Phosphorus metabolism in the laying hen with respect to egg shell formation

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Phosphorus metabolism in the laying hen with respect to egg shell formation
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ix, 75 leaves : graphs ; 28 cm.
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Choi, Jin Ho, 1945-
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Chickens -- Feeding and feeds   ( lcsh )
Phosphorus in animal nutrition   ( lcsh )
Eggshells   ( lcsh )
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theses   ( marcgt )
non-fiction   ( marcgt )

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Thesis:
Thesis--University of Florida.
Bibliography:
Includes bibliographical references (leaves 68-74).
Statement of Responsibility:
by Jin Ho Choi.
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Typescript.
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Vita.

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University of Florida
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PHOSPHORUS METABOLISM IN THE LAYING
HEN WITH RESPECT TO EGG SHELL FORMATION
















By

JIN HO CHOI


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


1978














ACKNOWLEDGEMENTS


The author is grateful to Dr. R. H. Harms, Chairman of the

Department of Poultry Science and chairman of his supervisory committee,

for his guidance, assistance, and encouragement in the planning and

conducting of this research. The author would also like to thank the

other members of his committee, Drs. H. R. Wilson, C. R. Douglas, C. B.

Ammerman, and P. W. Chun, for suggestions and advice.

Special appreciation goes to Dr. R. D. Miles who has been a good

friend and of great help in accomplishing the research by discussing

the problems, arranging materials, and sharing the work.

Thanks are extended to Mr. D. P. Eberst and other members of the

farm crew, and to the laboratory technicians for their friendly coordi-

nation with the author for the entire period he was a graduate student

at the University of Florida.














TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS . .... ii

LIST OF TABLES . . ..... v

LIST OF FIGURES . . . vi

ABSTRACT. ..... . .... .vii

INTRODUCTION AND LITERATURE REVIEW . .. 1

CHAPTER I EFFECTS OF DIFFERENT DIETARY PHOSPHORUS LEVELS
ON EGG SPECIFIC GRAVITY AND BLOOD PHOSPHORUS
OF HENS . . 10

Experimental Procedure . .. 12

Experiment 1 . . 12
Experiment 2 . . 14

Results and Discussion . . 15

Experiment 1 . . .. 15
Experiment 2 . ... 18

Summary . . 22

CHAPTER II THE RESPONSE OF SERUM INORGANIC PHOSPHORUS
LEVEL IN LAYING HENS FED LOW LEVELS OF
DIETARY PHOSPHORUS . . 24

Experimental Procedure . .... .24
Results and Discussion . . 26
Summary. . .. .. 33

CHAPTER III BLOOD PHOSPHORUS LEVELS OF LAYING HENS AT
VARIOUS TIME INTERVALS AFTER DOSING WITH
PHOSPHORIC ACID . . ... .35

Experimental Procedure . .... 36
Results and Discussion . .. 38
Summary . . .. 51








TABLE OF CONTENTS (Continued)


Page


CHAPTER IV THE PHOSPHORUS EXCRETION PATTERN AND BALANCE
DURING ONE EGG CYCLE OF THE LAYING HEN FED A
PHOSPHORUS DEFICIENT DIET WITH OR WITHOUT A
SINGLE DOSE OF PHOSPHORIC ACID .

Experimental Procedure . .
Results and Discussion . .
Summary . . .

SUMMARY . . . .

REFERENCES . . .

BIOGRAPHICAL SKETCH . . .













LIST OF TABLES


Table Page

1 Composition of the basal diet . .... 13

2 Specific gravity of eggs from hens fed different
levels of phosphorus and calcium after three days
on a phosphorus deficient diet (Experiment 1) .. 16

3 Serum phosphorus level (mg./lOO ml.) of hens fed
different levels of phosphorus and calcium after
three days on a phosphorus deficient diet
(Experiment 1) . . ... 17

4 Serum phosphorus level of hens fed diets contain-
ing different phosphorus levels at different
times of the day (Experiment 2) . .. 19

5 Specific gravity of eggs from hens fed diets
containing different phosphorus levels at
different times of the day (Experiment 2) .. 21

6 Serum inorganic phosphorus level of laying hens
fed different levels of dietary phosphorus
(Experiment 3) . . 29

7 Composition of experimental diet . .... 37

8 Comparison of the strength of association of
the two different regression models of the serum
phosphorus . . .. ..... .46

9 Composition of experimental diet . ... 54

10 Recovery of chromic oxide with laying hens for a
three-day period . . ... .. 56

11 Dietary phosphorus intake and amount of phosphorus
excreted by the dosed and control hens ... 58

12 Percent excretion of phosphorus by control birds
(% of phosphorus excreted out of the amount
ingested) . . .. .59

13 Corrected excretion of phosphorus (total phosphorus
excreted minus unabsorbed dietary phosphorus) ...... 62

V













LIST OF FIGURES


Figure Page

1 Serum inorganic phosphorus level of laying hens
after different periods of feeding a phosphorus
deficient diet (Experiment 1 = days 0-3;
Experiment 2 = days 14-28) . .... 28

2 Graphical presentation of serum inorganic
phosphorus level of laying hens fed different
levels of dietary phosphorus (Experiment 3) ... 31

3 Serum inorganic phosphorus level of hens at
oviposition at various time intervals after
dosing with 100 mg. of phosphorus . ... 40

4 Logarithmic presentation of the elevated portion
of serum phosphorus of hens at various time
intervals after dosing with 100 mg. of phosphorus .... .42

5 Logarithmic model of regression on serum
phosphorus of hens at various time intervals
after dosing with 100 mg. of phosphorus ... 44

6 Serum phosphorus level of hens after specific
time intervals following dosing with 100 mg. of
phosphorus (each vertical line represents mean
S.E.) . . ... .. .. .48

7 Hypothetical curve for the change of serum
phosphorus level of the laying hen after dosing
with 100 mg. of phosphorus . . 50








Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy



PHOSPHORUS METABOLISM OF THE LAYING HEN
WITH RESPECT TO EGG SHELL FORMATION

By

Jin Ho Choi

August, 1978

Chairman: Robert H. Harms
Major Department: Animal Science

A series of experiments was conducted to investigate phosphorus

metabolism in the egg laying cycle of hens.

Different dietary levels of calcium and phosphorus, as limestone

and dicalcium phosphate, were fed to laying hens to observe effects on

egg specific gravity and blood inorganic phosphorus. Both high phos-

phorus and/or low calcium levels decreased the specific gravity of eggs

and increased the serum phosphorus level. Feeding hens different levels

of phosphorus at different times of the day gave an indication that the

hen's requirement for phosphorus varies during the egg laying cycle.

Birds receiving 1.40% phosphorus between 7:00 a.m. -9:30 a.m. and 0.30%

phosphorus between 9:30 a.m. -8:00 p.m. laid eggs with higher specific

gravity than hens fed 0.30% phosphorus between 7:00 a.m. -9:30 a.m. and

1.40% phosphorus between 9:30 a.m. -8:00 p.m. Serum phosphorus levels

of hens fed 1.40% phosphorus between 7:00 a.m.- 9:30 a.m. were lower

prior to oviposition and higher after oviposition than those of hens

fed 1.40% phosphorus between 9:30 a.m. -8:00 p.m.

The relationship between dietary phosphorus and serum phosphorus

levels was studied. There were no significant differences in serum








phosphorus levels immediately after oviposition of hens fed levels of

dietary phosphorus between 0.375% and 1.40%. Serum phosphorus level of

hens fed a diet containing 0.30% phosphorus, all from plant sources,

significantly decreased within 24 hours and remained at that level for

four weeks. Decreasing dietary phosphorus from 0.30 to 0.22% resulted

in a further decrease in serum phosphorus level. It is concluded that

the hen is capable of maintaining a normal blood phosphorus level over

a wide range of dietary phosphorus intake but decreases her serum phos-

phorus level immediately when fed suboptimal levels of phosphorus.

A single dose of 100 mg. of phosphorus in 10 ml. of a phosphoric

acid solution was orally administered to hens fed a 0.30% phosphorus

diet and serum phosphorus levels were measured. Serum phosphorus level

of undosed hens was 2.5 mg./l00 ml. at oviposition. After dosing serum

phosphorus level increased sharply and reached a peak between 30 and

60 minutes, after which the level decreased logarithmically. A hypo-

thetical curve was obtained which depicted the serum phosphorus level

of the hen fed a phosphorus deficient diet and dosed with 100 mg. of

phosphorus.

A balance trial was conducted to study the phosphorus excretion

pattern of laying hens in relation to the egg cycle. Hens fed a diet

containing 0.30% phosphorus excreted 24.3 mg. less phosphorus than was

ingested during the day, indicating that they had to withdraw approxi-

mately 100 mg. of phosphorus from their body reserves to produce an egg.

