E-134-6M-Ll80 TEXASAHRICULTURAL EXPERIMENT snnou A. B. CONNER, DIRECTOR COLLEGE STATION, BRAZOS COUNTY. TEXAS BULLETIN NO. 513 AUGUST, 1935 DIVISION OF CHEMISTRY AND DIVISION OF RURAL HOME RESEARCH The Relation of the Color and Carotene Content of Butter Fat to Its Vitamin A Potency L i E; A :5? Y‘ Azricultusal iiéillilil-Iiésaii iTieztharar-P 0* "B"! \ Gollage Station, Iexafi AGRICULTURAL AND MECHANICAL COLLEGE OF TEXAS T. O. WALTON, President Administration : A. B. Conner, M. S., Director R. E. Karper, M. S., Vice Director Clarice Mixson, B. A., Secretary M. P. Holleman, Chief Clerk D. R. McDonald, Asst. Chief Clerk Chester Higgs, Executive Assistant Howard Berry, B. S., Technical Asst. Chemistry: . S. Fraps, Ph. D., Chief; State Chemist E. Asbury, M. S., Chemist ~ F. Fudge, Ph. D., Chemist C. Carlyle, M. S., Asst. Chemist L. Ogier, B. S., Asst. Chemist . J. Sterges, M. S., Asst. Chemist Velma Graham, Asst. Chemist Jeanne F. DeMottier, Asst. Chemist W. H. Garman, M. S., Asst Chemist A. R. Kemmerer, Ph. D., Asst.’ Chemist A. W. Walde, Ph. D., Asst. Chemist F. J. McClure, Ph. D., Asst. Chemist Horticulture: S. H. Yarnell, Sc. D., Chief Range Animal Husbandry: J. M. Jones, A. M. Chief B. L. Warwick, Ph. D., Breeding Investiga. S. P. Davis, Wool and Mohair Specialist J. H. Jones, B. S., Animal Husbandman Entomology: F. L. Thomas, Ph. D., Chief ; State Entomologist H. J. Reinhard, B. S., Entomologist R. K. Fletcher, Ph. D., Entomologist W. L. Owen, Jr., M. S., Entomologist J. N. Roney, M. S., Entomologist J. C. Gaines, Jr., M. S., Entomologist S. E. Jones, M. S., Entomologist F. F. Bibby, B. S., Entomologist "R. W. Moreland. B. S., Asst. Entomologist C. E. Heard, B. S., Chief Inspector C. J. Bilrgin, B. S., Foulbrood Inspector Agronomy: ~ E. B. Reynolds, Ph. D., Chief R. E. Karper, M. S., Agronomist P. C. Mangelsdorf, Sc. D., Agronomist D. T. Killough, M. S., Agronomist J. O. Beasley, M. S., Asst. Agronomist Publications: A. D. Jackson, Chief SUBSTATIONS No. l, Beeville, Bee County: R. A. Hall, B. S., Superintendent No. 2, Tyler, Smith County: P. R. Johnson, M. S., Superintendent "B. H. Hendrickson, B. S., Sci. in Soil Erosion "R. W. Baird, M. S., Assoc. Agr. Engineer No. 3, Angleton, Brazoria County: . R. H. Stansel, M. S., Superintendent H. M. Reed, B. S., Horticulturist No. 4, Jefferson County: R. H. Wyche, B. S., Superintendent "H. M. Beachell, B. S., Junior Agronomist No. 5, Temple, Bell County: Henry Dunlavy, M. S., Superintendent - C. H. Rogers, Ph. D., Plant Pathologist H. E. Rea, B. S., Agronomist “E. B. Deeter, B. S., Soil Erosion .-A-.--v_. Veterinary Science: - ‘M. Francis, D. V. M., Chief H. Schmidt, D. V. M., Veterinarian "F. P. Mathews, D. V. M., M. S., V = Plant Pathology and Physiology: J. J. Taubenhaus, Ph. D., Chief W. L. B. Loring, M. S., Asst. Plant Pathol G. E. Altstatt, M. S., Asst. Plant Pathol "Glenn Boyd, B. S., Asst. Plant Pathologis Farm and Ranch Economics: L. P. Gabbard, M. S., Chief W. E. Paulson, Ph. D., Marketing C. A. Bonnen, M. S., Farm Management ic"W. R. Nisbet, B. S., Ranch Management "A. C. Magee, M. S., Farm_Management Rural Home Research: ~ Jessie Whitacre, Ph. D., Chief Mary Anna Grimes, M. ‘S., Textiles Sylvia Cover, Ph. D., Foods Soil Survey: "W. T. Carter, B. S., Chief E. H. Templin, B. S., Soil Surveyor J. W. Huckabee, B. S., Soil Surveyor I. C. Mowery, B. S., Soil Surveyor Botany: V. L. Cory, M. S., Acting Chief Swine Husbandry: Fred Hale, M. S., Chief Dairy Husbandry: O. C. Copeland, M. S., Dairy Husbandman Poultry Husbandry: R. M. Sherwood, M. S., Chief , - Poultry Hush. Z E11 s m E Q- “U i’ J. R. Couch, M. S., Assoc. Paul D. Stu kie, B. S., Asst. Poultry Husb. Agricultural ngineering: H. P. Smith, M. S., Chief l‘ ,' Main Station Farm: G .T. Mc ess, Superintendent Apiculture ( an Antonio): H. B. Parks, B. S., Chief A. H. Alex, B. S., Queen Breeder Feed Control Service: F. D. Fuller, M. S., Chief James Sullivan, Asst. Chief. . D. Pearce, Secretary . H. Rogers, Feed Inspector L. Kirkland, B. S., Feed Inspector D. Reynolds, Jr., Feed Inspector A. Moore, Feed Inspector J. Wilson, B. S., Feed Inspector G. Wickes, D. V. M., Feed Inspector J. K. Francklow, Feed Inspector Fgypgum No. 9, Balmorhea, Reeves County: J. J. Bayles, B. S., Superintendent No. 10, College Station, Brazos County: R. M. Sherwood, M: S., In Charge L. J. McCall, Farm Superintendent No. ll, Nacogdoches, Nacogdoches County: H. F. Morris, M. S. Superintendent "No. 12, Chillicothe, Hardeman County: "J. R. Quinby, M. S. Superintendent "J. C. Stephens, M. A., Asst. Agronomist No. 14, Sonora, Sutton-Edwards Counties: W. H. Dameron, B. S., Superintendent I. B. Boughton, D. V. M., Veterinarian W. T. Hardy, D. V. M., Veterinarian O. L. Carpenter, Shepherd "O. G. Babcock, B. S., Asst. Entomologist No. l5, Weslaco, Hidalgo County: y: "P. L. Hopkins, B. S., Junior Civil Engineer W, H, Friend, B, S., Superintendent l No. 6 Denton, Denton County: P. B. Dunkle, M. S., Superintendent “I. M. Atkins, B. S., Junior Agronomist No. 7, Spur, Dickens County: R. E. Dickson, B. S., Superintendent B. C. Langley, M. S., Agronomist No. 8, Lubbock, Lubbock County: D. L. Jones, Superintendent Frank Gaines, Irrig. and Forest Nurs. Members of Teaching Staff Carrying G. W. Adriance, Ph. D., Horticulture S. W. Bilsing. Ph. D., Entomology _D. Scoates, A. E., Agricultural Engineering A. K. Mackey, M. S., Animal Husbandry R. G. Reeves, Ph. D., Biology J. S. Mogford, M. S., Agronomy F. R. Brison, M. S., Horticulture School of Veterinary Medicine. ‘Dean, S. W. Clark, B. S., Entomologist W. J. Bach, M. S., Plant Pathologist J. F. Wood, B. S., Horticulturist No. 16, Iowa Park, Wichita County: C. H. McDowell, B. S., Superintendent L. E. Brooks, B. S., Horticulturist No. 19, Winterhaven, Dimmit County: E. Mortensen, B. S., Superintendent "L. R. Hawthorn, M. S., Horticulturist Cooperative Projects on the Station: W. R. Horlacher, Ph. D., Genetics H. Knox, M. S., Animal Husbandry A. L. Darnell, M. A., Dairy Husbandry R. O. Berry, B. S., Biology 1 R. T. Stewart, Ph.D., Agronomy V. A. Little, M. S., Entomology F‘ 111.85: August 1, 1935 "In cooperation with U. S. Department of Agriculture. , $In cooperation with Texas Extension Service. ‘In eoomrat ion with State Department of Agriculture. Consumers demand a butter of a rich yellow color. The natural yellow color 0f butter fat is chiefly due to carotene, a precursor of vitamin A. Jersey cows on grass pasture secreted rich yellow butter fat high in carotene content and vitamin A potency, but when these cows were removed from pasture there was a decrease in propor- tion to the length of time the grass was withheld. It was found that yellow corn enabled cows to secrete butter fat containing a little more carotene and of a little higher vitamin A potency than did White corn, nevertheless the vitamin A and carotene decreased during the period of feeding the yellow corn. The decrease was less when 3 pounds of alfalfa leaf meal was added daily to the yellow corn ration, but even 6 pounds daily was not sufficient to check the decrease. With sorghum silage as the sole source of vitamin A, the butter fat was pale in color and low in carotene content and vitamin A potency. The natural yellow color of butter fat is directly proportional to the carotene content. The carotene content of the butter fat is not an accurate measure of the vitamin A potency, but butter fats highly colored are probably‘ high in vitamin A potency and butter fats of a light yellow or nearly white color are low in vita- min A. The relation between the carotene content and the vita- min A potency depends upon the ration of the cow, the quantities of vitamin A and carotene stored at the beginning of the lactation period, the length of time which the ration deficient in vitamin A or carotene has been fed, the extent of such deficiency, and perhaps upon the individual cow. Cows that were on a carotene-poor, vitamin A deficient diet, and that were secreting pale butter fats low in carotene content and vitamin A potency, after only 3 days of 5 hours ‘each on pasture secreted yellow butter fats as high in vitamin A potency but with a carotene content approximately one-third that of butter fat from cows continuously on a diet adequate in carotene. Goats on green grass pasture were found to produce nearly white butter fats, of low carotene content but of high vitamin A potency. The goat evidently varies greatly from the cow in ability to convert the carotene of the feed into vitamin A, and the color of goat butter fat cannot be used as an indication of its vita- min A potency. l CONTENTS o - Page Introduction ‘ A Related Previous Work Source of Material y Method Developed for Estimating Carotene in Butter .................................. -_10 Examination of the Butter Fat-_-__-. , 12 Influence of Yellow and White Corn on Carotene Content and Vitamin A Potency __12 Influence of Alfalfa Leaf Meal and Yellow Corn on Carotene Content and Vitamin A Potency i 14 Influence of Pasture Grass on Carotene Content and Vitamin A Potency 18 Relation of Carotene Content and Vitamin A Potency to the Stage of Depletion 20 Percentage of Vitamin A Potency Due to Carotene ..................................... -22 Relation of Carotene Content to Vitamin A Potency ................................... ..22 \ Relation of Color of Butter Fat to Carotene Content and Vitamin A Potency ..24 Goat Butter Fat l a 29 Acknowledgments I 30 Summary 30 Literature Cited i ..... -. 31 BULLETIN NO.