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Phosphorus in the Nutrition of the Adult Hen1
Phosphorus in the Nutrition of the Adult Hen 1 3. THE INFLUENCE OF PHOSPHORUS SOURCE AND LEVEL ON CAGE LAYER OSTEOPOROSIS (CAGE LAYER FATIGUE) E. P. SINGSEN, C. RIDDELL, L. D. MATTERSON AND J. J. TLUSTOHOWICZ Departments of Poultry Science and Animal Diseases, University of Connecticut, Storrs, Connecticut 06268 (Received for publication August 13, 1968)
D
1 Scientific Contribution No. 331, Agricultural Experiment Station, University of Connecticut, Storrs.
be responsible. Simpson et al. (1964) found histological evidence of bone damage in caged pullets fed diets low in phosphorus. These birds exhibited symptoms similar to CLF, and the authors suggested that phosphorus deficiency may be implicated in the syndrome. Riddell et al. (1968) presented evidence that the basic pathological defect responsible for the typical posterior paralysis symptom of CLF was a compression fracture in the fourth or fifth thoracic vertebra, with associated compression and degeneration of the spinal cord. They also concurred with Urist (1960) that the syndrome could more appropriately be called cage layer osteoporosis. The objective of this report is to present the nutritional aspects of three experiments involving dicalcium phosphate and Curacao rock phosphate as dietary phosphorus supplements for caged layers, the second and third of which produced the osteoporosis cases studied by Riddell et al. (1968). MATERIALS AND METHODS
The low phosphorus basal ration (0.20% total P) used in these experiments was the same as described by Singsen et al. (1969). Three percent calcium was maintained in all diets. Experiments 1, 2, and 3A were conducted in 11"X18" cages in a windowless, heated, force-ventilated room. The cage laying room was kept under strict quarantine, and there was no evidence of contagious disease,
394
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URING a series of experiments conducted in this laboratory between 1961 and 1967 on the relative availability of phosphorus from various sources for caged hens, unexpected mortality was observed to which osteoporosis appeared to be a major contributing factor. The syndrome resembled "cage layer fatigue" (CLF) as first described by Couch (1955) and later in greater detail by Grumbles (1959). A similar condition has since been reported elsewhere in the world by Bell and Siller (1962, Scotland), Gardiner (1964, Ireland), Smetana (1965, Australia), and Hartwigk (1966, Germany). Genetic differences in susceptibility were reported by Francis (1957) based on variations in incidence of CLF from 0.65% to 3.95% in seven strains of birds. Several reports have implicated skeletal weakness or damage in the condition. Urist and Deutsch (1960) described severe osteoporosis in White Leghorn hens in heavy egg production and noted that it did not occur in immature and young mature pullets or cockerels. Urist (1960) suggested that the name "cage layer fatigue" be changed to cage layer osteoporosis (CLO). He noted similarities between field cases of CLF and osteoporosis as studied in his laboratory and suggested that a hereditary disease of bone rather than nutitional or endocrine factors may
395
PHOSPHORUS SOURCE AND OSTEOPOROSIS TABLE 1.—The influence of phosphorus source and level on egg production and mortality {Experiment 1, combined data, 2 replicates) Dicalcium phosphate1
Source of phosphorus Total phosphorus, % Added phosphorus, %
0.34 0.14
0.48 0.28
Average egg production, % (hen-day basis) Combined data, total % 0.34+0.48% P vs. 0.62+0.76% P Mortality, % Combined data, total % 0.34+0.48% P vs.
0.62+0.76% P 1
Curacao rock phosphate* 0.76 0.56
0.34 0.14
0.48 0.28
69.7
0.62 0.42
0.76 0.56
72.8
73.2
68.7 13.3
16.7
3.3
16.7
25.0
20.0
12.50 15.0
10.0
22.5
15.0
18.3 19.58 16.7
Dynafos, International Minerals and Chemical Corporation, Skokie, Illinois, containing 18.79% phosphorus and 21.30% calcium. Curacao Island rock phosphate, containing 14.60% phosphorus and 35.34% calcium.
other than leucosis, during these experiments. Thirty Leghorn-type pullets, housed two in a cage comprised each replicate in Experiments 1, 2, and 3A, and all treatments were replicated twice. Part B of Experiment 3 was conducted in 12"X12" colony houses on the University poultry farm. Four pens of 40 pullets each were housed on raised wire floors, and five pens, three of 40 and two of 50 pullets each, on litter. The treatments were not replicated. Arbor Acres stock was used in Experiments 1 and 3, and Hy-Line stock in Experiment 2. Several bags of each phosphate source were obtained, blended, and used throughout each experiment. The phosphorus sources and mixed diets were analyzed periodically for calcium and phosphorus (Table 1). Phosphorus was determined according to the method of Fiske and Subbarow (1925) and calcium by the A.O.A.C (1950) method. The samples were solubilized by the wet oxidation method of Reitz et al. (1960). Analysis of variance followed the method of Snedecor (1956). EXPERIMENTAL AND DISCUSSION
Experiment 1 was designed to explore the relative effectiveness of phosphorus from dicalcium phosphate and Curacao rock phosphate when fed at both sub-
optimum and adequate levels. Singsen et al. (1969) had noted increased mortality in birds receiving diets supplemented with 0.14% phosphorus (0.34% total) from the Curacao product, and therefore levels of 0.34, 0.48, 0.62, and 0.76% total phosphorus were chosen for this experiment. The results, summarized in Table 1, indicate a trend toward higher egg production as phosphorus level increased from either source, but the differences were not statistically significant. Average mortality was higher for the groups receiving the Curacao rock phosphate, confirming the observation of Singsen et al. (1969). Analysis of variance indicated that the differences in mortality due to both source and level of phosphorus were statistically significant with F values of 9.11 and 4.92 respectively where 5.32 and 4.07 represented the 5% level of significance. Combining the data on a total by source basis and on the two higher vs. the two lower levels within sources brings out these differences more clearly. I t was apparent therefore that increasing the total phosphorus to an adequate level had not eliminated the mortality differential due to phosphorus source. During the course of Experiment 1, periodic necropsy reports from the Department of Animal Diseases indicated
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2
0.62 0.42
396
E.
P.
SINGSEN,
C.
RIDDELL, L.
D.
MATTERSON AND J.
