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The Effect of Antibiotics on Hatchability of Hens’ Eggs and Progeny Growth Performance1,2,3,4
The Effect of Antibiotics on Hatchability of Hens' Eggs and Progeny Growth Performance1,2,3'4 ORVILLE G. BENTLEY AND T. V. HERSHBERGER Department of Animal Science, Ohio Agricultural Experiment Station, Wooster (Received for publication October 3, 1953)
INTRODUCTION
T
1 Published with the approval of the Director of the Ohio Agricultural Experiment Station. 2 This work was supported in part by Grants-inAid from the Commercial Solvents Corporation, Terre Haute, Indiana, and Merck and Company, Inc., Rahway, N. J. 3 The antibiotics used in this study were kindly supplied by the following: Bacitracin and Baciferm, Commercial Solvents Corp.; Procaine Penicillin by Merck and Company, Inc.; Aureomycin • HC1, Lederle Laboratories, Pearl River, N . Y.; Terramycin, Chas. Pfizer and Company, Brooklyn, N. Y. Merck and Company, Inc., also supplied the crystalline Vitamin B12. 4 The study reported herein was initiated by Dr. R. M. Bethke, formerly Chairman, Department of Animal Science, Ohio Agricultural Experiment Station.
EXPERIMENTAL Each year about 250 White Leghorn hens from the Station flock that had completed one year's production were transferred indoors from pasture in midOctober and molting was forced by feeding shelled corn and water for five days. A vitamin B12 deficient all-plant ration (Table 1) similar to the one used by Pensack et al. (1949) was fed for the next three months. Preliminary hatchability data were obtained with eggs saved for 5 to 8 weeks after the hens came into production. Hatchability data and production records were used as a basis to select 144 hens which were allotted equally into 12 lots or pens. Two male birds were placed in each pen and, to minimize the effect of the males on the experiment, they were rotated from pen to pen at about two week intervals. Lights were turned on in the morning to provide a 14 to 15 hour day. Oyster shells and water were fed ad libitum. Wood shavings were used for litter and it was changed at 4 to 6 week intervals throughout the experiment to reduce the possibility of vitamin B12 being
641
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HE growth promoting action of antibiotics in chick starting rations has been well established. However, the use of antibiotics in breeder rations, with respect to hatchability of hens' eggs and their effect on progeny growth performance, has not been investigated extensively. Several investigators, Petersen et al. (1952), Lillie and Bird (1952), Sunde et al. (1952), and Berg et al. (1952) have fed various antibiotics to hens and observed no beneficial effects. Nevertheless Elam et al. (1951), Mariakulandai et al. (1952), Carlson et al. (1952), and Sizemore et al. (1953) have reported that antibiotic supplementation of breeder rations improved hatchability of the fertile eggs. Slinger, Ferguson, and McConachie (1952) and Pepper et al. (1952) have reported that penicillin in the maternal ration exerts a growth promoting effect in the progeny, indicating a carry-over from the dam via the egg. ^ ^
During the course of studies conducted since 1949 on the effect of "A.P.F." supplementation of all-plant basal hen rations on hatchability and progeny growth performance, additional experiments with crystalline antibiotics have been carried out. The results to be presented in this paper are concerned primarily with the effect of antibiotics on hatchability, and a possible carry-over effect of antibiotics from the maternal ration on progeny growth performance.
642 TABLE 1.-
0. G. BENTLEY AND T. V. HERSHBERGER -Composition of vitamin Bn deficient basal breeder ration Percent
Ground yellow corn Ground oats Wheat middlings Wheat bran Dehydrated alfalfa meal Soybean oil meal, 44% protein solvent extracted Steamed bone meal Ground limestone Iodized salt Butyl fermentation solubles* Feeding oil (1,500 A-^00 D) Manganese sulfate, monohydrate, 6 gm. per 100 lb. Oyster shells, ad libitum
40.75 20.00 7.50 7.50 2.50 17.00 2.50 1.00 0.50 0.50 0.25
* Contained 500 ng. riboflavin per gm.
supplied via the litter. Hatchability Studies: The basal hen ration described in Table 1 was supplemented with vitamin B12 or antibiotics singly or with a combination of vitamin B12 and an antibiotic, as indicated in Tables 3, 4, and 5. The effect of the various supplements on the hatchability of the eggs was determined by pedigree hatching the eggs weekly for a period of 20 weeks in 1950,17 weeks in 1951, and 16 weeks in 1952. Progeny Growth Studies: To determine the effect of the maternal ration on the growth performance of the progeny, day old chicks were grown to six weeks of age in electrically heated batteries having raised screen bottoms. A vitamin B12 deficient all-plant chick ration which contained 50 percent soybean meal was fed alone or supplemented with vitamin B12 or bacitracin plus vitamin B12. The composition of the ration is given in Table 2. Microbiological Assays for Vitamin B^: Liver samples from hens, day old chicks, six week old chicks, and egg yolks were analyzed for vitamin B12 using Lactobacillus leichmanii ATCC 7830. The medium of Skeggs et al. (1950) with the following modification was used: sodium
TABLE 2.-
-Composition of vitamin Bn deficient basal chick ration
Ingredient Ground yellow corn Alfalfa leaf meal Soybean oil meal, 44 percent protein Steamed bone meal Ground limestone Iodized salt Feeding oil (1,500 A-100 D) Manganese sulfate, monohydrate Niacin Riboflavin
Percent 43.25 3.00 50.00 2.00 1.00 0.50 0.25 6 gm. per 100 lb. 0.8 gm. per 100 lb. 0.2 gm. per 100 lb.
RESULTS AND DISCUSSION
Hatchability Data: An all-plant breeder ration supplemented with fermentation residues which contained vitamin B12 or with two percent condensed fish solubles resulted in a 19 percent increase in the hatchability of hens' eggs over those from the unsupplemented hens (see Table 3). Both levels of vitamin B12 fed (7.5 or 15.0jug./lb.) were equally effective, indicating that the lowest level was above the minimum requirement for hatchability in this experiment. Two of the supplements were found, subsequently, to contain aureomycin as well as vitamin B12. However, hatchability was no better in the groups fed aureomycin and vitamin B12 than in the groups which did not receive aureomycin. In the 1950 and 1951 experiments the effect of feeding crystalline antibiotics on hatchability was investigated. The results are given in Table 4. Of the four antibiotics used in the
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Ingredient
ascorbate replaced sodium thioglycolate as a reducing agent. Water extracts of the samples were prepared by autoclaving a homogenized sample for 3 minutes at 15 pounds pressure and then filtering to remove the insoluble material. Potassium cyanide was added to protect the vitamin during the process of sample preparation.