‘ 513 ' AUGUST, 1935 THE RELATION OF THE COLOR AND CAROTENE CON- TENT OEBUTTER FAT TO ITS VITAMIN A POTENCY RAY TREICHLER, DIVISION or CHEMISTRY; MARY ANNA GmMEs, Division or RURAL HOME RESEARCH; G. S. FRAPs, DIVISION or CHEMISTRY For many years consumers of butter have preferred yellow butter to white butter. This preference has been met by coloring the white butter to suit the demand of the consumer. Since it was believed that the addition of artificial coloring did not affect the food value of the butter, the prac- tice of coloring butter has been recognized and is permitted by the pure food laws of the States and of the National government so long as a harmless color is used. Many early investigators reported observations of the intimate relation- ship between the yellow color of various animal materials and their vitamin A activity, while others found evidence to the contrary. Later work showed that this relationship was apparent only when carotene was the source of the yellow color. This latter observation was further strengthened by the discovery that carotene is the precursor or parent sub- stance of vitamin A. Related Previous Work Osborn and Mendel (25), 1913, suggested a probable relationship be- tween the yellow color of butter fats and egg yolks and their vitamin A content. Steenbock and Boutwell (36), 1920, recognized that butter rich in yellow pigment contained large amounts of vitamin A and that oleo oils highly pigmented with yellow were good sources of vitamin A. They stated that vitamin A accompanies the yellow pigment in carrots, sweet potatoes, corn, and other vegetable material, and that in those cases where the vitamin A was found in low amounts or was lacking, the ma- terials were devoid, or nearly so, of yellow color. Hoagland and Snider (19), 1926, found that yellow oleo oil was rela- tively high in vitamin A, while the lighter colored oils were deficient in the vitamin in direct proportion to the lack of yellow color. A number of years ago, Palmer (26), 1912, found from a study of the chemical and physical properties of butter fat pigment that it was identical with carotene regardless of the degree of pigmentation of the butter fat from which the pigment was isolated. Yellow corn, a xanthophyll-rich i fsubstance, did not exert an appreciable influence on the color of the butter The color of butter fat seems to be chiefly due to carotene. Palmer and In Eckles (27), 1914, varied the ration of cows so as to regulate the amount of carotene ingested and measured the color of the butter fat with a Lovibond tintometer. The results showed that the carotene color in the A butter fat was related to the carotene content of the ration. Yellow maize did not exert an appreciable influence on the butter fat color even when fed in amounts ranging from six to ten pounds per day per cow. The same authors (28) found that pigment of human milk fat consisted of l6 BULLETIN NO. 513, TEXAS AGRICULTURAL EXPERIMENT STATION carotene and xanthophyll and that the degree of pigmentation was in- fluenced by the carotinoids in the diet. The importance of the yellow color in butter was emphasized by Moore’s evidence (22), 1930, that the carotene in plant materials is more or less completely converted by the animal body into vitamin A, and Green and Mellanby (17), 1930, claimed that the vitamin A activity of butter, egg yolk, carrots and green vegetables was due to carotene. Palmer and Kempster (29), 1919, stated that the fat-soluble content of butter fat does not closely parallel the yellow color, but that highly pig- mented butters are rich in vitamin A and butters poor in yellow color are generally low in vitamin content. Other workers, Stephenson (37), 1920, and Willimot and Wokes (41), 1927, after removing the yellow pigment from butter fat by absorption, found no decrease in the vitamin A po- tency by the methods then used. Baumann and Steenbock (2), 1933, reported that the vitamin A activity of butter fat is due to carotene and vitamin A although the biological activity of the carotene accmHN E UQSmMHHHHH-H fl UGN m" M950?“ MOM .32. QmUHAM-H ....... 33% 853$ A: 3 5H3 3 33s.: 3 3.3: 3 min: 3 8.3: oH .......................................................... 1 ca»: “was? é: w é: 3 .................................... .6333 32: .32 3:83. .2: 3: .2: 3H .2: 3 é: 3 .2: 3 d: 3 d: 3 E5 as: 5E H5 in HE d: H3 AH: H5 d: H5 5E w.» i: H5 d: HE ................................................ .215 H63 c0300 33H H 93A H 3mm H H35 H Hana H 93m H 3am H ....... .. :3 BEE 3 n33: m @235 w $3.: 3 min: m BB3 m @355 m .................... .1 ..... i ............................. .. wcofiwfifi \ A: H5 min: 3 at»: 3 3.8: 3 $3: 3 $.23 3 mfiwn 3 m3“: 3 ................................................... 43E H33 .6236 mHHwQ 3 mt“: 3 wanna ow F30 35>? wanna Ow mHHNQ 0w $.35 Ow muHwQ Ow ...... 2 CHOU B050? "£33m NH NH. cw HQ w mm 3.. m 038.3 3.3m o» cowfinsunwn 59G 93D m w H. m m N. m H. was?» E mun ouwcmxopna< 3H HHm :3 H3» 3m 3H 8 3 nwnfis: >50 ‘HQ HM N nonaflfi QHHOHU QBOQ M5 GOmuNH vim GOmHNQONm HO ouaam -Q.M4 in Qmfiflhw 10 BULLETIN NO. 513, TEXAS AGRICULTURAL EXPERIMENT STATION alone as a source of vitamin A; cow 322, receiving daily 3 pounds 0L4; heat-dried alfalfa leaf meal of high potency, in addition to yellow corng] and cow 329, receiving daily 6 pounds of the alfalfa leaf meal with the: yellow corn. The grain mixture consisted of 60 parts yellow corn, 2770‘! parts cottonseed meal, 10 parts wheat bran, 2 parts ground limestone,“ and 1 part salt, fed at the rate of approximately 1 pound to 21/2 pounds 7 of milk. All three cows were also fed daily approximately 20 poundsJZ. of wet beet pulp and cottonseed hulls ad libitum. Considerable difficultyj was encountered in getting the cows to eat all the alfalfa meal. It was thoroughly mixed with the beet- pulp, and the grain was spread over the t? mixture in small quantities at a time in order to get the cows to eat it. ~ In the third group, cows 301 and 311 were depleted of vitamin A by h receiving daily for 60 days 14 pounds of a grain mixture consisting of 60 parts white hominy feed, 27 parts cottonseed meal, 10 parts wheat bran, p 2 parts limestone, 1 part salt, and 12 pounds wet beet pulp with cotton- seed hulls ad libitum. They were turned on young sudan grass pasture for 5 hours daily for the remainder of the experimental period. Butter was prepared just before the cows were placed on pasture, and thereafter at regular intervals. A fourth group provided butter fats from cow 182, and from goats. Cow 182 had been on a ration consisting of sorghum silage, cottonseed meal, and cottonseed hulls ad libitum for approximately 5 years. Goat butter fat, sample No. 37741, was obtained from a group of 25 Angora goats in a late stage of lactation and giving little milk. No grain or hay was being fed and the pasture was poor. Sample No. 39740 was obtained from a group of Angora goats at from 45 to 60 days after the lactation period began. No grain or hay was fed, but these goats were on good pasture. Method Developed for Estimating Carotene in Butter ‘Several methods have been used for estimating carotene in butter. Palmer (27), Wilbur, Hilton, and Hauge (40), and others compared the color of the melted butter fat with a 0.2 per cent solution of potassium dichromate in a colorimeter. The color is assumed to be the same as that of carotene in petroleum ether, but as was shown by Barnett (1), the color intensity of carotene in butter fat is several times that in petroleum ether. Gillam et al (13) saponified the butter with caustic soda, ex~ tracted the carotene with ether, evaporated to dryness, dissolved in chloro- form, and estimated it by measuring the absorption of light at 450-460 millimicrons. Baumann and Steenbock (2) measured the intensity of absorption of melted butter fat at 460 and 485 millimicrons and compared the results with the absorption of a standard solution of carotene in purified cottonseed oil (Wessen oil). Shrewsbury and Kraybill (34) mixed 7 grams of butter fat with 100 cc. of petroleum ether and determined the absorption at 436 millimicrons. Barnett (1)\ diluted the butter fat with cocoanut oil and measured the absorption at 455 to .500 millimicrons. He also estimated the carotene by comparing the color in a colorimeter with a 0.2 per cent RELATION OF COLOR AND CAROTENE OF BUTTER FAT TO VITAMIN A 11 solution of potassium dichromate, using a factor to correct for the greater intensity of color of the carotene in butter fat. The butter fat samples in this study were prepared from freshly made butter. The butter was melted at a temperature not exceeding 60° C., and the butter fat was decanted off, filtered into brown glass containers, and stored in an electric refrigerator. The sample was removed from the refrigerator only for feeding or making transmission tests. No butter fat was diluted for color analysis. Since it was shown by Barnett (1) that carotene in butter absorbs more light than carotene dissolved in petroleum ether or cocoanut oil, it was found necessary to devise a method for calculating the carotene from absorption of light by the melted butter fat. The color produced by carotene in butter fat was standardized against its absorption of light by the following procedure. Solutions of carotene in butter fat were made from S. M. A. carotene (a mixture of approximately 15% Alpha and 85% Beta). To purify this carotene, 0.1 gram was dissolved in 20 cc. of chloroform, precipitated by the addition of 20 cc. of methyl alcohol, filtered, washed, and dried under reduced pressure over calcium chloride. Twenty milligrams of the purified carotene were dissolved in 50 cc. of petroleum benzine (73-76° C. boiling