Experiment 2 was designed to test the possibility that sodium fluoride was contributing to the increased mortality caused by Curacao rock phosphate. Quantitative chemical analyses of the dicalcium and Curacao phosphates were conducted in the laboratory of Dr. H. J. Fisher, chief chemist, the Connecticut TABLE 2.
TLTJSTOHOWICZ
Agricultural Experiment Station, New Haven, Connecticut. The dicalcium and Curacao phosphates contained 0.039 and 0.22% sodium and 0.075 and 0.132% fluorine respectively. These tests indicated sodium to be present in the Curacao product in excess of that which could be accounted for as sodium chloride, suggesting the presence of approximately 0.12% sodium fluoride, a much more toxic material than the normally present calcium fluoride. Qualitative spectrographic analyses yielded no further leads. The plan of experiment and results are shown in Table 2. Sodium fluoride, at the levels fed in this experiment, had no effect on any of the performance characteristics measured. Analysis of variance indicated that the differences in both egg production and mortality due to phosphorus source were statistically significant, with F values of 9.025 and 8.015 respectively where 5.99 represented the 5% level of significance. Since sodium fluoride exerted no influence on results, the mortality and necropsy data from all groups were combined as shown in Table 3 to permit study of the relationship between phosphorus source and cause of death. The total mortality was 11.25% for the birds receiving dicalcium phosphate and 20.83% for those receiving Curacao rock phosphate. The difference was due entirely to the inci-
The effect of phosphorus source and sodium fluoride on the performance of caged layers (Experiment 2, combined data, 2 replicates) Dicalcium phosphate
Phosphorus source Total phosphorus, % Sodium fluoride, m g . A g . diet* Egg production, % (hen-day basis) Mortality, % Live body weights, gms. Final Gain Feed consumed, g m s . / h e n / d a y Average egg weights, gms. (252) days)
0.62 0 71.2
0.62 12 69.7
0.62 24 66.7
Curacao rock phosphate 0.62 48 67.4
0.62 0 64.8
0.62 12 65.9
0.62 24 65.7
0.62 48 65.9
8.3
11.6
16.6
8.3
23.4
16.6
15.0
28.4
1,619 1,776 157 96.3
1,623 1,829 206 95.2
1,638 1,805 167 93.1
1,637 1,840 203 91.8
1,612 1,789 177 90.5
1,614 1,712 98 91.9
1,636 1,798 162 95.7
1,664 1,742 78 89.5
61.8
60.9
60.6
61.3
61.0
60.5
60.8
60.7
* J. T. Baker Chemical Company, reagent sodium fluoride, assay 100%.
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that osteoporosis was a frequent finding. In Experiments 2 and 3, therefore, a detailed necropsy of all birds that died was undertaken by one of the authors (C.R.). A few birds in which excessive deterioration had occurred prior to necropsy are reported as "not examined" in the tabular summaries. Most birds diagnosed as CLO were exhibiting typical symptoms of paralysis, and all CLO diagnoses were confirmed at necropsy by either (1) gross examination, (2) histological study of sections from the sixth rib, femur, and tibia, or (3) radiographs of the spinal column. Details of the bone pathology have been reported by Riddell et al. (1968). Unfortunately, the complete necropsy and histological data were not available until the end of Experiment 3 and therefore could not be used in planning the experiments. When available, however, they permitted a complete reexamination of the experiments from a cause of death point of view, and the findings are presented with each experiment.
J.
397
PHOSPHORUS SOURCE AND OSTEOPOROSIS TABLE 3.—The relationship between phosphorus and cause of death, based on necropsy (Experiment 2, combined data, 8 replicates) Phosphorus source
Diagnosis
%
Dicalcium phosphate (0.62% P)
Cage layer osteoporosis Leucosis Miscellaneous diagnoses Not examined
1.25 2.92 5.42 1.66
Curacao rock phosphate (0.62% P)
Cage layer osteoporosis Leucosis Miscellaneous diagnoses Not examined
Total
11.25 10.00 4.17 3.33 3.33
Total
20.83
TABLE 4.—The effect of phosphorus source and level on the performance of caged layers [Experiment 3A, combined data, 4 replicates) Phosphorus source Total phosphorus, % Average egg production, % (hen-day basis) Mortality, % Total by P source, % Feed consumed, gms./hen/day
Dicalcium phosphate
Curacao phosphate
0.34 66.8
0.76 67.3
0.34 61.5
0.7( 65.8
22.5
8.3
35.0
24.2
90.2
91.7
15.4 90.8
29.6 91.1
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dence of CLO, 1.25% vs. 10.00%, while mortality from all other causes amounted to 10.00% and 10.83% respectively for the dicalcium phosphate and Curacao rock phosphate. Gain in body weight averaged 183 grams and 123 grams respectively on the two phosphate sources, again confirming the observations of Singsen el al. (1969). Experiment 3 was conducted in two parts, A and B, and involved (1) vitamin D source, (2) phosphorus source and level, and (3) housing environment. In Part A, conducted in cages, an irradiated animal sterol and a combination of cod liver oil and tuna liver oil of known potencies, were fed to provide 1323 I.C.U. of vitamin D per kg. of diet. Two levels of total phosphorus, 0.34% and 0.76% (0.14% and 0.56% added) from dicalcium phosphate and Curacao rock phosphate, were fed with each vitamin D source. The diets were mixed every 14 to 21 days in order to minimize vitamin D destruc-
tion. At the end of the experiment it was obvious that both vitamin D sources were equally effective, since total mortality was identical, 22.4% on each source, and the egg productions, 66.7% (dry D3) and 63.9% (oils), were not significantly different. The experimental plan and results presented in Table 4 have therefore been simplified by combining data from both vitamin D sources. Average egg production was somewhat lower than in previous experiments primarily because there was no significant increase at the 0.76% level of total phosphorus. The differential in mortality, however, followed the same pattern observed in previous experiments, totaling 15.4% for the dicalcium phosphate fed groups and 29.6% for those receiving Curacao rock phosphate. The cause of death based on necropsy has been summarized in Table 5. Total mortality was effectively decreased by increasing total phosphorus from either source, but the effect was much more pronounced with the birds fed dicalcium phosphate. Furthermore, 0.56% supplemental phosphorus from Curacao rock phosphate was no more effective than 0.14% from dicalcium phosphate. Again, as in Experiment 2, the incidence of CLO was the major factor involved. Increasing the total phosphorus from 0.34% to 0.76% (0.14 and 0.56% supplemental) from Curacao rock phosphate decreased CLO from 23.3% to 13.3%, whereas the same amount of phosphorus
