Assessing the phosphorus status of growing beef heifers

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Title:
Assessing the phosphorus status of growing beef heifers
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xiv, 314 leaves : ill. ; 28 cm.
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Williams, Scot N., 1958-
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Heifers   ( lcsh )
Phosphorus in animal nutrition   ( lcsh )
Phosphorus -- Physiological effect   ( lcsh )
Beef cattle -- Growth   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1987.
Bibliography:
Includes bibliographical references.
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by Scot N. Williams.
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Typescript.
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Vita.

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University of Florida
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oclc - 19712572
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Full Text

















ASSESSING THE PHOSPHORUS STATUS
OF GROWING BEEF HEIFERS







BY







SCOT N. WILLIAMS


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


UNIVERSITY OF FLORIDA


1987

















Dedicated to

My Mom
with all my love from Pitsy Popper's best friend

My Gramp
If I can be half the person you are,
I will have been a great success

My fam ly (old and new)

My friends

And the fond memory of my friend
Thomas Andrew Shay

















ACKNOWLEDGMENTS


My sincere appreciation is expressed to Dr. Lee R. McDowell,

chairman of the supervisory committee, for directing my academic

program and research. His kindness, humor and friendship have made a

long project an enjoyable one.

Acknowledgments are also due to Dr. Clarence B. Ammerman, Dr.

Joseph H. Conrad, Dr. Alvin C. Warnick and Dr. Rachel M. Shireman for

their valuable time and advice as members of the supervisory

committee.

Special recognition is due to Dr. P.V. Rao for his numerous

hours of assistance in statistical analysis and Dr. James F. Hengtes

for providing animals and advice for carrying out this project.

Special thanks are offered to Dr. Donnie Ray Campbell for his

assistance throughout with SAS.

Sincere thanks are expressed to Dr. Rogene Tesar and Dr. Frank

Pipers for assistance with photon absorption and ultrasound

techniques. Special thanks go to Dr. Gary Miller for his expertise

with mechanical analysis of bone in this study.

No words can express the author's appreciation to Dr. Larry A.

Lawrence for his unending encouragement and advice in completing all

bone aspects of this project.

Special thanks are extended to the Agency for International

Development, Washington, DC, and Occidental Chemical Corporation,

Chicago, IL, for funding this project.











The author would also like to express his sincere thanks to

Nancy Wilkinson and Pam Miles for their tremendous assistance in the

field and laboratory portions of this project. Extra special thanks

go to Nancy Wilkinson whose help through every phase of this project

was paramount to its completion. This author will always be indebted

to her for all the hours of warmth, laughter, friendship and help.

The author has been blessed with many friendships during his

stay at Florida. Nancy, Pam, Debbie and Wendy; this author lacks the

words to describe his feelings for you. These friendships will last

a lifetime.

Larry (Larunga), Donnie (it's a duck), Mark (the cool man),

Charlie (Guy) and Jeff (Cappy); times together and feelings for you

cannot be expressed herein. It has been this author's pleasure and

privilege to be considered your friend--one should not be so lucky.

Special thanks and thoughts are extended to friends who gave

both socially and professionally and helped this author round out his

education: Bill, Martha, David, Mariano, Siobhan, Francis, Greg,

Badinga, Joe, Yousif, Donna (Earl Rayette), Jose, Carlos, Mike,

Cindy, Rick and Gary.

Also, special thanks go to Cindy Zimmerman and Ken Sourbeer for

their proficiency in helping prepare this manuscript.

Special thanks go to my wife, Vanessa, for support and love

throughout the preparation of this manuscript. This author is

blessed with a lifetime of your companionship.

Mom, Dad, Gram, Gramp, Bob, Cathy, Donna, Michael, Lee, Rick,

Jean, Pete, Jane, Georgia, Dana, Tom, Buddy, Penny, Bernie and all

the kids--thanks for being.





















TABLE OF CONTENTS


ACKNOWLEDGMENTS..............


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


LIST OF FIGURES..............


ABSTRACT......................


CHAPTERS


ONE INTRODUCTION.....................


TWO LITERATURE REVIEW................


Introduction ....................
Metabolic Functions..............
Metabolism ... ....................
Deficiency.........................
Requirements....................
Assessment of Phosphorus Status..


THREE MATERIALS AND METHODS............


Page


.........iii


.........vii


...........x


........xiii


.................... ..... 1


.................. ....... 4


............... .......... 4
........................ .5
.........................7
........................ 13
........................ 15
........... ............ 34


........................ 72


General Protocol...................................... 72
Noninvasive and Mechanical Analysis of Bone..............83
Chemical Analyses........................................89
Statistical Analyses.....................................93


FOUR INFLUENCE OF DIETARY PHOSPHORUS LEVEL ON GROWTH AND
REPRODUCTION OF GROWING BEEF HEIFERS.....................97


Introduction............................................. 97
Materials and Methods....................................98
Results and Discussion.................................103
Conclusions.............................................131


FIVE CHEMICAL AND PHYSICAL PROPERTIES OF BONE IN BEEF
HEIFERS AS INDICATORS OF PHOSPHORUS STATUS.............. 133


Introduction............................................133
Materials and Methods...................................134


..............


..............


..............


..............


..............


..............


..............


..............












SIX


SEVEN


Results and Discussion ................................ 141
Conclusions............................................ 172

NONINVASIVE TECHNIQUES TO ASSESS PHOSPHORUS STATUS
AND PREDICT BONE MINERAL CONTENT AND BONE STRENGTH
IN THE BOVINE...................................... ..... 175

Introduction............................................ 175
Materials and Methods....................................176
Results and Discussion..................................182
Conclusions............................................... 202

SUMMARY AND CONCLUSIONS................................209


APPENDICES

A TABLES........................... .............

B RAW EXPERIMENTAL DATA.........................

LITERATURE CITED.........................................
BIOGRAPHICAL SKETCH.................... .................


.219

.233

.293
.314


1111
















LIST OF TABLES


Table Page

1 Influence of type of diet and dietary mineral level
(mg-kg-1) on P content (mg-L1) of rumen fluid
(RF).................................... .................. 20

2 Composition of basal diets fed during phosphorus
depletion and supplementation phases.......................73

3 Bleeding dates and blood constituents sampled during
depletion phase........................................... 76

4 Samples obtained at slaughter of experimental animals......78

5 Sample collection during P supplementation phase...........80

6 Mineral analyses performed on samples......................90

7 Influence of dietary phosphorus level on total gain,
average daily gain, total dry matter intake and feed:
gain of heifers during 210 d ad libitum feeding period
of phosphorus supplementation phase....................... 106

8 Blood serum, plasma and whole blood P and Ca
concentrations, hemoglobin and packed cell volume--
phosphorus depletion phase.................................110

9 Influence of dietary phosphorus level and sampling
period on blood serum, plasma and whole blood P and
Ca concentrations, hemoglobin and packed cell volume--
phosphorus supplementation phase...........................112

10 Influence of dietary P level on selected tissues,
ruminal contents, abomasal contents and rumen fluid
phosphorus concentration..................................118

11 Influence of dietary phosphorus level and sampling
period on fecal, hair and saliva phosphorus
concentration............................................ 120

12 Influence of dietary phosphorus level and time of
palpation on right, left and total ovary volume............123











13 Influence of dietary phosphorus level on calf birth
weight, calf weight change and calf average daily
gain from parturition to 3 wk postpartum (21 d
period ................................................. 125

14 Influence of dietary phosphorus level on cow blood P
and Ca concentrations, hemoglobin, packed cell volume
and colostrum/milk P and Ca concentrations at
parturition and 3 wk postpartum...........................127

15 Influence of dietary phosphorus level on calf blood P
and Ca concentrations, hemoglobin and packed cell
volume at day of birth and 3 wk postpartum.................128

16 Influence of dietary phosphorus level and sampling
period on various 12th rib cortical bone parameters........142

17 Influence of dietary phosphorus level on the breaking
load (kg) of the 12th rib.......... ........................ 146

18 Influence of dietary phosphorus level on various
coccygeal vertebrae bone parameters........................148

19 Influence of dietary phosphorus level on various whole
third metacarpal (McIII) physical and chemical
properties.............................................. .152

20 Influence of dietary phosphorus level on various
mechanical and chemical properties and area indices
of third metacarpals......................... ............. 156

21 Correlation coefficients between various third
metacarpal (McIII) diaphysis mechanical, chemical
and physical properties (measured at point of failure)....157

22 Influence of dietary phosphorus level on bone mineral
content (BMC), photon absorptiometry, radiographic
photometry and ultrasound measurements at two locations
along the bovine third metacarpal (McIII) diaphysis.......183

23 Correlation coefficients between noninvasive
techniques, bone mineral content (BMC), ash and
various cross-sectional area indices from two 2 cm
third metacarpal (McIII) cross-sections....................189

24 Correlation coefficients between noninvasive
techniques versus mechanical and physical properties
of third metacarpals (McIII) (measured at point of
failure) ................................................. 193


viii











25 Correlation coefficients between ultrasound versus bone
mineral content and mechanical properties of third
metacarpals... ........................................... 203

26 Influence of dietary phosphorus level on average
gain-animal1 (kg-14d ) during the 210d
ad libitum feeding period of the phosphorus
supplementation phase.................................... 219

27 Dry matter feed intake (DMFI) during the 210d
ad libitum feeding period of the phosphorus
supplementation phase....................................220

28 Blood serum mineral concentrations--phosphorus
depletion phase......................................... 221

29 Blood plasma mineral concentrations--phosphorus
depletion phase......................................... 222

30 Whole blood mineral concentrations--phosphorus
depletion phase...........................................223

31 Influence of dietary phosphorus level and sampling
period on blood serum mineral concentrations--
phosphorus supplementation phase..........................224

32 Influence of dietary phosphorus level and sampling
period on blood plasma mineral concentrations--
phosphorus supplementation phase...........................225

33 Influence of dietary phosphorus level and sampling
period on whole blood mineral concentrations--
phosphorus supplementation phase..........................226

