CHICKS from dams fed balanced rations

The Variable Response of Normal Chicks to Stress Rations and a Vitamin Bi 2 Supplement* N. D. MAGRUDER, 1 J. K. BLETNER, 2 T. B. CLARK, 2 AND C. E. WEAKLEY, JR. 3 West Virginia Agricultural Experiment Station, Morgantown CHICKS from dams fed balanced rations containing adequate amounts of vitamin B12 have demonstrated consistently a desirable carry-over effect of the vitamin resulting in superior livability and growth. In many studies this effect should be eliminated when early chick growth is used in studies involving vitamin B12. Elimination of this effect has been accomplished by using chicks from dams on a vitamin B12 deficient ration and/or by using stress rations for chicks from normal dams. The term "stress ration" has been used either to indicate a ration that will deplete the chick more rapidly than a balanced ration deficient only in the vitamin studied or to indicate a ration that will increase the chick's requirement for the factor. Rubin and Bird (1947) and Bird et al. (1948) developed a high protein stress ration containing 70 percent soybean oil meal to be used in a short assay procedure for studying the growth factor in cow manure. Later this factor was identified as vitamin B12 (Ott et al., 1948; Lillie et al., 1948). A ration containing iodinated casein was used by Robblee et al. (1948) and Nichol et al. (1949) as a stress ration in studies involving unidentified growth factors and vitamin Bu. * Published with the approval of the Director of the West Virginia Agricultural Experiment Station as Scientific Paper No. 473. 1 Present address: Department of Animal Nutrition, Pennsylvania State College, State College, Pennsylvania. 2 Department of Animal Husbandry, Poultry Husbandry Section. 3 Department of Agricultural Biochemistry. (Received for publication August 24, 1953) The purpose of the experiments reported here was to compare the efficacy of the two types of stress rations used in vitamin B12 studies when the experimental chicks are from hens fed a balanced ration containing sources of vitamin B i2 and housed on unchanged litter. EXPERIMENTAL Four series of experiments were conducted using normal, day-old New Hampshire chicks. The chicks were obtained from this Station's pullet flock which had been fed a ration containing sources of vitamin B i2. These pullets were housed during August and September on new litter which was not removed from the house during the laying year. The chicks were TABLE 1. Percentage composition of basal experimental rations Ration No. 1 2 Ingredients Corn Soybean oil meal 1 Alfalfa meal Bonemeal Limestone MnS0 4 Fish liver oil 2 Fortified whey 3 Salt Niacin Total Calculated analysis: Crude protein Fat Crude fiber Percent 55.6 35.0 3.0 1.5 1.0 0.028 0.2 3.0 0.67 454.0 mg. 4 99.998 21.97 2.56 4.10 Percent 20.6 70.0 3.0 1.5 1.0 0.028 0.2 3.0 0.67 454.0 mg. 4 99.998 34.79 1.30 5.95 1 44 percent protein, solvent extracted. 2 3,000 I.U. vitamin A, 300 I.C. units vitamin D per gram. 3 Riboflavin, not less than 23 milligrams per pound (50 micrograms per gram). 4 Per 100 pounds of ration. 511

512 MAGRUDER, BLETNER, CLARK AND WEAKLEY, JR. TABLE 2. Outline of the procedures used in all series Treatment number Series 1 1 2 Series 2 3 4 Series 3 5 6 7 8 Series 4 9 10 11 12 Stress period Rations and supplement 0-2 weeks Ration 2 0-2 weeks plus I.C. 2 0-6 weeks Ration 2 Ration 2 plus Bn l Ration 2 plus PGA* Ration 2 plus B > plus PGA* 0-4 weeks plus I.C. S Ration 2 Ration 2 plus I.C. 2 Treatment number la lb 2a 2b 3a 3b 4a 4b Growth period Rations and supplement 3-6 weeks plus Bis 1 plus Bia 1 3-6 weeks plus Bis' plus Bi* J 1 22.7 grams Merck's APF Supplement No. 3 per 100 pounds ration. 2 45.4 grams iodinated casein per 100 pounds ration. 3 32.93 grams Merck's APF Supplement No. 3 per 100 pounds ration. 4 0.09 grams folic acid per 100 pounds ration. maintained in electrically heated battery brooders having raised wire floors. The experimental laboratory has been described by Bletner et al. (1953). The chicks were weighed individually at weekly intervals beginning at the end of the second week in series 1 and 2 and at the end of the first week in series 3 and 4. The experimental basal rations are listed in Table 1. The treatment numbers, rations and supplements are indicated in Table 2. The rations used in each series were prepared from the same supply of ingredients except for the corn. Series 1 Procedure. The purpose of this series was to compare the response of normal chicks to stress rations containing 35 and 70 percent soybean oil meal fed for two weeks and the subsequent response of the chicks to a 35 percent soybean