,x-grr; (La;— . . 5.». , 1 n u\.: = 1 .1. ed .‘éJ .s‘ .3. ,c. .g v - t L {“5“ i . 4r ‘1’ mad 5 ”.5,an THESE LIBRARY Michlgan State Univermty ROOM USE ONLY ABSTRACT THE EFFECT OF CRUDE COTTONSEED OIL AND VITAMIN E ON THE ABSORPTION OF FATTY ACIDS FROM THE GASTROENTESTINAL TRACT OF THE LAXING HEN by Stephen W. Baum Addition of crude cottonseed oil to a hen's ration caused cold storage eggs to develop pink whites and salmon colored yolks.‘ The stearic acid to oleic acid ratio of these "cottonseed" eggs was higher than that of normal eggs. The Change in the stearic acid to oleic acid ratio could have been caused by changes in the selective deposition of fatty acids in the egg, prevention of dehydrogenation of stearic acid to oleic acid in the liver, changes in the selective absorption of fatty acids through the intestine, or a combina— tion of these. Evans 3; a1. (1962), also confirmed in this thesis, have shown that plasma, livers, and ovaries from hens fed rations containing crude cottonseed oil contained more stearic acid and less oleic acid than similar tissues from hens fed the basal ration only.‘ The ovary contained a smaller percentage of stearic acid than the other tissues. As the percentage of these fatty acids in the total fatty acids of the livers, plasma, and ovaries changed in a like Dtephen W. Baum manner and did not change by the greatest amount in the ovary, the "active” factor in crude cottonseed oil is not only causing changes in the selective deposition of stearic acid and oleic acid in eggs. Laying hens were fed a basal ration, the basal ration plus 3% crude cottonseed oil, the basal ration plus dl-CY-tocopheryl acetate, or the basal ration plus crude cottonseed oil and dl-cr-tocopheryl acetate. The hens were sacrificed, intestines removed and divided into three equal lengths, the contents removed, and the relative concentrations of fatty acids in the intestinal contents measured. Absorp- tion was followed by measuring the disappearance of fatty acids from the intestinal contents. Ovaries, livers, and plasma from hens fed the supple- mented rations contained more stearic acid and less oleic acid than those from hens fed the basal ration. Fatty acid distribution in tissues from hens fed the basal ration plus crude cottonseed oil and hens fed the basal ration only usually differed more from each other than that from hens on the other rations. More stearic acid and less oleic acid was generally absorbed from the intestines of hens fed crude cottonseed oil containing rations than were absorbed by hens fed the basal ration. The relative stearic acid and oleic acid absorption agreed with the relative amounts of these acids found in the plasma, livers, and ovaries. Stephen W. Baum When laying hens were fed a ration containing dl—a — toc0pheryl acetate, there was a rise in the stearic acid to oleic acid ratio of their plasma, livers, and ovaries over the ratio found in similar tissues from hens fed the basal ration. nens fed the basal plus crude cottonseed oil and dl-cr-toc0pheryl acetate had a higher stearic acid to oleic acid ratio in their tissues than that from hens fed the basal ration plus dl-CY-tOCOpheryl acetate. 1 TH' EF"ECT OF CRUDE COTTOKSEED OIL AND f VITAMIN E ON THE ABSORPTION OF FATTY ACIDS FROM THE GASTROENTESTIEAL TRACT OF THE LAYING~ HEN By Stephen W. Baum A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Biochemistry 1965 ACKNO WLEDGIVEENTS I have a deep appreciation and sense of gratitude to Dr. R. J. Evans and Dr. S. L. Bandemer for their encourage— ment, assistance, and criticisms. Their help throughout the duration of my present studies was invaluable and can not be adequately acknowledged. I should like to extend my sincere thanks to Dr. R. W. meke, Dr. R. G. Hansen, and Professor J. A. Davidson for their help and suggestions. I am deeply grateful to the National Cottonseed Products Association for financing the research project. Cooperation and encouragement by my other colleagues in the laboratory is deeply appreciated. ii Table of Contents Page nQflvomEmm-«IELQTS 0.00.00.00.00.000000000000000.0.0.0... ii List of Tables ........................................ iv List of Figures ....................................... V I-"J'.st of Appendices .................................... Vi INTRODUCTION .......................................... l EXPERIMENTH. .......................................... 9 Organization ........................................ 9 Collection of Samples ............................... lo Extraction and Methylation .......................... lO Gas-liquid Chromatography ........................... 12 RESULTS ............................................... 13 Crude Cottonseed Oil ................................ 13 dl-a-toc0pheryl acetate ............................. 29 Minor Peaks ......................................... 34 VISCUSSION ............................................ 35 .‘Fatty Acid “bsorption ............................... 35 Fatty “Cids in Tissues OCOOOOOOOOOOOOOOOO0.......0... 37 dl-a-tOCOthIYI acetate o...o0.0.00.00000000000000000 39 SUMMARY oooooooooooooooooooooooooooooooo0.000.000.0000. Tl 1.192;;ka um cxmn .00......0.0.00.00000000000000‘.0...... 4‘) ‘QPPELQDICES OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOQOOOOI 47 iii TASLE 10. 11. LIST OF TASLES {atty “Cid Composition of yotal fatty ticj-ds in the “ations .C....................... Fatty Acid Composition of Total Fatty flCids in the Qlood Plasma of Hens fed Various Rations .QQQ‘0.0.COOOOOOOCOCOOCOOOOOOCO fatty Acid Composition of Total “atty Acids in the Livers of Hens fed Various £"3tions 00.0.0.0....OOOCOOODOOOOOOOOOO00...... Fatty “Cid Composition of Total Fatty Acids in the Ovaries of Hens fed various Rations O..0........O......................... Eatty Acid Composition of Total Fatty Acids in bgg lolks of hens fed Various Rations ..... DiStribution Of bl? fiat—t}, dCidS .ooogooooooooo ‘atty Acid Composition in the Contents of the uastroentestinal Tract and Selected Tissues from nens fed the Basal Ration only... batty “cid Composition in the Contents of the uastroentestinal Tract and Selected Tissues from nens fed a Ration containing “rude Cottonseed Oil ......................... batty “Cid Composition in the Contents of the Uastroentestinal Tract and Selected Tissues from nens fed a Ration containing dlw(-Tocopheryl Acetate ...................... fiatty Acid Composition in the Contents of the uastroentestinal Tract and Delected Tissues from Hens fed a Ration containing “rude K"ottonseed Oil and dl«(—Toc0pheryl acetate 00.0.00...00......0.000000000000000... The Effect of the “uration of feeding “rude “ottonseed Oil on the tatty “Cid A Distribution in Plasma, Liver, and Uvary ..... iv 15 17 18 19 2O 21 25 27 28 FIGURE 1. List of Figures The Percentage of Total Fatty ACids Stearic Acid in the in the J-ntestinal Contents and some Tissues ................ 22 The Percentage of Total Fatty Acids Lontents and some The Percentage of Total Fatty Acids Contents and some Oleic Acid in the in the Intestinal Tissues OOOCOOOOOCOOOOOO 23 Linoleic-Acid in the in the Intestinal Tissues ................ 24 APPENDIX 1. LIST OF APPLSJICLS *atty Acid Composition of Total batty ACids in the “atiOnS 0.0000000000000000. ‘atty Acid Vomposition of Total L“atty Acids in the “lood Elasma of “ens fed fiarious “ations Satty Acid Composition of Total Fatty Acids in the Livers of Hens fed Various Rations 0.00.0000...OOOOOOOOOOOOOOOCOOOO batty ncid Composition of Total Fatty acids in the Ovaries of Hens fed Various Aations fatty Acid Composition of Total I-‘atty “cids in bgg iolks of dens fed Various Aations ooooooooooooooooooooooooooooocoo zatty Acid Composition in the Contents of the gastroentestinal Tract and from Hens fed the Sasal nation only ...................... Qelected Iissues ratty “Cid K"Opposition in the “ontents .of the bastroentestinal Tract and ”elected Tissues from £1ens fed a Basal nation plus 35 Crude Cottonseed Oil O000.......0....OOOOOOOOOOOOOCCOOOOC Eatty dCid Composition in the Contents of the °astroentestinal Tract and Selected Tissues from “ens fed Basal Ration plus dl- -Toc0pheryl Acetate .... fatty Acid Composition in the Contents of the Sastroentestinal Tract and Selected Tissues from hens fed Dasal Ration plus 3% Crude Cottonseed Uil and dl- -‘oc0pheryl “cetate ................ vi 47 51 52 58 INTRODUCTION The discoloration of egg yolks from hens fed ground cottonseed was first reported by Roberts and Rice (1891). A black, brown, or olive color develOped in the yolks during cold storage (New Mexico, 1927). Schaible, Moore, and Moore (1934) showed that gossypol, a yellow toxic pigment in cottonseed, gave rise to the olive color. Ferric iron from the yolk proteins formed an olive colored complex with gossypol (Swensen g; a;., 1942). Sherwood (1928, 1931) observed salmon colored yolks and pink egg whites in cold storage eggs. He showed that the substance causing the discoloration was in crude cotton- seed oil and partially refined cottonseed oil, but not in extracted cottonseed meal, soapstock, or highly refined cottonseed oil. Upp (1932) showed that there were no bacteria or molds in the discolored eggs. Almquist and Lorenz (1932) observed that when pink egg whites were fractionated with ammonium sulfate, a pink colored substance showed up in the albumin fraction. These~ same researchers showed that coagulation of the egg proteins caused this color to disappear. Sherwood (1931) observed that the water content of the salmon yolks increased over that of normal yolks while the percentage of protein 2 remained constant. He concluded that albuminous material was probably traveling from the white to the yolk. Schaible e§_a1. (1946a) confirmed the previous observation. They showed that eggs frozen in the shell or separated yolks and egg whites from hens fed crude cottonseed oil did not discolor on cold storage. They concluded that something was traveling from the yolk into the white and.yig§ Eggga. They showed (1946b, 1946C) that conalbumin from egg white combined with the ferric iron from the yolk to form a com- plex. The salmon color is produced by the blending of the pink color of the ferric-conalbumin complex with the normal yellow pigments of the yolk. They, also, showed (1946a) in contrast to the observations of Sipe (1931), that pink egg white developed in eggs from hens fed non-cottonseed rations when in cold storage for a period of twenty to forty-two months. This is five to ten times longer than is necessary to develOp the same color in eggs from hens having crude cottonseed oil in their rations. Schaible 3;.Ql- (1946c) believed that there was something in crude cotton- seed oil that caused the weakening of the vitelline membrane. The Halphen test (A.O.A.C., 1955) is an empirical color reaction used to identify cottonseed oil in the presence of other oils. It is carried out by heating a sample of oil with isoamyl or amylalcohol and a solution of sulfur in carbon disulfide. “ red color indicates a positive result. Lorenz (1939) isolated a fraction from crude cottonseed oil that gave a positive Halphen test and caused 3 eggs from hens fed it to develop pink whites and salmon colored yolks on storage. No other type of egg discoloration was noted. Dijkstra and Duin (1955) reported that ‘Etezgulla fgetida seed oil gave a positive Halphen test. Nunn (1952) and Faure (1956) proved that the material in E. fgegida seed oil that gave the positive Halphen test was a cyclOprOpene fatty acid, sterculic acid, with a structure Ca3(c32)7_C§g-(CH2)7COOH, MacFarlane et a1. (1957) discovered 2 a Halphen positive cyclopropene fatty acid from Malvg gfigyijlgga, mavalic acid. They showed that its structure C—(CH2)6COOH. Masson et a1. (1957) reported H 2 that when sterculic acid was fed to hens it caused pink A CH — = was 3(CI-12)7 Cb; whites and salmon colored yolks of eggs kept in cold storage for at least one month. According to Shenstone and vickery (1959) about one-quarter of the ingested cyclOprOpene fatty acids was deposited in the egg yolk. They found that cyclo- prOpane fatty acids did not give positive Halphen reactions and when fed to hens did not cause pink whites and salmon colored yolks in eggs on cold storage. They, also, observed that cyclopropene fatty acids caused some egg white proteins to diffuse into the yolk. Shenstone and Vickery (1961) later found that crude cottonseed oil contained 0.7% - 1.5% malvalic acid and 0.3% - 0.5% sterculic acid. Shenstone and Vickery (1956) reported that cycloprOpene fatty acid soaps have strong surface action prOperties that might weaken the vitelline membrane. 4 Rinehart g; a1. (1959) and Masson (1959) observed that sterculic acid slowly polymerized at room temperature and the polymers formed underwent no reaction with the Halphen reagent. Evans 3; 3;. (1960) showed that crude cottonseed oil that had been heated to 2000C gave a negative Halphen reaction, but still produced pink whites and salmon colored yolks in cold storage eggs. Evans g; a1. (1960) said that this showed that either cycloprOpene polymerization products or something unrelated to cyclopropenes could be the "active" factor(s) in crude cottonseed oil that produces pink‘whites. Ellis ggyal. (1931) reported that feeding Crude cottonseed oil to hogs caused the stearic acid and linoleic acid of their backfat to increase at the eXpense of the palmitic and oleic acids. Evans gt a1. (1961) observed that stored eggs produced by hens fed crude cottonseed oil or Stercglia fggtiga seed oil in their ration contained more stearic acid and less oleic acid than stored eggs produced by hens fed the basal ration. The stearic acid to oleic acid ratio was 1:4 or 1:3 in normal eggs and 181 or 181.5 in eggs with pink whites and salmon colored yolks. This change in the stearic acid to oleic acid ratio could be caused by the "active" fraction in crude cottonseed oil causing a change in the selective deposition of fatty acids (by the ovary) in the egg, changing the pattern of bio- synthesis of fatty acids in the liver, changing the selective absorption of fatty acids through the intestine, 5 or by any combination of these. when Evans 33 a1. (1962) fed crude cottonseed oil or 3. figetida seed oil in the rations of hens it caused an increase in the stearic acid to oleic acid ratios in the livers, ovaries, and plasma as compared with the ratios found in similar tissues from hens fed the basal ration. They showed that the ovaries had a lower stearic acid to oleic acid ratio than the plasma or livers from hens on the same ration. One would have eXpected the reverse of this data if crude cottonseed oil acted by changing the selective deposition of fatty acids (by the ovary) into the egg. Mice and rats can dehydrogenate stearic acid to oleic acid (Schoenheimer and Rittenberg, 1936: and Bernhard.§§ al., 1959), Evans g; a1. (1963) have theorized and experimented on how crude cottonseed oil could change the fatty acid metabolism of hen's liver. Stearic acid might be formed from acetate (which comes from carbohydrate catabolism) and then be dehydrogenated to oleic acid. If this dehydrogenation is prevented by the crude cottonseed oil "active" factor, addition of large amounts of fats to a crude cottonseed oil containing ration should prevent more stearic acid from being laid down in eggs from hens fed this ration than in those eggs from hens fed the basal ration. Rations with 10% crude cottonseed oil or 2.5% crude cottonseed oil plus 7.5% corn oil caused eggs to be formed with large amounts of stearic acid (19% as compared to 9% 5 from basal and 11% from 10% corn oil rations). If linoleic acid is metabolized to acetate and then built up into stearic acid which is prevented from being dehydrogenated to oleic acid by crude cottonseed oil, feeding high levels of oleic acid and low levels of linoleic acid should cause normal amounts of oleic acid and stearic acid to be deposited in eggs. Eggs from hens fed 0.5%.3. foetida seeds plus 10% olive oil in their rations contained less oleic acid (3376 as compared to 43%) than eggs from hens fed 10% olive oil in their rations. Eggs from hens fed olive oil had more oleic acid than eggs from hens on the basal ration \49% as compared to 39%). Linoleic acid might be hydrogenated by the hen or by microorganisms in the gastro— entestinal tract to oleic acid or stearic acid, and the equilibrium between stearic acid and oleic acid unbalanced by the "active" fraction in crude cottonseed oil.The author has never heard of this happening in the non-ruminant or of being tested for in fowl. Linoleic acid is not synthesized ,dg,ngyg by the laying hen (Murty §£.él-: 1960). When diet- ary linoleic acid was increased by feeding safflower oil in the hen's ration, the oleic content of the eggs was reduced and the linoleic acid content increased as compared with eggs from hens fed a basal ration (Choudhury and Reiset; 1959). This decline in oleic acid was not to as low a level as when only crude cottonseed oil was added to a basal ration (Evans g; al., 1961). 