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Vitamin B12. The Absorption of 57 Co or 60 Co-B12 by the Laying Hen and Mature Rooster12
Vitamin B12. The Absorption of "Co or 00Co-Bi2 by the Laying Hen and Mature Rooster1'2 HARDY M. EDWARDS, JR. Department of Poultry Science, University of Georgia, Athens, Georgia 30601 (Received for publication December 15, 1967)
EXPERIMENTAL Twelve mature Single Comb White Leghorn chickens of each sex were utilized for this study. The animals were housed in individual cages. The records kept on a larger group of hens from which the females were selected indicated that all of the hens chosen for the study were producing eggs. The hens received a practical 1 University of Georgia, College of Agriculture Experiment Stations, Journal Series Paper number 198, College Station, Athens. 2 This investigation was supported in part by a Public Health Service research career program award No. 18, 411 from the Arthritis and Metabolic Disease Institute.
type ration3 that calculated to contain 6.6 micrograms of Bi2 per kilogram. Five females and five males were given 67 Co-Bi2 orally and 5 females and 5 males given 67Co-Bi5 intramuscularly in the first experiment. They were fasted overnight prior to receiving radioisotope and were then allowed feed for 30 minutes immediately prior to receiving the radioisotope dose. The other two males and females were treated identically except they did not receive any isotope and were to serve as background birds in the counting procedure. Immediately after dosing, the birds were placed in a whole body counter and were counted for 4 minutes. The whole body counter is equipped with a 400 channel analyzer which allows for counting background birds in channels 2-200 prior to counting the controls in channels 202-400. A resolver integrator attachment permits subtraction of the counts of the control bird (channel 2-200) from 3 The ration contained the following ingredients expressed as gm./lOO gm.: ground yellow corn, 67.5; soybean meal (dehulled), 15.0; alfalfa meal (17% dehydrated), 3.75; poultry by-product meal, 5.0; poultry fat, 1.0; sodium chloride, 0.5; defluorinated phosphate (18% P), 1.25; ground limestone, 5.75; vitamin premix, 0.15; and trace mineral mixture, 0.1. The vitamin mixture provided the following amounts per 100 gm. of finished feed: vitamin A, 2001.U.; vitamin D3, 88,1.C.U.; vitamin Bi2, 0.66 meg.; riboflavin, 0.22 mg.; calcium pantothenate, 0.44 mg.; niacin, 1.0 mg.; choline chloride, 10.0 mg.; and 1, 2, dihydro-6-ethoxy-2, 2, 4,-trimethylquinoline, 12.4 mg. The mineral mixture provided the following amounts per 100 mg. of finished feed: Ca, 26 mg.; Mn, 6.0 mg.; Co, 0.04 mg.; Fe, 2.5 mg.; Cu, 0.2 mg.; Zn, 2.0 mg.; and I, 0.12 mg.
414
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Y
ACOWITZei al. (1952) demonstrated that eggs from the same hen showed remarkable individuality with respect to their content of vitamin Bi2. Eggs from individual hens depleted of vitamin B12 for one month contained from 0.77 to 4.06 millimicrograms of B12 per gram of egg yolk. Evans et al. (1955) also noted that individual hens differed in the ability to place vitamin Bi2 in their eggs. They also raised the question as to whether this reflected a difference in digestion, absorption, or metabolism of vitamin BX2 by individual hens. The studies reported in this paper were conducted to gain information on the absorption and retention of 67Co-Bi2 and M Co-Bi2 by laying hens and mature roosters and to determine the amounts of these tagged vitamins placed in eggs by laying hens.
