A Study of the Vitamin a Content of Colostrum

A STUDY OF THE VITAMIN A CONTENT OF COLOSTRUM

AND MILK OF DAIRY COWS

MARGARET DILLON BAIR

B. A., Friends University, 1926

A THESIS

submitted in partial fulfillment of the

requirements for the degree of

MASTER OF SCIENCE

KANSAS STATE COLLEGE

OF AGRICULTURE AND APPLIED SCIENCE

1936 ii

TABLE OF CONTENTS

Introduction 1

Review of Literature 1

Procedure 5

Discussion 18

Summary and Conclusions 24

Acknowledgment 27

Literature Cited 28

119134 INTRODUCTION

Milk is now recognized as an important source of vi-

tamin A. Colostrum, the first milk secreted by a mammal after parturition, has long been considered important for

the young of the species, but its value as a source of vitamin A, more potent than normal milk, has only recently commanded attention. It seemed of interest, therefore, to compare the vitamin A content of colostrum and of normal milk produced by individual cows with the total vitamin A yield per cow per day at different stages of lactation.

REVIEW OF LITERATURE

In 1921 Drummond and co-workers (5) found that rats fed colostrum in addition to a vitamin A -free diet made more rapid growth than those fed normal milk in the same amounts. They reported that colostrum was rich in vitamin

A, and that there was a gradual fall in the amount of vitamin A in the secretion as the colostrum was replaced by the normal milk. These preliminary experiments also indicated that milk obtained from Jersey cows and closely 2

related breeds tended to be richer in vitamin A than that of Shorthorn and Black Angus cows.

Later, in 1931, Davis and Hathaway (4) used the

Sherman and Munsell rat growth method to determine the vitamin A content of pooled samples of milk from Holstein,

Ayrshire, Jersey and Guernsey cows. No significant differences were found between the vitamin A content of the milks of these breeds. In 1933 Wilbur, Hilton and Hauge

(16) tested biologically the vitamin A activity of samples of butter made from the milk of 4 Guernsey and 2 Ayrshire cows. These authors reported that the vitamin A poten- cies of these samples of butter were similar without regard to the breed of cow or the color of the butter. In the same year Hathaway and Davis (6) again studied the vitamin A content of milk from different breeds of cows and found that the vitamin A content is largely associated with the butterfat, that the standardization of the butterfat content of the milk by addition of skim milk reduces the vitamin A potency, and, that Holstein cream contains more vitamin A than Jersey cream.

In 1933 Baumann and Steenbock (1) reported on the carotene and vitamin A content of butter, using spectro- graphic methods of analysis. Carotene accounted for only 3

15 per cent of the total biological activity of the butter.

Variations in carotene and vitamin A contents were seasonal

and regular, the largest amounts appearing in the summer

butter. Later, Baumann, Steenbock, Beeson, and Rupel (2) used much the same methods to study the carotene and vi-

tamin A contents of butter made from pooled milk produced by Holstein, Ayrshire, Jersey, Guernsey and Brown Swiss

cows, all on the same ration. They found that Jersey and

Guernsey butters contained larger proportions of carotene

and smaller amounts of vitamin A while the Holstein and

Ayrshire butters contained little carotene and more vitamin A than did the other samples. There was little difference in the total activity of the butter produced from the milks of the different breeds. Semb, Baumann and

Steenbock (10) used spectroscopic methods to study butterfat prepared 'from colostrum produced by cows on a uniform ration. The values in carotene and vitamin A fell rapidly during the first week of lactation, after which the de- creases were slow. Breed tendencies were not definite, although the carotene content of colostrum from Guernseys was markedly higher than that from the other breeds. Car- otene in the blood plasma and vitamin A storage were considered.. It is suggested that the high concentration of 4

carotene and vitamin A in the butterfat of colostrum might

be attributed to an accumulation of these factors in the

tissues during the later phases of pregnancy when the

milk flow has ceased. In 1934 Dann (3) studied samples of

colostrum of 14 individual dairy Shorthorns receiving the

same ration, which contained a good supply of carotene.

