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Studies on Phosphorus Assay Techniques
Studies on Phosphorus Assay Techniques 1. THE ASSAY OF SOFT PHOSPHATE WITH CHICKS I. MOTZOK Department of Nutrition, Ontario Agricultural College, University of Guelph, Guelph, Canada (Received for publication October 26, 1967)
A
component of the calcium:phosphorus ratios. With increasing increments of added phosphorus in the assay diets there was a decrease in the proportion of unavailable to available phosphorus and a concomitant decrease in the ratio of calcium to available phosphorus. Examples calculated from the data of Dilworth and Day (1964) and Waldroup et al. (1965) are presented in the discussion of this paper. The main part of the current study was designed to investigate the effect of calcium: phosphorus ratios based on total available or inorganic phosphorus (Pz)1 in a phosphorus assay procedure. An experiment was also performed to determine the level of calcium required with the basal diet for maximum growth combined with a low bone ash response. The objective was to devise a procedure in which growth would be uniform with all levels of dietary phosphorus and bone calcification would serve as the sole response criterion. EXPERIMENTAL
The composition of the basal diet (Table 1) used in these experiments was similar to that employed in a previous study (Motzok et al, 1965). The basal diet was analyzed prior to the addition of calcium and phosphorus supplements. Sufficient amounts of the mineral supplements were added to provide the desired levels of calcium and phosphorus in the diets and ground cellulose was used as the additional variable to maintain the nutrient content at
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1
See footnote 3 in Table 1.
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NUMBER of chick assay procedures have been devised and tested for the estimation of availability of phosphorus in various mineral supplements. Invariably the assays have indicated a relatively poor availability of phosphorus from soft phosphate, the range extending from 25% (Gillis et al, 1954) to 58% (Waldroup et al, 1965). These low values for the utilizable phosphorus of soft phosphate are in marked contrast with the relatively good growth and bone calcification of chick reported recently with this type of supplement when specific calcium: phosphorus ratios and high levels of vitamin D 3 were used (Motzok et al, 1965, 1967; Fritz and Roberts, 1966; McKnight and Watts, 1966). The literature concerned with various aspects of phosphorus biosassay has been reviewed by Nelson and Peeler (1964), Waldroup et al. (1965) and Sullivan (1966). In many of the assay diets the calcium content has been adjusted to give either a constant level of calcium or a constant or variable calcium:phosphorus ratio for all levels of phosphorus. In the latter type of assays the calcium:phosphorus ratios were usually based on total calcium and total phosphorus. In cases where the basal diet contained large amounts of plant products, such as soybean meal and corn, a major portion of the phosphorus was organic in nature, a form of phosphorus which usually has been considered to be only partly available to the chick. Consequently, a constant amount of unavailable phosphorus was included in the phosphorus
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I . MOTZOK TABLE 1.—Composition of diets Ingredients
Percent
Ground wheat Soybean meal (44% protein) Ground yellow corn Casein Dried buttermilk Dehydrated alfalfa Salt (iodized) Soybean oil Micro-ingredients1 Variable ingredients 2
42.0 31.0 15.0 1.0 1.0 1.5 0.25 2.5 0.35 5.4 22.2 0.17 0.39 0.13
1 Supplied per kilogram of diet: 42 mg. manganese, 3.3 mg. riboflavin, 1.54 g. choline chloride (25%), 0.5 g. DL-methionine, 6,150 I.U. vitamin A, 1.0 g. Merck's antibiotic—vitamin B I2 supplement (containing 2 g. procaine penicillin G and 3.0 mg. vitamin B,2 per pound). 2 Required amounts of calcium and phosphorus supplements, vitamin D 3 and ground cellulose (Solka Floe). 3 Calculated on the basis that all of the phosphorus of non-plant feed ingredients is considered to be inorganic and that 30% of the phosphorus of plant products is non-phytin phosphorus and may be considered as part of the inorganic phosphorus required (N.R.C., 1966).
a constant level. A commercial preparation of vitamin D 3 supplement2 of known potency was used to supply the desired level of this vitamin. In the assay experiments the standard and test supplements (dicalcium phosphate, chemical grade CaHP0 4 -2H 2 0, and soft phosphate) were added to the diets at increment levels of phosphorus which were equal in magnitude on the logarithmic scale to the base 10 (Summers e( al., 1959). Calcium carbonate (U.S.P. grade) was used to adjust the calcium content of the diets to the levels specified. The complete diets were analyzed for calcium and phosphorus as a check on the accuracy of mixing.
EXPERIMENTAL AND RESULTS
Preliminary Experiment. Prior to conducting assays on soft phosphate, a study was made on the response of chicks to the basal diet supplemented only with calcium carbonate. A control diet containing 1.0% calcium and 0.45% inorganic phosphorus, with dicalcium phosphate as the source of additional phosphorus, was included to provide an index of the response of chicks to the calcium-supplemented basal diet. Two levels of vitamin D 3 , 450 and 7500 I.C.U./kg., were used and the responses of chicks to various treatments are given in Table 2. The diet with the lowest level of calcium (0.4%) promoted the best growth among the treatments without added phosphorus. The growth of chicks decreased progressively as the dietary calcium was increased, the decrease in growth being highly significant when the diets contained 0.8 or 1.0% calcium. Similarly, there was a significant decrease in the ash content of tibiae of chicks fed diets containing more
2
We are indebted to Dawe's Laboratories, Inc., Chicago, 111., for the determination of the potency of the vitamin D 3 supplement.
s The number of replicates and chicks per groups are given in the tables.
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Average analysis Crude protein Calcium Phosphorus Total (analyzed) Inorganic (calculated) 3
Day-old White Plymouth Rock pullets were randomized into lots of 15-20 chicks each. Replicated lots3 of chicks were fed each ration for 4 weeks. They were reared in electrically heated wire-floored battery brooders and the pens and diets were allocated at random. The chicks were weighed at regular intervals and at the end of the experiment the feed was removed from the troughs in the evening of the day before the final weights were recorded and the chicks killed for the removal of tibiae. The ash content of the bones was determined according to the A.O.A.C. (1955) procedure for vitamin D assay. The data were subjected to analysis of variance as outlined by Snedecor (1957) and parallel line assay as specified by Bliss (1952).
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PHOSPHORUS ASSAY
TABLE 2.—Effect of varying amounts of calcium, phosphorus and vitamin Dz on -weight and bone ash of chicks1 at 4 weeks Vitamin Ds (I.C.U./kg.) Mineral supplements
Ca!