B 12
ABSORPTION AND R E T E N T I O N
All eggs were collected daily, identified, and stored in a cooler at 2°C. A quantity of 67Co-Bi2 equal to 1 0 % of the oral and intramuscular dose was injected into the center of an egg from one of the control hens and this egg counted in a well type sodium iodide scintillation counter. This count, corrected for background with a control egg, was then multiplied by 10 and the product taken as 100% of the dose for purposes of studying the amount of 57 Co-Bi 2 t h a t had been transferred to the eggs by the laying hens. All of the eggs collected for 23 days after dosing in the first experiment and for 18 days after dosing in the second experiment were then counted in the well type scintillation counter. T h e quantity in each egg was calculated as a percent of the dose. T h e second experiment was conducted 70 days after the start of the first experiment utilizing the same animals with the following exceptions: (1) 60 Co-Bi 2 was used instead of 67 CO-B 12 , (2) the counting days differed slightly and the experiment was terminated 18 days after dosing, (3) the channels chosen for integration were different due to the difference in energy of
emission of the isotope utilized, (4) the size of the dose was smaller and (5) the intramuscular dose was smaller than the oral dose. I n experiment one, the 2, 5, 8, 16, 23, and 35 day data for the females and the 8, 16, 27, and 35 day d a t a for the males were used to calculate the regression lines and these lines were extrapolated to 0 days and this intercept with the Y axis taken as the absorption values. The 2 and 4 day d a t a for males in experiment one were not utilized since these points were definitely not components of the same linear regression t h a t develops from the later observations. In experiment two, the 2, 4, 8, and 15 day d a t a for the females and the 3, 5, 8, and 16 day d a t a from the males were used to calculate the regression lines. T h e isotope d a t a and dose level utilized in the two experiments were as follows: 67
Co-B 12 -assayed 0.5 microcuries/ml. and contained 0.568 micrograms of Bi 2 /ml. Oral and intramuscular dose 1.0 ml. 60 Co-B] 2 -assayed 0.6 microcuries/ml. and contained 0.53 micrograms of B 1 2 /ml. Oral dose 0.2 ml., intramuscular dose 0.12 ml. RESULTS AND DISCUSSION A summary of the d a t a from the whole body counting of the birds in experiment one is presented graphically in Figure 1. T h e d a t a from the birds given the 57 CoBi 2 intramuscularly indicate t h a t the biological half-life of Bi 2 in the male is approximately 35 days while in the female it is approximately 14 days. The d a t a from the females also indicate t h a t the curve m a y have two components. The d a t a from the orally dosed birds, both male and female, show very erratic curves for the first 5 days, and then the curves behave in a more predictable manner when com-
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the experimental bird (channels 202-400). T h e energy peak of the particular isotope is selected and subsequently integrated and this total is printed. These counts are then corrected for radioisotope decay and differences in electronics by counting a standard of the isotope being used at the beginning, middle, and end of a counting period. After being counted the birds were replaced in individual pens and allowed free access to feed and water. The females were counted on days, 0, 2, 5, 8, 16, 23, and 35 days from dosing. T h e males were counted on days, 0, 2, 4, 8, 16, 27, and 35 days from dosing. These counts were all corrected b y the count from the standard and then calculated as a percent of the dose administered.
415
416
H. M.
100, 9Q 80 70 60 50
EDWARDS, J R . TABLE 1.—The percent of^Co-Bn absorbed by individual laying hens and mature roosters
\ \ \ ^ ~ " ~ ~ ^ ^
Laying hens
40 3Q
20.
10
15 20 25 DAYS AFTER DOSING
30
35
FIG. 1. The retention of "C0-B12 by laying hens and mature roosters. The C0-B12 was administered to the hens and roosters on day 0 either orally or intramuscularly.
pared to the curves obtained with the intramuscularly dosed birds. The absorption data show that there is a tremendous difference in the individual animal's ability to absorb the administered 67Co-Bi2 (Table 1). The females, as a group, absorbed more than the males. The retention data on individual female birds are presented in Figure 2. The individual retention data on the males are not presented since there were very little differences in the curves for the individual animals. It is apparent from this figure that while the absorption varies among the individual F E M A L E S ORALLY
Absorption
276 282 283 290 296
33 59 56 29 77
%
Rooster number
15 DAYS A F T E R
20
%
18 29 28 11 45
277 278 286 288 297
hens the manner in which the 67Co-B12 is metabolized is very similar with the result that the slope of the curves for the individual birds show very little variations. The data on transfer of the 57Co-Bi2 to the eggs by the laying hens are presented in Figure 3. Hens number 282, 283, and 296 absorbed considerably more 57Co-Bi2 than hens 276 and 290; and they also deposited much larger amounts of 67Co-Bi2 in the eggs. The largest amounts were present in the eggs laid 5 or 6 days after receiving the dose. The hens that absorbed relatively large amounts of the dose deposited between 6.6% and 8.3% in the eggs containing the maximum amounts of "Co-B^. The hens that only absorbed 25 to 30% of the dose had much lower levels of "C0-B12
DOSED FEMALES INTRAMUSCULARLY
10
Absorption
25 DOSING
6
10 15 20 25 DAYS A F T E R DOSING
DOSED
30
35
FIG. 2. The retention of "C0-B12 by individual laying hens. The 67Co-Bi2 was administered to the hens on day 0 either orally or intramuscularly.
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m
Hen number
Roosters
417
B12 ABSORPTION AND R E T E N T I O N INTRAMUSCULARLY DOSED
68.2 70.1 28.6 41 .6 53.0
FIG. 3. The percent of
57
8 9 10 11 12 13 14 15 16 1> 18 19 20 21 22 23 DAYS AFTER DOSING
DAYS AFTER DOSING
Co-Bu that appears in the eggs laid for 23 days after dosing hens either orally or intramuscularly with 67Co-Bi2.