Carotene and vitamin A were determined with a Lovibond

tintometer and the richest samples were also assayed bio-

logically. For every animal but one the colostrum had a maximum vitamin A content immediately after birth of the

calf or in the next 12 hours. Experiments while the cows were on winter feed indicated that the colostrum is en- riched with vitamin A at the expense of the large store in the body. Heifers generally produced richer colostrum than cows, probably because the body reserves have not been depleted by previous lactation periods. Dann concluded that the vitamin A content of colostrum may be 10 to 100 times that of the later milk of the same cow, independent of season. He said that on the first day of life the calf receives supplies of vitamin A greater than the later milk could give in 20 to 50 days.

In 1932 Moore (7) concluded that carotene must be changed into vitamin A in the body of the cow, an herbiv- orous animal, because he found large amounts of vitamin A 5

in the liver and in the milk fat, whereas he found only

small amounts of carotene in the body fat. This is con-

sistent with the finding that feeding large amounts of

carrots to the cow results in an increase in the carotene

and also in the vitamin A content of the milk fat.

PROCEDURE

Colostrum and milk for these experiments were obtain-

ed from cows of the dairy herd, Department of Dairy Hus- bandry, Kansas State College of Agriculture and Applied

Science.. These cows were fed the regular herd ration, which included a good quality alfalfa hay, Atlas sorgo or corn silage, and a grain mixture of corn, bran and cotton seed meal. All the cows used in these tests freshened in the spring of the year. They had access to limited rye pasture in April and early May.

Composite samples from the 24 -hour yield of colostrum or milk from individual cows representing Guernsey, Ayr- shire and Holstein breeds were saved by the Department of

Dairy Husbandry through the courtesy of W. H. Riddell, and stored at 0o centigrade until needed for the experiment. According to Osborne and Mendel (8) butterfat may 6

be stored at a temperature of 80 - 18o centigrade for at

least as long as a year with no destructive effects on

the vitamin A content. The total yield of milk, in

pounds, the per cent of butterfat and the per cent of to-

tal solids were determined for the individual cow. The

original plan called for taking samples on the first,

second, fourth, seventh, fourteenth and twenty-eighth days

of lactation. Unavoidable circumstances altered the time

of the collection of the samples somewhat. Table 1 gives

details concerning the cows used, the dates of collection

of samples, the yields, and the percentages of butterfat

and total solids, as well as calculated total yields of butterfat and total solids.

Biological assay is no longer necessary in estimating the vitamin A activity of a product. The yellow color of carotene and the blue color of antimony trichloride with vitamin A have been used to estimate the amounts of carotene and vitamin A present, in spite of the fact that the blue color is not specific for vitamin A. For example,

Dann (3) used the Lovibond tintometer to determine the yellow units of carotene and the blue units of vitamin A in samples of colostrum and milk. Dann mentioned that this method measured carotene and xanthophyll together but that 7

the error was small, with only about 7 per cent of the

yellow color due to xanthophyll. He stated that this

material need not be removed because the error in the col-

orimetric method used would begreater than that due to

this amount of xanthophyll. The use' of more painstaking

spectroscopic methods marks recent advances in the tech-

nique of determinations of vitamin A activity. For ex-

ample, Semb, Baumann and Steenbock (10) reported carotene

readings made with a spectrophotometer, and spectrophoto-

metric determinations of vitamin A made with a quartz

spectrograph.

Biological methods of assay continue to be used for

estimations of the total vitamin A value of a product, when

it is desirable not to subject the sample to unusual pro- cesses which might change the vitamin A activity. The prolonged biological methods have lost popularity where quick results are necessary. On the other hand, animal experiments provide results not dependent upon the assump- tion that the total vitamin A activity of a material may be accounted for by the vitamin A plus the (3 -carotene present. Many laboratories have used the biological method of Sherman and Munsell (12). A modification of this, the single feeding method of Sherman and Todhunter (14), 8

presents advantages which led to its selection for these

experiments.