Pis
(%) (%)
450 |7,500
450
|7,500
Av. wt. (g.) Bone ash (%) CaCOs CaC03 CaC03 CaCOs CaHP04-2Hs0 and CaCOs
0.4 0.6 0.8 1.0
0.13 0.13 0.13 0.13
460 444 386 301
461 445 391 321
38.6 37.9 35.2 31.0
39.8 40.5 36.3 33.6
1.0
0.45
495
505
46.8
45.9
than 0.6% calcium. The highest growth and bone ash obtained with the basal diet supplemented only with calcium were significantly lower than the responses produced by the diet containing 1.0% calcium and 0.45% inorganic phosphorus. However, good livability was obtained and the mortality which did occur was not related to the dietary treatments. Thirteen chicks died; of these, three deaths occurred among the birds fed the diet containing 1.0% calcium and 0.45% inorganic phosphorus. Assay 1. In the preliminary study with the basal diet, maximum growth was obtained when the calcium content of the basal diet
Assay 2. In this experiment the Ca:Pz ratio was maintained at approximately 2.2:1. The choice of this ratio was based on the observation by Motzok et al. (1965) that good growth and bone ash were obtained in chicks fed diets with soft phosphate when the total calcium:total phosphorus ratio was 1.4:1, which is equivalent to a ratio of
TABLE 3.—Effect of varying amounts of dicalcium phosphate and soft phosphate (Ca:Pi approximately 3.1:1 in all diets) on weight, feed utilization and bone ash of chicks1 at 4 weeks Phosphorus supplements
P Added
Pi 2
Ca3
(%)
(%)
(%)
Weight (g.)
CaHPOv2H 2 0
0.044 0.070 0.111 0.176
0.174 0.200 0.241 0.306
0.54 0.62 0.75 0.95
444 440 433 444
1.99 1.88 1.89 1.89
797 744 731 757
41.0 41.5 42.4 45.2
Soft phosphate
0.044 0.070 0.111 0.176
0.174 0.200 0.241 0.306
0.54 0.62 0.75 0.95
444 438 413 423
2.12 1.88 1.98 1.92
829 737 728 726
40.6 42.4 42.4 42.1
Feed/Gain Feed per Bone ash bird (g.) (%)
Least significant difference for P = 0.05 and P = 0.01 respectively: Growth—28 g. and 39 g. Bone ash -1.6% and 2.2%. 1 Duplicate groups, 20 chicks per group. 2 Total inorganic phosphorus (calculated as in Table 1, footnote 3). 3 Total calcium.
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Least significant difference tor P=0.05 and P=0.01 respectively: Weight—20 g. and 28 g. Bone ash—2.7% and 3.8%. 1 Duplicate groups, 20 chicks per group. 2 Total calcium by analysis. • Total inorganic phosphorus (calculated as described in Table 1, footnote 3).
without added phosphorus was 0.4% and the Ca:Pz ratio was approximately 3.1:1. Accordingly, an assay of soft phosphate was conducted in which a Ca:P« ratio of about 3.1:1 was maintained in all diets. Vitamin D 3 was provided at a level of 6600 I.C.U./kg. of diet and the responses of chicks to the various dietary treatments are given in Table 3. Analysis of the data showed that there was no significant difference in the growth of chicks among the dietary treatments employed in this experiment. Although there was a marked increase in bone ash with increasing levels of dicalcium phosphate, there was no response in the bone ash of chicks fed diets with soft phosphate beyond the second increment of the supplement. Consequently, no attempt was made to estimate the availability of phosphorus from the test supplement in this trial.
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I . MOTZOK
TABLE 4.-
soft phosphate was calculated to be 63%, with 95% approximate fiducial limits of 50-79%. Assay 3. In the previous trial the high growth response with the two upper levels of dicalcium phosphate suggested that perhaps the amount of calcium was suboptimal for growth in the diets with the two low levels of dicalcium phosphate and in the diets supplemented with soft phosphate. This assumption was tested in a third trial in which the Ca:Pz ratio was increased to 2.5:1. In all other respects the assay procedure was similar to that of Assay 2 and the responses of chicks to these treatments are given in Table 5. Feed utilization was relatively uniform and there was no significant difference in the weight of chicks among any of the treatments. The slopes of the response curves for bone ash obtained with the standard and test supplements were highly significant, linear and parallel, and the comparative biological availability for phosphorus from soft phosphate was estimated to be 71%, with 95% approximate fiducial limits of 63-81%. DISCUSSION
Soft phosphate was chosen as the test
-Effect of varying amounts of dicalcium phosphate and soft phosphate (Ca:Pi at 2.2:1 in all diets) on weight, feed utilization and bone ash of chicks1 at 4 weeks
Phosphorus supplements
P added
Pi 2
Ca3
(%)
(%)
(%)
Weight (g.)
CaHP0 4 -2H 2 0
0.044 0.070 0.111 0.176
0.385 0.440 0.535 0.675
407 407 444 444
1.93 1.96 1.88 1.87
703 710 752 752
37.8 38.0 40.6 43.0
Soft phosphate
0.044 0.070 0.111 0.176
0.174 0.200 ' 0.241 ro.306 P* 0.174 0.200 0.241 0.306
0.390 0.440 0.535 0.675
367 393 398 395
2.10 1.95 1.95 1.89
658 683 683 671
35.7 36.6 38.2 40.3
Feed/Gain Feed per bird (g.)
Bone ash
(%)
Least significant difference for P = 0.05 and P = 0.01 respectively: Growth—29 g. and 44 g. Bone ash1.8% and 2.8%. 1 Duplicate groups, 20 chicks per group. 2 Total inorganic phosphorus (calculated as in Footnote 3, Table 1). 3 Total calcium.
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2.2:1 on the basis of total inorganic phosphorus. A four point assay on soft phosphate was conducted with dicalcium phosphate as the standard and 7500 I.C.U. of vitamin D 3 /kg. in all diets. The levels of phosphorus employed and the responses of the birds to the dietary treatments are given in Table 4. The average weights of the chicks which received diets with the two low levels of dicalcium phosphate were significantly lower than those of birds fed diets with the two high levels of the standard phosphate. The weights of chicks fed diets with soft phosphate, with the exception of the lowest level, were comparable and not significantly different from those of chicks fed the two low levels of dicalcium phosphate. The growth of chicks appeared to be governed largely by the amount of the diet consumed. When the data for bone ash were plotted, the first point in each case appeared to be on the induction part of the response curve. Consequently, only the data for the three upper levels were used in the estimate of availability. The slopes of the response curves for bone ash were found to be highly significant, linear and parallel, and the relative availability of phosphorus in
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PHOSPHORUS ASSAY
TABLE 5.—Effect of varying amounts of dicalcium phosphate and soft phosphate {Ca'.Pi approximately 2.5:1 in all diets) on weight, feed utilization and bone ash of chicks1 at 4 weeks Average Feed per weight Feed/Gain bird (g.) (g.)