34 Influence of dietary phosphorus level on concentration
of liver, kidney, heart and muscle........................227

35 Influence of dietary phosphorus level on ruminal and
abomasal grab sample mineral concentrations..............228

36 Influence of dietary phosphorus level and sampling
period on fecal mineral concentration.....................229

37 Influence of dietary phosphorus level and sampling
period on hair Zn concentration...........................230

38 Influence of dietary phosphorus level and time of
sampling on milk mineral concentrations...................231

39 Influence of dietary phosphorus level on liver biopsy
mineral concentrations....................................232
















LIST OF FIGURES


Figure Page

1 Hormonal regulation of phosphorus homeostasis...............12

2 Diagram illustrating 3-point flexure test of bovine
third metacarpal: a) pretest and b) during testing
with force applied to the posterior surface................55

3 Force-deformation curve resulting from a flexure test......56

4 Diagram illustrating the use of ultrasound on bovine
third metacarpals.......................... ..... .......... 86

5 Cross-section of bovine third metacarpal (McIII)
illustrating the anterioposterior (AP) outer (B) and
inner (b) diameters and the lateromedial (LM) outer
(D) and inner (d) diameters used in calculating moment
of inertia of an ellipse (MIE), circular area index
(CAI) and elliptical area index (EAI)......................88

6 Average body weights of heifers in the unsupplemented
P group (.12%P, DMB) and P supplemented group (.20%P,
DMB) over the 210 d ad libitum feeding period during
the P supplementation phase of the experiment.............105

7 Regression analysis of liveweight gain (Y) and
experimental period (X) over the 210 d ad libitum
feeding period in the P supplementation phase by
treatment group .......................................... 108

8 Typical dual photon absorptiometry (DPA) scans from
animals fed either .12% P (DMB) on the left side (a)
or .20% P (DMB) on the right side (b).....................153

9 Relationship between breaking load (Y) and bone
mineral content (BMC) (X) of third metacarpal
break section............................................ 160

10 Relationship between bone mineral content (Y) and
radiographic area index (XRI) (X)--third metacarpal
break section ............................................ 161











11 Relationship between bone mineral content (Y) and
circular area index (CAI) (X)--third metacarpal
break section............................................ 162

12 Relationship between bone mineral content (Y) and
elliptical area index (EAI) (X)--third metacarpal
break section .......................................... 163

13 Relationship between bone mineral content (Y) and
planimeter area index (PAI) (X)--third metacarpal
break section ............................................ 164

14 Relationship between breaking strength (Y) and percent
bone ash (PBA) (X)--third metacarpal break section........169

15 Relationship between breaking strength (Y) and bone
mineral content (BMC) (X)--third metacarpal break
section .................................................. 170

16 Relationship between bone mineral content (Y) and
radiographic area index (XRI) (X) from two 2 cm
third metacarpal cross-sections...........................185

17 Relationship between bone mineral content (Y) and
circular area index (CAI) (X) from two 2 cm
third metacarpal cross-sections...........................186

18 Relationship between bone mineral content (Y) and
elliptical area index (EAI) (X) from two 2 cm
third metacarpal cross-sections...........................187

19 Relationship between bone mineral content (Y) and
planimeter area index (PAI) (X) from two 2 cm
third metacarpal cross-sections...........................188


20 Relationship between bone mineral content (Y) from two
2 cm third metacarpal cross-sections and radiographic
bone aluminum equivalents (RBAE) (X).....................


.191


21 Relationship between bone mineral content (Y) from two
2 cm third metacarpal cross-sections and photon
absorption bone mineral content (PBMC) (X)................195


22 Relationship between bone mineral content (Y) from two
2 cm third metacarpal cross-sections and photon
absorption bone mineral density (PBMD) (X).............


..196


23 Relationship between bone mineral content (Y) from two
2 cm third metacarpal cross-sections and mediolateral
ultrasonic velocity (US) (X)...............................199











24 Relationship between bone mineral content (Y) and
radiographic area index ultrasonic velocity (XRAY-US)
(X)..................................................... 204

25 Relationship between bone mineral content (Y) and
circular area index ultrasonic velocity (CIR-US)
(X) ..................................................... 205

26 Relationship between bone mineral content (Y) and
elliptical area index ultrasonic velocity (ELL-US)
(X) ..................................................... 206

27 Relationship between bone mineral content (Y) and
planimeter area index ultrasonic velocity (PLAN-US)
(X)...................................................... 207
















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

ASSESSING THE PHOSPHORUS STATUS
OF GROWING BEEF HEIFERS

BY

SCOT N. WILLIAMS

December, 1987


Chairman: L.R. McDowell
Major Department: Animal Science


Effects of dietary phosphorus (P) on growth and reproduction and

the suitability of various criteria for assessing P status of growing

beef heifers were investigated. Relationships between bone mineral

content (BMC), cortical area and mechanical properties of bovine

third metacarpals (McIII) were defined for this class and age of

animal. Additionally, photon absorption, radiographic photometry and

ultrasound were tested to determine their ability to predict BMC and

bone strength properties of bovine McIII.

The duration of the experiment ranged from 525 to 772 d

depending on slaughter date of individual animals. Initially,

fifteen weaned Angus heifers were fed ad libitum a low P diet (.10%,

dry basis) for 270 d. Heifers were then randomly allotted into two

groups (7 animals-group-1) and received either .12 (LP) or .20% P

(HP) (dry basis). Heifers were fed ad libitum for the initial 210 d


xiii










of this period. Heifers were bred naturally via 120 d exposure to a

bull. Common experimental endpoint was day 245 of the treatment

phase for nonpregnant and 3 wk postpartum for pregnant heifers.

Total gain and average daily gain were greater (p<.01) in HP

than LP heifers during the treatment phase (257 vs 205 kg and 1.22 vs

.98 kgd-1, respectively). Onset of puberty and conception rate were

not affected by dietary P level. Five and six heifers in the HP and

LP groups, respectively, were found to be pregnant.

Serum, plasma and whole blood P concentrations varied throughout

the experiment. Twelfth rib bone biopsies provided the clearest

indication of P status of all samples collected in vivo. Rib bone

density (g-cm-3) (p<.05) and P concentration (mg.cm-3) (p<.01) were

greater in HP heifers.

Breaking load (p<.05), breaking strength (p<.05) and Young's

modulus (p<.10) of McIII were all greater in HP heifers. Bone

mineral content was greatest (p<.01) in each of two 2 cm McIII cross-

sections derived from HP heifers.

Correlations between photon absorption, radiographic photometry

and ultrasound with BMC of the two 2 cm McIII cross-sections removed

at the midpoint and 3 cm proximal to the midpoint were .908

(p<.0001), .967 (p<.0001) and .565 (p<.0001), respectively.

Improvements in correlations between BMC and ultrasonic velocity were

obtained by using cortical area estimates for distance traveled by

generated sound pulses.
















CHAPTER ONE
INTRODUCTION


A broad spectrum of economic, cultural and technological factors

have caused dramatic changes within many segments of the livestock

industry over the past 25 yr, especially with respect to practices

implemented to increase and maximize production in livestock

operations.

Recent advances in such areas as genetic research for increasing

economic traits of breeding herds, crossbreeding systems to optimize

average genetic merit and utilize hybrid vigor, improved reproductive

efficiency (as evidenced by estrus synchronization, pregnancy

diagnosis, superovulation and embryo transfer), rumen regulating

drugs, implants, feed additives and genetic engineering to produce

more useful microflora are just a few of the areas in which

scientists have sought methods to increase ruminant productivity.

However, the utility of these advances is doubtful unless the

basic nutritional needs of the animal can be met for a given level of

production. Therefore, knowledge concerning specific nutrient

requirements is a necessity if both economical and optimal beef

production are to be realized, as undernutrition is commonly accepted

to be the most important limitation to ruminant livestock worldwide.

Phosphorus (P), the second most abundant mineral element in the

animal body, has long been recognized for its practical importance in






2



ruminant nutrition worldwide. Phosphorus is intimately associated

with the function of all animal tissues by virtue of its role in the

processes of energy exchange. Thus, it may be expected that any

limitation to P supply will be reflected in impairment of body

function.

Phosphorus deficiency is primarily a condition of grazing

ruminants with deficiencies under field conditions having been

recognized for many years. Phosphorus has been reported to be the

most prevalent mineral deficiency of cattle and sheep in the United

States (Mitchell, 1947) and in the world (Underwood, 1981; McDowell,

1985). Because P deficiency is of such great importance, it has been

intensively studied for the past 60 yr. Much of the early work on P

requirements is still applicable and crucial to current understanding

of P nutrition.

Since these early studies, a considerable volume of research has

been published on the P requirements of beef cattle. However, there

is no clear consensus on the P needs of ruminants. Also, it is

obvious that determination of P requirements is only as good as the

sensitivity of the response criteria measured. After years of

research, reliable and objective methods to determine P status in

ruminants are still sought.

The objectives of this study were to compare the influence of

two dietary levels (.12 and .20%, dry basis) of P fed to beef heifers

in a long-term study on 1) growth; 2) reproductive performance; 3)

delineating P status as gauged by various parameters; 4) various






3



chemical, physical and structural properties of selected bones; and

5) the use of several commercially available noninvasive bone density

techniques to predict bone mineral content and bone strength in third

metacarpals of these animals.
















CHAPTER TWO
LITERATURE REVIEW


Introduction

Phosphorus (P), a member of Group V-B of the periodic system

along with antimony (Sb), arsenic (As), bismuth (Bi) and nitrogen

(N), plays a more varied role in the chemistry of living organisms

than any other element (NRC, 1980). Although widely distributed in

nature, P is not found in a free elemental state, but rather in the

form of phosphates. The earth's crust contains approximately .12% P,

whereas the P content of whole cattle is estimated to be .7% (Miller,

1979).

Phosphorus is of great applied importance in ruminant nutrition

throughout the world and is probably the nutrient most frequently

given as a supplement to grazing ruminants (Cohen, 1980).