Rats from Wistar Institute stock were fed the diet

described by Sherman ana Crocker (11) which consisted of:

Dried whole milk - 1 part

Ground whole wheat - 2 parts

Sodium chloride 2 per cent of the

weight of the wheat

This food and distilled water were supplied ad libitum.

The young rats were weaned when 38 to 40 grams in weight

and placed on the vitamin A -free diet recommended by

Sherman and Munsell (12). The diet contains:

Vitamin A -free casein - 18 per cent

Dried brewer's yeast - 10 per cent

Osborne and Mendel salt mixture - 4 per cent

Cornstarch - 67 per cent

Sodium chloride - 1 per cent

The Osborne and Mendel salt mixture was made by a short

cut method, according to the suggestions of Wesson (15).

As recommended by the U. S. P. and Druggists Committee (9)

Squibbs' viosterol was used to furnish the vitamin D. The amount fed was 0.9 gram viosterol to 1,000 grams of food.

The original recommendation was for "Viosterol in Oil 9

250 D", which is approximately equivalent to the viosterol

in oil now on the market, containing 10,000 U. S. P. vi-

tamin D units per gram.

During the first part of the depletion period, the

animals were kept in groups of 5 or less in a cage with

raised wire floors. The rats were weighed individually

at frequent intervals and were placed in separate cages

and weighed daily as the end of the depletion period ap-

proached. When the weight of the rat remained stationary

for a week or the rat developed xerophthalmia, it was con-

sidered depleted and ready for the experiment. The de-

pleted animals were divided into groups comparable as to weight, sex'and litter. Weekly weighings of all rats were made during the experimental period, which lasted from the

time of depletion until death of the animal.

Carotene (International Standard for vitamin A, held

for the Health Organization of the League of Nations by the National Institute for Medical Research, Hampstead,

London, N. W. 3) was secured through the Bureau of Chemis- try and Soils, United States Department of Agriculture.

The carotene was dissolved in cottonseed oil and stored in the dark below 0 centigrade, until needed. TheS-carotene content of this solution was determined in the spectropho- 10

tometer, using the value for log I/I0 of 0.44 as given by

Baumann and Steenbock (1) for a solution containing 0.002

mg. of carotene per cc. of cottonseed oil measured at a

depth of 1 cm. The carotene was given in single feedings

to 2 groups of rats. The first group received 17 N(pure

t5 -carotene or 28.3 International units, and the second

group, 34 N( pure VS -carotene or 56.6 International units.

A '(is equal to 0.001 milligram or 0.000001 gram. The weight records of these animals furnished data for the

carotene reference curves.

A group of negative control rats was maintained,

matched as for the other series but receiving only water

and the basal vitamin A -free diet.

Six groups of rats were used for testing the samples

of colostrum and milk from each cow. Ten or more animals were started in each group, but a few were later dropped

from consideration, as they were in some manner not suited

to the experiment. In a few instances the rat refused to eat the weighed portion of sample presented to it, as, for

example, some of those fed on the colostrum of Maggie, which showed presence of blood. In other instances the rats appeared to have been depleted too much or too little.

Table 2 gives data concerning the size of portion present- 11

ed for a single feeding and the number of rats supplying

acceptable data in each group.

Weight records of the animals were used to compile

data for composite growth curves. According to the sug- gestion of Sherman and Todhunter (14), the curves were plotted on cross section paper, 10 squares to the inch,

(figure 1), and a planimeter used for measuring the areas.

For each area the base line was the horizontal line representing the weight of the negative control rats at one week. The left boundary was the curve of the first week for the negative control animals and the remainder of the boundary was the composite curve of the group in question.

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A - Crystal

B - Maggie

C - Lady

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Table 2. Summary of data f m rat feeding experiments.