Phosphorus supplements
P added
Pi 2
Ca3
(%)
(%)
(%)
CaHP0 4 -2H 2 0
0.044 0.070 0.111 0.176
0.174 0.200 0.241 0.306
0.435 0.500 0.600 0.776
400 389 403 396
1.96 1.96 1.95 1.96
701 680 693 715
39.6 40.7 42.9 44.1
Soft phosphate
0.044 0.070 0.111 0.176
0.174 0.200 0.241 0.306
0.435 0.500 0.600 0.776
375 378 396 404
1.98 1.97 1.96 1.95
675 660 706 716
38.3 39.5 41.5 43.0
Bone ash
(%)
material on the basis that it represented natural inorganic phosphorus supplements possessing relatively low solubility (Hill et al., 1945), a property which may be the main reason for the low availability of its phosphorus estimated by many previous assay procedures. The basal diet used in the current investigations had the same composition as the one with which a good response in chicks was obtained with soft phosphate by Motzok et al. (1965). The present data show that this diet supplemented with a small amount of calcium (0.21% as calcium carbonate) supported good livability and growth. The utilizable phosphorus provided by the basal ingredients obviated the need of adding "available" inorganic phosphorus to a basal diet low in phosphorus, such as the semipurined diet employed by Nelson and Peeler (1961). Nelson and Peeler (1961) also used a practical diet for the assay of soft phosphate. Employing a calcium: available phosphorus ratio of 2:1, they obtained a response in growth and bone ash with added increments of phosphorus. The diets were supplemented with 750 I.C.TJ. of vitamin D 3 /kg. and the availability of phosphorus from soft phosphate was estimated
to be 36%. In the present study there was also a response in bone ash and growth to added phosphorus when the Ca:Pi was maintained at 2.2:1 and the diets contained 7500 I.C.U. vitamin D 3 /kg. The higher level of vitamin D 3 used in this trial was probably the main reason for the estimated phosphorus availability of 63%, in comparison with the low availability of this mineral from soft phosphate obtained by Nelson and Peeler (1961). The data of Assays 1 and 3 provide evidence that the dietary Ca:Pj ratio in an assay procedure is a critical factor governing the availability of phosphorus from a supplement of relatively low solubility, even with an abundance of vitamin D 3 in the diet. A Ca:Pi ratio of 2.5:1 with the high level of vitamin D 3 enabled chicks to utilize 71% of the phosphorus from soft phosphate and promoted uniform growth on all levels of the supplements. On the other hand, a Ca:P« ratio of 3.1:1 gave uniform growth but the response in bone ash to increasing levels of soft phosphate was negligible. It appears that, with a Ca:Pz ratio of 3.1:1, the extra calcium provided by calcium carbonate was in excess of the amount required for maximum utilization of minerals from soft phosphate in
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Least significant difference for P = 0.05 and P = 0.01 respectively: Growth—38 g. and 54 g. Bone ash1.3% and 1.8%. 1 Triplicate groups, 15 chicks per group. 2 Total inorganic phosphorus (calculated as in Footnote 3, Table 1). 8 Total calcium.
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I. MOTZOK
the highest level of supplemental phosphorus which provide a Ca:Pz ratio of about 2.6:1. In the present study a difference in bone ash of 1.1% was obtained between birds fed dicalcium phosphate and those fed soft phosphate at the highest level of phosphorus when the Ca:Pi ratio was 2.5:1 (Table 5). Perhaps the amount of vitamin D 3 employed (792 I.C.U./kg.) by Waldroup et al. was inadequate for maximum utilization of soft phosphate even with the narrow Ca:P? ratio. A comparison of the availabilities of 63 and 71% by application of the t test revealed no significant difference (P > 0.05) between the two estimates obtained with assays 2 and 3 in the present study. However, the difference in availabilities was wide enough to suggest that differences in growth at different phosphorus levels in assay 2 may have been a complicating factor which resulted in a lower estimate of phosphorus availability. The relationship between growth and bone ash in response to supplemental phosphorus and calcium appears to be quite complex. For example, Nelson and Walker (1964) showed that a plot of the percentage bone ash and the logarithm of the percentage dietary phosphorus was a straight line, whereas the weights of chicks showed a highly significant curvature over the same range of dietary phosphorus. Indeed, there appears to be no precise information in the literature on the relative requirements of calcium and phosphorus for each of these responses in birds. In an attempt to resolve this problem in an assay procedure, Sullivan (1966) proposed the use of three criteria, body weight, percent bone ash and gain:feed ratio, to compute a single biological value of a phosphorus supplement. Practical usage, precision and simplicity were considered in the derivation of a formula for four-week poult data. The approximate
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an assay procedure, producing a condition analogous to that obtained with high calcium in studies with adequate amounts of phosphorus (Motzok et al., 1965, 1967; Fritz and Roberts, 1966; McKnight and Watts, 1966). Dilworth and Day (1964) obtained uniform growth and a response in bone ash with increasing levels of phosphorus from several phosphorus supplements in a chick assay in which the diets were supplemented with 2010 I.C.U. vitamin D 3 /kg. However, the responses in growth and bone ash with soft phosphate were appreciably lower than those obtained with the standard supplement and the availability of phosphorus from soft phosphate was calculated to be only 38%. These workers employed a total calcium:total phosphorus ratio of 1.7:1. If it is assumed that their basal diet contained about 0.09% available phosphorus, the Ca:Pz° ratios calculated from their data decreased from about 3.2:1 to 2.8:1 when the supplemental phosphorus was increased from 0.15 to 0.25%. Waldroup et al. (1965) used three regimes of supplemental calcium in a comparison of phosphorus assay techniques and the comparative availability of phosphorus from soft phosphate was 58.9, 52.5 and 51.5% with a constant level of 1% calcium, a 2:1 Ca:P ratio and a "sliding" Ca:P ratio, respectively. On the assumption that the basal diet contained about 0.09% available phosphorus, calculations reveal that with a constant 1% calcium in all diets the Ca:Pz ratios dropped from about 7.1:1 to 2.6:1 with increasing levels of phosphorus; with a constant Ca:P ratio (2:1) the Ca:P« ratios decreased from 5:1 to 3.2:1; with the "sliding" Ca:P ratios the Ca:P« ratios diminished from 3:1 to 2.8:1. In the assay with the constant level of 1% calcium these workers obtained a difference in bone ash of 3.2% between chicks fed dicalcium phosphate and soft phosphate at
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PHOSPHORUS ASSAY
ratios in the diets were too narrow or too wide and in some cases the level of vitamin D 3 was inadequate for maximum utilization of the minerals in soft phosphate. Even the comparative availability of the phosphorus in soft phosphate, obtained in the present study, appears to be somewhat lower than the degree of utilization which has been achieved with optimum amounts of calcium and vitamin D 3 in practical diets containing 0.45-0.48% inorganic phosphorus (Motzok et ah, 1965,1967). SUMMARY
Studies were made on the effect of different ratios of calcium anorganic phosphorus (Pi) on the response of chicks in an assay for the availability of phosphorus from soft phosphate with dicalcium phosphate as the standard. The following observations were made: 1. When the Ca:P« ratios were either 2.2:1 or 2.5:1, a highly significant increase in bone ash was obtained with increasing levels of the standard and test supplements. The responses were linear and parallel for the three upper levels of phosphorus when the Ca:P? ratio was 2.2:1 and for all four points when the ratio was 2.5:1. 2. There were some significant variations in growth produced by the Ca:Pz ratio of 2.2:1 and the comparative availability of phosphorus in soft phosphate, calculated from the bone ash data, was 63%. 3. When the Ca:Pi ratio was 2.5:1, growth was relatively uniform at all levels of phosphorus and the availability of phosphorus from soft phosphate was 71%. 4. A wide Ca:Pz" ratio (3.1:1) produced uniform growth with graded levels of phosphorus but a significant response in bone ash was obtained only with dicalcium phosphate. There was no increase in bone ash of chicks fed soft phosphate beyond the second level of phosphorus in a four point assay.
