....................... Vegetables.............................................. Juice................................................... COffeeCOOCOOOCOOOOOOO0.00.0...O...OOOOOOOOOOOOOOOOOOCOOO Milk and Milk PI‘OduCtSooooooso...oooooooOOQOoooooooooooo ~J \n ¢~ t~ \o \o \o meatSOOOOOOOOOOOOOOOOOOIOOOO0.0.0.0....OOOOOOOOOOOOOOOOO mutton FlavorCOOOOCOOOOOOOOOOOOOOOOOOOO0.0.0.00000900... 13 METHODS EMPLOYED IN THE SEPARATION, CONCENTRATION, AND IDENTI- FICATION OF FOOD FLAVOR AND ODOR COMPONENTS.................. 14 Chemical................................................ 14 Column and Paper Chromatography......................... 17 Distillation............................................ 18 Gas Chromatography, Mass Spectrometry, and Infrared Spectroscopy............................................ 2O EXPERIMENTAL PROCEDURE............................................ 23 PRELIMINARY STUDIES.......................................... 23 Source of Experimental.Material......................... 23 Method of Sex Odor Detection............................ 23 Determination of Sex Odor‘Volatilization.Temperature.... 23 IEzamination of Boar Tissues and Organs for the Presence Of Boar Odor”.......................................... 21'. Effect of Curing Boar Meat on Intensity of Sex 0dor..... 25 The Presence and Solubility Properties of Components Responsible for sex morOOOOOOOOOOOOOOOOO000.00.00.00... 25 iii TABLE OF CONTENTS (CONTINUED) Page Nitrogen determination of Rendered Boar and Barrow Fat.. 26 Saponification.......................................... 27 Injection of Live Boars with."Diquel" Animal Tranquilizer 28 METHODS EMPLOIED FOR ISOLATION AND COLLECTION OF SEX ODOR CMONEMSOOOOOOOOOOOOOOOOOO0.0.00.0000...OOOIOOOOOOOOOOOOOOO 28 LOW Temperature " High vacum Distillation. o o e o o o o o o o o o o 28 Gas Chromatography of‘Volatiles Obtained by High.Tempera- ture Atmospheric Pressure Distillation.................. 29 Direct Sampling MethOdSOOOOOOOO0.00.00000000000000000000 33 Steam Distillation Of Boar Fat"....................u.o 33 Examination of Contents of the Preputial Diverticulum of BoarSOOO...OOOOCOOOOOOOOOOOOOO0.0......OOOOOOOOOOOOOOOOO 33 Tests for the Presence of Ammonia, Carbonyls, and Sulfur Compounds in the Volatile Distillate from Boar Fat...... 34 Gas Chromatography of the Unsaponifiable Matter from.Boar am Barrow FatOOOOOOOO0.0.0.000....OOOOOOOOOOOOOOOOCO... 34 Column Chromatography of Unsaponifiable Matter from Boar FatOOOOOOOOOOOOOOQOO...0.00.00.00.000000000000000000COOC 35 Paper Chromatography of Unsaponifiable Matter and Prep- aration of Chemical.Derivatives......................... 35 RESUETS AND DISCUSSION............................................ 37 PRELIMINARY STUDIES.......................................... 37 Production of Sex Odor by Heating Boar Fat.............. 37 Sex Odor Volatilization Temperature..................... 37 Intensity of Sex Odor in Various Boar Tissues and Organs 37 Effects of Curing Boar Meat on Intensity of Sex Odor...; 38 Presence of Sex Odor and its Solubility Properties...... 39 Nitrogen Content of Rendered Boar and Barrow Fat........ 40 Effect of Injecting Live Boars with."Diquel" Animal TranquilizerOOOOOOOOCOO00....OOOOOOOOOOOQOCOOOOOOO0.0... 41 iv TABLE OF CONTENTS (CONTINUED) Page ISOLATION AND COLLECTION OF SEX ODOR COMPONENTS............. 41 Low Temperature - High'Vacuum Distillation of Boar Fat. 41 Gas Chromatographic Analysis of Volatiles Obtained from Boar and Barrow Fat by High Temperature - Atmospheric Pressure Distiuationaooooeooo00000000000000000000.0000 42 Analysis of Volatiles Obtained by Direct Sampling methMSOOOOOOOOOOOOOOO00.000.00.000.0....00.000.00.000. 47 Analysis of Steam Distillate of Boar Fat............... 50 Analysis of the Preputial Glands and Preputial Diverti- culm from BoarSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 50 Analysis of the Contents of the Preputial Diverticulum from BoarSOeooooeo0.0000000000000000...0000000000000... 5]. Presence of Ammonia, Carbonyls and Sulfur Compounds in the VOlatile DiStillate from Boar Fall-10000000000000.0000 51 Presence of the Sex Odor Component(s) in the Unsaponi- fiable Fraction......0000......0......00...........0... 53 Fractions Obtained by Column Chromatography of Boar Fat Unsaponifiable MatterOOOOOOOO000......00.000.000.000... 60 Paper Chromatography and Chemical Derivatives of Unsa- ponifiable matter...OOOOOOOOOOOOOOOOO..0...00.0.0000... 61 sumvmmCONCLUSIONSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 62 LHEMUM CITE....0.0.0....OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO... 65 LIST OF TABLES Table 1. 2. 3. Boar weights and amount of tranquilizer administered........... Incidence of sex odor in various boar tissues and organs....... Solubility of fat and sex odor components in various organic solventSOOOOOOOI0.0...D....00...0..OOOUOOIOICOOOOICOO0.00...... vi Page 28 38 40 ...... . n ‘__. ,_,,......»-~-' ..._,.. 1..‘.. . I .V..... .... ......- ........-.~ .. .... .. . - ‘- LIST OF TABLES Table l. Boar weights and amount of tranquilizer administered........... 2. Incidence of sex odor in various boar tissues and organs....... 3. Solubility of fat and sex odor components in various organic salventSOOOOOOOOOOO...0.0.0.0000...OOOOOOOOOOOOOOOOOOOOOO00.... vi Page 28 38 40 LIST OF FIGURES Figure I. Gas chromatogram of ether extract of aqueous distillate from boar fat.............................................. II. Gas chromatogram of ether extract of aqueous distillate from barrow fat............................................ III. Gas chromatogram of unsaponifiable matter from boar fat.... IV. Gas chromatogram of unsaponifiable matter from barrow fat.. V. Gas chromatogram of cholesterol in ether................... VI. Gas chromatogram of squalene in acetone.................... vii Page .48 49 56 57 58 59 In Harris Bradford Craig candidate for the degree of Doctor of PhilOSOphy Final examination: December 19, 1960 Dissertation: Fractionation of the Component(s) Responsible for Sex Odor in Pork Outline of Studies: Major Subject: Animal Husbandry Minor Subjects: Biochemistry and Physiology Biographical Items: Born: February 9, 1928, Liberty, South Carolina Undergraduate Studies: Clemson Agricultural College Clemson, South Carolina 1945 - 1949 Graduate Studies: North Carolina State College Raleigh, North Carolina 1954 - 1956 Michigan State University East Lansing, Michigan 1958 - 1960 Experience: Graduate Research Assistant, Department of Animal Industry North Carolina State College, 1954 ~ 1956 Instructor, Department of Animal Industry North Carolina State College 1956 - 1958 Graduate Research Assistant, Department of Animal Husbandry Michigan State University 1958 - 1960 Member: Society of the Sigma Xi, Gamma Sigma Delta, American Society of Animal Production, and Institute of Food Technologists. viii INTRODUCTION During the past several years considerable effort has been directed toward isolation and identification of flavor and odor components pres- ent in various foodstuffs. Prior to this time advances were, of neces- sity, slow and difficult due to lack of adequate research instruments. With the develOpment of research methods such as gas chromatography, in- frared spectroscOpy, and mass spectrometry the problem of flavor research has become much less tedious and time consuming. Solutions to seemingly impossible problems have been obtained with relative ease. Heat is one of the food commodities that has been the subject of ex- tensive flavor investigations. Studies on beef flavor have been under- taken largely in regard to isolation and identification of compounds pro- duced when beef is irradiated. Such flavor might be referred to as an “induced" flavor and is quite objectionable to certain individuals. As contrasted to "induced" off flavor, some meat animal carcasses possess what might be called "natural" off flavors. Since pork and mutton are claimed by some to have objectionable odors, they can be placed in this category, The problem of mutton flavor has not been investigated to any great extent, but studies are currently underway (Jacobson 1960) to iden- tify some of the responsible components. Off odor in pork, more commonly referred to as sex odor or boar o- dor, has been a plague to the meat processor for many years. Currently, about 3,000 boars and stage are condemned under Federal Meat Inspection each year in the United States because of sex odor. Probably a much larger number go undetected. Consequently, many hog slaughtering and pork processing establishments avoid slaughtering boars or processing 2 boar carcasses because of the commonness of sex odor. Sex odor has been described variously as pungent, sickly, onionlike, or unpleasantly per- spirative. According to Self (1957) it is not confined to boars, but 15 present in stage, cryptorchids, gilts, barrows, and sows as well. Self (1957) also reported that only a certain percent of the carcasses check- ed actually possessed off odor. Some were completely free of any trace of sex odor, as detected by exposing a tissue sample to a heat test, which caused the odor to volatilize. Percentagewise, the number of of- fenders was approximately the same with sows and gilts as for barrows and bears. In view of the annual economic loss due to condemned carcasses and the adverse processor-consumer relationship, when.meat and meat products possessing sex odor are utilized, it was deemed important to study the cause of sex odor in pork. The primary Objectives of this investigation were to determine if sex odor was present in pork fat, or lean, or both; to study its solubility characteristics; and to attempt to collect, iso- late and identify the responsible component(s). REVIEW OF LITERATURE m Egon AND _c_p_9_a PROBLEMS g rows Flavor and/or odor of foods are extremely important from the stand- point of consumer acceptance. A food might rate high in every aspect exp cept flavor, but because flavor is lacking or is objectionable, the cone sumer hastily refuses the product. Flavor was defined by Cracker (1937) as that property of a food or beverage that makes it excite the senses of taste and smell. Kremlich ( 1959) emphasized the importance of fla- vor and pointed out that little is known concerning its chemical nature or contributing components. means: Since off odors and flavors make a product unacceptable, a number of workers have made a study of these undesirable traits in vegetables. Sondheimer‘gt'§;., (1955) stated that the bitter flavor present in some carrots could be removed for study by hydrocarbon extraction. In a study of bitter flavor in canned carrots, Shallenberger 35 gl., (1960) divided