Rats used Sq. in.:. Yield :Yield of vitamin A Amt. fed:Date pro-: :Av. Wt. when :Area in:for each:Value per gram :for day:per day, computed ::17-cavot ene I.U. Cows : grams :duced :No .:depleted.grs fed:. carotene I.U.:grams 2 :3- 4-38 §6 : 8.88,0P, 4.44 10.52 17.49: 3629 : 38,177 63 471 122,571 : 2 :3- 5-35 : 9 : 92 : 4.97 : 11.78 19.58: 6260 : 73,743 Elmbar 7 :3- 7-35 :10 : 93 7.0311." 1.00 2,73 3.84;12928 : 35,293 50,936 : 40,006 Crystal :.15 :3-10-35 : 7 94 10,63'i''10.71, 1.68 2.60a4288 24,004 8.27 0.97 1.62:14742 :. 14,300 23,882 Lt_qi : 20 :3-17-35 : 6 90 20 :3-28-35 6 87t : 11.10' 0.56 1.33 2.21;15695 : 20,874 34,686

2 :5- 8-35 : 9 96' 12.95/Y6.48 15.36 25.53: 4990 : 76,646 127,395

2 :5- 9-35 : 7 : 97' 5.82-q'', 2.91 6.90 11.47:14515 :100,154 166,487

0.48 1.14 1.89:24812 :. 28,286 46,895 S. L. 7 :5-13-35 : 7 : 92 3.57.]i Maggie 15 :5-19-35 : 6 : 95 : 7.99 %,..a.53 1.26 2.09:25311 : 31,892 52,900 20 :6- 1-35 7 90 7.07C 0.35 0,83 1.38:26853 : 22,288 37,057 20 :7-31-35 6 : 90 10.37/1i340.52 : 1.23 2,05:21183 :. 26,055 43,525

2 :5-28-35 : 8 : 94 : 13.89-L 6.95 16.47 27.38; 2359 : 38,853 64,589

2 :5-27-35 :10 : 92 p-10.39/',5.20 12.32 20.49: 8392 :103,389 171,952 7 :5-29-35 :10 ; 93 z "7.79 -It 1.11 2.63 4.37:14334 : 37,698 62,610 E. M. V. 15 :6- 3-35 : 8 91 4-9.55 ?,' 0.64 1.52 2.52:14878 : 22,615 37,493 Lady 20 :6-10-35 : 9 : 90 : 9,041:e',0.47 1.11 1.85:15150 : 16,817 28,028 20 :6-30-35 : 5 : 90 : 8.28 0.41 0.97 1.62:14878 : 14,432 24,102 1 7/

2 :2- 3-35 .10 : 90 ;1--8.31/40 4.17 9.88 16.43: 4309 : 42,573 70,797 2 :2- 4-35 : 7 : 90 : 4.4741" 2.24 5.31 8.83: 3357 : 17,826 29,642 7 :2- 6-35 : 8 : 91 5.584; 0.80 0 1.90 3.15 :17055 : 32,405 53,723

E. P. V. 15 :2- 9-35 : 6 93 . 9 416. 0 * 63 1.49 2.48:19142 : 28,522 47,472 Ella 20 :2-16-35 : 8 : 90 : 9.9'7q1b,(00.50 1.19 1.97:21729 : 25,855 42,802 '70 20 :2-28-35 : 8 91 : 11.5811i' 0.58 1.37 2.29:19958 : 27,342 45,704

Value of 1 sq. in. 34/ -puree-carotene 2.486 -carotene or Carotene or 56.6 I. U. 9 92 13.69 4.13 I. U.

Carotene 17Y-pures-carotene or 28.3 I. U. 11 91 7.55 2.25 ;eV -carotene Average value for 1 sq. inch 3.75 I. U. = 2.37Y pure49-carotene or 3.94 I. U.

Negative controls A -free diet only 93 17 18

DISCUSSION

Table 1 presents the records for the cows used in the experiments. It will be noted that none of the cows from which the samples of colostrum or milk were taken was heifers. Dann (3) stated that colostrum from heifers tended to be richer in vitamin A than colostrum from cows.

He accounted for this difference by the supposition that the vitamin A of the heifer had not been exhausted by previous lactations.