643
E F F E C T OF ANTIBIOTICS ON REPRODUCTION
TABLE 3.—Hatchability of eggs from hens fed vitamin Bn supplements or condensed fish solubles and the growth performance of their progeny
Ration
Basal Basal Basal Basal Basal Basal
Preliminary hatchability (8 weeks)
Total number fertile eggs
Hatchability 20 weeks (fertile eggs)
Progeny growth Sex av., 6 wks. 3 (final weight)
24 24 24 24 24 24
percent 74.3 76.6 71.1 73.7 72.6 73.1
1,292 1,321 1,318 1,207 1,267 1,351
percent 68.4 83.4 79.5 80.3 83.4 80.7
299 400 348 454 402 447
Merck's APF No. 3 which contained 12.5 mg. vitamin Bi2 activity per pound. Lederle's APF supplement No. 5 which was later shown to have antibiotic activity. Chicks fed Bi2 deficient ration (Table 2); 40 chicks per lot.
experiment completed in 1951 aureomycin* and bacitracin appeared to increase hatchability by 8 to 9 percent. However, when the experiment was re* Aureomycin in all experiments was supplied as aureomycin • HC1.
peated in 1952, with different hens, none of the antibiotic supplements, including aureomycin and bacitracin, improved hatchability. Unfortunately, it was decided to lower the amount of antibiotic fed from 20 gm. per ton, as used in 1951, to 10 gm. per ton in 1952 to keep in line
TABLE 4.—The effect of antibiotics, vitamin Ba, and combinations of antibiotics and vitamin B& on hatchability of fertile eggs 1951 Hen ration
Basal (vit. B12 deficient) Basal plus aureomycin2 Basal plus bacitracin Basal plus vitamin Bi23 Basal plus vitamin B I2 and bacitracin Basal plus vitamin B12 and Baciferm(2.0 lb./ton) Basal plus vitamin B12 and aureomycin Basal plus vitamin B12 and procaine penicillin Basal plus vitamin Bi2 and terramycin Basal plus vitamin B12 and B-complex vitamins 4 1
No. of hens 1
1952
Preliminary Total Average number hatchhatchfertile ability ability eggs (17 weeks) (5 weeks) percent 86.7
percent 49.6
Preliminary Total Average hatchnumber hatchfertile ability ability eggs (16 weeks) (5 weeks) percent 77.9 76.7 77.3 77.8
434 492 449 920
percent 87.3 70.9 72.8 83.3
24
87.7
837
78.2
—• —
12 12 12 24
24
86.8
1,075
84.6
12
79.1
456
73.5
—
—
—
—
24
77.8
673
86.7
1
86.9
781
85.1
24
76.7
962
76.8
24
86.9
936
76.2
12
78.6
313
82.4
24
86.3
949
79.4
—
—
—
—
—
12
79.3
317
85.2
— —
•
—
—• —
—
738
No. of hens
—
•
—
•
—
In the 1951 experiment these groups were divided after ten weeks; thus the eggs were obtained from 24 hens (10 weeks) and for only 12 hens for the last 7 weeks (See Table 5). 2 Antibiotics added at the rate of 20 gm./ton of feed in 1951. In 1952, 10 gm./ton was used. 3 In the 1951 experiment ten,ug./lb. of vitamin B12 added as Merck's APF No. 3. A 0.1 percent triturate of vitamin B12 on mannitol (Merck's) which supplied 7.5 Mg-/lb. of vitamin B12 was used in 1952. 4 Following amounts of B-complex vitamins added (gm./lOO lb. feed): niacin, 0.8; calcium pantothenate, 0.91; choline chloride, 6.8; vitamin B6, 0.2; folic acid, 0.5; and vitamin Bi, 0.09.
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1 2 3
plus 2.00% condensed fish solubles plus 7.50 jug./lb. vitamin Bi 2 l plus 15.00 Mg-/lb. vitamin Bi 2 l plus 0.25% APF supplement No. 5 2 plus 0.50% APF supplement No. 5 2
No. of hens
644
O. G. BENTLEY AND T. V. HERSHBERGER
TABLE 5.—The effect of feeding aureomycin to vitamin Bn deficient hens on hatchability of fertile eggs 1951 Experiment* Ten weeks No. of hens
12 12
Hen ration
Basal (Bn def.) Basal
Av. hatchability of eggs percent 53.5 57.2
Seven weeks Change in hen ration None 20 gm./ton aureomycin added
Av. hatchability of eggs percent 44.1 66.7
with levels frequently used in chick growth experiments. Also, crystalline vitamin B12 was used in place of the crude A.P.F. supplement. To what extent these changes may have influenced the results is not known. In 1951 aureomycin was added to the vitamin B12 deficient basal hen ration after the experiment had been in progress for ten weeks (Table 5). Hatchability increased from 57 percent to 67 percent over a seven-week period, while another lot of hens continued on the basal ration dropped in hatchability from 54 percent to 44 percent. Prior to feeding aureomycin, the hens had been on a vitamin B12 deficient ration for about 20 weeks. Since a marked response in hatchability was obtained by feeding vitamin B12 in the 1951 experiment, a vitamin B12 sparing effect is suggested as a possible explanation for the action of aureomycin in this experiment. However, Petersen et al. (1952) failed to obtain a response in hatchability by feeding either streptomycin or aureomycin to hens severely depleted of vitamin B12 (36 weeks). Previous reports indicated that aureomycin spares vitamin B12 for growth in chicks (Oleson et al., 1950; Stokstad and Jukes, 1951). Cravioto-Munoz et al. (1951), and Linkswiler, Baumann and Snell (1951) found that feeding aureomycin to rats would exert a sparing effect on vitamin B i2 and vitamin B 6 .
A mixture of six B-vitamins in addition to vitamin B12 and riboflavin failed to improve hatchability in this experiment. TABLE 6.—Summary of analysis of variance of chick growth data Mean square
F value
359 2 2 1 1 2
112,329 17,225 266,995 262,116 62,066
46.4f 7.It 110.3f 108.3f 25.6f
4 2
2,321 6,586
.96 2.7
4 341
40,595 2,421
of Source of variation Degrees freedom Total Chick rations Antibiotics B12
Sex Chick ration XB12 Chick ration X antibiotics Bi2Xantibiotics Chick ration XB12 X antibiotics Error
t Significant at 1% level.
16.8f
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* For the complete results of the experiment see Table 4.
In the second experiment (1952), the basal ration was supplemented with aureomycin or bacitracin and fed to two lots of 12 hens each in an attempt to duplicate the 1951 results. Because the expected depression in hatchability due to the lack of dietary vitamin B12 did not occur, it was not possible to get additional information concerning the effect of antibiotics in vitamin B12 deficient rations on hatchability. No explanation can be given for the good hatchability with the basal ration as the vitamin B12 content of the day old chick livers, egg yolk, and hen livers was low (see Table 9), and the carry-over of the vitamin to the progeny was not indicated by the results of progeny growth experiments (see Table 7). Hens that laid more than 200 eggs in their first years' production were used in 1952, while in the two earlier experiments hens with individual records of less than 200 eggs per year were used. Lillie, Olsen and Bird (1951) noted that high producing hens were more difficult to deplete of vitamin B12, as determined by hatchability of their eggs, than were the lower producing hens.
645
EFFECT OF ANTIBIOTICS ON REPRODUCTION
TABLE 8.—Progeny growth from hens fed vitamin Bn with penicillin or baciferm1* Chick ration Supplement to vitamin Bu deficient basal hen ration Vitamin Bis Vitamin Bi:+Baciferm 2.0 lb./ton Vitamin Bia+Procaine Penicillin 10 gm./ton
Total weight, 6 weeks, sex av., gm.