3 9 8 E . P . SlNGSEN, C . RlDDELL, L . D . MATTERSON AND J . J . TLUSTOHOWICZ TABLE 5.—The relationship between phosphorus level and source and cause of death, based on necropsy {Experiment 3A, combined data, 4 replicates) % Phosphorus Necropsy diagnosis
Phosphorus source
0.34
%
0.83
Leucosis
3.33
2.50
Miscellaneous diagnosis
1.67
3.33
Not examined
2.50
1.67
Total
22.50
8.33
23.33
13.33
Leucosis
2.50
5.83
Miscellaneous diagnosis
4.17
5.00
Not examined
5.00
Total
35.00
Cage layer osteoporosis Curacao rock phosphate
from dicalcium phosphate decreased CLO from 15.0% to 0.83%. Total mortality from all causes other than CLO were essentially the same regardless of phosphorus source or level. On the basis of these experiments, it would appear that the phosphorus in Curacao rock phosphate is less available to the laying hen than that in dicalcium phosphate. Furthermore, egg production apparently takes precedence over the maintenance of skeletal integrity and, over a period of time, the use of a phosphorus source of lower availability results in a gradual erosion of the skeleton and increased mortality from CLO. Within the range of phosphorus levels used in these experiments, it was not possible to compensate for the lower availability by increasing the total phosphorus. The basic reason for the lower availability is not known. It is clear, however, that in formulating rations for laying hens, phosphorus source and availability must be given careful consideration. Part B of Experiment 3 was undertaken to permit a direct comparison of the causes
0 24.17
of death in birds housed on (1) raised wire floors, and (2) on litter with those of birds housed in cages (Part A) when the same diets were fed in all three environments. The four diets fed, containing 0.14% or 0.56% supplemental phosphorus (0.34% and 0.76% total) from either dicalcium phosphate or Curacao rock phosphate and the dry vitamin D 3 source, were identical with those fed in Part A of Experiment 3. They were all mixed at one place and aliquots delivered to the three environment locations. The two groups of 50 birds housed on litter received the 0.34% total phosphorus diet containing dicalcium phosphate, and from these pens five healthy specimens were sacrificed at bimonthly intervals for necropsy. The results of the histological study will be reported elsewhere. The data in Table 6 indicate that birds housed on either wire floors or litter achieved higher egg production and suffered less mortality than their counterparts housed in cages. Only one case of CLO was diagnosed in any of these groups. The data on cause of death in all treat-
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15.00
Cage layer osteoporosis
Dicalcium phosphate
%
0.76
399
PHOSPHORUS SOURCE AND OSTEOPOROSIS
The data presented provide strong evidence that, in our laboratory, both the level and the source of dietary phosphorus, as well as the housing environment, are
TABLE 7.—The influence of housing environment on the incidence of cage layer osteoporosis and other causes of death {Experiment 3, combined data, 2 phosphorus levels and sources in all 3 experiments) Necropsy diagnosis, % Environment p^rosTs Cages (480)» Wire floors (160)1 Litter floors (220)1
Leucosis
0ther
3.55 3.12 2.73
5.84 6.21 4.08
13.12 0.63 0.00
1 ( ) The total number of birds started in each environment.
involved in the etiology of CLO. This confirms and extends the postulation of Simpson et al. (1964). The modifying aspect of genetics as reported by Francis (1957) and supported by Urist (1960) is clearly recognized. It is our opinion that CLO is a complex syndrome involving several factors, the most important of which are (1) heavy egg production, (2) level of available phosphorus, (3) some aspect of stress induced by confinement in the cage, and (4) genetics. It is possible that the genetic factor involves a difference in phosphorus requirement or ability to utilize phosphorus from different sources. It should be noted, however, that under the conditions of these experiments, adequate available phosphorus reduced CLO o2-Or STRAIN A STRAIN B A
TABLE 6.—The effect of phosphorus source and level on egg production, mortality, and incidence of cage layer osteoporosis in birds housed on wire floors or litter {Experiment 3B) Phosphorus source
Dicalcium phosphate
Curacao rock phosphate
0.34
0.76
0.34
0.76
Housing Environment Wire floors Egg production, % Mortality, % Incidence CLO, %
74.6 15.0 0
79.9 5.0 0
72.8 10.0 2.5
68.9 12.5 0
Litter floors Egg production, % Mortality, % Incidence CLO, %
73.4 4.0 0
71.4 10.0 0
75.0 10.0 0
77.4 7.5 0
5 CAG
Total phosphorus, %
—
16
TIME
24
32
(WEEKS)
]FIG. 1. The relationship between time and the incidence of CLO in 2 strains of laying hens.
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ments were therefore combined according to housing environment and are shown in Table 7. It is immediately obvious that osteoporosis was a major cause of mortality only in birds housed in cages. Mortality from leucosis and all other causes was similar in all three environments. The observation of Francis (1957) on strain differences in susceptibility to CLO appears to be supported by the data reported here. Examination of the time distribution of mortality due to CLO in these experiments revealed that Experiment 2 (strain B) appeared to differ from Experiments 1 and 3 (strain A). All data from these experiments were therefore combined on the basis of strain and the relationship between time and the incidence of death from CLO. The results, shown in Figure 1, suggest that strain A suffered earlier and continuously from CLO, whereas strain B suffered most heavily during the third (8 to 12 weeks of lay) and last three (28 to 40 weeks of lay) periods. The authors recognize, however, that since these experiments were conducted in three successive years, this strain difference aspect should be investigated further.