the flavor into volatile and nonvolatile fractions. The bitter ‘volatile fraction was present in very low concentration and could be re- moved by steam distillation. After extraction of the distillate with ethyl ether and evaporation, an oil having a bitter, harsh, hot flavor was noted. It was postulated that the volatile component was an essen- tial oil made up of a single sesquiterpene or a mixture of sesquiter- penes. It is a well known fact that certain vegetables must be blanched be- fore freezing. Lee gt.gl., (1955) showed definite development of off flavor in two to four weeks in frozen unblanched peas, corn, and snap 3- 4 beans. The crude lipid matter extracted from these vegetables showed a definite increase in acid after one week's storage. The increase con- tinued throughout the storage period, but was more pronounced during the early months of storage. Spencer and Stanley (1954) found that tomato odor was at least part- ly due to alcohols, carbonyls, and unsaturated compounds. They further noted that changes in the unsaturated compounds may be involved in fla- vor deterioration of stored tomato products. isles Blair g§’§1., (1952) stated that the characteristic orange-like flavor of orange juice was due to the presence of a small amount of peel oil. Without peel oil, the juice was relatively insipid. When canned orange juice was heated to a temperature higher than 70 degrees F., an off flavor developed due to some substances produced by chemical inter- action of the terpenes of the peel oil with the acid in the juice. Qailea Coffee bean flavors and aromas are products of a number of treat- ments such as roasting, storing, and brewing. Kaufman (1951) stated that the crude protein fractions of the coffee bean seemed to be the major source of coffee aroma. Off flavors were due to caffeine or to pyridine which was formed during roasting from trigonelline. Johnson at al., (1938) in a study of roasted coffee found the following constituents: diacetyl, methyl acetylcarbinol, furan, furfuraldehyde, furfuryl alco- hol, acetaldehyde, pyridine, and hydrogen sulfide. They postulated that stale flavor of coffee was probably concerned with changes in the volatile aroma and flavor substances and did not involve fat rancidity. Segall and Proctor (1959) noted a relationship between the content of /‘D 5 the mercaptans in a coffee brew and the organoleptic rating of these brews. They suggested that the content of the mercaptans in the coffee brew was closely associated with flavor and flavor changes. manganese Milk and milk products are subject to a variety of off flavors. Patton gtflgl., (1957) in a review article on flavor of milk and its products stat- ed that flavor control was concerned with (a) prevention of off flavor, (b) maintenance of desired flavor and (c) deve10pment of characteristic flavor. They emphasized that feed flavors which gain entry to milk through the digestive tract of the cow represented the most important off flavors in market milk. Other important flavor defects of milk were termed: oxi- dized, rancid, and sunlight. The oxidized flavor was due to a series of unsaturated aldehydes which arose through autoxidation of the milk lipids. Rancid flavor was due to hydrolytic splitting of milk fat. Butyric acid was known to be partly responsible for this flavor. According to these authors, hydrogen sulfide, a compound which arose from heat denaturation of milk serum proteins, was evident in milk after momentary heating to ap- proximately 170 degrees F. Oxidation in relation to off flavor has been studied by a number of workers. Thurston (1937) stated that the most important chemical changes causing off flavors in milk and.milk products were due to oxidation ins volving butterfat or constituents of milk closely associated with butter- fat. Peres g§,gl., (1955a) described oxidized flavors in milk as "card- board", "oily", "metallic", ”tallowy", and ”emery". They recovered a number of carbonyl compounds from oxidized milk and deduced that they were produced by the preferential oxidation of di- and polyunsaturated fatty acids. In a subsequent paper, (Pores g§,gl., 1955b), it was 6 claimed that the two main constituents of oxidized (cardboard) flavor in milk were 2 - 4 heptadienal and 2 - 4 nonadienal. Patton (1954) investigated the mechanism of sunlight flavor forma- tion in milk and listed methional as a compound of importance. He did not show the presence of this compound in milk, in which naturally induced sun? light flavor was evident, but it was shown by means of infrared spectral data that methional was a product of the sunlight catalyzed reaction be- tween methionine and riboflavin. He concluded that the amino acid methio- nine is a specific source of the flavor. Patton et_gl., (1956) studied the relationship between methyl sulfide and milk flavor and stated that a high concentration of this compound could account for certain "cowy'I and feed type odors; whereas, abnormally low amounts of methyl sulfide, such as might occur in concentrated or dried milk products, may be responsible in part for the lack of so called fresh milk flavor. Rooney and Patton (1956) involved delta-decalactone as the agent re- sponsible for the coconut-like off flavor, which developed in butter oil during storage or during heating. Patton and Tharp (1959) studied the ef- fects of steam distillation or saponification on milk fat and identified a homologue series of ketones (acetone through pentadecanone - 2) in milk fat so treated. Fresh milk fat was found to be devoid of such ketones with the exception of acetone. The authors stated that trace amounts of methyl ketones can alter the flavor of milk products. Flavor and odor problems arise when milk and milk products are ex- posed to ionizing radiations. Bierman.§t‘al,,(l956) found that radia- tionpinduced off flavors in.milk and cream increased with increasing moisture and fat content. .However, as the total solids increased, the product became less susceptible to flavor change. Day gt gl., (l957a,1957b) 7 stated that milk can be sterilized readily by ionizing radiations, but off flavors and odors resulting from.this treatment make the product unpalat- able. They further noted that of the carbonyls produced in skim milk by irradiation, only acetaldehyde seemed to be of flavor significance (malty). They concluded that the principle radiation induced off flavor appeared to arise from a group of potent, disagreeable smelling sulfur compounds. Methyl mercaptan, methyl sulfide, and methyl disulfide were postulated to be the dominant members of this group. These compounds primarily origi- nate from the milk protein, casein. East: (is with milk and milk products, ionizing radiation also produces ob- jectionable flavors and odors in other foods. Proctor‘gt‘gl., (1955) re- ported that high levels of cathode ray treatment may induce changes in food flavor, that are difficult to mask or modify so that the food will be organoleptically acceptable. Similar results were noted by Pratt and Ecklund (1956). These authors studied the effect of irradiation on sever- al meat items and vegetables. Groninger'gt al., (1956) evaluated the orga- noleptic properties of beef, pork, cured ham, salmon, tuna, and halibut and found significant flavor changes in the irradiated product. ‘Witting and Batzer (1957) prepared a crude reaction mixture of me- thional from methyl mercaptan and acrolein. They stated that this mix- ture had an odor typical of ground beef, which had received 2 - 4 mega- rep of gamma irradiation. Pearsonlgt gl., (1959) stated that the most critical problem in con- nection with the acceptance of irradiated meats is the development of a characteristic flavor, commonly referred to as "irradiation flavor“. Their study indicated that hydrogen sulfide, methyl mercaptan, and carbonyls 8 were responsible for a considerable part of the poor acceptability of ir- radiated beef, pork, and veal. Similar results were reported by Hedin gt 3;" (1960). They reported that the irradiated or “wet dog" odor was most prominent in an undialyzed meat slurry. When a sample was dialyzed, the most intense irradiated odor was produced by the nondialyzable frac- tion. It was postulated that the odor was associated with sulfhydryl or closely related compounds, since cysteine and methionine could no longer be detected in the protein after irradiation and since the odor was quenched by sulfhydryl reagents. Batzer and Doty (1955) also implicated sulfur compounds as the source of some undesirable odors in irradiated meat. Batzer 31; 31., (1957) reported that meats with a high fat content do not develop off odors to the same extent as do leaner meats. Their ‘ results indicated that carbonyl compounds produced in irradiated ground beef muscle differed from those obtained from irradiated beef or pork fat. The amounts of these compounds produced were shown to increase with increasing irradiation dosage. Animal flesh is known to acquire off flavor and odor from certain ra- tions. Raine 93; 3., (1953) fed spoiled meat scraps and horse manure to growing pigs. Taste and odor tests of the flesh showed that the meat scrap group had taken on repulsive tasting and smelling substances. This was especially true of the lard where more volatile aldehydes, methyl ke- tones, secondary amines, trimethyl amine, skatole, primary amines, and phe- nols were found as compared to lard from control pigs. Kemp gt 3;" (1952) and Grmor 93 pl" (1950) examined the flesh of hogs which had been treated with benzene hexachloride prior to slaughter and found that this cupound produced off flavors and odors when applied immediately before “*7 9 death. Both the lean and fat were involved. Vacuum packed, stored, dehydrated pork developed an off flavor and/or odor during storage at elevated temperatures and/or for long time intervals (Burnett 33 gl., 1955). These authors reported that the volatile constit- uents obtained from this product were basic in reaction and exhibited re- ducing properties. Acetaldehyde was present in dehydrated pork samples stored at both 20 degrees F. and 94 degrees F., whereas, ammonia was de- tected in meat samples stored at 100 degrees F. and 160 degrees F. The problem of sex odor in pork has been recognized for some time. However, there is little information available as to the nature of the odor and the agents responsible for its production. Lerche (1936) re- ported that as soon as the male hog was sexually mature and the testes became capable of functioning, there appeared a specific sexual odor in the meat and fat of the animal. He described the odor as being onionlike or unpleasantly perspirative