oil meal ration containing a vitamin B12 supplement. In this series, January-hatched, day-old cockerel chicks were used. Approximately twice as many chicks were started as were used during the growth period. The chicks were divided into eight lots of 39 chicks each. Four of the lots were fed ration 1 during the two-week stress period. This was designated as treatment 1. The other four lots were fed ration 2 which was designated as treatment 2. A four-week growth period followed the stress period. The chicks selected for further study during this period were those whose weights were nearest the mean for their treatment. Eighty treatment 1 chicks were divided into eight lots having approximately the same average weight. Four of these lots were assigned to treatment la and continued on ration 1. The four remaining lots were assigned to treatment lb and fed ration 1 supplemented with 0.05 percent of Merck's APF Supplement No. 3, 4 a source of vitamin B12. The chicks from treatment 2 were handled exactly as those from treatment 1. During the growth period the chicks in treatment 2a received the same ration as those in treatment la, and those in treatment 2b the same ration as those in treatment lb. For the growth period, the four replicate lots in each treatment were assigned to the batteries by using a standard form for a four-by-four Latin Square Layout. Results At the end of the two-week stress period the chicks in treatment 1 averaged 131 grams while those in treatment 2 averaged 101 grams. The 70 percent soybean oil meal ration severely retarded the growth of the chicks in this series. The data obtained during the growth period are summarized in Table 3. Analysis of covariance was used to determine 4 Supplied by Merck & Co., Rahway, New Jersey, 12.5 mg. vitamin B12 activity per pound.

VARIABLE RESPONSE TO STRESS RATIONS AND VITAMIN BI 2 513 TABLE 3. Summary of the results of the growth- period for series 1 and 2 (Four lots per treatment) Gain in Adjusted1 Treat- i. Bgain in. weight,, ment?ji weight number veeks,.. 3-6 weeks Feedgain ratio Mortality (growth period) grams feed grams grams per gram percent of gain Series 1 la 442 411 2.56 2.5 lb 543 514 2.32 0.0 2a 399 429 2.59 0.0 2b 516 545 2.30 0.0 Series 2 3a 479 469 2.54 0.0 3b 539 528 2.44 0.0 4a 441 453 2.56 0.0 4b 501 511 2.42 0.0 1 Adjusted by covariance method. the significance of the differences in body weight gains. This appeared necessary because the two-week weights of the chicks in treatment la and lb differed greatly from those in treatment 2a and 2b. The effect of the vitamin B12 supplement was highly significant (1 percent level) when treatments lb and 2b were compared with treatments la and 2a, respectively. This highly significant response appeared as early as the end of the first week of the growth period for both lb and 2b treatments. The chicks in treatments lb and 2b utilized their feed more efficiently than the chicks in treatments la and 2a. The differences were highly significant. The mortality during the stress period was about 4 percent in both treatments 1 and 2. No specific lesions could be noted in the dead chicks from treatment 1 but toward the end of the second week several of the dead chicks from treatment 2 showed evidence of nutritional gout. Only one chick died during the four-week growth period. When the chicks were four weeks old (the end of the second week of the growth period) those in treatments 2a and 2b began to show an abnormal feather condition. In some chicks a white bar appeared about the middle of all primary and secondary wing feathers and the main tail feathers. In other chicks the area where the white bar would be expected was characterized by the absence of barbs although the ends of the feathers appeared normal. In still other chicks these feathers either broke off at the area of the white bar or failed to grow beyond it. This condition appeared chiefly in the chicks that had been fed the 70 percent soybean oil meal ration during the stress period and not until after the chicks had been on a 35 percent soybean oil meal ration for two weeks. Only three of the 80 chicks in treatments la and lb showed evidence of a white bar across the wing feathers while a chick with normal color and feather structure was the exception in treatments 2a and 2b. The vitamin B i2 supplement fed during the growth period had no effect on the occurrence of this feather abnormality. Figure 1 shows a typical chick from treatment 2a or 2b. Series 2 Procedure. The purpose of this series was to compare the response of normal FIG. 1. A chick with the abnormal feathering produced with the 70 percent soybean oil meal ration.

