Citation
Development of a chick assay for determining availability of phosphorus from various phosphate materials

Material Information

Title:
Development of a chick assay for determining availability of phosphorus from various phosphate materials
Creator:
Damron, Bobby L. ( Dissertant )
Harms, Robert H. ( Thesis advisor )
Fry, Jack L. ( Reviewer )
Wilson, Henry R. ( Reviewer )
Grigsby, S. E. ( Reviewer )
Ammerman, C. B. ( Reviewer )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Copyright Date:
1968
Language:
English
Physical Description:
62 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Ashes ( jstor )
Birds ( jstor )
Body weight ( jstor )
Bones ( jstor )
Calcium ( jstor )
Diet ( jstor )
Phosphates ( jstor )
Phosphorus ( jstor )
Poultry ( jstor )
Tibia ( jstor )
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Abstract:
Abbreviated introduction: This series of experiments was designed to develop a standard assay curve for each of the commercial phosphate sources used in poultry diets. Such a standard curve should be of great value in eventually establishing a standard phosphorus assay that will give more realistic values of utilization, especially for test materials.
Thesis:
Thesis (Ph. D.)--University of Florida, 1968.
Bibliography:
Bibliography: leaves 58-60.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Bobby Leon Damron.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
029884141 ( AlephBibNum )
24664216 ( OCLC )
ACF3872 ( NOTIS )

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DEVELOPMENT OF A CHICK ASSAY FOR
DETERMINING AVAILABILITY OF
PHOSPHORUS FROM VARIOUS
PHOSPHATE MATERIALS









By
BOBBY LEON DAMRON












A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY










UNIVERSITY OF FLORIDA
1968












ACKNOWLEDGEMENTS


The author is grateful to Dr. Robert H. Harms,

Chairman of the Supervisory Committee, for his constant

guidance, patience, and assistance in the planning and

execution of the research reported in this dissertation.

Appreciation is also expressed to Dr. J. L. Fry, Dr. H. R.

Wilson, Dr. S. E. Grigsby, and Dr. C. B. Ammerman for

their valuable suggestions and encouragement.

The author is indebted to Miss Kathleen Wall for

her technical assistance, and to the undergraduate students

for their assistance.

The cooperation of the Smith-Douglass Company,

Norfolk, Virginia, is gratefully acknowledged for technical

and financial contributions.













TABLE OF CONTENTS


Page
ACKNOWLEDGEMENTS ... .................................... ii

LIST OF TABLES ....................................... v

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW .......... 1

2 DEVELOPMENT OF A CALCIUM STANDARD CURVE
FOR MONOSODIUM PHOSPHATE .................. 8

Experiment 1 ................................ 8
Experimental Procedure ................. 8
Results and Discussion ................ 11
Experiment 2 ................................ 15
Experimental Procedure ................. 15
Results and Discussion ........ .......... 15
Summary ................................... 18

3 DEVELOPMENT OF A CALCIUM STANDARD CURVE
FOR DEFLUORINATED PHOSPHATE ............... 19

Experimental Procedure .... ................ 19
Results and Discussion .................... 21
Summary ................................... 23

4 DEVELOPMENT OF A CALCIUM STANDARD CURVE
FOR DICALCIUM PHOSPHATE ................... 25

Experimental Procedure .... ................ 25
Results and Discussion ...................... 27
Summary ................................... 29

5 DEVELOPMENT OF A CALCIUM STANDARD CURVE
FOR SOFT PHOSPHATE .... .......... ......... 31

Experimental Procedure ... ................. 32
Results and Discussion .................... 33
Summary ................................... 36










TABLE OF CONTENTS--Continued


CHAPTER Page

6 INFLUENCE OF DIET COMPOSITION ON THE
UTILIZATION OF SOFT PHOSPHATE IN
BROILER DIETS ............................... 38

Experimental Procedure .... ................ 39
Experiment 1 ............................. 42
Experiment 2 ............................. 43
Experiment 3 ............................. 43
Results and Discussion .................... 44
Experiment 1 ............................. 44
Experiment 2 ............................. 45
Experiment 3 ............................. 48
Summary ................................... 52

7 SUMMARY ...................................... 54


REFERENCES .......................................... 58












LIST OF TABLES


Table Page

1 Composition of basal diet .................... 9

2 Tibia ash and body weight of chicks fed
various levels of phosphorus and calcium
supplied from monosodium phosphate and
reagent grade calcium carbonate .............. 12

3 Tibia ash and body weight of chicks fed
various levels of phosphorus and calcium
supplied from monosodium phosphate and
reagent grade calcium carbonate .............. 17

4 Tibia ash and body weight of chicks fed
various levels of phosphorus and calcium
supplied from defluorinated phosphate and
reagent grade calcium carbonate .............. 22

5 Tibia ash and body weight of chicks fed
various levels of phosphorus and calcium
supplied from dicalcium phosphate and
reagent grade calcium carbonate .............. 28

6 Tibia ash and body weight of chicks fed
various levels of phosphorus and calcium
supplied from soft phosphate and reagent
grade calcium carbonate ..................... 34

7 Composition of basal diets .................. 41

8 Four-week body weights of chicks fed
different levels of soft phosphate and
ground limestone (experiment 1) ............. 46

9 Four-week body weights, feed conversion,
and tibia ash of chicks fed different
levels of soft phosphate and ground
limestone (experiment 2) ..................... 47

10 Four- and eight-week body weights, feed
conversion, and tibia ash of chicks fed
different levels of soft phosphate and
ground limestone (experiment 3) ............. 49










CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW


The element phosphorus was first prepared in the

free state by Brandt, a German chemist, in 1669 and first

recognized as an essential bone component by Gahn, a

Swedish chemist, in 1769. Phosphorus probably plays a

more varied role in the chemistry of living organisms

than any other single element. It is also an essential

constituent of proteins and fats occurring in muscular

tissues, vital organs and brain. Phosphates are also

known to be important buffers in tissue fluids.

Deficiencies of mineral elements in animal rations

began to be recognized about 100 years ago when weak bones

in cattle grazing in certain localities began to be as-

sociated with mineral inadequacies of the soil. According

to Ewing (1963) the earliest recorded use of a phosphate

feed supplement for the specific purpose of preventing a

phosphorus deficiency in ruminants occurred in 1861 when

Von Gohren reported that the weak bones of cattle grazing

near the Rhine River could be prevented and cured by

feeding small amounts of bone meal. Analysis of the soil

and grass in these areas revealed a very low phosphorus

and calcium content.


- 1 -





- 2 -


Weak bones in non-ruminants, particularly in swine,

was noted as a serious problem around 1885, and it was

found that feeding bone meal to pigs as a supplement to

corn more than doubled the breaking strength of deficient

bones.

Rock phosphate is the most abundant source of

phosphorus, with approximately half of the 26 billion ton

world reserve located in the United States. These

phosphates were used as sources of phosphorus in plant

nutrition as early as 1860. The belief that only phospho-

rus from plant and animal sources was available to animals

accounted for the delay in its use in animal nutrition,

and as late as 1914 it was still felt that at least a part

of the animal's phosphorus requirement should be supplied

in organic form.

The danger of fluorine toxicity began to be

recognized as a serious problem around 1930, and, as a

consequence, raw rock phosphates have now been largely

replaced by sources that have been treated for the removal

of fluorine. Defluorinated super phosphate, which was

one of the first of these sources to be produced in

commercial quantities, was in general use about 1940.

Considerable research has been conducted in recent

years to determine which phosphorus supplements are suita-

ble for use in poultry feeds. Among the sources recently





- 3 -


evaluated was colloidal phosphate, which is also called

soft phosphate. Soft phosphate is obtained as a by-product

during the mining of rock phosphate, and is an inexpensive

source of supplemental phosphorus. The use of such a

supplement in poultry diets would be of great importance

from a commercial point of view.

The latest addition to the list of commercial

phosphate feed supplements is a dicalcium phosphate which

is made by treating phosphoric acid with the proper amount

of lime. It is practically free of fluorine and contains

up to 22 percent phosphorus as compared to approximately

15 percent in the best grades of bone meal and about 8.5

to 14.5 percent in the original defluorinated rock

phosphates.

The National Research Council (1966), in a recent

revision of its suggested nutrient requirements for chickens,

increased the total dietary phosphorus requirement for

starting chickens from 0.60 percent to 0.70 percent of the

diet. Numerous studies have been conducted to establish

this requirement for the chick, and there has been vari-

ation in reported requirements. McGinnis et al. (1944)

reported that levels of greater than 0.58 percent phosphorus

were required for maximum calcification. The available

phosphorus requirement necessary for satisfactory





- 4 -


calcification was found by Singsen et al. (1947) to be

between 0.38 and 0.47 percent of the diet. Gillis et al.

(1949) and O'Rourke et al. (1952) found a requirement of

approximately 0.50 percent. Grau and Zweigart (1953)

indicated that maximum tibia ash of chicks was obtained

with a level of not more than 0.45 percent phosphorus.

Maximum calcification of chick bones was produced by a

level of 0.58 percent total phosphorus in studies conducted

by Fisher et al. (1953). A level of 0.76 to 0.81 percent

phosphorus was suggested by Couch et al. (1937) as being

adequate for normal growth and bone calcification of

chicks up to 12 weeks of age. Additional studies were

reviewed by Singsen at al. (1948), Gillis ot al. (1949),

and O'Rourke et al. (1952), and Nelson and Walker (1964).

Much of this variability in reported phosphorus

requirements can probably be attributed to differences in

availability of the phosphorus to the chick. The National

Research Council (1966) states that at least 0.50 percent

of the total feed of starting chickens should be inorganic

phosphorus. All the phosphorus of non-plant feed ingredients

is considered to be inorganic in nature. Approximately 30

percent of the phosphorus of plant products is non-Phytin

phosphorus and may be considered as part of the inorganic

phosphorus requirement. A portion of the phosphorus





- 5 -


requirement of growing chickens and laying and breeding

hens must also be supplied in inorganic form, but this

requirement for inorganic phosphorus is lower and not as

well defined as it is for starting chickens. The inter-

action of vitamin D, has long been recognized as one of

the factors governing the utilization of calcium and

phosphorus from mineral sources. It has been demonstrated

under certain conditions that levels of vitamin D3 con-

siderably in excess of those suggested by the National

Research Council (1966) may improve the utilization of the

phosphorus from certain low grade phosphate sources

(Motzok et al., 1965; Fritz and Roberts, 1966; and McKnight

and Watts, 1966).

The advent of the computer has afforded the re-

searcher and nutritionist with a valuable tool for the

formulation of least-cost feeds. In return, the machine

has increased the burden of its operator by demanding

exacting specifications and analytical values for feeds

and feedstuffs in order to function properly. In order

for the nutritionist to formulate diets in the most

economical and profitable manner he must have an accurate

evaluation of the phosphorus content of these supplements

and its availability. In the usual assay of phosphate

supplements, the test sources are added to a phosphorus-

deficient diet to supply graded sub-optimum levels





- 6 -


of phosphorus. The response, generally expressed in terms

of bone calcification or body weight, is then compared to

that from a standard source fed at equivalent total dietary

phosphorus levels to establish a relative biological value

for the test phosphate.

The interaction of calcium and phosphorus has

long been recognized. Adverse Ca:P ratios limit the

utilization of phosphorus, especially at sub-optimal

levels such as are used in phosphorus assay diets. However,

there is no standard calcium level or Ca:P ratio in general

use. Nelson and Peeler (1964) indicated that one of the

problems involved in the development of a biological assay

for feed phosphates was whether to hold the calcium level

constant or have a constant Ca:P ratio in the assay diet.

In certain assays, constant calcium to phosphorus ratios

were employed (Matterson et at., 1945; Singsen and Scott,

1946; Creech et al., 1956; Nelson and Peeler, 1961), while

in others, constant calcium levels of 1.0 percent (Ammerman

et at., 1960) or 1.2 percent (Gardiner et at., 1959) were

used.

Recent studies by Waldroup et al. (1965a) pointed

out that variation in the calcium content of the diet used

in assaying phosphorus sources may influence significantly

the relative biological value of a particular phosphate





- 7 -


source. In this report it was suggested that the use of

a specific calcium level at different phosphorus levels

appears to be desirable in order to elicit maximum response

of the chick and allow full utilization of a particular

phosphorus source. It is reasonable to assume that the

most valid biological value would be obtained if the chick

was able to maximally utilize the phosphorus present in

the test diet. Therefore, a calcium level should be used

which produces maximum growth and bone ash.

This series of experiments was designed to develop

a standard assay curve for each of the commercial phosphate

sources used in poultry diets. Such a standard curve

should be of great value in eventually establishing a

standard phosphorus assay that will give more realistic

values of utilization, especially for test materials.












CHAPTER 2

DEVELOPMENT OF A CALCIUM STANDARD CURVE
FOR MONOSODIUM PHOSPHATE


Monosodium phosphate has for many years been

recognized and utilized as a standard phosphorus source

in the determination of biological values and phosphorus

requirements for poultry. These experiments were conducted

to develop a standard calcium curve for this phosphorus

reference material.


Experiment 1


Experimental Procedure

The basal diet used for this study was composed

primarily of degerminated corn and soybean meal (Table 1).

It was calculated to contain 22 percent protein and 2200

Calories of productive energy per kilogram of diet.

Twenty-two hundred International Chick Units of supplemental

vitamin D, were provided per kilogram of diet in order to

eliminate any effect of this vitamin upon utilization.

This diet was found, by analysis, to contain 0.30 percent

total phosphorus and 0.26 percent calcium. This is a

considerably higher calcium level than previously reported

by Waldroup et al. (1965a) for this diet. The reason for this


- 8 -




- 9 -


TABLE 1

Composition of basal diet


Ingredient Percent of diet

Degerminated corn meal 51.70

Cerelose 5.40

Soybean meal (50% protein) 34.00

Alfalfa meal (20% protein) 3.00

Iodized salt 0.40

Micro-ingredients' 0.50

Variable2 5.00


Percent protein 22.00

Productive energy (Cal./kg.) 2200

Percent phosphorus 0.30

Percent calcium 0.26


ISupplied per kilogram of diet: vitamin A, 6600
I.U.; vitamin D3, 2200 I.C.U.; vitamin K, 2.2 mg.; ribo-
flavin, 4.4 mg.; pantothenic acid, 13.2 mg.; niacin, 39.6
mg.; choline, 499.4 mg.; vitamin B12, 22 mcg.; Santoquin,
0.0125%; manganese, 71.4 mg.; iron, 19.8 mg.; copper,
1.98 mg.; cobalt, 198 mg., iodine, 1.1 mg.; and zinc,
99 mcg.

2Calcium and phosphorus levels were obtained by
altering the levels of reagent grade calcium carbonate,
phosphate source, and yellow builders sand.




- 10 -


difference was not determined; however, it points out the

necessity of chemical analysis of the basal diet used with

each set of ingredients. Reagent grade monosodium phosphate

(NaH2PO4 H20) was used to supply supplemental phosphorus

in 0.05 percent increments to a total of 0.55 percent total

phosphorus.

Within each phosphorus level, reagent grade calcium

carbonate was used to supply supplemental calcium. Five

calcium levels were used at each phosphorus level. Levels

were selected which were estimated from previous studies

(Waldroup et al., 1965a) to result in a response line and

plateau within each phosphorus level so that the appropriate

calcium requirement for that particular phosphorus level

could be selected.

Three successive trials were conducted. In each

trial, three replicate groups of five male and five female

broiler-type chicks (Vantress x White Plymouth Rock) per

group were fed each diet from 1 to 21 days of age. The

chicks were sexed, debeaked, and randomly assigned to

treatment groups at one day of age.

The chicks were brooded in Oakes 801-A five deck

thermostatically controlled, electrically heated, battery

brooders with raised wire floors. At the termination of

each trial, all birds were individually weighed, and two

males and two females from each group were sacrificed for





- 11 -


tibia ash determinations. The vitamin D determination

procedure of the Association of Official Agricultural

Chemists (1965) was followed in ashing the bones. Sta-

tistical analysis (Analysis of Variance; Snedecor, 1956)

of the data failed to reveal a treatment x trial inter-

action; therefore, the data from the three trials were

combined. Significant differences between tibia ash and

body weight treatment means were determined by Duncan's

multiple range test (1955). Based on these measurements,

the ideal calcium level was selected for each level of

phosphorus, and a standard curve of calcium levels

determined for the various additions of monosodium

phosphate.