Deficiencies under practical conditions have been recognized for some

time (Henderson and Weakley, 1930; Eckles et al., 1932; Becker et

al., 1933). Theiler and Green (1932) cited the observations of

LaVaillant in his African travels from 1780 to 1785, which they

believed to be the earliest known chronicle of P deficiency signs in

cattle.

Mitchell (1947) stated that P deficiency was the most prevalent

mineral deficiency of cattle and sheep in the world. Underwood

(1966) again pointed out the importance of P in ruminant nutrition






5



when he stated that ". there is no doubt that phosphorus

deficiency is the most widespread and economically important of all

the mineral disabilities affecting grazing livestock" (p. 34). Blake

et al. (1976) concluded that research to better define the dietary P

requirement is needed as a failure to provide adequate P to cattle

would be deleterious in terms of not only impaired growth and

performance, but costly in that providing excess supplemental P to

ruminants is substantial and much greater than for any other mineral

element. These authors further point out that an overuse of 20%, for

example, would mean an unnecessary expenditure of $80 million

annually in the United States.



Metabolic Functions

Phosphorus has more known functions than any other mineral

element in the body. Extensive examination of all functions is

impractical in the present review; however, it seems appropriate in

this study to summarize the major functions.

Phosphorus along with Ca plays an intregal role in bone

formation and maintenance as well as formation of teeth.

Approximately 83% of the P in young cattle is in the skeleton

(Duncan, 1958), present as bone mineral (i.e., calcium phosphate

[Ca3(PO4)2] and hydroxyapatite [Ca10(PO4)6(OH)2]) (Harrison, 1984).

This percentage is higher in adult than young animals. The large

concentration of P within the skeleton provides an excellent

storehouse of the mineral which can be mobilized during times of

metabolic demand (Guyton, 1981).










Nonskeletal P (approximately 14% of the total body P in cattle)

is found in the cell and extracellular fluids as organic phosphoric

acid esters, phosphoproteins, phospholipids and the inorganic

-1 -2
phospate ions, H2P04 and HP042. Although the concentrations of

these ions are usually expressed in terms of P content, there is no

elemental P in the body as such. Accordingly, some prefer to use the

term phosphate in nutrition and metabolism.

The nonskeletal P has many crucial roles in the ruminant

animal. It is a key element in energy metabolism as high energy

phosphate bonds, such as in adenosine triphosphate (ATP) and

adenosine diphosphate (ADP) are essential for providing energy to

drive most metabolic reactions.

Nucleic acids, important in genetic and metabolic components,

consist of phosphoric acid, a pentose sugar and purine and/or

pyrimidine bases. Examples of these include deoxyribonucleic acid

(DNA) and ribonucleic acid (RNA).

Phospholipids are an important constituent of the cell wall and

function in the transfer of materials across the cell wall as well as

being important in the transport of lipid materials in blood and

lymph. Phospholipids consist of fatty acids, phosphate and a

nitrogenous base, such as choline, ethanolamine or serine (Lehninger,

1982).

Phosphorus is an essential component of buffer systems in the

blood and other body fluids, including the rumen. Phosphorus is also

essential for the proper functioning of rumen microbes, especially










those involved in cellulose digestion (Anderson et al., 1956).

Because of its many roles within the ruminant animal body, P is

important in virtually every biochemical aspect of the ruminant

animal.



Metabolism

Absorption of P from the rumen and omasum is negligible and it

has not been clearly shown that P is absorbed from the abomasum

(Cohen, 1980). Intestinal absorption of phosphate occurs only in the

small intestine, mainly in the proximal (duodenal) end, and both a

sodium-requiring active transport process and diffusion are involved

(Harrison and Harrison, 1963; Walling, 1977). The precise role of

the phosphohydrolases present in the intestinal brush border in the

active transport process is not known (Harrison, 1984). Passive

diffusion is greatest at high luminal concentrations of P; thus in a

P deficiency, absorption will have a greater energy demand on the

animal.

Absorption of P is stimulated by the active form of vitamin D,

1,25 dihydroxycholecalciferol (1,25 (OH)2D3) (Wasserman, 1981).

Wasserman (1981) also reported that the percentage of P absorbed is

partially dependent on the level of P intake, but Miller (1983)

reported that the percentage absorbed is not closely tied to need as

is Ca absorption. Phosphorus absorption percentage seems to be quite

variable among individual animals and from day to day for the same

animal (ARC, 1980).









The amount of P absorbed is dependent on source, Ca:P ratio,

intestinal pH, lactose intake, and dietary levels of Ca, P, vitamin

D, iron (Fe), aluminum (Al), manganese (Mn), potassium (K), magnesium

(Mg), zinc (Zn) and fat (Irving, 1964; Miller, 1983). Recently, Blum

et al. (1974) reported that the effects of high dietary levels of Ca,

Mg, Al and Fe are less pronounced in ruminants than simple-stomached

animals in regards to decreasing P absorption due to a lower pH in

these animals at the site of absorption. Field (1981) reported that

absorption of P is largely a function of that part of intake of the

mineral which is solubilized in the digestive tract prior to the site

of absorption.

Ruminants secrete copious amounts of P in saliva which is in

contrast to monogastric animals, with the secretion apparently

regulated by parathyroid hormone (PTH) (Tomas, 1974, cited by Cohen,

1980). The daily turnover of P in ruminant saliva is similar to or

greater than that of daily P intake (Cohen, 1980). Tomas et al.

(1967) reported that total daily P secretion from both parotid glands

of sheep exceeded P intake by a factor of 7.2 to 1.3 for P intakes

from 0.4 to 4.0 g Pd -, respectively. It has been estimated that

80% of the phosphate secreted into the digestive tract is salivary in

origin (Care et al., 1980), making it the main contributor of P in

the gut. The majority of phosphorus in saliva is inorganic

phosphate, concentrations of which range from 3 to 20 times that in

blood (Preston, 1976). The salts of phosphoric acid in saliva are

the most important buffering system in the rumen (Annison and Lewis,

1959). The amount of P in saliva may be involved in the regulation










of endogenous fecal excretion of this element (Miller, 1983).

Phosphorus output in saliva is dependent on P intake, and salivary P

concentration is directly related to plasma P concentration

(McDowell, 1985).

The rate of salivary flow in ruminants is quite variable. It

may be reduced with pelleted diets, concentrate diets and in young

animals fed a high proportion of cereal grains. There is generally an

inverse relationship between the concentration of phosphate and rate

of secretion in saliva; therefore, quantity of P entering the rumen

may not greatly decrease when salivary flow is reduced.

The role of the endocrine system in control of P absorption from

the digestive tract has been of particular interest in recent

years. Fox et al. (1978) have reported that pigs fed diets low in P

adapted by a hormonal mechanism such that the efficiency of the

mechanism by which P is absorbed (as well as Ca) from the small

intestine is increased. Earlier work by Young et al. (1966)

indicated that sheep increased the efficiency of P absorption when

fed diets low in P. The adaptive change to a diet low in either P or

Ca has been shown to be related to increased production of

1,25 (OH)2D3 in the kidney in nonruminants (Hausslar et al., 1976),

but has not been clearly demonstrated in ruminants (Care et al.,

1980).

As previously mentioned, PTH increases the P concentration in

the saliva. The concentration of blood P has no direct regulatory

influence on the synthesis and secretion of PTH; however, certain












disease conditions with hyperphosphatemia in both animals and man are

associated clinically with hyperparathyroidism. An elevated blood P

level may lead indirectly to parathyroid stimulation by virtue of its

ability to lower blood Ca according to the mass-law equation when the

serum is saturated with respect to the two ions (Krook and Lowe,

1964). Parathyroid hormone is the major regulator of Ca metabolism

and maintains plasma Ca concentrations by stimulating bone resorption

(Kronfeld et al., 1976). The secretion of PTH is inversely related

to Ca levels in the blood that is perfusing the parathyroid gland and

is not affected by changes in P levels in the plasma (Sherwood et

al., 1968). Cohen (1980) concluded that the major regulatory

influence on P turnover was dependent mainly on Ca status of the

animal.

The action of PTH on bone resorption is dependent on the

presence of 1,25 (OH)2D3 (Omdahl and DeLuca, 1973) and is inhibited

by calcitonin (CT), secreted by the thyroid gland (Rasmussen and

Tenenhouse, 1967; Guyton, 1981). The active metabolites of vitamin

D3 make bone cells more sensitive to the direct effects of PTH

("permissive effect") and greatly enhance the gastrointestinal

absorption of Ca, thus amplifying the effect of PTH upon Ca plasma

concentration. Estrogens may also be involved in this mechanism

(Guyton, 1981).

Absorption of P from the intestine is enhanced by 1,25 (OH)2D3

as previously noted (Wasserman, 1981), which is a product of










sequential hydroxylation of vitamin D3 in the liver and kidney. The

second hydroxylation which occurs in the kidney (1a-hydroxylase step)

is stimulated by low serum (and intracellular) inorganic phosphate

levels and depressed by high serum calcium levels (DeLuca and

Schnoes, 1976). Figure 1 is a schematic representation of the

hormonal control of phosphorus homeostasis in the ruminant.

In contrast to monogastric animals, ruminants excrete most of

the endogenous P through the feces (ARC, 1980); however, a greater

amount has been reported in the urine of cattle fed high concentrate

diets (Preston, 1976). These findings are related to the fact that

ruminants have a higher renal threshold for P than do monogastrics

(Mayer et al., 1968). Advantages of this fact for the ruminant lie

in their evolutionary adaptation to low quality roughage diets, which

generally contain little P. At various times, the amount of P

required for salivary production and intake are opposed. If the

renal threshold for P was low, then at times when intestinal P

reabsorption is occurring, thus causing an increase in plasma P

concentration, P would be excreted in the urine. This P would not be

"surplus" to the requirement of the animal and would then have to be

met by a diet originally low in available P.

Mayer et al. (1966) have shown that PTH decreases the daily

excretion of fecal P by cows, although it was not proven if this was

due to enhanced P absorption. These cows had a marked increase in

urinary excretion of P when given an I.V. dose of PTH.