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contribution of each response criterion to the calculated biological value was: body weight, 55%; percent bone ash, 40%; gain:feed ratio, 5%. However, Nelson and Walker (1964) examined a vast amount of their assay data and presented a critical appraisal of the value of growth of chicks as a criterion of response in a phosphorus assay procedure. These investigators analyzed 82 experiments using only the steepest part of the growth response curves and calculated that 5.9 times as many pens of chicks would be required to obtain comparable accuracy when body weight instead of bone ash is used to measure phosphorus quality. It appears from their data that a higher degree of accuracy in a phosphorus bioassay can be achieved when bone ash serves as the sole response criterion than when 55% of the biological value of phosphorus is based on body weight and only 40% on bone ash, as recommended by Sullivan (1966). The maintenance of uniform growth of chicks given various levels of calcium and phosphorus in assay 3 of the current work eliminated the need of taking into account a response in growth in the estimation of phosphorus availability. It is recognized that the range in bone ash was not as wide as that obtained by other investigators, but the responses in bone ash to increasing increments of the standard phosphate and test material were linear and parallel and conformed to the assay requirements outlined by Bliss (1952). Furthermore, these responses were at the upper portion of the full range in bone ash obtainable in chicks and they represent the area of response in which supplemental phosphorus is utilized under practical feeding conditions. The present results suggest that the low values for the availability of phosphorus from soft phosphate which have been reported in the literature were due mainly to assay procedures. It appears that the Ca:Vi
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I . MOTZOK ACKNOWLEDGMENTS
REFERENCES Association of Official Agricultural Chemists, 1955. Methods of Analysis. 8th Ed., Washington, D.C. Bliss, C. I., 1952. The Statistics of Bioassay. Academic Press, Inc., New York. Dilworth, B. C , and E. J. Day, 1964. Phosphorus availability studies with feed grade phosphates. Poultry Sci. 43 : 1039-1044. Fritz, J. C , and T. Roberts, 1966. Influence of levels of vitamin D and calciu mon the utilization of phosphorus by the growing chick. Poultry Sci. 45: 1085-1086. Gillis, M. B., L. C. Norris and G. F. Heuser, 1954. Studies on the biological value of inorganic phosphates. J. Nutrition, 52: 115-125. Hill, W. L., D. S. Reynolds, S. B. Hendricks and K. D. Jacobs, 1945. Nutritive evaluation of defluorinated phosphates and other phosphorus supplements. I. Preparation and properties of the samples. J. Assoc. Off. Agric. Chem. 28:
105-118. McKnight, W. F., and A. B. Watts, 1966. The effect of vitamin D s on the utilization of phosphorus from various sources. Poultry Sci. 45: 1104. Motzok, I., D. Arthur and H. D. Branion, 1965. Factors affecting the utilization of calcium and phosphorus from soft phosphate by chicks, Poultry Sci. 44: 1261-1270. Motzok, I., D. Arthur and S. J. Slinger, 1967. Utilization of calcium and phosphorus from soft phosphate by chicks. Poultry Sci. 46: 985-991. National Research Council, 1966. Nutrient requirements for domestic animals. 1. Nutrient requirements of poultry. Nelson, T. S., and H. T. Peeler, 1961. The availability of phosphorus from single and combined phosphates to chicks. Poultry Sci. 40: 1321-1328. Nelson, T. S., and H. T. Peeler, 1964. Current status of biological testing of feed phosphates. Feedstuffs, 36: 64-69. Nelson, T. S., and A. C. Walker, 1964. The biological evaluation of phosphorus compounds. Poultry Sci. 43: 94-98. Snedecor, G. W., 1957. Statistical Methods. The Iowa State College Press, Ames, Iowa. Sullivan, T. W., 1966. A triple response method of determining biological value of phosphorus sources with young turkeys. Poultry Sci. 45: 1236-1245. Summers, J. D., S. J. Slinger, W. F. Pepper, I. Motzok and G. C. Ashton, 1959. Availability of phosphorus in soft phosphate and phosphorus acid and the effect of acidulation of soft phosphate. Poultry Sci. 38: 1168-1179. Waldroup, P. W., C. B. Ammerman and R. H. Harmus, 1965. A comparison of phosphorus assay techniques with chicks. Poultry Sci. 44:10861089.
NEWS AND NOTES (continued from page 915) its Fact Finding Conference. He was selected for the honor by an independent committee which considered 49 candidates nominated by people in the industry. The award is made "in recognition of service to the poultry and egg industry above and beyond self-gain." In reporting its choice, the committee credited Johnson with being an "idea man" who had done as much as any one person to make it possible for the poultry and egg business to become a modern, auto-
mated food business. Dedicated to the growth and progress of the industry, he repeatedly has invested the capital, time, talent, and energy of himself and his company, with no guarantee of compensation, to give the industry answers to many of its problems in production, processing, and marketing. A native of Maine, he graduated in 1922 from Iowa State College, and became a Poultry Extension Man in Iowa. He soon moved to the Buckeye Incubator Company as a salesman.
(continued on page 999)
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The author is indebted to the Ontario Department of Agriculture and Food for financial aid and the Soft Phosphate Research Institute Inc., Ocala, Florida, and The National Research Council of Canada for grants in support of this work. He wishes to thank Merck, Sharp and Dohme of Canada Ltd. for the antibiotic and vitamin B12 supplement and Pfizer Canada Ltd. for the vitamin A. He is grateful to Dr. S. J. Slinger and Dr. D. C. Hill for valuable discussions and advice and acknowledges the supervision of the analytical laboratory by Professor D. Arthur.