in the eggs laid. Hen number 296, which laid a large number of eggs, showed very high levels of 57 Co-Bi 2 in the eggs. Hen 290 only laid two eggs during the critical period of the experiment and they were lower in 67 Co-Bi 2 content t h a n those eggs from the three hens t h a t absorbed greater amounts of the 67 Co-Bi 2 . When the hens received the "C0-B12 intramuscularly, three hens (279, 280, and 293) showed very similar curves for deposition of the 87 Co-Bi2 in the eggs. T h e curves peaked on days 5 and 6 and the eggs on these days contained between 9.6% and 1 0 . 3 % of the dose. Two hens (number 281 and 285) deposited a much lower level of 67Co-Bi2 in the eggs. T h e peak quantities were only 4 . 6 % and 4 . 8 % . I t should be noted in Figure 2 t h a t these two hens had curves similar to hens t h a t deposited much more 67 Co-Bi2 in their eggs during the period t h a t the eggs were collected and counted. Thus these hens were excreting either 57 Co-Bi2 or 6 7 Coinfeces, urine, or b y some other route. From Figure 2, it is apparent t h a t intramuscularly dosed hen, number 279, continued to lose 67 Co-Bi 2 from its body at a faster rate the other 4 hens between the 15th and 25th day. I t is interesting to note t h a t this hen was also depositing more 57 Co-Bi 2 in the eggs laid
between the 8th and 23rd day than any of the other hens. A s u m m a r y of the results of the whole body counting of the birds in experiment two is presented in Figure 4. T h e retention curves for the various groups are similar to those obtained in the first experiment. T h e percent of 60 Co-B 12 absorbed b y the individual hens and roosters is presented in Table 2. T h e absorption of the e0 Co-Bi 2 did not follow exactly the same p a t t e r n among the individual birds t h a t it did in experiment one. H e n number 276 showed very low absorption in the first experiment (33%) while she absorbed 6 3 % of the dose in the second trial. However, hen number 100 £
8Q
<
60
I
5Q
£ m °
40
z
2Q
«" INTRAMUSCUI Aff
"^^^^^^ -•*—2?4L —^L^ORA^
30
J a. 1Q C
1
2
3
4
5
6
7
6
DAYS AFTER
9
10 l'l
12
13 14 15 16
DOSING
FIG. 4. The retention of 60Co-Bi2 by laying hens and mature roosters. The 60Co-Bi2 was administered to the hens and roosters on day 0 either orally or intramuscularly.
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7
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
418
H. M. EDWARDS, JR.
once the initial loss (unabsorbed 60Co-Bi2) occurred. The data on retention of 60Co-Bi2 by Laying liens Roosters laying hens (Figure 5) are more revealing when considered along with the data on Rooster AbsorpHen Absorpnumber tion number tion transfer of 60Co-Bi2 to eggs laid by these hens (Figure 6). Hen number 296 absorbed % % 276 63 277 32 a large amount of the oral dose of the 282 65 288 32 labeled vitamin. She then laid an egg on 283 61 297 41 290 43 278 41 the second day after dosing which con296 84 tained 7.2% of the dose. She did not lay again until 14 days later when she laid a 290 absorbed relatively low amounts of clutch of three eggs, all containing ap60 the labeled Bj2 in both experiments while proximately 3 % of the dose of Co-Bi2. hen number 296 absorbed the largest The retention curve for this hen subamounts in both experiments. The av- stantiates these data. The initial early 60 erage absorption by both hens and roosters drop due to nonabsorbed Co-Bi2 loss is in experiment two was 12% greater than followed by a period where the curve tends to flatten when compared to the other in the first experiment. 60 hens who continued to lay during this The four roosters that received the Coearly part of the experiment. Hen number B12 intramuscularly showed very little dif290 absorbed much less 60Co-Bi2 than the ference in the rate of loss of the vitamin from the body and the individual reten- other hens; however, she had deposited tion data are, therefore, not presented. The rather large amounts in the eggs laid rate of loss was, however, much greater during the 18 days after dosing. It would during the first 5 days of the experiment appear that this hen is extremely efficient (Figure 4) once again indicating that there in the utilization of absorbed B12. Another may be two components to the curve. The interpretation is that she has a very small roosters that received the 90Co-B12 orally but dynamic pool of B12 in her body. The had very similar absorpalso showed little variation in the rate at other three hens 60 which the vitamin was lost from the body tion of the Co-Bi2 and deposited large TABLE 2.—The percent of ""Go-Bit absorbed by individual laying hens and mature roosters
5 6 7 8 9 10 (1 DAYS A F T E R DOSING
60
l'a
13
DOSED
14 15 DAYS AFTER
60
DOSING
FIG. 5. The retention of Co-Bi2 by individual laying hens. The Co-Bi2 was administered to the hens on day 0 either orally or intramuscularly.
