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Influence of Diet Composition on the Response of Chicks to Vitamin B12
892
D . C. H I L L AND H . D .
SUMMARY Vitamin D is required for phosphorus absorption from the digestive tract. Calcium p a n t o t h e n a t e , niacin, paraamino benzoic acid, pyridoxine, vitamin B12 a n d folic acid play a role in bone mineralization. REFERENCES
Driggers, J. C, R. L. Sherley, G. K. Davis and N. R. Mehrhof, 1951. The transference of radioactive calcium and phosphorus from hen to chick. Poultry Sci. 30: 199-204. Hubbel, R. B., L. B. Mendel and A. J. Wakeman, 1937. A new salt mixture for use in experimental data. J. Nutrition 14:273-285. O'Neil, J. B., J. R. Jowsey, C. C. Lee, M. A. Reade and J. W. T. Spinks, 1948. Determination of the fate of phosphorus in the laying hen by means of radio-phosphorus (P-32). Science 107: 295-196. Scott, M. L., 1951. Studies on the enlarged hock disorder in turkeys. 2. Factor affecting the excretion and retention of creatinine by young poults. Poultry Sci. 30: 839-845. Sherley, R. L., J. C. Driggers, J. McCall, G. K. Davis and N. R. Mehrhof, 1951. Excretion and retention of P-32 and Ca-45 by laying hens. Poultry Sci. 30: 730-734.
Influence of Diet Composition on the Response of Chicks to Vitamin B12 D . C. H I L L AND H . D .
BRANION
Department of Nutrition, Ontario Agricultural College, Guelph, Canada (Received for publication March 13, 1952)
TV T A N Y investigators have found t h a t *•*•*• a high level of soybean oil meal in the diet tends to increase the growth response of chicks to vitamin B 12 supplementation. I t has been common practice, therefore, in chick assays for vitamin B 12 to use a basal diet containing 60 to 70 percent of soybean oil meal. Such diets have been used by Stokstad et al. (1949), Lillie et al. (1948) and Peeler et al. (1951) as well as by others. H a r t m a n et al. (1949) have shown t h a t increasing the protein level in the diet of r a t s increases the response to vitamin B , r Such observations have led to the belief t h a t vitamin B 12 , in a t least one of its functions, is intimately concerned with the metabolism of protein.
On the other hand, Chow and Barrows (1950), using rats as experimental animals, found evidence t h a t vitamin B 12 plays an important role in carbohydrate or fat metabolism, and t h a t nitrogen retention on vitamin B 12 deficient diets was not improved b y supplementation with vitamin B 12 . Interest in this problem led us to conduct an experiment in B 12 supplementation wherein the protein level of the diet was raised while an a t t e m p t was made to avoid altering the productive energy of the diet. T h e two basal diets are shown in Table 1. T h e protein content of basal no. 2 was approximately 29 percent. This level of protein was obtained b y including in the
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Association of Official Agricultural Chemists, 1945. Official and Tentative Methods of Analysis: 625626. Branson, H., H. W. Banks, Jr. and L. B. Dodson, 1947. The effects of vitamins on phosphorus metabolism in the chick embryo. 1. Vitamin D and the utilization of inorganic phosphorus. Science 106: 637-638.
BRANION
D I E T COMPOSITION AND VITAMIN B I 2
893
TABLE 1.—Basal diets used infirstexperiment
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5 percent. T h e calculated productive energy contents of Basal no. 1 a n d Basal Basal Basal Ingredient no. 2 were 82.6 Therms and 83.0 Therms No. 2 No. 1 per 100 lbs. diet respectively. lb. lb. Day-old female New Hampshire chicks Ground yellow corn 27.50 — Ground oats 12.50 — obtained from a commercial hatchery Rolled oat groats 6.50 26.50 were used in this experiment. T h e experiGround barley6.00 — Ground wheat 15.00 12.00 ment was replicated 4 times. T e n birds Dehydrated alfalfa meal 3.00 3.00 were allotted a t random to a group a n d Soybean oil meal (solvent) 26.00 50.00 Corn oil 5.00 each group was confined to a single pen. — Salt (iodized) .60 .60 There were thus 32 pens in this experiGround limestone 1.00 1.00 Ca 3 (P0 4 ) 2 1.50 1.50 ment. Fortified fish oil (2,400AThe vitamin B 1 2 supplements used in 400D) .24 .24 DL-methionine .20 .20 this experiment were A P F no. 3 a n d A P F MnS0 4 -4H 2 0 9.00 gm. 9.00 i no. 8 supplied b y Merck & Co. L t d . a n d Choline chloride (25% concentrate) 85.60 gm. 85.60 s said to contain 12.5 mg. of vitamin B 12 Riboflavin concentrate (1 per lb. A P F no. 3 does not contain an antibiotic. A P F no. 8 contains 2.5 mg. Percent protein 20.3 29.1 procaine penicillin per lb. T h e diets were Productive energy per 100 lb. in therms 82.6 83.0 fed ad libitum. The diets and results are given in Table TABLE 2.