7 Neither vitamin A, carotene, nor ferric chloride caused a decrease in the number of pink whites and salmon colored yolks in eggs from hens fed crude cottonseed oil in their rations (Evans £3 al.,l957). Lauryl peroxide plus heat destroyed 87% of the total tocopherols in refined cottonseed oils (Machlin, 1961). There was 7 mg. of a peroxide-resistant non-toc0pherol compound in refined cottonseed oil which reacted with the Emery-Engel reagent used in vitamin E analysis (Machlin, 1961). Refined cotton- seed oil had the most d-CI—tOCOpherOl per gram of unsaturated fatty acid of any vegetable oil tested by Herting and Drury (1963). They reported that crude cottonseed oil had two- thirds as much d-cr-toc0pherol per gram of unsaturated fatty acid as refined cottonseed oil (0.23 mg. of d—or-tocopherol per gram of unsaturated fatty acid in crude cottonseed oil). It is most probable that crude cottonseed oil prevents the dehydrogenation of stearic acid to oleic acid in the liver. The effect of crude cottonseed oil on the absorption of fatty acids from intestinal contents has not been investigated. The emphasis of this thesis will be changed in the selective intestinal absorption of stearic, oleic, and linoleic acids caused by crude cottonseed oil. It is well known that vitamin E and tocopherols protect unsaturated fatty acids. dl- —toc0pheryl acetate‘was ‘added .to rations of hens fed the basal ration or the basal ration plus 3% crude cottonseed oil in order to determine if 8 it would Change the effects of the ”active" fraction of crude cottonseed oil on the stearic acid to oleic acid ratio in fatty acids of the plasma, ovary, and liver or on intestinal absorption by the laying hen. EXPERIMENTAL O '2 !° Two groups of four Single Comb White Leghorn hens per group were kept in individual laying cages and fed a basal ration wdth the following percentage compositiont ground corn, 34.5: ground oats, 20.0; wheat bran, 15.0: flour middlings, 10.0; dehydrated alfalfa (15% protein), 3.0; dried milk, 2.0: menhaden fish meal (60% protein), 2.5: soybean oil meal (44% protein), 2.5: ground oyster shell flour, 5.0: steamed bone meal, 1.5: salt, 0.6: and fish oil (3,000 units of vitamin A and 400 ICU of vitamin D3), 0.4. This ration contained 4.1% of ether extractable material. *n EXperiment Ia one group of hens was fed the basal ration only. The second group was fed the basal ration plus 3.0% crude cottonseed oil added at the expense of an equalweight of basal ration. Each group of hens was fed 3,000 grams of ration during the experiment. “Xperiment Ib, a repeat of EXperiment Ia, was started two weeks after the start of Experiment Ia. During the one week between the running of Experiments Ia and Ib, Experiment IIa was con- ducted. Experiment IIa was a repeat of Experiments Ia, with the exceptions that 0.48 grams of dl-cr-tocopheryl acetate was added to the basal ration and 8.91 grams of 10 d—cr-tocopheryl acetate on corn meal was added to the ration containing crude cottonseed oil. Experiment IIb was started two weeks after the start of Experiment IIa. Experiment IIb was a repeat of Experiment IIa, with the exception that both rations contained 0.67 grams of dl-CI-tocopheryl acetate. The hens were on experiment for 72 hours, at the end of which time they were sacrificed. (All experiments were of the same duration.) CQllQQtiQn 9: Samples The following samples were collected: blood, liver, ovary, and content of the gastrointestinal tract (crop, gizzard, and intestine divided into three sections of equal length). Blood was collected in citrate, and the plasma was separated from the cells by centrifugation. All tissues were analyzed on an individual basis. Liver and ovary samples were homogenized in a Waring Blender. Livers, ovaries, and gastroentestinal contents were shell frozen andlqophilized, and the dried samples were stored at -150F until analyzed. Blood plasma was frozen at -60°C until analyzed. Extraggj QD and Methylatign The results of the fatty acid analyses were pooled according to the ration of the hens that the samples were taken from and the type of material taken (plasma, liver, and ovary: or contents of crop, gizzard, lst third of ll intestine, 2nd third of intestine, or 3rd third of intestine). All livers, ovaries, and plasma samples were analyzed. The gastrointestinal contents of one hen from each of the eight groups were analyzed. The samples, except for the plasma, were extracted three times each by shaking with 50 ml. portions of chloro- form-methanol (181) at room temperature for 30 minutes each time. The extracts were evaporated to dryness under nitrogen, and the oil (residue) was saponified by mixing with 10 ml. of ethanol containing 1.5 grams of potassium hydroxide and allowing to stand over night at room temperature. The soaps were dissolved in 25 ml. of water and sulfuric acid to convert them to the free fatty acids. Fatty acids were extracted with peroxide-free ether and made to a volume of 100 ml. Three ml. aliquots of fatty acids were converted to the the methyl esters with diazomethane. The solutions were evaporated under nitrogen and the reSidues dissolved in benzene. Plasma fatty acid methyl esters were prepared by the procedure of Michaels (1958). One ml. of plasma was added to 25 m1. of ethanol-acetone (1:1) mixture. After bringing to a boil, the mixture was filtered through Watman #40 paper, the precipitate washed with solvent, and the filtrate evaporated to dryness under nitrogen. The residue was taken up in 10 ml. of 1% sulfuric acid in methanol and refluxed one hour in a test tube capped with a marble. 12 This mixture was then evaporated to about 1 ml. under nitrogen and diluted with 4 ml. of distilled water.- The water suspension was extracted twice with 10 ml. portions of petroleum ether (300 - 60°C). The petroleum ether was evaporated under nitrogen and the residue was dissolved in 100 microliters of benzene. G — ro t ra Fatty acid methyl esters were separated in a six foot by 1/4 inch (inside diameter) glass column that was filled with diethylene glycol succinate (20%) on Anakrom 3531, 110-120 mesh. A Barber-Colman Model Series 5000 gas- 1iquid chromatographic machine was used. Nitrogen gas flowed at a rate of 90 ml. per minute at a pressure of 25 pounds per square inch through the glass column (which was kept at a temperature of 182°C). The effluent peaks were detected with a flame ionization detector (compressed air at 40 pounds per square inch and hydrogen gas at 19 pounds per square inch) that was kept at 210°C in an insulated bath. Fatty acid methyl esters were vaporized by an injector port heater that was kept at 300°C. A Barber-Colman single point dual range 8000 series recorder set at 5—mv. with a chart speed of 1/3 of an inch per minute was used. The area of each peak was determined by triangulation. lTradename of Analytical Engineering Laboratory Inc. acid and base washed diatomaciatious earth that has been coated with silicone, RESULTS Crude CQtthseed Oil ' The total fatty acids of a ration containing 3% crude cottonseed oil had 4 and 6 percentage points more of palmitic acid and linoleic acid, reapectively, and l, 4, and 2 percentage points less of stearic acid, oleic acid, and linolenic acid, respectively, than the total fatty acids from the basal ration (Table 1). The percentage of stearic acid in the total fatty acids from ovaries, livers, and plasma of hens increased when crude cottonseed oil was added to the basal ration (Table 2). There was more_palmitic acid (3 percentage points), more stearic acid (6 percentage points), more'I linoleic acid (3 percentage points), and less oleic acid (8 percentage points) in the total fatty acids of plasma from hens on the basal plus crude cottonseed oil ration than in those from hens fed the basal ration only (Table 2). There was more stearic acid (6 percentage points in liver and 5 percentage points in ovary), more linoleic aCid (4 percentage points in both), and less oleic acid (10 percent— age points in both) in the tbtal fatty acids in livers and ovaries from hens fed the basal ration plus crude cottonseed oil than in those from hens fed the basal ration (Tables 3 13 l4 and 4). The fatty acids in yolks from hens fed the basal plus crude cottonseed oil ration were within one percentage point of their correSponding fatty acid in yolks from hens fed the basal ration (Table 5). The Crude cottonseed oil containing ration (CSO) caused the percentages of some minor fatty acids in some of the tissues to change from the levels in hens fed the basal ration: myristic aCid changed only in the plasma (1.2% in basal fed hens and 0.6% in C50 fed hens): palmitoleic acid decreased by half in the ration, half in plasma, half in livers, and one-third in yolks, and more than doubled in ovaries: heptadecanoic acid doubled in plasma and livers: and arachidonic acid increased in the ovaries (2.0% in basal fed hens to 3.2% in C30 fed hens) and decreased in plasma (2.6% in basal fed hens to 1.6% in C80 fed hens) (Tables 2,3,4, and 5). The crude cottonseed oil containing ration caused some drastic changes in the percentages of some fatty acids in the intestinal contents as Compared with the correSpond— ing fatty acids in the contents of similar sections of intestines from hens fed the basal ration (Tables 7 and 8; and figures 1,2, and 3). The addition of crude cottonseed oil to the basal ration caused the percentage of palmitic acid to increase in the 3rd third of the intestine, decrease