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F E M A L E S INTRAMUSCULARLY
F E M A L E S ORALLY DOSED
419
B 12 ABSORPTION AND R E T E N T I O N INTRAMUSCULARLY DOSED
HEN NO. "/.OF DOSE IN NO. EGGS TOTAL EGGS 2 7 9 14 83.6 280 5 30-5 281 15 67.3 285 14 69.4
FIG. 6. The percent of
60
15 16 17 18
Co-Bi2 that appears in the eggs laid for IS days after dosing hens either orally or intramuscularly with 60Co-Bi2.
amounts in the eggs. T h e biological halflives of the 60Co-Bi2 was similar in these although hen number 276 appeared to be holding on to the labeled B 12 more tenaciously than the other hens toward the end of the experiment. Hens number 281 and 285 in the second experiment showed no problem in tranferring labeled Bi 2 to eggs after being given the 60Co-B]2 intramuscularly. These same hens showed very poor transfer of the labeled isotope in the first experiment. This would indicate t h a t the very rapid excretion of labeled Bi 2 t h a t must have taken place in the first experiment was not taking place at this time in these hens. I t is not known whether the " C o in the first experiment was excreted as 67 Co-B 12 or 67 Co. Also, the route of elimination of the 57 Co is unknown; however, it is apparent t h a t it was not excreted in the eggs and it was not in the hens (Figure 2 and 3). I n the second experiment, hen number 280 stopped laying on the 7 th day after dosing. As a result of this she showed very slow loss of 60Co-Bi2 from her body from the 8th to 15th day (Figure 5). T h e present report does not answer all the questions raised b y the studies of
Yacowitz et al. (1952) and Evans et al. (1955). This report does indicate t h a t absorption, excretion from the hen in other than the egg, and possibly endogenous metabolism are factors in determining the quantity of B ] 2 t h a t may be transported from the feed to the egg. T h e important question of whether eggs from hens t h a t have B12 absorption problems can hatch still needs investigation. However, the variation over a period of time in the ability of hen both to absorb Bi 2 and to place it in the egg indicates that it will not be a simple question to answer. SUMMARY
Experiments were conducted utilizing laying hens and m a t u r e roosters to determine the absorption and excretion of 67 Co-Bi2 and 60 Co-Bi2. T h e experiments utilized the same animals at different times. When 57 Co-Bi 2 was utilized, absorption values from 2 9 % to 7 7 % were obtained for the laying hens and values from 1 1 % to 4 5 % were obtained with the roosters. In the experiment, utilizing 60 CoBi 2 , the absorption values for the hens were between 4 3 % and 8 4 % and between 3 2 % and 4 1 % for the males. I n most cases
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6 7 8 9 10 11 12 13 DAYS AFTER DOSING
420
H.
M.
EDWARDS, JR.
and 4.7% in the eggs laid on the seventh day. The average biological half life of 57 Co-Bi2 in laying hens is much shorter (14 days) than in the mature roosters (35 days). REFERENCES Evans, R. J., S. L. Bandemer, D. H. Bauer and J. A. Davidson, 1952. The vitamin Bi2 content of fresh and stored shell eggs. Poultry Sci. 34: 922-928. Yacowitz, H., R. F. Miller, L. C. Norris and G. F. Heuser, 1952. Vitamin B12 studies with the hen. Poultry Sci. 31: 89-94.
The Metabolism of 36Cl-Clopidol (3,5-Dichloro-2,6-Dimethyl-4-Pyridinol) in Chickens G R A N T N . SMITH
Biomechanisms Laboratory, Agricultural Products Center, The Dow Chemical Company, Midland, Michigan 48640 (Received for publication August 14, 1968)
INTRODUCTION
C
LOPIDOL (3,5-dichloro-2,6-dimethyl-4-pyridinol) is a new anti-coccidial compound which is being used for the control of coccidiosis in chickens (Stevenson, 1965; Stock el al., 1967). As part of the biochemical studies on this compound a series of investigations on the distribution and fate of the compound within the chicken were undertaken using 36Cl-clopidol. In an initial study on the distribution of the compound in chicken tissues (Bauriedel, 1964) it was observed that a radioactive compound or compounds were present in the various tissues when the bird was fed a feed containing 0.0125% of the 36Cl-clopidol. If the radioactivity was calculated in terms of the original compound fed to the bird, the muscle tissue contained approximately 3 to 4 p.p.m. of the compound while the liver tissue contained from 10 to 12 p.p.m. of the compound.
When the treated feed was withdrawn from the birds, there was a rapid decrease in the level of radioactivity in all the tissues. If the data were plotted on semi-log paper with the concentration of radioactivity in the tissues vs. time since the compound was withdrawn, there was a singificant change in the slope of the curve with time. The data indicated that the radioactivity in the tissue was associated with at least two components. One component had a very short biological halflife since about 90% of the radioactivity present in the tissues at the time the birds were taken off medication was eliminated within 2 days. The remaining radioactivity was slowly eliminated during the remaining portion of the observation period. By the 14th day the 36Cl-activity in the tissues was at or below the limits of detection (0.01 p.p.m.). Preliminary investigations indicated that the short biological half-life component (the major component) could be
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where a laying hen did not absorb large amounts of the labeled vitamins, small amounts of the labeled vitamin appeared in the eggs. The eggs laid 6-8 days after dosing with either 67Co-Bi2 or 60Co-Bi2 contained the greatest percent of the dose. When the dose was given intramuscularly, from 9-11% of the dose appeared in the egg containing the maximum amount in all except two hens. These two hens in one experiment deposited a maximum of 4.6%
EXPERIMENTAL Twelve mature Single Comb White Leghorn chickens of each sex were utilized for this study. The animals were housed in individual cages. The records kept on a larger group of hens from which the females were selected indicated that all of the hens chosen for the study were producing eggs. The hens received a practical 1 University of Georgia, College of Agriculture Experiment Stations, Journal Series Paper number 198, College Station, Athens. 2 This investigation was supported in part by a Public Health Service research career program award No. 18, 411 from the Arthritis and Metabolic Disease Institute.