These hens excreted 63.1 27.0 mg. of endogenous phosphorus during the

24-hour period, most of which was excreted during the period between

12-24 hours after oviposition. The excretion pattern of endogenous

phosphorus was closely related to the egg laying cycle of the hen.


viii








Hens dosed with 100 mg. of phosphorus, as a phosphoric acid solu-

tion, excreted more phosphorus than undosed hens. Approximately 45 mg.

out of 100 mg. of phosphorus dosed were excreted during the 24-hour

period.













INTRODUCTION AND LITERATURE REVIEW


Numerous data have been published suggesting the dietary phosphorus

requirement of laying hens, ranging from as low as 0.30% (Salman and

McGinnis, 1968) to as high as 0.80% (Norris et al., 1933; Miller and

Bearse, 1934; Evans and Carver, 1942). Pepper et al. (1959) indicated

that a level of 0.38% phosphorus was adequate for maximum egg production

by laying hens, and that the total requirement could be supplied by

plant sources. Owings et al. (1977) reported that egg production of

hens fed a diet containing 0.30% phosphorus, all from plant sources,

decreased significantly within four weeks, while hens fed diets supple-

mented with 0.09% or more inorganic phosphorus continued normal

production. Lee et al. (1968) reported that laying hens required

between 0.58 and 0.81% phosphorus for normal egg production, and

recommended a level of 0.70% total phosphorus.

Hurwitz and Griminger (1962) showed by their balance method that

a phosphorus intake between 0.24 and 0.36 gram per day was required to

maintain phosphorus balance in the caged hen laying at a rate of 79%.

Edwards (1974) concluded that the phosphorus requirement for caged

layers did not exceed 0.45% of the diet when a corn-soy based diet was

fed.

There have been indications that the phosphorus requirement of

hens maintained in cages is higher than for hens maintained on the

floor (Harms et al., 1961; Marr et al., 1961; Singsen et al., 1962).







Singsen et al. (1962) found that the phosphorus requirement of laying

hens maintained on litter was 0.53% and that increasing the phosphorus

levels to 0.70% resulted in decreased egg production. Significantly

depressed egg production has been reported with both phosphorus levels

too low (O'Rourke et al., 1954) and too high (Harms et al., 1965) in

the diet.

It is obvious that the shell quality of eggs is influenced by the

level of dietary calcium. Several investigators have reported that

the shell quality of chicken eggs is significantly decreased when low

levels of dietary calcium are fed (Deobald et al., 1936; Evans et al.,

1944b; Hurwitz and Bar, 1966; Mehring, 1964; Nevalainen, 1969).

Peterson et al. (1960) reported significant improvements in shell

quality at dietary calcium levels above 2.25%. They found that a level

of 3.38% calcium resulted in a highly significant increase in shell

quality, as measured by specific gravity. They also reported further

improvements at levels of 4.50 and 5.25% calcium, but on a reduced

scale. Similar results have been reported by several other

investigators (Pepper et al., 1968; Reddy et al., 1968; Sullivan and

Kingan, 1962; Mueller, 1961; Harms et al., 1961).

Since the major component of the egg shell is calcium, it is

natural that most efforts to improve egg shell quality have been con-

centrated on manipulating the calcium level in the diet. Comparatively

less work has been done with phosphorus metabolism in this regard.

The existing data in the literature exhibit conflicting results as to

the effects of phosphorus on egg shell quality.

Several researchers have reported that there was no significant

influence of dietary phosphorus levels on egg shell quality as







measured by specific gravity of eggs (Pepper et al., 1959; Walter and

Aitken, 1962), by shell weight per unit surface area and breaking

strength (Hurwitz and Bornstein, 1963), or by shell thickness (Hinners

et al., 1963). Evans et al. (1944a) found significantly thicker egg

shells in one experiment with 0.80% phosphorus than with 0.60% phos-

phorus at a constant calcium level of 2.50%.

However, Taylor (1961) demonstrated that a substantial increase

in egg shell thickness occurred when hens were transferred from a high

phosphorus (0.80%) to a low phosphorus (0.10%) diet. Arscott et al.

(1962) reported that increasing the phosphorus level to 0.90% reduced

shell thickness and increased blood phosphorus. Mostert and Swart

(1968) found that increasing dietary phosphorus levels from 0.45 to

0.65% resulted in decreased egg shell quality. Gerry and Bird (1967)

reported similar results.

Bletner and McGhee (1975) reported that egg specific gravity gen-

erally improved as the phosphorus content of the diet decreased and

the calcium content increased. These authors indicated that caged

layers may be fed a diet containing as little as 0.35% phosphorus with

an improvement in egg specific gravity without seriuos detrimental

effects on other production parameters. Hamilton and Sibbald (1977)

also reported that the dietary phosphorus level had a significant

effect on specific gravity of eggs. However, they further stated that

the effect was not consistent, and the difference was so small as to

be of questionable practical importance. A reduction in egg shell

quality due to dietary phosphorus levels above that required for maxi-

mum production has been reported by several other workers (Kovac, 1973;







Damron et al., 1974; Shirley, 1974; Charles and Jensen, 1975; Summers

et al., 1976).

Taylor (1965) obtained eggs with better shell quality in terms of

shell weight (mg. per cm2 of surface area) with a low phosphorus

(0.46%) diet than with a high phosphorus (1.0%) diet. This author

suggested that the increase in shell thickness on the low phosphorus

diet was due to increased absorption and retention of dietary calcium.

Summers et al. (1976) also reported that the level of dietary phos-

phorus influenced calcium retention. However, the significance of the

increased retention of dietary calcium in explaining the thicker egg

shell obtained from lower dietary phosphorus is questionable. Taylor

(1965) also failed to confirm his suggestion experimentally in the

balance trials.

There may be some other metabolic factors related to the egg

shell formation of hens fed low levels of phosphorus. Bachra et al.

(1963) reported that in vitro precipitation of calcium carbonate was

prevented by the presence of phosphate ions.

It has been well established that egg shell membrane formation

takes place in the isthmus and shell formation occurs in the shell

gland (Richardson, 1935). This author also indicated that shell

deposition was initiated in the tubular region between the isthmus and

the shell gland. However, there are reports suggesting that initia-

tion of shell deposition may occur in the isthmus (Robinson and King,

1963; Taylor and Hertelendy, 1960; Schraer and Schraer, 1965).

Recently, Creger et al. (1976) reported, in their electron

microscopic study, the existence of the calcium rich mucopolysaccharide

material deposited over the shell membrane of the egg removed from the







isthmus 3.5 hours after oviposition of the previous egg. At 3.75 hours

post-oviposition, they found that the egg was in the shell gland and a

rapid secretion of calcium-rich material had begun. Stemberger et al.

(1977) confirmed that the initial stage of shell calcification occurs

in the isthmus.

The majority of normal chicken egg shell contain from 1.6 to 2.4

grams of calcium, although the actual amount depends on the size of

the egg and the thickness of the shell. The egg remains in the shell

gland for about 20 hours (Sturkie, 1976). During the first five hours,

calcium carbonate deposition increases gradually and then remains at a

rate of approximately 300 mg. per hour until about two hours before

oviposition. This rate of active shell deposition is equivalent to

approximately 120 mg. of calcium transferred from the shell gland to

the egg shell per hour. This amount of calcium must eventually be

supplied by the feed. In order to meet the increased demand for

calcium, a greater amount of calcium is absorbed from the intestines

during shell deposition while large quantities of endogenous phos-

phorus are extreted into the duodenum (Hurwitz and Bar, 1965).

Simkiss (1961) suggested that intestinal absorption alone cannot

supply all the calcium needed during active shell formation and bone

reserves have to be utilized. During the daylight hours when hens are

eating, most of the calcium necessary for shell formation can be sup-

plied by that absorbed from the intestines. However, during the night

when birds are not eating, which is the time when most shell calcifica-

tion occurs, the primary source of calcium must be bone resorption.

When laying hens are fed a diet low in calcium, their skeleton will







provide essentially all the calcium necessary for egg shell formation

(Hurwitz and Griminger, 1961; Weikel and Neuman, 1961).

It has been known that during the ten days before sexual maturity

female birds develop a new tissue in the bone marrow cavity of certain

bones which is called medullary bone. This new type of bone is most

easily observed in the femur and tibia. Medullary bone serves as a

readily available source of calcium for the egg shell and is exten-

sively resorbed during shell calcification (Candlish, 1971).

Zallone and Mueller (1969) and Simkiss (1967) stressed that

medullary bone is not simply a calcium reservoir to be replenished and

depleted cyclically, but that it is a continuously labile tissue.

Hurwitz (1965) reported that medullary bone is renewed 10-15 times as

fast as cortical bone. In an earlier study, Hurwitz (1964) administered

4Ca intravenously to laying hens near the time of ovulation and found

that radioactivity of bone segments increased to two hours after dosing

and then declined markedly, paralleling egg shell deposition.