Results and Discussion

The level of supplemental calcium fed with each

level of phosphorus significantly influenced tibia ash

and body weight (Table 2). However, the greatest influence

was noted with tibia ash since responses were obtained

with higher levels of calcium, especially at the higher

phosphorus levels. The ideal calcium level was different

for the five different phosphorus levels.

Maximum tibia ash and body weight were obtained

with 0.18 percent supplemental calcium when the diet

contained 0.30 percent phosphorus (Table 2). However,





12 -


TABLE 2

Tibia ash and body weight of chicks fed various levels
of phosphorus and calcium supplied from monosodium
phosphate and reagent grade calcium carbonate


Calcium1
(%) Tibia Body
Total phosphorus' ash2 weight2
(%) Supplemental Total (%) (grams)


0.30





0.35





0.40





0.45





0.50





0.55


0.03
0.08
0.13
0.18
0.23
0.03
0.09
0.15
0.21
0.27
0.03
0.10
0.17
0.24
0.31
0.03
0.11
0.19
0.27
0.35
0.03
0.12
0.21
0.30
0.39
0.03
0.13
0.23
0.33
0.43


0.29
0.34
0.39
0.44
0.49
0.29
0.35
0.41
0.47
0.53
0.29
0.36
0.43
0.50
0.57
0.29
0.37
0.45
0.53
0.61
0.29
0.38
0.47
0.56
0.65
0.29
0.39
0.49
0.59
0.69


28.6a
32.1de
33.6fgh
35.3ij
33.7fgh
29.8bc
32.6efg
36.7jk
36.9k1
36.5jk
33.0efg
34.3ghi
37.3k1
38.71m
39.2mn
29.2ab
33.4efgh
39.9no
38.51m
39.1mm
30.8cd
35.3ij
38.31m
40.1no
40.2no
31.3d
34.5hi
39.6mm
41.2o
41.4o


210a
250bcd
265cde
271ef
280efg
242cd
267de
293g
298gh
290g
240bc
280efg
302h
299gh
303h
225ab
281efg
302h
310h
313h
237ab
291g
314h
316h
309h
220a
2S4fg
297gh
361h
309h


IThe basal diet contained 0.30 percent phosphorus and
0.26 percent calcium.

2Means with different letters are significantly
different according to Duncan's multiple range test.





- 13 -


body weight was not different with levels of 0.13 or 0.23

percent supplemental calcium. Levels of 0.15, 0.21, and

0.27 percent supplemental calcium with 0.35 percent phospho-

rus resulted in maximum tibia ash and body weights

(Table 2). Supplemental calcium levels of less than

0.15 percent resulted in a significant lowering of the

tibia ash and body weight.

Maximum growth was obtained with supplemental

calcium levels of 0.17, 0.24,and 0.31 percent,when the

diet contained 0.40 percent phosphorus (Table 2). However,

a level of 0.17 percent supplemental calcium did not

support maximum tibia ash. This would indicate a level

of 0.24 percent supplemental calcium as being ideal for

this level of phosphorus.

Levels of 0.19 and 0.35 percent supplemental calcium

supported both maximum tibia ash and body weight (Table 2)

when the diet contained 0.45 percent phosphorus. The

level of 0.27 percent supplemental calcium supported

maximum body weight; however, tibia ash was significantly

lower than for the group receiving 0.19 percent supple-

mental calcium. The lowered tibia ash was attributed to

chance since the tibia ash was also numerically greater

for groups receiving 0.35 percent supplemental calcium.

When the diet contained 0.50 percent phosphorus,

levels of 0.21, 0.30, and 0.39 percent supplemental calcium





- 14 -


supported maximum body weight (Table 2). However, tibia

ash was significantly lower when the diet contained only

0.21 percent supplemental calcium.

Maximum body weight was obtained with supplemental

calcium levels of 0.23, 0.33, and 0.43 percent when the

diet contained 0.55 percent phosphorus (Table 2). However,

a significantly lower tibia ash was obtained with the diet

containing 0.23 percent calcium.

Based on the above results,a standard curve has

been suggested for use in selection of the proper level of

calcium to use in order to elicit maximum response,

measured as both tibia ash and body weight. The suggested

calcium levels are 0.44, 0.47, 0.50, 0.53, 0.56, and 0.59

percent for phosphorus levels of 0.30, 0.35, 0.40, 0.45,

0.50, and 0.55 percent, respectively, when supplemental

phosphorus is supplied as monosodium phosphate (NaH2PO

* HO2). It should be emphasized, however, that these

levels are only for the basal diet employed in this

experiment and may not apply when dietary modifications

are made.

Increasing the phosphorus:calcium ratio by the ad-

dition of phosphorus to diets containing calcium levels of

0.29, 0.39, 0.49, and 0.53 percent did not depress performance

of chicks when measured as tibia ash or growth rate

(Table 2). An improvement of tibia ash was produced





- 15 -


by widening the P:Ca ratio with the addition of phosphorus

to the diet containing the low level of calcium (0.29

percent total calcium). This was in contrast to the

effect of widening the calcium:phosphorus ratios at low

levels of phosphorus as reported by Vandepopuliere et

al. (1961).


Experiment 2


Experimental Procedure

A second experiment was conducted to determine

whether the suggested calcium levels would produce maximum

tibia ash and body weight when phosphorus was fed at other

levels. The same general procedure was followed as used

in experiment 1 except that four, instead of three,

replicates of five males and five females were used per

treatment. Also, tibia ash was determined on five males

per treatment instead of two males and two females per

treatment as in experiment 1. Levels of 0.07, 0.14, and

0.21 percent phosphorus from monosodium phosphate

(NaH PO4 H20) were added to the basal diet and five

suitable levels of calcium were fed at each of the three

levels of phosphorus.


Results and Discussion

A level of 0.19 percent supplemental (0.45 percent

total) calcium gave maximum tibia ash and body weight when




- 16 -


0.07 percent supplemental phosphorus was added (Table 3).

This is in fair agreement with the 0.485 percent suggested

on the standard curve (experiment 1), and a level of 0.49

percent calcium did not significantly reduce either body

weight or tibia ash.

When 0.14 percent phosphorus was added, a level

of 0.26 percent supplemental calcium produced maximum

tibia ash and body weight. However, when the diet

contained 0.21 percent supplemental phosphorus, a level

of 0.30 percent supplemental calcium gave maximum per-

formance. These levels agree very well with those sug-

gested on the standard curve. These data indicated that

the suggested levels of calcium (experiment 1) would

elicit maximum tibia ash and body weight. Therefore, it

is suggested that such a curve be utilized in phosphorus

assays for determining the level of calcium to use with

this diet.

Based on the data in experiment 2 (Table 3), it

would appear that these levels have a sufficient margin

of safety for normal variation in calcium content of the

basal diet. Although the chicks grew at a higher rate in

this experiment, the response to the various dietary

phosphorus and calcium levels was essentially the same

as observed in the first experiment.





- 17 -


TABLE 3

Tibia ash and body weight of chicks fed various levels
of phosphorus and calcium supplied from monosodium
phosphate and reagent grade calcium carbonate


Calcium'
(%) Tibia Body
Total phosphorus' ash2 weight2
(%) Supplemental Total (%) (grams)

0.13 0.39 36.1f 344cd
0.16 0.42 35.8f 347bcd
0.37 0.19 0.45 36.1f 339de
0.23 0.49 34.6f 325de
0.27 0.53 34.8f 313e

0.19 0.45 39.9cde 364abc
0.22 0.48 39.7cde 375a
0.44 0.26 0.52 40.2bcd 369abc
0.30 0.56 39.1de 350bcd
0.34 0.64 38.2e 311e

0.23 0.49 41.2abc 372ab
0.26 0.52 41.2abc 382a
0.51 0.30 0.56 42.4a 381a
0.34 0.60 41.4abc 372ab
0.42 0.68 41.7ab 345cd


IThe basal diet contained 0.30
and 0.26 percent calcium.


percent phosphorus


SMeans with different letters are significantly
different according to Duncan's multiple range test.





- 18 -


Summary


An experiment, involving three trials, was conducted

using reagent grade monosodium phosphate and calcium carbon-

ate to establish a calcium standard curve for use in

phosphorus availability studies. Six levels of supplemental

phosphorus (0 to 0.25 percent) were added to the degermi-

nated corn-soybean meal basal diet, with five calcium

levels per phosphorus level.

Broiler-type chicks were fed the experimental diets

for 21 days, and tibia ash data and body weights were used

to determine the calcium requirement at each phosphorus

level. These data were used to construct a curve giving

the suggested calcium level for any level of monosodium

phosphate when using a diet based largely upon degerminated

corn and soybean meal.

Results from a second experiment indicated that

calcium levels selected from the standard curve for other

levels of monosodium phosphate would produce maximum bone

ash and body weight.













CHAPTER 3

DEVELOPMENT OF A CALCIUM STANDARD CURVE
FOR DEFLUORINATED PHOSPHATE


Defluorinated phosphate is one of the most widely

used sources of phosphorus in poultry diets because of the

abundance of raw material, its low fluorine content, and

the fact that it has a high concentration of readily

available calcium and phosphorus. The Ca:P ratio of

defluorinated phosphate is quite similar to that found in

bone, and this is considered to be another advantage for

this material.

Gillis et al. (1954), using biological assay

techniques and beta tricalcium phosphate as a standard,

found defluorinated phosphate to have a biological value

varying from 82 to 99 percent, depending upon the method

of manufacture.

The experiment reported herein was conducted to

develop a standard calcium curve which will allow re-

searchers to more accurately evaluate the biological

availability of this feed grade phosphorus supplement.


Experimental Procedure


Two successive trials were conducted. Three repli-

cate groups, each containing five male and five female


- 19 -





- 20 -


broiler-type chicks (Vantress x White Plymouth Rock),

received each dietary treatment. The chicks were sexed,

debeaked, and randomly assigned to the treatments at one

day of age. The birds received the experimental diets

and tap water ad libitum from 1 to 21 days of age.

The basal diet used for this study was identical

to the one described in Chapter 2 (Table 1). Commercial

grade defluorinated phosphate was used to supply supple-

mental phosphorus in 0.07 percent increments to a total

dietary level of 0.51 percent phosphorus (Table 4).

Within each phosphorus level, reagent grade calcium

carbonate was used to supply supplemental calcium. Five

equally spaced calcium levels were used at each phosphorus

level in an attempt to select the appropriate calcium level

which would give a maximum response with each particular

phosphorus level.

The battery brooders used in this experiment were

the same as those described in Chapter 2. At the termi-

nation of each trial the birds were separated and weighed

according to sex. Two males and two females from each

group were sacrificed for tibia ash determinations. The

vitamin D determination procedure of the Association of

Official Agricultural Chemists (1965) was followed in

ashing the bones. Statistical analysis of the data





- 21 -


(Analysis of Variance; Snedecor, 1956) failed to reveal a

treatment x trial interaction; therefore, the data from

the two trials were combined.

Significant differences between tibia ash and

body weight treatment means were determined by Duncan's

multiple range test (1955). Based on these measurements,

the ideal calcium level was selected for each level of

phosphorus, and a standard curve of calcium levels

determined for the various additions of defluorinated

phosphate.


Results and Discussion


The combined results of the two trials (Table 4)

indicate that the level of supplemental calcium was more

critical at the lowest level of phosphorus supplementation.

There were no significant differences between tibia ash

values within a given level of phosphorus; however, each

increment of phosphorus resulted in a significant increase

of tibia ash values. Body weight also tended to increase

numerically as the level of phosphorus increased; however,

trends of statistical significance were not as clear cut.

When the diet contained 0.37 percent phosphorus,

the highest levels of bone ash resulted from the feeding

of 0.475 and 0.520 percent total calcium. Since these

two treatments also produced the heaviest body weights of





- 22 -


TABLE 4

Tibia ash and body weight of chicks fed various levels
of phosphorus and calcium supplied from defluorinated
phosphate and reagent grade calcium carbonate



Calciumi
(%) Tibia Body
Total phosphorus' ash2 weight2
(%) Supplemental Total (%) (grams)

. 0.385 33.7a 295a
0.045 0.430 33.9a 293a
0.37 0.090 0.475 34.4a 313bc
0.135 0.520 34.8a 310b
0.180 0.565 34.1a 305ab

. 0.510 37.3b 333de
0.050 0.560 38.7b 337de
0.44 0.100 0.610 37.5b 327cd
0.150 0.660 38.3b 331de
0.200 0.710 38.1b 332de

. 0.635 40.9c 347e
0.025 0.660 40.3c 337de
0.51 0.050 0.685 41.1c 347e
0.075 0.710 40.6c 344e
0.100 0.735 41.1c 346e


IThe basal diet contained 0.30
and 0.26 percent calcium.


percent phosphorus


Means with different letters are significantly
different according to Duncan's multiple range test.






- 23 -


any diet containing 0.37 percent phosphorus, an intermediate

calcium level of 0.498 percent was selected as one providing

an adequate safety margin for this level of phosphorus.

Changing the calcium level when the diet contained

0.44 percent phosphorus did not produce a significant

increase of body weight or bone ash, but the group that

received 0.560 percent total dietary calcium had numerically

superior body weight and bone ash. In order to allow for

a margin of safety around the value selected for the curve,

0.590 percent total calcium was selected for this level of

phosphorus.

When the diet contained 0.51 percent phosphorus,a

level of 0.685 percent calcium resulted in a combination

of the highest body weight and greatest percent bone ash

of any of the calcium levels tested. Since there were no

significant differences between either the body weights

or bone ash of this series and very little fluctuation

among numerical values, it was felt that the choice of

0.685 percent total calcium would afford an ample margin

of safety on either side of the standard curve.


Summary


An experiment consisting of two trials was conducted

to establish a calcium standard curve for use in phosphorus

availability studies involving defluorinated phosphate.




- 24 -


Three levels of supplemental phosphorus (0.07, 0.14, and

0.21 percent) supplied from defluorinated phosphate were

added to the degerminated corn-soybean meal basal diet.

Five equally spaced calcium levels were fed with each

level of phosphorus.

Broiler-type chicks received the experimental

diets from 1 to 21 days of age; tibia ash and body weight

data were used as the criteria for determining the

calcium requirement at each phosphorus level. The

optimum calcium levels selected were 0.498, 0.590, and

0.685 percent, respectively, for total phosphorus levels

of 0.37, 0.44, and 0.51 percent.












CHAPTER 4

DEVELOPMENT OF A CALCIUM STANDARD CURVE
FOR DICALCIUM PHOSPHATE


Dicalcium phosphate is a highly available phosphate

material that is widely utilized as a source of inorganic

phosphorus for poultry diets. Gillis et al. (1954) and

Nelson and Peeler (1961), using beta-tricalcium phosphate

as the reference material, found dicalcium phosphate to

have a biological value of 98 and 97 percent, respectively.

This experiment was conducted to determine the

levels of calcium necessary for the maximum expression of

biological availability by this commercial phosphate source

at various levels of phosphorus supplementation.


Experimental Procedure


Three successive trials were conducted. In each

trial three replicate groups containing five male and

five female broiler-type chicks (Vantress x White Plymouth

Rock) received each dietary treatment and cap water a_

libitum from 1 to 21 days of age. The chicks were sexed,

debeaked, and randomly assigned to treatment groups az

one day of age.

The basal diet used was identical to the one

described in Chapter 2 (Table 1). Commercial grade


- 25 -





- 26 -


dicalcium phosphate was used to supply supplemental

phosphorus in 0.07 percent increments to a total dietary

level of 0.51 percent (Table 5). Within each phosphorus

level reagent grade calcium carbonate was used to provide

five equally spaced supplemental calcium levels. This

range of calcium supplementation was provided for each

phosphorus level in order that the appropriate calcium

requirement for each level might be determined.

