KENNARD and Chamberlin (1948)

Transcription

1 The Effect of Management on the Vitamin B 12 Content of Poultry House Litter II. AS DETERMINED BY MICROBIOLOGICAL ASSAY 1 E. R. HALBROOK, 2 T. S. SUTTON AND A. R. WINTER Departments of Poultry Husbandry and Agricultural Biochemistry, Ohio State University, Columbus KENNARD and Chamberlin (1948) have shown that chicks reared on poultry house litter which has been "built-up" or used with additions from time to time, rather than being cleaned out, grow more rapidly on an all-vegetable diet deficient in the "aniknal protein factor" than when the litter is changed frequently. Similarly, higher hatchability of eggs from hens kept on built-up litter was observed by Kennard, Bethke, and Chamberlin (1948) as compared to eggs from hens kept on frequently changed litter and fed an all-vegetable diet. (Received for publication March 13, 1950) These observations relative to chick growth have been confirmed by Halbrook, Winter and Sutton (1950) and on hatchability by Schlamb and Winter (1948). Rubin, Bird and Rothchild (1946) observed that the "animal protein factor" was present in hen feces, and McGinnis et al. (1947) that incubating hen feces for 72 hours at 30 C. stimulated further synthesis of the factor as determined by chick growth tests. EXPERIMENTAL PROCEDURE In a series of experiments in which a study was made of the relationship of 1 Part I of this series gave the results of chick growth assays: 2 Present address: Montana State College, Bozeman. 679 litter management to the vitamin Bi 2 requirement in the diet of chicks and as reported in Part I of this series (Halbrook et al., 1950) an analysis was made of the vitamin Bi 2 content of the litter and of the chick droppings. The microbiological assay procedure used was essentially as described by Skeggs et al. (1948) and modified by Capps et al. (1949). A modification in the procedure was to add 0.1 percent of sodium thioglycollate to the assay medium as recommended by Stokstad el al. (1949) and a second modification was to adjust the medium to ph 5.7 instead of 6.8 in accord with observations by Wright (1949) and Lewis et al. (1949). Lactobacillus leichmannii (ATCC 4797) was used as the assay organism. Turbidimetric determination of growth was made with a Coleman Universal spectrophotometer with a wavelength of 610 millimicrons. Crystalline vitamin B12 in saline solution (Cobione*) was used as the standard of comparison. Both droppings and litter were first diluted 1 to 10 with distilled water, autoclaved for 15 min. at 15 lb. pressure, and then the supernatant used for making further dilutions to give the desired range for best assay. In general a dilution of 1 part in 10,000 to 50,000 was required to * Supplied by Merck and Co. Inc., Rahway, N.J., through the courtesy of Dr. D. F. Green.

2 680 E. R. HALBROOK, T. S. SUTTON AND A. R. WINTER Date of collection Age of chicks give the desired assay range. With the use of L. leichmannii (ATCC 4797) and an assay medium ph of 5.7, a range of 0.01 to millimicrograms of vitamin Bi 2 per tube (10 ml.) was found to be best for assay readings. The litter used was ground corn cobs. The built-up litter used by the chicks had previously been used for approximately one year by hens, dams of the chicks, fed an all-vegetable vitamin Bi2-deficient diet so that there was little chance of an accumulation of vitamin B12 in the litter due to feed content. The assays on droppings were made on fresh droppings collected on wax paper from birds placed on a wire floor with the paper beneath. The droppings were handled with a sterile spatula and placed in sterile containers in a manner suitable for making microflora counts, so there was little opportunity for appreciable microflora change or vitamin B i2 synthesis in view of the fact that samples were also autoclaved immediately. Litter was removed from the brooding pens from time to time during the eightweek brooding period and assayed microbiologically for its vitamin B J2 content. New litter and laying house litter were also analyzed. Fresh droppings were collected from these experimental chicks under different litter management and assayed from time to time during the eight-week brooding period. RESULTS AND DISCUSSION It will be observed from figure 1 that fresh cobs have a very low vitamin B ]2 content. The average of five analyses was 1.1 millimicrograms per gram of litter with a range of 0.75 to 1.5. A short period of use by chicks or hens increased this content rapidly as shown in table 1. Cob litter in use by pullets for a few weeks analyzed 24 millimicrograms per gram of litter with analyses varying from 15 to 37. Chick litter 5 weeks old showed an average value of 50 millimicrograms of vitamin Bi 2 per gram. At seven weeks this content was increased to 107 and at eight weeks to 124. Built-up litter over 1 year old from the parent breeding flockpen averaged 261 millimicrograms per TABLE 1. Summary of litter vitamin Bn assays on basis of diet and management (Vitamin B12 indicated in millimicrograms per gram of litter) Diet and Litter Management 1. Basal. Litter changed wkly. 2. Basal. Litter unchanged 3. Basal. Litter unchanged-r-h Basal. Litter unchanged-i-h 2 0+CoCl2 5. Basal. Built-up litter 6. Basal. Built-up litter+h Basal.+B12 supplement litter unchanged 8. Basal.+CoCk litter unchanged A. Experimental Chick Pens Dec. 1 5 wks. 55/56 28/ / /175 78/52 68/52 Dec wks. 105/131 73/86 143/ /192 87/ /145 Dec / / / / / /147 94/141 Dec /133/103 78/75 272/210/ /255/ Fresh corn cobs 2. New cob pullet litter 3. Built-up hen litter B. Other Litter Assays 0.75; 0.96; 0.80; 1.5; ; 37; 30; 20; ; 233; 250; 263; 255 Av. m/ig. Bi2/gram