4 0 0 E . P . SlNGSEN, C. RlDDELL, L . D . MATTERSON AND J . J . TLTJSTOHOWICZ
to a minimum, but did not entirely eliminate it. It is conceivable that the conditions of severe stress imposed by a high rate of egg production and multiple bird housing in cages may make it impossible to eliminate all losses from CLO. The authors also recognize that other factors, including both nutrition and disease, could become involved and modify a specific case of CLO. SUMMARY
Sodium fluoride and source of vitamin D had no effect on performance. ACKNOWLEDGMENT
The authors are indebted to Mr. Peter McManus for technical assistance with diet preparation and care of the experimental birds, to Dr. and Mrs. Joseph
REFERENCES Association of Official Agricultural Chemists, 19S0. Methods of Analysis. Washington, D. C. Bell, D. J., and W. G. Siller, 1962. Cage layer fatigue in Brown Leghorns. Res. Vet. Sci. 3: 219-230. Couch, J. R., 1955. Cage layer fatique. Feed Age, 5: 55-57. Fiske, C. H., and Y. Subbarow, 1925. The colorimetric determination of phosphorus. J. Biol. Chem. 66: 375. Francis, D. W., 1957. Strain differences in the incidence of cage layer fatigue. Poultry Sci. 36: 181-183. Gardiner, T., 1964. Cage layers' fatigue. Agr. North Ireland, 39: 81. Grumbles, L. H., 1959. Cage layer fatigue (cage paralysis). Avian Diseases, 3: 122-125. Hartwigk, H., 1966. "Kafigmiidigkeit" oder Kafiglahme des Legehuhnes, ein neues syndrom bei im Kafig gehalten Hiihnern. Deut. Tierarztl. Wochschur. 277-279. Reitz, L. L., W. H. Smith and M. P. Plumlee, 1960. A simple oxidation procedure for biological materials. Analytical Chem. 32: 1728. Riddell, C , C. F. Helmboldt, E. P. Singsen and L. D. Matterson, 1968. Bone pathology of birds affected by cage layer fatigue. Avian Diseases, 12: 285-296.
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Three 40-week experiments with caged layers explored the effect of dietary phosphorus source and level on egg production, mortality, and incidence of cage layer osteoporosis (cage layer fatigue). Birds receiving supplemental phosphorus from dicalcium phosphate had significantly higher egg production in one experiment and significantly lower mortality in all three experiments than birds fed diets supplemented with Curacao rock phosphate. The increased mortality due to phosphorus source, which ranged from 50 to nearly 100%, was due entirely to cage layer osteoporosis. Increasing supplemental phosphorus to 0.56% (0.76% total) from either source decreased total mortality and incidence of cage layer osteoporosis, but Curacao rock phosphate was markedly less effective than dicalcium phosphate. It was concluded that the phosphorus in Curacao rock phosphate was less available to laying hens than that in dicalcium phosphate. Birds receiving the same diets, but housed on wire floors or litter, showed little or no cage layer osteoporosis.
Lucas for aid on the statistical analyses, and to Dr. W. J. Pudelkiewicz for analytical work. The assistance of Dr. H. J. Fisher, the Connecticut Agricultural Experiment Station, New Haven, Connecticut, is also gratefully acknowledged. The trace mineral mixture used in these experiments was contributed by the Limestone Products Corporation of America, Newton, New Jersey. Financial and/or product contributions were received at various times from International Minerals and Chemical Corporation, Skokie, Illinois; J. H. Baker and Bro., New York, New York; and the Central Connecticut Cooperative Farmers Association, Manchester, Connecticut. The senior author is also indebted to Dr. R. H. Harms, Poultry Science Department, University of Florida, Gainesville, Florida, for both critical evaluation and the office facilities used during the preparation of this manuscript.
PHOSPHORUS SOURCE AND OSTEOPOROSIS Simpson, C. F., P. W. Waldroup, C. B. Ammerman, and R. H. Harms, 1964. Relationship of dietary calcium and phosphorus levels to the cage layer fatigue syndrome. Avian Diseases, 8: 92-100. Singsen, E. P., L. D. Matterson, J. J. Tlustohowicz and W. J. Pudelkiewicz, 1969. Phosphorus in the nutrition of the adult hen. 2. The relative availability of phosphorus from several sources. Poultry Sci. 48: 387-393.
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Smetana, P., 1965. "Fatigue" in caged layers. West ern Australia Dept. of Agr. J . 6: 176-177. Snedecor, G. W., 1956. Statistical Methods. The Iowa State College Press, Ames, Iowa. Urist, M. R., 1960. Cage layer osteoporosis. Endocrinology, 67: 879-880. Urist, M. R., and N. M. Deutsch, 1960. Osteoporosis in the laying hen. Endocrinology, 66: 377391.
The Prevention of Lymphoid Leukosis with Androgens B. R.
BURMESTER
(Received for publication August 14, 1968)
T
HE bursa of Fabricius plays a key role in the development of lymphoid leukosis (LL) (Peterson et ah, 1964, 1966). Surgical removal of the bursa, even though delayed up to 4-5 months of age, prevented the occurrence of this neoplasm. However, bursectomy had no effect on occurrence of erythroblastosis or osteopetrosis, neoplasms also caused by LL virus. Removal of the thymus, a lymphoid organ similar in several respects to the bursa of Fabricius, did not significantly affect the occurrence of any of these neoplasms. This is consistent with the hypothesis (Cooper et ah, 1968) that LL is a neoplasm of the bursa-dependent lymphoid system. The primary neoplasm occurs in the bursa follicles and the malignant cells metastasize to the vital visceral organs eventually causing death. Androgens have been used to prevent the development of the bursa of Fabricius when administered to embryos (Meyer et ah, 1959; Glick and Sadler, 1961; May and Glick, 1964) or to cause rapid involution in the chicken when given after hatching (Glick, 1957; Kirkaptrick and * Poultry Research Branch, Animal Husbandry Research Division, ARS, Regional Poultry Research Laboratory, East Lansing, Michigan 48823.