in the 2nd third of the intestine, and possibly decrease in the 1st third of the intestine (taking into con- sideration the differences in the percentage of palmitic acid 15 in the two rations). Crude cottonseed oil caused the per— centage of stearic acid in the 3rd third of the intestine to increase, in the lst third of the intestine to decrease slightly, and in the 2nd third of the intestine to decrease greatly as compared to the contents of similar sections of intestines from hens fed the basal ration. Taking in considering the difference in the percentage of oleic acid in the total fatty acids of the ration, the addition of crude cottonseed oil to the basal ration caused the percentage of oleic acid to increase slightly in the 3rd third of the intestine, increase greatly in the 2nd third of the intestine, and to decrease in the lst third of the intestine. The crude cottonseed oil containing ration caused the percentage of linoleic acid in the total fatty acids to increase in the lst third of the intestine, in- crease in the 2nd third of the intestine, and decrease slightly in the 3rd third of the intestine (taking in con- sideration the difference in the percentage of linoleic acid in the total fatty acids of the two rations) as com- pared to the percentages present in the intestines of hens fed the basal ration. The percentages of some minor fatty acids in the total fatty acids of the intestinal contents Changed when hens fed the crude cottonseed oil containing ration were compared with hens fed the basal ration: myristoleic acid decreased in the 3rd third of the intestine: palmitoleic acid increased greatly in the lst and 2nd thirds, as compared to the increase in the 3rd third: arachidic 16 Table 1. Fatty acid “omposition of the l"otal Jfatty Acids in the rations (Percent of total fatty acids). .— —. “xperiment I Experiment II dl- -toc0pheryl acetate Fatty acid Basal Cottonseed basal Cottonseed “yristic 0.8 0.8 0.8 0.8 Myristoleic 0.3 0.1 0.2 0.2 Palmitic 15.6 19.4 13.1 18.5 Palmitoleic 1.3 1.0 0.4 2.0 “eptadecanoic 0.1 0.1 0.0 0.0 Dtearic 4.3 3.4 3.0 3.5 Oleic 27.8 23.4 25.0 22.6 Linoleic 40.3 46.8 42.0 45.0 Arachidic 0.2 0.2 0.0 0.0 Linolenic 4.3 2.4 4.0 2.8 “rachidonic 0.4 0.4 0.7 0.4 Lignoceric 0.2 0.4 0.8 0.6 aydroxystearic 0.0 0.4 0.6 0.4 Ricinoleic 0.3 0.2 0.6 0.0 17 Table 2. ‘atty acid composition of the total fatty acids in theblood plasma from hens fed various rations (Percent of total fatty acids). #— Experiment I Experiment II dl- -tocopheryl wasal Cottonseed Jasal Cottonseed oilfi_~ oil myristic 1.2 0.6 0.7 0.4 Myristoleic 0.2 0.2 0.7 0.4 Palmitic 25.8 23.9 25.4 28.9 ‘almitoleic 2.0 1.0 1.8 0.9 Heptadecanoic 0.6 1.0 0.9 0.6 Stearic 15.7 22.0 20.2 19.8 L«'leic 33.5 25.4 29.8 25.4 Linoleic 13.0 16.1 14.2 16.2 AraChidiC 1.1 1.0 0.3 0.8 Linolenic 2.3 2.2 1.5 1.5 “rachidonic 2.6 1.6 1.8 1.7 18 Table 3. Fatty acid composition of the total fatty acids in the livers from hens fed various rations \Percent of total fatty acids). - m AT— .— EXperiment 1 .Experiment II dl- -tocopheryl acetate Sasal LJottonseed Dasal Cottonseed ~ _oil oil myristic 9.4 0.4 0.4 0.5 nyristoleic 0.2 0.2 0.2 0.2 Palmitic 20.2’ 20.0 21.4 21.7 Palmitoleic 2.0 0.7 1.4 1.0 Heptadecanoic 0.2 0.4 0.4 0.2) °tearic 13.4 19.5 18.5 19.5 oleic; 35.4 25.5 29.2 27.8 . Linoleic 15.9 19.6 18.0 18.8 flraChidic 0.2 0.2 0.2 0.2 Linolenic 0.9 0.8 0.8 0.8 Arachidonic 4.8 4.8 4.2 3.4 Hignoceric 0.2 0.3 0.1 0.2 Hydroxystearic 2.7 2.7 2.3 2.0 “icinoleic 0.6 0.7 0.1 0.3 19 Table 4. “atty acid composition of the total fatty acids in the ovaries from hens fed various rations (Percent of total fatty acids). Experiment I Experiment II dl- -toc0pheryl acetate Jatty acid Jasal Cottonseed nasal Cottonseed oil oil karistic 0.4 0.4 0.4 0.4 .{yristoleic 0.2 0.1 0.2 0.2 Palmitic 21.5 21.0 21.1 22.1 Palmitoleic 0.4 1.0 1.0 0.7 leptadecanoic 0.0 0.1 0.0 0.1 5tearic 10.8 15.6 12.2 16.0 Oleic 42.1 32.4 37.8 33.2 Linoleic 17.2 21.2 19.0 18.4 Arachidic 0.2 0.2 0.4 0.1 Linolenic 1.2 1.0 1.2 0.8 Arachidonic 2.0 3.2 2.4 2.2 Lignoceric 0.1 0.1 0.1 0.2 Hydroxystearic 1.9 1.8 1.6 1.2 “icinoleic 0.2 0.1 0.1 0.2 20 Table 5. fatty acid composition of the total fatty acids in egg yolks from hens fed various rations. kercent of total fatty acids). Experiment I Experiment 11 d1- -toc0phery1 acetate _ fatty acid Dasal Cottonseed basal Cottonseed oil oil xyristic 0.4 0.4 0.4 0.4 nyristoleic 0.2 0.2 0.2 0.2 Palmitic 20.4 21.4 20.6 21.0 Palmitoleic 1.3 1.2 3.4 1.4 ‘ -fleptadecanoic 0.0 0.0 0.0 0.0 stearic 9.9 10.8 9.8 13.4 Oleic 41.2 41.8 40.0 40.8 Linoleic 18.2 17.2 18.8 16.5 Arachidic 0.0 0.4 0.0 0.0 Linolenic 1.5 1.2 1.2 1.2 drachidonic 2.4 2.3 2-3 201 Lignoceric 0.0 0.1 0.1 0.0 Hydroxystearic 1.8 1.9 1.9 1.6 Ahinoleic 0.4 0.1 0.1 0.0 21 Table 6. Distribution of C Fatty dcids (Percent of total fatty acids). W Stearic Acid Oleic ACid B CSO B+E CSO+E B CSO B+E CSO+E Ovary 10.8 15.6 12.2 16.0 42.1 32.4 37.8 33.2 Liver 13.4 19.6 18.5 19.5 35.4 25.6 29.2 27.8 Blood 15.7 22.0 20.2 19.8 33.5 25.4 29.8 25.4 Ration 4.3 3.4 3.0 3.5 27.8 23.4 25.0 22.6 Crop 5.7 5.2 4.8 3.3 26.5 23.8 25.2 23.2 Contents Gizzard 5.7 3.9 3.8 3.4 23.9 25.8 24.9 25.4 Contents Intestine 23.9 18.1 18.6 14.0 19.1 13.6 16.7 12.4 C'ontents Intestine 23.1 13.2 16.2 11.4 14.6 26.3 13.8 14.2 Contents ' Intestine 12.6 14.7 12.3 12.8 19.1‘ 19.9 21.8 20.9 Contents Feces 9.0 9.4 7.6 5.8 22.4 15.7 Vl9.8 20.6 Linoleic “Cid __ Linolenic Acid Ovary 17.2 21.2 19.0 18.4 1.2 1.0 1.2 0.8 eiver 15.9 19.6 18.0 18.8 0.9 0.8 0.8 0.8 Blood 13.0 16.1 14.2 16.2 2.8 2.2 1.5 1.5 Ration 40.8 46.8 42.0 45.0 4.3 2.4 4.0 2.8 Crop 30.6 34.7 38.6 36.7 3.2 1.8 3.4 2.1 Contents Gizzard 34.8 41.8 38.0 34.0 2.8 2.6 2.4 2.5 Contents ' Intestine 21.0 38.1 39.4 35.8 0.7 1.0 1.4 1.8 Uontents Intestine 20.6 31.8 32.6 30.4 1.6 1.9 1.5 1.6 C’ontents Intestine 29.2 29.4 35.8 21.6 2.2 1.1 2.0 1.9 C'ontents Feces 19.4 14.6 20.1 27.6 2.4 1.6 2.0 3.1 A B = basal 5+5 = Basal T dl-a-tocopheryl CbO = Basal + 3% crude acetate. CSO+E Basal + 3% Crude cotton- seed oil + dl—G-tOCOpheryl acetate. cottonseed oil 22 1Fig. 1. The percentage of stearic acid in the total fatty acids in the intestinal contents and some tissues. 40 38 34 32 30 28 24 22 20 16 14 12 10 Ration Crop Gizzard 11 ill iIll Feces “<2 Hm<0 Hm r10 ovAn 24 Fig. 3. The percentage of linoleic acid in the total fatty acids in the intestinal contents and some tissues. W 60 57 54 51 48 45 42 39 36 33 30 27 24 21 18 15 12 Ration Crop Gizzard iI iII iIII Feces MHm4w>o um>44 msmmam mmomm m N 4 UHMNNHG mono C04umm 0408 muumm wcmumCUCH H ucmsfiummxm .AmUHUm wuumu ammo» mo unmonmmv .haco c04umn Hanan any new new: 604m mmsmmHu omuomamm pcm uomuu 48:4ummuc4ouummm gnu mo mucmucoo any :4 nso«U4moquu p4um muumm .4 04969. 6.6 6.6 6.6 6.4 4.6 6.6 6.6 4.6 0464064646 6.6 6.4 4.4 4.4 4.6 4.6 6.6 6.6 04466066404666 4.6 4.6 6.4 6.6 4.6 6.6 6.6 6.6 6.6 0446006644 4.4 4.6 6.4 6.6 6.4 4.4 6.4 6.6 6.6 6.6 04606460644 4.4 6.6 6.4 6.4 4.4 6.4 6.4 6.4 6.4 6.4 046640644 4.6 6.6 4.6 6.4 6.6 6.6 6.6 6.6 6.6 4.6 046460646 6.64 6.44 6.44 6.64 6.64 6.46 4.66 6.46 4.66 6.66 04640644 6.66 6.64 6.64 4.64 6.64 6.64 6.64 6.64 6.64 6.64 04640 6.44 6.64 6.44 6.6 4.64 4.64 4.64 6.6 4.6 6.6 0446666 6.6 6.6 6.4 6.6 4.6 4.6 6.6 4.6 6.6 4.6 0406606660666 6.4 4.4 6.6 6.4 4.4 6.4 6.6 6.4 6.4 6.4 04640446466 6.64 6.64 6.64 6.46 6.44 6.44 6.64 4.64 6.64 6.64 04449466 4.6 4.6 4.6 6.6 4.6 4.6 4.6 4.6 4.6 4.6 04640064448 6.6 6.6 6.6 4.4 4.6 6.6 6.6 6.6 6.4 6.6 0446444: >46>0 46>44 6Em64d mmomm m N 4 646N440 mono s04u6m 6406 >uu6m 6C4ummuCH H unmE4Hmme .Amp4o6 muu6m Hmuou M0 ucwunmmv .HHO pmwmcouuoo mpsuo Rm mC4C46uCoo C04464 6 new new; 804m mmnmmfiu pmuomamm 666 uU6uu H664466UC4044666 6£u mo mucmunou 63» C4 C04u4momsou. 6406 44466 .6 64969 27 0.0 m.m m.m o.o m.m o.o N.H m.o o.o 0464064044. 6.4 6.4 6.4 4.4 4.4 4.4 6.6 4.6 6.6 04466066xon646 4.6 4.6 6.6 6.6 6.6 6.6 4.6 6.6 6.6 4 0446006644 6.6 6.4 4.4 6.4 6.4 6.4 6.6 6.6 , 4.6 04606460646. 4.6 6.6 6.4 6.4 6.4 6.4 6.4 6.6 6.6 046640644 4.6 6.6 6.4 6.4 6.6 4.6 6.6 4.6 6.6 046460644 6.64 4.44 4.64 6.66 6.46 6.66 6.66 6.66 6.46 04640644 4.64 6.64 6.64 6.44 6.64 4.64 6.64 4.64 6.64 04640 6.44 6.44 6.4 6.44 4.64 6.64 6.6 6.6 6.6 0466646 4.6 6.4 6.6 6.6 6.6 6.6 4.6 6.6 6.6 0406606660666 6.4 4.4 4.4 6.6 6.6 6.6 4.4 6.4 6.6 04640446466 4.64 4.64 6.44 6.44 6.64 6.64 6.64 6.44 4.64 04446466 4.6 6.6 6.6 6.6 6.6 4.6 4.6 4.6 4.6 046400644»: 6.0 0.4 o.4 m.o o.o m.o 0.4 m.o w.o 049644»: um>49 666699 mmumm m N 4 pnmww4w @040 60.49664 1. p406 muumm 064ummuCH HH unmEHHwaxm II 1.! .466406 >996m 46909 no 96004645 . .mumumom Hmhw9moooulanap m64646usoo 604964 6 new 6609 E044 mmomm4u 00900406 6:6 90649 466496096404966m 09» no 69669600 099 64 60494609500. 6406 >996m .0 64969 23 4.0 6.0 6.6 6.6 4.6 0.6 6.6 6.4 6.0 0.0 0464066046 6 6.0 6.4 6.4 0.6 0.6 6.4 6.6 6.0 6.0 6.0 04066066600666 4.0 6.0 . 