Parathyroidectomy was followed by a decline in urinary P and

subsequent rise in fecal P.















SALIVA
P







INTESTINA
P
ABSORPTIO


LOW
r BLOOD
S Co

PTHI


I


L
N


'
\
I

LOW
BLOOD \

P


CT

tf,


BONE


BLOOD P
4l BLOOD P


'BLOOD Ca


Figure 1. Hormonal regulation of phosphorus homeostasis. Solid
line (-) represents positive stimuli, broken lines (---)
negative feedback. PTH = parathyroid hormone; CT =
calcitonin. Adapted from Cohen, 1980.











Barrow and Lambourne (1962) indicated that organic P excretion

remains relatively constant and independent of the amount of P

consumed, whereas fecal inorganic P varies directly with P intake in

cattle (Cohen, 1974).



Deficiency

Phosphorus deficiency is predominantly a condition of grazing

ruminants (McDowell, 1976; Underwood, 1981). Cattle long restricted

upon the native forage grown on any fenced pasture area reflect in

their physical condition and performance the nutritive value of the

forages and the fertility of the soil. A combination of soil and

climatic effects on forage P concentrations are the major

contributors in defining the P deficient areas throughout the world

for grazing livestock (Underwood, 1981). Soils low in plant

available P result in forage with below normal P contents and the

occurrence of a dry period in each year, when the plants are dry and

mature and the seed is shed, exacerbates or prolongs this effect.

Whole-plant P contents fall rapidly with increasing maturity; this

coupled with low initial P concentrations in the plant and long dry

periods will result in low P concentrations in forage for long

periods (Underwood, 1981). Additionally, under field conditions, the

protein content of the forage generally parallels that of P, such

that a protein deficiency, and many times a deficiency of available

energy, are all contributing factors in the malnutrition of livestock

in P deficient areas (Underwood, 1981; McDowell, 1985).











Pioneer P supplementation studies in South Africa in the early

1900s (Van Niekerk, 1978) revealed the cause of bovine botulism and

aphosphorosis. Both conditions were the result of a severe P

deficiency, with cattle exhibiting subnormal growth and reproduction

and a depraved appetite or "pica" as illustrated by bone chewing.

The syndrome of bovine P deficiency, according to Theiler and Green

(1932), appears as follows: hypophosphoremia, osteophagia,

osteoporosis, osteomalacia (in the adult) or rickets (in the young),

cachexia and exitus. Eckles and Gullickson (1927) and Riddell et al.

(1934) observed a depraved appetite as exhibited by chewing rocks,

soil consumption and chewing stall chains. A general lack of

thriftness and poor condition were also observed. Kleiber et al.

(1936) described the signs in beef heifers as similar to those

above. Pica was manifested largely as coprohagy and marked

osteophagia when tested with sun-bleached bones. Abnormal chewing of

objects may also occur with other dietary deficiencies however (e.g.,

Na and Ca) (McDowell, 1985). Often the pica as represented by bone

chewing has been misinterpreted to suggest that P deficient ruminants

selected substances containing P. Many materials sought by P

deficient animals contain little or no P (Becker et al., 1933;

Preston, 1976).

Almost every phase of performance in relation to cattle may be

depressed by a P deficiency. In addition to those P deficiency signs

previously mentioned, others reported include lethargy, stiffness,

decreased voluntary feed intake, lower feed efficiency, reduced

lactation, impaired reproduction and bone abnormalities











(Eckles et al., 1932; Becker et al., 1933; Huffman et al., 1933;

Preston, 1976; Miller, 1979; Underwood, 1981).



Requirements

Knowledge concerning specific nutrient requirements is a

necessity for optimum or economical beef cattle production. A

considerable volume of research work has been published on the

requirements of P in beef cattle nutrition; however, there is no

clear consensus on the needs of ruminants for macro-elements,

including that of P (Field, 1981). Braude (1978), addressing this,

stated that the best we can expect from requirements is that they

provide us with guidelines, which if interpreted intelligently, are

useful in practical livestock feeding, but are by no means absolute.

Underwood (1981) reported adequate Ca and P nutrition depends

not only upon sufficient total dietary supplies, but also on the

chemical forms in which they occur in the diet, vitamin D status of

the diet or animal, as well as the Ca:P ratio in the diet. However,

other factors that should be taken into account when determining the

actual nutrient level in the diet include variability of ingredient

nutrients, animal performance potential, energy and protein level of

the feed, ambient temperatures, stress (i.e., disease, overcrowding,

poor ventilation, inadequate temperature control), nutrient avail-

ability, present status or need for the nutrient, compensatory

growth, interaction of nutrients, variability in animal response and

management variability. It is, therefore, essential that as many











factors as possible be taken into consideration when assessing

requirements so that proper interpretations can be made.

The main basis for mineral requirements of ruminant livestock

today are the National Research Council (NRC) publications on

nutrient requirements of beef cattle (NRC, 1984) and dairy cattle

(NRC, 1978), in addition to the British publication on nutrient

requirements of ruminants [Agricultural Research Council] (ARC,

1980). The following discussion reviews published results as they

pertain directly and indirectly to minimum P requirements in beef

cattle nutrition.

Growth

Using a ration calculated to contain .26% P on a dry matter

basis (DMB), composed of equal parts of corn and alfalfa hay, Forbes

et al. (1929) fed 2 yr old steers four planes of nutrition from one-

half through twice maintenance energy intake to establish changing P

balance. The steers in this trial were in positive P balance when

fed a maintenance energy intake or above, indicative that P

requirements relative to energy requirements may be greater for near

maintenance compared to fattening in more mature steers.

Kleiber et al. (1936) found that a diet containing .13% P (DMB)

was not adequate for beef heifers which ceased to grow after 6 mo,

only maintained their weight when P level was reduced to .09% and

lost weight when the level was further reduced to .07% when compared

to controls fed .4% P. The authors suggested that P deficiency was

associated with impaired utilization of energy (i.e., they found that










P deficiency lowers total efficiency mainly by lowering appetite and,

secondly, by lowering the availability of metabolizable energy for

net energy purposes; digestibility of the feed was not affected).

Kleiber et al. (1936) also reported that bone chewing and coprophagy

developed in the low P groups. Serum P decreased approximately 67%

in heifers fed .07% P vs controls receiving .4% P.

Henderson and Weakley (1930) reported that dairy calves fed

.13% P with an adequate level of Ca grew as well as calves fed

.22% P. However, lower serum inorganic P levels (3.89 vs 4.69

mg.dl-1) and bone ash (55.7 vs 60.7%, dry-fat-extracted) were

observed for the calves fed .13% vs .22% P, respectively. Huffman et

al. (1933) reported that .20% P was insufficient for satisfactory

growth in dairy heifers from 3 to 18 mo; however, .41% P appeared

sufficient. In a similar study with two groups of 6 mo old calves,

Archibald and Bennett (1935) indicated that approximately .18% P was

as effective as .31% P in promoting growth.

Beeson et al. (1937, cited in Preston, 1976) reported that a

ration containing .12% P (DMB) (8.23 g P-d-1) fed to growing calves

resulted in poor condition, rough hair coats, wood chewing and soil

consumption. They concluded that the "physiological minimum"

requirement was .19% P and recommended .21% P (DMB) (2 g P-45.5 Kg-1

body weight.d-1) as the P requirement for growing-finishing calves.

Mitchell (1947) stated that a P level of less than .12% (DMB) in

forage will not supply adequate P for growth of cattle, consistent

with the work of Beeson et al. (1937, cited in Preston, 1976).











Burroughs et al. (1956) reported an increase in feedlot

performance of growing-finishing steers when ration P levels were

increased from .18 to .25%. In this same study, the authors

postulated a higher P requirement for rumen microbes than for the

growing host. This was based on the observation that steer calves

fed a ration containing .28% P (DMB) gained faster than those fed

.20% P, even though blood inorganic P levels were the same. However,

it has been reported by South African workers (Clark, 1953) that,

even during clinical hypophosphorosis, concentrations of water

-1
soluble P in rumen fluid do not fall below 200 mg-L Preston and

Pfander (1964) reported that growing lambs fed rations containing

either .12, .15, or .29% P (air-dry basis, 91% DM) had proportional

increases in total P concentration in rumen contents (.692, .931 and

1.17 mg*g" respectively) as well as total rumen fluid P (1.37, 2.04

and 3.20 g, respectively). Average daily gain of lambs for the 3

rations was 5, 28 and 168 g, respectively, over the first 49 d of the

experiment. Cohen (1978; cited in Cohen, 1980) has reported rumen

inorganic P concentrations between 150 and 183 mg P.L-1 rumen fluid

-1
in heifers with low intakes of dietary P (5.0 and 15.3 g*d-,

respectively).

There are still few reports in the literature on the P

requirements of rumen microorganisms, although these levels are in

excess of the minimum requirements of 60 mg P.L1 for Bacteroides

succinogenes (Bryant et al., 1959). It has been suggested from in

vitro results that a mean level of about 100 mg.L-1 of available P in

the rumen is adequate for growth of rumen bacteria and for










cellulolytic activity (Durand and Kawashima, 1980). The soluble P

content of rumen fluid in vivo is usually well above this suggested

level unless P intakes are very low (Table 1). It would seem from

this discussion that the P requirement of rumen microbes is high;

however, satisfaction of the microbial requirement depends more on

salivary P, plasma P and bone resorption than on the temporary supply

of available P. Witt and Owens (1983) reported no difference between

9.5 or 18.3 g P*d- fed to mature cattle on rate or extent of

digestion and suggested there was some evidence to indicate that P

may be mobilized before digestibility of nutrients is reduced.