B„
B„+
deficient
+B„
bacitracin
447
445
444
461
436
435
467
466
464
i Combined results of two growth experiments. Each group represents the average of from 23-49 chicks. 2 Baciferm contained 5 gm. per pound bacitracin.
the vitamin B12 deficient chick ration was fed. Pensack et al. (1949) and Petersen et al. (1950) reported similar results. A highly significant carry-over effect from bacitracin in the hen ration occurred when the chicks were fed a vitamin B12 deficient ration. Under similar conditions, aureomycin improved growth and the difference due to treatment approached significance but it did not exceed the 5% level. With the vitamin B12 or vitamin B12 and bacitracin chick rations, the chicks from the hens fed the vitamin B12 basal ration supplemented with antibiotics generally grew better but the differences were not significant at the 5% level. TABLE 7.—The effect of vitamin Bn and antibiotics in If the hens received vitamin B12 in their hens' ration on progeny growth1'2'3 rations there was no statistically significant carry-over effect on progeny growth Chick rations from feeding either aureomycin or baciTotal weight, 6 weeks, tracin. For additional data concerning this sex av., gm. Hen ration supplement observation see Table 3. B12 plus B12 B12 bacitracin Feeding bacitracin to chicks hatched deficient added added from hens fed the vitamin B12 deficient ration tended to improve growth while Vitamin Ba deficient 421 306 405 None there was little effect from bacitracin on 447 401 337 Aureomycin 452 the growth of chicks from hens fed vita428 367 Bacitracin min B12 in these experiments (last 2 Vitamin Bn added columns Table 7). 447 445 444 None 456 428 460 Aureomycin In addition to aureomycin and baci470 446 Bacitracin 451 tracin, procaine penicillin and Baciferm 1 Ten males and ten females per group. were fed to hens as a part of the experi2 Vitamin Bu (0.1% triturate on mannitol) added to chick ration at level of 15^g./lb.; bacitracin ment reported in Table 7. Because cerand aureomycin added at rate of 10 gm./ton. tain dietary treatments were not made 'L.S.D. between groups=33 gm. (5% level); with these antibiotics, i.e., not fed to hens 44 gm. (1% level).
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Progeny Growth Experiments: To determine whether or not bacitracin, aureomycin, or vitamin B12 in the maternal diet would influence the growth performance of the progeny, male and female chicks from hens fed these antibiotics with or without vitamin B12 were placed on three different chick rations, i.e., basal (vitamin B12 deficient), basal plus vitamin Bi2, and basal plus vitamin B12 and bacitracin. The composition of the basal chick ration is given in Table 2. This 4-way factorial experiment was analyzed by an analysis of variance, the results of which are given in Table 6. The addition of either vitamin Bt2 or antibiotics to the basal hen ration had a highly significant effect on the growth performance of their progeny. An inspection of the chick growth data in Table 7 indicates that the effect of vitamin B12 in the hen ration on progeny growth was observed when either a vitamin B12 deficient or a vitamin B12 supplemented chick ration was used; although the most pronounced effect was demonstrated when
646
O. G. BENTLEY AND T. V. HERSHBERGER TABLE 9.—Levels of vitamin Bu in chick and hen livers and egg yolk, ng./100 gm. wet weight (1952 hatchability experiment)
Hen ration supplements 1
Six week old chick livers
Weeks hens were on test rations
Chick rations 2
5
13
17
—B12
+B 1 2
.6 .6 .6 3.4 1.5 3.2
1.7 1.5 2.7 13.1 12.8 12.8
1.1 3.8 1.7 18.0 16.0 26.0
5.5 5.6 4.0 7.6 6.8 5.8
3.5
11.7
15.0
3.6
14.7
2.6
8.7
Egg 3 yolk
Hen livers4
66.0 41.0 54.0 47.0 71.0 71.5
0.25 0.5 0.4 5.5 5.7 4.7
21.9 25.7 22.6 84.7 136.3 74.3
6.1
73.5
5.1
104.3
19.5
6.1
95.0
4.1
58.3
18.5
7.2
88.0
4.6
77.9
1
See Table 1 for complete composition. See Table 2 for complete composition. Yolks from 2 dozen eggs homogenized to obtain samples. 4 Livers taken from 4 hens per dietary group for a composite sample. Both egg and hen liver sample taken at end of 1952 test. 2 3
on a vitamin B12 deficient ration, the results were not treated in the previously mentioned statistical analysis. The data are reported separately in Table 8. Feeding procaine penicillin to the hens appeared to have some effect on chick growth—the growth increase over the controls being about 20 gm. This may indicate a difference in behavior of penicillin in the hen ration, as aureomycin, bacitracin, and Baciferm appeared to have no effect on progeny growth if the hen ration contained vitamin B i2 . These results suggest that bacitracin and probably aureomycin in a breeder ration can exert a carry-over effect on the progeny under certain conditions, namely: when the antibiotics are fed with a vitamin B12 deficient breeder ration, and the chick ration is also deficient in vitamin B12. If adequate vitamin B12 is present in either ration the effect is either not observed or in demonstrated to a limited extent. A vitamin B i2 sparing effect of
antibiotics is suggested as an explanation for some of the growth data obtained under the conditions of our experiments. To determine the effect of feeding antibiotics on the amount of vitamin B12 available to the chicks, vitamin B12 analyses were made on samples of egg yolks, dayold chick and hen livers. The results are given in Table 9. Vitamin Bn Analyses: The concentration of vitamin B12 in composite samples of day-old chick livers obtained from hatches on the 5th, 13th, and 17th week of the 1952 hatchability experiment was unaffected by bacitracin or aureomycin in the maternal ration, while the addition of vitamin Bi2 to the hen ration increased the level of Bi2 in the day old chick livers 6 to 10 times. Likewise, samples of egg yolks and livers from each of the dietary groups of hens revealed no increased vitamin BX2 storage as a result of feeding antibiotics. Tappan et al. (1950) reported that feeding aureomycin to rats did not influence
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Basal (B12 deficient) Basal plus aureomycin Basal plus bacitracin Basal plus vitamin Bi2 Basal plus vitamin B12 plus bacitracin Basal plus vitamin B12 plus baciferm Basal plus vitamin B ]2 plus aureomycm Basal plus vitamin B12 plus procaine penicillin Basal plus vitamin Bi2 plus B-vitamin mix
Day old chick livers
EFFECT OF ANTIBIOTICS ON REPRODUCTION
SUMMARY Three hatchability experiments, each of from 16 to 20 weeks' duration, have been carried out using an all-plant breeder ration for hens. Progeny growth studies to determine the carry-over effect of the supplements in the maternal ration on progeny growth performance were also made. In addition, the vitamin B12 content of day-old chick livers, egg yolks, and 6 week old chick livers, as determined with L. liechmannii ATCC 7830, was obtained. The results are summarized as follows: 1. Feeding antibiotics (bacitracin, terramycin, aureomycin • HC1, or procaine penicillin) to hens did not consistently improve hatchability of the fertile eggs. However, in one experiment there was some increase in hatchability with either bacitracin or aureomycin • HC1, both in the presence and absence of supplementary vitamin B i2 . 2. Bacitracin and probably aureomycin • HC1, in the maternal diet appeared to exert a significant carry-over effect on the progeny growth if the antibiotics were added to a vitamin B12 deficient breeder ration and the chicks were fed a vitamin B12 deficient ration. Little or no effect on progeny growth was observed when antibiotics were added to the vitamin B12 supplemented hen ration, although there appeared to be a trend toward better growth performance in chicks from hens fed vitamin B12 and procaine penicillin. 3. The level of vitamin B12 in day-old chick livers, egg yolks, and hens' livers was not increased by feeding the antibiotics to hens. This would indicate that any carry-over effect from bacitracin or aureomycin from the hen to the progeny was probably not mediated directly through the release of more vitamin B12 from the egg yolk to the chick.