4.0 6.4 6.4 6.4 6.0 6.0 6.0 6.0 0406006644 0.6 6.6 6.6 4.4 0.4 6.4 6.4 6.0 6.0 6.0 04600460666 0.4 0.4 6.4 4.6 6.4 6.4 6.4 6.6 4.6 6.6 046640644 4.0 6.0 6.0 6.0 0.0 0.0 0.0 6.0 6.0 0.0 040460666 4.64 6.64 6.44 6.66 6.46 6.06 6.66 0.66 6.66 0.66 04640644 6.66 6.66 0.66 6.06 6.06 6.64 6.64 6.66 6.66 6.66 04640 6.64 6.64 6.64 6.6 6.64 6.44 0.64 6.6 6.6 6.6 0466606 0.0 6.0 6.0 6.0 0.0 6.0 6.0 m.o N.o o.o 0406606660m6m 6.0 6.4 0.4 6.0 6.4 6.0 6.0 6.4 6.4 0.6 04640046466 0.66 6.66 6.66 6.64 4.46 6.64 6.06 6.64 4.66 6.64 04046466 4.0 4.0 6.0 6.0 6.0 6.0 4.0 6.0 6.0 6.0 04640064662 6.0 6.0 6.0 6.0 6.0 6.0 6.0 0.4 0.4 6.0 0406406: .mu6>0 06>44 6fim64m 66066 m N .4 6066640 QOHUil,60406m 6406 >006m 66406606H HH 066E406mxm Ill Elfin-“Hg! i .566406 60060 46000 00 06600660 .0060606 4%06300000I0I46 666 440 6666600000 66500 Rm @646460600 604060 6 660 666: 3000 6656640 66006466 666 00600 4664066064000666 600 00 60660600 660 64.:0404600E00 ,06406 >006m .04 64068 29 acid decreased in the 3rd third; linolenic acid probably decreased in the 3rd third; arachidonic acid increased in the lst and 2nd thirds; lignoceric acid decreased through— out; unknown fatty acid number 16 decreased in the 2nd third: and :ricinoleic acid decreased greatly in the 2nd and 3rd thirds, and possibly in the lst third. dl—a-TOCQoheryl Acetate The basal ration plus dl-Q-tocopheryl acetate had more palmitic acid, slightly more linoleic acid, less oleic acid, less stearic acid, more lignoceric acid, more hydroxy- stearic acid, more ricinoleic acid, and less palmitoleic acid than the basal ration (Table l). The ration containing crude cottonseed oil and dl-a—toc0pheryl acetate had less palmitic acid, more palmitoleic acid, slightly less oleic acid, and less linoleic acid than the cottonseed oil ration. The ration containing crude cottonseed oil and dl-a-tocopheryl acetate had less linolenic acid, more palmitoleic acid, less oleic acid, and more linoleic acid than the ration contain- ing only dl-a-tocopheryl acetate. There was more stearic acid (4 percentage points), more linoleic acid (1.2 percentage points), and less oleic acid (4 percentage points) in the total fatty acids of plasma from hens fed dl-G-tOCOpheryl acetate than in those of plasma from hens fed the basal ration only (Table 2). There was less stearic acid (2 percentage points) in plasma from hens fed a ration containing crude cottonseed oil and 3O dl- -tocopheryl acetate than in plasma from hens fed the basal ration plus crude cottonseed oil. 0f the two dl- - tocopheryl acetate containing rations, the radon that also contained crude cottonseed oil caused the fatty acids of the plasma to contain more palmitic acid, more linoleic acid, and less oleic acid than the plasma fatty acids of hens fed the ration supplemented with dl- -toc0pheryl acetate only. There was more stearic acid (5 percentage points), more linoleic acid (2 percentage points), and less oleic acid (6 percentage points) in the total fatty acids of livers from hens fed the basal ration plus dl- —toc0pheryl acetate than in those of livers from hens fed the basal ration. (Table 3). There was more oleic acid (2 percentage points) in livers from hens fed a ration containing crude cottonseed oil and dl- —toc0pheryl acetate than in livers of hens fed the basal ration plus crude cottonseed oil. There was slightly more stearic acid and slightly less oleic acid in liVers from hens fed a ration containing crude cottonseed oil and d1— -t060pheryl acetate than in those from hens fed the basal ration plus dl- -toc0pheryl acetate. There was more stearic acid (1 percentage point), more linoleic acid (2 percentage points), and less oleic acid (4 percentage points) in the total fatty acids of ovaries from hens fed the basal ration than the other rations (Table 4). There was slightly more palmitic acid and less linoleic acid in ovaries from hens fed the basal ration plus crude cottonseed 31 oil and dl-a-tocopheryl acetate than in those from hens fed a ration containing crude cottonseed oil. There was more stearic acid, slightly more palmitic acid, and less oleic acid in ovaries from hens fed a ration containing crude cottonseed oil and dl—a-tOCOpheryl acetate than in those from hens fed the basal ration plus dl-a-toc0pheryl acetate. There was more palmitoleic acid and less oleic acid in the total fatty acids of egg yolks from hens fed the basal ration plus dl—a-toc0pheryl acetate than those from hens fed the basal ration (Table 5}. There was more stearic acid (2.6 percentage points) and less oleic acid (1 percent— age point) in the total fatty acids of egg yolks from hens fed crude cottonseed oil and dl—a-toc0pheryl acetate in their ration than in those of egg yolks from hens fed the basal ration plus crude cottonseed oil. There was more stearic acid and less linoleic acid in egg yolks from hens fed crude cottonseed oil and dl-a-tocopheryl acetate than in those from hens fed the basal ration plus dl-a—tocopheryl acetate. The addition of dl-a—tocopheryl acetate to the basal ration caused Changes in the percentages of some fatty acids of the intestinal contents (Tables 7 and 9: and figures 1, 2, and 3). dl-a-tocopheryl acetate caused the percentages . in the total fatty acids of palmitic acid to decrease in the lst and 2nd thirds of the intestines, of stearic acid to decrease in the lst and 2nd thirds of the intestine, of linoleic acid to increase throughout the intestine, of 32 myristoleic acid to decrease in the 3rd third of the intes— tine, of linolenic acid to increase in the lst third of the intestine, of palmitoleic acid to increase in the lst third of the intestine, of lignoceric acid to decrease throughout the intestine, of arachidonic acid to increase in the 2nd third of the intestine, and of rnicinoleic acid to decrease in the lst and 3rd thirds and to increase in the 2nd third of the intestine. The addition of dl—a-toc0pheryl acetate to a ration containing crude cottonseed oil caused Changes in the per- centages of some fatty acids of the intestinal contents (Tables 8 and 10: and figure 1,2, and 3). The addition of dl—a—toc0pheryl acetate to the crude cottonseed oil containing ration caused the percentage of palmitic acid to increase in the 3rd third of the intestine, of stearic acid to decrease throughout the intestine, of oleic acid to decrease in the 2nd third of the intestine, of linoleic acid to decrease in the lst and 3rd thirds of the intestine, of palm- itoleic acid to decrease in the 2nd third of the intestine, of arachidic acid to decrease throughout the intestine, of linolenic acid to increase in the lst and 3rd thirds and possibly to decrease in the 2nd third of the intestine, of arachidonic acid to decrease in the lst and 3rd thirds of the intestine, of lignoceric acid to increase in the 2nd and 3rd thirds of the intestine, and of hydroxystearic acid and ridinoleic acid to increase throughout the intestine. 33 The addition of crude cottonseed oil to a basal ration plus dl—a—toc0pheryl acetate caused changes in the percentages of some fatty acids in the total fatty acids of the intestinal contents (Tables 9 and 10; and figures 1,2, and 3). The addition of crude cottonseed oil to the d141- tocopheryl acetate containing ration caused the percentage of palmitic acid to increase in the lst and 3rd thirds and to decrease in the 2nd third of the intestine, of stearic acid to decrease in the lst and 2nd thirds of the intestine, of oleic acid to decrease in the lst third of the intestine, of linoleic acid to decrease throughout the intestine, of myristic acid to decrease in the lst and 2nd thirds of the intestine, of palmitoleic acid to increase in the 3rd third, of aradhidic acid to decrease throughout (particularly in the 3rd third) of the intestine, of lignoceric acid to in- crease in the 2nd and 3rd thirds of the intestine, and of hydroxystearic acid and Mriainoleic acid to increase through- out the intestine. The fatty acid distribution in tissues from hens fed rations containing crude cottonseed oil or dl—a—toc0pheryl acetate were very similar (Tables 2,3,4, and 5). The addition of dl4I-tocopheryl acetate to a basal ration caused a change in the fatty acid distribution of the tissues of the hens that ate it to a position between that of hens fed the basal ration plus crude cottonseed oil and of hens fed a basal ration only. (The percentage of oleic acid in total fatty acids when basal ration plus dl-a—tocopheryl acetate was fed, 34 was midway between the percent of this fatty acid in the two rations that did not contain dl- -tocopheryl acetate. Hing; Peaks Many of the minor fatty acids could not be identified as they did not correSpond to any standards that could be bought commercially. Dome of the peaks that appeared in the same place as the fatty acid standards are lé—methylhepta— decanoic (iso-stearic) acid, rincinoleic acid, lZ—hydroxy- stearic acid, heptadecanoic