point), and 0.25 cc. of this was made up to 5.000 grams with melted butter fat. This constituted the stock solution. Desired amounts of the stock solution were weighed and added to sufficient butter fat to make 5.0 grams. The butter fat was melted, mixed by rotation, and placed in transmission tubes 10 millimeters deep. Then the percentage of transmission of light was read on a Keuffel and Esser spectrophotometer at 470, 480, and 490 millimicrons with a sample of the original butter fat in the comparison tube, to correct for its color. The average of at least 20 readings made at each of the three wave lengths for each sample was used. Two separate samples were made up each day for a period of 4 days, making a total of 8 samples for each weight of carotene in. butter fat examined. A series of solutions was made with 0.6, 1.0, 1.5, 1.75, 2.0, 2.25, 2.5, 3.0, and 4.0 micrograms of carotene per gram of butter fat. As the resulting densities when plotted. did not form a regular curve, it was considered best to base the method of calculating unknown butter fats upon the actual results, instead of upon a theoretical curve. The density at each point was calculated to 1 microgram of carotene per gram, and those close together were arbitrarily selected and averaged. The reciprocal of the average densities was then calculated so that the results of any reading can be expressed in micrograms of carotene per gram of butter fat by multiplying the density by the appropriate factor. These are given in Table 2. The density at 490 millimicrons was not used, because the absorption at this point is not as closely related to the carotene in the butter fat as at other parts of the spectrum. 12 BULLETIN NO. 513, TEXAS AGRICULTURAL EXPERIMENT STATION Examination of the Butter Fat Each butter fat sample was prepared for examination 0n the Keuffel and Esser spectrophotometer by being melted at 40° C. and placed in a 10 _millimeter transmission tube. For samples from cows 59 and 61 the portion of the spectrum examined ranged from 440 to 700 millimicrons. All other samples were read at 470, 480, and 490 millimicrons. The densities at 470 and 480 millimicrons were multiplied by the cor- responding factors in Table 2, and the average of the two was expressed as the micrograms of carotene per gram of butter fat. No corrections Were made for the presence of coloring materials other than carotene. The results are given in Table 3. All vitamin A determinations were made by the modified Sherman- Munsell method employed in this laboratory, the details of which were given in a previous paper (11). The results are expressed in units of vitamin A potency (Table 3). Table 2. Factors for conversion of color density, read in a 10 mm. tube, to micrograms of carotene per gram, or parts per million For 470 millimicrons Factor If density is less than .240 multiply density by ______________________________________________ _. 2.92 If density is from .240 to .310 multiply density by ....... ._ 3.72 If density is from .310 to _560 multiply density by ___________________________________ 47 4.43 If density is from .560 to .710 multiply density by ........................ .1. ........ ._ 5.16 If density is over .710 multiply density by 5.55 For 480 millimicrons Factor . . . . l If density 1s less than .210 multiply density by ......... .. . I 3.53 If density is from .210 to .260 multiply density by .................................. ..| 4.63 If density is from .260 to .505 multiply density 5.00 If density is over .505 multiply density by . _' 5.75 l Influence of Yellow and White Corn on Carotene Content_and Vitamin A Potency The carotene content of the butter fat samples from cow 59 (fed white corn) decreased from 9.18 micrograms per gram of butter fat at the beginning to 0.06 micrograms per gram in 28 weeks, a total decrease of 99 per cent. Ninety-three per cent of the total decrease occurred during the first six weeks. After the cow had been 2 weeks on pasture, the carotene content rose to 4.44 micrograms per gram of butter fat, or 48 per cent of the initial content. This increase amounted to 7300 per cent of the carotene content of the 28th week (Table 3 and Figure 1). Although there was a marked decrease in the quantity of both‘ carotene and units of vitamin A potency in the butter fats, there was an increase in the ratio of units of vitamin A potency to carotene content. It is evident that white corn does not provide sufficent vitamin A or carotene I -11. RELATION OF COLOR AND CAROTENE OF BUTTER FAT TO VITAMIN A 13 for a cow to secrete milk of appreciablewitaimin A potency and carotene content. ' The carotene content of the butter fat samples from cow 61 (on yellow corn) decreased from 10.66 micrograms per gram of butter fat to 0.29 micrograms per gram, this decrease amounting to 97 per cent. Of the total decrease during the period of 28 weeks, 93 per cent occurred in the first Units Vitamin A Micrograms Carotene 4o , 20 O 35 18 32 . 16 29 T’ 14 24 ' 12 3° ‘ 1O I \ O 16 \ \ 12 \ 4 6 O \ O \ 8 o aid} \ v 4 \ . g/ \ .34 4 . . 8/ 2 \ . g \ m. / " ~ 0- _ _ _ 1 LII Q __—-—O——-——o--__q—— -a Q Weeks 4 8 12 16 2O 24 28 32 Fig. 1. The carotene content and vitamin A potency of butter fats from cow 59, receiving white corn. 8 weeks. After the cow had been on grass pasture for 2 weeks, the carotene content increased t0 4.53 micrograms per gram of butter fat, or 43 per cent of the initial content and 1462 per cent of the carotene content of the 28th week (Table 3 and Figure 2). The decrease in units of vitamin A potency and carotene content of the butter fat produced by this cow on yellow corn was extensive but slightly less than that of the cow on white corn. The percentage of vitamin A potency due to carotene was higher and the ratio of vitamin A units to carotene lower at all times than was that of cow 59. The carotene content of the butter fat samples from cow 196 (fed yellow corn) decreased from an initial content of 12.78 micrograms per gram of butter fat to 1.03 micrograms per gram during the experimental period. The total decrease in carotene content in 20 weeks amounted to 92 per cent of the initial content, of which decrease 97 per cent occurred during the first 8 weeks (Table 3). Of the three cows receiving a vitamin A deficient ration containing corn, the two receiving yellow corn produced butter fat. containing more carotene and having a higher vitamin A potency than the cow receiving white corn. This is shown in Table 3, and in Figures 1 and 2. At the be- 14 BULLETIN NO. 513, TEXAS AGRICULTURAL EXPERIMENT STATION ginning of the feeding and at the higher levels of vitamin A potency of the butter fats there is little difference in the carotene content, but at the lower levels the cows receiving yellow corn give a butter fat con- taining more carotene than that from the cow receiving white corn. This shows that both the coloring matter and the vitamin A potency of the butter fat are influenced by the yellow corn, although the influence is not great. Both butter fats were practically white in later stages of the experiment. \ Units Vitaznin A llieresrw- first” 4o Z0 U I 35 18 3g 16 3g 14 24 a 12 ~ <1 20 n 1° I1’ \ 4-? 16 \ ' g 8 \ c \ 12 \ \, 6 O \\ a g \ l’ 4 \. o________ o ~ \ ..______, . 57 4 \I\ .1’? é 2 ~ . _ ~ \ ri./ (l; Q _.____.--—-o--——I——""" 0 Week: 4 a 12 1e zo m 2a a2 Fig. 2. The carotene content and vitamin- A potency of butter fats from cow 61, receiving yellow corn. Influence of Alfalfa Leaf Meal and Yellow Corn on Carotene Content and Vitamin A Potency The carotene content of the butter fat from Cow 322, receiving yellow corn and 3 pounds of alfalfa leaf meal, decreased from an initial value of 12.18 micrograms per gram of butter fat to 1.78 micrograms per gram in the 32 weeks. The total decrease amounted to 85 per cent of the" initial content, while 82 per cent of the total decrease occurred during the first 4 weeks. The cow on the vitamin A deficient ration, receiving 3 pounds alfalfa meal in addition to yellow corn, produced butter fat containing more carotene for the same vitamin A potency than the cows receiving the yellow corn alone. After the initial sample, which was colostrum (33), there was less loss in vitamin A potency and carotene content of the butter fats from cow 322 (getting 3 pounds of alfalfa leaf meal in addition to yellow corn) than from cows 61 and 196 (on yellow corn alone). The ratio of vitamin A potency to carotene did not show the rapid increase 15 RELATION OF 001.011 AND CAROTENE OF BUTTER FAT TO VITAMIN A 3N 6.3555 w m.» wan mam? .4 was mfiomow QTHENMN no mxwwB o?» ~3w4~ 6.2525 mmwnm E m 24 wN.0 ww.0 m3 0N $3 .0 fiwm wNwwm .011 Nfiw 0w w v0.0 NN.0 mN.0 v2 0H £2 .NH N54 35m m % 09m 0m m m: 00.0 ww.0 02 w 0N2 .5 wish $5M 0 3N 3 w mm.” $5 w0.0 3 w £2 .0N .32 2.5m mm/ 00A mm mN wwfi 0m.N NN.N 3 0 mmfi .NN $.54 5.3m m\....w. 3m 0m mN 3w $3 Nw.0 5N AN $2 .N|H .32 $5..“ $4: 0N m 0N0 00.0 00.0 mNN wN $3 £7: £00 Q05.“ 50H 0H w wN.0 :2 w0.0 m3 wN $2 .0N|0H 590m 2.5m U .32 2 N 2.0 w0.0 2s >2 0N N02 .NN|NN $54 ~35 ) 56H 3 w. ww.0 00.0 :0 0.2. 0H $2 .0N|mN 35.0 000mm .A ww.0 S 0 N0.0 N40 0N.0 H: NH $3 .wN..>N 2.2. fl 3%” W ww.0 wN N. N04 NN.0 0N0 00 w $3 0.3: mag _ mmwwm o ma... wN NN w0.N Nw.0 ww.0 m... 0 N02 d-N 5x2 _ Nwmwm m ma... 0m E NNN ww.0 00.0 ww w N02 dNLMN 1.54 3&5 w 23 0H mm wNw N50 2.... .3 m N00." .0.TwH $.34 32m 1 m0...“ mm S g...“ w0.0 NOA wm N N02 NT: $.54 203m 1W ma.” 5. mm 00.3 .22 vN H 32 .m $.34 mmNwm fiNé mN Nm ww.w $5 0w.0 2N HN $3 NA .>oZ $9..“ $3 H H 00.0 N0.0 N0.0 2: wN N00.