type ration3 that calculated to contain 6.6 micrograms of Bi2 per kilogram. Five females and five males were given 67 Co-Bi2 orally and 5 females and 5 males given 67Co-Bi5 intramuscularly in the first experiment. They were fasted overnight prior to receiving radioisotope and were then allowed feed for 30 minutes immediately prior to receiving the radioisotope dose. The other two males and females were treated identically except they did not receive any isotope and were to serve as background birds in the counting procedure. Immediately after dosing, the birds were placed in a whole body counter and were counted for 4 minutes. The whole body counter is equipped with a 400 channel analyzer which allows for counting background birds in channels 2-200 prior to counting the controls in channels 202-400. A resolver integrator attachment permits subtraction of the counts of the control bird (channel 2-200) from 3 The ration contained the following ingredients expressed as gm./lOO gm.: ground yellow corn, 67.5; soybean meal (dehulled), 15.0; alfalfa meal (17% dehydrated), 3.75; poultry by-product meal, 5.0; poultry fat, 1.0; sodium chloride, 0.5; defluorinated phosphate (18% P), 1.25; ground limestone, 5.75; vitamin premix, 0.15; and trace mineral mixture, 0.1. The vitamin mixture provided the following amounts per 100 gm. of finished feed: vitamin A, 2001.U.; vitamin D3, 88,1.C.U.; vitamin Bi2, 0.66 meg.; riboflavin, 0.22 mg.; calcium pantothenate, 0.44 mg.; niacin, 1.0 mg.; choline chloride, 10.0 mg.; and 1, 2, dihydro-6-ethoxy-2, 2, 4,-trimethylquinoline, 12.4 mg. The mineral mixture provided the following amounts per 100 mg. of finished feed: Ca, 26 mg.; Mn, 6.0 mg.; Co, 0.04 mg.; Fe, 2.5 mg.; Cu, 0.2 mg.; Zn, 2.0 mg.; and I, 0.12 mg.
414
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Y
ACOWITZei al. (1952) demonstrated that eggs from the same hen showed remarkable individuality with respect to their content of vitamin Bi2. Eggs from individual hens depleted of vitamin B12 for one month contained from 0.77 to 4.06 millimicrograms of B12 per gram of egg yolk. Evans et al. (1955) also noted that individual hens differed in the ability to place vitamin Bi2 in their eggs. They also raised the question as to whether this reflected a difference in digestion, absorption, or metabolism of vitamin BX2 by individual hens. The studies reported in this paper were conducted to gain information on the absorption and retention of 67Co-Bi2 and M Co-Bi2 by laying hens and mature roosters and to determine the amounts of these tagged vitamins placed in eggs by laying hens.