acid, and pentadecanoic acid (l4—methylpentadecanoic acid appeared at about the same place). (See appendices). Wheeler 3; a1. (1959) found heptadecanoic acid in yolks of eggs from hens fed 10% cotton- seed oil in their rations. Time did not allow for the identification of more minor peaks. DISCUSSION Patti agid absgrption There have been a few studies on the sites of fat and fatty acid absorption through the intestine. Renner (1965’ observed that the fat of tallow, lard, and soybean oil was mostly absorbed from the third—fifth of the intestine of four week old chicks, while almost all of the remaining fat was absorbed in the fourth-fifth of the intestine. She did not test for absorbability through the duodenum, as its contents were a thin paste. Benson (1956) reported that olive oil was preferentially absorbed from the third quarter of the small intestine of the rat. Cottonseed oil fatty acids in the form of triglycerides was "efficiently absorbed” (60%) in the proximal intestine of the dog (“noebe1, 1962). Renner and.Hill (1961) reported that, in hens, as the Chainlength of saturated fatty acids increased, their absorbability decreased; while unsaturated fatty acids were readily absorbed: myristic, 29%: palmitic, 12”: stearic, 4%: and oleic, 94%. In the eXperiments that I conducted, stearic acid was not as well absorbed as the other C18 fatty acids (Table 6). Noyan at al. (1964) reported that about 92% of the labeled palmitic acid fed to chickens was absorbed from the duodenum. They observed that 35 36 the palmitate was released by the intestinal mucosa into the blood as triglycerides of very low density lipoproteins. From tables 7,8,9 and 10 the sites of absorption of the major fatty acids were determined by the decrease in the percentage of each fatty acid in the total fatty acids of the intestinal contents. stearic acid was absorbed through the 3rd third and the 2nd plus the 3rd thirds of the) intestine when the basal ration and the ration containing crude cottonseed oil, reSpectively, were fed. some oleic acid was absorbed in the lst third of the intestine, but was mostly absorbed in the 2nd third of the intestine unless crude cottonseed oil was included in the ration (absorption from the 2nd third then drOpped drastically). Linoleic acid was absorbed from the lst and 2nd thirds of the intestine when the basal ration was fed and the 2nd and 3rd thirds of the intestine when the basal ration plus crude cottonseed oil was fed. Crude cottonseed oil containing rations in- creased slightly the absorption of palmitic acid through the lst and 2nd thirds of the intestine, while it halved the absorption of palmitic acid from the 3rd third of the intestine (Comparison with the results of feeding the basal ration) (Tables 7 and 8). Crude cottonseed oil could have expanded the total area of absorption of stearic acid by increasing the absorption of stearic acid by the 2nd third of the intestine. Young and Garnett (1963) observed that stearic acid is absorbed to a much higher degree in the chick when oleic acid 37 is added to the ration. The crude cottonseed oil plus basal ration decreased the absorption of oleic acid from the 2nd third of the intestine by 80% (Table 6). It is possible that the "active" factor in crude cottonseed oil prevents oleic acid from being absorbed from the 2nd third of the hen's intestine, this in turn allowing more stearic acid to be absorbed there. "1 r t ci 8 tis ues According to table 11 my data compares favorably with the data of Evans gt al. (1962) with respect to the fatty acid composition of plasma and livers from hens on the basal rations and the basal plus 3% crude cottonseed oil ration. The changes between the fatty acid composition of ovaries from hens fed the basal plus crude cotton seed oil ration and the basal ration only is about half as much in the hens on my Experiment I as on their experiment. Their hens were on eXperimental rations for six weeks before being sacrificed, while my hens were on experimental rations for three days. The fatty acid composition of the plasma and livers seem to come to equilibrium at least within three days, while the fatty acid equilibrium of the ovaries takes longer to establish. The eggs, laid the second day of my experiments, had the same fatty acid composition irreSpective of ration (Table 5). This was eXpected, as these eggs were in the oviduct for the entire duration of the experiment. I do not know why our data on the myristic acid composition of the rations and plasma disagree. Table 11. 33 The effect of the duration of feeding crude cottonseed oil on the fatty acid distribution in plasma, liver, and ovary. \Percent of total fatty acids). ____‘-__f r Fatty acid “ation Plasma Diver Ovary B two 5 050 B cso B cs0 Myristic 0.8 0.8 1.2 0.6 0.4 0.4 0.4 0.4 *1.7 1.5 .o.1 0.2 0.5 0.6 0.5 0.5 Palmitic 15.6 19.4 25.3 23.9 20.2 20.0 21.5 21.0 *16.6 20.9 31.3 33.2 23.4 26.5 25.7 23.5 Palmitoleic 1.8 1.0 2.0 1.0 2.0 0.7 0.4 1.0 *2.7 2.1 2.9 2.3 3.1 2.3 3.6 1.8 stearic 4.3 3.4 15.7 22.0 13.4 14.6 10.8 15.6 *3.2 2.9 17.5 22.0 14.0 22.2 10.4 21.4 Oleic 27.8 23.4 33.5 25.4 35.4 25.6 42.1 32.4 *27.4 23.4 34.5 24.3 36.2 27.7 42.4 26.6 Linoleic 40.8 46.8 13.0 16.1 15.9 19.6 17.2 21.2 *45.1 47.3 13.7 18.1 22.9 20.8 17.1 21.2 *row of data from Evans g; g;.,(l962). My data will be lower than Evans et al., (1962) as I have based my numbers on more fatty acids. 5 F basal ration Cot) = 3% crude cottonseed oil ration. 39 dl-g-tgcooheryl acetate The amount of dl-a—tocopheryl acetate used (0.667 grams in 3,000 grams of rations) is the same, 0.022%, as that reported by hachlin sf al. (1962) to be sufficient to keep hatchability, fertility, and production of eggs at normal levels when hens were fed high linoleic acid rations (about 70/‘3 of the total fatty acids). The rations fed to my hens had a linoleic content of 40% ~ 48% of the total fatty acids. The dl-a-tocopheryl acetate level fed should have insured that there was a surplus of vitamin E precursor. The surplus dl-d—tocopheryl acetate was sufficient to change the fatty acid absorption from the laying hen's intestine.’ dl-a—toc0pheryl acetate had little effect on the site of absorption Of stearic acid but it seemed to partially counteract the effect of crude cottonseed oil on the absorp- tion of oleic acid from the intestine. It lowered the per- centage of oleic acid in the total fatty acids of the contents in the 2nd third of the intestine (showing increased absorption) and increased slightly the percentage of oleic acid in the total fatty acids of the liver (Table 6 and figure‘ 2). dl—a-toc0pheryl acetate changed the site of absorption of palmitic acid and linoleic acid from the lst and 2nd thirds (much absorbed from the basal ration here) to only the 2nd third of the intestine. If crude cottonseed oil was in the ration, whether or not dl-a-tocopheryl acetate was present, palmitic acid and linoleic acid was absorbed in the 40 2nd and 3rd thirds of the intestine (Tables 6,8, and 10; and figure 3). s Ub'li‘iARY Fatty acids from ovaries, livers, and plasma from hens fed the basal ration contained less stearic acid, less linoleic acid, and more oleic acid than the fatty acids of similar tissues from hens fed the other rations. Tissues from hens fed the basal ration plus crude cottonseed oil had the largest stearic acid to oleic acid ratio and tissues from hens on the basal ration only had the smallest stearic acid to oleic acid ratio of hens on the four experimental rations. Generally more stearic acid, less linoleic acid, and less oleic acid was absorbed from the intestines of hens fed the basal plus 3% crude cottonseed oil ration than from the intestines of hens fed the basal ration. The relative absorption of stearic acid and oleic acid correSponded with the relative amounts of these fatty acids found in the plasma, livers, and ovaries. This does not seem to hold true in the case of linoleic acid. In those hens where the percentage of stearic acid in the total fatty acids of the intestinal contents was high, the percent— age of stearic acid in the total fatty acids of the plasma, livers, and ovaries was lowest. 