“ d7: JuO O25 00.0w m N w0.0 N0.0 N0.0 F: wN N02 .0N|0~ 30m 3N5 w omflm w m w0.0 N0.0 2... m3 0N N004 fiN-NN .M§4 N35 wwam m N. 3.0 w0.0 v0.0 m: 3 N03 dNéN 3:0 @215 m, 0N0m m Z wN.0 w0.0 0H0 mm NH $2 .wN|~_N wish 35:.“ u.. 00.2 0 w ww.0 3.0 2.0 mw w fir: .513 52.2 wmwwm n». 3.2 wH w w0.0 2.0 wN.0 .3.» 0 $3 5N hug 933 a ww.0 m: 0N IN ww.0 ww.0 wN w $3 dNéN $.34 95%. w 2.0 m: mm 3w 00.0 E20 5 m N23 .072 $.54 Nwmwm m NN.w 0m mN N06 w0.0 :4 0H N $2 N73 $.34 Nomwm .1. wmw mm 0w 2.0 EA 3.4 w H $2 6 $.34 NaNwm Q. wnouoawo 55w»? fin: wfiwnwonumfi HHOQQPOHNU 0mm nmfibn paw aouuzn mcouoib mnonoin cwuwn 5036M .3285: m op min: miwnwonofih Edkw non Edhw 3a A215 0ww 122E 00w nofiwuofifl fiwunmniuwnxo moflnfidw mo wHmQ macaw m m“ 4 aria»? 0.0 wfloaoawo 4 c0503?» wcwuouwo an mfimcmfi an 35:00“ nvuww wzwfi flo mxoo? lnonwQ Wm 03.3w S. m3. zoiwuon 32D wfiwuwoaumwfi 1 < awfiw»? N. 0am uQ-i-A 0a 0:355 uiuuauwu 0:: hOGQuOQ 4 5E5?» 63cc we £03303 u: ¢M$u win 5.35m 5 01in. n 5 NE! ... wit»! N 0 I T A T S T N E M I R E P X E L A R U T L U c TL R G A S A X E T s. 1 5 0 N N 1 T E L L U B 16 duflwxmfi a mu 0mm 0.3.3 .4 HE.» mHHomow wifiwwm duzumufl wwwuw Go 930B o3» .534? 8 w é-.- .... -. i- S» mam $2 fi .30 £2; I “v0.0 0H l. Hw.0 HN.0 #N.0 0wN NvN Ex: Q. .250 4:53. fi WW 0H6 0N mHH 0N0 00.0 Ea HwN .32 .0 >000 M233 9 m. 00.»: 0H m 00.0 00.0 H0.0 NH 2N $3 .0H .000 g0” I. B 00 m0.N 5 0H 00.0 N00 N00 ma“ N0 $3 .N 60G $300 M HM 00.0 0m. 0H 00.0 00.0 00.0 3w 0N £3 J. 5oz 00000 m8) 00H m0 0H 00.0 00.H H0.H 00H 0N $3 .0 .35 000.00 M $9 NwH N0 HH H00 v0.H >0.H wNH 0H m»? .NH $54 $5» 0 Jwu. 00H w0 NH .000 ¢N.H mN.H 00H m mm0H .3 wiflh. 93.5 m m 1 Nm.N 00 0N m0$ mNH 09H N0 v $3 .0N >62 265 u 9 n. HlflN m0 mm .32 0¢.N 0HXN .3 0 £3 .NN E54 05000 ("W21 T? 0¢€ >0 w 00H 00.0 0Y0 wNN N0 m00H .N 60G $15 K. 2d 0H 0H NH.H HQN.0 00.0 00N 0N mmfi J. .32 @0000 %wwvZ v0.0 mm. v 0w.N 0Y0 00.0 0HH 0N $3 .0 fiww @805 0 .l@ a3. 3 S w?“ m3. $5 ww 3 £2 .5 .34 $5 M m I Hw.m 0N vH HHXN 00.0 n00 00 w 000H QH widh 35m m 1am... 00.0 .2. 3 00.0 00.0 00.0 Nm H» £2 .0N 0G2“ H0000 M m . EB 0N 0b 2.2 mH.N mH.N H. 0 $3 .NN mS< $3M (m V H4 mcoaonso AS830?» fin: N mfiwawouomfi Hflwaouonau us.“ .332: an“ pofisn mnonomfi mnoaumfi awmwn 903mm umnfin: m 8. $15 mawawonowfi EHNHM .80 8.3m non I215 00¢ IMZME 0 S. nompwuum: Hwwflwimaonwo 335$ mo wpwQ E3.» w nod Q c053? 0.0 wcoaopwu 4 c083? ocwuonwo as zfimcofl an hnzmcmfl .800.» mmwfl no wave? Lonaq u. m ofiwfl 3 2% hucouofl BED wiauwonumfi n Q ASEQfiHP e0 eoufiuuaOleam uofi-E no 2525a anon-ease e5- 55:3 4 58a»? 6B3 we c.5302 we swan-m can 5.03M .0 visa. RELATION OF COLOR AND CAROTENE OF BUTTER FAT TO VITAMIN A 17 occurring in the cases of cows 59 and\61, but was greater than that for cow 196. The addition of 3 pounds of alfalfa leaf meal to the ration including yellow corn evidently prevented as rapid and extensive a decrease in vitamin A potency and carotene content as occurred when yellow corn alone was fed. The carotene content of the butter fat from cow 329 (fed 6 pounds of alfalfa leaf meal) decreased from 13.55 micrograms per gram of butter fat in the initial sample to 3.39 micrograms per gram in the sample taken after 32 weeks. The total decrease amounted to 75 per cent. Fifty-five per cent of the total decrease occurred during the first 4 weeks (Table 3, and Figure 3). Units vitamin A ‘ Micrograms Carotene 44 22 4Q 2o 56 1e 32 I 1s 28 14 24 12 2o 1o 1e 12 8 ~ ~ “caiolian. 4 s § q 1.. _ _ _ l 4 2 0 l A g “GEES 4 8 12 l6 20 Z4 2B 32 Fig. 3. The carotene content and vitamin A potency of butter fats from cow 329, receiving daily 6 pounds of alfalfa leaf meal and yellow corn. The butter fats from cow 329 underwent a much smaller percentage loss in vitamin A potency than did those from cows 59, 61, and 196. The carotene content was approximately twice that of butter fats from cow 322, which received half as much alfalfa leaf meal. The percentage of vitamin A potency due to carotene was higher and the ratio of vitamin A to carotene lower than in the butter fats from all other cows. Six pounds of alfalfa leaf meal in addition to yellow corn did not prevent a decrease in vitamin A potency but enabled the cow to maintain her butter fat at a higher level than did the rations fed the other cows. The cow on the vitamin A deficient ration which received 6 pounds of alfalfa leaf meal in addition to the yellow corn produced butter fat F 18 BULLETIN NO. 513, TEXAS AGRICULTURAL EXPERIMENT STATION containing more carotene at the corresponding period of lactation and more carotene at the same level of vitamin A than did the cow receiving 3 pounds of alfalfa leaf meal and yellow corn. There are greater dif- ferences between the carotene contents of these butter fats than between the vitamin A potencies at similar stages of depletion. It may be questioned whether the coloring matter in the butter fat from the cow receiving the 6 pounds of alfalfa meal and yellow corn is all carotene. Influence of Pasture Grass on Carotene Content and Vitamin A Potency The cows in group 3 were depleted of vitamin A by being fed for sixty days on a ration very low in vitamin A. At the end of that time, they were put for five hours each day upon good pasturage of sudan grass. The details of this work are given in Table 4. As shown by Copeland, Fraps and Treichler (unpublished work), butter fat samples from cow 301 and cow 311, Table 4, increased in vitamin A potency from 12 units per gram of butter fat to a maximum of 40 or 50 units per gram after the cow had been on pasture for 15 hours during three days. Further samples showed a decrease in potency to 40 units per gram after the cows had been on grass pasture for a total of 70 hours covering a period of 14 days. The increase in vitamin A potency of the 14 day sample was 233 per cent of the potency of the initial sample. The carotene content showed a gradual increase from 0.53 micrograms per gram of butter fat for the first sample to 8.03 micrograms per gram for the sample taken after 70 hours on pasture (Table 4). The increase in carotene content of the last sample amounted to 1415 per cent of that of the initial sample. Approximately 15 hours on pasture during 3 days were required to restore the vitamin. A potency of the butter fat to the probable level exist- ing before the cows were fed a diet deficient in vitamin A. The vitamin A potency varied little as the hours on pasture extended beyond 15, but the carotene content increased throughout the entire 70 hours. Conse- quently the percentage of vitamin A due to carotene increased and the ratio of vitamin A to carotene decreased as the hours on pasture increased . After l0 to 15 hours on pasture the carotene content had reached approximately the same figure as had the butter fats from cows 59, 61, and 322 after 4 weeks on their experimental rations. The restora- tion by cows 301 and 311 of carotene and vitamin A potency was more rapid. than was the decrease in the case of cows 59, 61, and 322. At the stage of depletion reached by cows 301 and 311 recovery of the butter fat in vitamin A potency and carotene content seems comparatively rapid. For cows 301 and 311, with a vitamin A potency of 12 units per gram of butter fat at the beginning of the experiment, a maximum of 4O to 50 units was reached after 3 days (15 hours) of grazing. Thereafter the vitamin A potency remained relatively constant. The cause of the decrease occurring after 7 days on pasture is not known. 19 RELATION OF COLOR AND CAROTENE OF BUTTER FAT TO VITAMIN A Snwnwnmfim 33D mo no???“ écfiwnoO .0 .0 .5 wnwsBwwzw compwuflnsn we wonwiow cm wwswmcznw v.63 N28». 23 mo >252: ma» uficnooflou .32. wan; d $63 N om S... 2o 2o ooé. £2 .N .32 .52 w m o m. oo o2 N S. of. N2. 2o o3: oos $2 .2 2:2. 2E5 mm u m N3. s. S. 22.. EA SA mm 2 22 .... oom N22 w om... o... NN m3. wmA £2 2 N N22 .NN .>oz N22 ... a wm So oN o... ooo No.2 3A B. o £2 ....N .>oz N22 w w. w M moo 2 S. N3. NE. 2.6 S. N N22 oN 5oz o22 m w Woo No.2 2 3 2N of. moo .2. N £2 .NN .>oz N22 o o. H NNJN N 2 $2 2N... ¢N.o fi. 2 2x2 .NN .>oz wNSm $0 m. NNsN o 2 $1. NZ 2o o» o N22 .oN .>oz n22 mu PW ooo NN 3 3N o: 3.2 mo 3 $2 o .89 N22 2o 5 NN 2o 3.2 2.2 2. N .22 .NN .>oz N22 o. a W m. 5N oN 3 o5. Nwo 5o E. N. NN2 5N .>oz N22 m mwm . 3.2 2 on 2.... 5o 3o 3 N N22 4N .>oz 525 o o o 2 o3“ 2 oN 3N 3o 5d 2 N No.2 .NN 5oz NNSN w Wvm ooHmN o oN NE. 2o oNo fi. 2 £2 .NN .>oz $22 w o o . o... mu N. N2 N“ o 3d 5.0 S. o mmmH 6m 5oz wmflmm m u 4 W QQQQOHNU CMEQMAT» “SS5. Ahdfi hwmm mfiauwozoih HHUCQPOHNU paw nmpuin 2mm .5325 mcouomofizfimfi wionomfiflflfi Gamma dun mo .3255: Ow wig MEGHMOMUME Edhw hwQ ENFM hQQ Owfi ad Obfi #6 QOMQNAONM 02,592» wwfinchwm MO 008g hHOaN 12215 4 55w»? 