Feed intake, weight gains and plasma inorganic P concentration

showed linear increases (Long et al., 1956) over the range of .07,

.11 and .15% dietary P in yearling steers (177 Kg average initial

weight) fed diets containing cottonseed hulls, dried beet pulp,

dehydrated alfalfa meal, corn gluten meal and urea. Supplemental P

was added as NaH2PO4 and Ca:P ratios were standardized over all diets

at 4:1. Slight increases in gains were observed when the P level was

further increased to .19%; however, this was not significantly

different from gain observed in steers fed the .15% P diet. Wise et

al. (1958) reported that ruminating calves (91-125 Kg, initially) had

a growth requirement of about .22% P, air-dry (.24% P, DMB). These

calves were fed a semi-purified basal diet (.09-.10% P, air-dry)

composed of corn meal, beet pulp, dried molasses, glucose, corn oil,

corn starch, blood meal, urea, minerals and vitamins A and D.

Phosphorus in the oasal diet was supplemented as dicalcium

phosphate. At P levels below .18%, feed consumption and growth were













Table 1. Influence of type of diet and dietary mineral level
(mg-kg DM) on P content (mg*L ) of rumen fluid (RF).



P

Animal Diet Diet RF


Sheep (4)a'b Urea-corn concentrate 3600 1300
Alfalfa hay 2800 370

Cattle (3)b Milo 800 1243
Alfalfa hay 400 50

Steer (1)c Grass hay 2900 360

Steer (1)b Mixed grass hay + wheat bran 6800 831

Steers (4)b Mixed hay semi-purified 400 200
1600 420
5400 540

Lambs (4)b High beet pulp 1300 690
1600 930
3200 1170

Wethers (3)b Semi-purified 0 80
(4)b 7000 760


Adapted from Durand

a Number of animals.

b Strained RF.

c Clarified RF.


and Kawashima (1980).











depressed. Also calves fed below this level had rough hair coats and

were unthrifty as compared to calves fed diets containing .22% P or

more. Bone growth appeared satisfactory at the .22% P level,

although rib bone ash was numerically higher at levels of .30 and

.38% P (48.9, 51.4 and 50.1, respectively). Tillman et al. (1959)

fed 18 Hereford steers (146-182 Kg, initially) a basal ration

containing .12% P for 47 d. A 70 d experimental period followed with

animals assigned by weight and age to three groups: either .14, .17

or .20% P. Supplemental P was provided as dibasic calcium

phosphate. Weight gain, feed consumption and feed efficiency

increased linearly, suggesting that the .17% P level was not adequate

for these criteria. Bone growth and plasma inorganic P, however,

differed from linearity in the .17 and .20% P groups indicating that

based on these criteria, the .17% P level was adequate. This result

was in agreement with that of Burroughs et al. (1956) and indicated

that the P requirement for weight gain and feed response is greater

than for bone growth or maintenance of plasma inorganic P level.

Pope et al. (1958) could not show improved performance in finishing

steers fed milo-sorghum silage rations when the P level of the ration

dry matter was increased from .23 to either .32 or .41%.

A dietary Ca:P ratio between 1:1 and 2:1 is assumed to be ideal

for growth and bone formation since this approximates the ratio of

the two minerals in bone. Ruminants can tolerate a wide range of

Ca:P ratios, especially when vitamin D levels are high (Underwood,

1981). In a trial with sheep, Young et al. (1966) fed a diet

containing .26% P with a total Ca:P ratio of 10:1, which had no











effect on P absorption. Wise et al. (1963), in a factorial

experiment, fed calves three levels of dietary Ca (.27, .81 and

2.43%) and three levels of dietary P (.17, .34 and .68%), yielding

nine Ca:P ratios ranging from .4:1 to 14.3:1. All ratios between 1:1

and 7:1 gave satisfactory results in regards to growth and feed

efficiency; however, ratios below 1:1 and above 7:1 caused

significant decreases in the above parameters. Similar results have

been reported by Call et al. (1978) when beef heifers fed for 2 yr on

diets containing Ca:P ratios of 7:1 to 9:1 had no adverse effects on

growth, feed efficiency or fertility. Results with Holstein steers

which were fed Ca:P ratios of 1:1, 4:1 and 8:1 (Ricketts et al.,

1970) indicated that gain and growth were less than the other groups

when an 8:1 ratio was fed.

When assessing the effects of Ca:P ratios, it is imperative that

one take into account the absolute levels of these minerals. When

either level is deficient, increasing the level of the other can

result in deleterious effects upon the animal. Although the number

of studies with cattle are limited, it is believed that a ratio in

which P exceeds Ca can be quite detrimental when levels of these

minerals are marginal (Wise et al., 1963; Miller, 1979; ARC, 1980).

This last consideration in lieu of absolute Ca and P intakes becomes

critical when researchers are looking for a positive response to P

supplementation. High Ca intakes may depress 1,25 (OH)2D3 production

with a subsequent decrease in P absorption, PTH secretion, salivary P

concentrations and mobilization of P from bone. Under these

conditions witn a low P intake, a growth response to P










supplementation could occur, provided no other nutrients are

limiting. If dietary Ca intake was low, the converse of the

aforementioned situation would occur, and possibly more P would be

available in tissue for growth. Therefore, under these conditions, a

growth response to P supplementation may not occur.

One must, therefore, keep in mind when assessing the P

requirement of various classes of beef cattle if all other nutrients

are in an adequate and available supply to meet the animals needs for

a given level of production. The tendency for seasonal fluctuations

of N and P levels to parallel each other in pastures has been

observed worldwide (Hemingway, 1967). From such considerations,

Eckles et al. (1935) concluded that the P deficiency occurring in the

field was a combined deficiency of N and P, rather than a P

deficiency alone. Du Toit et al. (1940, cited by Little, 1970) also

considered that the low protein content of pastures over much of the

year was the major problem and felt that the mobilization of skeletal

reserves masked appearance of any specific clinical deficiencies.

Becker et al. (1933) also reported similar findings (i.e., low P

content of pasture conjoined with low crude protein content of the

same pastures). Cohen (1974) concluded that P supplementation

(regardless of season) would not improve liveweight performance when

beef cattle graze pastures of low quality (i.e., low N, low energy or

limiting in other nutrients).

Little (1980) in a pair feeding regimen fed seven Hereford and

one Brahman cross steer and five Brahman cross heifers (average

initial weight 165 Kg) an ad libitum basal diet of Stylosanthes











humilis (1.8% N, .12% P and .77% Ca, DMB), with or without P

supplement at the rate of 5 g*d-1 (provided as NaH4PO4 three times

per wk) for 70 d. Irrespective of treatment, feed was consumed in

adequate amounts to support .5 Kg*d-1 gain. No differences were

found between groups with respect to voluntary feed intake (even when

expressed on a metabolic body size). Rib biopsy P concentration

(expressed as mg P.cm-3, fresh basis) and plasma inorganic P

concentration yielded no differences between groups. The author thus

concluded that the unsupplemented animals were not P-deficient and

that the .12% P (DMB) was adequate for this level of production

(i.e., for .5 Kg gain per day). This was in agreement with earlier

work (Little, 1968) in which 16 yearling Droughtmaster cattle (eight

heifers, eight steers) were fed a basal diet of S. humilis which

contained .07% P (DMB) but adequate in N and energy. Phosphorus was

added at the rate of 0, 4, 8 or 12 g*d-1 as potassium phosphate. The

unsupplemented P level was inadequate for growth compared to the

other groups. Little (1968) indicated that slightly less than .13% P

(DMB) would be adequate for the rate of gain obtained (.5 Kgd- ) as

there were no significant differences among gains made in the

supplemented groups.

In a similar study, Call et al. (1978) investigated the

influence of P on growth and reproduction in beef heifers. In this

study, two replicates of 48 Hereford heifers (initially 7 mo age and

165 Kg) were individually fed a basal ration (pelleted or chopped

alfalfa, chopped wheat straw and grass hay, top-dressed with beet

molasses) containing either .14% P (as-fed) or the basal ration top










dressed with sufficient monosodium phosphate to provide .36% P

(as-fed). Rations were adequate in protein, energy, minerals and

vitamins. Animals were fed ad libitum. The Ca:P ratio ranged from

3.5:1 to 7.1:1 for the high P and low P groups, respectively. The

experiment lasted approximately 2 yr. There was no difference

between groups in average daily gain (ADG); .44 vs .45 Kg-d-1 for the

.14 and .36% P groups, respectively; feed efficiency was also similar

in the two groups. Also the authors reported no signs of depraved

appetite or other clinical signs of P deficiency. These results were

similar to those of Little (1980) and Wise et al. (1963); however,

these results are in disagreement with the findings of Teh et al.

(1982) who reported increased gains in dairy calves fed .31 vs .24% P

(.84 vs .63 Kg*d-1, respectively).

In a follow-up study, Butcher et al. (1979) continued feeding

the animals reported on by Call et al. (1978) for an additional 2 yr

period. After 4 yr on the same diets, Butcher et al. (1979) reported

no significant differences between the .14 and .36% P groups with

respect to growth rate or voluntary intake. Numerical differences in

blood, bone and muscle tissue were seen; however, these were not

deemed as physiologically significant. After 4 yr, one-half the

animals on both the .14 and .36% P groups were reassigned to a .09% P

level. Appetite was somewhat depressed after 8 mo on the .09% P

diet, whereas, after 14 mo, one-half of the cows on this reduced

level showed reduced intake. Body weight changes were not different

between the .14 and .36% P groups at this time. At necropsy, the











cows from the .09% P group also showed reduced bone density and

increased likelihood of fractures.

Reproduction

The relationship between the nutrition of animals and their

fertility has offered researchers some of the most interesting and

important problems in animal production over the years. Evidence

(either circumstantial, practical or research in nature) has

demonstrated that dietary deficiencies may exert an adverse effect

upon one or more of the several stages in the reproductive cycle,

with the general observation that fertility and reproduction exhibit

abnormalities ranging from slight repression to complete cessation--

sometimes with the sudden interruption of pregnancy itself.

With beef cattle, breeding, pregnancy and lactation for the most

part take place on feeding regimes that primarily utilize pasture,

range or some type of preserved forage. It was under these types of

conditions that P was initially shown to be important in the

nutrition of cattle and sheep. Many studies have indicated

inadequate P levels in range and pasture, especially during the late

winter months. Phosphorus deficiency of grazing ruminants has been

reported in many areas of the world, including the United States,

South Africa and Australia, under similar conditions.