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liver vitamin B12 levels. Vitamin B12 levels for day-old chick livers and egg yolk, reported in Table 9, compare favorably with results published by Halick and Couch (1951) and Sunde, Halpin and Cravens (1952), but the value for egg yolks are somewhat higher than those reported by Yacowitz et al. (1952). A vitamin B12 sparing effect of antibiotics in a growing rat or chick could be explained by the action of antibiotics on the intestinal flora. However, if the antibiotic effect is to be transmitted to the progeny, as is indicated by the chick growth data (Tables 7 and 8), it would seem that either vitamin B12 or a factor that exerts a vitamin Bi2-like growth effect on chicks but which does not stimulate the growth of L. leichmannii must be deposited in the egg from whence the chick can obtain the nutrient. Since no increased storage of vitamin B12 in the eggs was observed, there is a possibility that a growth promoting factor, other than vitamin B12, was stored in the egg as a result of feeding antibiotics. This possibility is now under study using rats to assay the egg yolks. Waibel, Sunde and Cravens (1952) found that feeding penicillin to hens resulted in greater storage of folic acid and biotin in the egg yolk. Grieff and Pinkerton (1951) reported that the growth of Rickettsiae cultured in embryos from hens fed terramycin was inhibited. Wong et al. (1953) failed to corroborate this observation and reported no detectable storage of terramycin or penicillin in the egg even when 1,000 gms. per ton of feed was used. Nevertheless the results reported herein would confirm the findings of Slinger et al. (1952), Carlson et al. (1953) and Sizemore et al. (1953) which suggest that under certain conditions antibiotics in the maternal ration may influence progeny growth performance.
647
648
0. G. BENTLEY AND T. V. HERSHBERGER ACKNOWLEDGEMENTS
The authors wish to thank Mr. D. C. Kennard, Dr. E. N. Moore, and Mr. V. D. Chamberlin of the Poultry Science Department, Ohio Agricultural Experiment Station, for supplying the hens used in this study, and Dr. C. R. Weaver, Entomology Department, Ohio Agricultural Experiment Station, for his help with the statistical analyses.
Berg, L. E., J. S. Carver, G. E. Bearse and J. McGinnis, 1952. Antibiotics in the nutrition of laying hens. Washington Agr. Exp. Sta. Bui. No. 534: 1-10. Carlson, C. W., E. D. Jones, R. A. Wilcox and W. Kohlmeyer, 1952. Antibiotics in breeder diets. Poultry Sci. 31:910. Carlson, C. W., D. G. Jones, W. Kohlmeyer and A. L. Moxon, 1953. The effect of vitamin B l 2 and aureomycin on reproductive performance in turkeys. Poultry Sci. 32:984-989. Cravioto-Munoz, J., H. G. Poncher and H. A. Waisman, 1951. Vitamin B12 sparing action of aureomycin in the rat. Proc. Soc. Expt. Biol. Med. 77: 18-19. Elam, J. F., L. L. Gee and J. R. Couch, 1951. Effect of feeding penicillin on the life cycle of the chick. Proc. Soc. Expt. Biol. Med. 77: 209-213. Grieff, D., and H. Pinkerton, 1951. Rickettsiostasis in fertile eggs from the use of antibiotic residues in poultry feeds. Proc. Soc. Expt. Biol. Med. 78: 690-695. Halick, J. V., and J. R. Couch, 1951. Antibiotics in mature fowl nutrition. Proc. Soc. Expt. Biol. Med. 76: 58-62. Lillie, R. J., and H. R. Bird, 1952. Effect of antibiotic supplements upon hatchability and upon growth and viability of Progeny. Poultry Sci. 31: 513517. Lillie, R. J., M. W. Olsen and H. R. Bird, 1951. Variation in reproductive response of hens to dietary deficiency. Poultry Sci. 30: 92-97. Linkswiler, H., C. A. Baumann and E. E. Snell, 1951. Effect of aureomycin on the response of rats to various forms of vitamin Be. J. Nutrition, 43:565573. Mariakulandai, A., T. Myint and J. McGinnis, 1952. Effect of terramycin and vitamin Bu on hatchability. Proc. Soc. Expt. Biol. Med. 79: 242-244. Oleson, J. J., B. L. Hutchings and A. R. Whitehill,
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REFERENCES
1950. The effect of feeding aureomycin on the vitamin B12 requirement of the chick. Arch. Biochem. 29: 334-338. Pensack, J. M., R. M. Bethke and D. C. Kennard, 1949. The effect of fish meal and extracts of fish meal on hatchability of hens' eggs and growth of progeny. Poultry Sci. 28:398-405. Pepper, W. F., S. J. Slinger and I. Motzok, 1952. Effect of aureomycin on the manganese requirement of chicks fed varying levels of salt and phosphorus. Poultry Sci. 31: 1054-1061. Petersen, C. F., A. C. Wiese, R. V. Dahlstrom and C. E. Lampman, 1952. Influence of vitamin B12 and antibiotics on hatchability. Poultry Sci. 31: 129-132. Petersen, C. F., A. C. Wiese, C. E. Lampman and R. V. Dahlstrom, 1950. Role of crystalline vitamin B12 for hatchability. Poultry Sci. 29:618-619. Sizemore, J. R., R. J. Lillie, C. A. Denton and H. R. Bird, 1953. Influence of aureomycin in the chick diet upon subsequent reproductive performance of laying hens. Poultry Sci. 32: 618-624. Skeggs, H. R., H. M. Nepple, K. A. Valentik, J. W. Huff and L. D. Wright, 1950. Observations on the use of Lactobacillus leichmannii 4797 in the microbiological assay of vitamin B12. J. Biol. Chem. 184: 211-221. Slinger, S. J., A. E. Ferguson and J. D. McConachie, 1952. A carry-over effect of penicillin from dam to progeny. Poultry Sci. 31: 172-174. Stokstad, E. L. R., and T. H. Jukes, 1951. Effects of various levels of vitamin B12 upon growth response produced by aureomycin in chicks. Proc. Soc. Expt. Biol. Med. 76: 73-76. Sunde, M. L., J. G. Halpin and W. W. Cravens, 1952. The effect of vitamin B12 supplements and antibiotic feed supplements on egg production and hatchability. Poultry Sci. 31: 617-620. Tappan, D. V., U. J. Lewis, U. D. Register and C. A. Elvehjem, 1950. Further studies on the rat growth assay for vitamin Bi2 activity. Arch. Biochem. 29:408-412. Waibel, P. E., M. L. Sunde and W. W. Cravens, 1952. Effect of addition of penicillin to the hen's ration on biotin and folic acid content of eggs. Poultry Sci. 31:621-624. Wong, S. C , R. C. Kersey, J. R. McMahon, W. M. Reynolds, W. K. Warden and J. McGinnis, 1953. Effects of feeding terramycin and penicillin to laying hens on antibiotic deposition in eggs and growth of marine typhus rickettsiae in embryonated eggs. Fed. Proc. 12: 434. Yacowitz, H., R. F. Miller, L. C. Norris and G. F. Heuser, 1952. Vitamin B12 studies with the hen. Poultry Sci. 31:89-94.