9v 0550.25 4 £22.31» _ 0:30.30 >fimnwfi 352mg noawd mhdfi 33w vnonmmQ umwm. 03am o» o5. hoiowofi 232D wiwnwonomg .50 Q82 4 cmiwfi.» N$ amuflON EOMM 1G5 055mm.» FQ 95cc EQH% 3am HQHHQJ HO HGQHGOU OQQQQHGO win hQ-HQHOH 4 flmidfim> 3w Q—JN'F 20 BULLETIN NO. 513, TEXAS AGRICULTURAL EXPERIMENT STATION The carotene content of the butter fat increased during the whole feeding period of fourteen days, the carotene continuing to increase after the vitamin A potency became constant. Unless it is assumed that the continued feeding of the pasture grass resulted in a decrease in the true vitamin A at the same time that there was an increase in carotene, it is necessary to conclude that some of the coloring matter in butter fat considered carotene is not carotene and has -no vitamin A potency. A reasonable conclusion would be that the vitamin A remains constant after the third day, while increases in color is due to something which does not affect the vitamin A potency, even though it is measured as carotene. This requires further study. Samples of butter fat from cow 182, receiving sorghum silage (Table 3), varied in vitamin A potency from 5 units per gram of butter fat for the sample taken 204 weeks after the cow was placed on the experimental ration, to 7 units per gram 37 weeks later. The last sample taken after the experimental ration had been fed 256 weeks had a vitamin A potency of 8 units per gram. The carotene content varied from 0.30 micrograms per gram of butter fat for the initial sample to 1.3 micrograms per gram for a sample taken 37 weeks later. The sample taken 38 weeks after the first sample had a carotene content of 0.81 micrograms per gram. On a ration in which the sole source of vitamin A potency is sorghum silage a cow cannot produce butter fat containing a high carotene content or high vitamin A potency. Relation of Carotene Content and Vitamin A Potency To the Stage of Depletion The carotene content of the various samples of butter fats at cor- responding periods of feeding are given in Table 5. For the first three weeks of feeding, there is little difference in the carotene content of the butter fat from the cow fed white corn and from the one fed yellow corn. With the fourth week, however, a difference appears, and for the remainder of the experiment there are appreciable differences in the carotene content of the butter fat of the two cows. ' At the fourth Week there was little difference between the carotene content of the butter fat from the two cows fed yellow corn and from the one receiving 3 pounds of alfalfa leaf meal daily, but the carotene in the butter fat from the cow receiving 6 pounds of alfalfa leaf meal was twice as much as that in the butter fat from the other three cows. At the eighth week, the differences between the butter fats of the cows were greater, and these differences continued to the end of the experiment. The carotene content of the butter thus depends upon the feed, and is high in the butter fats from cows receiving large amounts of carotene. None of the rations fed contained enough carotene to maintain the carotene content of the butter fat at a constant level. The carotene con- tent decreased from 13.5 parts per million at the beginning to 3.35 parts RELATION 01F COLOR AND CAROTENE OF BUTTER FAT TO VITAMIN A 21 per million in 28 weeks in the butter fat of the cow receiving as much as 6 pounds of dehydrated alfalfa leaf meal, or an average of 116,000 units of vitamin A per day. Examination of Table 3 shows that with all the cows except those 0n pasture the vitamin A potency of the butter fat decreased during the period of feeding. That the decrease in the vitamin A potency was due to the feed and not to the stage of lactation is shown by the rapid increase ' in the potency of the butter fat when the depleted cows were placed on pasture (Tables 3 and 4). The butter fats from cows 59 and 61 increased from a vitamin A potency of 1 and 5 to one of 32 and 25 units per gram respectively, after 2 weeks on pasture. The vitamin A potency of the butter fat from cows 301 and 311 was 12 units per gram before the cows were placed on pasture, and after 3 days of 5 hours each on pasture it increased to 33 and 50 units per gram respectively. This shows that the lower potency secured from Table 5. Micrograms of carotene per gram of butter fats at the same intervals during the experimental feeding period 3 lbs. alfalfa 6 lbs. alfalfa Weeks White corn Yellow corn Yellow corn and yellow corn and yellow corn on 0.0 units 17000 units 8432 units 60189 units 116101 units feed vitamin A vitamin A vitamin A vitamin vitamin per day per day per day per day per day I 1 9.18 10.66 12.78 12 18 13.55 2 5.92 5.57 . . . _ . . _ . . __ 3 4.41 4.24 .. .... _. 4 2.11 3.22 3.35 3.64 7.95 6 0.68 2.04 .... -. _ 8 0.43 1.02 1.43 2.41 7.05 12 0.28 0.61 . . . . . . . . . _ . . . . . _ _. 16 0.19 0.33 0.97 2.38 6.04 20 0.08 0.15 1.03 2.30 5.68 24 0.04 0.24 .. . . . . . . . _ . . .. 28 0.06 0.29 1.12 3.36 I (pasture) (pasture) 2 4.44 4.53 . . . . _ . . . . . . . _ . . . . _. 32 __ __ . _ _ _ _ . . . __ 1.78 3.39 the other cows was due to their depletion of vitamin A and the insufficiency of carotene or vitamin A potency in the feed. It therefore seems correct to refer to the cows on all the rations as being on a vitamin A deficient diet, and to refer to the stage of depletion rather than to the stage of lactation. According to the work previously cited (7) the colostrum may be much higher in vitamin A potency than ordinary milk, but it appears that Jersey cows receiving sufficient sup- 22 BULLETIN NO. 513, TEXAS AGRICULTURAL EXPERIMENT STATION plies of vitamin A potency should ordinarily produce butter fat having a potency of about 30 to 50 units per gram. Percentage of Vitamin A Potency Due To Carotene For the purpose of this discussion, it is assumed with Baumann and Steenbock (2) that 0.6 parts per million of carotene is equal to one unit of vitamin A potency. The use of a different factor would not alter the comparative relations here discussed. The percentage of the vitamin A potency due to carotene based on this assumption is found to decrease from 38 per cent at the beginning of the feeding to 1 per cent near the end in the case of cow 59 (receiving white corn), and from 47 per cent at the beginning to 9 per cent at the end in the case of cow 61 (receiving yellow corn). In the case of cow 196 (receiving yellow corn), the percentage of vitamin A potency due to carotene varied from 85 to 30. In that of cow 322 (receiving three pounds of alfalfa leaf meal as well as yellow corn), the percentage of vitamin A potency due to carotene varied from 55 to 30, and in the case of cow 329 (receiving yellow corn and six pounds of alfalfa leaf meal), the percentage varied from 96 to 56. Although the quantity of carotene in the butter fat is related to the quantity fed, this variation indicates that some of the coloring matter introduced by the alfalfa leaf meal is not carotene and has little or no vitamin A potency. This opinion is con- firmed by further examination of the data. On August 12, cow 322 (receiving 3 pounds of alfalfa leaf meal), produced butter fat having a vitamin A potency of 10 units per gram, while cow 329 (receiving 6 pounds of alfalfa) produced butter fat containing 11 units. However, the carotene in the first case was 2.38 parts per million and in the second case 6.04. The difference of 3.66 parts per million of carotene was ac- companied by a difference of only 1 unit of vitamin A potency. On September 9, a difference of 3.38 parts per million of carotene was accom- panied by a difference of 3 units of vitamin A potency. On November 4, a difference of 2.24 parts per million of carotene was accompanied by no difference in vitamin A potency, the carotene content of one butter fat being three times that of the other. Relation of Carotene Content To Vitamin A Potency The variation in carotene content of butter fats having the same vitamin A potency is shown in Table 6, in which the samples of butter fat are arranged in the order of their vitamin A potency regardless of the stage of depletion. This table shows that the carotene content at the same vitamin A potency varied with the food received by the cow. Butter fat from the cow fed white corn contained less carotene than the butter fats of approximately equal potency from the cows fed yellow corn. At equal vitamin A potency, butter fats from cow 329 (fed daily 6 pounds alfalfa leaf meal together with yellow corn), contained much more caro- tene than did butter fats from the other cows. The variation in the RELATION OF COLOR AND CAROTENE OF BUTTER FAT TO VITAMIN A 23 ratio of vitamin A potency to carotene content is shown in Tables 3 and 4. For cows 59 and 61 this ratio increases with the stage of deple- tion, but after the cows were placed on pasture this ratio decreased. These results are opento at least three hypotheses in explanation. One is that carotene and vitamin A per se may have different rates of’ excre- tion into the butter depending on the food eaten and the stage of depletion of the cow, the rate of excretion of carotene being quite sensi- tive to changes in the content of the ration and the stage of depletion while the rate of excretion of vitamin A per se is less sensitive. Thus when a cow on a ration low in carotene and vitamin A potency (17,000- 18,000 units daily) was given additional amounts of carotene, the carotene content of the butter fat increased more rapidly than its vitamin A_ potency Table 6. Carotene content of butter fats arranged by vitamin A potency Units Group 1 Group 2 Group 3 Group 4 vitamin A per gram 3 lbs. 6 lbs. butter White Yellow Yellow alfalfa leaf alfalfa leaf Grass Grass Sorghum fat corn corn corn meal and meal and pasture pasture silage yellow corn yellow corn 701, _ . . _ . . . . __ _- 12.18 . _ . . . . . . . _ . . _ . . . _. 50 .- ..-_. ._ -\-~ 3.73 . _ . . . . . . _ _- 40 9.18 . . . . . . . _ . 2 . . _ _ . _ . .- 4.79 2.43 ---- a ” a . . _ . . . . _ . _ _ . _ . _ _ . . . . _ _ .. 1 ..... _. 8.03 4-43 1 m. " | .... __ . __ . . . . . _ _ . . . .. _._..-. ..__. 6.09 ____ .. " _ _ _ _ _ _ _ _ , , _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ , _ , _ _ _ _ _ , _ __ 9.94 ____ __ 38 4.14 10.66 _ . . . . . . . . . . . _ . _ _ _ . _ . _- _. .... ._ ” ______ 4,24 ______ __ ____ _ _ _ _ _ _ _ _ _ _ _ _ _ _ , _ _ _ _ . _ _ _ __ 33 . . . . . . . _ . _ . . . . . . . . _ _ . . . .. 13.55 6 13 . _ _ _ . . . . . . __ 32 4.44 . . . 1 _ . . . -. .._-. _ . _ . _ . _ . . . . . _ _ . . . _ . . . . . _. 30 .____ ____ .. _.