The battery brooders used in this experiment were

identical to the ones described in Chapter 2. At the

termination of each trial the birds were separated and

weighed according to sex, and two males and two females

from each replicate group were sacrificed for tibia ash

determinations. The vitamin D determination procedure of

the Association of Official Agricultural Chemists (1965) was

followed in ashing the bones. Statistical analysis of the

data (Analysis of Variance; Snedecor, 1956) did not reveal

a significant treatment x trial interaction; therefore,

the data from the three trials were combined.

Significant differences between tibia ash and body

weight treatment means were determined by Duncan's multiple

range test (1955). Based on these measurements, the ideal

calcium level for each level of phosphorus was selected,

and a standard curve of calcium levels determined for the

various additions of dicalcium phosphate.





- 27 -


Results and Discussion


The combined results of the three trials with

dicalcium phosphate are presented in Table 5. A signifi-

cant increase of tibia ash and body weight resulted when

the total calcium level was increased from 0.35 to 0.43

percent in diets containing 0.37 percent total phosphorus.

The diet containing 0.43 percent calcium produced the

largest numerical bone ash value of any group receiving

0.37 percent phosphorus, and no further significant

improvement of tibia ash or body weight resulted from

feeding higher calcium levels with this level of phosphorus.

In order that a margin of safety might be allowed around

the calcium level proposed for this level of phosphorus, a

total calcium level of 0.47 percent was selected.

The tibia ash of birds receiving diets containing

0.44 percent total phosphorus was significantly increased

when the dietary calcium level was increased from 0.44 to

0.50 percent; however, there were no significant differences

noted among body weights. The diet containing 0.44 percent

phosphorus and 0.56 percent calcium resulted in tibia ash

and body weight values which were numerically superior to

any of the treatment groups receiving 0.44 percent phospho-

rus. However, it was considered that since consistent

numerical improvements of tibia ash and body weight





- 28 -


TABLE 5

Tibia ash and body weight of chicks fed various levels
of phosphorus and calcium supplied from dicalcium
phosphate and reagent grade calcium carbonate


Calcium'
(%) Tibia Body
Total phosphorus' ash2 weight2
(%) Supplemental Total (%) (grams)

0.02 0.35 30.8a 315a
0.10 0.43 36.2bc 346cd
0.37 0.18 0.51 35.1b 348cd
0.26 0.59 35.2b 328ab
0.34 0.67 35.8b 334bc

0.04 0.44 35.9b 337bcd
0.10 0.50 37.7cd 340bcd
0.44 0.16 0.56 38.7def 353d
0.22 0.62 38.6def 336bcd
0.28 0.68 38.3de 350cd

0.07 0.53 39.9ef 346cd
0.11 0.57 39.9ef 348cd
0.51 0.15 0.61 40.2f 346cd
0.19 0.65 40.3f 344bcd
0.23 0.69 39.7ef 349cd


IThe basal diet contained 0.30
and 0.26 percent calcium.


percent phosphorus


2Means with different letters are significantly
different according to Duncan's multiple range test.






- 29 -


resulted from the feeding of 0.44, 0.50, and 0.56 percent

calcium, a level somewhat in excess of 0.56 percent was

required in order to afford some factor of safety. Since

a dietary level of 0.62 percent calcium with 0.44 percent

phosphorus produced a tibia ash value that was almost

equal to that resulting from the feeding of 0.56 percent

calcium, an intermediate total calcium level of 0.58

percent was selected as the second point of the standard

curve for the assay of dicalcium phosphate.

Increasing the level of calcium with 0.51 percent

phosphorus produced essentially no response within either

criterion, as there were no significant differences (and

only very small numerical variations) between any of the

tibia ash or body weight values. These results were not

unexpected since 0.51 percent phosphorus is approaching

the bird's requirement, and it is well established that

dietary calcium levels become less critical as the phosphc-

rus content of the diet is increased. In order to provide

an adequate safety margin, 0.69 percent to-al dietary

calcium was selected for a level of 0.51 percent phosphorus.





This experiment, comprised of three trials, was

conducted to define a calcium standard curve for use in

studying the availability of phosphorus from commercial





- 30 -


grade dicalcium phosphate. Three levels of supplemental

phosphorus (0.07, 0.14, and 0.21 percent) were provided

from dicalcium phosphate, and five equally spaced levels

of supplemental calcium supplied from reagent grade

calcium carbonate were fed with each level of phosphorus

in a degerminated corn-soybean meal basal diet.

Broiler-type chicks housed in starting batteries

received the experimental diets from 1 to 21 days of age.

Tibia ash and body weight data were used as the criteria

for determining the optimum level of calcium to be fed

with each level of phosphorus. The calcium levels

selected were 0.47, 0.58, and 0.69 percent, respectively,

for total phosphorus levels of 0.37, 0.44, and 0.51

percent.












CHAPTER 5

DEVELOPMENT OF A CALCIUM STANDARD CURVE
FOR SOFT PHOSPHATE


In recent years there has been considerable

interest in the utilization of soft phosphate, also

known as colloidal phosphate or phosphatic clay, as a

source of phosphorus in poultry diets. Soft phosphate

is one of the materials with low phosphorus content

(approximately 18 percent calcium and 9 percent phospho-

rus), yet it is also one of the most inexpensive sources

of supplemental phosphorus.

The main problem associated with the use of this

material is the low biological availability of its

phosphorus and the wide range of reported availability

values. Nelson and Peeler (1961) reported an availability

of only 34 percent for the phosphorus of soft phosphate.

In contrast, Waldroup et al. (1965a) estimated that 51-59

percent of the phosphorus in soft phosphate was available

and suggested that the dietary calcium level could influ-

ence availability.

This experiment was conducted in order to determine

the levels of dietary calcium necessary for the maximum

expression of biological availability by this phosphate

source.


- 31 -





- 32 -


Experimental Procedure


In each of three successive trials, three replicate

groups containing five male and five female broiler-type

chicks (Vantress x White Plymouth Rock) received each

dietary treatment and tap water ad Zibitum from 1 to 21

days of age. The birds were sexed, debeaked, and randomly

assigned to treatment groups at one day of age.

The basal diet used was identical to the one

described in Chapter 2 (Table 1). Supplemental phospho-

rus supplied from commercial grade soft phosphate was

added in 0.07 percent increments to result in total

dietary phosphorus levels of 0.37, 0.44, and 0.51 percent.

Within each phosphorus level,reagent grade calcium

carbonate was used to supply five equally spaced supple-

mental calcium levels (Table 6). The levels of calcium

supplementation varied among the three phosphorus levels

and were provided in order that the appropriate calcium

requirement for each level of phosphorus might be determined.

The battery brooders used in this experiment were

identical to the ones described in Chapter 2. On the final

day of each trial, the birds were separated and weighed

according to sex, and two males and two females from each

replicate group were sacrificed for tibia ash determi-

nations. The vitamin D determination procedure of the





- 33 -


Association of Official Agricultural Chemists (1965) was

followed in ashing the bones. Statistical evaluation of

the data (Analysis of Variance; Snedecor, 1956) revealed

no significant treatment x trial interaction; therefore,

the data from the three trials were combined.

Significant differences between tibia ash and body

weight treatment means were determined by Duncan's multiple

range test (1955). Based on these measurements, the ideal

calcium level for each phosphorus level was selected, and

a standard curve of calcium levels determined for the

various additions of soft phosphate.


Results and Discussion


The combined results of the three trials with soft

phosphate are shown in Table 6. There were no significant

differences among the body weights of birds receiving 0.37

percent total phosphorus, regardless of the calcium level

fed. The addition of 0.06 and 0.12 percent supplemental

calcium to a diet containing 0.07 percent phosphorus from

soft phosphate (0.37 percent total phosphorus) resulted

in a significant increase of bone ash for each of these

calcium additions. No further significant improvements

were noted by increasing the supplemental calcium level

above 0.12 percent (0.52 percent total). The birds

receiving a diet containing 0.12 percent supplemental




- 34 -


TABLE 6

Tibia ash and body weight of chicks fed various levels
of phosphorus and calcium supplied from soft phosphate
and reagent grade calcium carbonate


Calcium1
(%) Tibia Body
Total phosphorus' ash2 weight2
(%) Supplemental Total (%) (grams)

S. 0.40 31.5a 311a
0.06 0.46 34.4b 318a
0.37 0.12 0.52 36.3cde 323ab
0.18 0.58 35.6bcde 313a
0.24 0.64 35.1bcd 326abc

S. 0.54 36.6de 338bcd
0.04 0.58 37.0e 341cd
0.44 0.08 0.62 36.3cde 352d
0.12 0.66 36.7de 339bcd
0.16 0.70 34.8bc 343d

0.68 40.1f 355d
0.02 0.70 39.6f 349d
0.51 0.04 0.72 40.3f 347d
0.06 0.74 39.5f 344d
0.08 0.76 38.9f 347d


iThe basal diet contained 0.30
and 0.26 percent calcium.


percent phosphorus


2Means with different letters are significantly
different according to Duncan's multiple range test.





- 35 -


calcium (0.52 percent total) had the greatest tibia ash

value resulting from any of the diets containing 0.37

percent phosphorus. Since a margin of safety was needed

on the total calcium level selected, it was proposed that

a dietary calcium level of 0.56 percent would be appropri-

ate for this phosphorus level.

Supplementing the diet containing 0.44 percent

total phosphorus with graded levels of reagent grade

calcium carbonate resulted in no significant improvement

of either tibia ash or body weight. However, a level of

0.16 percent supplemental calcium (0.70 percent total)

produced a statistically significant depression of tibia

ash. The addition of 0.04 percent supplemental calcium

(0.58 percent total) resulted in a tibia ash value which

was numerically superior to that of any other calcium

level. The diet containing 0.08 percent supplemental

calcium (0.62 percent total) produced the best body weight

of any diet containing 0.44 percent total phosphorus. A

level of 0.62 percent total calcium was selected for feeding

with 0.44 percent total phosphorus.

None of the calcium levels supplied with 0.51

percent total phosphorus provided a significantly different

response as measured by tibia ash or body weight; however,

there was a slight numerical decrease of tibia ash as the

total calcium level was increased above 0.72 percent.




- 36 -


Again, this indicates that the level of calcium becomes

less critical as the dietary phosphorus level approaches

the bird's requirement. A level of 0.68 percent total

calcium was selected for feeding with 0.51 percent total

phosphorus because this dietary combination produced the

greatest body weight of any diet containing 0.51 percent

total phosphorus, and near maximum tibia ash.


Summary


An experiment consisting of three trials was

conducted in order to define a calcium standard curve

which would be of value in studying the availability of

phosphorus from soft phosphate. Three levels of supple-

mental phosphorus (0.07, 0.14, and 0.21 percent) were

provided from soft phosphate, and five equally spaced

supplemental calcium levels supplied from reagent grade

calcium carbonate were fed with each level of phosphorus

in a degerminated corn-soybean meal basal diet. Both the

supplemental and total calcium levels varied among the

different levels of phosphorus.

Broiler-type chicks housed in starting batteries

received the experimental diets from 1 to 21 days of age.

Body weight and tibia ash data were used as the criteria

for determining the optimum level of calcium to be fed

with each level of phosphorus. With due consideration





37 -





having been given to safety margins, the total calcium

levels selected were 0.56, 0.62, and 0.68 percent,

respectively, for total phosphorus levels of 0.37, 0.44,

and 0.51 percent.













CHAPTER 6

INFLUENCE OF DIET COMPOSITION ON THE UTILIZATION
OF SOFT PHOSPHATE IN BROILER DIETS


Numerous studies have been conducted to evaluate

the biological value of soft phosphate for poultry. The

majority of these reports has indicated that the phospho-

rus from soft phosphate is poorly utilized. Nelson and

Peeler (1961) reported a biological value of only 34

percent for soft phosphate and found that the availability

of poor quality phosphates was not improved when fed in a

mixture with materials possessing a higher biological

availability.

In addition, Summers et al. (1959) reported the

availability of phosphorus from soft phosphate to be

approximately 47 percent, and Waldroup et aZ. (1965a)

estimated that 51-59 percent of the phosphorus in soft

phosphate was available.

Summers et al. (1959) found that the availability

of soft phosphate was improved by the addition of either

phosphoric or hydrochloric acid. In contrast, Waldroup

et al. (1965c) reported no apparent improvement in the

utilization of phosphorus from soft phosphate by combining

it with phosphoric acid. These workers concluded that the


- 38 -





- 39 -


apparent increased phosphorus content of the mixture could

be attributed to the loss of water vapor during the chemi-

cal reaction that occurred as the materials were combined.

Waldroup et al. (1963) found that when dietary

calcium and phosphorus levels were suboptimal, the

calcium:phosphorus ratio was more critical in diets

containing soft phosphate than in those diets containing

a more readily available phosphorus source. It has been

demonstrated under certain conditions that levels of

vitamin D3 considerably in excess of those suggested by

the National Research Council (1966) may improve the

utilization of the phosphorus from soft phosphate

(Motzok et al., 1965; Fritz and Roberts, 1966; and

McKnight and Watts, 1966).

The experiments reported in this chapter were

conducted to study the influence of various dietary

factors on the utilization of phosphorus from soft

phosphate, and to attempt to obtain maximum growth with

broiler-type chicks when soft phosphate was utilized as

the sole supplemental source of phosphorus.


Experimental Procedure


Day-old broiler-type chicks, obtained from a

commercial hatchery, were utilized in all experiments.

Ten male and ten female chicks were randomly assigned to





- 40 -


floor pens according to a randomized block design. Four

replicate pens were assigned to each dietary treatment.

All pens contained 2.32 square meters of floor area, and

were uniform in shape and construction. Each pen was

provided with one automatic waterer, one hanging cylindri-

cal feeder, and one infrared heat lamp. Dried peanut hulls

were used as litter.

Treatments consisted of graded additions of soft

phosphate to the basal diets shown in Table 7. In

experiments 1 and 2, and one-half of experiment 3, a

basal diet (Table 7, diet 1) containing 0.34 percent

phosphorus and 0.16 percent calcium was used. The fish

meal basal (Table 7, diet 2) of experiment 3 contained

0.46 percent phosphorus and 0.28 percent calcium. Since

the vitamin D3 level of feeds has been shown to influence

calcium and phosphorus utilization, all experimental diets

were fortified with 7920 I.C.U. of vitamin D3/kg.

All diets were kept iso-caloric (2093 Cal./kg.)

and iso-nitrogenous (22.05 percent protein) by varying

the level of corn, soybean oil meal, and animal fat.

Values of Maddy et al. (1963) were used in the calculation

of adjustments. The birds were given the experimental

diets and water ad libitum from one day of age until the

end of the test.





- 41 -


TABLE 7

Composition of basal diets


1 2

Ingredients (Percent of diet)

Yellow corn 47.41 51.60

Soybean meal (50%) 35.50 30.60

Fish meal (60%) . 2.50

Alfalfa meal (20%) 3.00 3.00

Animal fat 5.74 3.30

Micro-ingredients' 0.50 0.50

Iodized salt 0.40 0.40

Variable ingredients2 7.45 8.10


IMicro-ingredient mix supplied per kilogram of
diet: 6,600 I.U. vitamin A, 449.4 mg. choline, 39.6 mg.
niacin, 7,920 I.C.U. vitamin D3, 4.4 mg. riboflavin, 13.2
mg. pantothenic acid, 22 mcg. vitamin B12, 0.0275 percent
Santoquin, 19.8 mg. iron, 1.98 mg. copper, 198 mcg. cobalt,
11 mg. iodine, 99 mcg. zinc and 220 mg. of manganese
sulfate.

2Variable ingredients included sources of supple-
mental phosphorus and calcium and an inert filler.




- 42 -


Body weight and tibia ash were used as criteria of

evaluation. In addition, feed efficiency values were

calculated for experiments 2 and 3. The birds from each

pen were grouped by sex and weighed at four weeks of age

in experiments 1 and 2, and at four and eight weeks of

age in experiment 3. In experiments 2 and 3, two males

and two females from one pen of each dietary treatment

were sacrificed at four weeks of age for bone ash determi-

nations. The left tibia was removed, cleaned of adhering

tissue and individually ashed according to the method

outlined by the Association of Official Agricultural

Chemists (1965). Feed consumption was also determined

at four weeks of age in experiment 2, and at four and

eight weeks of age for experiment 3.

