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Vitamin B12 in Blood of Newborn and Colostrum-Fed Calves and in Colostrum and Normal Milk of Holstein and Jersey Cows1,2
V I T A M I N B12 I N B L O O D OF N E W B O R N A N D C O L O S T R U M - F E D C A L V E S A N D I N C O L O S T R U M A N D N O R M A L M I L K OF H O L S T E I N A N D J E R S E Y C O W S 1, 2 W. B. ANTHONY,S J. 1~. COUCH,4 I. W. RUPEL,3 W I T H TH'E T E C H N I C A L A S S I S T A N C E OF MARY B. H E N D E R S O N AND C H A R L E S T A BROWN Agricultural and Mechanical College of Texas and Texas Agricultural Experiment Station, College Station
Studies on the levels of various blood constituents have yielded considerable information relative to the dietary requirements and health of the y o u n g calf. Such records before and following ingestion of colostrum have helped define its role in establishing the normal blood picture in calves, l ' e a r s o n and Darnell (4) reported on the concentration of thiamin, riboflavin, niacin and pantothenie acid in eolostrum f r o m Holstein and J e r s e y breeds. Sutton (6, 7), H a n d (2), J o h n son (3), Theophilus (8) and W h i t n a h (9) with their respective co-workers have reported on the levels of riboflavin in colostrum and factors affecting its level in milk. To the a u t h o r s ' knowledge, however, no i n f o r m a t i o n has been published concerning the concentration of vitamin B1~ in eolostrum or in the blood of the dairy calf at birth and following the ingestion of colostrum. Accordingly, this investigation was carried out to determine the v i t a m i n B~-~ in eolostrum and in blood of calves. EXPIi~RINIENTAL PROCEDURE
Cob)strum and milk samples were collected f r o m p u r e b r e d tIolstein and J e r s e y cows calving in the A. & M. College herd d u r i n g J u l y , A u g u s t and September, I95(). The feeding regime for this herd consisted of a 15 to 16 per cent protein grain mixture, alfalfa hay, sorghum silage and some sudan-grass pasture. A few of the samples were f r o m first-calf heifers but most of the samples were f r o m older cows which freshened a f t e r a rest period of 6 to 8 wk. The cows were machine-milked, and a 0.5-pt. sample was taken immediately f r o m the milking machine bucket. The first milking always occurred within 6 hr. a f t e r the cow calved. The other samples were t a k e n 24, 48, 72, 96, 144 and 192 hr., and 15, 30, 45 and 60 days a f t e r calving. Calves were allowed to remain with their dams for the first day and, consequently, the first milking reported in this s t u d y represents milk r e m a i n i n g in the udders a f t e r calves had llnrsed.
I n most instances, vitamin 1112 analysis was begun a few hours a f t e r the samples were collected. I f the analysis was delayed, samples were frozen immediately afWr collection and stored until processed. Received for publication Feb. 15, 1951. Supported in part by Project RC 1862, I)ivision of Research Grants and Fellowships, U. S. Public. Health Scrvice~ National Institute of Health, Bethesda, Maryland. 2 The crystalline vitamin B~ used in the microbiological assays was generously supplied by D. F. Green, Vetcrint~ry Department, Merck and Co., Rahway, New Jersey. Department of ])airy Husbandry. 4 Biochemistry and Nutritio~ and Poultry Husbandry Departments. 749
750
W . B. A N T H O N Y ET AL
Stock solutions were p r e p a r e d f o r microbiological assay as indicated by Couch et al. (1). Ten ml. of colostrum or milk (in the case of the blood samples, 3 ml. of oxalated whole blood) were p i p e t t e d into 50 ml. of w a t e r in a 125-ml. flask. Ten ml. of a p a n c r e a t i n solution (3 rag. per milliliter) were added and the solution was covered with toluene. The cork-stoppered flask was shaken at 37.5 ° C. for a p p r o x i m a t e l y 24 hr. The samples t h e n were autoclaved for 5 rain. at 15 lb. pressure. A f t e r cooling, the samples were made up to 100 ml. and filtered. The filtrates