-_ _ ______ _. 3.03 .... .. 25 5.92 4.53 12 78 . . . T . . . . . _ . . _ . . . . . . 1 7.45 N.-. 20 2.11 . . . . _ . . . . . . .. 7.95 O 78 .... __ 18 .- . . . i _ _ . _ _- . . . 1 . . _ . . . __ 0.84 .... ._ 17 .... .. 5.57 _ . . . . _ _ . _ . . . . . . _ . _ .. -_._ . . _ . . . . . _ . a 15 ____ ._ 3.22 _ . . . . . _ _ _ _ . _ . . _ . . . . . . . _ . . . . . . _ -. 14 . . . , . . . . _ . . _ . . .. 3.64 . _ _ _ _ _ _ . . . . . _ . . . . . . . . _ _ . .. ” _ _ _ _ _ . _ _ _ _ _ _ _ _ _. 2.41 ______ ._ _ . . . . _ . . . _ __ 12 .... __ 2.04 . . _ _ . . _ . _ _ ._ 7.05 0.53 0 44 .... .. 11 0.28 . . . . . . . . . _ . . . . . . . -_ 6.04 . . . . _ . . _ _ .. 10 3.00 . . . 1 . _ . _ _. 2.38 5.68 .... ._ 1 12 .... .. " _ . _ . . _ . . . . 4~~ 1.12 3.36 . _ _ _ _ _ . . . . . . . _ . _ .. ” ____ ,_ _ _ _ __ _ ______ __ 3.39 ____ __ __ ____ -_ 8 0 68 .... __ 0.35 1 78 . . . _ . . _ . . _ _ . _ . . . _ . _ _ . . . . .. ” 0 43 ".1. 1.43 _ . _ _ . . . . _ . . . . . . . . . . . . . . . . .. 7 0 19 1.02 .... ._ 2.30 . . . _ . . . . . . . . . . . . . . .. 0 97 6 0.61 . . _ _ _ . _ . _ . _ . . . . . . . . . . . . . . _ _ . _ _ . . . . . . _ . 5 .... _. 0.33 __-__ _ _ _ _ _ _ _ _ _ _ . . _ ._ _. .... ._ 0 30 " 0.29 _ _ _ _ _ _ _ _ _ _ _ _ . _ _ _ _ _ _ _ _ _ , _ _ _ _ , _ _ _ _ _ _ . . _ __ 4 .... 7' 0.24 0.97 . . . . . _ . _ . . . . _ _ _ . . . _ _ . . _ . . . . . _ _ . .. 3 0.08 -. 1.03 . . _ . . _ . . . . . _ . . . . . . . _ _ _ . . _ _ _ . . . . . .. 2 0.04 0 15 _ . . . . . _ _ _ . _ _ . , _ _ _ _ . . . _ . _ _ . . _ . _ _ 1 . . . . . ._ 1 0.06 . _ _ . . _ . . . . . _ . . _ . . . . . _ . . . . _ . . . . . . . . . . . . . . . . . . .. ‘Colostrum and this increase was accompanied by a less rapid increase in vitamin A per se. Also when a cow with a good reserve of vitamin A potency was placed on a ration of low vitamin A potency, there was a more rapid decrease in carotene than in vitamin A potency of the butter fat, with a less rapid decrease in vitamin A per se. At a higher level of vitamin A 24 BULLETIN NO. 513, TEXAS AGRICULTURAL EXPERIMENT STATION potency in the feed (60,000 units daily), the difference in rates of excretionwas negligible. This. occurred with the cow receiving 3 pounds of alfalfa leaf meal and yellow corn. At a still higher level 0f vitamin A potency in the feed (6 pounds of alfalfa leaf meal and yellow corn, 116,000 units daily), the vitamin A potency decreased more rapidly with the stage of depletion than did the carotene, so that most of the vitamin A potency of the butter fat was due to carotene. A second possible explanation of the results is that the variations in carotene content of the butter fats from cows receiving different amounts of vitamin A potency may be accounted for by the ‘capacity of the cow to transform carotene into vitamin A. The cow may transform small amounts of carotene into vitamin A more completely than larger amounts. When the quantity of carotene fed exceeded the capacity of the cow to trans- form it into vitamin A, more of it went directly into the butter fat. A third hypothesis in explanation of the results is that in highly colored butters some of the coloring matter which has been considered carotene is not carotene and has no vitamin A potency. Relation of Color of Butter Fat To Carotene Content and Vitamin A Potency The color of melted butter fat is yellower than that of the unmelted fat, because of the difference in physical character. The butter itself has a lighter color than the butter fat, because of the presence of the curd and salt, and because of the physical structure of the butter. Since the carotene is the chief coloring matter in the butter fat, the quantity of carotene should be related to the color of the butter fat. . Butter fat samples from cows 59 and 61 were analyzed for color. The results expressed as percentage transmission of radiant energy at various wave lengths in the visible portion of the spectrum, are given in Table 7. In all‘ of the samples, maximum transmission occurs at approximately 540 millimicrons and remains near this level through 700 millimicrons. The greatest difference in transmission occurred between 460 and 480 millimicrons, with a rapidly rising percentage transmission as 540 was approached. The color differences in the various butter fat samples are most noticeable between 440 and 540 millimicrons, between which points the transmission becomes greater as the butters become less yellow and approach white. a It was thought advisable to convert the color data from these butter fats into terms by which the perceptible difference in color could be expressed. This conversion makes possible the reproduction of the color. The data from the complete analyses of the butter fats from cows 59 and 61 were therefore converted into elementary color excitations by the use of standard illuminate B, and the monochromatic equivalent was determined. The terms are defined in the Colorimetry Committee Re- port of the Optical Society of America (30). The method employed RELATION OF COLOR AND CAROTENE OF BUTTER FAT TO VITAMIN A "Table 7. Spectrophotometric analysis of butter fats from cows receiving white and yellow corn Q WWWqQ1@@Qq?Q .@fiwqH°*°°°w $ wwmwmmmmoooo mmmmmmdmoddd mmmmmmmmoooo mommmmmmooom P4r-lv-lv—l pap-ha Q Q@§WfiTWQQQqQ w9Qmw°P°°°°° vrmwmwmmcmoo wmmmmmddd"' m awmmwmmaomoo m m ooggg v-i I-lv-l I-(r-1v-4P4 ° ”Q”§Q@Q#QwQY @WQ@QW@QQ?Q@ $ rvmvmwmmowom wmwmmmwoooom mmmammmmomom mmmmmm oooom I-l I-l I—iI"|P-1I-1 6 H°fi€9@?@Q°9? w@w@@@@@@@Qw Q vwmwmwmmcoom $NNH©mH®@®OM wwmmmmmmooom wmmmmmmmamom HHM H B ° §QY@Q@QQ§Q?§ W1?Q@Q@QWQQq d Q wmumwmvvmoow fiwmwmmmwmwoa > @b®®®®@®®QO® mvwwmmwmmmow H r-4r-l I-l O 4-7 E ~ ° QQQ9YqQW§?Q§ §W@Y§§QW?T9@ 3 E mfivvmflmwmoar vmocmmmmmwww M mvmvwmmmmamm mmwvvwwmmmmm ‘g cp-q I E ° 9Q@§§?Q9QQfi9 wqwwwwwwwwww E 3 mowwmwommovm mm~w>>vvwwNw mmmbwmmmzmm mmwmvvwmmmw o a a “ ° @TfiQW§99?Qfi9 @WWW@§9?@Y9@ Q Q wwwvwmvmwoww HmHov>wwmmmv G Hmmwwmmm2m~ fimmwwwwmwmfl Q 0 $ Q ° WYWQQQQWWQTW TWQ@Qq§WWQQ9 $ moomwwmwmwmw aamwvawocmfiw E Hmmwhwwmmmfi MHNmmwwmwmH 5 __ __ _ 2 ~ g ° Q§W@QYQQ§q@q @9§?W@9Q@§Q° M Q HP@@P®@@N@m@ cmwmmmmwwmom 5 Hmmwvwmmmfl ~mmmmvwww~ U] Q ____ _ 2 H ° @@§YYW§Y?fiWW ‘§§QW§@@§YWm 3 Hrmvwvmmflmwm zrmmmvvwmmfim ~mmwvwmmma 1 ~NmwmvwwwH Q ‘fi?QWQq?W§Q? '9§“wqW@Q§@” ¢ irmflmwbfimmwfl :mmmoNmow®Hm z ~wmwwwwmmH : ammwmvwvwfl 3 ‘YQQQQQWQQYQ ‘@w°°@§Q°W@W w :v~¢wmo¢wmwm :wmmmvm>mmHw 2 Hmmwwwwmmfi : mmwwvwfi F! 3 ‘QWQQQWQQTWQ ‘QW?WW@mW§w* w zmcfirflmwwmbm twmwbwwmwmmm : Hwmwvwwmmfl : ~Nmmmwwvw~ H . Q ‘W@@@qQ9°WWW ‘W9m@NQW@Q@” Q zwomwmvwommo :wmmvmwmwm¢w : fiMm$b®@®®N I Hmwmmwwbwfl H __ _ L>w Nmwcmvmwmmom mflmmmmwmmwflw ohfi mcvmwmvowvmw mowwwmvowrmw JOE mmmmmwvmflmmm Nmmmmwhmfimmw “Q wwwmwwwwvvrv wwwwwwwwbvvb Qwg mmmmmmmmmmmm mmmmmmmwmmmm _ (H100 812w) 6s M00 (uxoa monas) [9 MOQ lThe values for 420 and 430 millimicrons were ilerived by extrapolation. % BULLETIN VNO. 513, TEXAS AGRICULTURAL EXPERIMENT STATION Q YQWWWQQQQQQQ 9W?W9Qfi9ww”m ¢ wwwwwmwwmmow wwwwawwwmmmw P mmmmmmmmmmam mmmmmmmmmmmm ° 99§fi@9fiQ§§?W q?fi9fi@§q°w“m $ wwwwrmmwwmow wwwwmmmmddéd mmmmmmmmmmam mmmmmmmmommm I-l ‘U 0 5 E O €QR@?@Q?QQ?@ QQQ@Nw€RQ@@~ # Q wwwwvwwmmoow mvwwmmwwmddd 5 wwmmmmmmmaam mmmmmmmmmm C563 I we 5 3 3 @@§@W€§Q9@Q? wqwwwqqqqwqq Q G {p %%GJQD§@%C3@@@D' ¢5%UJ@C5U3QQ@O5C§$@ u 3 mwmmmmmmamsm mmmmmmmmmmcm c I-l E s _ E 1 3 §@§@WQQ9?§?Q @€@?NN§°°W°° w wwwwrwwmomov mwwmdomdd$dd 1 E w ammmmmmmamam mmmmmmmmsmao a: > "‘ w $ ~63 § E 3 wQ@Qw@@QQQQ@ #w@@w@@@Q=Q¢ ; w wmwwwwmmoocm mwwwmmmmodod I-iv-(Iiv-l H 2* E E 3 ww@@w@w@qQQQ @w@@@w@wQ=¢¢ Q ~ wmmmwwmmoooo mmwwmmwmod6' 8 E Q mmmmmmmmaiaz mam mmamoocg I-(I-(Firi u M 3 E u g QWWQQQQWQQQQ @NQQ@NWW°°°° Q wmmmmwmmooco mmmwmmwmd'd' ° ° mmmmmm coco mmmmmmmmogog E g V"1I—‘Hf-4 I-iv-ir-iv-i E 3 I 3 3 Qwdwqwwwqqqq @@ww~Q§wQQ°Q mmmmmvmmmcao mmmwmmwm o Q Q Q mmmmmmmmmooc mama m gogg QII P4I-4F4I-I G1 3 M § § 3 QNQ”"Q@§9QQ? ww@~wwQwqQ¢= ‘ ammmawmacooo mmmmmmwmoo" é Q w mmmmmm magic mmmmmmm oogg ' H HHHH ‘h! = 5 m F: E E 8 QfiQ@W§Y“°°?? YQ?Y@W9°°°°w mmwmowmmo ""' % H Q mmmmmmm zggg $°@°$°“8§§8$ Y-l V-l i H § g WWQNYWQQQQQQ YW@@§“N°“°°@ mwwmmwmoocoo am mm’ "d" K W mmmmmmaaazaz mm@m $°8$¢€$ E Pl f-iv-i a '8 c rvmflmfloooooo Q i 3 @@@¢¢§¢a¢¢¢¢ §§&§§§E§§§§§ - 3 mmmmm giozo mmmmmm o coo ‘a F‘ F'| I-l I-iv-lr-i 3 w o wmro¢oNwQo¢o mwwfiwomomcoo p --------~-~~ .--.~.....