Several investigators have reported that large amounts of phos-

phorus are excreted during shell calcification (Common, 1932; Tyler,

1940; Hurwitz and Bar, 1965). Tyler (1946) reported that an increase

in phosphorus excretion was accompanied by a decrease in calcium and

carbonate excretion when eggs were laid. While the calcium liberated

as a result of bone resorption is utilized for egg shell formation,

much of the phosphorus released simultaneously is excreted in the

urine (Fussell, 1960).

Early reports have indicated that the level of inorganic phos-

phorus in the blood plasma of hens increases during egg shell formation

(Feinberg et al., 1937; Peterson and Parrish, 1939; Paul and







Snetsinger, 1969). These findings suggest that the rate at which phos-

phorus is released from bones as a result of bone resorption is faster

than the rate at which phosphorus is excreted by the kidney.

Sloan (1976) reported that the serum calcium level of the hens

was significantly lower four hours before and after oviposition than

it was at the time of lay. Paul and Snetsinger (1969) reported that

the serum calcium level gradually decreased from one through 23 hours

after oviposition. However, Miller et al. (1977b) found that serum

calcium was relatively constant before and after oviposition.

Miller et al. (1977a) found a definite cyclic pattern of serum

phosphorus level in the hen, which was associated with the egg laying

cycle. According to their findings, serum phosphorus level of the

hen increases gradually during the shell formation and reaches a peak

approximately three hours prior to oviposition. Serum phosphorus then

decreases sharply to its lowest level of the day and remains at this

level until about 5-6 hours after oviposition.

Maynard and Loosli (1969) indicated that an excess of either

calcium or phosphorus interferes with the absorption of the other

mineral from the intestines. Thus, a certain ratio of calcium and

phosphorus in feed is desired for the best absorption of both elements.

For most animals this ratio ranges from 1:1 to 2:1 of calcium:

phosphorus, which is approximately the same as the ratio in the bones.

In the diet of laying hens the ratio of calcium to phosphorus is

much wider due to the large requirement of calcium for egg shell

formation. The National Research Council (1977) suggests that the diet

of laying hens should contain 3.25% calcium and 0.50% phosphorus.

This gives 6.5:1 ratio of calcium to phosphorus. It would be a logi-

cal assumption that the desirable ratio for the hen is much wider







during egg shell calcification and returns to the 1:1 to 2:1 ratio of

other animals while bone deposition is occurring.

It is a widely accepted concept that increasing the calcium level

in a phosphorus deficient diet will aggravate the phosphorus deficiency.

In growing birds the calcium:phosphorus ratio is more critical when a

low level of phosphorus is fed than when an adequate level of phos-

phorus is fed (Harms, 1971; Choi and Harms, 1977). A high calcium

level is detrimental when the diet contains a low level of phosphorus.

However, in laying hens high levels of calcium seem to spare phos-

phorus under certain conditions of phosphorus deficiency (O'Rourke

et al., 1954).

It is postulated that a high level of dietary calcium reduces the

necessity of calcium being withdrawn from the bones for the egg shell

calcification. Thus, less phosphorus will be released from the bones

and less will be excreted during egg shell formation. Consequently, a

high level of calcium has a sparing effect on phosphorus. Harms et al.

(1961) and Waldroup et al. (1974) published data indicating that with

chickens and turkeys, respectively, egg production was improved by

elevating the dietary calcium level at suboptimal levels of supple-

mental inorganic phosphorus.

When reviewing the literature, it is apparent that phosphorus

plays an important role in storing and mobilizing calcium in laying

hens. A possible manipulation of phosphorus intake of hens might be

of value in the efforts to improve egg shell quality. Determining the

blood inorganic phosphorus level is indicative of the hen's status of

phosphorus metabolism. A series of experiments was conducted to study

interrelationships between phosphorus intake and blood phosphorus, and







the excretion of phosphorus in hens with regard to egg shell

formation.













CHAPTER I
EFFECTS OF DIFFERENT DIETARY PHOSPHORUS LEVELS
ON EGG SPECIFIC GRAVITY AND BLOOD PHOSPHORUS OF HENS


There is a contradiction in the literature concerning the relation-

ship of the phosphorus level in the laying hen's diet and egg shell

quality. Evans et al. (1944a) reported that hens fed 0.80% phosphorus

laid eggs with significantly thicker shells than those fed 0.60%

phosphorus. On the other hand, numerous researchers found no differ-

ence in egg shell quality when hens were fed different levels of

phosphorus. Walter and Aitken (1962) and Singsen et al. (1962)

reported that dietary phosphorus level did not significantly influence

egg specific gravity. Hinners et al. (1963) failed to show any

significant changes in egg shell thickness by increasing phosphorus

levels from 0.705 to 1.018%. Miller and Bearse (1934) and Garlich

et al. (1975) reported similar results.

Several recent reports indicate that an excess of dietary phos-

phorus decreases egg shell quality. Hunt and Chancey (1970) found

that when total dietary phosphorus was reduced from 0.72 to 0.39% there

was no effect on shell quality of eggs laid by caged birds. However,

in the second experiment, egg shell quality was improved by decreasing

the dietary phosphorus level of hens on litter. Mostert and Swart

(1968) found that increasing levels of dietary phosphorus from 0.45 to

0.65% resulted in reduced egg shell quality. Taylor (1965) reported

that the shell thickness of eggs laid by hens receiving 0.46%







phosphorus was greater than the shell thickness of eggs from hens fed

1.00% phosphorus. Hamilton and Sibbald (1977) obtained eggs of higher

specific gravity by reducing dietary phosphorus level from 0.58 to

0.45% without any change in egg production. Arscott et al. (1962)

reported that increasing the phosphorus content in the diet from 0.60

to 0.90% resulted in an increase in blood phosphorus, which was accom-

panied by a reduced specific gravity of eggs.

Miller et al. (1977a) reported that the serum phosphorus level

reached a peak approximately three hours prior to oviposition, and then

the serum phosphorus level decreased sharply, reaching the lowest level

of the day. In the earlier reports reviewed by Sturkie (1976), it was

shown that calcium carbonate deposition to the egg shell was completed

about two hours before oviposition.

These findings indicated that the requirement of phosphorus of

the laying hen changes according to the physiological status of the

hen during a 24-hour period. Holcombe et al. (1976) showed that

hens were able to regulate their intake of phosphorus when a choice

of two levels of dietary phosphorus was offered. Hens offered a

choice between 0.19 and 2.43% phosphorus diets exhibited a noon-time

peak in preferential consumption of the high phosphorus diet, fol-

lowed by a marked decline in the afternoon and evening.

The current study was conducted to determine the effects of

different levels of dietary phosphorus on egg specific gravity and

serum phosphorus level of laying hens.







Experimental Procedure


Two experiments were conducted using individually caged Babcock

B-300 laying hens. Experimental diets were mixed using a corn-soybean

meal basal diet which was formulated to contain 0.09% calcium and

0.30% phosphorus (Table 1). The variable portion of the basal diet

was composed of ground limestone, dicalcium phosphate, and white

builder's sand in portions required to provide the desired levels of

calcium and phosphorus in each diet. All diets were isocaloric and

isonitrogenous.

Specific gravity of eggs was measured following the procedures of

Voisey and Hamilton (1976). After deproteinization, the blood serum

inorganic phosphorus level was determined by the method of Harris and

Popat (1954).


Experiment 1


Since body storage of phosphorus in laying hens might vary

greatly at the beginning of the experiment, 120 hens were fed a phos-

phorus deficient diet (0.30% P) for three days prior to the initiation

of the experiment to obtain a more homogeneous condition. Another 120

hens continuously fed a diet containing 0.75% phosphorus served as a

control. On the first day of the experiment the hens previously fed

the phosphorus deficient diet were divided into 12 groups of 10 birds

each. Each group was assigned to one of the four dietary treatments

with three replicate groups per treatment. Diets 1, 2, and 3 contained

0.30, 0.75, and 1.40% phosphorus, respectively, with a fixed calcium

level of 3.50%. Diet 1 was the same as the diet fed during the














Table 1. Composition of the basal diet


Ingredient Percent of diet


Yellow corn meal 54.10

Soybean meal (50%) 21.50

Alfalfa meal (20%) 2.00

Animal fat 4.50

Microingredients 0.50

DL-Methionine 0.03

Iodized salt 0.35
**
Variable 17.02


Crude protein 15.5%

Metabolizable energy 2805 kcal./kg.

Calcium 0.09%

Phosphorus 0.30%


Supplied per kg. of diet: 6000 I.U. vit. A;
2200 I.C.U. vit. D3; 2.2 mg. menadione dimethyl-
pyrimidinol bisulfite; 4.4 mg. riboflavin; 13.2 mg.
pantothenic acid; 39.6 mg. niacin; 499.4 mg.
choline chloride; 22 mcg. vit. BI2; 125 mg.
ethoxyquin; 50 mg. manganese; 50 mg. iron; 6 mg.
copper; 0.198 mg. cobalt; 1.1 mg. iodine; 35 mg.
zinc.
**
Variable = ground limestone, dicalcium phos-
phate, and white builder's sand in varying
proportions.







preliminary period. Diet 4 contained 0.75% phosphorus and 1.40%

calcium. Specific gravity was measured on all the eggs laid. On the

first and fourth days of the experimental period, between 10:30 a.m.

and noon, blood samples were collected from the hens that had laid

that morning.