514 MAGRUDER, BLETNER, CLARK AND WEAKLEY, JR. chicks to 35 percent soybean oil meal rations with and without 0.1 percent iodinated casein 5 when fed for a two-week period and the subsequent response of the chicks to a vitamin B 12 supplement. Dayold cockerel chicks, hatched in early March, were used. The experimental design and procedure were identical with those of series 1. The treatment numbers, rations, and supplements are given in Table 2. Results The iodinated casein supplemented ration retarded the growth of the chicks during the stress period but not as severely as the 70 percent soybean oil meal ration of series 1. The average body weights of the chicks were 135 and 125 grams, respectively, for treatments 3 and 4 at the end of the two-week stress period. The data obtained during the growth period are summarized in Table 3. Analysis of covariance was used to determine the significance of the differences in body weight gains. The response to the vitamin B u supplement was highly significant at the end of the growth period. A significant response was not obtained until the end of the second week of the growth period one week later than the response occurred in series 1. The feed utilization followed a trend similar to that in series 1, but the differences were not statistically significant. The mortality during the stress period was about 0.6 percent for treatment 3 and about 7 percent for treatment 4. Autopsies revealed no specific lesions. The treatment 4 chicks died at the rate of about one each day beginning when they were two days old. There were no death losses during the growth-period. 6 Protamone, supplied by Cerophyl Laboratories, Inc., Kansas City, Mo. The analysis of covariance revealed a highly significant difference between the shelf levels of the battery stands. However, the design of the experiment was such that the effect of what may have been locational difference was removed in the analysis of the data and did not affect the interpretation of the results. Series 3 Procedure. The purpose of series 3 was to study the abnormal feather condition which appeared in the chicks in treatments 2a and 2b in series 1. Late Aprilhatched cockerel chicks were used. Since this was not a critical growth study no attempt was made to equalize lots according to body weights. The chicks were separated into eight lots of 15 or 16 chicks each. The treatment numbers, rations, and supplements are given in Table 2. All experimental rations were fed for a sixweek period beginning when the chicks were one day old. Results The feathering condition noted in series 1 appeared at the end of the fourth week in all lots of chicks in this series. There was no evidence that any of the supplements had any effect on the occurrence of the condition. There was but little change in the severity or the occurrence of the abnormality between the end of the fourth week and the end of the sixth week. In this series the abnormality was not as severe as it was in series 1. Even though this was not a critical growth study, data were obtained on body weights, feed efficiency, and mortality (Table 4). The chicks fed the vitamin Bi 2 supplemented rations were slightly larger than the other chicks at the end of the third week, but it was not until the end of the fourth week that the differences were large enough to be significant (5 percent

VARIABLE RESPONSE TO STRESS RATIONS AND VITAMIN BI 2 515 TABLE 4.- -Summary of the results of series 3 and 4 Treatment number Body weight Feed-gain ratio Mortality grams feed grams per gram percent of gain Series 3 (Av. 2 lots per treatments to 6 weeks of age) 5 506 2.82 6.S 6 591 2.62 0.0 7 494 2.95 6.5 8 628 2.69 0.0 Series 4 (Av. 4 lots per treatment to 4 weeks of age) 9 318 2.24 1.6 10 311 2.60 6.7 11 290 2.65 1.7 12 245 4.44 17.2 level). The effect of the vitamin B12 supplement on feed efficiency was significant. Mortality was extremely low. Two chicks died during the first week and two during the sixth week. Series 4 Procedure. The purpose of series 4 was to study further the effect of iodinated casein by adding it to both the 35 and 70 percent soybean oil meal basal rations. June-hatched chicks of mixed sexes were used. The chicks were divided into 16 lots of from 14 to 17 chicks each. The treatment numbers, rations, and supplements are given in Table 2. All rations were fed for a four-week period beginning