The dates on which samples of colostrum or milk were saved corresponded closely to the original plan. However, it will be observed that there was no fourth day milk for

Maggie, and the last sample tested for Maggie was taken three months after freshening. Also the fourth sample for Lady was taken on the ninth day after freshening in- stead of the seventh day, as was planned.

The percentages, of fat and the percentages of total solids were determined by the Department of Dairy Husband- ry. Comparisons of fat percentages show that 3 cows had much the same per cent of fat in the colostrum as in the 19

later milk. In one case the per cent of fat was much higher for colostrum than for later milk. Breed distinctions

were not evident, the percentages of fat for the cows used

being similar. The percentages of total solids were high-

er for colostrum than for later milk. Generally speaking,

there was a gradual decrease in the total solids from the

first day of lactation to the last day when samples were

collected. In this regard, no noteworthy differences were

found between the three breeds of cows.

The total yield in pounds was obtained by combining

the weights of the morning and evening milkings on the day when a sample was saved. The collection of colostrum is

beset by many difficulties. The total yield of colostrum

in pounds is as accurate as is possible to obtain. How-

ever, there is little doubt that the total yields for some

of the colostrum are low. For example, on the second day

Ella gave 7.0 pounds of colostrum in the morning, and only

0.4 pound of colostrum in the afternoon. Beginning with

the fourth day, the total yield of milk is considered re-

liable.

The calculated total yield of fat and of total solids

per day presents further data. In each case, the total

yield of fat for the first or second day is distinctly less 20

than the yield per day for the same cow later in the first month of lactation.

The original records have been studied and used to

compile data presented in table 2. Suitable amounts of

the sample to give the rats for single feedings were de-

termined by preliminary experiments. Two grams of colos-

trum proved satisfactory for a single feeding, but larger

amounts of milk had to be used as lactation progressed, 20 grams of the last samples being necessary.

For each group more than 10 rats were started but because of unavoidable circumstances it was necessary to eliminate some of the rats from the records. The bloody colostrum of the first and second day of Maggie's lactation seemed very repugnant to the rats and many refused to eat their portions. Other rats) seemed to have been depleted too much or too little. Slight amounts of mold in the first and second day's colostrum of Ella might have affected the results of these particular experiments. In other instances the rats also refused to consume the entire portion offered.

Depleted weights of the rats had a maximum range of

80 to 105 grams. The original plan was to use rats aver- aging about 92 grams for each group with sexes equally 21

divided. Because some rats with poor records had to be

dropped from the final summaries, the average weights for

the groups ranged from 87 grams for the smallest to 97

grams for the largest. At the same time some groups be-

came smaller in number than desired. The sex ratio remained well balanced in the groups.

The composite curves were plotted according to the method of Sherman and Todhunter (14). Each area, was measured in square inches with a planimeter in triplicate, and the average recorded as indicated in table 2. The areas under the standard carotene curves were measured for reference. Dividing the area in square inches by the weight of the single feeding gave the square inches for each unit fed. It is evident that the vitamin A activi- ties are high for the samples of the first 2 days of lactation of all 4 cows, because the areas produced by 1 gram of sample are much larger than those produced later. Per unit of weight, the product of the first or second day of lactation of the cow was from 8 to 19 times as rich as the less potent milk produced later in the month.

The results secured from the two carotene reference curves were considered reliable because they checked with one another. One group of rats received twice as much 22

carotene as did the other group, and the area under the

subsequent curve theoretically should have been twice as

large as for the group receiving the smaller amount. This

ideal condition was approached at least as well as in the

experiments considered satisfactory by Sherman and Tod -

hunter (14). In the present experiments, 34 \'`. pure

0 -carotene equivalent to 56.6 International units, pro-

duced an area of 14.40 square inches, using averages from

the two reference curves.