Experiment 2


It was assumed that most hens would lay in the morning. If the

average time of laying were considered to be 9:00 a.m., egg shell

formation would have ended at 7:00 a.m. Thus, the daylight period of

a day was roughly divided into two time periods; Period I (7:00 a.m.-

9:30 a.m.) and Period II (9:30 a.m. -8:00 p.m.). Period I presumably

represented the time during which bone remineralization occurred and

the hen required phosphorus from the diet, and Period II represented a

portion of the time when shell calcification occurred and phosphorus

was being released from the bones.

One-hundred-eighty laying hens were divided into 18 groups, and

each group of 10 hens was assigned to one of the three treatments with

six replications per treatment. Birds in Treatment 1 were fed 0.75%

phosphorus during both time periods. Birds in Treatment 2 were fed

1.40% phosphorus in Period I and 0.30% phosphorus in Period II, while

birds in Treatment 3 were fed 0.30% phosphorus in Period I and 1.40%

phosphorus in Period II. All diets contained 3.50% calcium. Hens had

no access to feed between 8:00 p.m. and 7:00 a.m. Specific gravity

was measured on all the eggs laid for three weeks. On the 15th day,

hens that had laid on the previous day and were expected to lay (as

determined by cloacal palpation) that morning were bled between 6:00







a.m. and 7:00 a.m. when the blood phosphorus was assumed to be at its

highest level of the daily cycle (Miller et al., 1977a). Between 9:30

a.m. and 11:30 a.m. on the same day the hens that had laid both on the

previous day and that morning were also bled. The second bleeding was

assumed to be about the time when blood phosphorus was at its lowest

level during the day.


Results and Discussion

Experiment 1


Hens fed Diet 1 laid eggs with higher specific gravity than con-

trol birds (Table 2). The eggs laid by the hens that were fed 0.75%

phosphorus (Diet 2), which had been previously fed the phosphorus

deficient diet for three days, had about the same specific gravity as

those laid by the control birds. Decreasing the dietary calcium to

1.40% (Diet 4) decreased (P < 0.01) specific gravity of eggs. Feeding

a high phosphorus level (Diet 3) resulted in a slight decrease in egg

specific gravity.

It is assumed that the serum phosphorus levels shown in Table 3

represent the lowest value of phosphorus in a daily cycle because all

of the blood samples were collected within four hours after oviposition.

The serum phosphorus level of the control hens fed 0.75% phosphorus

and 3.50% calcium was 4.20 mg./l00 ml. Miller et al. (1977a) found

that the blood serum phosphorus level of hens fed the same levels of

calcium and phosphorus was 4.88 mg./100 ml. at the time of the lowest

level of the day. Serum phosphorus level decreased to 1.32 mg./100 ml.

after three days of feeding the phosphorus deficient diet. As the






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dietary phosphorus levels increased to 0.75 and 1.40% (Diets 2 and 3,

respectively), increased levels of serum phosphorus were obtained.

It should be noted that the first day values represent the values

of serum phosphorus of hens fed each experimental diet for only 2.5-4

hours before bleeding. The serum phosphorus level of hens fed 0.75%

dietary phosphorus (Diet 2) was significantly lower the first day than

that of control birds; however, they returned to a normal level by the

fourth day. Hens fed 1.40% phosphorus (Diet 3), however, had a serum

phosphorus level higher than that of control birds on the first day--

more accurately, after 2.5-4 hours on that diet.

A significant increase in level of serum phosphorus was observed

with hens fed Diet 4, which contained a lower level of calcium. The

data suggest that due to the limited supply of dietary calcium, more

calcium and phosphorus were released from the bones while egg shell

formation was occurring and less were deposited into the bones during

bone recalcification. Thus, a portion of the excess phosphorus might

have remained in the blood to increase the serum phosphorus level.

This assumption is strongly supported by the decreased specific gravity

of eggs from this group of hens, as shown in Table 2.


Experiment 2


Serum phosphorus level of hens on Treatment 2 fed 1.40% phos-

phorus for Period I (7:00 a.m. -9:30 a.m.) and 0.30% phosphorus for

Period II (9:30 a.m. -8:00 p.m.) was 5.25 mg./100 ml. prior to ovi-

position, which was the lowest value of the three treatments (Table

4). A value of 3.42 mg./l00 ml. for Treatment 2 after oviposition was
















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slightly higher than that of birds fed 0.30% phosphorus for Period I

and 1.40% phosphorus for Period II (Treatment 3).

Hens on Treatment 2 consumed 17.0 grams of feed containing 238 mg.

of phosphorus per hen during Period I. The serum phosphorus level of

3.42 mg./l00 ml. for these hens is much higher than the level of 1.89

mg./l00 ml. for the hens fed 0.30% phosphorus continuously (Table 3).

This implies that 238 mg. of phosphorus given between 7:00 a.m. -

9:30 a.m. nearly met the daily requirement of hens. However, this

amount of dietary phosphorus did not appear to be sufficient to raise

the serum phosphorus level up to that of the hens fed 0.75% phosphorus

for both periods (Treatment 1).

Compared with Treatment 2, the serum phosphorus level of hens fed

Treatment 3 was higher prior to oviposition (at the time of shell for-

mation) and lower after oviposition (at the time of bone deposition)

which was assumed to be detrimental to the egg shell quality. This

assumption was supported by the egg specific gravity data (Table 5)

in which eggs from hens of Treatment 3 had the lowest specific gravity.

Hens of Treatment 2 produced eggs of about the same specific gravity

as those of Treatment 1. In this study, the time of oviposition was

considered to be approximately 9:00 a.m. Feeding different levels of

phosphorus at different times of the day (Treatment 2) might have had

an improving effect on specific gravity of the eggs laid near 9:00 a.m.

However, a number of hens, in fact, laid much later than this time.

For those hens the time of shell formation would have been extended to

some time in the afternoon, and the time of feeding high phosphorus

did not coincide with the time of bone calcification. If the feeding

schedule of Treatment 2 had given a desirable effect on egg shell



















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quality of some birds, it should also have affected adversely the shell

quality of other hens. It is apparent that the feeding schedule of

Treatment 3 had an adverse effect on the majority of the hens.

It is concluded that approximately 250 mg. of phosphorus per hen

seems adequate in supplying the total daily requirement of phosphorus

for the laying hen, provided it is given exactly when the hen physio-

logically requires phosphorus.

From both experiments it was apparent that dietary phosphorus was

involved in and affected egg shell quality.


Summary

In the first of two experiments, laying hens were fed a diet con-

taining 0.30% phosphorus for a preliminary period of three days. At

the beginning of the experimental period, hens were fed 0.30, 0.75,

or 1.40% phosphorus with 3.50% calcium. Another group of hens was fed

0.75% phosphorus and 1.40% calcium. Specific gravity of the eggs and

blood serum inorganic phosphorus levels were determined and compared

with those from control hens continuously fed a diet containing 0.75%

phosphorus and 3.50% calcium. Both high phosphorus and/or low calcium

levels in diet decreased the specific gravity of eggs and increased

the serum phosphorus level.

In Experiment 2, hens were fed different levels of phosphorus at

different times of the day. Birds receiving 1.40% phosphorus between

7:00 a.m. -9:30 a.m. and 0.30% phosphorus between 9:30 a.m. -8:00 p.m.

(Treatment 2) laid eggs with higher specific gravity than hens fed

0.30% phosphorus between 7:00 a.m. -9:30 a.m. and 1.40% phosphorus

between 9:30 a.m. -8:00 p.m. (Treatment 3). Serum phosphorus levels







of hens of Treatment 2 were lower prior to oviposition when shell

calcification was occurring and higher after oviposition when bone

redeposition was occurring than those of hens of Treatment 3. There

was no further improvement in egg shell quality over that of hens fed

0.75% phosphorus for both time periods (7:00 a.m. -8:00 p.m.).

In both experiments it was apparent that the dietary phosphorus

level affected egg shell quality.














CHAPTER II
THE RESPONSE OF SERUM INORGANIC PHOSPHORUS LEVEL
IN LAYING HENS FED LOW LEVELS OF DIETARY PHOSPHORUS


Early reports (Feinberg et al., 1937; Peterson and Parrish, 1939;

Hunsaker, 1959; Paul and Snetsinger, 1969) have indicated that blood

plasma inorganic phosphorus of the laying hen fluctuates with the daily

cycle of egg production. The cyclic change in blood phosphorus fol-

lows the cycle for egg shell formation and bone redeposition (Miller

et al., 1977a). These results imply that it is important to consider

the production status of the hen in experiments where serum phosphorus

is measured.