when the chicks were day-old. The assignment of treatments to the batteries was made by using a standard form for a four-byfour Latin Square Layout. Results The results of this series are summarized in Table 4. The growth retarding effect of iodinated casein in treatment 10 was highly significant when the chicks were two weeks old but non-significant when they were four weeks old. The difference in body weight between the chicks in treatments 9 and 11 increased throughout the experimental period but was, at the end of the experiment, barely significant at the 5 percent level. The iodinated casein in the 70 percent soybean oil meal ration (treatment 12) severely retarded growth throughout the experimental period. This effect was highly significant as early as the end of the first week of the experiment. Iodinated casein significantly reduced feed efficiency only in treatment 12. The mortality was highest in treatments 10 and 12. Approximately 75 percent of the mortality occurred after the second week. No specific lesions were observed. The abnormal feather condition noted in chicks in treatments 2a and 2b in series 1 and studied in series 3 was also noted in some chicks in this series. The incidence of the feather condition was highest in treatment 12 chicks. It was not as severe as it was in series 1. An increased melanism was observed in the chicks in treatments 10 and 12. The excessive amount of black color was observed in both the surface and undercolor of the feathers of chicks fed rations containing 0.1 percent iodinated casein. It was similar to that described by Irwin et al. (1943) for Rhode Island Red chicks fed a ration containing 0.5 percent iodinated casein. DISCUSSION The growth response of vitamin B12 supplementations was statistically significant in series 1, 2, and 3 regardless of the treatment used. Since the dams of the experimental chicks were fed rations containing sources of vitamin B12 and were housed on litter that was not changed during the laying year, the large responses in treatments lb and 3b were somewhat unexpected. This response was of sufficient magnitude to be statistically significant

516 MAGRUDER, BLETNER, CLARK AND WEAKLEY, JR. one week after supplementation in series 1 and two weeks after supplementation in series 2. This indicates that the chicks had a relatively low vitamin B i2 carryover. Series 3 was not designed for a critical growth study, however, the data are interesting. The vitamin B12 supplementation was higher in this series than in series 1 and 2 because the 70 percent soybean oil meal ration was used throughout the six-week experimental period. The level of vitamin B12 (approximately two micrograms per 100 grams of ration) was considered to be adequate for stress rations of this type (Lillie et al., 1948) but later work by Ott (1951) indicates that 2.7 micrograms of vitamin B12 per 100 grams of ration are needed for maximum early growth of depleted chicks fed a 70 percent soybean oil meal ration. A satisfactory growth response was obtained, but it did not appear until the end of the fourth week. This relatively slow response is interpreted to indicate a somewhat higher vitamin B 12 carry-over than was thought to be present in series 1 and 2 chicks. The growth retarding effect of the stress rations at the time the chicks were two weeks old is shown in Table 5. The very marked retarding effect of the 70 percent soybean oil meal ration in series 1 (treatment 2) and a very small but significant effect in series 4 (treatment 11) indicates Series TABLE 5. Comparison of the 2-week weights of chicks in all series Date of hatch Rations and Supplements 35% SBOM» 35% SBOM+I.C. 70% SBOM 70% SBOM+APF 70% SBOM+PGA 70% SBOM+APF+PGA 70% SBOM+I.C. 1 2 January March grams grams 131 (l)t 135(3) 125 (4) 101 (2) 3 April grams 135 (5) 136 (6) 136 (7) 141 (8) 4 June grams 136 (9) 124 (10) 129(11) 116(12) * SBOM =soybean oil meal, I.C. =iodinated casein, APF =Merck's APF Supplement No. 3, PGA=folic acid, t Numbers in parentheses are treatment numbers. that a difference existed in the amount of vitamin B12 carry-over in the chicks used in these two series. The extremely uniform growth of chicks on the 35 percent soybean oil meal ration (treatment 1, 3, and 9) could indicate that the series 1 chicks carried a borderline amount of vitamin B 12, the effect of which was quickly overcome by the