42 dlsr—toc0pheryl acetate seemed to have the same type of effect on the absorption of stearic acid and oleic acid and their presence in the plasma, liver, and ovary as the "active" factor in crude cottonseed oil. Varying the amount of a-tocOpheryl acetate from 0.43 grams to 0.67 grams or 3.91 grams to 0.67 grams, or using the d form instead of the dl form did not change the results between parts a and b of Experiment II. It would be advantageous to feedCI-toc0pheryl acetate to hens in large doses for several weeks, collect the hens' eggs and put them into cold storage for nine months, and discover whether this produces pink whites and salmon colored yolks. lO. LITERATURE CITED “lmquist, H. J.: and Lorenz, F. W. Some possible causes of pink egg white. U.S. Egg Poultry Mag., 38:48, 1932. A.O.A.C., Official Methods of Analysis, 8th Ed., WaShington, D.C., p. 475, 1955. Benson Jr., J.R., Chandler, G.N., Vansteenhuyse, F.E., and Gagnon, J.O. btudies concerning the site of fat absorption in the small intestine of the rat. Gastroenterology, 30853, 1956. Bernhard, K., von Bulowedeter, 9., and Wagner, H. Die enzymatsche Dehydrierung der Stearinsaure zu Olsare. Helvetica Chimica Acta. 428152, 1959. Choudhury, B.B.R. and Reiser, R. Interconversions of polyunsaturated fatty acids by the laying hen. J. Nutrition, 688457, 1959. Dijkstra, G. and Duin, H.J. Structure of sterculic acid and other analogous fatty acids. “ature, 176871, 1955. Ellis, N.“., Rothwell, C.S., and Pool, W.0. The effect of ingested cottonseed oil on the composition of body fat. J. Biol. Chem., 92‘385, 1931. Evans, R.J., Bandemer, 5.L., Anderson, M., and Davidson, J.A. Fatty acid distribution in tissues from hens fed cottonseed oil or Etergulia foetida seeds. J. Nutrition, 768314, 1962. Evans, R.J., Bandemer, S.L., Davidson, J.A., and Schaible, P.J. Studies on the occurrence of pink whites and salmon colored yolk in stored eggs from hens fed crude cottonseed oil or cottonseed meal. Poultry Sci., 368798, 1957. - Evans, R.J., Bandemer, S.L., and Davidson, J.A. Heat inactivation of substances in crude cottonseed oil causing pink whites and large discolored yolks in stored eggs. Poultry Sci., 3931478, 1960. 43 ll. 12. 15. l6. 17. 20. 21. 22. N (.0 o 44 Evans, R.J., Davidson, J.A., and Bandemer, 5.”. Fatty acid and lipids distribution in egg yolks from hens fed cottonseed oil or Stergulia foetida seeds. J. Nutrition, 738282, 1961. Evans, R.J., Davidson, J.A., LaRue, J.N., and Bandemer, 3.”. Interference in fatty acid metabolism of laying hens caused by cottonseed oil feeding. Poultry SCi., 428875, 1963. Faure, P.K. sterculic acid and its Halphen reaction. Nature, 1788375, 1956. Herting, D.C. and Drury, E.E. Vitamin E content of vegetable oils and fats. J. Nutrition, 818335, 1963. Knoebel, L.K. Fat digestion and absorption in the proximal intestine of the dog. Proc. Soc. EXptl. Biol. Med., 1098558, 1962. Lorenz, F.W. Egg deterioration due to ingestion by hens of malvaceous materials. Poultry Sci., 188295, 1939. MacFarlane, J.J., Shenstone, F.°., and Vickery, J.R. Malvalic acid and its structure. Nature, 1798830, 1957. Machlin, L.J. Destruction of vitamin E in cottonseed oil. Poultry Sci., 4081631, 1961. Machlin, L.J., Gordon, R.S., Marr, J.E., and POpe, C.W. Effect of antioxidants and unsaturated fatty acids on reproduction in the hen. J. Nutrition, 768284, 1962. Masson, J.G. Chemical reactions and biological effect of sterculic acid and analogous fatty acids. Ph.D. thesis, University of Arizona, Tucson, 1959. Masson, 4.0., Vavich, M.G., Heywang, B.W., and.Kemmerer, A.R. Pink discoloration in eggs caused by sterculic acid. science, 1268751, 1957. Michaels, G.D. Micro fat analysis. Aerograph Research Notes, Fall Issue, 1958. Murty, N.L., Williams, M.C., and Reiser, R. Non- synthesis of linoleic acid from acetate-1—Cl4 by the laying hen. ”. Nutrition, 728451, 1960. 24. 25. 32. (J) U) 0 U) 0\ o 45 New Mexico Poultry Project V. Cottonseed meal feeding experiments, New'Mexico Agr. EXpt. 5tat., 38th Ann. Rept.‘506, 1927. Noyan, A., Lossow, W.J., Brot, N., and Chaikoff, I.L. Pathway and form of absorption of palmitic acid in the chicken. J. Lipid Res., 58538, 1964. Nunn, J.R. The structure of sterculic acid. J. Chem. SOC., l952=3l3, 1952. Renner, R. Site of fat absorption in the chick. Poultry Sci., 448861, 1965. Renner, R. and Hill, F.W. Utilization of fatty acids by the chicken. J. Nutrition, 748259, 1961. Rinehart, K.L., Jr., Termic, C.L., and Culberts, T.P. Structure of sterculic acid polymer. A cyclOprOpene rearrangement. J. Am. Chem. soc., 8185007, 1959. Roberts, J.P. and Rice, J.“. The effect on fowls of nitrogenous and carbonaceous rations. EXpt. Sta. Record, 28506, 1891. Schaible, P.J., Bandemer, S.L., and Davidson, J.A, Composition of fresh and storage eggs from hens fed cottonseed and non—cottonseed rations. I. General observations. Poultry Sci., 258450, 1946a. Schaible, P.J. and Bandemer, S.L. Composition of fresh and storage eggs from hens fed cottonseed and non- cottonseed rations. III. Iron content. Poultry Sci., 258451, 1946b. schaible, P.J. and Bandemer, °.L. Composition of fresh and storage eggs from hens fed cottonseed and non- cottonseed rations. V. Cause of discoloration. Poultry Sci., 258456, 1946c. Schaible, P-J., Moore, L.A., and Moore, J.M. Gossypol, a cause of discoloration in egg yolks. science, 798372, 1934. Schoenheimer, R. and Rittenberg, D. Deuterium as an indicator in the study of intermediary metabolism V. The desaturation of fatty acids in the organism. J. Biol. Chem., 1138505, 1936. Shenstone, F.S. and Vickery, J.R. A biologically active fatty acid in Malvagae. Nature, 177894, 1956. 37. (_ O ) 39. Shenstone, 3.5. and Vickery, J.R. Substances in plants. of the order Malvale causing pink whites in stored eggs. Poultry Sci., 3381055, 1959. Shenstone, F.S. and Vickery, J.R. Occurrence of cyclo- prOpene acids in some plants of the order Malvales. Nature, 1908168, 1961. Sherwood, R.M. The effect of various rations on the storage quality of eggs. Texas Agr. Expt. Stat. Bu11., 37685, 1923. Sherwood, R,M, The effect of cottonseed meal and other feeds on the storage quality of eggs. Texas Agr. Expt. stat. Bull., 42985, 1931. Sipe, G.R. Cold storage of Southern eggs. Poultry Sci., 103224, 1931. Swensen, A.D., Fieger, E.A., and Upp, C.W. The nature of egg yolk discoloration produced by cottonseed meal. ’oultry Sci., 218374, 1942. UPP. C.W. Storage studies of Louisiana eggs. Louisiana Bull., 229, 1932. Wheeler, P., Peterson, D.W., and Michaels, G.D. Fatty acid distribution in egg yolk as influenced by type and level of dietary fat. J. Nutrition, 698253, 1959. Young, R.J. and Garrett, “uL. Effect of oleic and linoleic acids on the absorption of saturated fatty acid in the chick. J. Nutrition, 818321, 1963. Cottonseed oil acetate EXperiment II dl- -tocopheryl Basal 47 Experiment.I Cottonseed oil ' -— fatty acid composition of the total fatty acids (Percent of total fatty acids). Basal in the rations. “ppendix 1. 01008OZZSOOOlSrOOOOOBO/OFO09004016600101010401 oooooooooooooooooooooooo.ooooooooooooooooooo 011080211400100000000r00030070333».02201010662 00......coco-cocoooo,ooooooooooooooooooooooo Onunoononfino4xu110+iaf4nuqua24:0n02AUonAUA401092410n62n0n62nu174620 o o o O o o 0 o o o 0 o. o I o o o O O O O o o o o o O O o o O O O O o o O O O O o O . noonun30.Unoomw1:0n0013nw0“%nooaznVOAUnoonunoonunoonunoonunoonunoonunoo \ n0011n68.Usa126no1101123810Qo0a22301211n62.1no4a2119_ n60a2n6412111101362 O 0 O O O O O O O O 0 O 0 O O 0 O O O O 0 O O O O O 0 O O O .2 O O O O O O O I O O O 00000000510004700004000OOOOOOOOQOOOOOOOOOOO l 2 4 c c c i .1 .1 C r - floo atone .1 C a ilm. ilnsl o i c , e e .ear n cc n i tc c c 1_Cnc1.c.a a c iTi . o r spa 1 .1 onei.ooce c sci sun c d a. W oc t tdttdt i ee.ie i ic c ..o nil s eoa.141aao rncdsl .h11 n kui o , non ar1213it mmt .4aiooSco678e901cc23n45373901r12 cu r rnllpo eenn an h llaUllgllllllZZdCZ 81a Vi "Yo1a1apvs +t110.1 r.1 e r41 .1 v11 Db n MPEEHi SOub AL. o an L ha u #4 r0 pendix 2. Fatty acid composition of the total fatty acids in the blood plasma from hens fed various rations (Vercent of total fatty acids. iso-Stearic 4 stearic Oleic éonodecanoic Dinoleic 5 drachidic Linolenic 6 7 8 uehenic 9 10 11 “rachidonic F W “xperiment ' EXperiment II dl- —tocopheryl acetate Eatty acid Dasal Cottonseed Basal Cottonseed oil . oil Decanoic- . . . . Pauric . . . . 1 . . . . 2 O O O O myristic . . - ° 3 . . . . Eyristoleic . . . . fentadecanoic . . . . ralmitic 2 . 2 . 2 . 2 . Palmitoleic . neptadecanoic . NOOOOOOONHOWmeOOONU‘IOOOl-‘OI-‘OO O mOOOOOOO(DI—'OOOU‘INOOO‘xOCDONNONOObO H 091* O O O O . NM I-‘OOOOOOONHOGOWNOOHHUJOOOOOOOO 0.. O C [—1 O OOOOOOOONOOl—‘OAOOOOOObNOmONl-‘O MN (DCDNroh)htnk‘m(DPJNCDaHoFJO(Odapcnxuoxiwrohio l—l l—‘OOOOOOOI—‘OOubOKDOOOOl-‘(fiOOOOOOOO M84 . HOQOOOOOHOOmOmooooomOOOOOOOO O O \JOOOLOmHmmNNOACOHOmxoOP-‘bmDNNNO H Cottonseed oil fious rations. acetate Experiment II dl- -tocophery1 Basal oil iment I Cottonseed T bxper ”a Eatty acid composition of the total fatty acids basal in the livers from hens fed va (Percent of total fatty acids. 4".