In the previous study (Chapter I), the serum phosphorus level was

lower when hens were fed a phosphorus deficient diet. Hurwitz and

Griminger (1962) also observed that plasma inorganic phosphorus con-

centrations reflected dietary phosphorus intake. However, there is a

lack of data concerning the relationship between dietary phosphorus

and serum phosphorus levels of hens.

The present study was conducted to determine the effects of feed-

ing lower levels of dietary phosphorus on the serum phosphorus levels

of laying hens.


Experimental Procedure

Three experiments were conducted using Babcock B-300 laying hens.

All hens were fed a diet containing 0.75% phosphorus and 3.50%







calcium prior to the initiation of each experiment. In all experi-

ments, hens were housed in individual wire cages and were fed the

experimental diets ad libitum. Experimental diets were mixed using

the same corn-soybean meal basal diet as described in Chapter I

(Table 1) by substituting the necessary amounts of limestone, dicalcium

phosphate, and white builder's sand in place of the variable portion

to obtain the desired levels of calcium and phosphorus. The basal

diet contained 0.30% phosphorus, all from plant sources. The diet

containing 0.22% phosphorus was obtained by replacing the yellow corn

in the basal diet by an equal amount of degerminated corn meal. Each

experimental diet was formulated to contain 3.50% calcium.

Blood samples were collected by heart puncture immediately after

oviposition only from hens that had laid for two consecutive days in

each experiment. No birds were bled twice in this study.

In Experiment 1, 120 hens were fed a diet containing 0.30% phos-

phorus with no supplemental inorganic phosphorus. Serum phosphorus

level of the hens was determined zero, one, two, and three days after

feeding the deficient diet.

In Experiment 2, 210 hens were fed the same diet as in Experiment

1 for an overall period of four weeks to observe the effect of longer

periods of feeding a phosphorus deficient diet on the serum phosphorus

levels. Serum phosphorus level was measured after two, three, and

four weeks of feeding the experimental diet.

In Experiment 3, seven different levels of dietary phosphorus

were fed. A total of 280 laying hens was divided into 14 groups of 20

hens each. Each group of birds was assigned to one of seven different

dietary phosphorus levels (0.22, 0.30, 0.375, 0.45, 0.60, 0.75, and







1.40%) with two replications per treatment. Blood samples were col-

lected on days 7 and 28.


Results and Discussion

The serum inorganic phosphorus level immediately after oviposi-

tion of hens previously fed the diet containing 0.75% phosphorus was

4.12 mg./100 ml. in Experiment 1 (Figure 1) and 4.72 mg./100 ml. when

fed 0.75% phosphorus in Experiment 3 (Table 6). Feeding the diet con-

taining 0.30% phosphorus resulted in a significant decrease in the

serum phosphorus level of hens within one day (P < 0.01). Combining

the results of Experiments 1 and 2 (Figure 1), it was apparent that

the decreased serum phosphorus remained at a relatively constant level

between 2 and 3 mg./l00 ml. for the entire four weeks of experimental

period. The rapid decline in serum phosphorus after only one day of

consuming the low phosphorus diet may be an indication of the sensi-

tivity of the hen in responding to the dietary level of phosphorus.

The serumphosphorus levels of hens fed 0.30 and 0.22% phosphorus

were significantly lower (P < 0.01) than those of the hens fed higher

levels of phosphorus in the diet (Table 6). Hens fed 0.22% phosphorus

exhibited a lower (P < 0.05) serum phosphorus level than those fed

0.30% phosphorus. No significant differences were found among birds

fed 0.375 to 1.40% phosphorus. A graphical presentation of the same

data shows the trend more clearly (Figure 2). These results imply

that a dietary level of 0.375% phosphorus or 0.175% available phos-

phorus (0.075% supplemental inorganic phosphorus) may be sufficient to

maintain the normal serum phosphorus level of the laying hen.

























(1)
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The phosphorus requirement of the laying hen seems to be quite

low. Cotterill et al. (1977) reported that phosphorus content of an

egg (yolk + albumen) was 126.1 mg. Romanoff and Romanoff (1949) indi-

cated that there were 110 mg. of phosphorus in the yolk, 20 mg. in the

shell, and 6 mg. in the albumen of an egg. This totals 136 mg. of

phosphorus in the whole egg. Harms and Miles (1977) calculated that

only 0.09% phosphorus would be necessary in the feed of a hen laying

300 eggs per year and eating 100 pounds of feed to supply the neces-

sary phosphorus for that contained in the egg. Owings et al. (1977)

reported that egg production of hens fed 0.30% phosphorus including

no inorganic phosphorus decreased markedly within four weeks, while

hens fed a diet with 0.09% or more supplemental inorganic phosphorus

continued egg production at satisfactory rates. These earlier findings

appear to be in accordance with the results of the current study in

relation to serum phosphorus.

It should be noted that hens were capable of maintaining the

normal serum phosphorus level over a wide range of dietary phosphorus,

probably by means of excretion of the excess phosphorus. The serum

phosphorus level of hens fed 0.30% phosphorus decreased immediately

and remained relatively constant for four weeks (Figure 1), instead of

decreasing gradually. However, it is highly probable that these hens

had enough body storage of phosphorus to support egg production for a

while on the deficient diet. According to the data of Owings et al.

(1977), hens fed 0.30% phosphorus maintained normal egg production for

the first three weeks and then decreased in the fourth week.

It is concluded that the hen has a very efficient mechanism for
maintaining her normal level of blood phosphorus and sensitively







responds to the decreased dietary level of phosphorus below the

optimum level by decreasing her blood phosphorus level. Decreased

levels of serum phosphorus when a suboptimal level of phosphorus is

fed would be attributed to the homeostatic effort of the hen to con-

serve body stores of phosphorus, regardless of the progress of the

deficiency of phosphorus.


Summary

Three experiments were conducted to determine the effects of

feeding low levels of dietary phosphorus on the serum inorganic phos-

phorus level of laying hens. A phosphorus deficient diet containing

0.30% total phosphorus, all of plant origin, was fed to hens for

three days in Experiment 1 and four weeks in Experiment 2. In Experi-

ment 3, seven different levels of dietary phosphorus were fed. Blood

serum inorganic phosphorus level was determined at different durations

of feeding the experimental diets. All the blood samples were col-

lected immediately following oviposition from the hens that had laid

both on the day before and the day of bleeding in each experiment.

The serum phosphorus level of hens previously fed 0.75% phos-

phorus was 4.12 mg./100 ml. in Experiment 1 and 4.72 mg./l00 ml. when

fed 0.75% phosphorus in Experiment 3. Serum phosphorus level of hens

fed the diet containing 0.30% phosphorus decreased to between 2 and 3

mg./l00 ml. within 24 hours and remained at this level for four weeks.

There were no significant differences in serum phosphorus levels of

hens fed dietary levels of phosphorus between 0.375 and 1.40%.

Decreasing dietary phosphorus from 0.30 to 0.22% further decreased the

serum phosphorus level. It is concluded that the hen is capable of







maintaining a normal blood phosphorus level over a wide range of

dietary phosphorus but decreases her serum phosphorus level immediately

when fed suboptimal levels of phosphorus.













CHAPTER III
BLOOD PHOSPHORUS LEVELS OF LAYING HENS AT VARIOUS
TIME INTERVALS AFTER DOSING WITH PHOSPHORIC ACID


A 24 hour period in the life of the laying hen with regard to her

laying cycle can be divided into two different phases; egg shell forma-

tion and bone redeposition. The change in blood inorganic phosphorus

level during the laying cycle appears to have a definite pattern cor-

responding to the two different phases of phosphorus metabolism. In

earlier studies, it has been shown that the level of plasma inorganic

phosphorus increases during shell formation (Feinberg et al., 1937;

Peterson and Parrish, 1939; Hunsaker, 1959). Furthermore, a large

quantity of phosphorus is excreted during shell formation (Tyler, 1946;

Fussell, 1960). When egg shell calcification ceases and bone deposi-

tion begins, serum phosphorus drops dramatically to its lowest level

of the daily cycle and remains at that level for about five hours

(Miller et al., 1977a). During this stage of the cycle, the phos-

phorus in the feed must be utilized to supply the necessary phosphorus

for bone deposition.

It would appear to be desirable to feed phosphorus at various

levels throughout the day depending upon the hen's need for phosphorus.

That is, a high level would be fed during bone deposition while a low

level would be fed during shell formation.

Miller (1976) indicated that oral dosing of hens with 100 mg. of

phosphorus per hen per day in a phosphoric acid solution while they







were fed a diet containing 0.29% phosphorus significantly increased

egg production, hatchability of fertile eggs, and serum calcium and

phosphorus levels over the undosed hens. In Chapter II, it was shown

that the serum phosphorus of laying hens was between 2 and 3 mg./l00

ml. at oviposition when they were fed a diet containing 0.30% phos-

phorus, all of plant origin.