were covered with toluene a n d stored at a p p r o x i m a t e l y 20 ° C. The filtrates constituted the stock solutions f r o m which dilutions for the microbiological assays for v i t a m i n B~2 were made. These were made according to the procedure of Skeggs et al. (5), with Lactobacillus leichmannii 4797 ( A T C C ) as the test organism, and acid production was the criterion used to measure growth response. Crystalline vitamin B12 was used as the s t a n d a r d in all of the assays mentioned in this report. The calves in this s t u d y were d r o p p e d b y the cows f r o m which the colostrum and milk samples were taken. Blood samples were d r a w n f r o m the j u g u l a r vein immediately following birth and before ingestion of colostrum. Additional blood samples were taken 24, 48, 72, 96, 144 and 192 hr. a f t e r birth. E a c h calf was hand-fed the milk f r o m its dam a f t e r the first d a y of life for 4 days. A f t e r the fifth day, mixed milk f r o m Holstein cows was fed. D a t a were collected on seven Holstein and six J e r s e y calves. The n u m b e r of samples at respective ages is less t h a n the total n u m b e r of calves due to loss of certain samples and the fact that certain calves were unavoidably omitted at given ages. RESULTS
V i t a m i n B ~ values in the blood of tIolstein and J e r s e y calves at birth and following the ingestion of colostrum are presented in table 1. I n contrast to TABLE
1
V i t a m i n B12 in blood o f d a i r y calves b e f o r e and f o l l o w i n g i n g e s t i o n of colostrum A g e (hr.)
.......................................
~6
]No. s a m p l e d t h i s a g e ......... 5 R a n g e ( m ~ g . / m l . ) ............... 1 , 0 - 1 . 2 M e a n ( m ~ g . / m l . ) .................. 1.1
N o . s a m p l e d t h i s a g e ......... 5 R a n g e ( m ~ g . / m l . ) ............... 0 . 7 - 1 . 3 M e a n ( m ~ g . / m l . ) .................. 1.0
24
48
H o l s t e i n calves 5 5 0.9-1.7 ].2
0.6-1..2 1.0
J e r s e y calves 5 4 0.9-1.4 1.1
0,6-1.6 1.l
72
96
]44
192
6
4
6
6
0.6-1.9 1.1
0.7-1.4 1.0
0.8-].6 1.2
0.7-1.3 1.1
4
4
4
4
0.7-1.5 1.1
0.7-1.1 0.8
0.4-1.7 ].]
0.5-1.3 0.9
changes of some other constituents of blood of y o u n g calves following the ingestion of colostrum, these data indicate a n o r m a l blood level of v i t a m i n B12 for the dairy calf at birth. No increase was shown in blood BI~ following the ingestion of colostrum. B y r e f e r r i n g to table 2, one m a y observe the vitamin B12 content of the colostrum consumed b y the calves f r o m which the data in table 1 was
751
V I T A M I N B12 I N C A L F BLOOD
collected. Since only one n u t r i t i o n a l level was studied, these data, therefore, do not clearly indicate placental t r a n s f e r of v i t a m i n B:2. The results of analyses for v i t a m i n B~2 in colostrum a n d normal milk f r o m p u r e b r e d Holstein a n d J e r s e y cows arc shown in table 2. Samples were collected f r o m 15 Jerseys and 16 Holsteins in the College herd. The colostrum was sampled within 6 hr. following calving. The colostrum and milk were followed b y sampling at 24, 48, 72, 144, 192 hr., and 15, 30 a n d 60 days a f t e r calving to determine the v i t a m i n B~2 content. A few samples were lost before the assays were completed a n d some cows were u n a v o i d a b l y missed at given times. Hence, the n u m b e r of assays completed was lower at certain intervals a f t e r calving t h a n the total n u m b e r of cows included in the study, but the d a t a indicate something of the individual variability and t r e n d following calving. The high initial concentration of v i t a m i n B12 in colostrum f r o m Holstein cows was greater, in most instances, t h a n the concentration found in J e r s e y colostrum. Although variaTABLE
2
V i t a m i n B,~ in colostrum and normal m i l k f r o m H o l s t e i n and J e r s e y cows Holstein cows
Time after calving
< 6 24 48 72 144 8 15 30 45 60
hr.