The data from these experiments were subjected to

the analysis of variance as outlined by Snedecor (1956).

Significant differences between treatment means were

determined by the multiple range test of Duncan (1955).


Experiment 1

A 3 x 4 factorial arrangement of treatments was

used involving three supplemental phosphorus levels

(0.40, 0.50, and 0.60 percent) and four levels of supple-

mental calcium (0, 0.20, 0.30, and 0.40 percent) added to

the basal diet (Table 7, diet 1). Two positive control





- 43 -


diets consisting of diet 1 supplemented to provide adequate

levels of phosphorus (0.70 percent) and calcium (0.80 and

1.00 percent) from commercial grade dicalcium phosphate

(21.69 percent phosphorus and 20.92 percent calcium) and

ground limestone were also fed. The diets of this

experiment were fed from one day until four weeks of age,

at which time the test was terminated.


Experiment 2

This experiment was also of four weeks duration,

starting when the chicks were housed at one day of age.

The basal diet was supplemented with soft phosphate in

order to provide levels of 0.40, 0.50, and 0.60 percent

supplemental phosphorus. Ground limestone was added to

supply supplemental calcium levels ranging from 0 to 0.40

percent (Table 9) to diets containing each of the supple-

mental phosphorus levels. Two positive control diets,

identical to those used in experiment 1, were also fed.


Experiment 3

One-half of the treatments of this experiment were

essentially replicates of a portion of experiments 1 and 2

in that similar additions of soft phosphate and ground

limestone were made to the basal diet (Table 7, diet 1).

Supplemental phosphorus levels of 0.40 and 0.50 percent

and supplemental calcium levels as shown in Table 10 were





- 44 -


provided from these sources. Two positive control diets

containing adequate levels of phosphorus (0.70 percent)

and calcium (0.80 percent and 1.00 percent) supplied from

dicalcium phosphate and ground limestone were also fed.

These control diets were identical to the ones fed in

experiments 1 and 2.

The other half of the experiment consisted of soft

phosphate and ground limestone additions to a basal diet

containing 2.5 percent fish meal (Table 7, diet 2).

Supplemental levels of 0.30 and 0.40 percent phosphorus

were fed with the supplemental calcium levels shown in

the lower half of Table 10. Two positive control diets

containing 2.5 percent fish meal, 0.70 percent total

phosphorus with 0.71 percent and 0.91 percent total

calcium, respectively, were also fed. These diets were

composed of the basal shown in Table 7 (diet 2) and

supplemental phosphorus and calcium added from dicalcium

phosphate and ground limestone.


Results and Discussion


Experiment 1

A level of 0.50 percent supplemental phosphorus

supplied from soft phosphate combined with 0.20 percent

supplemental calcium produced the best growth rate of any

treatment group; however, higher calcium levels significantly





- 45 -


depressed growth (Table 8). Body weights were significantly

increased by the addition of 0.20 percent supplemental

calcium to the diet containing 0.40 percent phosphorus

supplied from soft phosphate. Increasing the supplemental

phosphorus level from 0.50 to 0.60 percent, with optimal

calcium levels, depressed growth. However, at the 0.60

percent phosphorus level it was necessary to feed a higher

level of supplemental calcium in order to depress growth.

Growth at each level of phosphorus supplementation

was significantly influenced by the calcium level of the

diet. Even though total calcium and phosphorus levels

were considerably above those normally felt to be adequate,

no combination of calcium and phosphorus levels utilized

resulted in body weights statistically equivalent to those

of the positive control diets (Table 8).


Experiment 2

No level of supplemental phosphorus from soft

phosphate, regardless of calcium level, supported body

weights at four weeks of age that were equal to those

obtained with the positive control diets (Table 9). All

tibia ash values except those for the extreme lowest and

highest calcium levels of the 0.40 percent supplemental

phosphorus series were not significantly different from

those for positive control diets. This effect is possibly





- 46 -


TABLE 8

Four-week body weights of chicks fed different levels of
soft phosphate and ground limestone (experiment 1)


Supplemental' Total 4 wk.
P Ca2 Calcium Body weight5
(%) (%) (%) (grams)

0.363 0.16 0.80 479a
0.36 1.00 491a

S. 0.92 380g
0.20 1.12 440cd
0.404 0.30 1.22 432cd
0.40 1.32 426de

S. 1.12 450bc
0.20 1.32 464b
0.504 0.30 1.42 439cd
0.40 1.52 430de

1.32 420ef
0.20 1.52 439cd
0.604 0.30 1.62 432de
0.40 1.72 408f


IBasal diet contained
0.16 percent calcium.


0.34 percent phosphorus and


2Supplied as ground limestone.

3Supplied as dicalcium phosphate, and considered
to be positive controls.

4Supplied as soft phosphate.

SMeans with different letters are significantly
different according to Duncan's multiple range test.





- 47 -


TABLE 9

Four-week body weights, feed conversion, and tibia ash
of chicks fed different levels of soft phosphate
and ground limestone (experiment 2)


Supplemental' 4 wk. Grams feed/
P Ca2 Total Body weights gram of body Tibia ash5
(%) (%) calcium (grams) weight (%)

0.363 0.16 0.80 510d 1.79 45.1bc
0.36 1.00 517d 1.85 45.1bc

S. 0.92 470bc 1.80 42.3a
0.20 1.12 467bc 1.86 43.2ab
0.25 1.17 463bc 1.82 43.8abc
0.404 0.30 1.22 464bc 1.90 42.9ab
0.35 1.27 446ab 1.86 43.9abc
0.40 1.32 430a 1.94 41.6a

1.12 456abc 1.83 43.3ab
0.10 1.22 476c 1.80 45.0bc
0.20 1.32 452abc 1.89 45.6c
0.504 0.25 1.37 468bc 1.84 45.6c
0.30 1.42 470bc 1.90 44.8bc
0.40 1.52 456abc 1.89 42.9ab

1.32 448abc 1.83 45.9c
0.20 1.52 469bc 1.88 46.Oc
0.604 0.30 1.62 477c 1.87 44.9bc
0.40 1.72 459bc 1.84 44.8bc


1Basal diet contained 0.34 percent phosphorus and 0.16
percent calcium.

2Supplied as ground limestone.

3Supplied as dicalcium phosphate, and considered to be
positive controls.

4Supplied as soft phosphate.

SMeans with different letters are significantly
different according to Duncan's multiple range test.





- 48 -


explained by-a changing calcium and phosphorus ratio. No

statistically significant differences were found in feed

efficiency values.


Experiment 3

No combination of supplemental phosphorus and

calcium in diets without fish meal supported body weights

at four or eight weeks of age that were equal to those

produced with the positive control diets (Table 10).

Highest four-week tibia ash values from diets without fish

meal were obtained with 0.40 percent and 0.50 percent

supplemental phosphorus from soft phosphate and 0.10

percent supplemental calcium. Both of these values were

significantly greater than the bone ash of birds fed the

positive control diet containing 0.70 percent total

phosphorus and 0.80 percent total calcium. None of the

tibia ash values from diets without fish meal were

significantly different from those of birds fed the 0.70

percent phosphorus and 1.00 percent calcium positive

control diet.

No combination of phosphorus and calcium supple-

mentation supported maximum four-week body weights when

the diet contained fish meal (Table 10). However, the

weights of birds receiving fish meal diets were much

closer to the controls than those of birds fed diets






- 49 -


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- 51 -


containing no fish meal. Birds fed the fish meal diets

containing 0.40 percent supplemental phosphorus from soft

phosphate, regardless of the calcium level fed, had a body

weight at eight weeks of age which was not statistically

different from those of birds receiving the positive

control diets (Table 10). The birds receiving fish meal

and 0.30 percent added phosphorus with no supplemental

calcium also had body weights at eight weeks of age that

were not significantly different from the control groups.

Four- and eight-week body weights of birds fed the diet

with 0.30 percent supplemental phosphorus and 0.20 percent

supplemental calcium were significantly lower than weights

of birds fed most other diets containing fish meal. These

weights are obviously not representative of the treatment

since both higher and lower levels of supplemental calcium

produced greater body weights. This variability was

attributed to an unexplainable low weight for two of the

replications within this treatment. None of the tibia

ash values for the fish meal group were statistically

different (Table 10).

Previous work (Waldroup et al., 1965b) indicated

that growth of chicks was greatly improved when a small

amount of phosphate with a high availability was added to

the diet. These data would indicate that the fish meal

furnished some supplemental phosphorus of high availability,





- 52 -


and that this was necessary to obtain maximum growth of

broiler-type chicks. The phosphorus in fish meal has

been previously reported to be highly available (Waldroup

et at., 1965a).

Some significant differences were noted among feed

efficiency values when four-week measurements were evalu-

ated; however, these trends had disappeared by the time

the birds reached eight weeks of age.


Summary


Three experiments were conducted to ascertain

factors influencing the growth of broiler-type birds

while utilizing soft phosphate as the sole source of

supplemental phosphorus. A range of supplemental calcium

levels was provided from ground limestone in order to

eliminate the dietary calcium level as a limiting factor.

Four-week body weights equal to positive control

diets could not be produced by diets containing soft

phosphate as the sole supplemental phosphorus source. The

addition of 2.5 percent fish meal to the basal diet

improved four-week body weights; however, they still were

not equal to the weights of birds receiving the positive

control diet containing 0.80 percent calcium. The addition

of 0.40 percent supplemental phosphorus and 0, 0.10, or





53 -




0.20 percent supplemental calcium to a diet containing 2.5

percent fish meal resulted in an eight-week average body

weight statistically equal to the weight of the control

groups. The improved response of the birds receiving fish

meal diets was attributed to the supplemental phosphorus

of high availability supplied by the fish meal.












CHAPTER 7

SUMMARY


At present, there are no standardized levels of

calcium to be fed in an assay conducted to determine the

biological value of feed grade phosphate materials. Some

researchers feed a level of 1 percent calcium, regardless

of the level of phosphorus in the diet; others utilize a

constant calcium:phosphorus ratio over the entire range

of phosphorus supplementation. The main thesis investi-

gated and reported in this dissertation is that the

calcium level necessary for expression of maximum bio-

logical availability by a phosphate source varies with

the level of phosphorus supplied. Experiments were also

conducted to investigate the influence of diet composition

on the utilization of soft phosphate in broiler diets.

A series of 15 trials involving approximately

15,000 chicks was conducted in order to develop a more

meaningful chick assay for determining the availability

of phosphorus from various commercial phosphate sources.

A diet primarily of all-plant origin, containing degermi-

nated corn meal and soybean meal, was utilized in all

assay studies. The diet was calculated to contain 22

percent protein, 0.30 percent phosphorus, 0.26 percent

calcium, and 2,200 Calories of productive energy per kilogram.


- 54 -






- 55 -


The vitamin premix supplied 2,200 I.C.U. of vitamin D3/kg.

of diet in order to eliminate the possibility of this

vitamin being a factor limiting chick performance. Two

corn-soybean meal type basal diets, differing only in

fish meal content, were used for the studies concerning

diet composition.

All assay studies were of three weeks duration,

starting when the chicks were randomly assigned to battery

brooders at one day of age. Body weight and bone ash were

used as the criteria of evaluation. On the twenty-first

day of age all birds were weighed and representative bone

samples taken for individual ash determinations. The

studies of diet composition were of either four or eight

weeks duration, starting when the chicks were housed in

floor pens at one day of age. Body weight, bone ash, and

feed conversion values were used for evaluation purposes.

Five supplemental phosphorus levels (0.05, 0.10,

0.15, 0.20, and 0.25 percent) were provided from reagent

grade monosodium phosphate, which is the standard source

that other phosphates are usually compared with in order

to determine the biological value of the material tested.

Three supplemental phosphorus levels (0.07, 0.14, and 0.21

percent) were furnished by each of the other sources

tested--defluorinated phosphate, dicalcium phosphate, and

soft phosphate. Five equally spaced supplemental calcium





- 56 -


levels, which varied with source and level of phosphorus,

were provided from reagent grade calcium carbonate for

each of the phosphorus additions.

All the data were evaluated statistically, and,

for monosodium phosphate, levels of 0.44, 0.47, 0.50,

0.53, 0.56, and 0.59 percent total calcium were suggested

for total phosphorus levels of 0.30, 0.35, 0.40, 0.45,

0.50, and 0.55 percent, respectively. A subsequent

experiment indicated that these proposed calcium levels

were adequate.

The total dietary calcium levels proposed as a

standard curve for each of the remaining sources were as

follows: 0.498, 0.590, and 0.685 percent for defluorinated

phosphate; 0.47, 0.58, and 0.69 percent for dicalcium

phosphate; and 0.56, 0.62, and 0.68 percent for soft

phosphate with total phosphorus levels of 0.37, 0.44, and

0.51 percent, respectively. It should be emphasized that

these curves are valid only when assay diets identical to

those employed in the development of the curves are used.

Results of studies concerning the influence of

diet composition on soft phosphate utilization in broiler

diets indicated that the addition of 2.5 percent fish meal

to a diet containing soft phosphate as the sole supple-

mentary phosphorus source could produce body weights and

bone ash equal to control groups supplemented with





- 57 -


dicalcium phosphate. Various dietary combinations of

phosphorus supplied from soft phosphate and calcium

supplied as calcium carbonate were fed in an attempt to

produce growth and bone ash equal to that produced with

commercial feeds. No combination of calcium and phospho-

rus levels alone produced the desired result; however,

altering the diet composition with the inclusion of 2.5

percent fish meal in feeds containing 0.40 percent supple-

mental phosphorus and 0-0.20 percent supplemental calcium

resulted in bone ash and body weight values not signifi-

cantly different from those of controls. The response of

the birds receiving fish meal diets was attributed to

the highly available supplemental phosphorus supplied by

the fish meal.












REFERENCES


Ammerman, C. B., H. W. Norton, and H. M. Scott, 1960.
Rapid assay of inorganic phosphates for chicks.
Poultry Sci. 39:245-250.

Association of Official Agricultural Chemists, 1965.
Official Methods of Analysis, 10th Ed., Washington,
D. C.

Couch, J. R., G. S. Fraps, and R. M. Sherwood, 1937.
Vitamin D requirements of growing chicks as af-
fected by the calcium content of the ration.
Poultry Sci. 16:106-108.

Creech, B. G., B. L. Reid, and J. R. Couch, 1956. Evalu-
ation of dicalcium phosphate supplement as a
source of phosphorus for chicks. 1. Comparison
of dicalcium and tricalcium phosphate as a source
of phosphorus in chick and poult rations.
Poultry Sci. 35:654-658.

Duncan, D. B., 1955. Multiple range and multiple F tests.
Biometrics, 11:1-42.

Ewing, W. R., 1963. Poultry Nutrition, 5th Ed. The Ray
Ewing Company, Pasadena, California.

Fisher, H., E. P. Singsen, and L. D. Matterson, 1953.
The influence of feed efficiency on the phospho-
rus requirement for growth and bone calcification
in the chick. Poultry Sci. 32:749-754.

Fritz, J. C., and T. Roberts, 1966. Influence of levels
of vitamin D and calcium on the utilization of
phosphorus by the growing chick. Poultry Sci.
45:1085-1086.

Gardiner, E. E., H. E. Parker, and C. W. Carrick, 1959.
Soft phosphate in chick rations. Poultry Sci.
38:721-727.

Gillis, M. B., L. C. Norris, and G. F. Heuser, 1949. The
effect of phytin on the phosphorus requirement of
the chick. Poultry Sci. 28:283-288.


58 -





- 59 -


Gillis, M. B., L. C. Norris, and G. F. Heuser, 1954.
Studies on the biological value of inorganic
phosphates. J. Nutrition 52:115-125.

Grau, C. R., and P. A. Zweigart, 1953. Phosphatic clay
as a phosphorus source for chicks. Poultry Sci.
32:500-503.

McGinnis, J., L. C. Norris, and G. F. Heuser, 1944. Poor
utilization of phosphorus in cereals and legumes
by chicks for bone development. Poultry Sci.
23:157-159.