The purpose of the current study was to observe the pattern of

change in serum phosphorus of hens fed a low phosphorus diet after

dosing with phosphorus.


Experimental Procedure

Two experiments were conducted using Babcock B-300 laying hens.

The experimental diet was a corn-soybean meal-type diet including no

supplemental inorganic phosphorus (Table 7). The phosphorus and cal-

cium levels in this diet were 0.30% and 3.50%, respectively.

In Experiment 1, 240 laying hens were fed the experimental diet

for six days. At 8:30 a.m. on the seventh day, all hens that had laid

an egg on the previous day were dosed with 10 ml. of a phosphoric acid

solution which contained 100 mg. phosphorus. A 25 ml. syringe with a

piece of Tygon tubing attached was used to inject the solution into

the crop. Blood samples were collected by heart puncture immediately

after oviposition from the hens laying an egg following dosing.

Twenty hens were dosed with 10 ml. of distilled water instead of the

phosphoric acid solution and served as controls.

In experiment 2, 300 hens were fed the experimental diet for five

days. The method of dosing was the same as in Experiment 1, except

that hens were dosed immediately following oviposition between 6:30 a.m.













Table 7. Composition of experimental diet


Ingredient Percent of diet


Yellow corn meal 54.10

Soybean meal (50%) 21.50

Alfalfa meal (20%) 2.00

Animal fat 4.50

Microingredients* 0.50

DL-Methionine 0.03

Iodized salt 0.35

Limestone 8.97

Sand 8.05


Crude protein 15.5%

Metabolizable energy 2805 kcal./kg.

Calcium 3.50%

Phosphorus 0.30%


Supplied per kg. of diet: 6000 I.U. vit.A;
2200 I.C.U. vit. D3; 2.2 mg. menadione dimethyl-
pyrimidinol bisulfite; 4.4 mg. riboflavin; 13.2mg.
pantothenic acid; 39.6 mg. niacin; 499.4 mg.
choline chloride; 22 mcg. vit. B12; 125 mg.
ethoxyquin; 50 mg. manganese; 50 mg. iron; 6 mg.
copper; 0.198 mg. cobalt; 1.1 mg. iodine; 35 mg.
zinc.







and 10:00 a.m. on the sixth day of feeding the experimental diet.

Blood samples were collected at specific time intervals after dosing

(10, 20, 30, 60, 120, and 180 minutes). Some birds were bled at the

time of oviposition without dosing to serve as a control (0 time).


Results and Discussion


In Experiment 1, the serum phosphorus level of control hens was

2.5 mg./100 ml. (Figure 3). The serum phosphorus levels of hens that

had been dosed with 100 mg. of phosphorus were higher than that of

control birds and decreased with time. A linear regression equation

was calculated to be y = 7.8099 0.9430x, where x is time after dos-

ing in hours. However, it is probable that the pattern of decrease

may be logarithmic. Theoretically, with the prolonged time, the serum

phosphorus of the dosed hens would approach 2.5 mg./l0 ml, which is

the serum phosphorus level of the control hens. The actual observed

value of serum phosphorus minus 2.5 is assumed to be the elevated

portion of the serum phosphorus due to dosing. The pattern of change

in this elevated portion should be logarithmic. Therefore, in Figure

4 the common logarithm of the elevated portion of serum phosphorus was

taken as the vertical axis and a straight regression line was obtained.

The half-life of the elevated portion of serum phosphorus was calcu-

lated to be about 100 minutes. This regression line was transferred

onto the original linear scale to obtain the exponential curve seen in

Figure 5.

Two different models of regression were obtained on the same

distribution. The model that fits better with the original distribu-

tion is of interest at this point. Sum of squares for error of the

































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logarithmic model was calculated between the observed values and the

corresponding predicted values on the curve in Figure 5. Although

both models were highly significant (P < 0.01), the logarithmic model

showed a higher F-value, less standard deviation, and a higher R2

value (Table 8). It is concluded that the logarithmic model is more

closely associated with the original distribution than the linear

model.

In Experiment 2, the serum phosphorus level of the undosed con-

trol birds was again 2.5 mg./100 ml. (Figure 6). After dosing the

level of phosphorus in the serum began to increase sharply and reached

a maximum level after 30 to 60 minutes. A linear regression line was

obtained for the first increasing portion of the graph. The decreas-

ing pattern after the peak did not show a logarithmic mode. The possi-

ble explanation of this is that even if each individual hen had a peak

in serum phosphorus and a logarithmic decrease, the time of the peak

would be expected to vary dmong birds, therefore, the curve for the

mean of many birds would be rounded and might not show a logarithmic

pattern. The curve in Figure 6 was obtained for only three hours

after dosing. It seems reasonable that if it had been observed for an

extended time, the curve might have eventually become logarithmic,

assuming that the serum phosphorus level of undosed hens remains con-

stant over this length of time.

Combining the results of Experiments 1 and 2, a hypothetical curve

was obtained (Figure 7) which shows the change in serum phosphorus of

an average hen dosed with 100 mg. phosphorus. The first increasing

straight line was taken from Experiment 2 and the logarithmic decrease

came from Experiment 1. The intercept of these two curves represents
























Table 8. Comparison of the strength of association of the two
different regression models of the serum phosphorus


Linear model Logarithmic model
Parameter
Y = 7.8099 0.9430X Y = 2.5 + 7.0960 (1.5124)-X


MSE 1.02 0.83

F 34.72 48.69

Prob. F < 0.01 < 0.01

SD 1.01 0.91
**0.57 0.65
R 0.57 0.65


Standard deviation.

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Total SS
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the hypothetical peak of serum phosphorus. The peak appeared at about

37 minutes after dosing and the serum phosphorus level at this peak

was calculated to be 8.0 mg./l0 ml. However, it is not known whether

the hen would have a sharp peak of serum phosphorus (as shown in

Figure 7) or a plateau at a certain level.


Summary

Two experiments were conducted to study the pattern of change in

serum inorganic phosphorus level in laying hens fed a diet containing

0.30% total phosphorus after dosing with a known amount of phosphorus.

Serum phosphorus level of undosed hens was 2.5 mg./100 ml. at

oviposition. After dosing with 100 mg. of phosphorus in 10 ml. of a

phosphoric acid solution, serum phosphorus level increased sharply and

reached a peak between 30 and 60 minutes, after which the level de-

creased logarithmically. A hypothetical curve was obtained which

depicted the serum phosphorus level of the hen fed a phosphorus de-

ficient diet and dosed with 100 mg. of phosphorus. The peak appeared

at approximately 37 minutes after dosing. The serum phosphorus level

at the hypothetical peak was 8.0 mg./l00 ml. The half-life of the

elevated portion of serum phosphorus due to the dosing was about 100

minutes.















CHAPTER IV
THE PHOSPHORUS.EXCRETION PATTERN AND BALANCE DURING
ONE EGG CYCLE OF THE LAYING HEN FED A PHOSPHORUS DEFICIENT
DIET WITH OR WITHOUT A SINGLE DOSE OF PHOSPHORIC ACID

Daily calcium and phosphorus balance studies (Common, 1932; Tyler,

1940; Common and Hale, 1941) showed that the amount of phosphorus ex-

creted in the droppings of laying hens varied greatly from day to day,

and excretion of large amounts of phosphorus was associated with egg

shell formation. This was particularly true when a low level of di-

etary calcium was fed (Common, 1932). Tyler (1946) observed that an

increase in phosphorus excretion was accompanied by a decline in cal-

cium and carbonate excretion when eggs were laid. While the calcium

liberated as a result of bone resorption is utilized for egg shell

formation, the phosphorus released simultaneously is largely excreted

in the urine (Fussell, 1960).

In the previous study (Chapter III), a known amount of phosphorus

was dosed orally into the crop of hens fed a phosphorus deficient diet

(0.30% phosphorus). The results showed that the serum inorganic phos-

phorus level of the deficient hens dosed with 100 mg. of phosphorus in

a phosphoric acid solution increased and reached a peak within one

hour and then decreased logarithmically for 5-6 hours thereafter.

The purpose of the current study was to determine the pattern of

excretion and balance of phosphorus in hens, either dosed with phos-

phorus or undosed, at various intervals following oviposition.








Experimental Procedure


Sixty laying hens of Babcock B-300 strain were fed a diet (Table

9) containing 0.30% phosphorus, all of plant origin, and 0.20% chromic

oxide (Cr203) for a preliminary period of three days. This diet was

formulated to contain 3.50% calcium. In the morning of the fourth day,

10 hens that had laid both on the third day and the morning of the

fourth day were selected. At oviposition, each of the 10 hens was

dosed, directly into the crop, with 10 ml. of a phosphoric acid solu-

tion containing 100 mg. of phosphorus or 10 ml. of distilled water

in an alternating sequence. The five hens dosed with distilled water

served as a control. Immediately after dosing, each hen was placed

in an individual cage and excreta were collected quantitatively at 4,

8, 12, and 24 hours thereafter. All of the 10 hens laid an egg on

the following day.