Andrews, 1944). The use of this procedure for ablation of the bursa of Fabricius and thereby preventing the induction of LL was investigated and is herein reported. EXPERIMENTAL
General Procedure: Day-old chicks of the inbred White Leghorn line 151 were inoculated intra-abdominally with 0.2 ml. of a 10 _s dilution of the virus preparation L31 of strain RPL12 (Burmester et ah, 1960). The chicks were brooded in pens with wire floors and infra-red electric lamps to provide supplemental heat and received a standard ration of the laboratory feed. The experimental period was 245 days post inoculation. Chickens that died during the period were autopsied and a diagnosis established by gross findings when possible, and by microscopic examination when necessary. A histologic examination was made of the bursa of Fabricius or, when it was not visible, the area around the atrophied bursa. The extent of bursal atrophy and presence or absence of tumors was particularly noted. Trial 1: The first experiment was designed to determine whether the egg dipping method of administering the hormone is satisfactory. Eggs at 3 days of
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United Stales Department of Agriculture*
D
1 Scientific Contribution No. 331, Agricultural Experiment Station, University of Connecticut, Storrs.
be responsible. Simpson et al. (1964) found histological evidence of bone damage in caged pullets fed diets low in phosphorus. These birds exhibited symptoms similar to CLF, and the authors suggested that phosphorus deficiency may be implicated in the syndrome. Riddell et al. (1968) presented evidence that the basic pathological defect responsible for the typical posterior paralysis symptom of CLF was a compression fracture in the fourth or fifth thoracic vertebra, with associated compression and degeneration of the spinal cord. They also concurred with Urist (1960) that the syndrome could more appropriately be called cage layer osteoporosis. The objective of this report is to present the nutritional aspects of three experiments involving dicalcium phosphate and Curacao rock phosphate as dietary phosphorus supplements for caged layers, the second and third of which produced the osteoporosis cases studied by Riddell et al. (1968). MATERIALS AND METHODS
The low phosphorus basal ration (0.20% total P) used in these experiments was the same as described by Singsen et al. (1969). Three percent calcium was maintained in all diets. Experiments 1, 2, and 3A were conducted in 11"X18" cages in a windowless, heated, force-ventilated room. The cage laying room was kept under strict quarantine, and there was no evidence of contagious disease,
394
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URING a series of experiments conducted in this laboratory between 1961 and 1967 on the relative availability of phosphorus from various sources for caged hens, unexpected mortality was observed to which osteoporosis appeared to be a major contributing factor. The syndrome resembled "cage layer fatigue" (CLF) as first described by Couch (1955) and later in greater detail by Grumbles (1959). A similar condition has since been reported elsewhere in the world by Bell and Siller (1962, Scotland), Gardiner (1964, Ireland), Smetana (1965, Australia), and Hartwigk (1966, Germany). Genetic differences in susceptibility were reported by Francis (1957) based on variations in incidence of CLF from 0.65% to 3.95% in seven strains of birds. Several reports have implicated skeletal weakness or damage in the condition. Urist and Deutsch (1960) described severe osteoporosis in White Leghorn hens in heavy egg production and noted that it did not occur in immature and young mature pullets or cockerels. Urist (1960) suggested that the name "cage layer fatigue" be changed to cage layer osteoporosis (CLO). He noted similarities between field cases of CLF and osteoporosis as studied in his laboratory and suggested that a hereditary disease of bone rather than nutitional or endocrine factors may
395
PHOSPHORUS SOURCE AND OSTEOPOROSIS TABLE 1.—The influence of phosphorus source and level on egg production and mortality {Experiment 1, combined data, 2 replicates) Dicalcium phosphate1
Source of phosphorus Total phosphorus, % Added phosphorus, %
0.34 0.14
0.48 0.28
Average egg production, % (hen-day basis) Combined data, total % 0.34+0.48% P vs. 0.62+0.76% P Mortality, % Combined data, total % 0.34+0.48% P vs.
0.62+0.76% P 1
Curacao rock phosphate* 0.76 0.56
0.34 0.14
0.48 0.28
69.7
0.62 0.42
0.76 0.56
72.8
73.2
68.7 13.3
16.7
3.3
16.7
25.0
20.0
12.50 15.0
10.0
22.5
15.0
18.3 19.58 16.7
Dynafos, International Minerals and Chemical Corporation, Skokie, Illinois, containing 18.79% phosphorus and 21.30% calcium. Curacao Island rock phosphate, containing 14.60% phosphorus and 35.34% calcium.
other than leucosis, during these experiments. Thirty Leghorn-type pullets, housed two in a cage comprised each replicate in Experiments 1, 2, and 3A, and all treatments were replicated twice. Part B of Experiment 3 was conducted in 12"X12" colony houses on the University poultry farm. Four pens of 40 pullets each were housed on raised wire floors, and five pens, three of 40 and two of 50 pullets each, on litter. The treatments were not replicated. Arbor Acres stock was used in Experiments 1 and 3, and Hy-Line stock in Experiment 2. Several bags of each phosphate source were obtained, blended, and used throughout each experiment. The phosphorus sources and mixed diets were analyzed periodically for calcium and phosphorus (Table 1). Phosphorus was determined according to the method of Fiske and Subbarow (1925) and calcium by the A.O.A.C (1950) method. The samples were solubilized by the wet oxidation method of Reitz et al. (1960). Analysis of variance followed the method of Snedecor (1956). EXPERIMENTAL AND DISCUSSION
Experiment 1 was designed to explore the relative effectiveness of phosphorus from dicalcium phosphate and Curacao rock phosphate when fed at both sub-
optimum and adequate levels. Singsen et al. (1969) had noted increased mortality in birds receiving diets supplemented with 0.14% phosphorus (0.34% total) from the Curacao product, and therefore levels of 0.34, 0.48, 0.62, and 0.76% total phosphorus were chosen for this experiment. The results, summarized in Table 1, indicate a trend toward higher egg production as phosphorus level increased from either source, but the differences were not statistically significant. Average mortality was higher for the groups receiving the Curacao rock phosphate, confirming the observation of Singsen et al. (1969). Analysis of variance indicated that the differences in mortality due to both source and level of phosphorus were statistically significant with F values of 9.11 and 4.92 respectively where 5.32 and 4.07 represented the 5% level of significance. Combining the data on a total by source basis and on the two higher vs. the two lower levels within sources brings out these differences more clearly. I t was apparent therefore that increasing the total phosphorus to an adequate level had not eliminated the mortality differential due to phosphorus source. During the course of Experiment 1, periodic necropsy reports from the Department of Animal Diseases indicated
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2
0.62 0.42
396
E.
P.
SINGSEN,
C.
RIDDELL, L.
D.
MATTERSON AND J.
Experiment 2 was designed to test the possibility that sodium fluoride was contributing to the increased mortality caused by Curacao rock phosphate. Quantitative chemical analyses of the dicalcium and Curacao phosphates were conducted in the laboratory of Dr. H. J. Fisher, chief chemist, the Connecticut TABLE 2.