McKnight, W. F., and A. B. Watts, 1966. The effect of
vitamin D3 on the utilization of phosphorus from
various sources. Poultry Sci. 45:1104.

Maddy, K. H., R. B. Grainger, W. A. Dudley, and F. Puchal,
1963. The application of linear programming to
feed formulation. Feedstuffs 35 (5):28-30.

Matterson, L. D., E. P. Singsen, and H. M. Scott, 1945.
Rock phosphates as phosphorus supplements for the
growing chick. Poultry Sci. 24:188-190.

Motzok, I., D. Arthur, and H. D. Branion, 1965. Factors
affecting the utilization of calcium and phospho-
rus from soft phosphate by chicks. Poultry Sci.
44:1261-1270.

National Research Council, 1966. Nutrient requirements of
domestic animals. Nutrient requirements for poultry.
Washington D. C.

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 (11):32.

Nelson, T. S., and H. C. Walker, 1964. The biological
evaluation of phosphorus compounds. A summary.
Poultry Sci. 43:94-98.

O'Rourke, W. R., P. H. Phillips, and W. W. Cravens, 1952.
The phosphorus requirements of growing chickens as
related to age. Poultry Sci. 31:962-966.






60 -


Singsen, E. P., L. D. Matterson, and H. M. Scott, 1947.
Phosphorus in poultry nutrition. III. The
relationship between the source of vitamin D and
the utilization of cereal phosphorus by poults.
J. Nutrition 33:13-26.

Singsen, E. P., and H. M. Scott, 1946. Phosphorus in
poultry nutrition. II. Sodium acid phosphate,
tri-calcium phosphate and bonemeal as sources of
phosphorus for the growing chick. Poultry Sci.
25:302-303.

Singsen, E. P., H. M. Scott, and L. D. Matterson, 1948.
The phosphorus requirement of the chick. Storrs
Agr. Exp. Sta. Bull. 260.

Snedecor, G. W., 1956. Statistical Methods, 5th Ed. The
Iowa State College Press, Ames, Iowa.

Summers, J. D., S. J. Slinger, W. F. Pepper, I. Motzok, and
G. C. Ashton, 1959. Availability of phosphorus in
soft phosphate and phosphoric acid and the effect
of acidulation of soft phosphate. Poultry Sci.
38:1168-1179.

Vandepopuliere, J. M., C. B. Ammerman, and R. H. Harms,
1961. The relationship of calcium:phosphorus
ratios to the utilization of plant and inorganic
phosphorus by the chick. Poultry Sci. 40:951-957.

Waldroup, P. W., C. B. Ammerman, and R. H. Harms, 1963.
Calcium and phosphorus requirements of finishing
broilers using phosphorus sources of low and high
availability. Poultry Sci. 42:752-757.

Waldroup, P. W., C. B. Ammerman, and R. H. Harms, 1965a.
A comparison of phosphorus assay techniques with
chicks. Poultry Sci. 44:1086-1089.

Waldroup, P. W., C. B. Ammerman, and R. H. Harms, 1965b.
The utilization of phosphorus from animal protein
sources for chicks. Poultry Sci. 44:1302-1306.

Waldroup, P. W., C. B. Ammerman, and R. H. Harms, 1965c.
Studies on the acidulation of soft phosphate.
Poultry Sci. 44:1519-1523.












BIOGRAPHICAL SKETCH


Bobby Leon Damron was born November 6, 1941, at

Ocala, Florida. He was graduated from Gainesville High

School in June, 1959. In May, 1963, he received the degree

of Bachelor of Science in Agriculture with high honors

from the University of Florida. He also completed the

requirements for teacher certification in Vocational

Agriculture in May, 1963. He worked as an undergraduate

assistant in the Department of Poultry Science from

September, 1961 to May, 1963.

In September, 1963, he became a Research Assistant

in the Poultry Science Department and received the Master

of Science in Agriculture degree in December, 1964.

In January, 1966, he accepted the position of

Interim Research Associate and Instructor and continues

in that capacity at the present time.

The author is a member of the Poultry Science

Association, Alpha Zeta, Gamma Sigma Delta, Alpha Tau

Alpha, the Poultry Science Club, a past Vice-President

of the Collegiate F.F.A. Chapter, and a past President of

the Collegiate 4-H Club.









This dissertation was prepared under the direction

of the chairman of the candidate's supervisory committee

and has been approved by all members of that committee.
It was submitted to the Dean of the College of Agriculture

and to the Graduate Council, and was approved as partial

fulfillment of the requirements for the degree of Doctor
of Philosophy.



March, 1968





,ean, College of Agriculture




Dean, Graduate School


Supervisory Committee:



Chairman



(jt'^At,




Full Text

PAGE 1

DEVELOPMENT OF A CHICK ASSAY FOR DETERMINING AVAILABILITY OF PHOSPHORUS FROM VARIOUS PHOSPHATE MATERIALS By BOBBY LEON DAMRON A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA EST PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1968

PAGE 2

ACKNOWLEDGEMENTS The author is grateful to Dr. Robert H. Harms, Chairman of the Supervisory Committee, for his constant guidance, patience, and assistance in the planning and execution of the research reported in this dissertation. Appreciation is also expressed to Dr. J. L. Fry, Dr. H. R. Wilson, Dr. S. E. Grigsby, and Dr. C. B. Airjr>erman for their valuable suggestions and encouragement. The author is indebted to Miss Kathleen Wall for her technical assistance, and -co the undergraduate students for their assistance. The cooperation of the Smith-Douglass Company, Norfolk, Virginia, is gratefully acknov/ledged for technical and financial contributions.

PAGE 3

TABLE OF CONTENTS Page ACKNOWLEDGEMENTS j_j_ LIST OF TABLES ^ CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW 1 2 DEVELOPiMENT OF A CALCIUM STANDARD CURVE FOR MONOSODIUM PHOSPHATE 8 Experiment 1 8 Experimental Procedure 8 Results and Discussion 11 Experiment 2 15 Experimental Procedure 15 Results and Discussion 15 Summary 2_g 3 DEVELOPMENT OF A CALCIUM STANDARD CURVE FOR DEFLUORINATED PHOSPHATE 19 Experimental Procedure 19 Results and Discussion 21 Summary 23 4 DEVELOPMENT OF A CALCIUM STANDARD CURVE FOR DICALCIUM PHOSPHATE 25 Experimental Procedure 25 Results and Discussion 27 Summary 29 5 DEVELOPMENT OF A CALCIUM STANDARD CURVE FOR SOFT PHOSPHATE 31 Experimental Procedure 32 Results and Discussion 33 Summary 35

PAGE 4

TABLE OF CONTENTS — Continued CHAPTER Page 6 INFLUENCE OF DIET COiMPOSITION ON THE UTILIZATION OF SOFT PHOSPHATE IN BROILER DIETS 38 Experimental Procedure 39 Experiment 1 42 Experiment 2 43 Experiment 2 43 Results and Discussion 44 Experiment 1 44 Experiment 2 45 Experiment 2 48 Summary 52 7 SUMMARY 54 REFERENCES 58

PAGE 5

LIST OF TABLES Table Page 1 Composition of basal diet 9 2 Tibia ash and body weight of chicks fed various levels of phosphorus and calcium supplied from monosodium phosphate and reagent grade calcium carbonate 12 3 Tibia ash and body weight of chicks fed various levels of phosphorus and calcium supplied from monosodium phosphate and reagent grade calcium carbonate 17 4 Tibia ash and body weight of chicks fed various levels of phosphorus and calcium supplied from def luorinated phosphate and reagent grade calcium carbonate 22 5 Tibia ash and body weight of chicks fed various levels of phosphorus and calcium supplied from dicalcium phosphate and reagent grade calcium carbonate 28 6 Tibia ash and body weight of chicks fed various levels of phosphorus and calcium supplied from soft phosphate and reagent grade calcium carbonate 34 7 Composition of basal diets 41 8 Four -week body weights of chicks fed different levels of soft phosphate and ground limestone (experiment 1) 46 9 Four-week body weights, feed conversion, and tibia ash of chicks fed different levels of soft phosphate and ground limestone (experiment 2) 47 10 Fourand eight-week body weights, feed conversion, and tibia ash of chicks fed different levels of soft phosphate and ground limestone (experiment 3) 49

PAGE 6

CHAPTER 1 INTRODUCTION AND LITERATURE REVIEV7 The element phosphorus was first prepared in the free state by Brandt, a Gerraan chemist, in 1669 and first recognized as an essential bone component by Gahn, a Swedish chemist, in 1769. Phosphorus probably plays a more varied role in the chemistry of living organisms than any other single element. It is also an essential constituent of proteins and fats occurring in muscular tissues, vital organs and brain. Phosphates are also known to be important buffers in tissue fluids. Deficiencies of mineral elements in animal rations began to be recognized about 100 years ago when weak bones in cattle grazing in certain localities began to be associated with mineral inadequacies of the soil. According to Ewing (1963) the earliest recorded use of a phosphate feed supplement for the specific purpose of preventing a phosphorus deficiency in ruminants occurred in 1861 when Von Gohren reported that the weak bones of cattle grazing near the Rhine River could be prevented and cured by feeding small amounts of bone meal. Analysis of the soil and grass in these areas revealed a very low phosphorus and calcium content. 1 -

PAGE 7

2 Weak bones in non-ruminants, particularly in swine, was noted as a serious problem around 1885, and it was found that feeding bone meal to pigs as a supplement to corn more than doubled the breaking strength of deficient bones . Rock phosphate is the most abundant source of phosphorus, with approximately half of the 26 billion ton world reserve located in the United States. These phosphates were used as sources of phosphorus in plant nutrition as early as 1860. The belief that only phosphorus from plant and animal sources was available to animals accounted for the delay in its use in animal nutrition, and as late as 1914 it was still felt that at least a part of the animal's phosphorus requirement should be supplied in organic form. The danger of fluorine toxicity began to be recognized as a serious problem around 1930, and, as a consequence, raw rock phosphates have now been largely replaced by sources that have been treated for the removal of fluorine. Def luorinated super phosphate, which was one of the first of these sources to be produced in commercial quantities, was in general use about 1940. Considerable research has been conducted in recent years to determine which phosphorus supplements are suitable for use in poultry feeds. Among the sources recently

PAGE 8

3 evaluated was colloidal phosphate, which is also called soft phosphate. Soft phosphate is obtained as a by-product during the mining of rock phosphate, and is an inexpensive source of supplemental phosphorus. The use of such a supplement in poultry diets would be of great importance from a commercial point of view. The latest addition to the list of corrimercial phosphate feed supplements is a dicalci-um phosphate v/hich is made by treating phosphoric acid with the proper amount of lime. It is practically free of fluorine and contains up to 2 2 percent phosphorus as compared to approxim.ately 15 percent in the best grades of bone meal and about 8.5 to 14.5 percent in the original def luorinated rock phosphates . The National Research Council (1966) , in a recent revision of its suggested nutrient requirements for chickens, increased the total dietary phosphorus requirem.ent for starting chickens from 0.60 percent to 0.70 percent of the diet. Num^erous studies have been conducted to establish this requirement for the chick, and there has been variation in reported requirements. McGinnis &t al . (1944) reported that levels of greater than 0.53 percent phosphorus were required for maximum calcification. The available phosphorus requirement necessary for satisfactory

PAGE 9

4 calcification was found by Singsen et al . (1947) to be between 0.38 and 0.47 percent of the diet. Gillis et al . (1949) and ' Rourke et al . (1952) found a requirerr.enu of approximately 0.50 percent. Grau and Zweigart (1953) indicated that maximum tibia ash of chicks was obtained with a level of not more than 0.45 percent phosphorus. Maximum calcification of chick bones was produced by a level of 0.58 percent total phosphorus in studies conducted by Fisher et al . (1953). A level of 0.76 to 0.81 percent phosphorus was suggested by Couch et al . (1937) as being adequate for normal growth and bone calcification of chicks up to 12 weeks of age. Additional studies were reviewed by Singsen ct al. (1948) , Gillis et al. (1949) , and O'Rourke et al . (1952), and Nelson and Walker (1964). Much of this variability in reported phosphorus requirements can probably be attributed to differences in availability of the phosphorus to the chick. The National Research Council (1966) states that at least 0.50 percent of the total feed of starting chickens should be inorganic phosphorus. All the phosphorus of non-plant feed ingredients is considered to be inorganic in nature. Approximately 30 percent of the phosphorus of plant products is non-Phytin phosphorus and may be considered as part of the inorganic phosphorus requirement. A portion of the phosphorus

PAGE 10

5 requirement of growing chickens and laying and breeding hens must also be supplied in inorganic form, but this requirement for inorganic phosphorus is lower and not as well defined as it is for starting chickens. The interaction of vitamin D3 has long been recognized as one of the factors governing the utilization of calcium and phosphorus fromi mineral sources. It has been demonstrated under certain conditions that levels of vitamin D3 considerably in excess of those suggested by the National Research Council (1966) may improve the utilization of the phosphorus from certain low grade phosphate sources (Motzok et al.^ 19 65; Fritz and Roberts, 1966; and XcKnight and Watts, 1966) . The advent of the computer has afforded the researcher and nutritionist with a valuable tool for the formulation of least-cost feeds. In return, the machine has increased the burden of its operator by demianding exacting specifications and analytical values for feeds and feedstuffs in order to function properly. In order for the nutritionist to formulate diets in the most economical and profitable manner he must have an accurate evaluation of the phosphorus content of these supplements and its availability. In the usual assay of phosphate supplements, the test sources are added to a phosphorusdeficient diet to supply graded sub-optimum levels

PAGE 11

6 of phosphorus. The response, generally expressed in terras of bone calcification or body weight, is then compared to that from a standard source fed at equivalent total dietary phosphorus levels to establish a relative biological value for the test phosphate. The interaction of calcium and phosphorus has long been recognized. Adverse Ca:P ratios limit the utilization of phosphorus, especially at sub-optimal levels such as are used in phosphorus assay diets. However, there is no standard calcium level or Ca:P ratio in general use. Nelson and Peeler (1964) indicated that one of the problems involved in the development of a biological assay for feed phosphates was whether to hold the calcium level constant or have a constant Ca:P ratio in the assay diet. In certain assays, constant calcium to phosphorus ratios were employed (Matterson et at., 1945; Singsen and Scott, 1946; Creech et al . , 1956; Nelson and Peeler, 1961), while in others, constant calcium levels of 1.0 percent (Ammerman et at., 1960) or 1.2 percent (Gardiner et at., 1959) were used . Recent studies by Waldroup et al . (1965a) pointed out that variation in the calcium content of the diet used in assaying phosphorus sources may influence significantly the relative biological value of a particular phosphate

PAGE 12

7 source. In this report it was suggested that the use of a specific calcium level at different phosphorus levels appears to be desirable in order to elicit maximum response of the chick and allow full utilization of a particular phosphorus source. It is reasonable to assume that the most valid biological value would be obtained if the chick was able to maximally utilize the phosphorus present in the test diet. Therefore, a calcium level should be used which produces maximum growth and bone ash. This series of experiments was designed to develop a standard assay curve for each of the commercial phosphate sources used in poultry diets. Such a standard curve should be of great value in eventually establishing a standard phosphorus assay that will give more realistic values of utilization, especially for test materials.