Three hens were used for a chromic oxide recovery test. Both

feed consumption and amount of excreta were measured for three days.

Percent recovery of chromic oxide was calculated by the following

formula:


Percent Amount of chromic oxide excreted (mg.)
recovery Amount of chromic oxide intake (mg.)


All hens were fed ad libitum during the entire experimental

period. The excreta samples were dried in an oven at 90 C. for 24

hours to determine the amount of dry matter and then were exposed to

the air for 48 hours to equilibrate with atmospheric moisture.

Samples were ground and placed in air-tight sample jars and were later

analyzed for moisture, chromic oxide, and phosphorus contents.













Table 9. Composition of experimental diet


Ingredient Percent of diet


Yellow corn meal

Soybean meal (50%)

Alfalfa meal (20%)

Animal fat

Limestone

Microingredients

DL-Methionine

Iodized salt

Chromic oxide

Sand


Crude protein

Metabolizable energy 2805

Calcium

Phosphorus


54.10

21.50

2.00

4.50

8.97

0.50

0.03

0.35

0.20

7.85


15.5%

kcal./kg.

3.50%

0.30%


Supplied per kg. of diet: 6000 I.U. vit. A;
2200 I.C.U. vit. 03; 2.2 mg. menadione dimethyl-
pyrimidinol bisulfite; 4.4 mg. riboflavin; 13.2 mg.
pantothenic acid; 39.6 mg. niacin; 499.4 mg.
choline chloride; 22 mcg. vit. B12; 125 mg.
ethoxyquin; 50 mg. manganese; 50 mg. iron; 6 mg.
copper; 0.198 mg. cobalt; 1.1 mg. iodine; 35 mg.
zinc.







Phosphorus in the feed and excreta samples was analyzed by the

method of Harris and Popat (1954) after ashing the samples for four

hours at 6000 C. and dissolving in a diluted HC1 solution.

Determination of chromic oxide content was made by atomic absorption

spectrophotometry as recommended by Williams et al. (1962).


Results and Discussion

The average percent recovery of chromic oxide was 73.97 0.56%

(Table 10). Vohra and Kratzer (1967) stated that 87.55 2.76% of

chromic oxide was excreted from a single dose of this compound in 48

hours with 5-week-old chickens. Dansky and Hill (1952), in a study

with growing chicks, recovered 95% of the chromic oxide fed. The per-

cent recovery of this material with laying hens in this experiment was

apparently lower than those with growing chicks as reported in the

literature. The data in this experiment revealed that values were

highly reproducible and there was little variation among replications.

The amount of feed consumed corresponding to the amount of

excreta collected for each collection period was calculated by the

following formula:


% Cr in
excreta Amount of
Amount of feed (dry matter excreta dry
consumed basis) x matter (g.) x 100
% Cr in feed 73.97
(dry matter basis)


Since percent recovery of chromic oxide was 73.97%, a correction
100
factor, 73.97, was multiplied to obtain the true amount of feed

consumed. The feed ingested at a specific time will be excreted over






















Table 10.


Recovery of chromic oxide with laying hens for a three-day
period


Hn numr Chromium Chromium Percent
intake (mg.) excreted (mg.) recovery


1 375 280 74.67

2 441 328 74.38

3 356 259 72.86

73.97 0.56

Mean standard error of the mean.







a dispersed period of time and vice versa. Therefore, the above

formula gives the amount of feed ingested that accounts for the amount

of feces collected for the specific time, regardless of the time the

feed was eaten.

Amounts of phosphorus intake and amounts excreted by the dosed

and control hens are shown in Table 11. The control birds that were

fed the 0.30% phosphorus diet and dosed with distilled water received

an average of 302.6 mg. of phosphorus and excreted 278.3 mg. for a

24-hour period. The difference, 24.3 mg. per hen, was retained and

might be assumed to have been deposited in the egg. Romanoff and

Romanoff (1949) indicated that an egg contains a total of 136 mg.

phosphorus. In this context, the hens in this experiment had to with-

draw more than 100 mg. of phosphorus from their body reserves during a

24-hour period to produce an egg.

Hens dosed with 100 mg. of phosphorus excreted more phosphorus

for all the collection periods than the control birds. The amount of

phosphorus excretion should be heavily influenced by phosphorus intake.

Since there was very little difference in the amounts of phosphorus

intake from feed between dosed and control birds, the difference in

the amounts of the phosphorus excreted was assumed to be the excreted

portion of the dosed phosphorus, either unabsorbed or once absorbed

and excreted. Of the 100 mg. of dosed phosphorus, 44.5 mg. were

excreted during the 24-hour period (Table 11).

Percent excretion of phosphorus out of the dietary phosphorus

ingested for the overall 24-hour period was 93.3 10.9% for the con-

trol hens (Table 12). Percent excretion of phosphorus increased with

time following oviposition, and during the 12-24 hour period the hens














Table 11. Dietary phosphorus intake and amount of phosphorus excreted
by the dosed and control hens


Collection period
(hours after oviposition)
Treatment Total

0-4 4-8 8-12 12-24



Dietary phosphorus intake (mg.)

Dosed 60.7 49.8 40.3 150.0 300.7

Control 64.8 48.5 38.4 151.0 302.6

Difference
(dosed control) -4.1 1.3 1.8 -1.0 -1.9


Phosphorus excreted (mg.)
**
Dosed 50.2 45.7 39.3 187.6 322.8

Control 45.4 39.1 36.1 157.7 278.3

Difference
(dosed control) 4.8 6.6 3.2 29.9 44.5


Includes only dietary phosphorus but not phosphorus furnished by
the dosing.
**
Hens received 300.7 mg. of phosphorus from the diet and 100 mg.
from the dosing.























Table 12.


Percent excretion of phosphorus by
control birds (% of phosphorus excreted
out of the amount ingested)


Collection period
(hours after oviposition)


0-4 71.1 3.1

4-8 82.4 4.8

8-12 93.2 8.2

12-24 104.0 19.0

Overall (0-24) 93.3 10.9







excreted more phosphorus than was ingested. As the egg shell calcifi-

cation for the next egg started, phosphorus should have been released

from the bones and blood inorganic phosphorus should have been in-

creased, accompanied by an increased excretion of endogenous phosphorus.

The egg remains in the shell gland for approximately 20 hours. During

the first five hours, calcium carbonate deposition increases gradually

and then remains at a constant rate until two hours before oviposition

(Sturkie, 1976). During the period of 12-24 hours after oviposition

of the previous egg, shell calcification should have occurred most

actively and more endogenous phosphorus was excreted for this period

than the earlier periods.

During the period of 0-4 hours after oviposition, 71.1% of the

amount of phosphorus ingested was excreted. This period was assumed

to be the time when the egg shell deposition is negligible and active

redeposition of bone minerals was occurring, thus serum phosphorus was

at its lowest level of the day (Miller et al., 1977a). Hens were

physiologically in maximum need of phosphorus during this period of

time and, therefore, the endogenous excretion of phosphorus should

have been at its minimum rate of the day. Thus, 71.1% might be con-

sidered as the appraent indigestibility digestibilityy = 28.9%) of the

phosphorus in the diet, all of which was from plant origin.

It is also noted that standard error of the mean of percent phos-

phorus excretion increased as time passed (Table 12). This could be

explained as follows: during the 0-4 hour period, all the hens were

at the stage of bone recalcification and required phosphorus. They

were all in the same condition and there was little variation among

hens. As time passed, the time at which they started egg shell







formation and the time of active shell deposition may have varied

among hens. There were greater variations in the physiological condi-

tion of the birds in the later periods. The amount of endogenous

phosphorus during shell calcification would also have varied among the

hens, depending upon the body reserve of phosphorus in the individual

hens. The more actively shell formation occurred, the more variation

in the endogenous excretion of phosphorus.

Almost all of the phosphorus excreted by the control hens during

the 0-4 hour period could be assumed to be the unabsorbed portion of

the dietary phosphorus. The amount of phosphorus excreted during the

later periods consists of the unabsorbed dietary phosphorus plus

endogenous excretion of phosphorus. Taking 71.1% as the indigesti-

bility of the dietary phosphorus, the amount of unabsorbed dietary

phosphorus was estimated and subtracted from the total amount of phos-

phorus excreted during each period (Table 13). The corrected amounts

of phosphorus excreted from the control birds could be interpreted as

endogenous phosphorus. The assumed endogenous excretion of phosphorus

increased as time passed after oviposition, as previously discussed.