—Diets and results. First experiment 2. The d a t a contain several interesting Mean Mean points. I t appears t h a t with both types of weight weight 4 weeks 39 days basal diets the requirement for supplemental B 1 2 was n o t greater t h a n 10 Supplement to basal No. 1 {26% S.B.O.M. with no extra corn oil) gamma per kilo of diet. N o greater growth gm. gm. was obtained by the addition of 30 gamma 1 None 251 386 2 107Bi2 per kilo (APF-3)1 258 436 than with 10 gamma. More recent work 3 30-yB12 per kilo (APF-3)2 264 426 in our laboratory under conditions which 4 30YB 12 per kilo (APF-8) 281 453 might be expected to increase the B 12 Supplement to basal No. 2 requirement over t h a t in the present ex(50% S.B.O.M. plus 5% corn oil) 5 None 235 346 periment showed t h a t a level not exceed6 10TB, 2 per kilo (APF-3)1 305 484 ing 6 gamma was adequate. I t is also 7 30YB12 per kilo (APF-3) 2 295 487 8 3O7B12 per kilo (APF-8) 312 480 worth noting t h a t penicillin stimulated growth with basal no. 1 b u t was ineffec1 Donated by Merck & Co. Ltd., Montreal, contive with basal no. 2. A possible explanatains 12.5 mg. Bi per lb. 2 2 Donated by Merck & Co. Ltd., Montreal, con- tion of this result might be an effect of the tains 12.5 mg. B12 per lb. plus 2.5 mg. procaine pencorn oil on the microflora. I t has been icillin per lb. shown b y N a t h et al. (1948) t h a t high diet 50 percent of soybean oil meal. T h e corn oil diets fed to rats decrease the numproductive energy as calculated b y t h e ber of coliform organisms. method of Fraps (1946), was kept approxiHowever, the most interesting observamately t h e same in t h e t w o basal diets b y tion t o be made from t h e d a t a is the supereplacing the oats and barley with o a t rior mean weights of the birds receiving groats and adding corn oil t o the level of the high protein basal plus B 1 2 supple-
894
D. C. HILL AND H. D. BRANION
TABLE 3.—Basal diet used in secpnd experiment Lb.
Ingredient
27.50 Ground yellow corn 5.00 Ground oats 6.50 Ground oat groats 29.50 Ground wheat 2.00 Dehydrated alfalfa meal 26.00 Soybean oil meal (solvent) 0.60 Salt (iodized) 1.00 CaC0 3 1.50 Ca 3 (P0 4 ) i! .20 DL-methionine .24 Fortified fish oil (2.400A-400D) 9.00 ( MnS0 4 -4H 2 0 85.60 ( Choline chloride (25% concentrate) 2.84 j Riboflavin concentrate (1 gm. per oz.) Productive energy per 100 lb. in therms 86.9
TABLE 4.—Diets and results. Second experiment Supplement to basal
content
(ivxs.2s) 1 2 3 4 5 6
None 30-y B,. per kilo' 10 percent Na proteinate* 10 percent Na proteinate+30y Bu per kilo 0.1% DL-methionine+10 percent Naraoteinate 0.1% Di-methionine+10 percent Na proteinate+30y Bu per kilo
percent 21.2 22.1 29.0 28.1
9 ?
2 5 w e; ks
gm. 241 275 196 249
28.6
186
28.5
262
i Crystalline BH supplied by Merck & Co. Ltd. as a 0.1% NaCl triturate. 2 Soybean protein product manufactured by Archer Daniels Midland Co., Minneapolis, Minn. Replaced an equivalent weight of corn.
The experimental diets and the results are presented in Table 4. The supplemental protein used was sodium proteinate (an isolated soybean protein manufactured by Archer Daniels Midland Co.). Ten percent of this protein was added, replacing an equivalent weight of corn. Vitamin B, 2 was added in the form of crystalline B12 rather than APF-3 as in the previous experiment. I t is evident that supplementation with B12 consistently produced a growth response. These responses were statistically significant. The experimental error was very low in this experiment, a fact which may be attributed to the. equalization of the starting weights of the 24 experimental groups. Increasing the protein level of the diet
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ments. Statistically the comparison, Diets 2, 3, 4 vs. Diets 6, 7, 8 was highly significant. The relatively poor growth on the unsupplemented high protein basal was not unexpected in view of literature reports. Ignoring the diets 4 and 8 containing the antibiotic, the maximum responses obtained on basal no. 1 and basal no. 2 by addition of B12 were 50 and 141 gm. respectively, approximately a 3-fold difference in response. The greater mean weights and the greater response to B12 obtained with basal no. 2 might reasonably be attributed either to the presence of large amounts of soybean oil meal in the diet or to the added corn oil or to a combination of these factors. In a second experiment it was decided to try the effect of increasing the level of soybean protein rather than soybean oil meal on the response to B12. Corn oil was not added in this experiment. The basal ration used in this experiment is given in Table 3. I t is similar to basal no. 1 used in the previous experiment, although it was somewhat higher in energy, containing a smaller proportion of oats and no barley. The birds used were New Hampshire males obtained from a commercial hatchery. In order to deplete to some degree their B12 carry-over, they were fed two weeks on the basal diet. At the end of this period they were divided into groups on the basis of weight and assigned to the various experimental groups from within each weight group. In this way each experimental groups started the experimental period with approximately the same mean weight. The experiment was replicated 4 times, each group kept in a single pen, 10 birds to a pen and the diets were assigned to the groups at random. The experiment was terminated after the experimental diets has been fed for 3 weeks.