" '' ~' '' d.
" ~' ~' ''
:No. a s s a y s
13 16 14 12 14 12 6 10 11 10
J e r s e y cows
:Range
Mean
(m~g./ml.)
(m~tg./ml.)
28-78 ]5-40 12-37 13-45 14-48 11-38 5-36 3-24 3-15 2-9
49 +_ 5.4 25_+ 2.3 24 + 2.2 27 _+ 3.0 25 + 2.5 22 + 2.5 16 + 4 . 4 10 -+ 1.9 9 _+ 1.0 5.9 + 0.6
No. assays
13 12 13 12 14 13 6 9 12 14
/~ange
Me:tn
(m~g./ml.)
(m~tg./ml.)
3-56 2-35 4-40 8-27 4-27 3-25 3-]5 2-14 2-11 2-13
21 18 17 ]6 ]4 ]0 7 7 7 5.6
+ 4.6 + 2.7 + 2.9 _+ 1.3 _+ 2.0 + 1.6 -+ 1.7 + 1.4 _+ 0.9 + 0.8
tions have been reported in vitamin content of colostrum f r o m different breeds (2, 7), the differences f o u n d in this experiment were more pronounced. While the daily decline in vitamin B12 was r a p i d in the IIolstein colostrum, on the eighth d a y the level remained as high as t h a t present initially in average J e r s e y colostrum. Although the ranges of the two breeds overlap considerably, the means (at 144 hr.) are significantly different at the 5 per cent level of probability. These data showing a greater concentration of v i t a m i n B12 in the colostrum and milk f r o m the Itolstein t h a n in corresponding secretions f r o m the J e r s e y are in contrast to findings with respect to other B vitamins in normal milk (2, 3, 6). The breed differences observed are not explained by the data f r o m this work. The milk f r o m 48 J e r s e y cows was sampled s e p a r a t e l y at the a.m. and p.m. milkings and analyzed for its vitamin B12 content. A t the time of sampling, the cows were fresh f r o m 6 to 351 days, averaging 139 days, the group thus furnishing a more or less typical sample of normal herd milk.
752
W. B. ANTI-IONY~ ET AL TABLE' 3
Vitamin B~z content of normal Jersey herd mil]~ Time milked
No. samples
A.M. P.M. Mean for day
48 48 48
Range, B~2
Mean
( m~g./ml. )
( m~g./ml. )
3-22 4-13 4-16
8.7 + 0.63 6.9 + 0.33 7.6 ± 0.47
The mean of the morning values was 8.7 millimierograms per milliliter of milk, while the evening milkings averaged 6.9, a difference of 1.8 millimicrograms. Analysis of variance showed this to be significant at the 5 per cent level of probability. The reason for the difference between a.m. and p.m. values is not apparent. I t will be noted that the mean of 7.6 millimierograms (daily averages, 48 cows) is somewhat higher than the means at 60 days shown in table 2. The samples in table 2 represent single milkings and the smaller numbers may merely reflect sampling differences. SUMI~ARY
¥ i t a m i n B12 determinations were made on the blood of calves before ingestion of colostrum and subsequently to 8 days of age. No significant differences were observed in relation to age (within this range), breed or the food ingested. IIolstein eolostrum showed nearly double the values observed on Jersey colostrum. The colostrum B,~ values for both breeds declined gradually. The J e r s e y milk, showing averages of 7.0 millimicrograms after the 15th day, appeared to have declined to " h e r d a v e r a g e " levels. The IIolstcin milk maintained a slightly higher value through the 45th day. Forty-eight ,lersey cows fresh from 6 to 351 days showed an average vitamin B,= value of 7.6 millimicrograms per milliliter. Morning milk averaged 8.7 versus 6.9 for evening milk (with significance at the 0.05 level). ACKNOWLEDGMENT