INTRODUCTION
T
1 Published with the approval of the Director of the Ohio Agricultural Experiment Station. 2 This work was supported in part by Grants-inAid from the Commercial Solvents Corporation, Terre Haute, Indiana, and Merck and Company, Inc., Rahway, N. J. 3 The antibiotics used in this study were kindly supplied by the following: Bacitracin and Baciferm, Commercial Solvents Corp.; Procaine Penicillin by Merck and Company, Inc.; Aureomycin • HC1, Lederle Laboratories, Pearl River, N . Y.; Terramycin, Chas. Pfizer and Company, Brooklyn, N. Y. Merck and Company, Inc., also supplied the crystalline Vitamin B12. 4 The study reported herein was initiated by Dr. R. M. Bethke, formerly Chairman, Department of Animal Science, Ohio Agricultural Experiment Station.
EXPERIMENTAL Each year about 250 White Leghorn hens from the Station flock that had completed one year's production were transferred indoors from pasture in midOctober and molting was forced by feeding shelled corn and water for five days. A vitamin B12 deficient all-plant ration (Table 1) similar to the one used by Pensack et al. (1949) was fed for the next three months. Preliminary hatchability data were obtained with eggs saved for 5 to 8 weeks after the hens came into production. Hatchability data and production records were used as a basis to select 144 hens which were allotted equally into 12 lots or pens. Two male birds were placed in each pen and, to minimize the effect of the males on the experiment, they were rotated from pen to pen at about two week intervals. Lights were turned on in the morning to provide a 14 to 15 hour day. Oyster shells and water were fed ad libitum. Wood shavings were used for litter and it was changed at 4 to 6 week intervals throughout the experiment to reduce the possibility of vitamin B12 being
641
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HE growth promoting action of antibiotics in chick starting rations has been well established. However, the use of antibiotics in breeder rations, with respect to hatchability of hens' eggs and their effect on progeny growth performance, has not been investigated extensively. Several investigators, Petersen et al. (1952), Lillie and Bird (1952), Sunde et al. (1952), and Berg et al. (1952) have fed various antibiotics to hens and observed no beneficial effects. Nevertheless Elam et al. (1951), Mariakulandai et al. (1952), Carlson et al. (1952), and Sizemore et al. (1953) have reported that antibiotic supplementation of breeder rations improved hatchability of the fertile eggs. Slinger, Ferguson, and McConachie (1952) and Pepper et al. (1952) have reported that penicillin in the maternal ration exerts a growth promoting effect in the progeny, indicating a carry-over from the dam via the egg. ^ ^
During the course of studies conducted since 1949 on the effect of "A.P.F." supplementation of all-plant basal hen rations on hatchability and progeny growth performance, additional experiments with crystalline antibiotics have been carried out. The results to be presented in this paper are concerned primarily with the effect of antibiotics on hatchability, and a possible carry-over effect of antibiotics from the maternal ration on progeny growth performance.
642 TABLE 1.-
0. G. BENTLEY AND T. V. HERSHBERGER -Composition of vitamin Bn deficient basal breeder ration Percent
Ground yellow corn Ground oats Wheat middlings Wheat bran Dehydrated alfalfa meal Soybean oil meal, 44% protein solvent extracted Steamed bone meal Ground limestone Iodized salt Butyl fermentation solubles* Feeding oil (1,500 A-^00 D) Manganese sulfate, monohydrate, 6 gm. per 100 lb. Oyster shells, ad libitum
40.75 20.00 7.50 7.50 2.50 17.00 2.50 1.00 0.50 0.50 0.25
* Contained 500 ng. riboflavin per gm.
supplied via the litter. Hatchability Studies: The basal hen ration described in Table 1 was supplemented with vitamin B12 or antibiotics singly or with a combination of vitamin B12 and an antibiotic, as indicated in Tables 3, 4, and 5. The effect of the various supplements on the hatchability of the eggs was determined by pedigree hatching the eggs weekly for a period of 20 weeks in 1950,17 weeks in 1951, and 16 weeks in 1952. Progeny Growth Studies: To determine the effect of the maternal ration on the growth performance of the progeny, day old chicks were grown to six weeks of age in electrically heated batteries having raised screen bottoms. A vitamin B12 deficient all-plant chick ration which contained 50 percent soybean meal was fed alone or supplemented with vitamin B12 or bacitracin plus vitamin B12. The composition of the ration is given in Table 2. Microbiological Assays for Vitamin B^: Liver samples from hens, day old chicks, six week old chicks, and egg yolks were analyzed for vitamin B12 using Lactobacillus leichmanii ATCC 7830. The medium of Skeggs et al. (1950) with the following modification was used: sodium
TABLE 2.-
-Composition of vitamin Bn deficient basal chick ration
Ingredient Ground yellow corn Alfalfa leaf meal Soybean oil meal, 44 percent protein Steamed bone meal Ground limestone Iodized salt Feeding oil (1,500 A-100 D) Manganese sulfate, monohydrate Niacin Riboflavin
Percent 43.25 3.00 50.00 2.00 1.00 0.50 0.25 6 gm. per 100 lb. 0.8 gm. per 100 lb. 0.2 gm. per 100 lb.
RESULTS AND DISCUSSION
Hatchability Data: An all-plant breeder ration supplemented with fermentation residues which contained vitamin B12 or with two percent condensed fish solubles resulted in a 19 percent increase in the hatchability of hens' eggs over those from the unsupplemented hens (see Table 3). Both levels of vitamin B12 fed (7.5 or 15.0jug./lb.) were equally effective, indicating that the lowest level was above the minimum requirement for hatchability in this experiment. Two of the supplements were found, subsequently, to contain aureomycin as well as vitamin B12. However, hatchability was no better in the groups fed aureomycin and vitamin B12 than in the groups which did not receive aureomycin. In the 1950 and 1951 experiments the effect of feeding crystalline antibiotics on hatchability was investigated. The results are given in Table 4. Of the four antibiotics used in the
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Ingredient
ascorbate replaced sodium thioglycolate as a reducing agent. Water extracts of the samples were prepared by autoclaving a homogenized sample for 3 minutes at 15 pounds pressure and then filtering to remove the insoluble material. Potassium cyanide was added to protect the vitamin during the process of sample preparation.