D I E T COMPOSITION AND VITAMIN BI 2
(diet 3) produced a marked reduction in the mean weight and increased the response to B„ supplement over that obtained in diets 1 and 2 in the ratio of 53 to 34. The mean weight on diet 4, however, did not reach that of diet 2. In diets 5 and 6 an additional 0.1 percent DL-methionine was added over the 0.2 percent already present in the
895
methionine appeared to depress growth slightly. Moreover, the additional methionine, instead of sparing B12, seemed to give an increased response to supplemental B12. The smaller response to B12 on the "high" protein diet obtained in experiment no. 2, and the failure to obtain superior growth as previously observed, led
TABLE 5.—Diets and restdts. Thtrd experiment Diet No.
Supplement to basal
Protein (NX6.25)
Mean weight at 5 weeks
None
iercent 21.1
gm. 374
2 3
3O7B12 per kilo 5 percent corn oil
21.6 21.0
426 399
4 5
5 percent corn oil+30-yBi2 per kilo 10 percent Na proteinate
21.0 27.8
433 351
6 7
10 percent Na proteinate+30YBi 2 per kilo 10 percent Na proteinate-j-5 percent corn oil
28.5 28.7
412 309
8
10 percent Na proteinate+5 percent corn oil +3O7B12 per kilo
28.4
gm. 52 34 61 137
446
Factorial analysis of diet means Effect (a) Bi 2
(b) (c) (d) (e) (f) (g) 1 8
Corn oil Protein level B12XCOTO oil Bi2Xprotein level Corn oil X protein level Bi2Xcorn oilXprotein level s 2 (df=16) =
Mean square 30,360* 228 4.891 1 1,233 4,7432 614 3,309' 398
Significant at 5 % point. Significant at 1% point.
basal diet. These two diets were included in view of the reports by Almquist (1949) that the methionine requirement of the chick is increased when the protein level of the diet is increased. A sparing action between B12 and methionine has also been reported (Schaefer et al., 1944; Gillis and Norris, 1949) and it was considered that part of the B12 effect might be explained on this basis. No evidence of an increased requirement for methionine was obtained with these diets, rather the additional
to the belief that corn oil might be in some way concerned. This prompted a third experiment in which combinations of high protein, B l 2 and corn oil were studied. The experiment was designed as a 23 factorial. Light Sussex X New Hampshire female chicks obtained from a commercial hatchery' were used. The experiment was replicated 3 times. Each experimental group consisted of 12 birds confined in a single pen, and the assignment of the
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1
Response to Bi2 supplementation
896
D. C. HILL AND H. D. BRANION
B12, corn oil and protein level. This interaction may be interpreted that the change in response to B12 supplementation when the protein level of the diet was altered depended on the presence or absence of the 5 percent of corn oil. The findings in experiment 3, therefore, support the conclusion that the high response to B12 supplementation, and the superior weight achieved by the birds fed the B12 supplemented diets in Experiment 1, was due to the combined effect of elevated levels of protein and corn oil. The extra growth effects obtained with such diets over a B12 supplemented "low" protein diet seem to be due chiefly to an independent effect of the added oil. Since in Experiment 1 the productive energy of the two basal diets was closely similar it seems unlikely that this effect of oil could be due to extra calories added. The corn oil in the presence of an increased protein level without supplemental B12 definitely depressed growth. This effect could be explained on the basis that in some manner the corn oil in the presence of the increased protein level speeded up depletion of the B12 which had been carried over from the egg. SUMMARY The addition of corn oil and extra protein to a diet deficient in vitamin B12 was found to influence the growth response of chicks to dietary supplements of the vitamin. The addition of 5 percent of corn oil to a diet containing approximately 20 percent protein did not increase the response and only a moderate increase occurred when the protein level was raised to approximately 30 percent with no added corn oil. However, a greater than two-fold response was observed when both extra corn oil and protein were included in the diet. This effect was partly due to a marked depression in growth
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birds to the pens and the pens to the diet was random. The basal diet was the same as that used in the preceding experiment. The birds were fed the experimental diets for 5 weeks. Diets and mean weights at 5 weeks are given in Table 5. Vitamin B12 was added as crystalline B l 2 and the sodium proteinate and corn oil replaced an equivalent weight of corn. Again a definite response to B I2 was obtained in all cases of supplementation. Addition of corn oil alone to the basal also stimulated growth. However, the effects of corn oil and B12 were not entirely additive since diet 4 with added corn oil and B12 gave a mean weight only slightly superior to that of diet 2 with only B12 added. As in the preceding experiment the addition of sodium