3 VITAMIN BI 2 IN POULTRY HOUSE LITTER 681 ' FRESH UAJSED COBS growth was stimulated some as reported elsewhere (Halbrook et al. 1950). The average of all analyses on 54 samples of fresh droppings was 92 millimicrograms per gram on a wet basis or approximately 460 millimicrogiams per gram on a dry weight basis, as shown in table 2. The vitamin B J2 content of the droppings varied with litter management. The average analysis of 24 samples of droppings coming from chicks receiving the basal vitamin B 12 -deficient diet on unchanged litter (used from 1 to 8 weeks) was only 56 millimicrograms as compared to 107 for 16 samples of droppings coming from chicks on built-up litter. These data are shown in table 2. THE EFFECT OF MOISTURE AND COBALT ADDITION TO LITTER ON THE VITAMIN B 12 CONTENT OF DROPPINGS The addition of moisture to the litter at the rate of 1 gallon per 24 sq. ft. of floor space weekly had no tendency to increase the vitamin B12 content of the droppings which is in agreement with the results reported for litter above. The average vitamin B 12 content of droppings was slightly lower where moisture was added. The addition of cobalt chloride either to the litter or to the diet at the levels previously indicated seemed to have a definite tendency to lower the vitamin Bi 2 content of the droppings. Since chick growth had been increased TABLE 2. Summary of the vitamin Ba assays of fresh droppings on the basis of diet and management (Vitamin B12 indicated in millimicrograms per gram wet basis) Date of collection Age of chicks COB LITTER USED BY CHICKS 5 WEEKS LITTER USED BY CHICKS 7 WEEKS Diet and Litter Management 1. Basal. Unchanged Litter 2. Basal. Unchanged Litter+H.0 3. Basal. Unchanged Litter-j-HiO+CoCh 4. Basal. Built-up litter 5. Basal. Built-up litter +H50 5. Basal.+B12 Litter unchanged 7. Basal.+CoCls Litter unchanged 8. Basal. On wire 9. Basal.+Bn on wire LITTER USED BY CHICKS 8 WEEKS BUILT-UP LITTER OVER I TS R OLD I FIG. 1. The effect of age of poultry house litter on the vitamin Bi 2 content. gram of litter in five assays with variations shown of 233 to 304. This value of 261 was also the exact figure secured for 22 assays of built-up litter in use by the experimental chicks. Neither the addition of water to the litter nor cobalt chloride to either litter or diet at the rate of 2 gms. per sq. ft. of floor space for litter and 3 gm. per 100 lb. of diet, seemed to have any effect on the vitamin Bi 2 content of the litter when assayed microbiologically, although chick Oct wks Nov wks. 35/48 182/202 Nov wks. 30/32/ / / /17 238/ Dec. 8 6 wks Dec wks Dec /110 83/93 42/51 69/83 27/36 151/ /98 80/91 Dec