- ‘2 do q “ppenoix Fatty acid 000050227022158080281340260400120040r0021030 o.o-cocooooooooooooooooooocooooooooo.o.o... 0000402244421520002800402r002001101405020310 ooooooooooo-oooooooooooooooooooooooooo.o-o. l 010n0014n02a2n21247fiao6xunoOaznoonusooaznoOnonoOaaqunueoonsn62117.7.1 o o o o o o o o O o o o o o o o o o o o o o o o o o o o o o o o o o o o O o o o o o o noOnunoOnunVOMwnoonuno9Mwnuo.OnunoonunoonunooaenoonunoonunoonunVOAUoznVO l l 010n00.4n02a2920e2nflne4nun.0.263011920a24JUoo0n0922nunf4nu4.192041201 OOOOOOOOM/wzooo35050000000000400000000000020 1.3 1 C C C .1 .1 i C r CO COC .1 C a in ini o i C eC ea ear n CC 0. .1 ti c C 11Cnt11C1a sec 411 o r S e .l .1 08.1088 C Ci dn C .d e Yl one +. itaatrtdft i see ale .1 ilc C vxo n.1 s s.aifisa5 vinfdui .nil n hui o o n a r12.13.1t mmt .4aiooSco673 e901cc23 n45673901 r12 ”cu r r1n111_PnV .eponau awn .n lain“u1111917111111112n2auCaz e a V1 "1e abuses +.1.osl r.1 e rel .1 .Vfii D L W...“ m P PP n .1 80 “ML A L B A a L 1...... R 50 batty acid composition of the total fatty acids in the ovaries from hens fed various rations (Percent of fatty acids). ‘ _\ Appendix 4. —_— r‘*'xperiment I Experiment II dl— -toc0phery1 acetate Vottonseed Cottonseed basal Basal Fatty acids oil oil 0000402117101020401.80050240200n3200203020220 o.o-cocoooooooooooooooooooooooooo.o-o.o...- 00004021100012800042004014040081002014030610 on...cocoa-000.00.000.00... cocoa-0000....- 00000000llOOOZWOOJOOlOOOOOOOn/COOOO00000000100 l .1. 0000401110010er402020004Olr302004100303020310 1 0n0n00.4n621lso4AUnolno110aznizazOnu9:0114nunibnu111n60.3no4n0230n9720 0000000010000020700100000002OOOOOOOOOOOOlOO 14 l C C C .1 .1 .1 C r CO CDC .1 C a iin i1n41 o .1 C doc e1a e.ar. n Cm» n .1 t.1 c c 11c cainca a c 571 w r ese .1 .1 oeioee C Ci in C .1 8 Y1 one it t.a+ttsa+t .1 ace ile .1 i1C C VAC n.1 s s a41i_ab rncdal .n11 n hui o on“ ar12131t mmt .4aiOOSCOC78e9OlCC23n4 673901r12 cu r rndflpo eenn an h llaullgl5111122dC2 boa Y vie ea. tt11041 r41 8 rlr .1 1 "Xi 1?» M mxro.P"n.i abnon.n a.“ o n.n L was Cottonseed oil acetate dl- —toc0pheryl Experiment II :asal I oil 51 Cottonseed EXperiment g yolks from hens fed various rations basal ercent of total fatty acids). 93 5. ‘atty acid composition of the total fatty acids in ( q Appendix fatty acid . A 0000402004000480500702001201001000202030500 OOOOOOOOHIOOOB.4OOMOOOOO000OOZOOOOOOOOOOOOlOO 0000402164000800800302000203nuOlloozozozoglo ooooooooooooooooooooooooooooooooooooocoo... OOoooooomBOoongnDOOloooooOOZOOOOOOOOOOOOlOO l nonoonua.0.2no4azn60nuno9n092014110a2noOnuozoaonooaziloguozoqsno4nuoalnu OOOOOOOOfllooomMuonl001...OOGOOOOZOOOOOOOOOOOOlOO l 0000402048000920200102000204002000«503040340 O. oooooooomloooglog00100000002000000000000100 4 11 c c c .1 i .1 c r O CDC .1 C a cn ini O i C eC ia ear n CC n .1 ti C c occnc11c1a a c iTi o r s e .1 .1 l e .1 0 e e C C .1 d n C n... e v.1 on. +t Oadtrtaft i see ile .1 41C C ”no n.1 s traiTiaus r.C:.l hnl n bni o o n a r12.13.1t mmt .4 aimoScoal/C e901cc23n45_b7.._,._901r..12 cu r rnllpo eenn an h llaullmbllll 1225C2 .ea Y Yeaaes thi r.1 8 rr .1 Vii b.b Uh n.ro.P.a.1 .omon_b a.b .o .A,z .b a.“ —~ W m hens on the basal ration onl IrO H 'composition in the contents of the gastrointestinal tract and elected tissues Percent of total fatty acids). Eatty acid ? Appendix 6. Experiment I Plasma Liver Ovary eces L? ‘- Intestine 2 CrOp Gizzard 1 Ration Fatty acid OCDCDO‘TC)N(VKV¢CDOrfiCDQCDK)OCVF10F4PHDCQNCD C)O(DC)O(DC>OCDPiOCDCDOCDC)mCDC)HCDC)OCDC)O(D N was H QQQQMQNNVWMNH®HONONmO¢VO¢®O O O O O OCDOCDOC)OCDOFJOCDOor4c>ocazr3c>o Odg<3g30r40h4c>oc3c>oOCDCDO(30\O(3C)O:30\OE:CDOCDCHDCDO(DCJO H H O(“Phnp‘OCVCWCCVwrimr‘Olo(DCDQCDCV¢<¥O(VUDH O O O OCDCHDCDOCDngF‘OCDCH0230:%C)O(VFHDCDO(3C)O O‘nC)OT\C)N(DC)m(DCJNF‘U)O\DCHDCVOr4F40‘¢CDO o o o to o 00 o o. OCDCJOr4C)O(3g3H(3C30Lng3053C30(nCh4CDO(DCDO OCDFiOCDCDMr4¢3mr4CDH(CG)OCDC)NCQCHVF40(VF10 O O 0 O OCDC)OCDC)O(DEr4CDOCDVW;CDS(DCD¢CDCDOCDCDOCD 0 0 -H -H 0 L10 0!) H sic L)Cv4 O m m H MLa c LJU U U P'U()m()m «50 whfl -a H 00HHO@ 0 OH Us 0 00 u uUuOUu H mm mm H C~4 m m quvamU) HIJUr4 £r4 c «SHP4N«4WLH+JEE§D Id'mwac>oU10<3®r~d30H us 14 HCHHCM) owns or: .2 AH 0:0 t» ma:m:ua)m LMACDH LwH o 9.4 '2 athcha.m H mcazsq £31 an Appendix 6 Continued ......... Liver Ovary ‘lasma Feces (Y) Intestine 2 l Gizzard Lrop ’\ fatty acid .1 53 fi‘r—lOOCOOV‘OVPOV‘ONOO NOOOOOOOOOOOONOO mOONNOOKDOKOOVI'r-lmmm 0000000000000... [\OOOOOOOOOOOOMOO KI‘YI‘HPPOHFONOONOHNCC‘ HOOOOOOHOu—lr—JOOHNO OHO‘DOC)r—JIOO10 +10 0 x c .Cw4 0 Did UtJnJm Co<3CJOCDCDOr4CJOr4U3or~c>Or4CDOO© O 0 O O I O O O O O O O 0 O O I O O O I O O O 0 O 0 O OCDCDO‘VF40C32*HCD O owflcajroriHrACDOCDCHDCDO chcgNrnc)m O O O O I 0 I O O O O O O 0 0000000000... OOOOOOOOFr—‘IO r-l VO‘OO‘OOHOOOOOOON I-l Fir-I N OHOOl-nQNv—lmKONNr-INMNCUOV‘CNHNNr—INNQN oooooooooooooooooooooooooooo OCDCJOCDCJOCDPwACJO<3"VwcoriOc3r40<3c>0<3c>or4 H rim “1 OOOOmHHNOmmmOHKONr-IOKDOOOmONNOm OOOOOOOOOCOOOG)MOQOOI—IOOOOOOON OOOOKOHNONmr-iOOO‘COOCDNOQNNNONVHV‘ OOOOOOOOmu—IOOOmmOI-SOONOOOOOOOO OOOC‘IKO(\l(\lr-i0mmOr--INCI)O|'\CDOC)C)mmOP-ir-IP-ICDQi .0 O OOOOHOOOQHOOOW30%OOHHOOOOOOO OOOOCOOr-‘lOfi‘Or-lOn-lfl‘VO‘DONV'OONONOOV‘ OOOOOOOOSHOOOMSOQOONOOOOOOOO U 0 H H 0 (JO ()0!) -H 0 ~48: HCH O H 0:0 0:08; c 0!) c (J o HqUIJHIUtU mt) -a++ 0 H -H c)m«4c>mmczc m c .S Hrdm mm m mmmwmm DHOH HH 0 84 Gig 3 szhtharu-d m<325q 8 A a '8 continued 000...... Appendix 7. Intestine Liver Ovary sma Pla Gizzard l 2 3 Feces h Cron Ration Fatty acid 55 OOONOOmONOr-lomoo OOOOOOOOOOOOOOO r-iOr-INOOCOONHNr-ICOOr—l OOOOOOOOHOOONOO NOV“O\OOOOON0)ON'<1‘H OOI—IHOON r—ic—{OOv—lmm NNBkOVT‘kOI‘NVI‘NNNNOI“ 00000000000000 0 OOOOOOOOOOOOHHO VQVNNONONOOer—la—l o o 0 00000000000. OOOOOOOOOOOHOOO HOQ'V‘VOmONt—l o o 0 o o o o o o 0 0000000000 NOQ‘LnNQNoNQNNbLnH 000000000000000 000000000000000 OHNV'Q‘HKDOV'V'NOMOKD 00000000000000. OOOOOOOOOOOOOOH OONV‘OONOONOHVNO 0000000 000 OOOOOOOOOOOOOOO 0 H H mo 0 0H H um H mH (D >1 0 0 0 X: H 0 8C UNMC¢m®hmmOH N SHHOHHHHHHNNGHN H ~r-l >10“ m A m ‘- Plasma “iver OvarV Feces plus dl-a-tocopharyl acetate 3 Lntestine 2 Experiment II 1 rom hens fed basal ration Gizzard I- ... .L. ~ Croo Ration I I-atty acni c0mpositflm1 in the contents of the gastrointestinal tract and selected tissues (Percent of tota1 fatty acids). Appendix 8. Fatty acid 56 OOOOV‘ONHMNOOHWVOCOQNNQHNQ OOOOOOIOOOIOOOOOOOOOO... OOOOOOOOr-II-IOOOOCOOCOOOHOOOO N HF) ,._.| OOOOVONNFV)NNNONOOQHI\OHQ‘\Q O OOOOOOOOOHOOOFKQOOOOOOOOO N HN N ONOOOV"C’:)(Y)[\NOOOV‘OJOHNVJ'KDr-imq'm ooooooooooooooooooooooo O OOOOHOOOkOr—IHOOr-IVS‘Or—lOOOOOOO N mm H CD¢CDF+®CDWHDCVNCD m m>mkor40CBCDOlOGJQ O O O O O O 0 O O O O O O O 0 O 0 0 0 O O O OCDCDOrfiodOCDF3NC3 o P~m<3ggordcdmc3c>o . H OOOOWmQ'NONOWKD (D mC‘OKDODOmOONV‘O 00000000000 0 000 0000500 0 H HN OHHNKOHMVODV‘KDMHNODMKOOLOmr-{NNH oooooooooooooooooooooooo H Hf—‘l m OOOOQONNKOGDU) r-l (Ol‘Nfi'Ov—{V‘QNNO O O OCDCDOCDCJOCDCVOCD o GDQCDG\OU<)©r~d)0 03 H ucHHmo 00cc IUC .C QJM >1 paw erQJm 4J~10~4 Law 0 0.4 :3 EChh mtnva uao;zt1 33.1 n 0.2 H o O (“’7 9 10 11 NO 0 0 00 \DO‘ 00 00 FIN 00 00 NN 00 OO r-lr-i OO 0“") 00 00 NO 00 CO CO CO Plasma Liver Ovary Feces Intestine CrOp Gizzard 1 Continued ........... Ration Appendix 8. Fatty acid 57 O‘OOCOOOOr-IOHOHOQ‘V‘H .0 NOONOOOOOOOOOOOO MOOr-INOOCDOCDONOODOO O. GOOOOOOOOOOOONOO mNQNNNQNQNr—lmo-immo 0...... 0 0 . 0 . 0 0 . . HOOOOOOOOOOOOF‘IMO NNmHQ‘V‘Or-IOOOKOOKOmLD I. r—IOOHOOOHOHOOOHNH oaaoomovmvomomoa NOOOOOOOOOOOOHOO KOOOCCQ‘VNmONKOkaNQKQ 000.00.000.00... HOOOOOOOOOOOOHNO LOONmKOOf—INONHNHHON 0 00 00 .0 000.000.. HOOOOOOOOOOOOHOO OOOQ‘hKONVOVmO'ZmQNt—l 0000.00.00...... OOOOOOOOOOOOOOv—IO VI‘HO’Q‘CONVI‘CDONOWONCDN OOOOOOOOOOOOOOOO p.0rnnwmxooqmcgpugr+ouou>m O O I I O O O O O O O O O O O O OOOOOOOOOOOOOOOO U H 5.. U rUU -:--l U 0).,4 C. H 4.10) O H (OI—1 '0 0) >0 H0 U Kr: L2H O Owd UUchv‘mwhmmOr-{HUN m:3Hr4(marqrh4r4afvowovim L1H -H >m' 4:31 A w: Ovary Liver l tract and Plasma astrointestina rv V .J Intestine l ). Experiment II Uizzard -composition in the contents of the Crop selected tissues from hen fed basal ration plus 3% crude cottonseed oil and dl ( -a-toc0pheryl acetate Percent of total fatty acids ‘atty acid Ration 13“ a. Appendix 9. Fatty acid 53 (>0<3c>v<3~h4c>vcarhaoavcorqor4c>orfi"Wocuuuo OOOOOOOOV‘OOOOKONOCOOOHOOOOOOO N Hm ._.| OOOOV‘OHHNmfi‘NHNBOmHHOHOOOmmO O 0 0000.00.00.00.0000000000. QNNNKQNmr-IV“QKOQQ*<1'OOOJLON‘oc3c>oor4or4c>ococno<3C) H Hr4 ‘“ OOOOV‘Ou—IHNKOV“NNOVI‘OGJOOODONOOHHO OOOOOOOOOOOOOENHU‘OOHOOOOOOO r-‘lr-l OCVHhACDOCVC)mo<$U7Nw :xa)m:uo)m 4J~10w4 qu m :14 g szmcna.m-H mcaa a 4414 3 Continued Appendix 9. Intestine Plasma Liver Ovary Feces 3 2 l CrOp Gizzard Ration Fatty acid 59 OOONHOONOMOONKDHO O .0 NOONOOOOOOOOOOOO KOHONNNOMOKDOMNV‘DH 0.00.00.00.0000 O NOOOOOOOOOOOOc—IO V‘OOOr-{Hv—IKOOkDVd'NNNN O O NOOr-IOOOOOOr-IOc-II—INO HHOC‘mOHmOV'mKOKOOmC‘ 000.00.00.00... 0 Hracw4r4c>or4c>ococ>o(“04H Ol—{NFV‘OHMOHONNOI—Im 0 0 0 HOHHHOOOOOOOONOO \O‘Y§NHKOONVHHHOI\WON HOOOHHOOOOOHHHmO C‘I—{OcjkCDOOKCOV'HNV‘NmCD 0. 00...... . 0 0... HOOOOOOOOOOOONO)O WHONO‘CflomOMMNQ‘mNN OOOOOHOOOOOOOOu—IN “lmomhtooNommlnomxoh IO... 0 0.0.0. .0. O OOOOOOOHOOO OOOO VWDH4©KQCDOr4CDHCDr10‘$CDH OOOOOOOOOOOOOOOO U H H U r50 H 0 “*4 c -H +:m 0 H mrq U m >10 +10 U x c .Cw4 O O-H ULJNfficjwtnUDNCDOWOrfiLaflrfl m SriFiUNfiF4F4F4FMH‘VCVTjT4N “H ’H >i/ axa J m‘“ S w. R A R m L Y W S R E W N U E T A” VIII»! 1 3 1|?"me