TLTJSTOHOWICZ
Agricultural Experiment Station, New Haven, Connecticut. The dicalcium and Curacao phosphates contained 0.039 and 0.22% sodium and 0.075 and 0.132% fluorine respectively. These tests indicated sodium to be present in the Curacao product in excess of that which could be accounted for as sodium chloride, suggesting the presence of approximately 0.12% sodium fluoride, a much more toxic material than the normally present calcium fluoride. Qualitative spectrographic analyses yielded no further leads. The plan of experiment and results are shown in Table 2. Sodium fluoride, at the levels fed in this experiment, had no effect on any of the performance characteristics measured. Analysis of variance indicated that the differences in both egg production and mortality due to phosphorus source were statistically significant, with F values of 9.025 and 8.015 respectively where 5.99 represented the 5% level of significance. Since sodium fluoride exerted no influence on results, the mortality and necropsy data from all groups were combined as shown in Table 3 to permit study of the relationship between phosphorus source and cause of death. The total mortality was 11.25% for the birds receiving dicalcium phosphate and 20.83% for those receiving Curacao rock phosphate. The difference was due entirely to the inci-
The effect of phosphorus source and sodium fluoride on the performance of caged layers (Experiment 2, combined data, 2 replicates) Dicalcium phosphate
Phosphorus source Total phosphorus, % Sodium fluoride, m g . A g . diet* Egg production, % (hen-day basis) Mortality, % Live body weights, gms. Final Gain Feed consumed, g m s . / h e n / d a y Average egg weights, gms. (252) days)
0.62 0 71.2
0.62 12 69.7
0.62 24 66.7
Curacao rock phosphate 0.62 48 67.4
0.62 0 64.8
0.62 12 65.9
0.62 24 65.7
0.62 48 65.9
8.3
11.6
16.6
8.3
23.4
16.6
15.0
28.4
1,619 1,776 157 96.3
1,623 1,829 206 95.2
1,638 1,805 167 93.1
1,637 1,840 203 91.8
1,612 1,789 177 90.5
1,614 1,712 98 91.9
1,636 1,798 162 95.7
1,664 1,742 78 89.5
61.8
60.9
60.6
61.3
61.0
60.5
60.8
60.7
* J. T. Baker Chemical Company, reagent sodium fluoride, assay 100%.
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that osteoporosis was a frequent finding. In Experiments 2 and 3, therefore, a detailed necropsy of all birds that died was undertaken by one of the authors (C.R.). A few birds in which excessive deterioration had occurred prior to necropsy are reported as "not examined" in the tabular summaries. Most birds diagnosed as CLO were exhibiting typical symptoms of paralysis, and all CLO diagnoses were confirmed at necropsy by either (1) gross examination, (2) histological study of sections from the sixth rib, femur, and tibia, or (3) radiographs of the spinal column. Details of the bone pathology have been reported by Riddell et al. (1968). Unfortunately, the complete necropsy and histological data were not available until the end of Experiment 3 and therefore could not be used in planning the experiments. When available, however, they permitted a complete reexamination of the experiments from a cause of death point of view, and the findings are presented with each experiment.
J.
397
PHOSPHORUS SOURCE AND OSTEOPOROSIS TABLE 3.—The relationship between phosphorus and cause of death, based on necropsy (Experiment 2, combined data, 8 replicates) Phosphorus source
Diagnosis
%
Dicalcium phosphate (0.62% P)
Cage layer osteoporosis Leucosis Miscellaneous diagnoses Not examined
1.25 2.92 5.42 1.66
Curacao rock phosphate (0.62% P)
Cage layer osteoporosis Leucosis Miscellaneous diagnoses Not examined
Total
11.25 10.00 4.17 3.33 3.33
Total
20.83
TABLE 4.—The effect of phosphorus source and level on the performance of caged layers [Experiment 3A, combined data, 4 replicates) Phosphorus source Total phosphorus, % Average egg production, % (hen-day basis) Mortality, % Total by P source, % Feed consumed, gms./hen/day
Dicalcium phosphate
Curacao phosphate
0.34 66.8
0.76 67.3
0.34 61.5
0.7( 65.8
22.5
8.3
35.0
24.2
90.2
91.7
15.4 90.8
29.6 91.1
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dence of CLO, 1.25% vs. 10.00%, while mortality from all other causes amounted to 10.00% and 10.83% respectively for the dicalcium phosphate and Curacao rock phosphate. Gain in body weight averaged 183 grams and 123 grams respectively on the two phosphate sources, again confirming the observations of Singsen el al. (1969). Experiment 3 was conducted in two parts, A and B, and involved (1) vitamin D source, (2) phosphorus source and level, and (3) housing environment. In Part A, conducted in cages, an irradiated animal sterol and a combination of cod liver oil and tuna liver oil of known potencies, were fed to provide 1323 I.C.U. of vitamin D per kg. of diet. Two levels of total phosphorus, 0.34% and 0.76% (0.14% and 0.56% added) from dicalcium phosphate and Curacao rock phosphate, were fed with each vitamin D source. The diets were mixed every 14 to 21 days in order to minimize vitamin D destruc-
tion. At the end of the experiment it was obvious that both vitamin D sources were equally effective, since total mortality was identical, 22.4% on each source, and the egg productions, 66.7% (dry D3) and 63.9% (oils), were not significantly different. The experimental plan and results presented in Table 4 have therefore been simplified by combining data from both vitamin D sources. Average egg production was somewhat lower than in previous experiments primarily because there was no significant increase at the 0.76% level of total phosphorus. The differential in mortality, however, followed the same pattern observed in previous experiments, totaling 15.4% for the dicalcium phosphate fed groups and 29.6% for those receiving Curacao rock phosphate. The cause of death based on necropsy has been summarized in Table 5. Total mortality was effectively decreased by increasing total phosphorus from either source, but the effect was much more pronounced with the birds fed dicalcium phosphate. Furthermore, 0.56% supplemental phosphorus from Curacao rock phosphate was no more effective than 0.14% from dicalcium phosphate. Again, as in Experiment 2, the incidence of CLO was the major factor involved. Increasing the total phosphorus from 0.34% to 0.76% (0.14 and 0.56% supplemental) from Curacao rock phosphate decreased CLO from 23.3% to 13.3%, whereas the same amount of phosphorus