PAGE 13

CHAPTER 2 DEVELOPMENT OF A CALCIUM STANDARD CURVE FOR MONOSODIUM PHOSPHATE Monosodiura phosphate has for many years been recognized and utilized as a standard phosphorus source in the determination of biological values and phosphorus requirements for poultry. These experiments were conducted to develop a standard calcium curve for this phosphorus reference material. Experiment 1 Experimental Procedure The basal diet used for this study was composed primarily of degerminated corn and soybean meal (Table 1) . It was calculated to contain 22 percent protein and 2200 Calories of productive energy per kilogram of diet. Twenty-two hundred International Chick Units of supplemental vitamin D3 were provided per kilogram of diet in order to eliminate any effect of this vitamin upon utilization. This diet v;as found, by analysis, to contain 0.30 percent total phosphorus and 0.26 percent calcium. This is a considerably higher calcium level than previously reported by Waldroup et at. (1965a) for this diet. The reason for this

PAGE 14

9 TABLE 1 Composition of basal diet Ingredie: Percent or ciet Degerminated corn meal Cerelose Soybean meal (50% protein) Alfalfa meal (20% protein) Iodized salt Kicro-ingredients ^ Variable^ 51.70 5.40 34.00 3.00 0.40 0.50 5.00 Percent protein Productive energy (Cal./kg.) Percent phosphorus Percent calcium 22.00 2200 0.30 0.26 'Supplied per kilogram of diet: vitamin A, 66 00 I.U.; vitamin D3, 2200 I.C.U.; vitamin K, 2.2 mg . ; riboflavin, 4.4 mg.; pantothenic acid, 13.2 mg.; niacin, 39.6 mg.; choline, 499.4 mg.; vitamin B^,, 22 meg.; Santoquin, 0.0125%; m.anganese, 71.4 mg.; iron, "19.8 mg.; copper, 1.98 mg.; cobalt, 198 mg., iodine, 1.1 mg.; and zinc, 99 meg. ^Calcium and phosphorus levels were obtained by altering the levels of reagent grade calcium carbonate, phosphate source, and yellow builders sand.

PAGE 15

10 difference was not determined; however, it points out the necessity of chemical analysis of the basal diet used with each set of ingredients. Reagent grade monosodium phosphate (NaH2P04 • H2O) was used to supply supplemental phosphorus in 0.05 percent increments to a total of 0.55 percent total phosphorus . Within each phosphorus level, reagent grade calcium carbonate was used to supply supplemental calcium. Five calcium levels were used at each phosphorus level. Levels were selected which were estimated from previous studies (Waldroup et al . , 1965a) to result in a response line and plateau within each phosphorus level so that the appropriate calcium requirement for that particular phosphorus level could be selected. Three successive trials were conducted. In each trial, three replicate groups of five male and five female broiler-type chicks (Vantress x White Plymouth Rock) per group were fed each diet from 1 to 21 days of age. The chicks were sexed, debeaked, and randomly assigned to treatment groups at one day of age. The chicks were brooded in Oakes 801-A five deck thermostatically controlled, electrically heated, battery brooders with raised wire floors. At the termination of each trial, all birds were individually weighed, and two males and two females from each group were sacrificed for

PAGE 16

11 tibia ash determinations. The vitamin D determination procedure of the Association of Official Agricultural Chemists (1965) was follov.'ed in ashing the bones. Statistical analysis (Analysis of Variance; Snedecor, 1956) of the data failed to reveal a treatment x trial interaction; therefore, the data from the three trials v;ere combined. Significant differences between tibia ash and body weight treatment means v;ere determined by Duncan's multiple range test (1955). Based on these measurements, the ideal calcium level was selected for each level of phosphorus, and a standard curve of calciuiti levels determined for the various additions of monosodium phosphate . Results and Discussion The level of supplemental calcium fed with each level of phosphorus significantly influenced tibia ash and body weight (Table 2) . However, the greatest influence was noted with tibia ash since responses were obtained with higher levels of calcium, especially at the higher phosphorus levels. The ideal calcium level was different for the five different phosphorus levels. Maximum tibia ash and body weight v:ere obtained with 0.18 percent supplemental calcium when the diet contained 0.30 percent phosphorus (Table 2). Hov;ever,

PAGE 17

12 TABLE 2 Tibia ash and body weight of chicks fed various levels of phosphorus and calcium supplied from monosodium phosphate and reagent grade calcium carbonate

PAGE 18

13 body weight was not different with levels of 0.13 or 0.23 percent suppleir.ental calciuin. Levels of 0.15, 0.21, and 0.27 percent suppleir.en-cal calciurr: v;ith 0.35 percent phosphorus resulted in raaxiinura tibia ash and body v/eights (Table 2) . Supplemental calcium levels of less than 0.15 percent resulted in a significant lov;ering of the tibia ash and body weight. Maxira^'um growth was obtained with supple~iental calcium levels of 0.17, . 24, and 0.31 percent, v:hen the diet contained 0.40 percent phosphorus (Table 2). Hov;ever, a level of 0.17 percent supplemental calcium did not support maximum tibia ash. This would indicate a level of 0.24 percent supplemiental calcium as being ideal for this level of phosphorus . Levels of 0.19 and 0.35 percent supplem.ental calcium supported both maximum tibia ash and body vreight (Table 2) when the diet contained 0.45 percent phosphorus. The level of 0.27 percent supplemental calcium, supported maximum body weight; however, tibia ash was significantly lower than for the group receiving 0.19 percent supplemiental calcium. The lowered tibia ash was attributed to chance since the tibia ash v;as also num>erically greater for groups receiving 0.35 percent supplement:al calcium. V7hen the diet contained 0.50 percent phosphorus, levels of 0.21, 0.30, and 0.39 percent supplem.ental calci-um.

PAGE 19

14 supported maximum body weight (Table 2). However, tibia ash was significantly lower when the diet contained only 0.21 percent supplemental calcium. Maximum body weight was obtained with supplemental calcium levels of 0.23, 0.33, and 0.43 percent when the diet contained 0.55 percent phosphorus (Table 2). However, a significantly lower tibia ash was obtained with the diet containing 0.23 percent calcium. Based on the above results, a standard curve has been suggested for use in selection of the proper level of calcium to use in order to elicit maximum response, measured as both tibia ash and body weight. The suggested calcium levels are 0.44, 0.47, 0.50, 0.53, 0.56, and 0.59 percent for phosphorus levels of 0.30, 0.35, 0.40, 0.45, 0.50, and 0.55 percent, respectively, when supplemental phosphorus is supplied as monosodium phosphate (NaH2P0^ • H2O) . It should be emphasized, however, that these levels are only for the basal diet employed in this experiment and may not apply when dietary modifications are made . Increasing the phosphorus : calcium ratio by the addition of phosphorus to diets containing calcium levels of 0.29, 0.39, 0.49, and 0.53 percent did not depress performance of chicks when measured as tibia ash or growth rate (Table 2) . An improvement of tibia ash was produced

PAGE 20

15 by widening the P:Ca ratio with the addition of phosphorus to the diet containing the low level of calcium (0.29 percent total calcium) . This was in contrast to the effect of widening the calcium: phosphorus ratios at low levels of phosphorus as reported by Vandepopuliere et; al. (1961). Experiment 2 Experimental Procedure A second experiment was conducted to determine whether the suggested calcium levels would produce maximum tibia ash and body weight when phosphorus was fed at other levels. The same general procedure was follov;ed as used in experiment 1 except that four, instead of three, replicates of five males and five females were used per treatment. Also, tibia ash was determined on five males per treatment instead of two males and two females per treatment as in experiment 1. Levels of 0.07, 0.14, and 0.21 percent phosphorus from monosodium phosphate (NaHjPO^ • H2O) were added to the basal diet and five suitable levels of calcium were fed at each of the three levels of phosphorus. Results and Discussion A level of 0.19 percent supplemental (0.45 percent total) calcium gave maximum tibia ash and body weight when

PAGE 21

16 0.07 percent supplemental phosphorus was added (Table 3). This is in fair agreement with the 0.485 percent suggested on the standard curve (experiment 1), and a level of 0.49 percent calcium did not significantly reduce either body weight or tibia ash. When 0.14 percent phosphorus was added, a level of 0.26 percent supplemental calcium produced maximum tibia ash and body weight. However, when the diet contained 0.21 percent supplemental phosphorus, a level of 0.30 percent supplemental calcium gave maximum performance. These levels agree very well with those suggested on the standard curve. These data indicated that the suggested levels of calcium (experiment 1) would elicit maximum tibia ash and body weight. Therefore, it is suggested that such a curve be utilized in phosphorus assays for determining the level of calcium to use with this diet. Based on the data in experiment 2 (Table 3) , it would appear that these levels have a sufficient margin of safety for normal variation in calcium content of the basal diet. Although the chicks grew at a higher rate in this experiment, the response to the various dietary phosphorus and calcium levels was essentially the same as observed in the first experiment.

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17 TABLE 3 Tibia ash and body weight of chicks fed various levels of phosphorus and calcium supplied from monosodium phosphate and reagent grade calcium carbonate

PAGE 23

18 Summary An experiment, involving three trials, v;as conducted using reagent grade monosodium phosphate and calcium carbonate to establish a calcium standard curve for use in phosphorus availability studies. Six levels of supplemental phosphorus (0 to 0.25 percent) were added to the degerminated corn-soybean meal basal diet, with five calcium levels per phosphorus level. Broiler-type chicks were fed the experimental diets for 21 days, and tibia ash data and body weights v;ere used to determine the calcium requirement at each phosphorus level. These data were used to construct a curve giving the suggested calcium level for any level of monosodium phosphate when using a diet based largely upon degerminated corn and soybean meal. Results from a second experiment indicated that calcium levels selected from the standard curve for other levels of monosodium phosphate would produce maximuiTi bone ash and body weight.

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CHAPTER 3 DEVELOPMENT OF A CALCIUM STANDARD CURVE FOR DEFLUORINATED PHOSPHATE Def luorinated phosphate is one of the most widely used sources of phosphorus in poultry diets because of the abundance of raw material, its low fluorine content, and the fact that it has a high concentration of readily available calcium and phosphorus. The Ca:P ratio of def luorinated phosphate is quite similar to that found in bone, and this is considered to be another advantage for this material. Gillis et al . (1954), using biological assay techniques and beta tricalcium phosphate as a standard, found def luorinated phosphate to have a biological value varying from 82 to 99 percent, depending upon the method of manufacture. The experiment reported herein was conducted to develop a standard calcium curve which will allow researchers to more accurately evaluate the biological availability of this feed grade phosphorus supplement. Experimental Procedure Two successive trials v/ere conducted. Three replicate groups, each containing five male and five female 19 -

PAGE 25

20 broiler-type chicks (Vantress x White Plymouth Rock) , received each dietary treatment. The chicks were sexed, debeaked, and randomly assigned to the treatments at one day of age. The birds received the experimental diets and tap water ad libitum from 1 to 21 days of age. The basal diet used for this study was idenrical to the one described in Chapter 2 (Table 1) . Commercial grade def luorinated phosphate was used to supply supplemental phosphorus in 0.07 percent increments to a total dietary level of 0.51 percent phosphorus (Table 4). Within each phosphorus level, reagent grade calcium carbonate was used to supply supplemental calcium. Five equally spaced calcium levels were used at each phosphorus level in an attempt to select the appropriate calciuir-i level which would give a maximum response with each particular phosphorus level. The battery brooders used in this experiment were the same as those described in Chapter 2. At the termination of each trial the birds were separated and v/eighed according to sex. Two males and two females from each group were sacrificed for tibia ash determinations. The vitamin D determination procedure of the Association of Official Agricultural Chemists (1965) was iollov;ed in ashing the bones. Statistical analysis of the data

PAGE 26

21 (Analysis of Variance; Snedecor, 1956) failed to reveal a treatment x trial interaction; therefore, the data from the two trials were combined. Significant differences between tibia ash and body weight treatment means were determined by Duncan's multiple range test (1955). Based on these measurements, the ideal calcium level was selected for each level of phosphorus, and a standard curve of calciuiri levels determined for the various additions of def luorinated phosphate . Results and Discussion The combined results of the two trials (Table 4) indicate that the level of supplemental calcium was more critical at the lowest level of phosphorus supplementation, There were no significant differences between tibia ash values within a given level of phosphorus; however, each increment of phosphorus resulted in a significant increase of tibia ash values. Body weight also tended to increase numerically as the level of phosphorus increased; however, trends of statistical significance were not as clear cut. When the diet contained 0.37 percent phosphorus, the highest levels of bone ash resulted from the feeding of 0.475 and 0.520 percent total calcium. Since these two treatments also produced the heaviest body weights of

PAGE 27

22 TABLE 4 Tibia ash and body weight of chicles fed various levels of phosphorus and calcium supplied from def luorinated phosphate and reagent grade calcium carbonate Total phosphorus^ (%) 0.37 0.44 0.51 Calcium^

PAGE 28

23 any diet containing 0.37 percent phosphorus, an intermediate calcium level of 0.498 percent was selected as one providing an adequate safety margin for this level of phosphorus. Changing the calcium level when the diet contained 0.44 percent phosphorus did not produce a significant increase of body weight or bone ash, but the group that received 0.560 percent total dietary calcium had numerically superior body weight and bone ash. In order to allow for a margin of safety around the value selected for the curve, 0.590 percent total calcium was selected for this level of phosphorus . When the diet contained 0.51 percent phosphorus, a level of 0.685 percent calcium resulted in a combination of the highest body weight and greatest percent bone ash of any of the calcium levels tested. Since there v/ere no significant differences between either the body weights or bone ash of this series and very little fluctuation among numerical values, it was felt that the choice of 0.685 percent total calcium would afford an ample margin of safety on either side of the standard curve. Summavy An experiment consisting of two trials was conducted to establish a calcium standard curve for use in phosphorus availability studies involving def luorinated phosphate.

PAGE 29

24 Three levels of supplemental phosphorus (0.07, 0.14, and 0.21 percent) supplied from def luorinated phosphate were added to the degerminated corn-soybean meal basal diet. Five equally spaced calcium levels were fed with each level of phosphorus. Broiler-type chicks received the experimental diets from 1 to 21 days of age; tibia ash and body weight data were used as the criteria for determining the calcium requirement at each phosphorus level. The optimum calcium levels selected were 0.498, 0.590, and 0.685 percent, respectively, for total phosphorus levels of 0.37, 0.44, and 0.51 percent.

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CHAPTER 4 DEVELOPMENT OF A CALCIUX STANDARD CURVE FOR DICALCIUM PHOSPHATE Dicalcium phosphate is a highly available phosphate rua"cerial that is widely utilized as a source of inorganic phosphorus for poultry diets. Gillis &:, al. (1954) and Nelson and Peeler (1961) , using beta-tricalciuiTi phosphate as the reference n-.aterial , found dicalciur. phosphate to have a biological value of 93 and 97 percent, respectively. This experiment was conducT:ed uO determine x.'r^e levels of calcium necessary for the maximum expression of biological availability by this comiT.ercial phosphate source at various levels of phosphorus supplem>entation. ExperimentaZ Prooedure Three successive trials were conducted. In each trial three replicate groups containing five m>ale and five female broilertype chicks (Vantress x \«rnite Plym.outh Rock) received each dietary treatment and cap water aa lihitur. from. 1 x.o 21 days of age. The chicks were sexed, debeaked, and randomly assigned to treatmient groups ar one day of age. The basal diet used was identical to the one described in Chapter 2 (Table 1) . Comjr.ercial grade 25

PAGE 31

26 dicalcium phosphate was used to supply supplemental phosphorus in 0.07 percent increments to a total dietary level of 0.51 percent (Table 5). Within each phosphorus level reagent grade calcium carbonate was used to provide five equally spaced supplemental calcium levels. This range of calcium supplementation was provided for each phosphorus level in order that the appropriate calcium requirement for each level might be determined. The battery brooders used in this experiment were identical to the ones described in Chapter 2. At the termination of each trial the birds were separated and weighed according to sex, and two males and two females from each replicate group were sacrificed for tibia ash determinations. The vitamin D determination procedure of the Association of Official Agricultural Chemists (1965) was followed in ashing the bones. Statistical analysis of the data (Analysis of Variance; Snedecor, 1956) did not reveal a significant treatment x trial interaction; therefore, the data from the three trials were combined. Significant differences between tibia ash and body weight treatment means were determined by Duncan's multiple range test (1955) . Based on these measurements, the ideal calcium level for each level of phosphorus was selected, and a standard curve of calcium levels determined for the various additions of dicalcium phosphate.