643
E F F E C T OF ANTIBIOTICS ON REPRODUCTION
TABLE 3.—Hatchability of eggs from hens fed vitamin Bn supplements or condensed fish solubles and the growth performance of their progeny
Ration
Basal Basal Basal Basal Basal Basal
Preliminary hatchability (8 weeks)
Total number fertile eggs
Hatchability 20 weeks (fertile eggs)
Progeny growth Sex av., 6 wks. 3 (final weight)
24 24 24 24 24 24
percent 74.3 76.6 71.1 73.7 72.6 73.1
1,292 1,321 1,318 1,207 1,267 1,351
percent 68.4 83.4 79.5 80.3 83.4 80.7
299 400 348 454 402 447
Merck's APF No. 3 which contained 12.5 mg. vitamin Bi2 activity per pound. Lederle's APF supplement No. 5 which was later shown to have antibiotic activity. Chicks fed Bi2 deficient ration (Table 2); 40 chicks per lot.
experiment completed in 1951 aureomycin* and bacitracin appeared to increase hatchability by 8 to 9 percent. However, when the experiment was re* Aureomycin in all experiments was supplied as aureomycin • HC1.
peated in 1952, with different hens, none of the antibiotic supplements, including aureomycin and bacitracin, improved hatchability. Unfortunately, it was decided to lower the amount of antibiotic fed from 20 gm. per ton, as used in 1951, to 10 gm. per ton in 1952 to keep in line
TABLE 4.—The effect of antibiotics, vitamin Ba, and combinations of antibiotics and vitamin B& on hatchability of fertile eggs 1951 Hen ration
Basal (vit. B12 deficient) Basal plus aureomycin2 Basal plus bacitracin Basal plus vitamin Bi23 Basal plus vitamin B I2 and bacitracin Basal plus vitamin B12 and Baciferm(2.0 lb./ton) Basal plus vitamin B12 and aureomycin Basal plus vitamin B12 and procaine penicillin Basal plus vitamin Bi2 and terramycin Basal plus vitamin B12 and B-complex vitamins 4 1
No. of hens 1
1952
Preliminary Total Average number hatchhatchfertile ability ability eggs (17 weeks) (5 weeks) percent 86.7
percent 49.6
Preliminary Total Average hatchnumber hatchfertile ability ability eggs (16 weeks) (5 weeks) percent 77.9 76.7 77.3 77.8
434 492 449 920
percent 87.3 70.9 72.8 83.3
24
87.7
837
78.2
—• —
12 12 12 24
24
86.8
1,075
84.6
12
79.1
456
73.5
—
—
—
—
24
77.8
673
86.7
1
86.9
781
85.1
24
76.7
962
76.8
24
86.9
936
76.2
12
78.6
313
82.4
24
86.3
949
79.4
—
—
—
—
—
12
79.3
317
85.2
— —
•
—
—• —
—
738
No. of hens
—
•
—
•
—
In the 1951 experiment these groups were divided after ten weeks; thus the eggs were obtained from 24 hens (10 weeks) and for only 12 hens for the last 7 weeks (See Table 5). 2 Antibiotics added at the rate of 20 gm./ton of feed in 1951. In 1952, 10 gm./ton was used. 3 In the 1951 experiment ten,ug./lb. of vitamin B12 added as Merck's APF No. 3. A 0.1 percent triturate of vitamin B12 on mannitol (Merck's) which supplied 7.5 Mg-/lb. of vitamin B12 was used in 1952. 4 Following amounts of B-complex vitamins added (gm./lOO lb. feed): niacin, 0.8; calcium pantothenate, 0.91; choline chloride, 6.8; vitamin B6, 0.2; folic acid, 0.5; and vitamin Bi, 0.09.
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1 2 3
plus 2.00% condensed fish solubles plus 7.50 jug./lb. vitamin Bi 2 l plus 15.00 Mg-/lb. vitamin Bi 2 l plus 0.25% APF supplement No. 5 2 plus 0.50% APF supplement No. 5 2
No. of hens
644
O. G. BENTLEY AND T. V. HERSHBERGER
TABLE 5.—The effect of feeding aureomycin to vitamin Bn deficient hens on hatchability of fertile eggs 1951 Experiment* Ten weeks No. of hens
12 12
Hen ration
Basal (Bn def.) Basal
Av. hatchability of eggs percent 53.5 57.2
Seven weeks Change in hen ration None 20 gm./ton aureomycin added
Av. hatchability of eggs percent 44.1 66.7
with levels frequently used in chick growth experiments. Also, crystalline vitamin B12 was used in place of the crude A.P.F. supplement. To what extent these changes may have influenced the results is not known. In 1951 aureomycin was added to the vitamin B12 deficient basal hen ration after the experiment had been in progress for ten weeks (Table 5). Hatchability increased from 57 percent to 67 percent over a seven-week period, while another lot of hens continued on the basal ration dropped in hatchability from 54 percent to 44 percent. Prior to feeding aureomycin, the hens had been on a vitamin B12 deficient ration for about 20 weeks. Since a marked response in hatchability was obtained by feeding vitamin B12 in the 1951 experiment, a vitamin B12 sparing effect is suggested as a possible explanation for the action of aureomycin in this experiment. However, Petersen et al. (1952) failed to obtain a response in hatchability by feeding either streptomycin or aureomycin to hens severely depleted of vitamin B12 (36 weeks). Previous reports indicated that aureomycin spares vitamin B12 for growth in chicks (Oleson et al., 1950; Stokstad and Jukes, 1951). Cravioto-Munoz et al. (1951), and Linkswiler, Baumann and Snell (1951) found that feeding aureomycin to rats would exert a sparing effect on vitamin B i2 and vitamin B 6 .
A mixture of six B-vitamins in addition to vitamin B12 and riboflavin failed to improve hatchability in this experiment. TABLE 6.—Summary of analysis of variance of chick growth data Mean square
F value
359 2 2 1 1 2
112,329 17,225 266,995 262,116 62,066
46.4f 7.It 110.3f 108.3f 25.6f
4 2
2,321 6,586
.96 2.7
4 341
40,595 2,421
of Source of variation Degrees freedom Total Chick rations Antibiotics B12
Sex Chick ration XB12 Chick ration X antibiotics Bi2Xantibiotics Chick ration XB12 X antibiotics Error
t Significant at 1% level.
16.8f
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* For the complete results of the experiment see Table 4.
In the second experiment (1952), the basal ration was supplemented with aureomycin or bacitracin and fed to two lots of 12 hens each in an attempt to duplicate the 1951 results. Because the expected depression in hatchability due to the lack of dietary vitamin B12 did not occur, it was not possible to get additional information concerning the effect of antibiotics in vitamin B12 deficient rations on hatchability. No explanation can be given for the good hatchability with the basal ration as the vitamin B12 content of the day old chick livers, egg yolk, and hen livers was low (see Table 9), and the carry-over of the vitamin to the progeny was not indicated by the results of progeny growth experiments (see Table 7). Hens that laid more than 200 eggs in their first years' production were used in 1952, while in the two earlier experiments hens with individual records of less than 200 eggs per year were used. Lillie, Olsen and Bird (1951) noted that high producing hens were more difficult to deplete of vitamin B12, as determined by hatchability of their eggs, than were the lower producing hens.
645
EFFECT OF ANTIBIOTICS ON REPRODUCTION
TABLE 8.—Progeny growth from hens fed vitamin Bn with penicillin or baciferm1* Chick ration Supplement to vitamin Bu deficient basal hen ration Vitamin Bis Vitamin Bi:+Baciferm 2.0 lb./ton Vitamin Bia+Procaine Penicillin 10 gm./ton
Total weight, 6 weeks, sex av., gm.