4 682 E. R. HALBROOK, T. S. SUTTON AND A. R. WINTER slightly in all tests where cobalt chloride was added to the litter, it might be possible that cobalt has a beneficial action by in some manner increasing the absorption of the vitamin Bi 2 available in the diet. Another possible explanation of the action of cobalt is suggested by the results of cobalt and vitamin B12 studies with lambs as reported by Smith (1949) combined with our own observation with chicks that growth stimulation was obtained by adding cobalt chloride to the litter but no vitamin Bj 2 increase in the litter was observed on microbiological assay. Smith observed that the feeding of cobalt or the feeding or injection of anti-pernicious liver extracts were beneficial in cobalt deficient lambs whereas the injection of crystalline vitamin B i2 was without beneficial effects. He assumed from his experiments that cobalt has a major function other than vitamin Bi 2 synthesis in the lambs. These results with both chicks and lambs and microbiological assays of litter suggest the possibility that cobalt may stimulate the growth or activity of certain organisms that produce an essential factor or factors other than vitamin Bi 2. The addition of commercial APF supplement to the basal diet on unchanged litter caused an increase in the vitamin Bi 2 content of the droppings. THE SYNTHESIS OF VITAMIN B 12 IN DROPPINGS BY INCUBATION The vitamin B 12 content of fresh droppings was higher than expected. However, as shown in figure 2, in one trial when droppings were stored at 30 C. for 1 week the vitamin B J2 content increased to 513 millimicrograms per gram (wet weight basis) as compared to 244 when stored at 37 C. and only 50 when stored at 4 C. In a second trial involving more samples FRESH DROPPINGS (AV. OF 54 SAMPLES) STORED FOR I WEEK AT 4»C. STORED FOR I WEEK AT 30 C. STORED FOR I WEEK AT 37 C. FIG. 2. The effect of storage at various temperatures on the vitamin Bi 2 content of poultry droppings. and with storage at laboratory temperature (approximately 25 C.) and at 4 C, the increase was from 41 millimicrograms per gram (wet weight) at 4 to 134 at room temperature storage. The room temperature of approximately 25 C. may have been too low for maximum synthesis. It is apparent, however, from the studies made that a marked increase in vitamin Bj 2 content of droppings does occur on storage at temperatures from 25 to 37 C. and substantiates the earlier work with chick growth assay by McGinnis et al. (1947). VITAMIN B 12 CONTENT OF LITTER VS. DROPPINGS Reference has already been made to the general tendency of the vitamin B J2 content of droppings to correlate with the litter on which the chicks run. For

5 VITAMIN BK IN POULTRY HOUSE LITTER 683 example, the average analysis of litter from unchanged litter pens in which the basal vitamin B12 deficient diet was fed showed a vitamin Bj2 value of 110 millimicrograms per gram as compared to 208 for built-up litter with the same basal diet. The average vitamin B12 values of droppings of these same chicks were 73 and 125 millimicrograms per gram respectively on a wet basis or approximately 5 times this on the dry weight basis. These values correlate with chick growth to 8 weeks of 514 grams on the basal diet on unchanged litter as compared to 634 for chicks fed the basal diet on built-up litter. TEST FOR MICROBIOLOGICAL ASSAY STIMULATING SUBSTANCES OTHER THAN VITAMIN B 12 It was realized that there was a possibility of other growth-stimulating substances other than vitamin B12 being present in the poultry house litter and droppings. This possibility was checked by the NaOH treatment used by Hoffman et al. (1949) in which assays were made before and after treating with NaOH. It was found that autoclaving built-up litter and droppings after adding NaOH to a ph of 12 destroyed 74% of the activity in litter and 86% in droppings and may represent the amount of activity due to vitamin Bi 2. On the other hand, this also represents approximately the destruction of crystalline vitamin B12 added to the litter and droppings. SUMMARY 1. Vitamin B i2 microbiological assays of built-up corn cob litter (used for over 1 year) showed an average content of 261 millimicrograms of vitamin B 12 per gram (118 micrograms per pound) as compared to only 1 millimicrogram per gram for new cobs and 110 for unchanged cob litter used by chicks for 1 to 8 weeks. 2. Five weeks of litter use for chick brooding increased the vitamin B12 content of new cobs from 1 to 50 millimicrograms per gram. Eight weeks of use for chick brooding increased it to 124 millimicrograms per gram. 3. Fifty-four microbiological vitamin B12 assays of fresh droppings of chicks on different diets and different litter managements showed an average analysis of 92 millimicrograms of vitamin B ]2 per gram wet weight or 460 on a dry weight basis. 4. The droppings of chicks fed a vitamin B] 2 -deficient basal diet on built-up litter was as high as those from chicks fed the basal plus a commercial vitamin B i2 supplement at the rate of 20 micrograms per kilo of diet and was 71 percent more potent than the droppings of chicks fed the basal only on new unchanged litter. (This corresponded with chick growth data.) 5. Storage of poultry droppings at 30 C. for 1 week showed over a 900 percent increase in vitamin Bi 2 as compared to 4 C. storage, while storage at 37 C. showed an increase of 390 percent. Storage at room temperature (approximately 25 C.) showed a 226 percent increase. REFERENCES Capps, B. F., N. L. Hobbs and S. H. Fox, A method for the microbiological assay of vitamin B, 2. J. Biol. Chem. 178: Halbrook, E. R., A. R. Winter and T. S. Sutton, The effect of management on the vitamin B12 content of poultry house litter. I. As measured by chick growth. Poultry Sci. 29: Kennard, D. C. and V. D. Chamberlin, Built-up floor litter as a source of dietary factors essential for the growth of chickens. Poultry Sci. 27: , R. M. Bethke and V. D. Chamberlin, Built-up floor litter a source of dietary factors esential for hatchability of chicken eggs. Poultry Sci. 27: Lewis, J. C, K. Ijichi, N. S. Snell and J. A. Baribaldi, Fermentation process for production of vitamin Bi 2. Bureau of Agri. and Industrial Chem. (U.S.D.A.) Mimeo. Pub. 254: 1-7.