In many studies where P supplementation on pasture or range has

proven beneficial, the inability to state the P intake from the

forage has been a major problem in studying the minimum P

requirements for pregnancy and lactation. Nonetheless, P

supplementation has been both beneficial and essential, especially










during the fall and winter periods (Black et al., 1949; Thomas and

VanHorn, 1967; Little, 1975; Loxton et al., 1983). Also, it is not

always clear from the literature whether adequate levels of all

nutrients other than P were available, when a so-called P deficiency

is reported.

Theiler et al. (1928), in an early study with 200 breeding

cattle on the P deficient ranges of South Africa, attributed the

strikingly subnormal calf crop of a control herd (51%) not receiving

bonemeal in comparison with that of a bonemeal fed herd (80%) to

irregular breeding brought on by the drain of milk secretion on the P

reserves of the body. Subnormal fertility was associated with

depressed or irregular estrus, thus preventing or delaying

conception. The calves from cows receiving the supplement at 15 mo

of age weighed 318 Kg, nearly twice that of calves born to the

control cows. However, it may be unwise to attribute all the benefit

from bonemeal supplementation to P due to its high protein content

(Cohen, 1975).

Eckles et al. (1926) observed a marked inhibition of estrus in

cattle showing the general signs of a P deficiency. Out of 14 cows

studied from a P deficient area, all of which had been in pasture

with a bull, two were with calf and seven had nonfunctioning

ovaries. After a period of 3 to 9 mo, all animals eventually showed

estrus. Exact cause of the inhibition of estrus, however, could not

be directly stated. Huffman et al. (1933) fed either .22 or .44% P

(DMB) to dairy heifers from 3 mo of age to first calving. The .22%

ration resulted in lower plasma inorganic P concentration, decreased










appetite and weight gain, but seemed to have no adverse effects in

regards to reproduction in these animals.

Eckles et al. (1935) in a critical study pertaining to an

uncomplicated P deficiency on various parameters associated with

estrous cycle and reproduction in dairy cows concluded that P

deficiency did not cause abnormal estrous cycles although it appeared

to reduce "breeding efficiency" (this, however, was complicated by

the incidence of brucellosis). These authors postulated that the

disturbances in estrus and the low calf crop of cattle in P deficient

areas, under natural conditions, were probably due to multiple

nutrient deficiencies.

This is in agreement with Guilbert and Goss (1932) who noted

both cessation as well as long or irregular estrous cycles of rats

fed low dietary protein levels (i.e., 3.5 to 5.0%). Grossly P

deficient rations fed to heifers (3 g P*d-1) did not affect estrus or

normal ovulations when fed for an extended period (Theiler et al.,

1937). Palmer et al. (1941) studied combined P and protein

deficiency, comparable to the problem seen under field conditions.

They observed irregularities in estrous (such that periods of estrus

appeared to be missed), but no adverse effect on cyclical ovulation

or conception. Some evidence of delayed puberty (average age at

first ovulation and estrus was 14.5 mo) and dystocia were observed.

However, heifers on a diet specifically deficient in P continued to

exhibit estrus normally.

Knox and Watkins (1942) reported that 42% of the range grass

samples collected throughout the year in New Mexico were deficient in










P, and 75% of those collected in late winter were deficient. These

authors found the use of a P and Ca supplement, such as bonemeal,

resulted in reduced death losses in newborn calves, larger numbers of

calves weaned and greater weaning weight of calves. Black et al.

(1943) reported only 64% of control cows produced calves on range in

southern Texas, compared to 85% in cows given supplemental P (as

either bonemeal or disodium phosphate); only slightly more than 30%

of control cows calved in two consecutive years, whereas 75% of the

supplemented cows had calved. Practically all of the control cows

were observed chewing bones, which did not occur in supplemented

cows. Supplemental P had no influence on birth weight of calves;

however, weaning weight per calf was 31.4 Kg more in the supplemented

vs cows.

Hignett (1950) observed anestrus with low P levels, whereas

silent and irregular estrus periods were associated with moderate

deficiencies and infertility with marginal levels. These same

authors (Hignett and Hignett, 1951) in a survey of 50 dairy farms

reported that conception rate was improved in cows fed rations with

increasing P content. They suggested for cows producing milk (4.5 to

6.8 Kg daily, which is common in beef cows) a requirement of about

30 g P.d-1. Hignett and Hignett (1952) claimed that an intake of at

least 6.6 g P-100 Kg-1 body weight was required for "reasonable"

conception rates in heifers and that a P deficiency decreased estrus

activity. Subsequently, however, Littlejohn and Lewis (1960)

obtained no difference in fertility between animals given 2.0 and

11.4 g P-100 Kg-1 body weight 2 mo prior to breeding.











O'Moore (1952) reported cattle herds with a history of

infertility were always associated with less than .15% P in the dry

matter of pastures. When sodium phosphate was supplemented with or

without protein, only those that received the combination exhibited

estrus during the feeding period. In contrast to this, Bisschop

(1964) reported that the highest level of P in South African pastures

in 23 yr of monthly sampling was .13%. Bisschop (1964) concluded

that at no time did the natural pasture provide sufficient P or crude

protein for optimum production; however, both dairy and beef type

cows were shown to have grown and reproduced under these conditions,

although rather inefficiently. Ward (1968) and Lamond (1970)

reported the effects of undernutrition on fertility from Zimbabwe and

northern Australia, respectively. Lactating cows, with few

exceptions, were not calving 2 yr in succession. In P deficient

areas, if a calf is produced, it was observed that cows may not come

into a regular estrus again until body P levels were restored, either

by supplementing P or by ending lactation.

Over a 5 yr period on the King Ranch in southern Texas (Black

et al., 1949), the percent calf crop decreased in control cows from

91 to 22%, whereas P supplemented cows maintained a fairly constant

calving percentage. Nelson et al. (1951) reported that heifers

consuming grass, hay and supplement providing 10 g P (total) daily

was adequate to meet the P requirement for reproduction and

lactation; however, less than this was inadequate. In Montana

(Thomas and Van Horn, 1967) heifers receiving increasing levels of P

supplementation in addition to winter range had a higher percentage










calf crop over a 5 yr period. Little (1975) found a shortened

interval between calving and first estrus (62.4 vs 74.9 d) and

increased proportion of cows in estrus by 12 wk after calving (70 vs

50%) for P and protein supplemented and unsupplemented cows,

respectively. McDowell (1985) in a review reported that the average

of 16 cattle reports from tropical countries on the effect of P

supplementation on calving percentage was 75.6% for those receiving a

P supplement versus 52.9% for controls.

Holzschuh et al. (1971) fed yearling heifers either 7.8 or 12.7

g P d-1 for 21 mo. They reported that the heifers did not differ in

age at first estrus, period between first insemination and

conception, proportion which became pregnant or duration of

pregnancy. These authors emphasized that the heifers became pregnant

despite P deficiency. Call et al. (1974) fed heifers rations

containing either .16 or .51% P (DMB) during the growing and breeding

period. The authors found no differences in weight gain or

conception rate between the groups. No adverse effects on estrus or

conception rate in dairy heifers fed .21 to .24% P led Noller et al.

(1977) to conclude that these levels were sufficient for

reproduction. Call et al. (1978), in a study with 96 Hereford

heifers that were fed a basal ration containing .14% or the basal

ration supplemented with monosodium phosphate to provide .36% P (air-

dry basis) for 2 yr, reported that the lower level was adequate for

growth, puberty, conception, gestation and lactation. In this study,

there was no difference in age at puberty between groups; the heifers

that received the .14% P ration had a 96% pregnancy rate with 91%










live calves compared to 100 and 93%, respectively, for the

supplemented heifers (these differences were not significant).

Butcher et al. (1979) reported on results from the study conducted by

Call et al. (1978). The 1979 report revealed no reproductive

differences between the .14 and .36% P groups through three

gestations. After this time (i.e., 4 yr on trial), one-half the

animals in each group was randomly assigned to a .09% P group. Cows

that were placed on the reduced P level (.09%) went through the last

two-thirds of pregnancy, lactation and rebreeding with no apparent

adverse effects. All cows were bred by natural service. Conception

rates of 60, 91 and 95% were reported for the .36, .14 and .09% P

groups, respectively. These data would indicate that P levels as low

as .09% are still adequate for maintaining fertility in mature beef

cows. Call et al. (1982), reporting on the same animals through

their eighth gestation, disclosed that cows on the .09% P ration for

an additional year developed a complex syndrome which included the

following: unthriftness, body weight loss, reduced feed consumption,

lameness and impaired reproductive performance (i.e., cows failed to

rebreed after sixth calving). When these cows were subsequently

changed to a diet containing .14% P, voluntary intake increased, the

animals developed a glossy hair coat and all were bred within their

first two cycles after bulls were introduced. These observations

were indicative not only of the insidious nature of P deficiency in

cattle, but also the great adaptive power of the animal to a wide

range of P intakes (accomplished mainly through renal and salivary P

conservation mechanisms and bone mobilization).











Hurley et al. (1982) conducted an experiment to study the

effects of varying P levels (from 73 to 246% of NRC recommendations)

on estrous cycle and endocrine status of dairy heifers and reported

that dietary P inadequacy did not alter reproductive endocrine

function or estrus intensity in heifers in this trial. No difference

in blood serum concentration of progesterone, estradiol or

luteinizing hormone was noted around the time of estrus in the

heifers.

Judkins et al. (1985) reported on a P supplementation trial

involving 78 Angus X Hereford range cows (39 control cows received

salt only, 39 cows received a free choice mineral supplement with P)

over a 5 yr period in New Mexico. These authors reported that

fertility was not altered by P supplementation. However, no data

were reported on forage intake or P content of forage available.