3 9 8 E . P . SlNGSEN, C . RlDDELL, L . D . MATTERSON AND J . J . TLUSTOHOWICZ TABLE 5.—The relationship between phosphorus level and source and cause of death, based on necropsy {Experiment 3A, combined data, 4 replicates) % Phosphorus Necropsy diagnosis
Phosphorus source
0.34
%
0.83
Leucosis
3.33
2.50
Miscellaneous diagnosis
1.67
3.33
Not examined
2.50
1.67
Total
22.50
8.33
23.33
13.33
Leucosis
2.50
5.83
Miscellaneous diagnosis
4.17
5.00
Not examined
5.00
Total
35.00
Cage layer osteoporosis Curacao rock phosphate
from dicalcium phosphate decreased CLO from 15.0% to 0.83%. Total mortality from all causes other than CLO were essentially the same regardless of phosphorus source or level. On the basis of these experiments, it would appear that the phosphorus in Curacao rock phosphate is less available to the laying hen than that in dicalcium phosphate. Furthermore, egg production apparently takes precedence over the maintenance of skeletal integrity and, over a period of time, the use of a phosphorus source of lower availability results in a gradual erosion of the skeleton and increased mortality from CLO. Within the range of phosphorus levels used in these experiments, it was not possible to compensate for the lower availability by increasing the total phosphorus. The basic reason for the lower availability is not known. It is clear, however, that in formulating rations for laying hens, phosphorus source and availability must be given careful consideration. Part B of Experiment 3 was undertaken to permit a direct comparison of the causes
0 24.17
of death in birds housed on (1) raised wire floors, and (2) on litter with those of birds housed in cages (Part A) when the same diets were fed in all three environments. The four diets fed, containing 0.14% or 0.56% supplemental phosphorus (0.34% and 0.76% total) from either dicalcium phosphate or Curacao rock phosphate and the dry vitamin D 3 source, were identical with those fed in Part A of Experiment 3. They were all mixed at one place and aliquots delivered to the three environment locations. The two groups of 50 birds housed on litter received the 0.34% total phosphorus diet containing dicalcium phosphate, and from these pens five healthy specimens were sacrificed at bimonthly intervals for necropsy. The results of the histological study will be reported elsewhere. The data in Table 6 indicate that birds housed on either wire floors or litter achieved higher egg production and suffered less mortality than their counterparts housed in cages. Only one case of CLO was diagnosed in any of these groups. The data on cause of death in all treat-
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15.00
Cage layer osteoporosis
Dicalcium phosphate
%
0.76
399
PHOSPHORUS SOURCE AND OSTEOPOROSIS
The data presented provide strong evidence that, in our laboratory, both the level and the source of dietary phosphorus, as well as the housing environment, are
TABLE 7.—The influence of housing environment on the incidence of cage layer osteoporosis and other causes of death {Experiment 3, combined data, 2 phosphorus levels and sources in all 3 experiments) Necropsy diagnosis, % Environment p^rosTs Cages (480)» Wire floors (160)1 Litter floors (220)1
Leucosis
0ther
3.55 3.12 2.73
5.84 6.21 4.08
13.12 0.63 0.00
1 ( ) The total number of birds started in each environment.
involved in the etiology of CLO. This confirms and extends the postulation of Simpson et al. (1964). The modifying aspect of genetics as reported by Francis (1957) and supported by Urist (1960) is clearly recognized. It is our opinion that CLO is a complex syndrome involving several factors, the most important of which are (1) heavy egg production, (2) level of available phosphorus, (3) some aspect of stress induced by confinement in the cage, and (4) genetics. It is possible that the genetic factor involves a difference in phosphorus requirement or ability to utilize phosphorus from different sources. It should be noted, however, that under the conditions of these experiments, adequate available phosphorus reduced CLO o2-Or STRAIN A STRAIN B A
TABLE 6.—The effect of phosphorus source and level on egg production, mortality, and incidence of cage layer osteoporosis in birds housed on wire floors or litter {Experiment 3B) Phosphorus source
Dicalcium phosphate
Curacao rock phosphate
0.34
0.76
0.34
0.76
Housing Environment Wire floors Egg production, % Mortality, % Incidence CLO, %
74.6 15.0 0
79.9 5.0 0
72.8 10.0 2.5
68.9 12.5 0
Litter floors Egg production, % Mortality, % Incidence CLO, %
73.4 4.0 0
71.4 10.0 0
75.0 10.0 0
77.4 7.5 0
5 CAG
Total phosphorus, %
—
16
TIME
24
32
(WEEKS)
]FIG. 1. The relationship between time and the incidence of CLO in 2 strains of laying hens.
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ments were therefore combined according to housing environment and are shown in Table 7. It is immediately obvious that osteoporosis was a major cause of mortality only in birds housed in cages. Mortality from leucosis and all other causes was similar in all three environments. The observation of Francis (1957) on strain differences in susceptibility to CLO appears to be supported by the data reported here. Examination of the time distribution of mortality due to CLO in these experiments revealed that Experiment 2 (strain B) appeared to differ from Experiments 1 and 3 (strain A). All data from these experiments were therefore combined on the basis of strain and the relationship between time and the incidence of death from CLO. The results, shown in Figure 1, suggest that strain A suffered earlier and continuously from CLO, whereas strain B suffered most heavily during the third (8 to 12 weeks of lay) and last three (28 to 40 weeks of lay) periods. The authors recognize, however, that since these experiments were conducted in three successive years, this strain difference aspect should be investigated further.