The authors wish to extend their sincere appreciation to James Huff and Claude Free for assistance in collecting m a n y of the blood and colostrum samples for this experiment. REFERENCES (1) Coucit, J. R., OL(H*]SE, O., WITTEN, P. W., AND COLBY, R . W . The Vitamin B,~ Content of Blood from Various Species. Am. J. Physiol., 163: 77-80. 1950. (2) HAND, D. B., AND SIIARP, P. F. Tlm Riboflaviu Content of Cow's Milk. J. Dairy Sei., 22: 779-783. ]939. (3) JOHNSON, P., MAYNARD, L. A., AND LOOSLI, J. K. The Riboflavin Content of Milk as Influenced by Diet. J. Dairy Sci., 24: 57-64. ]941. (4) PEARSON, P. B., AND ])ARNELL, A . L . The Thiamine, Riboflavin, Nicotinic Acid, and Pantothenie Acid Content of Colostrum and Milk of the Cow and Ewe. J. Nutrition, 31: 51-58. 1946. (5) SKEGGS, HELEN R.) NEPPLE, t]~ELGA M., VALENTII,[, KATIIERINE A., HUFF, JESSE W.) AND WRIGHT, L . D . Observations on the use of Lactobacillus leichmannii 4797 in the Microbiological Assay of Vitamin B,.~. J. Biol. Chem., 184: 211-221. J950.
VITAMIN B]2 IN CALF BLOOD
753
(6) SUTTON, T. S., AND KAESER, I-I. E. The Riboflavin Content of Cows' Colostrum. J. Dairy Sci.. 30: 581-582. 1947. (7) SUTTON, T. S., WARNER, R. G., AND KAESER, H. ~]. The Concentration and Output of Carotenoid Pigments, Vitamin A, and Riboflavin in the Colostrum and Milk of Dairy Cows. J. Dairy Sci., 30: 927-932. 1947. (8) THEOPHILUS, D. R., AND STAI~[BERG, 0. E. The Influence of Breed, Feed, and Processing on the Riboflavin Content of Milk. J. Dairy Sci., 28: 259-268. 1945. (9) WI-IITNAI-I, C. H., KUNEKTI-I, B. L., AND KRAMEK, M . M . Riboflavin Content of Milk Collected in Different Months, and Correlated with Other Constituents of the Milk. J. Dairy Sei., 21: 593-600. 1938.
Studies on the levels of various blood constituents have yielded considerable information relative to the dietary requirements and health of the y o u n g calf. Such records before and following ingestion of colostrum have helped define its role in establishing the normal blood picture in calves, l ' e a r s o n and Darnell (4) reported on the concentration of thiamin, riboflavin, niacin and pantothenie acid in eolostrum f r o m Holstein and J e r s e y breeds. Sutton (6, 7), H a n d (2), J o h n son (3), Theophilus (8) and W h i t n a h (9) with their respective co-workers have reported on the levels of riboflavin in colostrum and factors affecting its level in milk. To the a u t h o r s ' knowledge, however, no i n f o r m a t i o n has been published concerning the concentration of vitamin B1~ in eolostrum or in the blood of the dairy calf at birth and following the ingestion of colostrum. Accordingly, this investigation was carried out to determine the v i t a m i n B~-~ in eolostrum and in blood of calves. EXPIi~RINIENTAL PROCEDURE
Cob)strum and milk samples were collected f r o m p u r e b r e d tIolstein and J e r s e y cows calving in the A. & M. College herd d u r i n g J u l y , A u g u s t and September, I95(). The feeding regime for this herd consisted of a 15 to 16 per cent protein grain mixture, alfalfa hay, sorghum silage and some sudan-grass pasture. A few of the samples were f r o m first-calf heifers but most of the samples were f r o m older cows which freshened a f t e r a rest period of 6 to 8 wk. The cows were machine-milked, and a 0.5-pt. sample was taken immediately f r o m the milking machine bucket. The first milking always occurred within 6 hr. a f t e r the cow calved. The other samples were t a k e n 24, 48, 72, 96, 144 and 192 hr., and 15, 30, 45 and 60 days a f t e r calving. Calves were allowed to remain with their dams for the first day and, consequently, the first milking reported in this s t u d y represents milk r e m a i n i n g in the udders a f t e r calves had llnrsed.