and claimed that it occurred in all boars with normally developed testes and in cryptorchids, unless the testes lye ing in the abdominal cavity were atrophied. He stated that the tissue must be heated and the vapors smelled in order to detect the odor. This was accomplished by placing small pieces of tissue in water in an Erlen- meyer flask and boiling. The vapors were allowed to bathe the nostrils of the tester. Tissue was also heat tested by frying in a skillet. When the heated tissue had cooled, no odor was evident. He also noted that after male hogs had been castrated, the sexual odor disappeared from the tissue. The tissue was found to be devoid of odor 57 - 68 days post-cas- tration. Gereke (1936) postulated that a connection must exist between the testes and the parotid salivary gland which is looated near the ear. 10 According to this author, cryptorchid boars may often be detected.bw'an over production of saliva. This condition was also known to occur when boars are sexually excited. The parotid gland, according to him, was a good tissue in which to detect the odor. He stated that the odor was not dependent upon the size of the testes, but more upon the condition of the animal. He noted sex odor to be especially evident in the case of small, thick-skinned animals, which were lacking in fat and retarded in development. Kunze (1936) claimed that the tissue of cryptorchid boars, which had been condemned because of too strong a sexual odor could be freed of this odor by “pickling“. After pickling for three weeks, the tissue was completely free from odor. He claimed that the odor producing material(s) had been bound by the salt. Miller (1958) outlined the procedure to be followed in federally in- spected plants. Stage and bears should be separated from the regular kill during antemortem inspection and identified for checking for sexual odor on post-marten examination. The lean and fat are then tested for odor by heating. A carcass which exhibits sexual odor should be condemned as un- fit for food. Miller (1958) grouped carcass odors into two categories: (a) those traceable to material ingested by the animal and (b) the odor known as sexual odor in swine. Sexual odor is usually detected in car- casses of boars and recently castrated stage. He further points out that care should be exercised not to confuse this condition with odor which is imparted to a portion of the carcass by contamination with smegma from the propuco. Howe and Barbells (1937) claimed that occasionally during cooking, meat from very old animals, such as bulls, cows, or rams developed an ll extremely unpleasant odor, often characterized as amoniacal. They al- so reported that sexual odors in tissues were usually associated with bears and stage that had been slaughtered soon after castration. The origin of the usage of the words "sexual odor“ is not known. Since the odor has been shown to disappear several weeks post castration, it would seem to be sex linked in some way and hence the term "sex odor.” In view of this fact, the inplication of androgenic hormones has been postulated. Bratzler 31:, pl," (1954) found that meat from boars was def- initely inferior from a palatability standpoint. Cooking of pork chops fru boars by braising produced a definite "off“ or ”boar" odor, which according to them carried through to the cooked product. no objectionp able odor was detected in the chops from 180 pound pigs castrated 31. days before slaughter. One group of pigs was castrated 118 days before slaughter and implanted with a 193 milligram pellet of testosterone pro- pionate at the time of castration. Examination of the heated tissue from these pigs failed to show any evidence of sexual odor. Johnsunxg§,gl., (1957) fed two levels (9 or 15 mg. per 1b. of feed) of methyl testosterone to growing fattening pigs and found no obj action- able odor in the flesh of pigs fed this androgen. Perry 53; 51., (1956) obtained similar results when methyl testosterone was fed to pigs of similar weights and ages. Christian and Turk (1958), in a study of the cause of sexual odor in the boar, designed an experiment using the following groups of pigs: 1. untreated boars, 2. untreated barrows, 3. boars with the preputial diverticulum removed, 1.. barrows injected subcutaneously with 20 milli- grams of testosterone daily, 5. boars fed 10 milligrams of stilbestrol daily, and 6. boars fed 50 milligrams of stilbestrol daily. Boar odor l2 and flavor were determined by panel evaluation of the boneless roasted loin. Results indicated that the panel could detect the most odor in group 1 followed by groups 3, 5, and 4, in that order. Panel scores on samples from the above lots, which had been frozen and stored for five months, were generally somewhat lower. This indicated that the odor had diminished during freezing and storing. Probably one of the most extensive studies concerning the problem of sex odor in pork was conducted by Self (1957). His work was stimulated by the research department of Oscar Mayer and Company. This organization had received consumer complaints regarding a "boar odor" in pork. This odor was also noted along their kill line, although they do not slaughter boars. Tissue samples were taken from the diaphragm muscle of 343 pigs as they passed down the kill line. Samples were wrapped in aluminum foil and heated to approximately 200 degrees F. under infrared tubes. Testing was performed by breaking open the foil wrapper and allowing the volatile substances to bathe the nostrils of the tester. Odor intensity was classed as strong, medium, or none. The incidence of the odor (17 per cent of those checked) was found to be as high among female hogs as among castrated males. Another group of older female hogs in various stages of the repro- ductive cycle was examined for odor. Approximately 17 per cent of the group total had sexual odor, but there was no clearcut group of offenders when clgssified on this basis. Progesterone administered to 22 gilts did not appear to alter the incidence of the odor in this group. In a group of 31 sexually mature boars a total of eight, or 25.8 per cent had some sex odor. qt study of boars by breed failed to show that any genetic group had a greater incidence of the odor than any of the other breeds tested. The author concluded that on the basis of data presented it seems that , 13 sex and breed have little influence on the overall incidence of sex odor or bear odor in pork. butt 3'; 3;... (1959) removed the preputial diverticulum from boars and found that the characteristic ranting odor was reduced, but at ten months of age no difference in bear odor was found between carcasses from oper- ated and unoperated boars. Lean areas were free of the odor, but all fat areas sampled gave off boar odor when heated. The greatest concentra- tion was found in the fatty tissue of the prepuce where brownish orange areas were noted in the dorsal and lateral regions of the orifice. His- tological examination showed this tissue to be modified sebaceous glands containing colloid-like material. Similar glands in barrows appeared to be nonpfunctional when examined histologically. Alcohol-ether extraction of the body tissues showed that the agent responsible for the odor is lip- ophilic and these authors postulated that it is a muscone. These workers surgically removed the glandular area in five immature boars. The animals were slaughtered at weights varying from 290 to 350 pounds. No odor was detected in any portion of the carcasses of two boars from which the pre- putial glands had been completely removed. Some odor was noted in care oasses of‘boars, in which post-slaughter examination.revealed that not all of the glandular area had been removed. It was concluded that the prepup tial glands, which are dependent upon the male sex hormone for secretory activity, produced a fat diffusible material which was responsible for sexual odor in bear carcasses. mm Elam Certain individuals object to the smell and taste of lamb and mutton. 21.31.: (1958) and Kean (1959) painted out the fact that mutton flavor and odor-does exist. Undoubtedly this has an effect on lamb and mutton 14 consumption. The problem of mutton flavor has not been explored from the standpoint of volatile components. Jacobson (1960) is currently working on identifi- cation of the components of flavor in lamb and mutton and application of this information toward increased utilization of these meats. McInnes ‘32 gl., (1956) found that the steam volatile acids of mutton fat, which might have some influence on flavor, included those from formic (01) to an acid with ten carbon atoms. Isobutyric, isovaleric, an anteiso acid, and alpha methyl butyric acid were also found. This was the first time, ac- cording to the author that these four compounds, as well as formic acid, have been shown to be components of an animal depot fat. mama EMPLOYED _I_1~_I 2gb; sspggnoxq, CONCENTQTION, gm; IDENTIFICATION gr; FOCD FLAVOR AND CDOR COMPONENTS magnum; One of the most helpful tools the researcher has at his disposal is the use of chemical qualitative tests. Since a number of the volatile flavor and odor components of foods have been shown to be carbonyls, the most logical approach to the identification of these materials is by use of 2 - 4 - dinitrophenylhydrazine. This reagent, when united chemically with a carbonyl, forms the corresponding 2 - A dinitrophenylhydrazone. Henze gt,gl., (1954) employed this reagent for reaction with carbonyl com- pounds obtained from apple storage volatiles. A total of 18 compounds were obtained and these were separated by silicic acid chromatography and analyzed by use of a BeckmanlD. U. spectrophotometer. Wong gt 31., (1958) used this method in studying methyl ketones obtained from evaporated milk. Dutra‘gt al., (1959) also found this procedure was appropriate for study» ing the carbonyls obtained from evaporated milk. The 2 - 4 dinitrophenyl- hydrazones were identified by melting point determination and paper 15 chromatography. Methyl ketones formed from milk fat during steam.distil- lation and saponification were likewise identified as their 2 - 4 dinitro- phenylhydrazones by Patton and Tharp (1959). Similarly, Patton pt 5;” (1958) used this method for separation and aid in identification of the volatile carbonyls from cheddar cheese. [Pippen.gt‘gl., (1958) obtained 18 carbonyl compounds from a simmer- ing mixture of chicken meat and water by passing the volatile stream through a solution of 2 - 4 dinitrophenylhydrazine. The corresponding hydrazones obtained were separated by use of column chromatography. Kramlich (1959) also used 2 - 4 dinitrophenylhydrazine he an aid in identi- fication of the carbonyls in beef flavor volatiles. The hydrazones were chromatographed using paper. Dimick and. Makower (1956) reported on the use of 2 - 4 dinitrOphenyl- hydrazine as a method for determining the volatile carbonyls of strawb berries. Garbonyls were estimated by converting to their 2 - 4 dini- trophenylhydrazone derivatives, and forming colored complexes with these by addition of potassium hydroxide to an alcoholic solution of the hydro- zone. Monocarbonyls produced a red color, whereas dicarbonyls such as biacetyl were recognized by the appearance of a blue color. The organic and inorganic sulfur compounds present in a food, or those generated when it is cooked, are capable of imparting undesirable odors to the product. Kohman (1952) claimed that the characteristic onion odor (flavor) was due to allyl propyl disulfide which was liberated 'by'an enzyme when the raw onion cell was ruptured. This compound was steam distilled from the rest of the onion, oxidized to sulfate sulfur with.bromine, and determined by gravimetric or colorimetric means. 