-. wwmmmwmmoooo ®®®®GmbOQOO® g m mmmmmmmmszzz mmmmmm c 20¢ I-i v-i i a H flgg mmmomfimwmmom mflmmumwamwflv Oafi mcwmwmvowvmw mowwwmvovrmw Q05 Nmmmgvwm~Nmm,mmmmmwraflmmm “#5 wwwwmwwwvvvv wwwwwwwwvvvv qd: mmmm mmwmmmm mmmmmmmmmmmm \ (11100 aqyqm) 6g MOQ {tuoo mouazi) 19 MOQ RELATION OF COLOR AND CAROTENE OF BUTTER FAT TO VITAMIN A 2'7 was that reported by Judd (21). The color is expressed as dominant wave length, colorimetric purity, and luminosity (Table 8). The colorimetric purity is the ratio between the intensity of the dominant Wave length and the intensity of the sample. The intensity of the dominant wave length is adjusted in such proportions that when mixed with white light it will yield a color match with that of the butter fat sample. The decrease in carotene content of butter fats from cows 59 and 61, given in Table 8, is accompanied by a shifting of the dominant wave length from 570 millimicrons toward the more greenish yellow portion of the- spectrum. A decrease in colorimetric purity and an increase in luminosity occurs, indicating a decrease in yellow color. The maximum loss in color of butter fats from cow 59 occurred at the 28th week and for butter fats from cow 61 at the 20th week, at which periods the carotene content was at a minimum for cow 61 and nearly so for cow 59. It was at these same periods that the vitamin A potency reached the lowest levels. After the cows were placed on grass pasture the butter fats became yellower, with a decrease in luminosity and an increase in purity and carotene con- tent to approximately the same values as for those samples taken during the third week. In this investigation it was assumed, in accordance with other investiga- tions, that the color absorbed at 470 and 480 millimicrons was practically all due to carotene. The work here presented raises the question whether - this is true. After the depleted cows, 301 and 311, were placed on pasture, the vitamin A potency reached its maximum in 3 days of feeding, but the carotene continued to increase from 3.73 and 4.43 parts per million to 8.03 and 9.94 respectively in 11 days, and had probably not reached its maxi- mum at that time, since some butter fats received in this investigation contained as much as 13.55 parts per million of carotene and since other investigators have found as much. Apparently, the carotene content of the butter fat increased and the vitamin A decreased after the third i day on pasture, and the vitamin A continued to decrease while the carotene continued to increase. Such an occurrence would suggest the theory of a more or less regular decrease in the power of the cow to convert carotene into vitamin A, which does not seem plausible. The alternative theory is that the increase in color is not due to carotene, _ but due to some other coloring matter. Both theories require study. Table 6 shows that with each experimental animal the decrease in vitamin A potency was accompanied by a reduction in carotene content. With these Jersey cows, all butter fats containing 25 or more units of , vitamin A potency contained more than 4 parts per million of carotene. However, the cow on yellow corn and 6 pounds of alfalfa leaf meal pro- duced butter fat which had from 1O to 20 units of vitamin A potency and from 5.68 to 7.95 micrograms of carotene per gram—-slightly more carotene than the cow on white corn put into butter fat which contained from 25 to 38 units of vitamin A potency. Other things being equal, a naturally yellow butter fat probably will be higher in vitamin A potency than one less b 28 BULLETIN NO. 513, TEXAS AGRICULTURAL EXPERIMENT STATION 65550.05 mmfiHM HO 0x003 03H 500.2." mN $4. $0 mm 0E w... 0.5. 0...... H?! NmmH .N .H .52 0.35 M0 m NNd Wm» om 5m NdN Em 1a wN NmmH .2 .~.H .000 135 M H. vNd pa» Hm 5m wdN w...» 0.0m wN NmmH 6N 5H 0.6m 3N5 9 N 5.0 m? wN £5 wfiN 0.3 N6..." oN NmmH 5N .NN .M54 ~35 I m $5 $3 S S» NJN 0.0m 0.5 2 NmmH 6N 6N 35H. 00$» ) w 3.0 0.2“ S. 5m NAN 3a ma“ NH NMNH .wN §N 25H. 05.0w M H. No.H 0.? S Sm 0.5 3w 0.3 w NmmH .~.H 6H mug £10m m NH 3.“ 0.2 2. 03 0.2 HHH. QNH. w ~25 .m .N hwSH ~23 M mH NN.N 0.8 w» 2a mzHH Y? “.2. H. $2 6N 6N H354 .00.?” o mm Fwd Q2. mm 0S m... NEH. 0.3. m NmmH dH .2 $.34 33m o NH “b6 mam Hm 0S HiH. w? w? N NmmH .NH .HH $.54 82m M mm $0.2 3a mm HE H.H Zn. w? H NmmH 6 $.54 “$2” ( Nw $4. n50 mm 2E 0.... Név Q3. 5..-. NMEH .N .H $02 N35 w“ H mod 0.20 oN .23 .03 mg NAM wN NmmH .2 §H .000 33m m N 2.... Ea HN 2a 0.0m 0.0m mam vN NmmH 6N 6H fiwm $.05 m mod 0.2 mN 25 0.0m 15 0.5 oN N2: 5N .NN $54 2:5 M. H. 3.0 5a wN S... oSN 0.3 Hdm wH $2 6N 6N 35H. 20.0w HH wNd v.5 mm 2E 3N Nd» i.» NH $2 dN éN 2.3. 5.5m M w .23 w...» 3 m8 wdN 0....» Hi5 w NmmH .~.H 6H >02 H23 m. w ma... Em 2. 5m c.HN m? .8» w NmmH 6 .N >02 mwfim m... 0N HH.N £0 5 2B w.H.H 05H. 0.5. H. NmNH 6N 6N 2.54 33m w» vHJ. $0 2w Sb m.» .15. Q3. m $2 dH 6H 5.54 Néww m nN Nmd Q3 Hm H5 3. fix. o.~.H~ N N2: NH .HH H354 No.3» m S. wHd 3w 8 HE ma v.3. w? H $2 6 H254 83m ( 00w 5035a 0am 50.35: 0500 .80 m5050HE uoHofiw 50050 HEM 503.05 Edam n09 5.55M .55 @500 .55 .3359 122E . E0505 #5555 .8580.“ 0530500 hfimo 3550i _ fiwcfi A5055» mmHaaww v0 000G 253w 4 555$?» mfiwnmoaogn |5HE5A $5200 0.503 0500 .505 E 0030.503 50 wxwwg Lonwq @355 55055500“ @051? nomudqmoxfl afimoimiq-H H050 .5355 00.52.5003» fivu-SH 030B 5:05.501 m0 E553 E wvumounxo Bum .335: N0 5300 .w 030a. Q RELATION OF COLOR AND CAROTENE OF BUTTER FAT TO VITAMIN A 29 yellow, but color alone is not a dependable criterion of vitamin A potency of butter fats. Table 5 shows that Jersey butter fat containing over 25 units of vitamin A also contains more than 4 parts per million of carotene. Butter fat containing 10 to 20 units_ of vitamin A may contain 0.28 to 7.95 parts per million of carotene. Butter that is naturally yellow will probably con- tain more vitamin A potency than poorly colored butter, and poorly colored butter is probaly low in vitamin A potency. Goat Butter Fat Since it is known that the carotene content of butter fat from cows indicates to some extent its vitamin A potency, it was decided to examine samples of goat butter fat which are practically devoid of yellow color. The goat butter fat examined (Table 4) contained approximately 0.4 micrograms of carotene per gram and had a vitamin A potency of 40 units per gram 0f butter fat in one sample and 50 units per gram in another sample. When these are compared with samples of cow butter fat of the same vitamin A potency, there is a striking difference in carotene content. The difference is emphasized when a comparison is made of the ratio of units of vitamin A to micrograms of carotene in the case of 11 samples of cow butter fat, having approximately 38 to 50 units of vitamin A potency per gram of butter fat, with the ratio of vitamin A units to micrograms of carotene in the 2 samples of goat butter fat. These 11 samples of cow butter fat had an average ratio of approxi- mately 8 as compared with a ratio of approximately 103 for the goat butter fat. The vitamin A potency of goat butter fat was approximately the same as that from cows on similar rations. However, there is a marked dif- ference in the carotene content of the butter fats. With equal vitamin A potency the carotene content of the butter fats from the cows was ap- proximately 14 times as much as was that of the butter fats from the goats. The ratio of vitamin A potency to carotene at the same level is from approximately 3.6 to 16.5 for the cows in contrast to 103 for the goats. The goat is evidently able to transform a larger proportion of the carotene in the feed into vitamin A than is the cow. The goat is apparently able to secrete milk which contains vitamin A per se with but negligible quantities of carotene, whereas the cow secretes milk containing both carotene and vitamin A. The amount of the latter is governed by the amount of vitamin A potency in the ration above that used for maintenance, and probably by the physiological factors in the cow that influence the formation and excretion of vitamin A. The amount of carotene in cow butter fat may be governed by physiological factors influencing the maximum level at which carotene when fed in excess is excreted in butter fat. Below the level set by these physiological factors, the amount excreted in the butter fat is governed by the amount ingested in the ration. v 30 BULLETIN NO. 513, TEXAS AGRICULTURAL EXPERIMENT STATION ACKNOWLEDGMENTS The authors are indebted to J. F. Fudge of the Division of Chemistry for preparation of the carotene in butter fat solutions; to E. E. Vezey, associate professor of Physics, for suggestions concerning the color data; and to O. C. Copeland for use of samples of butter and the history of animals, in advance of his own publication. SUMMARY The carotene content of butter fat was estimated in several series of samples secured from cows receiving definite amounts of vitamin A potency. The vitamin A potency of the butter had been determined by biological tests with rats. The carotene content was estimated from the absorption of light by the melted butter fat at 470 and 480 millimicrons as measured with a spectrophotometer. 'The absorption was calculated to carotene by means of factors secured from measurements of the absorption of light by known amounts of purified carotene dissolved in butter fat. Both the carotene and the vitamin A decreased during the period of feeding of the cows, which had been on pasture before the period of lactation began. The carotene of butter fat from a cow fed on white corn decreased from 9.18 parts per million at the beginning to 0.06 parts per million in 28 Weeks. Most of the decrease occurred during the first 6 weeks. The carotene content of butter fat of cows on yellow corn decreased from 10.66 at the beginning to 0.29 parts per million in 28 weeks. The cows on yellow corn produced butter fat containing more carotene and vitamin A than the cows on white corn. The cow on a daily ration of 3 pounds of alfalfa leaf meal in addition to yellow corn produced butter fat which decreased from 12.1 parts per million to 1.78 parts per million of carotene in 32 weeks. The cow on 6 pounds of alfalfa leaf meal daily produced butter fat which decreased from 13.5 to 3.3 parts per million of carotene in 32 weeks. The decrease in the carotene and vitamin A potency of the butter was less with the cow receiving the higher amounts of carotene, but was still appreciable. Cows depleted of vitamin A and placed upon sudan grass pasture pro- duced butter fat in which the carotene content increased during the entire period of the experiment from 0.53 to 8.03 parts per million of carotene, but did not reach the quantity of 12 parts per million found in the butter fat at the beginning of lactation of the cows used in the previous ex- periment. The carotene content of the butter fat of the cows on pasture continued to increase after the vitamin