6 684 NEWS AND NOTES McGinnis, J., J. M. Stevens and K. Groves, The in vitro synthesis of a chick growth promoting [factor in hen feces. Poultry Sci. 26: Rubin, M., H. R. Bird and I. Rothchild, A growth promoting factor for chicks in the feces of hens. Poultry Sci. 25: Schlamb, K. F., and A. R. Winter, An evaluation of dried distillers feedstuffs fed to chickens throughout their life cycle. Poultry Sci. 27: Smith, J. E., Cobalt studies with livestock. Proceedings of the 1949 Cornell Nutrition Conference for Feed Manufacturers, pp Stokstad, E. L. R., A. C. Dornbush, A. L. Franklin, C. E. Hoffman, B. L. Hutchings and T. H. Jukes, Microbiological assay of vitamin B12 by Lactobacillus leichmannii. Federation Proc, Part I, 8: 257. Wright, Marjory H., Thymidine and vitamin Bi 2. Science 110: teacher recognition program started this year by the Alumni Association. MINNESOTA NOTES Peters Hall, the new animal and poultry husbandry building on the St. Paul campus of the University of Minnesota, was dedicated at ceremonies on August 30. The building is one of several being dedicated at the University of Minnesota during the academic year , which has been designated as "University of Minnesota Centennial Year." The University was chartered by the Minnesota territorial legislature February 25, Peters Hall is being named after the late W. H. Peters, chief of the division of animal husbandry at Minnesota until his death August 8, C. H. Bailey, dean of the University of Minnesota College of Agriculture, presided at the ceremonies. Speakers included J. L. Morrill, president of the University of Minnesota, who spoke in behalf of the University; C. E. Snyder, editor of the Drovers Journal, Chicago, an old friend of Professor Peters, representing the livestock industry; and L. E. Card, head of the department of animal science, University of Illinois, in behalf of the poultry industry. News and Notes {Continued from page 665) Open house was held following the dedication ceremony. Professor Peters devoted his life to improving the U. S. livestock industry. Honors which came to him included the hanging of his portrait in the world-famous gallery of the Saddle and Sirloin Club in Chicago, an honor conferred on members of the American Society of Animal Production who do outstanding work in teaching and research. His portrait also hangs in the Master Livestock Breeders' Gallery on the St. Paul Campus of the University of Minnesota. Professor Peters, born July 9, 1895 on an Iowa farm, was graduated with honors from Iowa State College in He served as head of the animal husbandry sections at Manitoba Agricultural College and North Dakota Agricultural College before coming to the University of Minnesota in During his years as chief at Minnesota, the work of his division increased nearly four-fold, and an extensive research program was developed. Professor Peters served as president of the American Society of Animal Production in He was widely known as a magazine livestock editor and was the author of textbooks on livestock production used in colleges {Continued on page 706)