The carotene reference curves were used to determine

the value of 1 square inch of area in terms of -carotene

or International units. These values were used to calcu-

late the vitamin A activity of 1 gram of each sample in

terms of (3 -carotene and also International units. In terms

of 0 -carotene or International units the secretions of the

first and second days of lactation of the cow were richest

in vitamin A per gram of weight. By the fourth day of lac-

tation, there was a considerable drop in the vitamin A con-

tent per gram. For each of the 4 cows there was some fur-

ther decrease in vitamin A content from the fourth to the

seventh day of lact'ation. In general, the vitamin A content

of the milk remained much the same from the seventh to the

twenty-eighth day. These values for the vitamin A content 23

of milk are 'slightly lower than those compiled by Sherman

(13) who lists milk as containing 65 Sherman units of vi-

tamin A per ounce.. According to Baumann and Steenbock (1)

1 Sherman unit is equal to about 1.3 International units.

On this basis several of the milk samples studied would

contain 40 to 50 Sherman units per ounce.

The sudden decrease in the amount of International units per gram and the total International units per day

is quite evident for 'all cows. The decline of vitamin A

activity for the total yield of the second day of lactation

for Ella is probably due to the difficulty in obtaining a

complete yield of colostrum. After the fourth day of lac-

tation the International units per gram remained on much

the same level through the month tested. On the eighty- fifth day for Maggie, or at the end of the third month, the International units per gram and the total Interna-

tional units per day are still very much on the same level as for the seventh day. In these experiments the cows with the heaviest yield of milk per day had also the great- est amount of vitamin A activity per day. Figure 2 presents these relationships in graphic form. It is inter- esting to note the relatively high levels of vitamin A output maintained by both Maggie and Ella throughout the 24 period of the experiment.

SUMMARY AND CONCLUSIONS

Samples of colostrum and normal milk from 1 Holstein,

1 Guernsey and 2 Ayrshire cows collected approximately on

:the first, second, fourth, seventh, fourteenth and twenty -

'eighth days were available for the experiments described.

Each sample represented a composite from the 24.hour milk- ing. Yield for the day, per cent of total solids and per cent of fat were determined in each case. All samples were stored in a frozen condition until needed.

Albino rats were depleted of their body reserve of vitamin A by feeding vitamin A -free diet. At the end of the depletion period, the experimental rats were divided into groups comparable as to weight, sex and litter; one group of rats served as negative controls and other groups received single feedings of colostrum or milk, following the method of Sherman and Todhunter (14). According to the single feeding method, the rats are given a single weighed portion of the food to be tested and then retained on the

A -free diet. Weekly weighings of all rats were made during the experimental period which lasted from the time of the 25

single feeding until death of the rat. Composite curves were plotted and a planimeter used for measuring the areas under the curves.

'Computations were made to show the total vitamin A

content per gram of, sample expressed in two ways, as Y-of

(-carotene and as International units. The total yield of vitamin A per day for each cow was also calculated and expressed in the same terms.

When comparisons were made on the basis of the vitamin A activity per gram, the secretions of the first or

second day of lactation of the cows were highest, from 8 to 19 times as rich as the less potent milk produced later in the month. By the fourth day of lactation, there was a considerable drop in the vitamin A activity per gram of

sample. For each of the 4 cows there was some further decrease in vitamin A content from the fourth to the

seventh day. In general, the vitamin A content of the milk remained much the same from the seventh day to the end of the experiment. Maggie and Lady produced the richest samples of colostrum tested. These two rich samples contained 25.53 and 27.38 International units per gram re- spectively. One sample of colostrum tested as low as 8.83

International units per gram. This was produced by Ella 26

on the second day of lactation. Several of the samples of milk contained about 2 International units per gram. The least potent sample tested was produced by Maggie and contained 1.38 International units per gram. It so happens

that the yield for Maggie on that particular day was the highest of the entire experiment. The number of animals used permits no statement regarding breed differences.