stress ration. Further evidence that the chicks in series 2 had a greater vitamin B12 carry-over than those in series 1 can be obtained by comparing the 2-6 week body weight gains (Table 2). Treatment 3a chicks made larger gains than those iri treatment la, but the chicks in treatments lb and 3b, fed the vitamin B12 supplemented ration, made about the same gains. The iodinated casein ration in series 2 retarded the growth of the chicks but to a much smaller degree than the 70 percent soybean oil meal ration in series 1. The amount of iodinated casein used in the rations for treatments 4, 10, and 12 was larger than that used by Robblee et al. (1948) and Nichol et al. (1949). A more severe retarding effect was expected. In treatment 12, series 4, the growth retarding effect of the iodinated casein in the 70 percent soybean oil meal ration was more severe than that of either the 70 percent soybean oil meal ration alone or the 35 per cent soybean oil meal ration with iodinated casein, but not as severe as the effect of the 70 percent soybean oil meal ration in series 1. Much of the mortality occurred during the first week of each experiment, and therefore, in most instances should not be associated with treatment effects. However, the iodinated casein rations appeared to produce mortality throughout the periods that they were fed. The highest mortality rate occurred with treatment 12 when the iodinated casein was fed in the 70 percent soybean oil meal ration. The

VARIABLE RESPONSE TO STRESS RATIONS AND VITAMIN B 12 517 relatively low mortality from the 70 percent soybean oil meal ration itself was unexpected in view of literature reports. Ott (1951) also has reported obtaining low mortality with a vitamin B12 deficient, high protein ration. Feed efficiency was improved whenever the vitamin B12 supplement was fed. In two of three series the differences were of sufficient magnitude to be statistically significant. This was in accord with literature reports. In series 4, iodinated casein reduced feed efficiency, the effect being most severe when it was added to the 70 percent soybean oil meal ration. The abnormal feathering condition (Figure 1) encountered when the 70 percent soybean oil meal ration was fed resembled in some respects that reported for chicks from vitamin B12 deficient dams (Lillie et al., 1949), for folic acid deficient chicks (Lillie and Briggs, 1947; Lillie et al., 1950; and others), for chicks fed lysine deficient rations (Milligan et al., 1951) and for chicks subjected to inanition (Briggs, 1946). In these experiments both faulty feather structure and faulty feather pigmentation appeared in the same chick during the fourth week even though the chick had been placed on a 35 percent soybean oil meal ration at the end of the second week. It is possible that the chicks in series 3 might have responded to higher levels of vitamin B12 or folic acid or both if they had been used. It may be that more than one deficiency is accentuated by the 70 percent soybean oil meal ration and that all must be satisfied before normal feather pigment and structure can develop. These experiments indicated that the stress or deficiency causing the feather abnormality need be present only during the first two weeks of the chick's life and that the 70 percent soybean oil meal ration used in these experiments produced the severest feather abnormalities in chicks whose growth was retarded readily. This may not be in agreement with the observations made by Ott (1951) who considered that his 70 percent soybean oil meal ration was deficient in only one growthpromoting nutrient, namely, vitamin B12 activity. Pure vitamins, minerals, and amino acids were added to his ration. No mention was made of a faulty feather structure, and since Single Comb White Leghorn chicks were used, he had no opportunity to observe feather pigmentation. The variability in the response of the chicks to a vitamin B i2 supplement (Tables 3 and 4) and to stress rations (Tables 3 and 5) appeared to be due to a greater carry-over of the vitamin as the seasons progressed from winter to summer. Variation in the response of chicks was noted in early work with the cow-manure factor (Bird et al., 1948) and more recently by others including Ott (1951) who used chicks from dams fed an all-vegetable ration for vitamin B12 studies. Undoubtedly, in the experiments reported here, the variability was increased because no attempt was made to deplete the breeding flock by employing an allvegetable ration, wire floors, or frequent changes of litter. SUMMARY Depleting chicks of vitamin B12 by the use of stress rations was studied. Four series of trials were conducted in battery brooders using New Hampshire chicks hatched from eggs produced by pullets on unchanged litter and fed normal rations. 1. The response of the chicks to stress rations and to rations containing a vitamin B 12 supplement varied between hatches. 2. The 70 percent soybean oil meal ration appeared to retard growth more

