VITAMIN B~2 VS. VITAMIN B12b FOR COBALT DEFICIENT SHEEP. BEss A. Koch and SEDGWICK E. SMITH

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1 S VITAMIN B~2 VS. VITAMIN B12b FOR COBALT DEFICIENT SHEEP BEss A. Koch and SEDGWICK E. SMITH Cornell University 1 MITH et al. (1951) have shown that vitamin B12 injected in sufficient quantity will alleviate the symptoms of cobalt deficiency in lambs. This paper summarizes further evidence confirming this and also compares the effects of injected vitamin B,2 with one of its derivatives; namely, B~2b. Evidence at hand indicates that vitamin B12b and Bt.~a are identical and that both differ from B12 in having a hydroxy group in place of a cyano group. Methods The 18 lambs used in this study were selected from the original group of 25 used in a previous experiment (Smith et al., 1951). All lambs which had responded to a previous treatment were again reduced to a state of serious cobalt deficiency. The methods of handling and the rations fed were the same as in the previous experiment (Smith et al., 1951) except for the hay. The hay used in this study was from a different source and was lower in cobalt (0.03 ppm.) than that used previously. Lambs were assigned to treatment groups by selective randomization. The lambs were first divided into groups of similar previous treatment. From these groups, lambs were randomized into three treatment groups. The three treatment groups used in this study were as follows: One group of six lambs received subcutaneous injections of vitamin B12b 2 during the first five weeks (the extent of our available supply) and vitamin B12 '~ during the last seven, weeks of the study. A second group of six lambs received subcutaneous injections of vitamin B124 throughout the twelve week study period. A third group of six lambs was used as both a negative and a positive control group. During the first three weeks they were carried as a negative control group and received no treatment. At that poiut, oral cobalt therapy was initiated and they were carried as a positive control group for twelve weeks. 1 Department of Animal Husbandry. 2 Courtesy of Lederle Laboratories. a Merck and Co., Inc. 4 Lederle during the first two weeks---slerck thereafter.

2 1018 BERL A. KOCK AND SEDGWICK E. SMITH The vitamin B12b and vitamin Ble were in a water solution which contained 20 mcg. of vitamin per ml. of solution. Both vitamin B12 b and vitamin B12 were injected subcutaneously twice per week at the rate of I25 mcg. per week during the first two weeks. Thereafter injections were made once per week at the rate of 100 mcg. per week. The control group received cobalt orally as COC12.6H20 in water solution at the rate of one rag. per day. All six lambs were treated twice weekly until they showed an appetite response. Thereafter each lamb received 7 mg. of cobalt orally once per week. As the lambs in the various groups responded to treatment, as shown by an increase in appetite and body weight, the ration offered was TABLE 1. WEIGHT RESPONSES OF COBALT-DEFICIENT LAMBS WHEN INJECTED WITH VITAMIN B12b OR VITAMIN B12 Average Body Weight Pretreatment 5th 12th Total vitamin Treatment No. lambs week week week per lamb lbs. lbs. lbs. mcg. Vitamin B12b Vitamin B : 1283 Cobalt (oral) For 5 weeks and then B12 for 7 weeks, increased. This increase was rather rapid until each lamb received a maximum of 420 grams of shelled corn, 80 grams of skim milk powder, and 500 grams of chopped low-cobalt hay. Results and Discussion A positive or negative response to treatment was determined by an increase or decrease in concentrate consumption (corn and milk powder) and body weight. Hemoglobin values were determined biweekly but they were not used as a primary measure of response to treatment. The hemoglobin responses were rather erratic and of a much lower magnitude than appetite and weight responses in all groups. This slowness of hemoglobin response in cobalt-deficient lambs treated with cobalt has been noted by others (Filmer, 1933; Filmer and Underwood, 1937; Becker and Smith, 1951). Response to treatment as measured by body weight increase is summarized in table 1. Weights are shown for the fifth week and the twelfth week of treatment. Weights for the fifth week are shown in

3 .." VITAMIN B12 VERSUS VITAMIN B1._,h LAMBS PER TREATMENT GROUP "r _J..-.., ~':...., -" ~41C C BI2b " " "" J#J j~ BI2 "'~..'~ w f COBALT -2 C VIT.BI2b VIT~BI2... COBALT... -3C IO IB 20 WEEK Figure 1. Average body weight changes of the three treatment groups during a sixteen week period. order that vitamin B12b and vitamin Bz~o therapy might be compared with each other and with oral cobalt therapy. Figure 1 shows the weight changes of the three treatment groups graphically during a sixteen week period. Note that all three treatment groups showed a definite decline in body weight prior to the time treatment was started. The animals in the control group continued to lose weight until after they were fed cobalt salts whereupon they showed a quick response. Response to treatment as measured by appetite is summarized in table 2. The lambs in all three groups showed a very definite appetite response. Those lambs that were injected with vitamin B12~ or vitamin B~2 appeared to respond a little more rapidly during the first TABLE 2. APPETITE RESPONSES OF COBALT-DEFICIENT LAMBS WHEN INJECTED WITH VITAMIN B12~ OR VITAMIN B,2 Average Daily Concentrate Intake Pretreatment 1st 2nd 3rd 5th 12th Treatment No. lambs week week week week week week gins. gms. gms. gins. gms. gins. Vitamin Bv2b t Vitamin BI~ Cobalt (oral) i For 5 weeks and then B12 for? weeks.