I n most instances, vitamin 1112 analysis was begun a few hours a f t e r the samples were collected. I f the analysis was delayed, samples were frozen immediately afWr collection and stored until processed. Received for publication Feb. 15, 1951. Supported in part by Project RC 1862, I)ivision of Research Grants and Fellowships, U. S. Public. Health Scrvice~ National Institute of Health, Bethesda, Maryland. 2 The crystalline vitamin B~ used in the microbiological assays was generously supplied by D. F. Green, Vetcrint~ry Department, Merck and Co., Rahway, New Jersey. Department of ])airy Husbandry. 4 Biochemistry and Nutritio~ and Poultry Husbandry Departments. 749
750
W . B. A N T H O N Y ET AL
Stock solutions were p r e p a r e d f o r microbiological assay as indicated by Couch et al. (1). Ten ml. of colostrum or milk (in the case of the blood samples, 3 ml. of oxalated whole blood) were p i p e t t e d into 50 ml. of w a t e r in a 125-ml. flask. Ten ml. of a p a n c r e a t i n solution (3 rag. per milliliter) were added and the solution was covered with toluene. The cork-stoppered flask was shaken at 37.5 ° C. for a p p r o x i m a t e l y 24 hr. The samples t h e n were autoclaved for 5 rain. at 15 lb. pressure. A f t e r cooling, the samples were made up to 100 ml. and filtered. The filtrates were covered with toluene a n d stored at a p p r o x i m a t e l y 20 ° C. The filtrates constituted the stock solutions f r o m which dilutions for the microbiological assays for v i t a m i n B~2 were made. These were made according to the procedure of Skeggs et al. (5), with Lactobacillus leichmannii 4797 ( A T C C ) as the test organism, and acid production was the criterion used to measure growth response. Crystalline vitamin B12 was used as the s t a n d a r d in all of the assays mentioned in this report. The calves in this s t u d y were d r o p p e d b y the cows f r o m which the colostrum and milk samples were taken. Blood samples were d r a w n f r o m the j u g u l a r vein immediately following birth and before ingestion of colostrum. Additional blood samples were taken 24, 48, 72, 96, 144 and 192 hr. a f t e r birth. E a c h calf was hand-fed the milk f r o m its dam a f t e r the first d a y of life for 4 days. A f t e r the fifth day, mixed milk f r o m Holstein cows was fed. D a t a were collected on seven Holstein and six J e r s e y calves. The n u m b e r of samples at respective ages is less t h a n the total n u m b e r of calves due to loss of certain samples and the fact that certain calves were unavoidably omitted at given ages. RESULTS
V i t a m i n B ~ values in the blood of tIolstein and J e r s e y calves at birth and following the ingestion of colostrum are presented in table 1. I n contrast to TABLE
1
V i t a m i n B12 in blood o f d a i r y calves b e f o r e and f o l l o w i n g i n g e s t i o n of colostrum A g e (hr.)
.......................................
~6
]No. s a m p l e d t h i s a g e ......... 5 R a n g e ( m ~ g . / m l . ) ............... 1 , 0 - 1 . 2 M e a n ( m ~ g . / m l . ) .................. 1.1
N o . s a m p l e d t h i s a g e ......... 5 R a n g e ( m ~ g . / m l . ) ............... 0 . 7 - 1 . 3 M e a n ( m ~ g . / m l . ) .................. 1.0
24
48
H o l s t e i n calves 5 5 0.9-1.7 ].2
0.6-1..2 1.0
J e r s e y calves 5 4 0.9-1.4 1.1
0,6-1.6 1.l
72
96
]44
192
6
4
6
6
0.6-1.9 1.1
0.7-1.4 1.0
0.8-].6 1.2
0.7-1.3 1.1
4
4
4
4
0.7-1.5 1.1
0.7-1.1 0.8
0.4-1.7 ].]