4 0 0 E . P . SlNGSEN, C. RlDDELL, L . D . MATTERSON AND J . J . TLTJSTOHOWICZ
to a minimum, but did not entirely eliminate it. It is conceivable that the conditions of severe stress imposed by a high rate of egg production and multiple bird housing in cages may make it impossible to eliminate all losses from CLO. The authors also recognize that other factors, including both nutrition and disease, could become involved and modify a specific case of CLO. SUMMARY
Sodium fluoride and source of vitamin D had no effect on performance. ACKNOWLEDGMENT
The authors are indebted to Mr. Peter McManus for technical assistance with diet preparation and care of the experimental birds, to Dr. and Mrs. Joseph
REFERENCES Association of Official Agricultural Chemists, 19S0. Methods of Analysis. Washington, D. C. Bell, D. J., and W. G. Siller, 1962. Cage layer fatigue in Brown Leghorns. Res. Vet. Sci. 3: 219-230. Couch, J. R., 1955. Cage layer fatique. Feed Age, 5: 55-57. Fiske, C. H., and Y. Subbarow, 1925. The colorimetric determination of phosphorus. J. Biol. Chem. 66: 375. Francis, D. W., 1957. Strain differences in the incidence of cage layer fatigue. Poultry Sci. 36: 181-183. Gardiner, T., 1964. Cage layers' fatigue. Agr. North Ireland, 39: 81. Grumbles, L. H., 1959. Cage layer fatigue (cage paralysis). Avian Diseases, 3: 122-125. Hartwigk, H., 1966. "Kafigmiidigkeit" oder Kafiglahme des Legehuhnes, ein neues syndrom bei im Kafig gehalten Hiihnern. Deut. Tierarztl. Wochschur. 277-279. Reitz, L. L., W. H. Smith and M. P. Plumlee, 1960. A simple oxidation procedure for biological materials. Analytical Chem. 32: 1728. Riddell, C , C. F. Helmboldt, E. P. Singsen and L. D. Matterson, 1968. Bone pathology of birds affected by cage layer fatigue. Avian Diseases, 12: 285-296.
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Three 40-week experiments with caged layers explored the effect of dietary phosphorus source and level on egg production, mortality, and incidence of cage layer osteoporosis (cage layer fatigue). Birds receiving supplemental phosphorus from dicalcium phosphate had significantly higher egg production in one experiment and significantly lower mortality in all three experiments than birds fed diets supplemented with Curacao rock phosphate. The increased mortality due to phosphorus source, which ranged from 50 to nearly 100%, was due entirely to cage layer osteoporosis. Increasing supplemental phosphorus to 0.56% (0.76% total) from either source decreased total mortality and incidence of cage layer osteoporosis, but Curacao rock phosphate was markedly less effective than dicalcium phosphate. It was concluded that the phosphorus in Curacao rock phosphate was less available to laying hens than that in dicalcium phosphate. Birds receiving the same diets, but housed on wire floors or litter, showed little or no cage layer osteoporosis.
Lucas for aid on the statistical analyses, and to Dr. W. J. Pudelkiewicz for analytical work. The assistance of Dr. H. J. Fisher, the Connecticut Agricultural Experiment Station, New Haven, Connecticut, is also gratefully acknowledged. The trace mineral mixture used in these experiments was contributed by the Limestone Products Corporation of America, Newton, New Jersey. Financial and/or product contributions were received at various times from International Minerals and Chemical Corporation, Skokie, Illinois; J. H. Baker and Bro., New York, New York; and the Central Connecticut Cooperative Farmers Association, Manchester, Connecticut. The senior author is also indebted to Dr. R. H. Harms, Poultry Science Department, University of Florida, Gainesville, Florida, for both critical evaluation and the office facilities used during the preparation of this manuscript.
PHOSPHORUS SOURCE AND OSTEOPOROSIS Simpson, C. F., P. W. Waldroup, C. B. Ammerman, and R. H. Harms, 1964. Relationship of dietary calcium and phosphorus levels to the cage layer fatigue syndrome. Avian Diseases, 8: 92-100. Singsen, E. P., L. D. Matterson, J. J. Tlustohowicz and W. J. Pudelkiewicz, 1969. Phosphorus in the nutrition of the adult hen. 2. The relative availability of phosphorus from several sources. Poultry Sci. 48: 387-393.
401
Smetana, P., 1965. "Fatigue" in caged layers. West ern Australia Dept. of Agr. J . 6: 176-177. Snedecor, G. W., 1956. Statistical Methods. The Iowa State College Press, Ames, Iowa. Urist, M. R., 1960. Cage layer osteoporosis. Endocrinology, 67: 879-880. Urist, M. R., and N. M. Deutsch, 1960. Osteoporosis in the laying hen. Endocrinology, 66: 377391.
The Prevention of Lymphoid Leukosis with Androgens B. R.
BURMESTER
(Received for publication August 14, 1968)
T
HE bursa of Fabricius plays a key role in the development of lymphoid leukosis (LL) (Peterson et ah, 1964, 1966). Surgical removal of the bursa, even though delayed up to 4-5 months of age, prevented the occurrence of this neoplasm. However, bursectomy had no effect on occurrence of erythroblastosis or osteopetrosis, neoplasms also caused by LL virus. Removal of the thymus, a lymphoid organ similar in several respects to the bursa of Fabricius, did not significantly affect the occurrence of any of these neoplasms. This is consistent with the hypothesis (Cooper et ah, 1968) that LL is a neoplasm of the bursa-dependent lymphoid system. The primary neoplasm occurs in the bursa follicles and the malignant cells metastasize to the vital visceral organs eventually causing death. Androgens have been used to prevent the development of the bursa of Fabricius when administered to embryos (Meyer et ah, 1959; Glick and Sadler, 1961; May and Glick, 1964) or to cause rapid involution in the chicken when given after hatching (Glick, 1957; Kirkaptrick and * Poultry Research Branch, Animal Husbandry Research Division, ARS, Regional Poultry Research Laboratory, East Lansing, Michigan 48823.
Andrews, 1944). The use of this procedure for ablation of the bursa of Fabricius and thereby preventing the induction of LL was investigated and is herein reported. EXPERIMENTAL
General Procedure: Day-old chicks of the inbred White Leghorn line 151 were inoculated intra-abdominally with 0.2 ml. of a 10 _s dilution of the virus preparation L31 of strain RPL12 (Burmester et ah, 1960). The chicks were brooded in pens with wire floors and infra-red electric lamps to provide supplemental heat and received a standard ration of the laboratory feed. The experimental period was 245 days post inoculation. Chickens that died during the period were autopsied and a diagnosis established by gross findings when possible, and by microscopic examination when necessary. A histologic examination was made of the bursa of Fabricius or, when it was not visible, the area around the atrophied bursa. The extent of bursal atrophy and presence or absence of tumors was particularly noted. Trial 1: The first experiment was designed to determine whether the egg dipping method of administering the hormone is satisfactory. Eggs at 3 days of
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United Stales Department of Agriculture*