proteinate depressed growth and a somewhat greater response was obtained from B12 supplementation of the "high" protein diet than the "low" protein diet. The lowest mean 5 week weight was obtained on diet 7 which contained both 5 percent of corn oil and the additional protein. The addition of the corn oil to the diet containing 10 percent sodium proteinate decreased the mean weight from 351 gm. to 309 gm. Statistically this difference was significant with P = 0.011 (diets 5 and 7). The mean weight for diet 8, which contained added B12, corn oil and sodium proteinate was greatest of the 8 diets although not statistically greater than that for diet 2. The comparison of the mean weights obtained with diets 7 and 8 resulted in the high response to B12 supplementation of 137 gms., over 2\ times the response obtained with diets 1 and 2. The factorial analysis of the diet means is given below the table. The chief point of interest is the effect (g), the second order interaction among
NEWS AND NOTES when the vitamin
B12 deficient
"high"
c o r n o i l — " h i g h " p r o t e i n d i e t w a s fed. REFERENCES
Lillie, R. J., C. A. Denton and H. R. Bird, 1949. Assay of feedstuffs and concentrates for vitamin B12 potency by a chick growth method. Poultry Sci. 28: 772. Nath, H., V. H. Barki, W. B. Sarles and C. A. Elvehjem, 1948. Micro-organisms in the cecal contents of rats fed various carbohydrates and fats. J. Bact. 56: 783-793. Peeler, H. T., H. Yacowitz, C. W. Carlson, R. F. Miller, L. C. Norris and G. F. Heuser, 1951. Studies on the B12 content of feedstuffs and other materials. J. Nutrition, 43: 49-61. Schaefer, A. E., W. D. Salmon and D. R. Strength, 1949. Interrelationship of vitamin B12 and choline. I. Effect on hemorrhagic kidney syndrome in the rat. Proc. Soc. Expt. Biol. Med. 71: 193-196. Stokstad, E. L.1 R., T. H. Jukes, J. Pierce, A. C. Page, Jr. and A. L. Franklin, 1949. The multiple nature of the animal protein factor. J. Biol. Chem. 180: 646-654.
NEWS AND NOTES (Continued from page 887) Director. Dr. J. H. Quisenberry, Head of the Poultry Department was master of ceremonies. President M. T. Harrington presented the new plant to the Texas poultry industry. BRITISH COLUMBIA NOTES Professor E. A. Lloyd recently retired as Head of the Department of Poultry Husbandry, University of British Columbia, Vancouver, B. C , Canada, after 32 years of service. He is succeeded by J. Biely. Professor Lloyd was honoured at a banquet on June 28, at which he was presented with a leather bound volume, of letters of tribute from some 60 of his former students, and a new car. He has been named Professor Emeritus. He was born in Aurora, Ontario in 1887, obtained a B.S.A. degree from the University of Saskatchewan in 1917 and a M.S.A. degree from the State College of Washington in 1926. He was made a Fellow of the Poultry Science Association in 1950. He taught in primary and secondary schools in Ontario and Saskatchewan for seven years. In 1917 he joined the extension service of the University of Saskatchewan and in 1918 and 1919 was Editor of the Grain Growers' Guide. From 1919 to 1920 he was
Instructor at the Soldiers' Civil Re-establishment School at the University of British Columbia. He was appointed Assistant Professor and Acting Head of the Department of Poultry Husbandry in 1920 and in 1923 was made Head. Professor Lloyd was responsible for the introduction of the Japanese method of chick sexing in this continent. He has been interested in all phases of the poultry industry, especially in breeding. In 19251926 his White Leghorns made a worlds' record in the laying contests. His work has also resulted in the development of two autosexing breeds, the Redbars and Hampbars. Jacob Biely was born in Bogopol, Russia in 1903. He obtained his B.S.A. degree from the University of British Columbia in 1926, a M.S. degree from Kansas State College in 1929, and a M.S.A. degree from the University of British Columbia in 1930. He joined the staff of the Department of Poultry Husbandry, University of British Columbia in 1926, becoming Associate Professor in 1944. He has been primarily concerned in nutrition. He is a member of the Poultry Science Association, Agricultural Institute of Canada, World's Poultry Science Association, American Association for the Advancement of Science and the Animal Nutrition Research Council.
(Continued on page 904)
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Almquist, H. J., 1949. Amino acid balance at supernormal dietary levels. Proc. Soc. Expt. Biol. Med. 72:179-180. Chow, B. F., and L. Barrows, 1950. Role of B12 on nitrogen retention of rats fed on soybean protein diets at different caloric levels. Fed. Proc. 9: part 1,354. Fraps, G. S., 1946. Composition and productive energy of poultry feeds and rations. Texas Agr, Expt. Sta. Bui. 678. Gifts, M. B., and L. C. Norris, 1949. Vitamin B12 and the requirement of the chick for methylating compounds. Poultry Sci. 28: 749-750. Hartman, A. M., L. P. Dryden and C. A. Cary, 1949. A role of vitamin B t 2 in the normal mammal. Arch. Biochem. 23: 165-168.