B 12
ABSORPTION AND R E T E N T I O N
All eggs were collected daily, identified, and stored in a cooler at 2°C. A quantity of 67Co-Bi2 equal to 1 0 % of the oral and intramuscular dose was injected into the center of an egg from one of the control hens and this egg counted in a well type sodium iodide scintillation counter. This count, corrected for background with a control egg, was then multiplied by 10 and the product taken as 100% of the dose for purposes of studying the amount of 57 Co-Bi 2 t h a t had been transferred to the eggs by the laying hens. All of the eggs collected for 23 days after dosing in the first experiment and for 18 days after dosing in the second experiment were then counted in the well type scintillation counter. T h e quantity in each egg was calculated as a percent of the dose. T h e second experiment was conducted 70 days after the start of the first experiment utilizing the same animals with the following exceptions: (1) 60 Co-Bi 2 was used instead of 67 CO-B 12 , (2) the counting days differed slightly and the experiment was terminated 18 days after dosing, (3) the channels chosen for integration were different due to the difference in energy of
emission of the isotope utilized, (4) the size of the dose was smaller and (5) the intramuscular dose was smaller than the oral dose. I n experiment one, the 2, 5, 8, 16, 23, and 35 day data for the females and the 8, 16, 27, and 35 day d a t a for the males were used to calculate the regression lines and these lines were extrapolated to 0 days and this intercept with the Y axis taken as the absorption values. The 2 and 4 day d a t a for males in experiment one were not utilized since these points were definitely not components of the same linear regression t h a t develops from the later observations. In experiment two, the 2, 4, 8, and 15 day d a t a for the females and the 3, 5, 8, and 16 day d a t a from the males were used to calculate the regression lines. T h e isotope d a t a and dose level utilized in the two experiments were as follows: 67
Co-B 12 -assayed 0.5 microcuries/ml. and contained 0.568 micrograms of Bi 2 /ml. Oral and intramuscular dose 1.0 ml. 60 Co-B] 2 -assayed 0.6 microcuries/ml. and contained 0.53 micrograms of B 1 2 /ml. Oral dose 0.2 ml., intramuscular dose 0.12 ml. RESULTS AND DISCUSSION A summary of the d a t a from the whole body counting of the birds in experiment one is presented graphically in Figure 1. T h e d a t a from the birds given the 57 CoBi 2 intramuscularly indicate t h a t the biological half-life of Bi 2 in the male is approximately 35 days while in the female it is approximately 14 days. The d a t a from the females also indicate t h a t the curve m a y have two components. The d a t a from the orally dosed birds, both male and female, show very erratic curves for the first 5 days, and then the curves behave in a more predictable manner when com-
Downloaded from http://ps.oxfordjournals.org/ at University of Massachusetts Medical School on April 13, 2015
the experimental bird (channels 202-400). T h e energy peak of the particular isotope is selected and subsequently integrated and this total is printed. These counts are then corrected for radioisotope decay and differences in electronics by counting a standard of the isotope being used at the beginning, middle, and end of a counting period. After being counted the birds were replaced in individual pens and allowed free access to feed and water. The females were counted on days, 0, 2, 5, 8, 16, 23, and 35 days from dosing. T h e males were counted on days, 0, 2, 4, 8, 16, 27, and 35 days from dosing. These counts were all corrected b y the count from the standard and then calculated as a percent of the dose administered.
415
416
H. M.
100, 9Q 80 70 60 50
EDWARDS, J R . TABLE 1.—The percent of^Co-Bn absorbed by individual laying hens and mature roosters
\ \ \ ^ ~ " ~ ~ ^ ^
Laying hens
40 3Q
20.
10
15 20 25 DAYS AFTER DOSING
30
35
FIG. 1. The retention of "C0-B12 by laying hens and mature roosters. The C0-B12 was administered to the hens and roosters on day 0 either orally or intramuscularly.
pared to the curves obtained with the intramuscularly dosed birds. The absorption data show that there is a tremendous difference in the individual animal's ability to absorb the administered 67Co-Bi2 (Table 1). The females, as a group, absorbed more than the males. The retention data on individual female birds are presented in Figure 2. The individual retention data on the males are not presented since there were very little differences in the curves for the individual animals. It is apparent from this figure that while the absorption varies among the individual F E M A L E S ORALLY
Absorption
276 282 283 290 296
33 59 56 29 77
%
Rooster number
15 DAYS A F T E R
20
%
18 29 28 11 45
277 278 286 288 297
hens the manner in which the 67Co-B12 is metabolized is very similar with the result that the slope of the curves for the individual birds show very little variations. The data on transfer of the 57Co-Bi2 to the eggs by the laying hens are presented in Figure 3. Hens number 282, 283, and 296 absorbed considerably more 57Co-Bi2 than hens 276 and 290; and they also deposited much larger amounts of 67Co-Bi2 in the eggs. The largest amounts were present in the eggs laid 5 or 6 days after receiving the dose. The hens that absorbed relatively large amounts of the dose deposited between 6.6% and 8.3% in the eggs containing the maximum amounts of "Co-B^. The hens that only absorbed 25 to 30% of the dose had much lower levels of "C0-B12
DOSED FEMALES INTRAMUSCULARLY
10
Absorption
25 DOSING
6
10 15 20 25 DAYS A F T E R DOSING
DOSED
30
35
FIG. 2. The retention of "C0-B12 by individual laying hens. The 67Co-Bi2 was administered to the hens on day 0 either orally or intramuscularly.
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m
Hen number
Roosters
417
B12 ABSORPTION AND R E T E N T I O N INTRAMUSCULARLY DOSED
68.2 70.1 28.6 41 .6 53.0
FIG. 3. The percent of
57
8 9 10 11 12 13 14 15 16 1> 18 19 20 21 22 23 DAYS AFTER DOSING
DAYS AFTER DOSING
Co-Bu that appears in the eggs laid for 23 days after dosing hens either orally or intramuscularly with 67Co-Bi2.