A
component of the calcium:phosphorus ratios. With increasing increments of added phosphorus in the assay diets there was a decrease in the proportion of unavailable to available phosphorus and a concomitant decrease in the ratio of calcium to available phosphorus. Examples calculated from the data of Dilworth and Day (1964) and Waldroup et al. (1965) are presented in the discussion of this paper. The main part of the current study was designed to investigate the effect of calcium: phosphorus ratios based on total available or inorganic phosphorus (Pz)1 in a phosphorus assay procedure. An experiment was also performed to determine the level of calcium required with the basal diet for maximum growth combined with a low bone ash response. The objective was to devise a procedure in which growth would be uniform with all levels of dietary phosphorus and bone calcification would serve as the sole response criterion. EXPERIMENTAL
The composition of the basal diet (Table 1) used in these experiments was similar to that employed in a previous study (Motzok et al, 1965). The basal diet was analyzed prior to the addition of calcium and phosphorus supplements. Sufficient amounts of the mineral supplements were added to provide the desired levels of calcium and phosphorus in the diets and ground cellulose was used as the additional variable to maintain the nutrient content at
967
1
See footnote 3 in Table 1.
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NUMBER of chick assay procedures have been devised and tested for the estimation of availability of phosphorus in various mineral supplements. Invariably the assays have indicated a relatively poor availability of phosphorus from soft phosphate, the range extending from 25% (Gillis et al, 1954) to 58% (Waldroup et al, 1965). These low values for the utilizable phosphorus of soft phosphate are in marked contrast with the relatively good growth and bone calcification of chick reported recently with this type of supplement when specific calcium: phosphorus ratios and high levels of vitamin D 3 were used (Motzok et al, 1965, 1967; Fritz and Roberts, 1966; McKnight and Watts, 1966). The literature concerned with various aspects of phosphorus biosassay has been reviewed by Nelson and Peeler (1964), Waldroup et al. (1965) and Sullivan (1966). In many of the assay diets the calcium content has been adjusted to give either a constant level of calcium or a constant or variable calcium:phosphorus ratio for all levels of phosphorus. In the latter type of assays the calcium:phosphorus ratios were usually based on total calcium and total phosphorus. In cases where the basal diet contained large amounts of plant products, such as soybean meal and corn, a major portion of the phosphorus was organic in nature, a form of phosphorus which usually has been considered to be only partly available to the chick. Consequently, a constant amount of unavailable phosphorus was included in the phosphorus
968
I . MOTZOK TABLE 1.—Composition of diets Ingredients
Percent
Ground wheat Soybean meal (44% protein) Ground yellow corn Casein Dried buttermilk Dehydrated alfalfa Salt (iodized) Soybean oil Micro-ingredients1 Variable ingredients 2
42.0 31.0 15.0 1.0 1.0 1.5 0.25 2.5 0.35 5.4 22.2 0.17 0.39 0.13
1 Supplied per kilogram of diet: 42 mg. manganese, 3.3 mg. riboflavin, 1.54 g. choline chloride (25%), 0.5 g. DL-methionine, 6,150 I.U. vitamin A, 1.0 g. Merck's antibiotic—vitamin B I2 supplement (containing 2 g. procaine penicillin G and 3.0 mg. vitamin B,2 per pound). 2 Required amounts of calcium and phosphorus supplements, vitamin D 3 and ground cellulose (Solka Floe). 3 Calculated on the basis that all of the phosphorus of non-plant feed ingredients is considered to be inorganic and that 30% of the phosphorus of plant products is non-phytin phosphorus and may be considered as part of the inorganic phosphorus required (N.R.C., 1966).
a constant level. A commercial preparation of vitamin D 3 supplement2 of known potency was used to supply the desired level of this vitamin. In the assay experiments the standard and test supplements (dicalcium phosphate, chemical grade CaHP0 4 -2H 2 0, and soft phosphate) were added to the diets at increment levels of phosphorus which were equal in magnitude on the logarithmic scale to the base 10 (Summers e( al., 1959). Calcium carbonate (U.S.P. grade) was used to adjust the calcium content of the diets to the levels specified. The complete diets were analyzed for calcium and phosphorus as a check on the accuracy of mixing.
EXPERIMENTAL AND RESULTS
Preliminary Experiment. Prior to conducting assays on soft phosphate, a study was made on the response of chicks to the basal diet supplemented only with calcium carbonate. A control diet containing 1.0% calcium and 0.45% inorganic phosphorus, with dicalcium phosphate as the source of additional phosphorus, was included to provide an index of the response of chicks to the calcium-supplemented basal diet. Two levels of vitamin D 3 , 450 and 7500 I.C.U./kg., were used and the responses of chicks to various treatments are given in Table 2. The diet with the lowest level of calcium (0.4%) promoted the best growth among the treatments without added phosphorus. The growth of chicks decreased progressively as the dietary calcium was increased, the decrease in growth being highly significant when the diets contained 0.8 or 1.0% calcium. Similarly, there was a significant decrease in the ash content of tibiae of chicks fed diets containing more
2
We are indebted to Dawe's Laboratories, Inc., Chicago, 111., for the determination of the potency of the vitamin D 3 supplement.
s The number of replicates and chicks per groups are given in the tables.
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Average analysis Crude protein Calcium Phosphorus Total (analyzed) Inorganic (calculated) 3
Day-old White Plymouth Rock pullets were randomized into lots of 15-20 chicks each. Replicated lots3 of chicks were fed each ration for 4 weeks. They were reared in electrically heated wire-floored battery brooders and the pens and diets were allocated at random. The chicks were weighed at regular intervals and at the end of the experiment the feed was removed from the troughs in the evening of the day before the final weights were recorded and the chicks killed for the removal of tibiae. The ash content of the bones was determined according to the A.O.A.C. (1955) procedure for vitamin D assay. The data were subjected to analysis of variance as outlined by Snedecor (1957) and parallel line assay as specified by Bliss (1952).
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PHOSPHORUS ASSAY
TABLE 2.—Effect of varying amounts of calcium, phosphorus and vitamin Dz on -weight and bone ash of chicks1 at 4 weeks Vitamin Ds (I.C.U./kg.) Mineral supplements
Ca!