16 Dateolgt‘gl., (1957) trapped the volatile sulfur components from cooked cabbage employing a train of traps filled with anhydrous calcium chloride, lead acetate, four per cent aqueous mercuric cyanide and three per cent aqueous mercuric chloride. This trapping system removed water and aided in separation of organic and inorganic sulfur compounds. Pippen and Eyring (1957) characterized the volatile sulfur fractions of cooked chicken broth. Hydrogen sulfide was determined by the methylene blue method, which is specific for sulfide sulfur. The quantity of hydro- gen sulfide was determined colorimetrically, and total sulfur was deter- mined gravimetrically as barium sulfate. Patton.g§_gl., (1956) trapped methyl sulfide from the air space in a cold wall milk tank using mercurous chloride (1% aqueous) as the trap- ping agent. When collection was complete, the sample was treated with an equal volume of normal hydrochloric acid and warmed slightly. The odor given off appeared identical with that of methyl sulfide. Lead acetate was employed by Kramlich (1959) to trap the volatile sulfur compounds ob- tained from simmering ground beef. Sliwinski and Doty (1958) determined the amount of methyl mercap- tan in irradiated meat by use of N, N'- dimethyl - p - phenylenediamine. This reagent forms a red colored complex with methyl mercaptan. The a- mount of’mercaptan present was determined spectrophotometrically. These ‘workers found that hydrogen sulfide interfered with the colorimetric de- termination of mercaptan, but that it could be removed by precipitation with.nercuric acetate. Hornstein‘gt,§1., (1960) found that chemical identification of beef flavor components represented a satisfactory ap- preach to the problem. They identified several carbonyls, hydrogen sul- fide, and ammonia by various chemical means. 17 Heegen - Suit ,5 31..., (1949) identified various classes of chemical compounds present in the volatile constituents of grapes by use of ap- propriate chemical tests. The volatiles were collected in traps immersed in.e dry ice - ethanol mixture. The trap contents were saturated with . chemically pure ammonium sulfate and extracted with peroxide free other. The ether extract, after drying over anhydrous sodium sulfate, was dis- tilled to remove the ether. This other free liquid was then fractionated and tests performed on the various fractions. Several alcohols, carbon- yls, and acids were identified by this method. Ilackay and Hewitt (1959) utilized.emzymes present in cabbage and mustard for flavor development of reconstituted dehydrated cabbage. Flavor of these products, due in part to isothiocyanates, was released by treatment with thioglucosidases. Assay for isothiocyanates was made using gas chromatography and paper chromatography. This work emphasized the importance of enzyme liberation of flavor components. Olsen gt‘gl., (1959) determined ammonia in a distillate of several processed meats by use of Nesslers' reagent. Appropiate tests for other amines yielded negative results. Bouthilet (1951) identified ammonia as a constituent of chicken flavor by reaction of a steam.distillate of chicken.meat with benzene sulfonyl chloride. This reaction produced the sulfonemide of ammonia. mmmw Kremlich (1959) and Bernstein‘gt‘gl., (1960) used paper chromato- graphy to separate and identify the 2 - 4 dinitrophenylhydrazones Obtained by reacting volatile carbonyls from'beef with 2‘- 4 dinitrophenylhydrazine. A Likewise, Pippen‘g§,gl., (1958) and Pippen and Eyring (1957) have studied the flavor volatiles from cooked chicken using column and paper chromato- graphy and ion exchange chromatography, respectively. p 18 Clements and Deatherage (1957) and Lentner and.Deatherage (1959) em, ployed both column and paper chromatography in studies of the volatile components of roasted coffee. In addition, some non-volatile constit- uents were separated and identified by use of these methods. ‘Various methp yl ketones and other flavor compounds from evaporated milk and from milk fat have been separated and identified by use of column and paper chromatog- raphy (Wong gt _a_l., 1958; Patton and Tharp, 1959; and Dutra 93 33.4: 1959). Silicic acid chromatography was employed by Dimdck and Makower (1956) for identification of carbonyls Obtained from strawberry essence and by Henze gtpgl., (1954) for the determination of carbonyl compounds present in apple storage volatiles. in assay method for mustard oils in cabbage and mustard, outlined by Mackay and Hewitt (1959), employed paper and gas chromatography in an ef- fort to determine the amount of oils in these products. Distillation Numerous workers have employed various distillation techniques as an aid in the separation of food flavor components. Dateo §§'§;., (1957); Pippen gt 51., (1958); and Kremlich (1959) utilized distillation at at- mospheric pressure for the separation of volatiles from cabbage, chicken, and beef, respectively. Steam distillation has been accepted as an excellent technique for distillation of certain substances. This method allows components which have boiling points higher than water to be distilled from an aqueous mix- ture. Dacre (1955) used it for a study on cheddar cheese. Kohman (1952) noted it was a satisfactory method for removal of allyl propyl disulfide from onion. He found that a temperature of 121 degrees C. was necessary. Olson 33 21., (1959) obtained some of the volatile flavor components 19 from processed meat by use of steam distillation. The condensables were trapped in special glass traps immersed in ice water. Patton and Tharp (1959) found that steam distillation of milk fat produced a number of methyl ketones. These and other volatiles were trap- ped in two dry ice - ethanol traps. They maintained a system pressure of less than one millimeter and a temperature of 200 degrees C. Dutra gt 31., (1959) removed some flavor compounds from evaporated milk by steam dis- tillation. The steam distillate was concentrated about 40 to one in a modified Oldershaw - bubble plate column. Bouthilet (1950) employed a low pressure steam distillation appara- tus to separate volatile chicken flavor components from broth. In a later paper (Bouthilet 1951) he found that this method was suitable as an aid in fractionation of chicken flavor constituents. In view of the fact that many compounds are altered when heated to the temperatures necessary in the preceding distillation methods, a nume ber of workers have resorted to high vacuum - low temperature distilla- tion - (Haagen - Smit gt,gl., 1949; Patton gt‘gl., 1958; Merritt gt 91., 1959; Hornstein st 91., 1960; Stahl, 1957; Niegisch and Stahl, 1956; and Wong gt 31., 1958). Most of these workers employed cold traps of various temperatures to trap the distillates. In some cases, the components collected were separated by taking advantage of their volatility at dif- ferent temperatures. Johnson and Frey (1938) distilled ground dry coffee under high vac- uum, gradually increasing the temperature to 100 degrees C. Some re- ceivers for collection of the volatiles were cooled in solid carbon diox- ids and some in liquid air. Lookhart (1957) also used dry vacuum distil- lation, but the product was not heated. 20 Dimdck and.flakower (1951) described a laboratory scale continuous vacuum flash evaporator, a type of distillation apparatus. With this unit a juice or purse was concentrated and a water solution of volatile flavor' components was obtained. Olson gt,gl., (1959) removed some of the flavor components from de- fatted samples of heat processed beef luncheon meat with methanol. This is an example of solvent extraction of flavor components. Where this method is used, the solvent is generally removed by distillation or evapo- ration. m W3M mm. m __am1nfr Met 0 co Gas chromatography probably evolved from a study made by Martin and Synge (1941) who used two liquid phases rather than a gas phase and a liq- uid phase. Since that time, the method has been used for a variety of separation and identification problems (James, 1952; James and Martin, 1952; Ray, 1954; and James and Martin, 1956). Since its inception, this method has been employed for a number of flavor and odor problems. The components of the gaseous emanation pro- ducts of the onion were examined by Niegisch and Stahl (1956) using gas chromatography, mass spectrometry, and infrared spectroscopy. They idenp tified a total of six compounds. Bernhard (1958) found a number of comp pounds present in cold pressed lemon oil by the use of gas chromatography and infrared spectroscopy. Mackay and Hewitt (1959) assayed mustard oils from cabbage and mustard by using a gas chromatography instrument equip- ped with a one foot column. A short column was deemed necessary so as to avoid exposing the oils to heat for an excessive period of time. Patton‘s; 51., (1958) used a combination of gas chromatography and mass spectrometry to separate and identify the volatile components of 21 cheddar cheese. Day'gt‘§;., (1957b) and Patton gt‘al., (1956) identified some milk flavor components by use of gas chromatography. Tentative iden- tities for a fractional component from the various volatile mixtures were obtained by smelling the component as it emerged from the column and by comparing its retention volumes with those of known compounds. Dayggt gl., (1957b) also employed mass spectrometry as a tool for identification of the volatile components obtained from gamma irradiated skim milk. Hankinson 91 $1., (1958) found that gas chromatography provided a means for rapid and accurate analysis of the volatile fatty acids in milk. Similarly, Jennings (1957) applied this method to a study of the volatile flavor components present in a distillate of commercial dairy starter. The procedure for proper packing of a column for use in a gas chromatographic instrument was given by both of these authors. wynn.g§,§l., (1960) reported that gas chromatography could be employb ed as a means of detecting odors in milk. The claim was made that this method offers a valuable tool for the study of the effectiveness of deo- dorizing equipment as a means of removing the undesirable volatile fla- voring components of milk. Recent attacks on the chemistry of coffee aroma have been made using gas chromatography (Lockhart, 1957). This author claimed that the techni- que was applicable to all types of problems involving micro-concentration of odorants. Likewise Rhoades (1958) and Zlatkis and Sivetz (1960) one ployed gas chromatography as a means of separation and aid in identifica- tion of the volatiles obtained from coffee. 