B„
B„+
deficient
+B„
bacitracin
447
445
444
461
436
435
467
466
464
i Combined results of two growth experiments. Each group represents the average of from 23-49 chicks. 2 Baciferm contained 5 gm. per pound bacitracin.
the vitamin B12 deficient chick ration was fed. Pensack et al. (1949) and Petersen et al. (1950) reported similar results. A highly significant carry-over effect from bacitracin in the hen ration occurred when the chicks were fed a vitamin B12 deficient ration. Under similar conditions, aureomycin improved growth and the difference due to treatment approached significance but it did not exceed the 5% level. With the vitamin B12 or vitamin B12 and bacitracin chick rations, the chicks from the hens fed the vitamin B12 basal ration supplemented with antibiotics generally grew better but the differences were not significant at the 5% level. TABLE 7.—The effect of vitamin Bn and antibiotics in If the hens received vitamin B12 in their hens' ration on progeny growth1'2'3 rations there was no statistically significant carry-over effect on progeny growth Chick rations from feeding either aureomycin or baciTotal weight, 6 weeks, tracin. For additional data concerning this sex av., gm. Hen ration supplement observation see Table 3. B12 plus B12 B12 bacitracin Feeding bacitracin to chicks hatched deficient added added from hens fed the vitamin B12 deficient ration tended to improve growth while Vitamin Ba deficient 421 306 405 None there was little effect from bacitracin on 447 401 337 Aureomycin 452 the growth of chicks from hens fed vita428 367 Bacitracin min B12 in these experiments (last 2 Vitamin Bn added columns Table 7). 447 445 444 None 456 428 460 Aureomycin In addition to aureomycin and baci470 446 Bacitracin 451 tracin, procaine penicillin and Baciferm 1 Ten males and ten females per group. were fed to hens as a part of the experi2 Vitamin Bu (0.1% triturate on mannitol) added to chick ration at level of 15^g./lb.; bacitracin ment reported in Table 7. Because cerand aureomycin added at rate of 10 gm./ton. tain dietary treatments were not made 'L.S.D. between groups=33 gm. (5% level); with these antibiotics, i.e., not fed to hens 44 gm. (1% level).
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Progeny Growth Experiments: To determine whether or not bacitracin, aureomycin, or vitamin B12 in the maternal diet would influence the growth performance of the progeny, male and female chicks from hens fed these antibiotics with or without vitamin B12 were placed on three different chick rations, i.e., basal (vitamin B12 deficient), basal plus vitamin Bi2, and basal plus vitamin B12 and bacitracin. The composition of the basal chick ration is given in Table 2. This 4-way factorial experiment was analyzed by an analysis of variance, the results of which are given in Table 6. The addition of either vitamin Bt2 or antibiotics to the basal hen ration had a highly significant effect on the growth performance of their progeny. An inspection of the chick growth data in Table 7 indicates that the effect of vitamin B12 in the hen ration on progeny growth was observed when either a vitamin B12 deficient or a vitamin B12 supplemented chick ration was used; although the most pronounced effect was demonstrated when
646
O. G. BENTLEY AND T. V. HERSHBERGER TABLE 9.—Levels of vitamin Bu in chick and hen livers and egg yolk, ng./100 gm. wet weight (1952 hatchability experiment)
Hen ration supplements 1
Six week old chick livers
Weeks hens were on test rations
Chick rations 2
5
13
17
—B12
+B 1 2
.6 .6 .6 3.4 1.5 3.2
1.7 1.5 2.7 13.1 12.8 12.8
1.1 3.8 1.7 18.0 16.0 26.0
5.5 5.6 4.0 7.6 6.8 5.8
3.5
11.7
15.0
3.6
14.7
2.6
8.7
Egg 3 yolk
Hen livers4
66.0 41.0 54.0 47.0 71.0 71.5
0.25 0.5 0.4 5.5 5.7 4.7
21.9 25.7 22.6 84.7 136.3 74.3
6.1
73.5
5.1
104.3
19.5
6.1
95.0
4.1
58.3
18.5
7.2
88.0
4.6
77.9
1
See Table 1 for complete composition. See Table 2 for complete composition. Yolks from 2 dozen eggs homogenized to obtain samples. 4 Livers taken from 4 hens per dietary group for a composite sample. Both egg and hen liver sample taken at end of 1952 test. 2 3
on a vitamin B12 deficient ration, the results were not treated in the previously mentioned statistical analysis. The data are reported separately in Table 8. Feeding procaine penicillin to the hens appeared to have some effect on chick growth—the growth increase over the controls being about 20 gm. This may indicate a difference in behavior of penicillin in the hen ration, as aureomycin, bacitracin, and Baciferm appeared to have no effect on progeny growth if the hen ration contained vitamin B i2 . These results suggest that bacitracin and probably aureomycin in a breeder ration can exert a carry-over effect on the progeny under certain conditions, namely: when the antibiotics are fed with a vitamin B12 deficient breeder ration, and the chick ration is also deficient in vitamin B12. If adequate vitamin B12 is present in either ration the effect is either not observed or in demonstrated to a limited extent. A vitamin B i2 sparing effect of
antibiotics is suggested as an explanation for some of the growth data obtained under the conditions of our experiments. To determine the effect of feeding antibiotics on the amount of vitamin B12 available to the chicks, vitamin B12 analyses were made on samples of egg yolks, dayold chick and hen livers. The results are given in Table 9. Vitamin Bn Analyses: The concentration of vitamin B12 in composite samples of day-old chick livers obtained from hatches on the 5th, 13th, and 17th week of the 1952 hatchability experiment was unaffected by bacitracin or aureomycin in the maternal ration, while the addition of vitamin Bi2 to the hen ration increased the level of Bi2 in the day old chick livers 6 to 10 times. Likewise, samples of egg yolks and livers from each of the dietary groups of hens revealed no increased vitamin BX2 storage as a result of feeding antibiotics. Tappan et al. (1950) reported that feeding aureomycin to rats did not influence
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Basal (B12 deficient) Basal plus aureomycin Basal plus bacitracin Basal plus vitamin Bi2 Basal plus vitamin B12 plus bacitracin Basal plus vitamin B12 plus baciferm Basal plus vitamin B ]2 plus aureomycm Basal plus vitamin B12 plus procaine penicillin Basal plus vitamin Bi2 plus B-vitamin mix
Day old chick livers
EFFECT OF ANTIBIOTICS ON REPRODUCTION
SUMMARY Three hatchability experiments, each of from 16 to 20 weeks' duration, have been carried out using an all-plant breeder ration for hens. Progeny growth studies to determine the carry-over effect of the supplements in the maternal ration on progeny growth performance were also made. In addition, the vitamin B12 content of day-old chick livers, egg yolks, and 6 week old chick livers, as determined with L. liechmannii ATCC 7830, was obtained. The results are summarized as follows: 1. Feeding antibiotics (bacitracin, terramycin, aureomycin • HC1, or procaine penicillin) to hens did not consistently improve hatchability of the fertile eggs. However, in one experiment there was some increase in hatchability with either bacitracin or aureomycin • HC1, both in the presence and absence of supplementary vitamin B i2 . 2. Bacitracin and probably aureomycin • HC1, in the maternal diet appeared to exert a significant carry-over effect on the progeny growth if the antibiotics were added to a vitamin B12 deficient breeder ration and the chicks were fed a vitamin B12 deficient ration. Little or no effect on progeny growth was observed when antibiotics were added to the vitamin B12 supplemented hen ration, although there appeared to be a trend toward better growth performance in chicks from hens fed vitamin B12 and procaine penicillin. 3. The level of vitamin B12 in day-old chick livers, egg yolks, and hens' livers was not increased by feeding the antibiotics to hens. This would indicate that any carry-over effect from bacitracin or aureomycin from the hen to the progeny was probably not mediated directly through the release of more vitamin B12 from the egg yolk to the chick.