Comparisons may also be made on the basis of the vi-

tamin A produced by the cow in a day. For each of the

cows, the largest total output of vitamin A for one day was on the first or second day of lactation. The samples

collected on approximately the fourth day of lactation

gave results which showed total daily yields of vitamin A

approaching one another. Total daily outputs of vitamin

A for the remainder of the experiment were near the same

level. The highest total output, that of Lady on the

second day, was more than seven times as great as the low-

est outputs, those of Crystal and Lady toward the end of

the experiment. 27

ACKNOWLEDGMENT

The writer wishes to express her appreciation to her major instructor, Dr. Martha M. Kramer, for her sugges-

tions and assistance in the preparation of this manuscript.

She also wishes to thank Bernice L. Kunerth for her help in

the laboratory, and Dr. W. H. Riddell, Department of Dairy

Husbandry, for his supervision in the collection of samples of colostrum and milk. 28

LITERATURE CITED

1. Baumann, C. A. and Steenbock, H. Fat-soluble vitamins. XXXVI. The carotene and vitamin A content of butter. Jour. Biol. Chem. 101: 547-560. July 1933.

2. Baumann, C. A., Steenbock, H., Beeson, W. M. and Rupel, I. W. Fat-soluble vitamins. XXXIX. The influence of breed and diet of cows on the Carotene and vitamin A content of butter. Jour. Biol. Chem. 105: 167-176. April 1934.

3. Dann, W. J. The transmission of vitamin A from parents to young in mammals. II. The carotene content of cow's colostrum. Biochem. Jour. 27: 1998-2005. 1933.

4. Davis, H. P. and Hathaway, I. L. The vitamin A content of the milk of Holstein, Ayrshire, Jersey, and Guernsey cows. Nebr. Agr. Exp. Sta. Res. Bul. 54. 14 p. Feb. 1931.

5. Drummond, J. C., Coward, K. H. and Watson, F. A. Factors influencing the value of Milk and butter as sources of vitamin A. Biochem. Jour. 15: 540-552. 1921.

6. Hathaway, I. L. and Davis, H. P. The vitamin A content of skim milk, standardited milk, and cream from different breeds of cows. Nebr. Agr. Exp. Sta. Res. Bul. 69. 16 p. Dec. 1933.

7. Moore, Thomas.. Vitamin A and carotene. IX. Notes on the con- version of carotene to vitamin A in the cow. Biochem. Jour. 26: 1-9. Jan. 1932. 29

8. Osborne, T. B. and Mendel, L. B. The stability of the growth-prototing substance in butterfat. 'Jour. Biol. Chem. 24: 37-39. Jan. 1916.

9. Report of the vitamin A assay committee of the Amer - loan Drug Manufacturers Asdociation - twentieth annual meeting - May 1931. by Arthur D. Holmes, Chairman. Jour. Amer. Phar. Soc. 20: 583-594. June 1931.

10. Semb, J., Baumann, C. A. and Steenbock, H. Fat-soluble vitamins. XLI. The carotene and vitamin A content Of colostruM. Jour. Biol. Chem. 107: 697-703. Dec. 1934.

11. Sherman, H. C. and Crocker, J. Growth and reproduction upon simplified food supply. III. The efficiency of growth as influ- enced by the proportion of milk in the diet. Jour. Biol. Chem. 53: 49-52. May 1922.

12. Sherman, H. C. and Munsell, H. E. The quantitative determinations of vitamin A. Jour, of Amer. Chem. Soc. 47: 1639-1646. June 1925.

13. Sherman, H. C. The chemistry of food and nutrition, fourth ed. The Macmillan Co., New York. 614 p. 1932.

14. Sherman, H. C. and Todhunter, E.,N. The determination of vitamin A values by a method of'single feedings. Jour. Nutr. 8: 347- 356. Sept. 1934.

15. Wesson, Lawrence G. A modification of the Osborne -Mendel salt mixture containing only inorganic constituents. Science 75:. 339-340. Mar. 1932.

16. Wilbur, J. W. Hilton, J. H. and Hauge, S. M. The vitamin A activity of butter produced by Guernsey and Ayrshire cows. Jour. Dairy Sci. 16: 153-156. Mar. 1933.