'Volatile mixtures were trap- ped in dry ice - ethanol or liquid air traps and subjected to analysis by infrared spectroscOpy and mass spectrometry as well as by gas chromatog- rephvo 22 Kremlich (1959) trapped the volatile components from simmering ground beef in traps immersed in liquid air, and subsequently analyzed the trap contents using gas chromatography. Infrared spectral analysis was also attempted but failed to yield any definite information. Merritt gtngl., (1959) separated the volatile components of irradiated beef into three fractions by use of a high vacuum - low temperature distillation assembly. This method makes use of the differences in the vapor pressure - tempera- ture relationships of the compounds present. Two of these fractions were separated by means of gas chromatography and the components identified by amass spectrometry. Stahl (1957) outlined a procedure for the investigation odfan un- known odor. He emphasized the fact that gas chromatography and mass spec- trometry are truly synergistic, the former being used as an elegant means for separation of a mixture, so that its components can be identified by mass spectrometry. He claimed that the identification of an unknown can be very difficult, when attempted by gas chromatography alone. The come bination of techniques employed by this author were adapted to a variety of flavor prdblems. During the past few years, gas chromatography as an analytical meth- od has been greatly improved. Column efficiency has been increased sever- al fold by use of coated capillary columns (Golay, 1958; Zlatkis and .Lovelock, 1959). More sensitive detectors have also been developed. The ionization detector described by Lovelock (1958) is claimed to be capable -12 of detecting concentrations of 2 x lo moles of most organic compounds. EXPERIMENTAL PROCEDURE The preliminary part of this study was undertaken to determine the tissue location, solubility characteristics, and volatilization tempera- ture of sex odor in pork. In addition, an experiment was conducted to de- termine if sex odor could be prevented in the carcass by an intramuscular injection of tranquilizer into the live hog. The final phase of this in- vestigation dealt with attempts to collect, isolate, and identify the agents responsible for sex odor in pork. PRELIMINARY STUDIES §o_m‘_g§ 9;, Marimgntal Material All boars used in this investigation were procured from the Michigan State University swine farm. Barrow and gilt fat was obtained from hogs slaughtered in the University Meats Laboratory. A considerable amount of the boar fat used was provided through the courtesy of Crown Packing Com- pany, Detroit, Michigan, while the remainder was obtained from boars slaugh- tered at the Michigan State University Abattoir. M g; m m Dgtection Tissues were examined for the presence of sex odor by heating small cubes in a skillet or boiling them in an Erlenmeyer flask with a small amount of distilled water. At least three individuals verified the pres- ence or absence of the odor. Wig; 2: m 993; Volatilizatiog Tempgraturg Lard, free of sex odor, was placed in a deep fat fryer and heated to an initial temperature of 60 degrees C. Boar fat samples were placed in a small amount of water in B'lenmeyer flasks (125 m1.) fitted with cork stop- pars. A short length of glass tubing was inserted into the stoppers and the flasks were partially immersed in the lard. The temperature of the lard was increased gradually and the heat equalized by agitating with an 23 24 electric stirrer. As the vapor emerged from the glass tube, the tempera- ture was recorded at which sex odor was first noted. Egamdnation g; Boar*Tissue§ Egg Organs £2; thg Pregencg g; Boar Odo; An 18 month old Yorkshire boar was slaughtered in the University Meats Laboratory. 'Various tissues and organs were removed for use in determine ing the location of sex odor. The carcass was boned and the lean and fat separated, frozen, and stored in a freezer at - 20 degrees F. Barrow lean and fat were similarly treated and used as controls. The following boar tissues and organs were examined for sex odor by use of the heat test described previously: loan from loin (devoid of vis- ual fat), heart, parotid gland, liver, lung, kidney, fat from the diaphragm, spleen, testes, Cowpers gland, seminal vesicles, sperm cord and its over- lying muscle, penis, diaphragm muscle (free of visual fat), tongue, and preputial diverticulum. 1.8ample of lean tissue from the loin of a boar was ground twice in a food chopper and blended with five parts of distilled water for three minutes in a waring blender. The resulting slurry was centrifuged for 20 minutes at 2100 revolutions per minute. After centrifugation, the super- natant was decanted, strained through cotton, and heated over a gas flame to determine the presence or absence of sex odor. The residue was removed from the centrifuge bottle by adding distilled water and stirring. It was subsequently tested for odor by heating. The lean tissues from a barrow were similarly treated. In order to determine the effect of water extraction on fatty tissue, a fat sample from a boar and fatty tissues from a barrow were blended with five parts of distilled water for three minutes. The resulting emulsion type slurry was centrifuged for 20 minutes at 2100 revolutions per minute. 25 The fat layer was broken and the aqueous extract strained through cotton. Both the fat layer and the aqueous extract were heated to ascertain the presence or absence of sex odor. mammahaiaamu nsit antacids: The two bellies from the Yorkshire boar were rubbed with three-fourths of an ounce of a curing mix per pound, which consisted of eight pounds of salt, two pounds of sugar, one ounce of sodium nitrate, and two ounces of sodium nitrite. After pressure curing for 14 days, the bellies were soaked for 30 minutes in water and dried. One bacon slab was stored in a cooler at 38 degrees F.; the other, after smoking, was stored similarly. Samples from both slabs were heat tested in a skillet after zero, 90, and 120 days of cooler storage. Thg‘gzgggggg‘ggg Solubility gropgrtieg g; Componentg Eggpongiblg fgg‘figg'ggg; ml frozen 100 gram sample of lean from the ham of a boar was shaved in- to small pieces and placed in a round, 500 milliliter flaSk. Moisture was removed from the sample by freeze drying for 26 hours employing a glass receiver immersed in a dry ice - ethanol mixture. The vacuum was produced using a Cenco Hyvac 7 vacuum pump. The frost which collected in the re- ceiver was allowed to thaw and was then heated to determine the presence or absence of sex odor. Similarly, some of the dried lean was rehydrated with distilled water and checked for sex odor. The remaining portion of the freeze dried lean was placed in a Goldfisch fat extractor and extracted for three hours using anhydrous diethyl other as the solvent. After ex? traction, the moisture-free, fat-free lean was rehydrated with distilled water and heated to check for the presence of sex odor. The extracted fat was heated in a small beaker over a gas flame and the volatiles coming off smelled to determine the presence or absence of sex odor. 26 In addition to the above procedure, samples of fat and loan from both boars and barrows were cubed, placed in 250 milliliter Erlenmeyer flasks and heated gently over a gas flame. The ensuing vapors were checked for the presence of sex odor. The solubility characteristics of the sex odor component(s) toward water and several organic laboratory solvents were determined. Extrac- tion of boar fat and lean with water was conducted according to the meth- od mentioned previously. Extraction of boar fat with organic solvents was accomplished by placing 200 grams of ground fatback in a Wering blend- er and blending for one and one-half minutes with an equal quantity of solvent. The blended samples were filtered first through two layers of cheese cloth and then through Whatman 41-H filter paper. water accumu- lated during the above process was removed by use of a separatory funnel. Solvents were removed by heating the solvent-fat mixture on a steam bath (1800 F. or less) until the odor of the solvent was no longer evident. The amount of extracted solvent-free fat was noted, and a portion was heated to determine the presence or absence of sex odor. TPhe following organic solvents were employed: acetone, carbon tetrachloride, chloro- form, diethyl ether, petroleum ether (B. P. 30 - 60° 0.), dioxane, and eth- yl alcohol. 