897
D . C. H I L L AND H . D .
SUMMARY Vitamin D is required for phosphorus absorption from the digestive tract. Calcium p a n t o t h e n a t e , niacin, paraamino benzoic acid, pyridoxine, vitamin B12 a n d folic acid play a role in bone mineralization. REFERENCES
Driggers, J. C, R. L. Sherley, G. K. Davis and N. R. Mehrhof, 1951. The transference of radioactive calcium and phosphorus from hen to chick. Poultry Sci. 30: 199-204. Hubbel, R. B., L. B. Mendel and A. J. Wakeman, 1937. A new salt mixture for use in experimental data. J. Nutrition 14:273-285. O'Neil, J. B., J. R. Jowsey, C. C. Lee, M. A. Reade and J. W. T. Spinks, 1948. Determination of the fate of phosphorus in the laying hen by means of radio-phosphorus (P-32). Science 107: 295-196. Scott, M. L., 1951. Studies on the enlarged hock disorder in turkeys. 2. Factor affecting the excretion and retention of creatinine by young poults. Poultry Sci. 30: 839-845. Sherley, R. L., J. C. Driggers, J. McCall, G. K. Davis and N. R. Mehrhof, 1951. Excretion and retention of P-32 and Ca-45 by laying hens. Poultry Sci. 30: 730-734.
Influence of Diet Composition on the Response of Chicks to Vitamin B12 D . C. H I L L AND H . D .
BRANION
Department of Nutrition, Ontario Agricultural College, Guelph, Canada (Received for publication March 13, 1952)
TV T A N Y investigators have found t h a t *•*•*• a high level of soybean oil meal in the diet tends to increase the growth response of chicks to vitamin B 12 supplementation. I t has been common practice, therefore, in chick assays for vitamin B 12 to use a basal diet containing 60 to 70 percent of soybean oil meal. Such diets have been used by Stokstad et al. (1949), Lillie et al. (1948) and Peeler et al. (1951) as well as by others. H a r t m a n et al. (1949) have shown t h a t increasing the protein level in the diet of r a t s increases the response to vitamin B , r Such observations have led to the belief t h a t vitamin B 12 , in a t least one of its functions, is intimately concerned with the metabolism of protein.
On the other hand, Chow and Barrows (1950), using rats as experimental animals, found evidence t h a t vitamin B 12 plays an important role in carbohydrate or fat metabolism, and t h a t nitrogen retention on vitamin B 12 deficient diets was not improved b y supplementation with vitamin B 12 . Interest in this problem led us to conduct an experiment in B 12 supplementation wherein the protein level of the diet was raised while an a t t e m p t was made to avoid altering the productive energy of the diet. T h e two basal diets are shown in Table 1. T h e protein content of basal no. 2 was approximately 29 percent. This level of protein was obtained b y including in the
Downloaded from http://ps.oxfordjournals.org/ at Seton Hall on April 27, 2015
Association of Official Agricultural Chemists, 1945. Official and Tentative Methods of Analysis: 625626. Branson, H., H. W. Banks, Jr. and L. B. Dodson, 1947. The effects of vitamins on phosphorus metabolism in the chick embryo. 1. Vitamin D and the utilization of inorganic phosphorus. Science 106: 637-638.
BRANION
D I E T COMPOSITION AND VITAMIN B I 2
893
TABLE 1.—Basal diets used infirstexperiment
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5 percent. T h e calculated productive energy contents of Basal no. 1 a n d Basal Basal Basal Ingredient no. 2 were 82.6 Therms and 83.0 Therms No. 2 No. 1 per 100 lbs. diet respectively. lb. lb. Day-old female New Hampshire chicks Ground yellow corn 27.50 — Ground oats 12.50 — obtained from a commercial hatchery Rolled oat groats 6.50 26.50 were used in this experiment. T h e experiGround barley6.00 — Ground wheat 15.00 12.00 ment was replicated 4 times. T e n birds Dehydrated alfalfa meal 3.00 3.00 were allotted a t random to a group a n d Soybean oil meal (solvent) 26.00 50.00 Corn oil 5.00 each group was confined to a single pen. — Salt (iodized) .60 .60 There were thus 32 pens in this experiGround limestone 1.00 1.00 Ca 3 (P0 4 ) 2 1.50 1.50 ment. Fortified fish oil (2,400AThe vitamin B 1 2 supplements used in 400D) .24 .24 DL-methionine .20 .20 this experiment were A P F no. 3 a n d A P F MnS0 4 -4H 2 0 9.00 gm. 9.00 i no. 8 supplied b y Merck & Co. L t d . a n d Choline chloride (25% concentrate) 85.60 gm. 85.60 s said to contain 12.5 mg. of vitamin B 12 Riboflavin concentrate (1 per lb. A P F no. 3 does not contain an antibiotic. A P F no. 8 contains 2.5 mg. Percent protein 20.3 29.1 procaine penicillin per lb. T h e diets were Productive energy per 100 lb. in therms 82.6 83.0 fed ad libitum. The diets and results are given in Table TABLE 2.