Pis
(%) (%)
450 |7,500
450
|7,500
Av. wt. (g.) Bone ash (%) CaCOs CaC03 CaC03 CaCOs CaHP04-2Hs0 and CaCOs
0.4 0.6 0.8 1.0
0.13 0.13 0.13 0.13
460 444 386 301
461 445 391 321
38.6 37.9 35.2 31.0
39.8 40.5 36.3 33.6
1.0
0.45
495
505
46.8
45.9
than 0.6% calcium. The highest growth and bone ash obtained with the basal diet supplemented only with calcium were significantly lower than the responses produced by the diet containing 1.0% calcium and 0.45% inorganic phosphorus. However, good livability was obtained and the mortality which did occur was not related to the dietary treatments. Thirteen chicks died; of these, three deaths occurred among the birds fed the diet containing 1.0% calcium and 0.45% inorganic phosphorus. Assay 1. In the preliminary study with the basal diet, maximum growth was obtained when the calcium content of the basal diet
Assay 2. In this experiment the Ca:Pz ratio was maintained at approximately 2.2:1. The choice of this ratio was based on the observation by Motzok et al. (1965) that good growth and bone ash were obtained in chicks fed diets with soft phosphate when the total calcium:total phosphorus ratio was 1.4:1, which is equivalent to a ratio of
TABLE 3.—Effect of varying amounts of dicalcium phosphate and soft phosphate (Ca:Pi approximately 3.1:1 in all diets) on weight, feed utilization and bone ash of chicks1 at 4 weeks Phosphorus supplements
P Added
Pi 2
Ca3
(%)
(%)
(%)
Weight (g.)
CaHPOv2H 2 0
0.044 0.070 0.111 0.176
0.174 0.200 0.241 0.306
0.54 0.62 0.75 0.95
444 440 433 444
1.99 1.88 1.89 1.89
797 744 731 757
41.0 41.5 42.4 45.2
Soft phosphate
0.044 0.070 0.111 0.176
0.174 0.200 0.241 0.306
0.54 0.62 0.75 0.95
444 438 413 423
2.12 1.88 1.98 1.92
829 737 728 726
40.6 42.4 42.4 42.1
Feed/Gain Feed per Bone ash bird (g.) (%)
Least significant difference for P = 0.05 and P = 0.01 respectively: Growth—28 g. and 39 g. Bone ash -1.6% and 2.2%. 1 Duplicate groups, 20 chicks per group. 2 Total inorganic phosphorus (calculated as in Table 1, footnote 3). 3 Total calcium.
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Least significant difference tor P=0.05 and P=0.01 respectively: Weight—20 g. and 28 g. Bone ash—2.7% and 3.8%. 1 Duplicate groups, 20 chicks per group. 2 Total calcium by analysis. • Total inorganic phosphorus (calculated as described in Table 1, footnote 3).
without added phosphorus was 0.4% and the Ca:Pz ratio was approximately 3.1:1. Accordingly, an assay of soft phosphate was conducted in which a Ca:P« ratio of about 3.1:1 was maintained in all diets. Vitamin D 3 was provided at a level of 6600 I.C.U./kg. of diet and the responses of chicks to the various dietary treatments are given in Table 3. Analysis of the data showed that there was no significant difference in the growth of chicks among the dietary treatments employed in this experiment. Although there was a marked increase in bone ash with increasing levels of dicalcium phosphate, there was no response in the bone ash of chicks fed diets with soft phosphate beyond the second increment of the supplement. Consequently, no attempt was made to estimate the availability of phosphorus from the test supplement in this trial.
970
I . MOTZOK
TABLE 4.-
soft phosphate was calculated to be 63%, with 95% approximate fiducial limits of 50-79%. Assay 3. In the previous trial the high growth response with the two upper levels of dicalcium phosphate suggested that perhaps the amount of calcium was suboptimal for growth in the diets with the two low levels of dicalcium phosphate and in the diets supplemented with soft phosphate. This assumption was tested in a third trial in which the Ca:Pz ratio was increased to 2.5:1. In all other respects the assay procedure was similar to that of Assay 2 and the responses of chicks to these treatments are given in Table 5. Feed utilization was relatively uniform and there was no significant difference in the weight of chicks among any of the treatments. The slopes of the response curves for bone ash obtained with the standard and test supplements were highly significant, linear and parallel, and the comparative biological availability for phosphorus from soft phosphate was estimated to be 71%, with 95% approximate fiducial limits of 63-81%. DISCUSSION
Soft phosphate was chosen as the test
-Effect of varying amounts of dicalcium phosphate and soft phosphate (Ca:Pi at 2.2:1 in all diets) on weight, feed utilization and bone ash of chicks1 at 4 weeks
Phosphorus supplements
P added
Pi 2
Ca3
(%)
(%)
(%)
Weight (g.)
CaHP0 4 -2H 2 0
0.044 0.070 0.111 0.176
0.385 0.440 0.535 0.675
407 407 444 444
1.93 1.96 1.88 1.87
703 710 752 752
37.8 38.0 40.6 43.0
Soft phosphate
0.044 0.070 0.111 0.176
0.174 0.200 ' 0.241 ro.306 P* 0.174 0.200 0.241 0.306
0.390 0.440 0.535 0.675
367 393 398 395
2.10 1.95 1.95 1.89
658 683 683 671
35.7 36.6 38.2 40.3
Feed/Gain Feed per bird (g.)
Bone ash
(%)
Least significant difference for P = 0.05 and P = 0.01 respectively: Growth—29 g. and 44 g. Bone ash1.8% and 2.8%. 1 Duplicate groups, 20 chicks per group. 2 Total inorganic phosphorus (calculated as in Footnote 3, Table 1). 3 Total calcium.
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2.2:1 on the basis of total inorganic phosphorus. A four point assay on soft phosphate was conducted with dicalcium phosphate as the standard and 7500 I.C.U. of vitamin D 3 /kg. in all diets. The levels of phosphorus employed and the responses of the birds to the dietary treatments are given in Table 4. The average weights of the chicks which received diets with the two low levels of dicalcium phosphate were significantly lower than those of birds fed diets with the two high levels of the standard phosphate. The weights of chicks fed diets with soft phosphate, with the exception of the lowest level, were comparable and not significantly different from those of chicks fed the two low levels of dicalcium phosphate. The growth of chicks appeared to be governed largely by the amount of the diet consumed. When the data for bone ash were plotted, the first point in each case appeared to be on the induction part of the response curve. Consequently, only the data for the three upper levels were used in the estimate of availability. The slopes of the response curves for bone ash were found to be highly significant, linear and parallel, and the relative availability of phosphorus in
971
PHOSPHORUS ASSAY
TABLE 5.—Effect of varying amounts of dicalcium phosphate and soft phosphate {Ca'.Pi approximately 2.5:1 in all diets) on weight, feed utilization and bone ash of chicks1 at 4 weeks Average Feed per weight Feed/Gain bird (g.) (g.)