4 _i__aa_Ntro nanomaanaaoimmaamaradmnl wet neutral litmus paper strips were held in the volatile stream from the heated boar and barrow fat and the color change of the paper noted. This indicated whether the vapor was acidic or basic. Since the test indicated a basic reaction, it was decided to conduct nitrogen determina- tions on the solvent rendered boar and barrow fat. These determinations ‘were made employing the Kjeldahl procedure outlined by the Association of Official Agricultural Chemists (1955). Saponification Both boar and barrow fat were saponified as an aid in their fraction- ation. Deuel (1951) suggested that fat could be saponified in the cold by several methods, one of which was treatment of the fat with sodium ethylate. This method was employed in this portion of the study since the sex odor appeared to be volatile. One hundred grams of boar fat were rendered by blending with 200 milliliters of diethyl ether for one and one-half min- utes in a flaring blender. The connective tissue was removed by filtering the ether extracted fat through four layers of cheese cloth. The filtered liquid was placed in a 2000 milliliter Erlenmeyer flaSk and 400 milli- liters of diethyl ether was added. Sodium ethylate was prepared by dis- solving 16 grams of kerosene free-metalic sodium in 200 milliliters of 95 per cent ethyl alcohol. Any alcohol that evaporated was replaced. When the sodium had dissolved, the resulting sodium ethylate was added and stir- red into the mixture of ether and fat. The flask was stoppered, shaken vigorously, and allowed to remain at room temperature for 24 hours or more. After saponification was complete, the liquid was filtered from the soap by auction filtration using Whatman 41 filter paper and a water aspirator. The soap was extracted once with ethyl ether, refiltered, and allowed to dry at room temperature. The ether filtrate, containing some soap and the unsaponifiable matter, was washed repeatedly with distilled water in a separatory funnel. If the ether and water layers were sluggish in sepa- rating, a small amount of ethyl alcohol greatly facilitated separation. If a complete emulsion formed, reagent grade sodium chloride was added to cause separation of the ether and water layers. After washing, the ether extract was dried overnight with anhydrous sodium sulfate, filtered, and reduced to a volume of a few milliliters. 'Volume reduction was 28 accomplished under vacuum at room temperature. Both the soap and the un- saponifiable matter were heated to determine the location of the sex odor. Injection 9; L112 m with "Digugl" my; Trflguiliger This portion of the study was conducted in order to determine the ef- fect, if any, of animal tranquilizer injection on sex odor suppression in live boars, and hence in the carcass. Four mature Yorkshire boars were restrained and a one and one-half by three and one-half inch fat sample taken by biopsy technique from the region of the last rib just off the mid line of the back. Five cubic centimeters of two per cent procaine hydro- chloride were used as an anesthetic. After removal of the biopsy sample, Diquel was injected through the biopsy wound into the‘LQggisgimgg‘ggggi muscle. The data are given in Table l. Table‘l Boa; weights and amount 2; tranguilizer administered miners; Milka). ammunecth+t 6 - 4 412 3.50 l - 6 407 3.75 17 - 9 583 4.50 13 - 11 392 3.75 * Each c.c. contained 50 mg. of ethyl isobutrazine (2 - ethyl - 3' - dimethyl - amino - 2' - propyl) lO phenothiazine hydrochloride) 9E §§§ 9993 COMPONENTS L91 Tgmfirgturg - gig}; W Distillgtion Attempts were made to collect the volatile sex odor component(s) from boar fat by a low temperature - high vacuum distillation assembly. The procedure was similar to that outlined by Merritt gt a;., (1959). One hundred and fifty grams of frozen boar fat were cubed and placed in a 29 round, 500 milliliter flask. The flask was attached to a glass receiver and then immersed in liquid nitrogen. The system was evacuated using a Oenco vacuum.pump. After evacuation of the system, the liquid air was re- moved from the sample container and placed around the receiver. Distil- lation was continued for a period of six hours. The frost and the dried fat were exposed to heat to check for the presence of odor. 9.8m Waste r 9.: 1.3413201 ’0 __a_e_0bt in d 1.31 __s_Hi h Teamsters AM Mar; Digtillation ‘Various modifications of this type of distillation procedure were at- tempted. Initially, boar fat and skin was placed in a round 500 milli- liter flask and heated for 60 minutes by means of an electric mantle. Temperature was controlled by use of a Power-Stat voltage rheostat main- tained at a setting of 60 volts. 'Volatile materials were passed through an air cooled condenser and the vapors trapped in a two normal solution of hydrochloric acid. The acid solution was heated and checked for sex odor. The apparatus was later modified by replacing the air cooled con- denser with a short length of insulated glass tubing. Instead of acid, the volatiles were trapped in a glass trap made of an Erlenmeyer flask containing a one-half inch layer of glass beads. The trap was immersed in a_dry ice - ethanol mixture. ‘Volatiles obtained in the trap were heated over a gas flame and the odor of the fumes noted. A.distillation system similar to that just described was assembled and the volatiles from boar and later from barrow fat were allowed to bubble through a glass tube immersed in diethyl ether. Heat was applied to the system for a 90 minute period. This procedure was repeated five times using a fresh supply of fat each time. The ether - water mixture was removed and separated by use of a separatory funnel. The recovered 30 ether layer was dried over anhydrous sodium sulfate and reduced to a vol- ume of one milliliter on a steam bath. The resulting solution was ana~ lyzed by gas chromatography using a 100 foot capillary column containing dinonyl phthalate oi‘Apiezon "L" as the liquid phase. Liquid samples were injected by means of a ten microliter Hamilton syringe. Additional distillation studies were conducted in an effort to obtain volatiles from boar and odor free barrow fat, which could be analyzed by gas liquid partition chromatography. A Barber-Colman, Model 20, Ioniza- tion Detection System, gas chromatograph was employed for this purpose. The instrument was equipped with a radium detector and a six way gas sam- pling valve. The latter device was manufactured by the Wilkens Instrument and Research Company, Walnut Creek, California. The design of the Barber- Colman instrument allows the use of packed or capillary columns. A distillation assembly similar to that employed by Kramlich (1959) was used in collecting the volatiles from boar and barrow fat. A charge of 500 grams of fat with skin was mixed with an equal quantity of dis- tilled water and placed in a three neck, 12 liter flask. The flask was fitted with a glass tube through which nitrogen gas was bubbled. Vola— tiles were passed through a small glass air cooled condenser and a short length of rubber hose into a glass spiral cold finger trap, which was immersed in a dry ice - ethanol mixture. The contents of the flask were heated for two hours using a gas flame. The volatiles were vaporized by heating in a hot oil bath at 145 degrees C. and were injected into the gas chromatography instrument by means of the six way gas sampling valve. Results of the above attempt dictated alteration of the assembly. Sample size was increased to about 2500 grams. An ice water trap was added between the condenser and the dry ice — ethanol trap to remove 31 water vapor from the volatiles. Glass beads were used in the ice water trap to increase the area for condensation. The nitrogen stream was re- placed by a stream of air from a laboratory supply line. After heating 'the fat for five to six hours, the trap contents were examined by gas chromatography. The wet ice trap contents were extracted with diethyl ether to obtain a sample suitable for analysis. A five foot, one-fourth inch capper column employing diethylene glycol succinate as the liquid phase and Chromasorb as the stationary inert phase was used for the sepa- ration of the volatile components. Other modifications of the above procedure included the following: 1. Replacement of the gas flame with a hot lard bath to prevent charring of the fat. The lard bath temperature varied between 140 to 150 degrees C. 2. Application of a mantle to the condenser to maintain the temperature at 105 to 110 degrees 0., and thus prevent premature condensation of the volatiles. 3. Removal of the skin from the fat before heating to avoid producing volatiles from this source. 4. Removal of the connective tissue from the fat to avoid production of volatiles from this source. Ten pounds of boar fat were ground through a one-fourth inch plate and then warmed slightly in a stainless steel, steamejacketed kettle. After heating, the slurry was strained through stockinette and two layers of cheese cloth. The connective tissue was dis- carded and the liquid fat placed in the 12 liter flask and heated as pre- viously described for the intact boar fat. 5. Removal of the wet ice trap to avoid trapping of the volatiles at this point. 32 6. Gas chromatographic separation of the volatiles obtained from boar fat and barrow fat using a ten foot, one-fourth inch copper column packed with diethylene glycol succinate on Chromasorb. Since it was postulated that the above described procedures might have exposed the fat to excess heat for a prolonged period of time, a dif- ferent type of collection assembly was set up. Boar fat without skin was cut into small cubes and 200 grams were placed in a one liter pyrex suc- tion flaSk. The tOp of the flask was closed with a cork stepper contain- ing an ”L" shaped glass tube. This tube was connected to a 100 milliliter suction flask immersed in ice water which acted as the wet ice trap. This trap was followed by a glass spiral trap immersed in dry ice - ethanol. The flask containing the sample was heated for 30 minutes on an electric stove using medium heat. Volatiles produced by heating were drawn through the traps by a slight vacuum. Components collected in the dry ice - eth- anol trap were separated using the ten foot diethylene glycol succinate column previously described. The preputial glands from the fat surround- ing the prepuce were also treated as outlined above. In other studies employing the above procedure, the dry ice~ethanol trap was removed. The flask containing the sample was recharged with 615 grams of fat for a total of ten times. The total aqueous distillate trapped in the wet ice trap at four to five degrees C. was saturated with sodium chloride and extracted with four - 50 milliliter portions of di- ethyl ether. The resulting ether extract was dried over anhydrous sodi- um sulfate. After drying, the ether solution was reduced under hydro- static vacuum to a volume of two-tenths of a milliliter and examined by gas chromatography using the following columns: 100 foot capillary di- nonyl phthalate; 100 foot capillary Apiezon "L"; or ten foot, one-fourth inch columns packed with flexol plasticizer, mannitol or silicone 200. 