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27 Results and Disaussion The combined results of the three trials v/ith dicalcium phosphate are presented in Table 5. A significant increase of tibia ash and body weight resulted v/hen the total calcium level was increased from 0.35 to 0.43 percent in diets containing 0.37 percent total phosphorus. The diet containing 0.43 percent calcium produced the largest numerical bone ash value of any group receiving 0.37 percent phosphorus, and no further significant improvement of tibia ash or body weight resulted from feeding higher calcium levels with this level of phosphorus. In order that a margin of safety m.ight be allowed around the calcium level proposed for this level of phosphorus, a total calcium level of 0.47 percent was selected. The tibia ash of birds receiving diets containing 0.44 percent total phosphorus was significantly increased when the dietary calcium level was increased from 0.44 to 0.50 percent; however, there were no significant differences noted among body weights. The diet containing 0.4 4 percent phosphorus and 0.56 percent calcium resulted in tibia ash and body weight values which were numerically superior to any of the treatment groups receiving 0.44 percent phosphorus. However, it was considered that since consis-cent numerical improvements of tibia ash and body weight

PAGE 33

28 TABLE 5 Tibia ash and body \\7eight of chicks fed various levels of phosphorus and calcium supplied from dicalcium phosphate and reagent grade calcium carbonate Total phosphorus^ (%) Calcium

PAGE 34

29 resulted from the feeding of 0.44, 0,50, and 0.56 percent calciuiTi, a level soniewhat in excess of 0.56 percent v;as required in order to afford so~.e factor of safety. Since a dietary level of 0.62 percent calciuir. with 0.44 percent phosphorus produced a tibia ash value that was alriCSw equal to rhat resulting fror* the feeding of 0.56 percent calciuiTi, an intermediate total calci'u^i level of C.5o percent was selected as tne secona point of tne standara curve for the assay of dicalciuTi phosphate. Increasing the level of calciur. v;ith 0.51 percent phosphorus produced essentially no response v'ithin either criterion, as there were no significant differences (and only very snail nuiTierical variations} between any of the tibia ash or body weight values. These results vrere not unexpected since 0.51 percent phosphorus is approaching the bird's requirement, and it is vre^l estaolisned tnat dietary calciumi levels become less critica^ as tne pnospncrus content of the diet is increased. In order to provide an adequate safety m.argin, 0.59 percent totax dietary calcium was selected for a level of 0.51 percent phospncrus Summary This experiment, comprised of three trials, was conducted to define a calcium standard curve for use m studying the availability of phosphorus fromi comjr.ercial

PAGE 35

30 grade dicalcium phosphate. Three levels of supplemental phosphorus (0.07, 0.14, and 0.21 percent) were provided from dicalcium phosphate, and five equally spaced levels of supplemental calcium supplied from reagent grade calcium carbonate were fed with each level of phosphorus in a degerminated corn-soybean meal basal diet. Broiler-type chicks housed in starting batteries received the experimental diets from 1 to 21 days of age. Tibia ash and body weight data were used as the criteria for determining the optimum level of calcium to be fed with each level of phosphorus. The calcium levels selected were 0.47, 0.58, and 0.69 percent, respectively, for total phosphorus levels of 0.37, 0.44, and 0.51 percent.

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CHAPTER 5 DEVELOPMENT OF A CALCIUM STANDARD CURVE FOR SOFT PHOSPHATE In recent years there has been considerable interest in the utilization of soft phosphate, also known as colloidal phosphate or phosphatic clay, as a source of phosphorus in poultry diets. Soft phosphate is one of the materials with low phosphorus content (approximately 18 percent calcium and 9 percent phosphorus), yet it is also one of the most inexpensive sources of supplemental phosphorus. The main problem associated with the use of this material is the low biological availability of its phosphorus and the wide range of reported availability values. Nelson and Peeler (1961) reported an availability of only 34 percent for the phosphorus of soft phosphate. In contrast, Waldroup et al . (1965a) estimated that 51-59 percent of the phosphorus in soft phosphate was available and suggested that the dietary calcium level could influence availability. This experiment was conducted in order to determine the levels of dietary calcium necessary for the maximum expression of biological availability by this phosphate source. 31 -

PAGE 37

32 Experimental Prooedure In each of three successive trials, three replicate groups containing five male and five female broiler-type chicks (Vantress x white Plymouth Rock) received each dietary treatment and tap water ad libitum from 1 to 21 days of age. The birds were sexed, debeaked, and randomly assigned to treatment groups at one day of age. The basal diet used was identical to the one described in Chapter 2 (Table 1) . Supplemental phosphorus supplied from commercial grade soft phosphate was added in 0.07 percent increments to result in total dietary phosphorus levels of 0.37, 0.44, and 0.51 percent. Within each phosphorus level, reagent grade calcium carbonate was used to supply five equally spaced supplemental calcium levels (Table 6) . The levels of calcium supplementation varied among the three phosphorus levels and were provided in order that the appropriate calcium requirement for each level of phosphorus might be determ.ined, The battery brooders used in this experiment were identical to the ones described in Chapter 2. On the final day of each trial, the birds were separated and weighed according to sex, and two males and two females from each replicate group were sacrificed for tibia ash determinations. The vitamin D determination procedure of the

PAGE 38

33 Association of Official Agricultural Cherrdsts (1965) was followed in ashing the bones. Statisrical evaluation of the data (Analysis of Variance; Snedecor, 1956) revealed no significant treatment x trial interaction; therefore, the data from the three trials were combined. Significant differences between tibia ash and body weight treatment means were determined by Duncan's multiple range test (1955) . Based on these measurements, the ideal calcium, level for each phosphorus level was selected, and a standard curve of calcium levels determiined for the various additions of soft phosphate. Results and Discussion The combined results of the three trials with soft phosphate are shown in Table 6 . There were no significant differences am^ong the body weights of birds receiving 0,37 percent total phosphorus, regardless of the calcium level fed. The addition of 0.06 and 0.12 percent supplemental calcium to a diet containing 0.07 percent phosphorus from soft phosphate (0.37 percent total phosphorus) resulted in a significant increase of bone ash for each of these calcium additions. No further significant improvements were noted by increasing the supplemental calcium level above 0.12 percent (0.52 percent total). The birds receiving a diet containing 0.12 percent supplem.ental

PAGE 39

34 TABLE 6 Tibia ash and body weight of chicks fed various levels of phosphorus and calcium supplied from soft phosphate and reagent grade calcium carbonate Total phosphorus^ (%) 0.37 0.44 0.51 Calcium^

PAGE 40

35 calcium (0.52 percent total) had the greatest tibia ash value resulting from any of the diets containing 0.37 percent phosphorus. Since a margin of safety v/as needed on the total calcium level selected, it was proposed that a dietary calcium level of 0.56 percent v/ould be appropriate for this phosphorus level. Supplementing the diet containing 0,44 percent total phosphorus with graded levels of reagent grade calcium carbonate resulted in no significant improvement of either tibia ash or body weight. However, a level of 0.16 percent supplemental calcium (0.70 percent total) produced a statistically significant depression of tibia ash. The addition of 0.04 percent supplemental calcium (0.58 percent total) resulted in a tibia ash value which was numerically superior to that of any other calcium level. The diet containing 0.08 percent supplemental calcium (0.62 percent total) produced the best body weight of any diet containing 0.44 percent total phosphorus. A level of 0.62 percent total calcium was selected for feeding with 0.44 percent total phosphorus. None of the calcium levels supplied with 0.51 percent total phosphorus provided a significantly different response as measured by tibia ash or body weight; however, there was a slight numerical decrease of tibia ash as the total calcium level was increased above 0.72 percent.

PAGE 41

36 Again, this indicates that the level of calcium becomes less critical as the dietary phosphorus level approaches the bird's requirement. A level of 0.58 percent total calcium was selected for feeding with 0.51 percent total phosphorus because this dietary combination produced the greatest body weight of any diet containing 0.51 percent total phosphorus, and near maximum tibia ash. Summaicy An experiment consisting of three trials was conducted in order to define a calcium standard curve which would be of value in studying the availability of phosphorus from soft phosphate. Three levels of supplemental phosphorus (0.07, 0.14, and 0.21 percent) were provided from soft phosphate, and five equally spaced supplemental calcium levels supplied from reagent grade calcium carbonate were fed with each level of phosphorus in a degerminated corn-soybean meal basal diet. Both the supplemental and total calcium levels varied among the different levels of phosphorus. Broiler-type chicks housed in starting batteries received the experimental diets from 1 to 21 days of age. Body weight and tibia ash data were used as the criteria for determining the optimum level of calcium to be fed with each level of phosphorus. With due consideration

PAGE 42

37 having been given to safety margins , the total calcium levels selected were 0.56, 0.62, and 0.68 percent, respectively, for total phosphorus levels of 0.37, 0.44, and 0.51 percent.

PAGE 43

CHAPTER 6 INFLUENCE OF DIET COMPOSITION ON THE UTILIZATION OF SOFT PHOSPHATE IN BROILER DIETS Numerous studies have been conducted to evaluate the biological value of soft phosphate for poultry. The majority of these reports has indicated that the phosphorus from soft phosphate is poorly utilized. Nelson and Peeler (1961) reported a biological value of only 34 percent for soft phosphate and found that the availability of poor quality phosphates was not improved when fed in a mixture with materials possessing a higher biological availability . In addition, Summers et al . (1959) reported the availability of phosphorus from soft phosphate to be approximately 47 percent, and Waldroup et al . (1965a) estimated that 51-59 percent of the phosphorus in soft phosphate was available. Summers et al . (1959) found that the availability of soft phosphate was improved by the addition of either phosphoric or hydrochloric acid. In contrast, Waldroup et al. (1965c) reported no apparent improvement in the utilization of phosphorus from soft phosphate by combining it with phosphoric acid. These workers concluded that the 38 -

PAGE 44

39 apparent increased phosphorus content of the mixture could be attributed to the loss of water vapor during the chemical reaction that occurred as the materials were combined. Waldroup et at. (1963) found that when dietary calcium and phosphorus levels were suboptimal, the calcium: phosphorus ratio was more critical in diets containing soft phosphate than in those diets containing a more readily available phosphorus source. It has been demonstrated under certain conditions that levels of vitamin D3 considerably in excess of those suggested by the National Research Council (1966) may improve the utilization of the phosphorus from soft phosphate (Motzok et al . , 1965; Fritz and Roberts, 1966; and McKnight and Watts, 1966). The experiments reported in this chapter were conducted to study the influence of various dietary factors on the utilization of phosphorus from soft phosphate, and to attempt to obtain maximum growth with broiler-type chicks when soft phosphate was utilized as the sole supplemental source of phosphorus. Experimental Procedure Day-old broiler-type chicks, obtained from a commercial hatchery, were utilized in all experiments. Ten male and ten female chicks were randomly assigned to

PAGE 45

40 floor pens according to a randomized block design. Four replicate pens were assigned to each dietary treatment. All pens contained 2.32 square meters of floor area, and were uniform in shape and construction. Each pen was provided with one automatic waterer, one hanging cylindrical feeder, and one infrared heat lamp. Dried peanut hulls were used as litter. Treatments consisted of graded additions of soft phosphate to the basal diets shown in Table 7 . In experiments 1 and 2, and one-half of experiment 3, a basal diet (Table 7, diet 1) containing 0.34 percent phosphorus and 0.16 percent calcium was used. The fish meal basal (Table 7, diet 2) of experiment 3 contained 0.46 percent phosphorus and 0.28 percent calcium. Since the vitamin Dj level of feeds has been shown to influence calcium and phosphorus utilization, all experimental diets were fortified with 7920 I.C.U. of vitamin Dj/kg. All diets were kept iso-caloric (2093 Cal./kg.) and iso-nitrogenous (22.05 percent protein) by varying the level of corn, soybean oil meal, and animal fat. Values of Maddy et al . (1963) were used in the calculation of adjustments. The birds were given the experimental diets arid water ad libitum from one day of age until the end of the test.

PAGE 46

41 TABLE 7 Composition of basal diets 1 Ingredients (Percent of diet) Yellow corn Soybean meal (50%) Fish meal (60%) Alfalfa meal (20%) Animal fat Micro-ingredients ^ Iodized salt Variable ingredients' 47.41 35.50 3.00 5.74 0.50 0.40 7.45 51.60 30.60 2.50 3.00 3.30 0.50 0.40 8.10 ^Micro-ingredient mix supplied per kilogram of diet: 6,600 I.U. vitamin A, 449.4 mg. choline, 39.6 mg. niacin, 7,920 I.C.U. vitamin D3, 4.4 mg. riboflavin, 13.2 mg. pantothenic acid, 22 meg. vitamin B^^, 0.0275 percent Santoquin, 19.8 mg. iron, 1.9 8 mg. copper, 19 8 meg. cobalt, 11 mg. iodine, 99 meg. zinc and 220 mg . of manganese sulfate. ^Variable ingredients included sources of supplemental phosphorus and calcium and an inert filler.

PAGE 47

42 Body weight and tibia ash were used as criteria of evaluation. In addition, feed efficiency values were calculated for experiments 2 and 3. The birds from each pen were grouped by sex and weighed at four weeks of age in experiments 1 and 2, and at four and eight weeks of age in experiment 3. In experiments 2 and 3, two males and two females from one pen of each dietary treatment were sacrificed at four weeks of age for bone ash determinations. The left tibia was removed, cleaned of adhering tissue and individually ashed according to the method outlined by the Association of Official Agricultural Chemists (1965). Feed consumption was also determined at four weeks of age in experiment 2, and at four and eight weeks of age for experiment 3. The data from these experiments were subjected to the analysis of variance as outlined by Snedecor (1956) . Significant differences between treatment means were determined by the multiple range test of Duncan (1955) . Experiment 1 A 3 X 4 factorial arrangement of treatments was used involving three supplemental phosphorus levels (0.40, 0.50, and 0.60 percent) and four levels of supplemental calcium (0, 0.20, 0.30, and 0.40 percent) added to the basal diet (Table 7, diet 1). Two positive control

PAGE 48

43 diets consisting of diet 1 supplemented to provide adequate levels of phosphorus (0.70 percent) and calcium (0.80 and 1.00 percent) from commercial grade dicalcium phosphate (21.69 percent phosphorus and 20.92 percent calcium) and ground limestone were also fed. The diets of this experiment were fed from one day until four v/eeks of age, at which tim^e the test was terminated. Experiment 2 This experiment was also of four weeks duration, starting when the chicks were housed at one day of age. The basal diet was supplemented with soft phosphate in order to provide levels of 0.40, 0.50, and 0.60 percent supplemental phosphorus. Ground limestone v/as added to supply supplemental calcium levels ranging from to 0.40 percent (Table 9) to diets containing each of the supplemental phosphorus levels. Two positive control diets, identical to those used in experiment 1, were also fed. Experiment 2 One-half of the treatm^ents of this experiment were essentially replicates of a portion of experim.ents 1 and 2 in that similar additions of soft phosphate and ground limestone were m^ade to the basal diet (Table 7, diet 1). Supplemental phosphorus levels of 0.40 and 0.50 percent and supplemental calcium levels as shown in Table 10 were

PAGE 49

44 provided from these sources. Tv/o positive control diets containing adequate levels of phosphorus (0.70 percent) and calcium (0.80 percent and 1.00 percent) supplied from dicalcium phosphate and ground limestone v/ere also fed. These control diets were identical to the ones fed in experiments 1 and 2. The other half of the experiment consisted of soft phosphate and ground limestone additions to a basal diet containing 2.5 percent fish meal (Table 7, diet 2). Supplemental levels of 0.30 and 0.40 percent phosphorus were fed with the supplemental calcium levels shown in the lower half of Table 10. Two positive control diets containing 2.5 percent fish meal, 0.70 percent total phosphorus with 0.71 percent and 0.91 percent total calcium, respectively, were also fed. These diets were composed of the basal shown in Table 7 (diet 2) and supplemental phosphorus and calcium added from dicalcium. phosphate and ground limestone. Results and Discussion Experiment 1 A level of 0.50 percent supplemental phosphorus supplied from soft phosphate combined with 0.20 percent supplemental calcium produced the best growth rate of any treatment group; however, higher calcium levels significantly

PAGE 50

45 depressed growth (Table 8) . Body weights were significantly increased by the addition of 0.20 percent supplemental calcium to the diet containing 0.40 percent phosphorus supplied from soft phosphate. Increasing the supplemental phosphorus level from 0.50 to 0.60 percent, with optimal calcium levels, depressed growth. However, at the 0.60 percent phosphorus level it was necessary to feed a higher level of supplemental calcium in order to depress growth. Growth at each level of phosphorus supplementation was significantly influenced by the calcium level of the diet. Even though total calcium and phosphorus levels were considerably above those normally felt to be adequate, no combination of calcium and phosphorus levels utilized resulted in body weights statistically equivalent to those of the positive control diets (Table 8) . Experiment 2 No level of supplemental phosphorus from soft phosphate, regardless of calcium level, supported body weights at four weeks of age that were equal to those obtained with the positive control diets (Table 9). All tibia ash values except those for the extreme lowest and highest calcium levels of the 0.40 percent supplemental phosphorus series were not significantly different from those for positive control diets. This effect is possibly

PAGE 51

46 TABLE 8 Four-week body weights of chicks fed different levels of soft phosphate and ground limestone (experiment 1) Supplemental^ Total 4 V7k. P Ca^ Calcium Eodv weiaht' (%) (%) (%) (grams) 0.36^ 0.16 0.80 479a 0.36 1.00 491a . . 0.92 380g 0.20 1.12 440cd 0.40'* 0.30 1.22 432cd 0.40 1.32 426de . . 1.12 450bc 0.20 1.32 464b O.SO"* 0.30 1.42 439cd 0.40 1.52 430de . . 1.32 420ef 0.20 1.52 439cd 0.60"* 0.30 1.62 432de 0.40 1.72 408f ^Basal diet contained 0.34 percent phosphorus and 0.16 percent calcium. ^Supplied as ground limestone. ^Supplied as dicalcium phosphate, and considered to be positive controls. '^Supplied as soft phosphate. ^Means with different letters are significantlydifferent according to Duncan's multiple range test.