Greene et al. (1985) conducted an experiment to study the

effects of either low P (.04%) or adequate P (.44%) on growth and

estrous activity of rats. These authors concluded that the effect of

a P deficiency during gestation, lactation or weaning in rats was

limited to pup growth and survival postpartum and did not appear to

be associated with the regularity of estrous cycles, conception rate

or embryo survival.

In a recent study, Call et al. (1987) reported on the clinical

effects of low dietary P fed to 34 lactating Holstein dairy cows.

Three dietary concentrations of P were fed (.24, .32 and .42%, DMB)

for up to 12 mo. These authors reported that the reproductive

performance in the .24%P group was superior to both other treatment










groups. Cows receiving .24%P in the diet achieved a 92% pregnancy

rate as compared to 87 and 76% for the .32 and .42%P groups,

respectively. Cows on the lower P diet also required fewer breeding

per pregnancy. Also, cows receiving the .24%P diet tended to resume

cycling of estrus earlier after calving was completed than did cows

in other treatment groups.

Based on published results in the literature, there appears to

be wide variation in the definition of minimum P requirements for the

various phases of reproduction in female beef cattle. Phosphorus

deficiency is accepted as a major nutritional factor depressing

reproductive efficiency in mammals. In some studies, ovarian

function and fertility of females appear to be quite sensitive to P

intake; however, other work has failed to show a diminished

reproductive performance of animals on P deficient diets. It must

also be stated again that many times P deficient diets are usually

low in protein and energy, which along with lowered feed intake,

generates a P, energy and protein deficiency. Therefore, it may be

suggested from some research that a P deficiency, per se, will not

affect reproductive performance until malnutrition occurs. Also,

although reproductive failures have been reported when animals

consume diets low in P, the precise physiological mechanisms involved

have not been identified.



Assessment of Phosphorus Status

Problems of P deficiency in cattle are widespread (Mitchell,

1947; Underwood, 1981; McDowell, 1985), yet there is no one











completely satisfactory method of objectively assessing the P status

of the bovine. Because the problem of P deficiency is so prevalent,

it is imperative to identify the most precise yet practical indicator

of P status in cattle.

The detection of mineral element deficiencies or excesses

involves clinical, pathological and analytical criteria as well as

response from specific element supplementation. Clinical signs of

mineral deficiencies along with soil, water, plant and animal tissue

analyses have all been used with varying degrees of success to

establish mineral deficiencies and toxicities (McDowell, 1985).

A goal of mineral nutritionists for many years has been the

development of simple and accurate biochemical measurements of the

status of animals for the minerals in which there are important

practical problems (Miller and Stake, 1974). When the evidence from

clinical, pathological and biochemical examinations of the animal and

from chemical analysis of the diet and its components is combined and

assessed, it is usually possible to detect and define any nutritional

abnormality of mineral origin, even when it is mild (Underwood,

1981).

Specific clinical signs like aphosphorosis may be useful in

diagnosing a mineral deficiency, although by the time these signs are

apparent, the deficiency is usually in an advanced state (Little,

1982). McDowell et al. (1984) have reported on the essentiality of

chemical analyses and biological assays when studying mineral

imbalances (particularly the borderline or subclinical cases which

may not produce signs specific to a single mineral).











Various criteria have been used as diagnostic aids in assessing

the P status of grazing ruminants: feed samples (both hand plucked

grab samples and samples collected from esophageally fistulated

animals), soil analyses, depraved appetite, bone composition, bone

growth, bone breaking strength, blood analyses, rumen contents, rumen

fluid, fecal P concentrations, salivary P concentrations, hair P

concentrations, P concentrations in milk, P balance studies, muscle P

concentration, analysis of body organs and the performance of animals

in terms of growth and reproductive efficiency. Many of these will

be reviewed in the following sections.

Feed

Forage, according to the Netherlands Committee on Mineral

Nutrition (NCMN, 1973), is the most suitable sample for diagnosing a

P deficiency. Under controlled feeding experiments where animals

have no access to any feed other than what is offered, this may be a

useful tool. However, it is a more complicated situation in the

pasture with the grazing ruminant due to their selective grazing

behavior. Selectivity of the grazer may vary with animal species,

availability of plant, stage of maturity, intensity of grazing and

weather condition (Cook, 1964). Hand plucked pasture samples may not

represent the diet selected by the animal. Other problems associated

with this type of sampling in assessing mineral adequacy is the

difficulty of estimating forage intake and digestibility. The use of

esophageally fistulated animals on pasture has made this procedure

far more reliable; however, salivary P contamination makes these

samples unsuitable for estimating dietary P intake levels (Langlands,











1966; Little, 1972a) unless salivary P is labelled with 32P prior to

collection (Little et al., 1977).

Blood

Whole blood or blood serum or plasma is widely used for studies

in mineral nutrition (McDowell, 1985) and since it is easily obtained

from live animals, it is an ideal tissue for study. However,

according to Underwood (1981) values significantly and consistently

above or below so-called normal concentration or ranges provide

suggestive but not conclusive evidence of a dietary excess or

deficiency of particular minerals.

The first known response to a dietary deficiency of P is a fall

in the inorganic P fraction of blood plasma (Underwood, 1981).

Changes in blood inorganic P can occur quite fast after changing

animals to P deficient rations. Henderson and Weakly (1930) noted a

change from 8.0 to 4.6 mg P.dl-1 plasma within 4 d after a ration

change from an adequate to an inadequate P intake. Several authors

have reported increases in the blood inorganic P concentration after

"adjustment" to a ration low in P. Bone P is mobilized to maintain a

normal blood inorganic P level in P deficient animals but this

mobilization is not rapid (Duncan, 1958); therefore, blood may only

reflect rapid changes in dietary P whereas P deficiency, caused by

prolonged low levels of dietary P, may be better detected in bone.

The usefulness of inorganic P in blood serum or plasma is still

a matter of contention in regards to it being an adequate criterion

for assessing P status in ruminants. The NCMN (1973) reported that

blood is most relevant for detecting only Zn deficiency, whereas, Ca











concentration in blood plasma is influenced only by severe deficiency

and that of inorganic P cannot be used as a practical criterion at

all due to its great variation and the poor understanding of the

factors that cause this variation. Underwood (1966), conversely,

indicated that serum inorganic P was a satisfactory criterion for

assessing the P status of ruminant animals. Conrad (1978) has also

suggested that most research groups consider serum inorganic P to be

sufficiently sensitive and recommend it for diagnosing P

deficiencies, while the NCMN (1973) as previously mentioned

recommends forage (i.e., feed) analysis.

Many factors have been identified in influencing blood serum or

plasma inorganic P levels. Irving (1973) reported that the P level

in blood is not nearly as constant as the Ca level. Exercise (Palmer

et al., 1930; Gartner et al., 1965) and excitement (Eckles et al.,

1932) in cattle prior to bleeding have been reported to increase

blood inorganic P concentrations. Burdin and Howard (1963) have

reported that increased storage time and/or temperature postsampling

increased blood P due to hydrolysis of organically bound P from

phosphoric acid esters such as ATP. An increase of 64% in organic P

during 1 wk storage at ambient temperature occurred compared to only

a 4% increase when blood was stored at 4 C for 12 d. The type of

protein precipitant in preparing the sample for analysis (Little

et al., 1971) and water restriction (Rollinson and Bredon, 1960) have

also been shown to increase blood P levels. Phosphorus values are

also affected by parasites. Blood P increased approximately 2-fold











in liver fluke-infested White Fulani cattle in Nigeria (Ogunrinade

et al., 1980, cited by Mtlmuni, 1982).

Recently, Perge et al. (1983) indicated that the effect of feed

on Ca and P content in blood serum and saliva of wethers is

influenced not only by dietary supplies (i.e., amounts of the

elements in the ration) but also by the time of sampling. As Forar

et al. (1982) have pointed out, time of sampling should be

standardized when collecting blood samples to reduce the effect of

diurnal variation in plasma inorganic P concentration in cattle.

Wise et al. (1963) also have reported rapid increases in blood P

content after feeding, which could lead to false readings.

Inorganic P concentrations in plasma differed between animals in

a herd and between months within animals (Blooser et al., 1951).

Wilson et al. (1977) reported a tendency for inorganic P

concentration in plasma to decrease prior to parturition, with the

lowest point at the time of calving. Blood plasma-mineral profiles

of lactating dairy cows showed a decrease in P concentration at

parturition, except in young cows fed plain salt versus those

receiving a mineral supplement (McAdam and O'Dell, 1982). Only minor

fluctuations were observed in P concentration for animals on each

treatment throughout lactation.

Reference has already been made to studies where blood inorganic

P level was used to study P adequacy. Theiler et al. (1937) stated

that blood inorganic P may drop to one-fourth of the normal value

even before deficiency signs can be observed. Murray et al. (1936)

suggested that blood inorganic P levels indicating P deficiency are










less than 3.8 mg P-dl-1 serum in mature cattle and 5.3 mg P.dl-1

serum in young cattle. Cunha et al. (1964) considered cows with

concentrations lower than 5 mg P.dl-1 serum as deficient. Thompson

(1978) indicated that, usually, the first evidence of P deficiency is

a drop in plasma inorganic P below the normal values of 4-6 mg P*dl-1

of plasma for adults and 6-8 mg P.dl-1 of plasma for young animals.

Becker et al. (1933), in Florida, reported that nursing cows

grazing P deficient pastures had an average of 2.55 mg P.dl-1 of

serum, whereas these same cows when supplemented with bone meal had

values of 4.02 mg P*dl-1 of serum. In growing-finishing steers

(Beeson et al., 1941), plasma inorganic P was directly correlated

with P level in the ration with maximum gain being observed on .21% P

(DMB) which resulted in a plasma inorganic level of 6.6 mg P-dl-1

plasma; controls fed a ration of .13% P (DMB) had a plasma inorganic

P level of 3.6 mg P-dl-1 plasma. In agreement with this study, Cohen

(1974) reported that blood plasma P concentration in cattle was

significantly related to P intake, but that the relationship varied

depending on the time of day at which the samples were collected.