518 NEWS AND NOTES severely than the 35 percent soybean oil meal ration containing 0.1 percent iodinated casein. 3. The 70 percent soybean oil meal ration containing 0.1 percent iodinated casein retarded growth more severely than the same ration without the iodinated casein. 4. The rations containing the vitamin B12 supplement were utilized more efficiently than the unsupplemented rations. 5. The mortality was low except for the lots fed the iodinated casein. 6. A faulty feather structure and a white barring in the primary and secondary wing feathers and the main tail feathers was accentuated by the 70 percent soybean oil meal ration and could not be prevented by the addition of 0.09 gram of folic acid or by the addition of approximately 0.9 milligram of vitamin B12 per 100 pounds of ration or by a combination of the same amounts of each supplement per 100 pounds of ration. 7. The continuous feeding of 0.1 percent iodinated casein for four weeks produced an increased melanism in both the surface and undercolor of the feathers. REFERENCES Bird, H. R., M. Rubin and A. C. Groschke, 1948. A chick growth factor in cow manure. VII. Its stability and solubility. J. Biol. Chem. 174: 611-619. Bletner, J. K., T. B. Clark, C. E. Weakley, Jr., and A. H. VanLandingham, 1953. The effect of sulfaquinoxaline in different ration formulations on growth and feed efficiency of coccidia-free chicks. Poultry Sci. 32: 733-739. tional Poultry Producers Federation; The Poultry and Egg National Board; and The Pacific States Dairy and Poultry Association. NEW HAMPSHIRE NOTES The New Hampshire Poultry Growers Associa- NEWS AND NOTES {Continued from page 507) Briggs, G. M., 1946. Observations on an unidentified feather-pigment factor necessary for chickens fed purified diets. Poultry Sci. 25: 41-46. Irwin, M. R., E. P. Reinke and C. W. Turner, 1943. Effect of feeding thyroactive iodocasein on growth, feathering, and weights of glands of young chicks. Poultry Sci. 22: 374-380. Lillie, R. J., and G. M. Briggs, 1947. Folic acid requirements of New Hampshire chicks receiving synthetic diets. Poultry Sci. 26: 295-298. Lillie, R. J., C. A. Denton and H. R. Bird, 1948. Relation of vitamin B12 to the growth factor present in cow manure. J. Biol. Chem. 176: 1477-1478. Lillie, R. J., M. W. Olsen and H. R. Bird, 1949. Role of vitamin B12 in reproduction of poultry. Proc. Soc. Exptl. Biol. Med. 72: 598-602. Lillie, R. J., G. F. Combs and G. M. Briggs, 1950. Folic acid in poultry nutrition. II. Effect of maternal diet and chick diet upon mortality, growth, and feathering of progeny. Poultry Sci. 29: 122-129. Milligan, J. L., L. J. Machlin, H. R. Bird and B. W. Heywang, 1951. Lysine and methionine requirements of chicks fed practical diets. Poultry Sci. 30:578-586. Nichol, C. A., L. S. Dietrich, W. W. Cravens and C. A. Elvehjem, 1949. Activity of vitamin B12 in the growth of chicks. Proc. Soc. Exp. Biol. Med. 70: 40-42. Ott, W. H., 1951. Further studies of the activity of crystalline vitamin B12 for chick growth. Poultry Sci. 30: 86-91. Ott, W. H., E. L. Rickes and T. R. Wood, 1948. Activity of crystalline vitamin B12 for chick growth. J. Biol. Chem. 174: 1047-1048. Robblee, A. R., C. A. Nichol, W. W. Cravens, C. A. Elvehjem and J. G. Halpin, 1948. Relation between induced hyperthyroidism and an unidentified chick growth factor. Proc. Soc. Exp. Biol. Med. 67: 40CM04. Rubin, M., and H. R. Bird, 1947. A chick growth factor in cow manure. V. Relation to quantity and quality of soybean oil meal in the diet. J. Nutrition, 34: 233-245. tion has contributed $11,000 to support poultry disease research at the University of New Hampshire. The funds will be used to continue the spray vaccination program against Newcastle disease and bronchitis. {Continued on page 584)