Phosphorus supplements
P added
Pi 2
Ca3
(%)
(%)
(%)
CaHP0 4 -2H 2 0
0.044 0.070 0.111 0.176
0.174 0.200 0.241 0.306
0.435 0.500 0.600 0.776
400 389 403 396
1.96 1.96 1.95 1.96
701 680 693 715
39.6 40.7 42.9 44.1
Soft phosphate
0.044 0.070 0.111 0.176
0.174 0.200 0.241 0.306
0.435 0.500 0.600 0.776
375 378 396 404
1.98 1.97 1.96 1.95
675 660 706 716
38.3 39.5 41.5 43.0
Bone ash
(%)
material on the basis that it represented natural inorganic phosphorus supplements possessing relatively low solubility (Hill et al., 1945), a property which may be the main reason for the low availability of its phosphorus estimated by many previous assay procedures. The basal diet used in the current investigations had the same composition as the one with which a good response in chicks was obtained with soft phosphate by Motzok et al. (1965). The present data show that this diet supplemented with a small amount of calcium (0.21% as calcium carbonate) supported good livability and growth. The utilizable phosphorus provided by the basal ingredients obviated the need of adding "available" inorganic phosphorus to a basal diet low in phosphorus, such as the semipurined diet employed by Nelson and Peeler (1961). Nelson and Peeler (1961) also used a practical diet for the assay of soft phosphate. Employing a calcium: available phosphorus ratio of 2:1, they obtained a response in growth and bone ash with added increments of phosphorus. The diets were supplemented with 750 I.C.TJ. of vitamin D 3 /kg. and the availability of phosphorus from soft phosphate was estimated
to be 36%. In the present study there was also a response in bone ash and growth to added phosphorus when the Ca:Pi was maintained at 2.2:1 and the diets contained 7500 I.C.U. vitamin D 3 /kg. The higher level of vitamin D 3 used in this trial was probably the main reason for the estimated phosphorus availability of 63%, in comparison with the low availability of this mineral from soft phosphate obtained by Nelson and Peeler (1961). The data of Assays 1 and 3 provide evidence that the dietary Ca:Pj ratio in an assay procedure is a critical factor governing the availability of phosphorus from a supplement of relatively low solubility, even with an abundance of vitamin D 3 in the diet. A Ca:Pi ratio of 2.5:1 with the high level of vitamin D 3 enabled chicks to utilize 71% of the phosphorus from soft phosphate and promoted uniform growth on all levels of the supplements. On the other hand, a Ca:P« ratio of 3.1:1 gave uniform growth but the response in bone ash to increasing levels of soft phosphate was negligible. It appears that, with a Ca:Pz ratio of 3.1:1, the extra calcium provided by calcium carbonate was in excess of the amount required for maximum utilization of minerals from soft phosphate in
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Least significant difference for P = 0.05 and P = 0.01 respectively: Growth—38 g. and 54 g. Bone ash1.3% and 1.8%. 1 Triplicate groups, 15 chicks per group. 2 Total inorganic phosphorus (calculated as in Footnote 3, Table 1). 8 Total calcium.
972
I. MOTZOK
the highest level of supplemental phosphorus which provide a Ca:Pz ratio of about 2.6:1. In the present study a difference in bone ash of 1.1% was obtained between birds fed dicalcium phosphate and those fed soft phosphate at the highest level of phosphorus when the Ca:Pi ratio was 2.5:1 (Table 5). Perhaps the amount of vitamin D 3 employed (792 I.C.U./kg.) by Waldroup et al. was inadequate for maximum utilization of soft phosphate even with the narrow Ca:P? ratio. A comparison of the availabilities of 63 and 71% by application of the t test revealed no significant difference (P > 0.05) between the two estimates obtained with assays 2 and 3 in the present study. However, the difference in availabilities was wide enough to suggest that differences in growth at different phosphorus levels in assay 2 may have been a complicating factor which resulted in a lower estimate of phosphorus availability. The relationship between growth and bone ash in response to supplemental phosphorus and calcium appears to be quite complex. For example, Nelson and Walker (1964) showed that a plot of the percentage bone ash and the logarithm of the percentage dietary phosphorus was a straight line, whereas the weights of chicks showed a highly significant curvature over the same range of dietary phosphorus. Indeed, there appears to be no precise information in the literature on the relative requirements of calcium and phosphorus for each of these responses in birds. In an attempt to resolve this problem in an assay procedure, Sullivan (1966) proposed the use of three criteria, body weight, percent bone ash and gain:feed ratio, to compute a single biological value of a phosphorus supplement. Practical usage, precision and simplicity were considered in the derivation of a formula for four-week poult data. The approximate
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an assay procedure, producing a condition analogous to that obtained with high calcium in studies with adequate amounts of phosphorus (Motzok et al., 1965, 1967; Fritz and Roberts, 1966; McKnight and Watts, 1966). Dilworth and Day (1964) obtained uniform growth and a response in bone ash with increasing levels of phosphorus from several phosphorus supplements in a chick assay in which the diets were supplemented with 2010 I.C.U. vitamin D 3 /kg. However, the responses in growth and bone ash with soft phosphate were appreciably lower than those obtained with the standard supplement and the availability of phosphorus from soft phosphate was calculated to be only 38%. These workers employed a total calcium:total phosphorus ratio of 1.7:1. If it is assumed that their basal diet contained about 0.09% available phosphorus, the Ca:Pz° ratios calculated from their data decreased from about 3.2:1 to 2.8:1 when the supplemental phosphorus was increased from 0.15 to 0.25%. Waldroup et al. (1965) used three regimes of supplemental calcium in a comparison of phosphorus assay techniques and the comparative availability of phosphorus from soft phosphate was 58.9, 52.5 and 51.5% with a constant level of 1% calcium, a 2:1 Ca:P ratio and a "sliding" Ca:P ratio, respectively. On the assumption that the basal diet contained about 0.09% available phosphorus, calculations reveal that with a constant 1% calcium in all diets the Ca:Pz ratios dropped from about 7.1:1 to 2.6:1 with increasing levels of phosphorus; with a constant Ca:P ratio (2:1) the Ca:P« ratios decreased from 5:1 to 3.2:1; with the "sliding" Ca:P ratios the Ca:P« ratios diminished from 3:1 to 2.8:1. In the assay with the constant level of 1% calcium these workers obtained a difference in bone ash of 3.2% between chicks fed dicalcium phosphate and soft phosphate at
973
PHOSPHORUS ASSAY
ratios in the diets were too narrow or too wide and in some cases the level of vitamin D 3 was inadequate for maximum utilization of the minerals in soft phosphate. Even the comparative availability of the phosphorus in soft phosphate, obtained in the present study, appears to be somewhat lower than the degree of utilization which has been achieved with optimum amounts of calcium and vitamin D 3 in practical diets containing 0.45-0.48% inorganic phosphorus (Motzok et ah, 1965,1967). SUMMARY
Studies were made on the effect of different ratios of calcium anorganic phosphorus (Pi) on the response of chicks in an assay for the availability of phosphorus from soft phosphate with dicalcium phosphate as the standard. The following observations were made: 1. When the Ca:P« ratios were either 2.2:1 or 2.5:1, a highly significant increase in bone ash was obtained with increasing levels of the standard and test supplements. The responses were linear and parallel for the three upper levels of phosphorus when the Ca:P? ratio was 2.2:1 and for all four points when the ratio was 2.5:1. 2. There were some significant variations in growth produced by the Ca:Pz ratio of 2.2:1 and the comparative availability of phosphorus in soft phosphate, calculated from the bone ash data, was 63%. 3. When the Ca:Pi ratio was 2.5:1, growth was relatively uniform at all levels of phosphorus and the availability of phosphorus from soft phosphate was 71%. 4. A wide Ca:Pz" ratio (3.1:1) produced uniform growth with graded levels of phosphorus but a significant response in bone ash was obtained only with dicalcium phosphate. There was no increase in bone ash of chicks fed soft phosphate beyond the second level of phosphorus in a four point assay.