A potency had reached a maximum of 50 units per gram. This would indicate that the ability of the cows to convert carotene into vitamin A decreased regularly, so that a decrease in vitamin A potency accompanied the increased carotene; or that some of the coloring matter supposed to be carotene is not carotene and has no vitamin RELATION OF COLOR AND CAROTENE OF BUTTER FAT TO VITAMIN A 31 A potency; or that the cows varied in their capacity to convert carotene into vitamin A. _ Sorghum silage fed in liberal amounts did not produce butter fat high in vitamin A or in carotene. The carotene content of butter fat depends upon the feed and was high in the butter fats of the cow receiving a high amount of carotene. If it is assumed that 0.6 parts per million of carotene is equal to one unit of vitamin A potency, in the case of the cow fed on white corn the percentage of vitamin A potency due to carotene decreased from 38% at the beginning of the experiment to 1% near the end. In that of the cow receiving yellow corn, it decreased in one case from 47% at the beginning to 9% at the end, and in the other case from 85 to 30. In that of the cow receiving daily 3 pounds of alfalfa leaf meal in addition to yellow corn, the percentage of vitamin A potency due to carotene varied from 30 to 55, and in that of the cow receiving 6 pounds of alfalfa leaf meal in addition to yellow corn daily, it varied from 56 to 90. The high vitamin A potency ascribed to carotene in the butter fat of the cow receiving daily 6 pounds of alfalfa leaf meal again raised the question as to whether all of the coloring matter is really carotene and has vitamin A potency. The decrease in the carotene and vitamin A content of butter fat during the lactation period of the cow is due to deficiency of vitamin A potency in the feed and not to the stage of lactation, since the vitamin A potency was restored when the cows were put on pasture and secured an abundant supply of vitamin A potency. The relation between the carotene content and the vitamin A potency of butter fat depends upon the quantities of vitamin A and carotene stored by the cow at the beginning of the lactation period, upon the length of time which the feed deficient in vitamin A and carotene has been fed, and upon the extent of such deficiency. The data resulting from the spectrophotometric analysis of the butter fats from two cows were converted into monochromatic equivalents and the color was expressed as dominant wave length, colorimetric purity, and luminosity. A decrease in carotene content was accompanied by a decrease in purity and an increase in luminosity, reflecting the loss of yellow color. The butter fat of the goat on green pasture is low in carotene but high in vitamin A potency. Goats seem to have a greater power to transform carotene into vitamin A than have cows. Butter high in vitamin A is likely to be highly colored, but all colored butter is not high in vitamin A. Light colored butter is likely to be low in vitamin A. The chances are high that butter that is naturally very yellow will contain more vitamin A potency than light colored butter. LITERATURE CITED 1. Barnett, H. M. 1934. The determination of carotene in butter fat. Jour. Biol, Chem. 105:259. 10. 11. 12. 13. 14. 15. 16. 17. 18. BULLETIN NO. 513, TEXAS AGRICULTURAL EXPERIMENT STATION Baumann, C. A., and Steenbock, H. 1933. Fat soluble vitamin A XXXVI. The carotene and vitamin A content of butter. Jour. Biol. Chem. 101:547. Baumann, C. A., Steenbock, H., Beeson, W. M., and Rupel, I. W. 1934. Fat soluble vitamins XXXIX. The influence of breed and diet of cows on the carotene and vitamin A content of butter. Jour. Biol. Chem. 1052167. ' Booth, R. H., Kon, S. K., Dann, W. G., and Moore, T. 1934. CLX. A study of seasonal variation in butter fat. I. Seasonal variations in carotene, vitamin A and the antimony trichloride reaction. Biochem. Jour. 27:1189. Carr, F. H., and Jewell, W. 1933. Characteristics of highly active vitamin A. Nature 131:92. Chem. Abs. 27:2185. Cook, E. F. 1933. Recent developments in pharmacopoeial vitamin standardization. Amer. Jour. Pharm. 105:583. Dann, W. G. 1933. CCLXXI. The transmission of vitamin A from parents to young in animals. II. The carotene and vitamin A content of cows’ colostrum. Biochem. Jour. 27 :1998. Dutcher, R. A. 1933. Penn. State College. private communication. Fasold, H., and Heideman, E. R. 1933. The yellow color of the milk of goats deprived of thyroid. Z. ges. Exptl. Med. 92:53. Chem. Abs. 1934. 28:3109. Fraps, G. S., and Treichler, R. 1932. Quantitative variations in vitamin A content of butter fat. Jour. Ind. and Eng. Chem. 24:1079. Fraps, G. S., and Treichler, R. 1933. Vitamin A content of foods and feeds. Tex. Agr. Exp. Sta. Bul. 477. Fraps, G. S., Copeland, O. C., and Treichler, R. 1934. The vitamin A requirements of dairy cows. Tex. Agr. Exp. Sta. Bul. 495. Gillam, A. E., Heilbron, I. M., Morton, R. A., Bishop, G., and Drum- mond, J. C. 1933. CVLV. Variations in the quality of butter, par- ticularly in relation to the vitamin A, carotene and xanthophyll content as influenced by feeding artificially dried grass to stall fed cattle. Biochem. Jour. 27: 878. Gillam, A. E. 1934. for the assay of carotene and vitamin A in butter. 28:79. Gillam, A. E., Heilbron, I. M., and Watson, S. J. 1934. CXLVII. The re- lation of color and vitamin A content of butter to the nature of-the ration fed. (II. The carotinoid and vitamin contents of the butter. Biochem. Jour. 28:1076. Goldblatt, H., and Barnett, H. M. 1932. Carotene, and vitamin A. Proceedings of the Society for Experimental Biology and Medicine, 30:201. Green, H. N., and Mellanby, E. 1930. Carotene and vitamin A. Brit. Jour. Exptl. Path. 11=s1. \ Hilton, J. H., Hauge, S. M., and Wilbur, J. W. 1933. Maintaining the Station Bulletin 477 and XIV. A modified spectrophotometric method Biochem. Jour. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. . 36. 1..FJ§RARY\ ‘L-llllfiglllrlfiillllllr l k‘ ‘ h .71 VHAYQ I RELATION OF COLOR AND CAROTENE OF BUTTER FAT TO VITAMIN A 33 vitamin A value of butter through winter feeding conditions. Jour. Dairy Sci. 16:355. Experiment Sta. Record, 71:378. Hoagland, R., and Snider, G. G. 1926. Vitamin A in oleo-oil and also stearin. Jour. Agr. Research. 32:397. International conference on vitamin standardization. 1934. Amer. Med. Assoc. 103:353. ' Judd, D. B. 1933. The 1931 I. C. I. standard observer and coordinate system for colorimetry. Jour. Optical Soc. 23:359. Moore, T. 1930. Vitamin A and carotene. Biochem. Jour. 24:585. Moore, T. 1932. I. Vitamin A and carotene. IX. Notes on the conver- sion of carotene to vitamin A in the cow. Biochem. Jour. 26:1. Moore, T. ‘1933. CXVI. Vitamin A and carotene. X. The relative minimum doses of vitamin A and carotene. Biochem. Jour. 27:898. Osborne, T. B., and Mendel, L. B. 1913-14. The influence of butter fat on growth. Jour. Biol. Chem. 15:423. Palmer, L. S. 1912. Carotinoids and related pigments. Society Monograph. Page 190. Palmer, L. S., and Eckles, C. H. 1914. Carotene. The principal natural yellow pigment of milk fat. Its relations to plant carotene and the carotene of the body fat, corpus luteum and blood serum. I. The chemical and physiological relation of the pigments of milk fat to the carotene and xanthophylls of green plants. Jour. Biol.‘ Chem. 17:191. Palmer, L. S., and Eckles, E. H. 1914. Carotene. The principal natural yellow pigment of milk fat. Its relations to plant carotene and the carotene of the body fat, corpus luteum and blood serum. V. The pigments of human milk fat. Jour. Biol. Chem. 17:245. Palmer, L. S., and Kempster, H. L. 1919. Relation of plant carotinoids to growth, fecundity, and reproduction of fowls. Jour. Biol. Chem. 39:299. Report of the Colorimetry Committee of the Optical Society of America, 1922. Jour. Optical Soc. of Amer. and Rev. of Sci. Inst. VI: No. 6. Russell, W. C. 1933. Vitamin A in milk. N. J. Agr., 15; 4:23. Schertz, F. M. 1923. Jour. Agr. Research, 26:383. Semb, J ., Bauman, C. A., and Steenbock, H. 1934. Fat soluble vitamins. XLI. The carotene and vitamin A content of colostrum. Jour. Biol. Chem. 107 :697. Shrewsbury, C. L., and Kraybill, H. R. 1933. vitamin A potency and antioxidants of butter fat. 101:701. Smith, J. H. C., and Milner, H. W. 1934. properties of carotenes from different plant sources. Chem. 104 :437. Steenbock, H., and Boutwell, P. W. 1920. The comparative nutritive value of white and yellow maizes. Jour. Biol. Chem. 41:81. Stephenson, ‘M. 1920. A note on the differentiation of the yellow plant pigments from the fat soluble vitamin. Biochem. Jour. 14:715. J our. Amer. Chem. The carotene content, Jour. Biol. Chem. Carotene. VII. Physical Jour. Biol. 34 38. 39. 40. 41. BULLETIN NO. 513, TEXAS AGRICULTURAL EXPERIMENT STATION Troland, L. T., and committee. 1922. Report of the colorimetry com- mittee of the optical society of America,\1920-21. Jour. of the Optical Society of Amer. and Review of Scientific Instruments. 6:527. Watson, S. G., Bishop, G., Drummond, J. G., Gillam, D. E., and Heil- bron, I. M. 1934. CXLVII. The relation of the color and vitamin A content of butter to the nature of the ration fed. I. Influence of ration on yellow color of butter. II. The carotinoid and vitamin A con- tents of the butter. Biochem. Jour. 2821076. Wilbur, J. W., Hilton, J. H., and Hauge, S. M. 1933. The vitamin A activity of butter produced by Guernsey and Ayrshire cows. Jour. Dairy Science. 16:153. Willimott, S. G., and Wokes, F. 1927. Their application to naturally occurring products. Color tests for vitamin A. Lancet. 2:8.