0.5-1.3 0.9
changes of some other constituents of blood of y o u n g calves following the ingestion of colostrum, these data indicate a n o r m a l blood level of v i t a m i n B12 for the dairy calf at birth. No increase was shown in blood BI~ following the ingestion of colostrum. B y r e f e r r i n g to table 2, one m a y observe the vitamin B12 content of the colostrum consumed b y the calves f r o m which the data in table 1 was
751
V I T A M I N B12 I N C A L F BLOOD
collected. Since only one n u t r i t i o n a l level was studied, these data, therefore, do not clearly indicate placental t r a n s f e r of v i t a m i n B:2. The results of analyses for v i t a m i n B~2 in colostrum a n d normal milk f r o m p u r e b r e d Holstein a n d J e r s e y cows arc shown in table 2. Samples were collected f r o m 15 Jerseys and 16 Holsteins in the College herd. The colostrum was sampled within 6 hr. following calving. The colostrum and milk were followed b y sampling at 24, 48, 72, 144, 192 hr., and 15, 30 a n d 60 days a f t e r calving to determine the v i t a m i n B~2 content. A few samples were lost before the assays were completed a n d some cows were u n a v o i d a b l y missed at given times. Hence, the n u m b e r of assays completed was lower at certain intervals a f t e r calving t h a n the total n u m b e r of cows included in the study, but the d a t a indicate something of the individual variability and t r e n d following calving. The high initial concentration of v i t a m i n B12 in colostrum f r o m Holstein cows was greater, in most instances, t h a n the concentration found in J e r s e y colostrum. Although variaTABLE
2
V i t a m i n B,~ in colostrum and normal m i l k f r o m H o l s t e i n and J e r s e y cows Holstein cows
Time after calving
< 6 24 48 72 144 8 15 30 45 60
hr.
" '' ~' '' d.
" ~' ~' ''
:No. a s s a y s
13 16 14 12 14 12 6 10 11 10
J e r s e y cows
:Range
Mean
(m~g./ml.)
(m~tg./ml.)
28-78 ]5-40 12-37 13-45 14-48 11-38 5-36 3-24 3-15 2-9
49 +_ 5.4 25_+ 2.3 24 + 2.2 27 _+ 3.0 25 + 2.5 22 + 2.5 16 + 4 . 4 10 -+ 1.9 9 _+ 1.0 5.9 + 0.6
No. assays
13 12 13 12 14 13 6 9 12 14
/~ange
Me:tn
(m~g./ml.)
(m~tg./ml.)
3-56 2-35 4-40 8-27 4-27 3-25 3-]5 2-14 2-11 2-13
21 18 17 ]6 ]4 ]0 7 7 7 5.6
+ 4.6 + 2.7 + 2.9 _+ 1.3 _+ 2.0 + 1.6 -+ 1.7 + 1.4 _+ 0.9 + 0.8
tions have been reported in vitamin content of colostrum f r o m different breeds (2, 7), the differences f o u n d in this experiment were more pronounced. While the daily decline in vitamin B12 was r a p i d in the IIolstein colostrum, on the eighth d a y the level remained as high as t h a t present initially in average J e r s e y colostrum. Although the ranges of the two breeds overlap considerably, the means (at 144 hr.) are significantly different at the 5 per cent level of probability. These data showing a greater concentration of v i t a m i n B12 in the colostrum and milk f r o m the Itolstein t h a n in corresponding secretions f r o m the J e r s e y are in contrast to findings with respect to other B vitamins in normal milk (2, 3, 6). The breed differences observed are not explained by the data f r o m this work. The milk f r o m 48 J e r s e y cows was sampled s e p a r a t e l y at the a.m. and p.m. milkings and analyzed for its vitamin B12 content. A t the time of sampling, the cows were fresh f r o m 6 to 351 days, averaging 139 days, the group thus furnishing a more or less typical sample of normal herd milk.