—Diets and results. First experiment 2. The d a t a contain several interesting Mean Mean points. I t appears t h a t with both types of weight weight 4 weeks 39 days basal diets the requirement for supplemental B 1 2 was n o t greater t h a n 10 Supplement to basal No. 1 {26% S.B.O.M. with no extra corn oil) gamma per kilo of diet. N o greater growth gm. gm. was obtained by the addition of 30 gamma 1 None 251 386 2 107Bi2 per kilo (APF-3)1 258 436 than with 10 gamma. More recent work 3 30-yB12 per kilo (APF-3)2 264 426 in our laboratory under conditions which 4 30YB 12 per kilo (APF-8) 281 453 might be expected to increase the B 12 Supplement to basal No. 2 requirement over t h a t in the present ex(50% S.B.O.M. plus 5% corn oil) 5 None 235 346 periment showed t h a t a level not exceed6 10TB, 2 per kilo (APF-3)1 305 484 ing 6 gamma was adequate. I t is also 7 30YB12 per kilo (APF-3) 2 295 487 8 3O7B12 per kilo (APF-8) 312 480 worth noting t h a t penicillin stimulated growth with basal no. 1 b u t was ineffec1 Donated by Merck & Co. Ltd., Montreal, contive with basal no. 2. A possible explanatains 12.5 mg. Bi per lb. 2 2 Donated by Merck & Co. Ltd., Montreal, con- tion of this result might be an effect of the tains 12.5 mg. B12 per lb. plus 2.5 mg. procaine pencorn oil on the microflora. I t has been icillin per lb. shown b y N a t h et al. (1948) t h a t high diet 50 percent of soybean oil meal. T h e corn oil diets fed to rats decrease the numproductive energy as calculated b y t h e ber of coliform organisms. method of Fraps (1946), was kept approxiHowever, the most interesting observamately t h e same in t h e t w o basal diets b y tion t o be made from t h e d a t a is the supereplacing the oats and barley with o a t rior mean weights of the birds receiving groats and adding corn oil t o the level of the high protein basal plus B 1 2 supple-
894
D. C. HILL AND H. D. BRANION
TABLE 3.—Basal diet used in secpnd experiment Lb.
Ingredient
27.50 Ground yellow corn 5.00 Ground oats 6.50 Ground oat groats 29.50 Ground wheat 2.00 Dehydrated alfalfa meal 26.00 Soybean oil meal (solvent) 0.60 Salt (iodized) 1.00 CaC0 3 1.50 Ca 3 (P0 4 ) i! .20 DL-methionine .24 Fortified fish oil (2.400A-400D) 9.00 ( MnS0 4 -4H 2 0 85.60 ( Choline chloride (25% concentrate) 2.84 j Riboflavin concentrate (1 gm. per oz.) Productive energy per 100 lb. in therms 86.9
TABLE 4.—Diets and results. Second experiment Supplement to basal
content
(ivxs.2s) 1 2 3 4 5 6
None 30-y B,. per kilo' 10 percent Na proteinate* 10 percent Na proteinate+30y Bu per kilo 0.1% DL-methionine+10 percent Naraoteinate 0.1% Di-methionine+10 percent Na proteinate+30y Bu per kilo
percent 21.2 22.1 29.0 28.1
9 ?
2 5 w e; ks
gm. 241 275 196 249
28.6
186
28.5
262
i Crystalline BH supplied by Merck & Co. Ltd. as a 0.1% NaCl triturate. 2 Soybean protein product manufactured by Archer Daniels Midland Co., Minneapolis, Minn. Replaced an equivalent weight of corn.
The experimental diets and the results are presented in Table 4. The supplemental protein used was sodium proteinate (an isolated soybean protein manufactured by Archer Daniels Midland Co.). Ten percent of this protein was added, replacing an equivalent weight of corn. Vitamin B, 2 was added in the form of crystalline B12 rather than APF-3 as in the previous experiment. I t is evident that supplementation with B12 consistently produced a growth response. These responses were statistically significant. The experimental error was very low in this experiment, a fact which may be attributed to the. equalization of the starting weights of the 24 experimental groups. Increasing the protein level of the diet
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ments. Statistically the comparison, Diets 2, 3, 4 vs. Diets 6, 7, 8 was highly significant. The relatively poor growth on the unsupplemented high protein basal was not unexpected in view of literature reports. Ignoring the diets 4 and 8 containing the antibiotic, the maximum responses obtained on basal no. 1 and basal no. 2 by addition of B12 were 50 and 141 gm. respectively, approximately a 3-fold difference in response. The greater mean weights and the greater response to B12 obtained with basal no. 2 might reasonably be attributed either to the presence of large amounts of soybean oil meal in the diet or to the added corn oil or to a combination of these factors. In a second experiment it was decided to try the effect of increasing the level of soybean protein rather than soybean oil meal on the response to B12. Corn oil was not added in this experiment. The basal ration used in this experiment is given in Table 3. I t is similar to basal no. 1 used in the previous experiment, although it was somewhat higher in energy, containing a smaller proportion of oats and no barley. The birds used were New Hampshire males obtained from a commercial hatchery. In order to deplete to some degree their B12 carry-over, they were fed two weeks on the basal diet. At the end of this period they were divided into groups on the basis of weight and assigned to the various experimental groups from within each weight group. In this way each experimental groups started the experimental period with approximately the same mean weight. The experiment was replicated 4 times, each group kept in a single pen, 10 birds to a pen and the diets were assigned to the groups at random. The experiment was terminated after the experimental diets has been fed for 3 weeks.