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contribution of each response criterion to the calculated biological value was: body weight, 55%; percent bone ash, 40%; gain:feed ratio, 5%. However, Nelson and Walker (1964) examined a vast amount of their assay data and presented a critical appraisal of the value of growth of chicks as a criterion of response in a phosphorus assay procedure. These investigators analyzed 82 experiments using only the steepest part of the growth response curves and calculated that 5.9 times as many pens of chicks would be required to obtain comparable accuracy when body weight instead of bone ash is used to measure phosphorus quality. It appears from their data that a higher degree of accuracy in a phosphorus bioassay can be achieved when bone ash serves as the sole response criterion than when 55% of the biological value of phosphorus is based on body weight and only 40% on bone ash, as recommended by Sullivan (1966). The maintenance of uniform growth of chicks given various levels of calcium and phosphorus in assay 3 of the current work eliminated the need of taking into account a response in growth in the estimation of phosphorus availability. It is recognized that the range in bone ash was not as wide as that obtained by other investigators, but the responses in bone ash to increasing increments of the standard phosphate and test material were linear and parallel and conformed to the assay requirements outlined by Bliss (1952). Furthermore, these responses were at the upper portion of the full range in bone ash obtainable in chicks and they represent the area of response in which supplemental phosphorus is utilized under practical feeding conditions. The present results suggest that the low values for the availability of phosphorus from soft phosphate which have been reported in the literature were due mainly to assay procedures. It appears that the Ca:Vi
974
I . MOTZOK ACKNOWLEDGMENTS
REFERENCES Association of Official Agricultural Chemists, 1955. Methods of Analysis. 8th Ed., Washington, D.C. Bliss, C. I., 1952. The Statistics of Bioassay. Academic Press, Inc., New York. Dilworth, B. C , and E. J. Day, 1964. Phosphorus availability studies with feed grade phosphates. Poultry Sci. 43 : 1039-1044. Fritz, J. C , and T. Roberts, 1966. Influence of levels of vitamin D and calciu mon the utilization of phosphorus by the growing chick. Poultry Sci. 45: 1085-1086. Gillis, M. B., L. C. Norris and G. F. Heuser, 1954. Studies on the biological value of inorganic phosphates. J. Nutrition, 52: 115-125. Hill, W. L., D. S. Reynolds, S. B. Hendricks and K. D. Jacobs, 1945. Nutritive evaluation of defluorinated phosphates and other phosphorus supplements. I. Preparation and properties of the samples. J. Assoc. Off. Agric. Chem. 28:
105-118. McKnight, W. F., and A. B. Watts, 1966. The effect of vitamin D s on the utilization of phosphorus from various sources. Poultry Sci. 45: 1104. Motzok, I., D. Arthur and H. D. Branion, 1965. Factors affecting the utilization of calcium and phosphorus from soft phosphate by chicks, Poultry Sci. 44: 1261-1270. Motzok, I., D. Arthur and S. J. Slinger, 1967. Utilization of calcium and phosphorus from soft phosphate by chicks. Poultry Sci. 46: 985-991. National Research Council, 1966. Nutrient requirements for domestic animals. 1. Nutrient requirements of poultry. Nelson, T. S., and H. T. Peeler, 1961. The availability of phosphorus from single and combined phosphates to chicks. Poultry Sci. 40: 1321-1328. Nelson, T. S., and H. T. Peeler, 1964. Current status of biological testing of feed phosphates. Feedstuffs, 36: 64-69. Nelson, T. S., and A. C. Walker, 1964. The biological evaluation of phosphorus compounds. Poultry Sci. 43: 94-98. Snedecor, G. W., 1957. Statistical Methods. The Iowa State College Press, Ames, Iowa. Sullivan, T. W., 1966. A triple response method of determining biological value of phosphorus sources with young turkeys. Poultry Sci. 45: 1236-1245. Summers, J. D., S. J. Slinger, W. F. Pepper, I. Motzok and G. C. Ashton, 1959. Availability of phosphorus in soft phosphate and phosphorus acid and the effect of acidulation of soft phosphate. Poultry Sci. 38: 1168-1179. Waldroup, P. W., C. B. Ammerman and R. H. Harmus, 1965. A comparison of phosphorus assay techniques with chicks. Poultry Sci. 44:10861089.
NEWS AND NOTES (continued from page 915) its Fact Finding Conference. He was selected for the honor by an independent committee which considered 49 candidates nominated by people in the industry. The award is made "in recognition of service to the poultry and egg industry above and beyond self-gain." In reporting its choice, the committee credited Johnson with being an "idea man" who had done as much as any one person to make it possible for the poultry and egg business to become a modern, auto-
mated food business. Dedicated to the growth and progress of the industry, he repeatedly has invested the capital, time, talent, and energy of himself and his company, with no guarantee of compensation, to give the industry answers to many of its problems in production, processing, and marketing. A native of Maine, he graduated in 1922 from Iowa State College, and became a Poultry Extension Man in Iowa. He soon moved to the Buckeye Incubator Company as a salesman.
(continued on page 999)
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The author is indebted to the Ontario Department of Agriculture and Food for financial aid and the Soft Phosphate Research Institute Inc., Ocala, Florida, and The National Research Council of Canada for grants in support of this work. He wishes to thank Merck, Sharp and Dohme of Canada Ltd. for the antibiotic and vitamin B12 supplement and Pfizer Canada Ltd. for the vitamin A. He is grateful to Dr. S. J. Slinger and Dr. D. C. Hill for valuable discussions and advice and acknowledges the supervision of the analytical laboratory by Professor D. Arthur.