4 1020 BERL A. KOCK AND SEDGWICK E. SMITH two weeks than those fed cobalt. However, all three groups reached maximum feed consumption at about the same time (fourth to fifth week). Changes in body weight of the three treatment groups are summarized in figure 1. Note that during the four week pretreatment period the lambs of each group were losing weight. Those lambs which were to serve as positive controls (cobalt fed) were carried for three weeks as negative controls. It should be noted that they continued to lose weight throughout the negative control period. When cobalt therapy was initiated the control lambs showed a quick and consistent increase in body weight which is characteristic of cobalt deficient animals. The two groups of lambs that received injections of vitamin B12b or vitamin B12 during the first five weeks of the study showed a rapid response as measured by both appetite and weight gains. These weight gains were analyzed using analysis of variance (Snedecor, 1946) with total weight gains and also individual regressions of gain on time as units of measure. The difference between the weight gains of these two groups of lambs was not statistically significant as measured by either method. This observation is in accord with those made by other investigators. Stokstad et al. (1950) reported that vitamin B1._, and vitamin Bl.~b have similar activity for both chicks and L. leichmannii. Kaczka et al. (1951) reported that the biological activities of vitamin B12a and vitamin B12 are similar in assays using L. lactis, L. leichmannii, chicks, rats and humans. Evidence at hand indicates that vramin B12a and vitamin Bl.~b are identical (Kaczka et al., 1950, 1951, and Hendlin and Soars, 1951) and both differ from vitamin B12 in that the cyano group of the latter is replaced by a hydroxy group (Brink et al., 1950). When the vitamin Ba2b injections were replaced at the end of five weeks with injections of vitamin Bae there was no apparent change in the weekly rate of gain of the lambs. Table 1 summarizes the total weight gains of the three treatment groups during the 12 week treatment period. The average daily gain for all three groups of approximately 0.35 pounds is a very satisfactory gain for fattening lambs. The total gains made by each of the three treatment groups were very similar. An analysis of variance (Snedecor, 1946) using either total weight gains or individual regressions of weight on time revealed no significant differences among them. This study demonstrates again that vitamin B12, when injected,

5 VITAMIN 812 VERSUS VITAMIN B12b 1021 will alleviate the symptoms of cobalt deficiency in lambs and furthermore, that vitamin B12b is as effective in this respect as vitamin B12. The amount of cobalt in these vitamins is so small that the activity can not be due to cobalt per se. Although cobalt may have other metabolic functions in the ruminant body, its utilization in the production of vitamin B~_~ is certainly one of its more important functions. Summary It has been demonstrated that vitamin B12b is as effective as vitamin B12 in curing a cobalt deficiency in lambs. Cobalt deficient lambs injected with mcg. per week of vitamin B12 made excellent gains in weight over a period of 12 weeks, a length of time comparable with the usual commercial fattening period. Literature Cited Becker, D. E. and Sedgwick E. Smith The metabolism of cobalt in lambs. Jour. of Nutr. 43:87. Brink, Norman G., Frederick A. Kuehl, Jr., and Karl Folkers Vitamin B1,.,: The identification of vitamin B12 as a cyano-cobalt coordination complex. Science 112:354. Filmer, J. F Enzootic marasmus of cattle and sheep. Aust. Vet. Jour. 9:163. Filmer, J. F. and E. J. Underwood Enzootic marasmus. Aust. Vet. Jour. 13:57. Hendlin, David and Margaret H. Soars Comparative microbiological studies with vitamins B12 and BL2,. Jour. Biol. Chem. 188:603. Kaczka, Edward, A., D. E. Wolf, Frederick A. Kuehl, Jr., and Karl Folkers Vitamin B~2. Reactions of cyano-cohalamin and related compounds. Science 112:354. Kaczka, Edward A., Robert G. Denkewalter, Arnold Holland and Karl Folkers Vitamin B,2 XIII. Additional data on vitamin B12,. Jour. Am. Chem. Soc. 73:335. Smith, S. E., Berl A. Koch and K. L. Turk Cobalt deficiency and vitamin B~. Jour. Nutr. 44:455. Snedecor, George W Statistical Methods. The Iowa State College Press. Ames, Iowa. Stokstad, E. L. R Relation of vitamin B~b to vitamin B~ and the biological activities of the compounds. Federation Proceedings. 9:122.