The control birds excreted 63.1 mg. of endogenous phosphorus for the

24-hour period. The endogenous excretion from the control birds for

the 0-4 hour period shows a negative value, which was due to the round-

ing errors in the process of calculations. On the basis of the assump-

tions this must be considered as zero. Here again, the difference

between dosed and control birds is considered to be the excreted por-

tion of the dosed phosphorus. Of the 100 mg. of phosphorus dosed,

45.8 mg. were excreted during the entire 24-hour period, which agrees

with the 44.5 mg. calculated from the total excretion (Table 11). It
is concluded that approximately 45 mg. of the 100 mg. of dosed


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phosphorus were excreted and 55 mg. were retained by the hen during

the 24-hour period.

It should also be noted that excretion of the dosed phosphorus

decreased during the first 12 hours and only 1.9 mg. was excreted for

the 4-hour period between 8-12 hours after oviposition (0.47 mg. per

hour). This could be interpreted as indicating that almost all of the

dosed phosphorus was either absorbed or excreted by the end of the

first 12-hour period. The dosed phosphorus excreted for the later 12-

hour period (30.6 mg.) had been absorbed and then excreted. Only a

total of 15.2 mg. of the dosed phosphorus was excreted for the first

12 hours, which suggested that at least 84.8 mg. of the dosed phos-

phorus had been absorbed for this period. It is reasonable to assume

that some portions of the dosed phosphorus excreted within 12 hours

had once been absorbed. It is very likely that the true absorption

rate of the dosed phosphorus was much faster than that calculated

above, but no further estimations could be made in this study.


Summary


A balance trial was conducted to study the phosphorus excretion

pattern of laying hens in relation to the egg cycle. Excreta were

collected quantitatively at 4, 8, 12, and 24 hours after oviposition.

The amount of feed consumed corresponding to the excreta collected at

specific time intervals was calculated using chromic oxide as a marker.

The percent recovery of chromic oxide with laying hens was 73.97

0.56%.

The apparent digestibility of the dietary phosphorus, all of

plant origin, was estimated to be 28.9 3.1%. Laying hens fed a diet







containing 0.30% phosphorus excreted 24.3 mg. less phosphorus than in-

take during the day, indicating that hens had to withdraw approximately

100 mg. of phosphorus from their body to produce an egg. These hens

excreted 63.1 27.0 mg. of endogenous phosphorus during the 24-hour

period, most of which was excreted during the period between 12-24

hours after oviposition of the previous egg. The excretion pattern of

phosphorus was closely related to the egg laying cycle of the hen.

Hens dosed with 100 mg. of phosphorus, as a phosphoric acid solu-

tion, excreted more phosphorus than the undosed control birds.

Approximately 45 mg. of 100 mg. of phosphorus dosed were excreted dur-

inq the 24-hour period. It was apparent that at least 84.8 mg. of the

dosed phosphorus had been absorbed within 12 hours and a part of it

was excreted during the later period. Data indicated that the true

absorption rate of the dosed phosphorus might have been much faster.













SUMMARY


A series of experiments was conducted to investigate phosphorus

metabolism in laying hens with respect to the egg laying cycle. It

was apparent that phosphorus is closely involved in egg shell

formation.

A high level of dietary phosphorus resulted in a slight decrease

of egg specific gravity, which also resulted from a low level of

dietary calcium. Feeding a deficient level of phosphorus decreased

the serum phosphorus level of hens.

Data from the second experiment in Chapter I indicate that the

laying hen has different requirements of phosphorus at different times

of the day. Birds receiving 1.40% phosphorus between 7:00 a.m. -9:30

a.m. and 0.30% phosphorus between 9:30 a.m. -8:00 p.m. laid eggs with

higher specific gravity than hens fed 0.30% phosphorus between 7:00

a.m. -9:30 a.m. and 1.40% phosphorus between 9:30 a.m. -8:00 p.m.

Although there was no further improvement in egg shell quality over

that from hens fed 0.75% phosphorus for both time periods, the effects

of different schedules of feeding phosphorus were reflected in the

data of serum phosphorus levels of hens.

In Chapter II, it was shown that serum inorganic phosphorus level

of hens fed a deficient level (0.30%, all of plant sources) of phos-

phorus decreased significantly within 24 hours and remained at that

level for four weeks. There were no significant differences in serum








phosphorus levels of hens fed levels of dietary phosphorus between

0.375 and 1.40%. Decreasing dietary phosphorus level from 0.30 to

0.22% further decreased the serum phosphorus level. Results indicated

that 0.075% of supplemental inorganic phosphorus was sufficient for

the hen to maintain a normal serum phosphorus level. It is concluded

that the hen is capable of maintaining a normal blood phosphorus level

over a wide range of dietary phosphorus levels but decreases her serum

phosphorus level immediately when fed suboptimal levels of phosphorus.

Dosing 100 mg. of phosphorus, in a phosphoric acid solution, to

hens fed a 0.30% phosphorus diet increased serum phosphorus level

(Chapter III). Serum phosphorus level of dosed hens reached a peak

between 30 and 60 minutes after dosing and decreased logarithmically

thereafter. A hypothetical curve was obtained.

A balance trial was conducted using chromic oxide as a marker to

study the phosphorus excretion pattern of laying hens in relation to

the egg cycle. Hens fed a diet containing 0.30% phosphorus, all

of plant origin, excreted 24.3 mg. less phosphorus than intake, indi-

cating that hens had to withdraw approximately 100 mg. of phosphorus

from their body to produce an egg. Endogenous excretion of phosphorus

increased as time passed after oviposition. These hens excreted 63.1

+ 27.0 mg. of total endogenous phosphorus during the 24-hour period,

most of which was excreted during the period between 12-24 hours after

oviposition, which was the time of shell calcification for the next

egg. Hens dosed with 100 mg. of phosphorus, as a phosphoric acid

solution, excreted more phosphorus than undosed hens. Approximately

45 mg. of 100 mg. of dosed phosphorus were excreted during the 24-hour

period.







The data strongly support the hypothesis that laying hens require

a high level of dietary phosphorus during bone deposition and very

little phosphorus during egg shell formation.













REFERENCES


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Bachra, B. N., 0. R. Trautz, and S. L. Simon, 1963. Precipitation of
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Bletner, J. K., and G. C. McGhee, 1975. The effect of phosphorus on
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Candlish, J. K., 1971. The formation of mineral and organic matrix
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Charles, 0. W., and L. Jensen, 1975. Effect of phosphorus levels on
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Common, R. H., and R. W. Hale, 1941. Observations on the mineral
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Deobald, H. J., E. J. Lease, E. B. Hart, and J. G. Halpin, 1936.
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Edwards, H. M., Jr., 1974. Phosphorus levels need reappraising.
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Evans, R. J., J. S. Carver, and A. W. Brant, 1944a. The influence of
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Feinberg, J. G., J. S. Huqhes, and H. M. Scott, 1937. Fluctuations of
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Fussell, M. H., 1960. Studies on calcium and phosphorus metabolism in
the hen. Ph.D. dissertation, University of Cambridge, Cambridge
CB2 3DX, England.

Garlich, J. D., R. L. James, and J. B. Ward, 1975. Effects of short
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Gerry, R. W., and F. H. Bird, 1967. The performance of Red x Rock sex
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Hamilton, R. M. G., and J. R. Sibbald, 1977. The effects of dietary
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Harms, R. H., 1971. How important are calcium:phosphorus ratios for
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Harms, R. H., B. L. Damron, and P. W. Waldroup, 1965. Influence of
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BIOGRAPHICAL SKETCH


The author, Jin Ho Choi, was born in Korea on June 13, 1945. He

was graduated from Chung Dong High School in 1963. From May, 1966,

to April, 1969, he served in the Korean Army. His B.S.A. in Animal

Science was obtained in February, 1971, from the Seoul National

University.

He was employed by Purina Korea, Inc. from May, 1971, to

December, 1972. In February, 1974, he obtained the M.S. degree in

Animal Nutrition from the Seoul National University and continued his

studies there for another year as a Ph.D. candidate.

Since March, 1975, he has been a Ph.D. candidate in Poultry

Nutrition at the University of Florida.

He is a member of the Korean Society of Animal Science and the

Korean Scientists and Engineers Association in America, and served as

secretary of the Florida Chapter of the latter association. He is a

member of the Poultry Science Club at the University of Florida and

received the 1977 "Poultry Digest" scholarship.







I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




Robert H. Harms, Chairman
Professor of Poultry Science



I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




Henry R. 0ilson
Professor of Poultry Science



I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




Carroll R. Douglas
Associate Professor of Poultry Science



I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.



S <-,- ,, .17 ) ^ ^L^'^-? .r
Clarence B. Ammerman
Professor of Animal Science







I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




PAuil W. Chun
professor of Biochemistry and Molecular
Biology




This dissertation was submitted to the Graduate Faculty of the College
of Agriculture and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.

August, 1978




Dean, College of Agricultd~ e
I


Dean, Graduate School









































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