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liver vitamin B12 levels. Vitamin B12 levels for day-old chick livers and egg yolk, reported in Table 9, compare favorably with results published by Halick and Couch (1951) and Sunde, Halpin and Cravens (1952), but the value for egg yolks are somewhat higher than those reported by Yacowitz et al. (1952). A vitamin B12 sparing effect of antibiotics in a growing rat or chick could be explained by the action of antibiotics on the intestinal flora. However, if the antibiotic effect is to be transmitted to the progeny, as is indicated by the chick growth data (Tables 7 and 8), it would seem that either vitamin B12 or a factor that exerts a vitamin Bi2-like growth effect on chicks but which does not stimulate the growth of L. leichmannii must be deposited in the egg from whence the chick can obtain the nutrient. Since no increased storage of vitamin B12 in the eggs was observed, there is a possibility that a growth promoting factor, other than vitamin B12, was stored in the egg as a result of feeding antibiotics. This possibility is now under study using rats to assay the egg yolks. Waibel, Sunde and Cravens (1952) found that feeding penicillin to hens resulted in greater storage of folic acid and biotin in the egg yolk. Grieff and Pinkerton (1951) reported that the growth of Rickettsiae cultured in embryos from hens fed terramycin was inhibited. Wong et al. (1953) failed to corroborate this observation and reported no detectable storage of terramycin or penicillin in the egg even when 1,000 gms. per ton of feed was used. Nevertheless the results reported herein would confirm the findings of Slinger et al. (1952), Carlson et al. (1953) and Sizemore et al. (1953) which suggest that under certain conditions antibiotics in the maternal ration may influence progeny growth performance.
647
648
0. G. BENTLEY AND T. V. HERSHBERGER ACKNOWLEDGEMENTS
The authors wish to thank Mr. D. C. Kennard, Dr. E. N. Moore, and Mr. V. D. Chamberlin of the Poultry Science Department, Ohio Agricultural Experiment Station, for supplying the hens used in this study, and Dr. C. R. Weaver, Entomology Department, Ohio Agricultural Experiment Station, for his help with the statistical analyses.
Berg, L. E., J. S. Carver, G. E. Bearse and J. McGinnis, 1952. Antibiotics in the nutrition of laying hens. Washington Agr. Exp. Sta. Bui. No. 534: 1-10. Carlson, C. W., E. D. Jones, R. A. Wilcox and W. Kohlmeyer, 1952. Antibiotics in breeder diets. Poultry Sci. 31:910. Carlson, C. W., D. G. Jones, W. Kohlmeyer and A. L. Moxon, 1953. The effect of vitamin B l 2 and aureomycin on reproductive performance in turkeys. Poultry Sci. 32:984-989. Cravioto-Munoz, J., H. G. Poncher and H. A. Waisman, 1951. Vitamin B12 sparing action of aureomycin in the rat. Proc. Soc. Expt. Biol. Med. 77: 18-19. Elam, J. F., L. L. Gee and J. R. Couch, 1951. Effect of feeding penicillin on the life cycle of the chick. Proc. Soc. Expt. Biol. Med. 77: 209-213. Grieff, D., and H. Pinkerton, 1951. Rickettsiostasis in fertile eggs from the use of antibiotic residues in poultry feeds. Proc. Soc. Expt. Biol. Med. 78: 690-695. Halick, J. V., and J. R. Couch, 1951. Antibiotics in mature fowl nutrition. Proc. Soc. Expt. Biol. Med. 76: 58-62. Lillie, R. J., and H. R. Bird, 1952. Effect of antibiotic supplements upon hatchability and upon growth and viability of Progeny. Poultry Sci. 31: 513517. Lillie, R. J., M. W. Olsen and H. R. Bird, 1951. Variation in reproductive response of hens to dietary deficiency. Poultry Sci. 30: 92-97. Linkswiler, H., C. A. Baumann and E. E. Snell, 1951. Effect of aureomycin on the response of rats to various forms of vitamin Be. J. Nutrition, 43:565573. Mariakulandai, A., T. Myint and J. McGinnis, 1952. Effect of terramycin and vitamin Bu on hatchability. Proc. Soc. Expt. Biol. Med. 79: 242-244. Oleson, J. J., B. L. Hutchings and A. R. Whitehill,
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1950. The effect of feeding aureomycin on the vitamin B12 requirement of the chick. Arch. Biochem. 29: 334-338. Pensack, J. M., R. M. Bethke and D. C. Kennard, 1949. The effect of fish meal and extracts of fish meal on hatchability of hens' eggs and growth of progeny. Poultry Sci. 28:398-405. Pepper, W. F., S. J. Slinger and I. Motzok, 1952. Effect of aureomycin on the manganese requirement of chicks fed varying levels of salt and phosphorus. Poultry Sci. 31: 1054-1061. Petersen, C. F., A. C. Wiese, R. V. Dahlstrom and C. E. Lampman, 1952. Influence of vitamin B12 and antibiotics on hatchability. Poultry Sci. 31: 129-132. Petersen, C. F., A. C. Wiese, C. E. Lampman and R. V. Dahlstrom, 1950. Role of crystalline vitamin B12 for hatchability. Poultry Sci. 29:618-619. Sizemore, J. R., R. J. Lillie, C. A. Denton and H. R. Bird, 1953. Influence of aureomycin in the chick diet upon subsequent reproductive performance of laying hens. Poultry Sci. 32: 618-624. Skeggs, H. R., H. M. Nepple, K. A. Valentik, J. W. Huff and L. D. Wright, 1950. Observations on the use of Lactobacillus leichmannii 4797 in the microbiological assay of vitamin B12. J. Biol. Chem. 184: 211-221. Slinger, S. J., A. E. Ferguson and J. D. McConachie, 1952. A carry-over effect of penicillin from dam to progeny. Poultry Sci. 31: 172-174. Stokstad, E. L. R., and T. H. Jukes, 1951. Effects of various levels of vitamin B12 upon growth response produced by aureomycin in chicks. Proc. Soc. Expt. Biol. Med. 76: 73-76. Sunde, M. L., J. G. Halpin and W. W. Cravens, 1952. The effect of vitamin B12 supplements and antibiotic feed supplements on egg production and hatchability. Poultry Sci. 31: 617-620. Tappan, D. V., U. J. Lewis, U. D. Register and C. A. Elvehjem, 1950. Further studies on the rat growth assay for vitamin Bi2 activity. Arch. Biochem. 29:408-412. Waibel, P. E., M. L. Sunde and W. W. Cravens, 1952. Effect of addition of penicillin to the hen's ration on biotin and folic acid content of eggs. Poultry Sci. 31:621-624. Wong, S. C , R. C. Kersey, J. R. McMahon, W. M. Reynolds, W. K. Warden and J. McGinnis, 1953. Effects of feeding terramycin and penicillin to laying hens on antibiotic deposition in eggs and growth of marine typhus rickettsiae in embryonated eggs. Fed. Proc. 12: 434. Yacowitz, H., R. F. Miller, L. C. Norris and G. F. Heuser, 1952. Vitamin B12 studies with the hen. Poultry Sci. 31:89-94.