in the eggs laid. Hen number 296, which laid a large number of eggs, showed very high levels of 57 Co-Bi 2 in the eggs. Hen 290 only laid two eggs during the critical period of the experiment and they were lower in 67 Co-Bi 2 content t h a n those eggs from the three hens t h a t absorbed greater amounts of the 67 Co-Bi 2 . When the hens received the "C0-B12 intramuscularly, three hens (279, 280, and 293) showed very similar curves for deposition of the 87 Co-Bi2 in the eggs. T h e curves peaked on days 5 and 6 and the eggs on these days contained between 9.6% and 1 0 . 3 % of the dose. Two hens (number 281 and 285) deposited a much lower level of 67Co-Bi2 in the eggs. T h e peak quantities were only 4 . 6 % and 4 . 8 % . I t should be noted in Figure 2 t h a t these two hens had curves similar to hens t h a t deposited much more 67 Co-Bi2 in their eggs during the period t h a t the eggs were collected and counted. Thus these hens were excreting either 57 Co-Bi2 or 6 7 Coinfeces, urine, or b y some other route. From Figure 2, it is apparent t h a t intramuscularly dosed hen, number 279, continued to lose 67 Co-Bi 2 from its body at a faster rate the other 4 hens between the 15th and 25th day. I t is interesting to note t h a t this hen was also depositing more 57 Co-Bi 2 in the eggs laid
between the 8th and 23rd day than any of the other hens. A s u m m a r y of the results of the whole body counting of the birds in experiment two is presented in Figure 4. T h e retention curves for the various groups are similar to those obtained in the first experiment. T h e percent of 60 Co-B 12 absorbed b y the individual hens and roosters is presented in Table 2. T h e absorption of the e0 Co-Bi 2 did not follow exactly the same p a t t e r n among the individual birds t h a t it did in experiment one. H e n number 276 showed very low absorption in the first experiment (33%) while she absorbed 6 3 % of the dose in the second trial. However, hen number 100 £
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3
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13 14 15 16
DOSING
FIG. 4. The retention of 60Co-Bi2 by laying hens and mature roosters. The 60Co-Bi2 was administered to the hens and roosters on day 0 either orally or intramuscularly.
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7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
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H. M. EDWARDS, JR.
once the initial loss (unabsorbed 60Co-Bi2) occurred. The data on retention of 60Co-Bi2 by Laying liens Roosters laying hens (Figure 5) are more revealing when considered along with the data on Rooster AbsorpHen Absorpnumber tion number tion transfer of 60Co-Bi2 to eggs laid by these hens (Figure 6). Hen number 296 absorbed % % 276 63 277 32 a large amount of the oral dose of the 282 65 288 32 labeled vitamin. She then laid an egg on 283 61 297 41 290 43 278 41 the second day after dosing which con296 84 tained 7.2% of the dose. She did not lay again until 14 days later when she laid a 290 absorbed relatively low amounts of clutch of three eggs, all containing ap60 the labeled Bj2 in both experiments while proximately 3 % of the dose of Co-Bi2. hen number 296 absorbed the largest The retention curve for this hen subamounts in both experiments. The av- stantiates these data. The initial early 60 erage absorption by both hens and roosters drop due to nonabsorbed Co-Bi2 loss is in experiment two was 12% greater than followed by a period where the curve tends to flatten when compared to the other in the first experiment. 60 hens who continued to lay during this The four roosters that received the Coearly part of the experiment. Hen number B12 intramuscularly showed very little dif290 absorbed much less 60Co-Bi2 than the ference in the rate of loss of the vitamin from the body and the individual reten- other hens; however, she had deposited tion data are, therefore, not presented. The rather large amounts in the eggs laid rate of loss was, however, much greater during the 18 days after dosing. It would during the first 5 days of the experiment appear that this hen is extremely efficient (Figure 4) once again indicating that there in the utilization of absorbed B12. Another may be two components to the curve. The interpretation is that she has a very small roosters that received the 90Co-B12 orally but dynamic pool of B12 in her body. The had very similar absorpalso showed little variation in the rate at other three hens 60 which the vitamin was lost from the body tion of the Co-Bi2 and deposited large TABLE 2.—The percent of ""Go-Bit absorbed by individual laying hens and mature roosters
5 6 7 8 9 10 (1 DAYS A F T E R DOSING
60
l'a
13
DOSED
14 15 DAYS AFTER
60
DOSING
FIG. 5. The retention of Co-Bi2 by individual laying hens. The Co-Bi2 was administered to the hens on day 0 either orally or intramuscularly.