D I E T COMPOSITION AND VITAMIN BI 2
(diet 3) produced a marked reduction in the mean weight and increased the response to B„ supplement over that obtained in diets 1 and 2 in the ratio of 53 to 34. The mean weight on diet 4, however, did not reach that of diet 2. In diets 5 and 6 an additional 0.1 percent DL-methionine was added over the 0.2 percent already present in the
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methionine appeared to depress growth slightly. Moreover, the additional methionine, instead of sparing B12, seemed to give an increased response to supplemental B12. The smaller response to B12 on the "high" protein diet obtained in experiment no. 2, and the failure to obtain superior growth as previously observed, led
TABLE 5.—Diets and restdts. Thtrd experiment Diet No.
Supplement to basal
Protein (NX6.25)
Mean weight at 5 weeks
None
iercent 21.1
gm. 374
2 3
3O7B12 per kilo 5 percent corn oil
21.6 21.0
426 399
4 5
5 percent corn oil+30-yBi2 per kilo 10 percent Na proteinate
21.0 27.8
433 351
6 7
10 percent Na proteinate+30YBi 2 per kilo 10 percent Na proteinate-j-5 percent corn oil
28.5 28.7
412 309
8
10 percent Na proteinate+5 percent corn oil +3O7B12 per kilo
28.4
gm. 52 34 61 137
446
Factorial analysis of diet means Effect (a) Bi 2
(b) (c) (d) (e) (f) (g) 1 8
Corn oil Protein level B12XCOTO oil Bi2Xprotein level Corn oil X protein level Bi2Xcorn oilXprotein level s 2 (df=16) =
Mean square 30,360* 228 4.891 1 1,233 4,7432 614 3,309' 398
Significant at 5 % point. Significant at 1% point.
basal diet. These two diets were included in view of the reports by Almquist (1949) that the methionine requirement of the chick is increased when the protein level of the diet is increased. A sparing action between B12 and methionine has also been reported (Schaefer et al., 1944; Gillis and Norris, 1949) and it was considered that part of the B12 effect might be explained on this basis. No evidence of an increased requirement for methionine was obtained with these diets, rather the additional
to the belief that corn oil might be in some way concerned. This prompted a third experiment in which combinations of high protein, B l 2 and corn oil were studied. The experiment was designed as a 23 factorial. Light Sussex X New Hampshire female chicks obtained from a commercial hatchery' were used. The experiment was replicated 3 times. Each experimental group consisted of 12 birds confined in a single pen, and the assignment of the
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1
Response to Bi2 supplementation
896
D. C. HILL AND H. D. BRANION
B12, corn oil and protein level. This interaction may be interpreted that the change in response to B12 supplementation when the protein level of the diet was altered depended on the presence or absence of the 5 percent of corn oil. The findings in experiment 3, therefore, support the conclusion that the high response to B12 supplementation, and the superior weight achieved by the birds fed the B12 supplemented diets in Experiment 1, was due to the combined effect of elevated levels of protein and corn oil. The extra growth effects obtained with such diets over a B12 supplemented "low" protein diet seem to be due chiefly to an independent effect of the added oil. Since in Experiment 1 the productive energy of the two basal diets was closely similar it seems unlikely that this effect of oil could be due to extra calories added. The corn oil in the presence of an increased protein level without supplemental B12 definitely depressed growth. This effect could be explained on the basis that in some manner the corn oil in the presence of the increased protein level speeded up depletion of the B12 which had been carried over from the egg. SUMMARY The addition of corn oil and extra protein to a diet deficient in vitamin B12 was found to influence the growth response of chicks to dietary supplements of the vitamin. The addition of 5 percent of corn oil to a diet containing approximately 20 percent protein did not increase the response and only a moderate increase occurred when the protein level was raised to approximately 30 percent with no added corn oil. However, a greater than two-fold response was observed when both extra corn oil and protein were included in the diet. This effect was partly due to a marked depression in growth
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birds to the pens and the pens to the diet was random. The basal diet was the same as that used in the preceding experiment. The birds were fed the experimental diets for 5 weeks. Diets and mean weights at 5 weeks are given in Table 5. Vitamin B12 was added as crystalline B l 2 and the sodium proteinate and corn oil replaced an equivalent weight of corn. Again a definite response to B I2 was obtained in all cases of supplementation. Addition of corn oil alone to the basal also stimulated growth. However, the effects of corn oil and B12 were not entirely additive since diet 4 with added corn oil and B12 gave a mean weight only slightly superior to that of diet 2 with only B12 added. As in the preceding experiment the addition