33 The inert phase used in the packed columns was Chromasorb "W". m ngling Methodg A 200 gram sample of boar fat was cubed, placed in a 500 milliliter Erlenmeyer flaSk and was heated on a hot plate. When the sex odor emerged from the flask, a five milliliter hypodermic syringe was inserted into the neck of the flask and a one milliliter sample of the gas drawn into the syringe. This was injected into the gas chromatographic instrument equipped with a 100 foot capillary column using Apiezon "L" as the liquid phase. A ten foot diethylene glycol succinate column was also employed and the gas sample size increased to five milliliters. This portion of the study was repeated and the fat was heated in a skillet on an electric stove rather than in the Erlenmeyer flask. §tg§QfDistillgtion.g§ Egg; Egg Chopped boar fat (500 g.) was placed in a two liter double neck round bottom flask. A steam distillation apparatus was assembled and the boar fat steam distilled for four hours, or until a distillate volume of about 900 milliliters was obtained. The distillate was divided into five portions and extracted with 50 milliliter portions of diethyl ether. The ether extract was dried over anhydrous sodium sulfate and reduced to a volume of one milliliter under vacuum at a temperature of 40 degrees C. or less. Barrow fat was similarly distilled and the extracts from both fats analyzed by gas chromatography using a five foot, one-fourth inch cOpper column packed with polyethylene glycol on Celite and a ten foot, one-fourth inch copper column packed with diethylene glycol succinate on Chromasorb. museums mmmnammmeimmm The contents of the preputial diverticulum of several boars were com- bined, filtered and the filtrate extracted with an equal quantity of 34 ether. This extraction was repeated and the combined ether extracts were washed with distilled water. After evaporation of the ether, some of the residue was smelled before and after heating, and some analyzed by gas chromatography using a 100 foot capillary column coated with Apiezon "L". ngtg fgg‘thg Egggencg of Agmonia, Carbonyls, gpg Splfg; Compounds ip.th§ 'Volatilg‘Distillatg Eggg Egg; Egg Ammonia test paper was used to test for ammonia in the volatile stream coming from the distilling flask containing boar fat with connec- tive tissue. The paper was prepared by mixing ten milliliters of 20 per cent silver nitrate solution with five drOps of 40 per cent formalin and a few drops of dilute sodium hydroxide. This mixture was filtered and the filtrate immediately absorbed on strips of Whatman 1 filter paper. After drying, the paper was then ready for use. In the presence of am— monia the paper turns black. Carbonyls distilled from intact and from rendered boar fat were de- tected by paséhu; the volatile stream through a solution of 2 - 4 dini- trophenylhydrazine (2 g. per liter in 2 N HCl) and by mixing the contents of the dry ice trap with the reagent. A yellow precipitate indicated the presence of carbonyls. Sulfur compound(s) were detected by passing the volatile stream through or mixing the dry ice trap contents with a saturated solution of basic lead acetate. ae—Lasucmmtor h mmwmmmmmm The process of saponification and recovery of the unsaponifiable mat- ter was given in the first section of the experimental procedure. The unsaponifiable matter from boar and barrow fat was heat tested and ana- lyzed by gas chromatography using a ten foot, one-fourth inch copper 35 column packed with diethylene glycol succinate on Chromasorb or silicone SE - 30 on Chromasorb "W". Several compounds suspected of being present in unsaponifiable matter were obtained in pure form and subjected to gas chromatographic analysis. Their retention times were compared with those of peaks produced by similar analysis of the unsaponifiable matter. 92% Chromatography of Ungaponifiable Mam m M £31. A 161 milligram sample of unsaponifiable matter obtained from boar fat was subjected to silicic acid chromatography employing the method of Hirsch and Ahrens (1958). The column was 253 millimeters long and 23 millimeters in inside diameter. It was packed to a depth of 150 milli- meters with 325 mesh silicic acid. The bottom of the tube was fitted with a sintered glass disk. The type and volume of eluents were the same as those recommended by Hirsch and Ahrens (1958) for the stepwise elution of complex lipid mixtures. m—mXCh-‘t'omator h mwmmwmm W Paper chromatograms of the intact unsaponifiable matter were prepared on sheets of Whatman 1 filter paper. The chromatographs were made in an attempt to separate any free alcohols and aldehydes and/or ketones. The solvent system for the alcohols was n - butanol, ethanol, and water (4:1:5) while hexane and chloroform (9:1) was the solvent system for the free alde- hydes and ketones. The free carbonyls were chromatographed on paper sprayed with a dilute solution of sodium bisulfite. Attempts to prepare chemical derivatives of the alcohols were made by reacting three aliquots of unsaponifiable matter with 3 - nitrOphthalic anhydride,<> Hfloo .0 OAVmHOOOOOOOOOOO:0O CO waHOeeooooooggn—Hoo .o OOmH...mopmon mmmah .ohspmaomeoa : seemesosso so assesses .sa m.x .sa oa...sssaoo .afls\.ds on...opma scam nomad ha...AHmmv onswmoam mmo maevwaoaoaa m.H...oaam ngEsm new moon scam opsaafipmfip msoosws mo possess heave...oamsmm #H NH OH w 0 N 9.-.: . T1. » . L _ . b o m e m s OH mm _ Us a _ I MOOOoooohpnfipHPngmm esuodSeg xepxooeg . 0 at ‘ n . a . . 49 new resume scam opsHHHpmfiv pee msooswm mo poempxo Heaps mo Esamopcfionmo mmo Amopsmfisv oaflp mowpmopom OH (F NH r—OO LO Q. m.......asa>apaasem case...emepaoe Haoo 00 oquOOOOOOOOOOOHu—Hmo OD CONN-HOOOOOOOOOQEHOO .o OmH...mopao: mmmam o «oasesmmmmoe a npomsaoazo so Hepfisasz .cfi M_K .pm oa...:asfloo .cfis\.as ow...opea scam momma ha...AHmmv redeemed mew mmopfifionofia m.H...oNfim magnum acumen 50am opmaafipmfip mzoosdm mo possess Ho:9o...oamasm .HH omswwm w-‘ dents “H.- _ pl.- esuodseg iepxooeg 50 a ten foot packed column. No peaks were Obtained when the latter column was used. It is questionable whether capillary columns are capable of handling gas samples, since the volume of free space in columns of this type is so small. It would seem that if the sex odor component(s) were trapped at all by the direct sampling methods, they were not sufficient- ly concentrated for detection. mums 2!: $222 D_______sis’°illat 9.11 has: £319. When 500 grams of boar fat were steam distilled for four hours, a distillate of about 900 milliliters was obtained. The ether extract of the distillate appeared to be colorless, but a yellow color was noted when the volume was reduced to one milliliter. Barrow fat was also light yellow when similarly treated. .The aqueous distillate had a "sulfury" or "eggy" smell with overtones of ammonia. When heated, no indication of sex odor was noted. After ether extraction, the aqueous distillate still retained the sulfur-ammonia odor. Attempts to reconstitute sex odor by injecting a small volume of the ether extract into sex odor-free barrow fat and subsequently applying heat were unsuccessful. These re- sults, coupled with an absence of sex odor in the steam distillate, in- dicated that sexual odor pg; pp. could not be isolated and concentrated by the steam distillation method employed in this study. Gas chromatographic analysis of the ether extract from the steam distillate did not reveal any differences between boar and barrow fat. In fact, only one peak other than the solvent peak was noted. This was true using both a five foot polyethylene glycol column and a ten foot diethylene glycol succinate column. Matthew uti amadaemaamtmilmmmmmmm Dutt gt 3;" (1959) claimed that the preputial glands were the site of odor production. If this is true, heating of these glands should 51 produce a more intense odor, unless it is distributed in the fatty tis- sues as rapidly as it is produced. On heating of the preputial glands or the fat surrounding the preputial diverticulum, the apparent sex odor was no more intense than that Obtained from heating boar fat alone. Gas' chromatographic analysis of an aqueous distillate produced from the pre- putial glands and the surrounding fatty tissues failed to show any difb ference from that Observed when backfat from a boar was similarly ana- lyzed. Apglypig pf ppg Contents pfpphg Preputig1.Diverticulum from fipgpg The preputial diverticulum contains a liquid with a foul smelling odor. As previously mentioned, this odor is sometimes designated as "the" sexual odor, although it appears to be distinctly different. Since the present study had indicated that the sex odor compOnent(s) was/were sol- uble in ether, this solvent was used to extract the contents of the pre- putial diverticulum. After evaporation of the ether, a urine-like smell was Obvious. When the residue was heated, the same odor was noted except it was more intenSe. There was no indication that sex odor was present. When the extract was analyzed by gas chromatography, only the selvent peak was evident. Most of the material in the preputial diverticulum is prObably water soluble, and one would expect a rather small quantity of ether soluble material. The urine smell was present in the ether extract, prObably due to the fact that some of the odorous compounds in the urine were soluble in ether and were removed by the extraction process. Presence pf Ammoniaz Carbonyls and Sglfgg Compounds $3.223'Volatile Distillate from Boar ESE When ammonia test paper was held in the volatile stream emerging from heated boar fat, a black color was immediately noted on the paper. The 52 color reaction indicated that ammonia was being evolved from the heated fat. The ammonia, no doubt, arose from the decomposition of some of the amino acids which make up the connective tissue of the fat. Tests for ammonia were negative when the vapors from connective tissue-free boar fat were checked. It was suspected that a number of carbonyls were formed during the dis- tillation procedure in which the fat was heated for five hours. When the contents of a dry ice - ethanol trap were reacted with a solution of 2 - 4 dinitrOphenylhydrazine, a yellow precipitate was immediately Obtained, in- dicating the presence of one or more carbonyls. Paper chromatography of the 2 - 4 dinitrOphenylhydrazones, using petroleum ether (35 - 600 C.) and ethyl ether (95:5 V/V) as the solvent system, produced three spots. No attempt was made to identify the compounds responsible for the spots. When the volatile stream from heated, rendered, boar fat was passed through a solution of 2 - 4 dinitrOphenylhydrazine before being trapped in the dry ice - ethanol trap, a yellow precipitate was produced. Again, no attempt was made to identify these hydrazones, but the presence of carbonyls was definitely indicated. Gas chromatographic analysis of the volatiles af- ter removal of the carbonyls showed that several peaks had disappeared. Tests for the presence of sulfur compounds were conducted similarly by reacting the trap contents with lead acetate solution. The absence of a black precipitate indicated that sulfur compounds were not present. Gas chromatographic analysis of the volatiles after passage through the lead acetate solution indicated that no components had been removed. These results were not unexpected since the fat used in these determinations was free of connective tissue, and there were prObably no sulfur containing compounds present. 