752
W. B. ANTI-IONY~ ET AL TABLE' 3
Vitamin B~z content of normal Jersey herd mil]~ Time milked
No. samples
A.M. P.M. Mean for day
48 48 48
Range, B~2
Mean
( m~g./ml. )
( m~g./ml. )
3-22 4-13 4-16
8.7 + 0.63 6.9 + 0.33 7.6 ± 0.47
The mean of the morning values was 8.7 millimierograms per milliliter of milk, while the evening milkings averaged 6.9, a difference of 1.8 millimicrograms. Analysis of variance showed this to be significant at the 5 per cent level of probability. The reason for the difference between a.m. and p.m. values is not apparent. I t will be noted that the mean of 7.6 millimierograms (daily averages, 48 cows) is somewhat higher than the means at 60 days shown in table 2. The samples in table 2 represent single milkings and the smaller numbers may merely reflect sampling differences. SUMI~ARY
¥ i t a m i n B12 determinations were made on the blood of calves before ingestion of colostrum and subsequently to 8 days of age. No significant differences were observed in relation to age (within this range), breed or the food ingested. IIolstein eolostrum showed nearly double the values observed on Jersey colostrum. The colostrum B,~ values for both breeds declined gradually. The J e r s e y milk, showing averages of 7.0 millimicrograms after the 15th day, appeared to have declined to " h e r d a v e r a g e " levels. The IIolstcin milk maintained a slightly higher value through the 45th day. Forty-eight ,lersey cows fresh from 6 to 351 days showed an average vitamin B,= value of 7.6 millimicrograms per milliliter. Morning milk averaged 8.7 versus 6.9 for evening milk (with significance at the 0.05 level). ACKNOWLEDGMENT
The authors wish to extend their sincere appreciation to James Huff and Claude Free for assistance in collecting m a n y of the blood and colostrum samples for this experiment. REFERENCES (1) Coucit, J. R., OL(H*]SE, O., WITTEN, P. W., AND COLBY, R . W . The Vitamin B,~ Content of Blood from Various Species. Am. J. Physiol., 163: 77-80. 1950. (2) HAND, D. B., AND SIIARP, P. F. Tlm Riboflaviu Content of Cow's Milk. J. Dairy Sei., 22: 779-783. ]939. (3) JOHNSON, P., MAYNARD, L. A., AND LOOSLI, J. K. The Riboflavin Content of Milk as Influenced by Diet. J. Dairy Sci., 24: 57-64. ]941. (4) PEARSON, P. B., AND ])ARNELL, A . L . The Thiamine, Riboflavin, Nicotinic Acid, and Pantothenie Acid Content of Colostrum and Milk of the Cow and Ewe. J. Nutrition, 31: 51-58. 1946. (5) SKEGGS, HELEN R.) NEPPLE, t]~ELGA M., VALENTII,[, KATIIERINE A., HUFF, JESSE W.) AND WRIGHT, L . D . Observations on the use of Lactobacillus leichmannii 4797 in the Microbiological Assay of Vitamin B,.~. J. Biol. Chem., 184: 211-221. J950.
VITAMIN B]2 IN CALF BLOOD
753
(6) SUTTON, T. S., AND KAESER, I-I. E. The Riboflavin Content of Cows' Colostrum. J. Dairy Sci.. 30: 581-582. 1947. (7) SUTTON, T. S., WARNER, R. G., AND KAESER, H. ~]. The Concentration and Output of Carotenoid Pigments, Vitamin A, and Riboflavin in the Colostrum and Milk of Dairy Cows. J. Dairy Sci., 30: 927-932. 1947. (8) THEOPHILUS, D. R., AND STAI~[BERG, 0. E. The Influence of Breed, Feed, and Processing on the Riboflavin Content of Milk. J. Dairy Sci., 28: 259-268. 1945. (9) WI-IITNAI-I, C. H., KUNEKTI-I, B. L., AND KRAMEK, M . M . Riboflavin Content of Milk Collected in Different Months, and Correlated with Other Constituents of the Milk. J. Dairy Sei., 21: 593-600. 1938.