of sodium proteinate depressed growth and a somewhat greater response was obtained from B12 supplementation of the "high" protein diet than the "low" protein diet. The lowest mean 5 week weight was obtained on diet 7 which contained both 5 percent of corn oil and the additional protein. The addition of the corn oil to the diet containing 10 percent sodium proteinate decreased the mean weight from 351 gm. to 309 gm. Statistically this difference was significant with P = 0.011 (diets 5 and 7). The mean weight for diet 8, which contained added B12, corn oil and sodium proteinate was greatest of the 8 diets although not statistically greater than that for diet 2. The comparison of the mean weights obtained with diets 7 and 8 resulted in the high response to B12 supplementation of 137 gms., over 2\ times the response obtained with diets 1 and 2. The factorial analysis of the diet means is given below the table. The chief point of interest is the effect (g), the second order interaction among
NEWS AND NOTES when the vitamin
B12 deficient
"high"
c o r n o i l — " h i g h " p r o t e i n d i e t w a s fed. REFERENCES
Lillie, R. J., C. A. Denton and H. R. Bird, 1949. Assay of feedstuffs and concentrates for vitamin B12 potency by a chick growth method. Poultry Sci. 28: 772. Nath, H., V. H. Barki, W. B. Sarles and C. A. Elvehjem, 1948. Micro-organisms in the cecal contents of rats fed various carbohydrates and fats. J. Bact. 56: 783-793. Peeler, H. T., H. Yacowitz, C. W. Carlson, R. F. Miller, L. C. Norris and G. F. Heuser, 1951. Studies on the B12 content of feedstuffs and other materials. J. Nutrition, 43: 49-61. Schaefer, A. E., W. D. Salmon and D. R. Strength, 1949. Interrelationship of vitamin B12 and choline. I. Effect on hemorrhagic kidney syndrome in the rat. Proc. Soc. Expt. Biol. Med. 71: 193-196. Stokstad, E. L.1 R., T. H. Jukes, J. Pierce, A. C. Page, Jr. and A. L. Franklin, 1949. The multiple nature of the animal protein factor. J. Biol. Chem. 180: 646-654.
NEWS AND NOTES (Continued from page 887) Director. Dr. J. H. Quisenberry, Head of the Poultry Department was master of ceremonies. President M. T. Harrington presented the new plant to the Texas poultry industry. BRITISH COLUMBIA NOTES Professor E. A. Lloyd recently retired as Head of the Department of Poultry Husbandry, University of British Columbia, Vancouver, B. C , Canada, after 32 years of service. He is succeeded by J. Biely. Professor Lloyd was honoured at a banquet on June 28, at which he was presented with a leather bound volume, of letters of tribute from some 60 of his former students, and a new car. He has been named Professor Emeritus. He was born in Aurora, Ontario in 1887, obtained a B.S.A. degree from the University of Saskatchewan in 1917 and a M.S.A. degree from the State College of Washington in 1926. He was made a Fellow of the Poultry Science Association in 1950. He taught in primary and secondary schools in Ontario and Saskatchewan for seven years. In 1917 he joined the extension service of the University of Saskatchewan and in 1918 and 1919 was Editor of the Grain Growers' Guide. From 1919 to 1920 he was
Instructor at the Soldiers' Civil Re-establishment School at the University of British Columbia. He was appointed Assistant Professor and Acting Head of the Department of Poultry Husbandry in 1920 and in 1923 was made Head. Professor Lloyd was responsible for the introduction of the Japanese method of chick sexing in this continent. He has been interested in all phases of the poultry industry, especially in breeding. In 19251926 his White Leghorns made a worlds' record in the laying contests. His work has also resulted in the development of two autosexing breeds, the Redbars and Hampbars. Jacob Biely was born in Bogopol, Russia in 1903. He obtained his B.S.A. degree from the University of British Columbia in 1926, a M.S. degree from Kansas State College in 1929, and a M.S.A. degree from the University of British Columbia in 1930. He joined the staff of the Department of Poultry Husbandry, University of British Columbia in 1926, becoming Associate Professor in 1944. He has been primarily concerned in nutrition. He is a member of the Poultry Science Association, Agricultural Institute of Canada, World's Poultry Science Association, American Association for the Advancement of Science and the Animal Nutrition Research Council.
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Almquist, H. J., 1949. Amino acid balance at supernormal dietary levels. Proc. Soc. Expt. Biol. Med. 72:179-180. Chow, B. F., and L. Barrows, 1950. Role of B12 on nitrogen retention of rats fed on soybean protein diets at different caloric levels. Fed. Proc. 9: part 1,354. Fraps, G. S., 1946. Composition and productive energy of poultry feeds and rations. Texas Agr, Expt. Sta. Bui. 678. Gifts, M. B., and L. C. Norris, 1949. Vitamin B12 and the requirement of the chick for methylating compounds. Poultry Sci. 28: 749-750. Hartman, A. M., L. P. Dryden and C. A. Cary, 1949. A role of vitamin B t 2 in the normal mammal. Arch. Biochem. 23: 165-168.
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