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47 TABLE 9 Four-week body weights, feed conversion, and tibia ash of chicks fed different levels of soft phosphate and ground limestone (experiment 2) Supplemental^

PAGE 53

48 explained by a changing calcium and phosphorus ratio. No statistically significant differences were found in feed efficiency values. Experiment 2 No combination of supplemental phosphorus and calcium in diets without fish meal supported body weights at four or eight weeks of age that were equal to those produced with the positive control diets (Table 10) . Highest four-week tibia ash values from diets without fish meal were obtained with 0.40 percent and 0.50 percent supplemental phosphorus from soft phosphate and 0.10 percent supplemental calcium. Both of these values were significantly greater than the bone ash of birds fed the positive control diet containing 0.70 percent total phosphorus and 0.80 percent total calcium. None of the tibia ash values from diets without fish meal were significantly different from those of birds fed the 0.70 percent phosphorus and 1.00 percent calcium positive control diet. No combination of phosphorus and calcium supplementation supported maximum four-week body weights when the diet contained fish meal (Table 10) . However, the weights of birds receiving fish meal diets were much closer to the controls than those of birds fed diets

PAGE 54

49 u ^ O -P o e CO 0) -H (U •U G O -a -P (d o B o !^ 3 0) o > M C Cr> O O TJ o -^ w a -P w x: o •H Om >i o t3 en O O .H 5 0) 1 > -P (U •H -P
PAGE 55

50 K K O Co I I o r-i w < Eh

PAGE 56

51 containing no fish meal. Birds fed the fish meal diets containing 0.40 percent supplemental phosphorus from soft phosphate, regardless of the calcium level fed, had a body weight at eight weeks of age which was not statistically different from those of birds receiving the positive control diets (Table 10) . The birds receiving fish meal and 0.30 percent added phosphorus with no supplemental calcium also had body weights at eight weeks of age that were not significantly different from the control groups. Fourand eight-week body weights of birds fed the diet with 0.30 percent supplemental phosphorus and 0.20 percent supplemental calcium were significantly lower than weights of birds fed most other diets containing fish meal. These weights are obviously not representative of the treatment since both higher and lower levels of supplemental calcium produced greater body weights. This variability was attributed to an unexplainable low weight for two of the replications within this treatment. None of the tibia ash values for the fish meal group were statistically different (Table 10) . Previous work (VJaldroup et al . , 1965b) indicated that growth of chicks was greatly improved when a small amount of phosphate with a high availability was added to the diet. These data would indicate that the fish meal furnished some supplemental phosphorus of high availability,

PAGE 57

52 and that this was necessary to obtain maximum growth of broiler-type chicks. The phosphorus in fish meal has been previously reported to be highly available (Waldroup et at. , 1965a) . Some significant differences were noted among feed efficiency values when four-week measurements were evaluated; however, these trends had disappeared by the time the birds reached eight weeks of age. Summary Three experiments were conducted to ascertain factors influencing the growth of broiler-type birds while utilizing soft phosphate as the sole source of supplemental phosphorus. A range of supplemental calcium levels was provided from ground limestone in order to eliminate the dietary calcium level as a limiting factor. Four-week body weights equal to positive control diets could not be produced by diets containing soft phosphate as the sole supplemental phosphorus source. The addition of 2.5 percent fish meal to the basal diet improved four-week body weights; however, they still were not equal to the weights of birds receiving the positive control diet containing 0.80 percent calcium. The addition of 0.40 percent supplemental phosphorus and 0, 0.10, or

PAGE 58

53 0.20 percent supplemental calcium to a diet containing 2.5 percent fish meal resulted in an eight-week average body weight statistically equal to the weight of the control groups. The improved response of the birds receiving fish meal diets was attributed to the supplemental phosphorus of high availability supplied by the fish meal.

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CHAPTER 7 SUMMARY At present, there are no standardized levels of calcium to be fed in an assay conducted to determine the biological value of feed grade phosphate materials. Some researchers feed a level of 1 percent calcium, regardless of the level of phosphorus in the diet; others utilize a constant calcium:phosphorus ratio over the entire range of phosphorus supplementation. The main thesis investigated and reported in this dissertation is that the calcium level necessary for expression of maximum biological availability by a phosphate source varies with the level of phosphorus supplied. Experiments were also conducted to investigate the influence of diet composition on the utilization of soft phosphate in broiler diets. A series of 15 trials involving approximately 15,000 chicks was conducted in order to develop a more meaningful chick assay for determining the availability of phosphorus from various commercial phosphate sources. A diet primarily of all-plant origin, containing degerminated corn meal and soybean meal, was utilized in all assay studies. The diet was calculated to contain 22 percent protein, 0.30 percent phosphorus, 0.26 percent calcium, and 2,200 Calories of productive energy per kilogram. 54 -

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55 The vitamin premix supplied 2,200 I.C.U. of vitamin D^/kg. of diet in order to eliminate the possibility of this vitamin being a factor limiting chick performance. Two corn-soybean meal type basal diets, differing only in fish meal content, were used for the studies concerning diet composition. All assay studies were of three weeks duration, starting when the chicks were randomly assigned to battery brooders at one day of age. Body weight and bone ash were used as the criteria of evaluation. On the twenty-first day of age all birds were weighed and representative bone samples taken for individual ash determinations. The studies of diet composition were of either four or eight weeks duration, starting when the chicks were housed in floor pens at one day of age. Body weight, bone ash, and feed conversion values were used for evaluation purposes. Five supplemental phosphorus levels (0.05, 0.10, 0.15, 0.20, and 0.25 percent) were provided from reagent grade monosodium phosphate, which is the standard source that other phosphates are usually compared with in order to determine the biological value of the material tested. Three supplemental phosphorus levels (0.07, 0.14, and 0.21 percent) were furnished by each of the other sources tested--def luorinated phosphate, dicalcium phosphate, and soft phosphate. Five equally spaced supplemental calcium

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56 levels, which varied with source and level of phosphorus, were provided from reagent grade calcium carbonate for each of the phosphorus additions. All the data were evaluated statistically, and, for monosodium phosphate, levels of 0.44, 0.47, 0.50, 0.53, 0.56, and 0.59 percent total calcium were suggested for total phosphorus levels of 0.30, 0.35, 0.40, 0.45, 0.50, and 0.55 percent, respectively. A subsequent experiment indicated that these proposed calcium levels were adequate. The total dietary calcium levels proposed as a standard curve for each of the remaining sources were as follows: 0.498, 0.590, and 0.685 percent for def luorinated phosphate; 0.47, 0.58, and 0.69 percent for dicalcium phosphate; and 0.56, 0.62, and 0.68 percent for soft phosphate with total phosphorus levels of 0.37, 0.44, and 0.51 percent, respectively. It should be emphasized that these curves are valid only when assay diets identical to those employed in the development of the curves are used. Results of studies concerning the influence of diet composition on soft phosphate utilization in broiler diets indicated that the addition of 2.5 percent fish meal to a diet containing soft phosphate as the sole supplementary phosphorus source could produce body weights and bone ash equal to control groups supplemented v;ith

PAGE 62

57 dicalcium phosphate. Various dietary coiribinations of phosphorus supplied from soft phosphate and calciuiri supplied as calcium carbonate were fed in an attempt to produce growth and bone ash equal to that produced v/ith coiTuTiercial feeds. No corribination of calcium and phosphorus levels alone produced the desired result; hov/ever , altering the diet composition with the inclusion of 2.5 percent fish meal in feeds containing 0.40 percent supplemental phosphorus and 0-0.20 percent supplemental calcium resulted in bone ash and body weight values not significantly different from those of controls. The response of the birds receiving fish meal diets was attributed ro the highly available supplemental phosphorus supplied by the fish mieal.

PAGE 63

REFERENCES Aminerman, C. B., H. W. Norton, and H. M. Scott, 1960. Rapid assay of inorganic phosphates for chicks. Poultry Sci. 39:245-250. Association of Official Agricultural Chemists, 1965. Official Methods of Analysis, 10th Ed., Washington, D. C. Couch, J. R., G. S. Fraps, and R. M. Sherwood, 1937. Vitamin D requirements of growing chicks as affected by the calcium content of the ration. Poultry Sci. 16:106-108. Creech, B. G., B. L. Reid, and J. R. Couch, 1956. Evaluation of dicalcium phosphate supplement as a source of phosphorus for chicks. 1. Comparison of dicalcium and tricalcium phosphate as a source of phosphorus in chick and poult rations. Poultry Sci. 35:654-658. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11:1-42. Ewing, W. R. , 1963. Poultry Nutrition, 5th Ed. The Ray Ewing Company, Pasadena, California. Fisher, H. , E. P. Singsen, and L. D. Matterson, 1953. The influence of feed efficiency on the phosphorus requirement for growth and bone calcification in the chick. Poultry Sci. 32:749-754. Fritz, J. C, and T. Roberts, 1966. Influence of levels of vitamin D and calcium on the utilization of phosphorus by the growing chick. Poultry Sci. 45:1085-1086. Gardiner, E. E., H. E. Parker, and C. W. Soft phosphate in chick rations, 38:721-727. Carrick, 1959 Poultry Sci. Gillis, M. B., L. C. Norris, and G. F. Heuser, 1949. The effect of phytin on the phosphorus requirement of the chick. Poultry Sci. 28:283-288. 58 -

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59 Gillis, M. B., L. C. Norris, and G. F. Heuser, 1954. Studies on the biological value of inorganic phosphates. J. Nutrition 52:115-125. Grau, C. R. , and P. A. Zweigart, 1953. Phosphatic clayas a phosphorus source for chicks. Poultry Sci. 32:500-503. McGinnis, J., L. C. Norris, and G. F. Heuser, 1944. Poor utilization of phosphorus in cereals and legumes by chicks for bone development. Poultry Sci. 23:157-159. McKnight, W. F., and A. B. Watts, 1966. Tlie effect of vitamin D3 on the utilization of phosphorus from various sources. Poultry Sci. 45:1104. Maddy, K. H., R. B. Grainger, W. A. Dudley, and F. Puchal, 1963. The application of linear programirjning to feed formulation. Feedstuffs 35 (5):28-30. Matterson, L. D., E. P. Singsen, and H. M. Scott, 1945. Rock phosphates as phosphorus supplements for the growing chick. Poultry Sci. 24:188-190. 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. National Research Council, 1966. Nutrient requirements of domestic animals. Nutrient requirements for poultry, Washington D. C. 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 (11):32. Nelson, T. S., and H. C. Walker, 1964. The biological evaluation of phosphorus compounds. A summary. Poultry Sci. 43:94-98. O'Rourke, W. R. , P. H. Phillips, and W. W. Cravens, 1952. The phosphorus requirements of grov;ing chickens as related to age. Poultry Sci. 31:962-966.

PAGE 65

60 Singsen, E. P., L. D. Matterson, and H. M. Scott, 1947. Phosphorus in poultry nutrition. III. The relationship between the source of vitamin D and the utilization of cereal phosphorus by poults. J. Nutrition 33:13-26. Singsen, E, P., and H. M. Scott, 1946. Phosphorus in poultry nutrition. II. Sodium acid phosphate, tri-calcium phosphate and bonemeal as sources of phosphorus for the growing chick. Poultry Sci. 25:302-303. Singsen, E. P., H. M. Scott, and L. D. Matterson, 1948. The phosphorus requirement of the chick. Storrs Agr. Exp. Sta. Bull. 260. Snedecor, G. W. , 1956. Statistical Methods, 5th Ed. The Iowa State College Press, Ames, Iowa. Summers, J. D., S. J. Slinger, W. F. Pepper, I. Motzok, and G. C. Ashton, 1959. Availability of phosphorus in soft phosphate and phosphoric acid and the effect of acidulation of soft phosphate. Poultry Sci. 38:1168-1179. Vandepopuliere, J. M. , C. B. Ammerman, and R. H. Harms, 1961. The relationship of calcium:phosphorus ratios to the utilization of plant and inorganic phosphorus by the chick. Poultry Sci. 40:951-957. Waldroup, P. W. , C. B. Ammerman, and R. H. Harms, 19 63. Calcium and phosphorus requirements of finishing broilers using phosphorus sources of low and high availability. Poultry Sci. 42:752-757. Waldroup, P. W. , C. B. Ammerman, and R. H. Harms, 1965a. A comparison of phosphorus assay techniques with chicks. Poultry Sci. 44:1086-1089. Waldroup, P. W. , C. B. Ar.m-.erman, and R. K. Harms, 1965b. The utilization of phosphorus from animal protein sources for chicks. Poultry Sci. 44:1302-1306. Waldroup, P. W. , C. B. Ammerman, and R. H. Harms, 1965c. Studies on the acidulation of soft phosphate. Poultry Sci. 44:1519-1523.

PAGE 66

BIOGRAPHICAL SKETCH Bobby Leon Damron was born November 6, 1941, at Ocala, Florida. He was graduated frcra Gainesville High School in June, 1959. In Xay, 1963, he received the degree of Bachelor of Science in Agriculture with high honors from the University of Florida. He also completed the requirements for teacher certification in Vocational Agriculture in May, 1963. He worked as an undergraduate assistant in the Department of Poultry Science from September, 19 61 to May, 1963. In September, 1963, he became a Research Assistant in the Poultry Science Departm.ent and received the Master of Science in Agriculture degree in December, 1964. In January, 1966, he accepted the position of Interim Research Associate and Instructor and continues in that capacity at the present tim.e . The author is a member of the Poultry Science Association, Alpha Zeta, Gamjua Sigma Delta, Alpha Tau Alpha, the Poultry Science Club, a past Vice-President of the Collegiate F.F.A. Chapter, and a past President of the Collegiate 4-H Club.

PAGE 68

This dissertation was prepared under the direction of the chairman of the candidate's supervisory committee and has been approved by all members of that committee. It was submitted to the Dean of the College of Agriculture and to the Graduate Council, and was approved as partial fulfillment of the requirements for the degree of Doctor of Philosophy. March, 1968 :^^^"t< liean. College of Agriculture Dean, Graduate School Supervisory Committee: Vt t ^ Chairman ^^ 7jdM..


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METS:behaviorSec VIEWS Options available to the user viewing this item
METS:behavior VIEW1 STRUCTID Default View
METS:mechanism Viewer JPEGs Procedure xlink:type simple xlink:title JPEG_Viewer()
VIEW2 Alternate
zoomable JPEG2000s JP2_Viewer()
VIEW3
Related image viewer shows thumbnails each Related_Image_Viewer()
INTERFACES Banners or interfaces which resource can appear under
INT1 Interface
UFDC_Interface_Loader