53 Presence 9; the §§§ Qgg; Componentfsl in the Unsaponifiable Fraction Initial saponification of boar and barrow fat produced an unsaponi- fiable fraction which was oily and appeared much like liquid fat. In addition, the amount of this fraction obtained was in excess of that ex- pected. It was assumed that complete saponification had not been accoms plished. Analysis of the fractions from the two types of fat by heat test and gas chromatography failed to show any distinct differences. Both fractions smelled rather sickly sweet, somewhat suggestive of esters. Heating of the soap by either wet or dry heat did not produce any olfac- tory response reminiscent of sexual odor. However, since complete saponi- fication had apparently not been attained, the results of the above analy- ses were probably unreliable. The saponification method used in this study was considered quite suitable since at no time during the procedure was it necessary to heat the saponification mixture. This gentle treatment prevented destruction of the apparently heat-labile sex odor component(s). Thus, subsequent saponification of boar and barrow fat was conducted so as to attain com- plete formation of soap from the fatty acids. The unsaponifiable matter obtained was yellow in color, and the yield amounted to one to two-tenths of one per cent of the fat. This fraction was stored in a refrigerator, samples being removed for use as required. Chilling caused the entire fraction to solidify and after a time patches of white crystals appeared dispersed throughout the solid mass. It was postulated that these crys- tals were cholesterol, and microsc0pic examination showed them to be identical in color and shape to known cholesterol crystals. Unsaponifiable matter was tested for the presence of sex odor by placing a few small draps (about ten microliters) on the hot inlet port 54 of the gas chromatograph. When the unsaponifiable matter was heated, a permeating, concentrated sex odor was immediately noted. .A small amount of this fraction was injected into a piece of sex odor-free barrow fat. When this fat was heated in a skillet, the characteristic sex odor was detected. Heated unsaponifiable matter from barrow fat produced an odor I which was somewhat sweetish, but it was not the characteristic sex odor and could not be classed as objectionable. Analysis of the unsaponifiable fraction from boar and barrow fat using a ten foot diethylene glycol succinate column was not successful. On one occasion, several peaks were obtained but they could not be re- produced in subsequent trials. In addition, it was impossible to mainp tain a stable base line on the recorder. This was thought to be due to bleeding of the liquid phase caused by exposing the column to a tempera- ture of 240 degrees C. The maximum temperature recommended for this type of column is 250 degrees 0., but considerable column bleed probably occurs at a temperature of 240 degrees C. The six foot, silicone SE~30 column, which can Operate at tempera- tures up to 300 degrees 0., indicated a number of compounds were present in the unsaponifiable fraction. However, essentially no differences could be noted between the two fats even though the sex odor was defi- nitely present in the unsaponifiable matter from boar fat. A few minor differences were noted at times, but no real and consistent difference seemed to exist. These results were rather surprising. Perhaps no peaks were obtained because the heat of the column chamber destroyed the sex odor components and produced fragments which could not be observed on the chromatogram. Several chromatograms were run at 110 degrees 0.; however, only a few peaks were produced at this temperature and those Obtained were 55 similar for both types of fat. At the lower temperature poor resolup tion was obtained, apparently, due to the presence of the high boiling components which would mask other compounds. Typical chromatograms using a 250 degree column temperature are shown in.Figures III and IV. [All peaks shown are represented as actual size except peak number 13. The width of this peak was reduced to one- half its original size. The component responsible for this peak seemed to constitute the major portion of the unsaponifiable fraction. This was assumed to be cholesterol since this compounds is known to be a component of the unsaponifiable fraction of fats. Analysis of pure cholesterol by gas chromatography produced a peak which compared in shape and retention time to the unknown peak observed in the unsaponifiable matter (Figure V). Further evidence for the presence of cholesterol was shown by the fact that a positive test for £3 - 5 sterols was obtained, when the white crys- tals from the unsaponifiable matter were dissolved in chloroform and re- acted with acetic anhydride and concentrated sulfuric acid (Liebermann - Burchard reaction). The positive test was shown by the color of the re- action mixture, which varied from red to purple to bluish green. Cholesterol is a known component of the unsaponifiable matter from both boar and barrow fat. Hence, it was not expected to be one of the agents responsible for sex odor in boars. When cholesterol was heated, the undesirable sex odor was not observed. Squalene is also known to be a component of the unsaponifiable frac- tion of some fats. Analysis of a sample of squalene by gas chromatography (Figure VI) indicated that this compound had a retention time which come pared to that of peak number 12 (Figures III and IV) present in the chro- matograms of the unsaponifiable matter from both boar and barrow fat. No 9mm anon scam Havens oHQeHMfimommmos mo amawopssoano mac .HHH cadmfim Amopsnfisv asap cowpmopom mm mm 0H NH 0H m o ill—ill} . 5.--)»: LI . --.-!- . _ _ - . .. - .d e m. m. mu , as U. a it 9 NH O 1. m I S T: Z 9 6 _. 5 l __ ma Axmmm pmsa pom m op emoswmav OH......th>fl¢Hmnmm omsa...mwapao> Heme Co ONQNOOOOOOOOOOOHHQO Co HmNOOOOOOOOOEHOO .o ommm...ampaas eases o «manpenmgame a naommsoamo no omnmm Ammv mucosafim .sfi «.K .pm w...:ssfioo .sfis\.ds Mb...owma seam nomad NN...AHmmV masmmoam mac Hepadoaods o.H...onam camsnm amppma mapefimfinommmcs new anon...oamamm 'nl. IIJIII . -—u--. nn-as- a..- u . esuoosdfi;xepxooeg 57 mm ezrs Tendon %.qqprm x995 ma paw schema scam scenes oprHMHcomsmn: Mo amamopesoamo mac .>H ohsmfim Ammpsnfisv asap soapsopom mm 0H NH OH w o [m p a.“ L In . ll' ..Lfl..f|alll'i'. J. I‘ll-I II..- III I. 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I “-.- -- -‘_ -—_-.—_--—v*— I I esuooseu xepxooeu .amgpm oH HoaopmmHono mo smawopmsoano new .>.masmHm AmopscHsv osHp GOHpsopom mm mm NH 0H m 0 Q N 1 till P _ n n p p L I.- 1111 Home pampHom HoampmmHono 58 m.......spa>apamemm cemH...mmasHo> HHmo .0 OHONOOOOOOOOOOOHHwO 00 ONLNOOOOQOoecHHF—HHHOU .o Ome...nmpwmn ammHm «waspwaemsma a snowmaoneo so Ammv om-mm meooaaam .ea «.x .pe e...aasHoo .qHs\.HE hm...opma aon cown¢ mN...AHmmv wasmmoam mac mampHHOAOHa o.N...mNHm oHQEmm HoaopmmHomo...onsmm @1943 AJ— asuousai xepxooeg .ocopcom 2H msonsem mo asamopmsoaso new .H> oHsMHm Ammpansv csHp oOHunopmm oH qH NH 0H m o q N . L i .. L , LEE - r 111 t {I’lkllizi a e . Tm” [— a a. T. T. as e A n... u W 9 9 n... M. 8T M .m m m _ m > _ V I a s i. m e e u I\ I\ m H a f m w m u m a . _ a g T “.1 _m a m S o n u m.......§atm§ _ OOmH...owapHo> HHoo m_ 00 OHONOOoOOOOOOOO-fiufimo _ 00 Obfioooeooooognfioo . .o me...hmpwon AmmHm I "i o «macadamgsme _ s psoaaaoaeo so omumm ocooaHam .ea a.x .se e...essaoo .:H£\.HE 5w...mpwa BOHM nomad . mN...AHmmV wasmmonm new _ myopHHoaoHa m.o...onHm onswm m oconmwm...ngsmm i234 60 attempts were made to identify squalene by other qualitative means. Heat- ing the known compound produced a rather odd, sweet odor, but it in no way resembled sex odor. Thus, of the compounds present in the unsaponifiable fraction, it seems justifiable to eliminate cholesterol and squalene pg; fig, as being responsible for sex odor. It is still possible, however, that they may contribute to the deve10pment of sex odor by combination with other component(s) present in this fraction. Since squalene and cho- lesterol were present in both boar and barrow fat, it seems unlikely that they are responsible for sex odor. Fractions Obtained by Column Chromatography 2f Boar Eat Ungaponifigblg Mgttg; Hirsch and Ahrens (1958) list eight eluents varying in polarity from one per cent ethyl ether in petroleum ether to absolute methanol. When the unsaponifiable matter from boar fat was separated on a silicic acid column, three fractions were obtained. The first fraction was eluted with 200 mil- liliters of 25 per cent ethyl ether in petroleum ether (step 6). It was yellow in color and was present in a very small amount. Pure ethyl ether (step 7) eluted the largest fraction of the three. This fraction also was yellow in color and amounted to about 85-90 per cent of the charge placed on the column. On using pure methanol as the eluent (step 8), a small amount of yellowish brown liquid was obtained. When the three fractions were exposed to a heat test, it was found that the fraction eluted with ethyl ether contained the sex odor component(s). The fraction eluted in step 6 had a trace of the odor but it was absent in the material eluted in step 8. Gas chromatographic (silicone SE-BO column) analysis of the three fractions did little to increase our knowledge of the problem. A few peaks were noted from the material eluted with 25 per cent ethyl ether 61 in petroleum ether and with absolute methanol (steps 6 and 8). The frac- tion obtained by elution with ether (step 7) appeared to be very similar to the unfractionated unsaponifiable matter. Thus, the separation of un- saponifiable matter was not complete when undertaken with the eluents em- ployed. It is possible that a different series of eluents could be used to achieve a more efficient separation of the unsaponifiable fraction in- to its components. nggg Chromatography agd Chemical Derivatives 2f Eggaponifigble Mattgg Paper chromatography of the intact unsaponifiable matter yielded one large yellow spot with a Rf of 0.94 when using a solvent system recom- mended for free alcohols. Two dimentional paper chromatography of this spot (Rf 0.93) failed to show that other spots were present. Paper chro- matography of the 3-nitrophthalates of the alcohols produced one spot, which moved only a short distance from the base line and had a Rf value of 0.012. The crude derivatives of the unsaponifiable matter obtained with 3-nitr0phthalic anhydride and o