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F E M A L E S INTRAMUSCULARLY
F E M A L E S ORALLY DOSED
419
B 12 ABSORPTION AND R E T E N T I O N INTRAMUSCULARLY DOSED
HEN NO. "/.OF DOSE IN NO. EGGS TOTAL EGGS 2 7 9 14 83.6 280 5 30-5 281 15 67.3 285 14 69.4
FIG. 6. The percent of
60
15 16 17 18
Co-Bi2 that appears in the eggs laid for IS days after dosing hens either orally or intramuscularly with 60Co-Bi2.
amounts in the eggs. T h e biological halflives of the 60Co-Bi2 was similar in these although hen number 276 appeared to be holding on to the labeled B 12 more tenaciously than the other hens toward the end of the experiment. Hens number 281 and 285 in the second experiment showed no problem in tranferring labeled Bi 2 to eggs after being given the 60Co-B]2 intramuscularly. These same hens showed very poor transfer of the labeled isotope in the first experiment. This would indicate t h a t the very rapid excretion of labeled Bi 2 t h a t must have taken place in the first experiment was not taking place at this time in these hens. I t is not known whether the " C o in the first experiment was excreted as 67 Co-B 12 or 67 Co. Also, the route of elimination of the 57 Co is unknown; however, it is apparent t h a t it was not excreted in the eggs and it was not in the hens (Figure 2 and 3). I n the second experiment, hen number 280 stopped laying on the 7 th day after dosing. As a result of this she showed very slow loss of 60Co-Bi2 from her body from the 8th to 15th day (Figure 5). T h e present report does not answer all the questions raised b y the studies of
Yacowitz et al. (1952) and Evans et al. (1955). This report does indicate t h a t absorption, excretion from the hen in other than the egg, and possibly endogenous metabolism are factors in determining the quantity of B ] 2 t h a t may be transported from the feed to the egg. T h e important question of whether eggs from hens t h a t have B12 absorption problems can hatch still needs investigation. However, the variation over a period of time in the ability of hen both to absorb Bi 2 and to place it in the egg indicates that it will not be a simple question to answer. SUMMARY
Experiments were conducted utilizing laying hens and m a t u r e roosters to determine the absorption and excretion of 67 Co-Bi2 and 60 Co-Bi2. T h e experiments utilized the same animals at different times. When 57 Co-Bi 2 was utilized, absorption values from 2 9 % to 7 7 % were obtained for the laying hens and values from 1 1 % to 4 5 % were obtained with the roosters. In the experiment, utilizing 60 CoBi 2 , the absorption values for the hens were between 4 3 % and 8 4 % and between 3 2 % and 4 1 % for the males. I n most cases
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6 7 8 9 10 11 12 13 DAYS AFTER DOSING
420
H.
M.
EDWARDS, JR.
and 4.7% in the eggs laid on the seventh day. The average biological half life of 57 Co-Bi2 in laying hens is much shorter (14 days) than in the mature roosters (35 days). REFERENCES Evans, R. J., S. L. Bandemer, D. H. Bauer and J. A. Davidson, 1952. The vitamin Bi2 content of fresh and stored shell eggs. Poultry Sci. 34: 922-928. Yacowitz, H., R. F. Miller, L. C. Norris and G. F. Heuser, 1952. Vitamin B12 studies with the hen. Poultry Sci. 31: 89-94.
The Metabolism of 36Cl-Clopidol (3,5-Dichloro-2,6-Dimethyl-4-Pyridinol) in Chickens G R A N T N . SMITH
Biomechanisms Laboratory, Agricultural Products Center, The Dow Chemical Company, Midland, Michigan 48640 (Received for publication August 14, 1968)
INTRODUCTION
C
LOPIDOL (3,5-dichloro-2,6-dimethyl-4-pyridinol) is a new anti-coccidial compound which is being used for the control of coccidiosis in chickens (Stevenson, 1965; Stock el al., 1967). As part of the biochemical studies on this compound a series of investigations on the distribution and fate of the compound within the chicken were undertaken using 36Cl-clopidol. In an initial study on the distribution of the compound in chicken tissues (Bauriedel, 1964) it was observed that a radioactive compound or compounds were present in the various tissues when the bird was fed a feed containing 0.0125% of the 36Cl-clopidol. If the radioactivity was calculated in terms of the original compound fed to the bird, the muscle tissue contained approximately 3 to 4 p.p.m. of the compound while the liver tissue contained from 10 to 12 p.p.m. of the compound.
When the treated feed was withdrawn from the birds, there was a rapid decrease in the level of radioactivity in all the tissues. If the data were plotted on semi-log paper with the concentration of radioactivity in the tissues vs. time since the compound was withdrawn, there was a singificant change in the slope of the curve with time. The data indicated that the radioactivity in the tissue was associated with at least two components. One component had a very short biological halflife since about 90% of the radioactivity present in the tissues at the time the birds were taken off medication was eliminated within 2 days. The remaining radioactivity was slowly eliminated during the remaining portion of the observation period. By the 14th day the 36Cl-activity in the tissues was at or below the limits of detection (0.01 p.p.m.). Preliminary investigations indicated that the short biological half-life component (the major component) could be
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where a laying hen did not absorb large amounts of the labeled vitamins, small amounts of the labeled vitamin appeared in the eggs. The eggs laid 6-8 days after dosing with either 67Co-Bi2 or 60Co-Bi2 contained the greatest percent of the dose. When the dose was given intramuscularly, from 9-11% of the dose appeared in the egg containing the maximum amount in all except two hens. These two hens in one experiment deposited a maximum of 4.6%