Normal values for plasma inorganic P are 4-6 mg P-dl-1 for adults and

somewhat higher, often 6-8 mg-dl-1, for very young animals

(Underwood, 1981).

Kiatoko et al. (1982) in a study of the mineral status of beef

cattle herds from four soil order regions of Florida found that mean

plasma P in all regions was above 4.5 mg P-dl-1 during the wet

season, while during the dry season, concentrations in the Histosol

soil order were below this level. Of all the animals studied, 13%











had low plasma P during the dry season. Read et al. (1986a) recently

concluded that low plasma P levels adequately reflected low P

intakes; however, plasma P concentration was a poor indicator when

higher levels of P intake are concerned.

With regards to usefulness of whole blood as a criterion in

assessing P status in the bovine, the literature is limited. There

appears to be great variation in what is the accepted normal range of

values. Rodgers (1975) reported normal whole blood P values in

cattle of 35 to 45 mg P-dl-1; whereas, Call et al. (1978) have

reported values of 18 to 21 mg P.dl-1 in heifers fed .36% P (as-

fed). Perhaps a great deal of this variation is a product of the

method used in preparing the whole blood sample for P analysis. Many

of the factors affecting either serum or plasma inorganic P level,

such as water restriction and parasite load which would affect the

packed cell volume also greatly influence whole blood P

concentration. It should also be considered that whole blood P

levels are a combination of inorganic P and significant amounts of

organically bound P, the latter which is not "available" to the

animal as such. Miller (1983) has stated that total plasma P (i.e.,

includes plasma proteins) is not a very sensitive measure of P

deficiency for this very reason.

Hair

Hair has many innate properties that make it a likely tissue for

biopsy; it is easily collected with little trauma to the animal and

can be stored until analysis is convenient because it does not

deteriorate rapidly (Combs et al., 1982). For several years, it has










been proposed that body stores of minerals could possibly be

estimated from hair analyses, as growing hair is metabolically active

and a sequestering tissue. Hair may, therefore, mirror

concentrations of minerals that were in the hair follicle at the time

the hair was formed. However, mineral content of hair may also

reflect surface contamination by minerals in urine, feces, sweat,

feed and air-borne matter (Combs et al., 1982). Also, it has been

shown that the mineral content of hair is affected by season, breed,

hair color within and between breeds, sire, age and body location

(Miller et al., 1965; O'Mary, 1969; 1970; Hambidge et al., 1972;

Kiatoko et al., 1982).

The use of hair analyses to monitor the intake of Ca and P has

for the most part been unsuccessful. Anke (1966) reported that

dietary supplementation with Ca and P significantly increased

concentrations of Ca and P in pigmented hair of dairy cattle. A

positive correlation (Anke, 1967; cited in Combs et al., 1982) was

obtained between P content of feed and that of black hair of

cattle. In the latter study, Anke (1967; cited in Combs et al.,

1982) concluded that the supply of P to cattle was well reflected in

hair P content. This finding is in agreement with Legel (1971) who

reported that hair was suitable as an indicator of P status at

certain times of the year. These authors reported that P content of

hair showed seasonal variations which were greater when P intake was

low (7.8 vs 12.7 g P.d-1) in this trial. The P concentration of

pigmented hair had fallen significantly after 8 wk on the low P

diet. In contrast, Wysocki and Klett (1971) reported low











correlations between Ca and P intake in ponies and the Ca and P

content of their hair. Cohen (1973a) sampled 15 Angus and Angus X

Hereford yearling steers five times from December, 1969, to December,

1970 (3 mo apart). He found no difference in P content of hair

between sampling times and no significant relation between P content

of pasture and hair. This is in contrast to Kiatoko et al. (1982)

who reported higher P concentrations in dry versus wet season (219 vs

104 ppm, respectively). Cohen (1973b) found that drenching growing

steers with either 35 or 70 g P (given six times per wk as sodium

dihydrogen orthophosphate) did not change hair P or Ca

concentrations. From his two studies, Cohen (1973a, b) concluded

that hair P was not a satisfactory indicator of P status in cattle.

Bone

General characteristics. Bone is a highly specialized

connective tissue that consists of cells embedded within a gel-like

substance that becomes mineralized to varying degrees. The method of

secretion and the manner in which the cells become embedded in the

matrix are similar to those phenomena observed in cartilage.

Although the fibrous and ground substance components of bone are

similar to those of cartilage (collagen and acid mucopoly-

saccharides), the mineral portion of the matrix makes bone different

from other connective tissues (Banks, 1981). This mineral fraction

consists of amorphous calcium phosphate and the hydroxyapatite

crystal.

Cortical bone is composed of approximately 69% inorganic matter,

22% organic matter, and 9% water (Urist, 1980). Over 99% of the











body's Ca and 80% of the P are found in bone and, in conjunction,

these minerals comprise the major portion of the inorganic fraction

as hydroxyapatite. Initially, unstable forms of amorphous calcium

phosphate are formed, presumably because hydroxyapatite itself cannot

be formed from its constituent ions due to its large size and

crystallographic structure. As conditions change (particularly pH),

other forms are precipitated until hydroxyapatite is formed.

Generally, more recent precipitates have a low Ca:P ratio of 1 to

1.5:1, while aged precipitates have a ratio of 1.4 to 2:1.

Magnesium, Na, K, F, Sr, citrate and carbonate ions are also present

among bone salts. It is believed that these are adsorbed to

hydroxyapatite crystals, rather than forming crystals of their own.

It is the mineral fraction of bone which contributes to the great

compressional strength of bone (Guyton, 1981).

Collagen comprises 90% of the organic fraction of bone. The

remaining noncollagenous portion consists of other proteins,

proteoglycans and lipids. Collagen is the major structural protein

and displays great tensile strength, requiring a load of 10 to 40 Kg

to break a fiber 1 mm in diameter (Urist, 1980).

Although the exact mechanisms) of bone calcification is not

completely understood, it is believed that certain inhibitors, for

example, pyrophosphates, are present initially. When this inhibitor

is removed, crystallization is initiated. Collagen fibers have an

affinity for Ca salts and there are nucleation sites along the

molecule which encourage formation of a crystal seed and then further

precipitation of the salts. Although many other theories on the











mechanism of calcification exist, this nucleation theory is the best

attempt at explanation of the bone mineralization process to date.

Contrary to perceptions that might be formed from handling a

dried portion of the skeleton, bone is not a "dead" static tissue,

but rather a dynamic, metabolically active tissue in a constant state

of change. The extensive blood supply to bone gives some insight to

its viability and metabolic activity. Wilson (1972) reported that

every gram of bone has an exposed surface of several square meters

providing it with tremendous surface area for vascular exchange. The

large surface area is important to both Ca and P metabolism. Through

storage, resorption and mobilization of these elements, bone is

responsible for the long term homeostasis of these minerals.

Bone consists of three types of osteogenic cells: 1)

osteoblasts, 2) osteoclasts, and 3) osteocytes. The bone cells are

responsible for both structural and metabolic functions of bone.

Osteoblasts (bone forming cells) secrete collagen and ground

substance and lie on the surface of bone. Osteoclasts (which are

multi-nucleated) are responsible for resorption of bone. Generally,

osteoclasts resorb bone from fully mineralized tissue rather than

from osteoid surfaces. An adaptive modification in the plasma

membrane, the brush border, is thought to be capable of removal of

bone mineral as well as digestion of the organic matrix (Banks,

1981). Osteocytes are osteoblasts that have become embedded in

bone. Microscopically osteocytes are similar to osteoblasts. They

continue the same osteoblastic activity of maintaining the matrix











only to a lesser degree (Ham and Cormack, 1979). Osteocytes may also

possibly function in the resorption of bone.

Osteogenic cells are usually found on the endosteal and

periosteal surfaces of compact bone (Vaughan, 1970). Bands of

lamellar bone are adjacent to these layers, which contain osteocytes

and Haversian canals. These canals, which are lined with osteogenic

cells, are the pathways for blood vessels supplying bone. They

predominate internal to the lamellar bone. Each Haversian system is

surrounded by osteocytes communicating with each other through their

canaliculi. A cement line separates each Haversian canal from other

canaliculi, across which there is no communication. The area around

each canal bounded by a cement line is known as an osteon, or

resorption cavity. Some osteons show no resorption-apposition

activity for some time, while others show varying amounts of

remodeling. Lacunae are the areas of mineralized matrix left by some

earlier remodeling involving Haversian canals or lamellar bone.

Trabecular bone contains fewer Haversian systems and the remodeling,

which is more active than that in compact bone, takes place on the

surface of the trabeculae. Growth in length occurs by endochondral

ossification, which takes place at the epiphysis, while growth in

diameter occurs by concurrent resorption on the endosteal portion of

the bone and apposition on the periosteal side.

The turnover of bone is important to the performance of its

structural and metabolic functions. Mature bone is constantly being

resorbed and reformed. The normal process of internal remodeling may

occur with no change in the gross anatomic structure of the bone.











Banks (1981) has reported that internal remodeling may occur in

response to altered vascular relationships that require the

repositioning of vessels within compact bone for adequate osteocyte

nutrition, biomechanical stress, dietary deficiencies, immobilization

and/or disuse, and altered endocrine function.

Trabecular bone is more labile than compact bone. Trabecular

remodeling activity may be as great as three times higher than

compact bone. Remodeling though not a rapid process is dynamic.

Banks (1981) has reported the time required from resorption to

reformation of a mature Haversian system may require months. For

maintenance of normal skeletal volume, resorptive and formative

processes must be in balance. Homeostatic control of remodeling is

achieved by balanced activity of osteoblasts and osteoclasts.

Wolff's Law states that remodeling takes place so that skeletal

form and mass adjust to provide optimum bone strength (Lanyon,

1982). Frost (1973), building on Wolff's Law of structural

remodeling, developed the Flexure Drift Law which stated that during

bone formation, lamella orient parallel to the direction or resultant

of the greatest tension and/or compression stress. Frost (1973) also

proposed that this force response characteristic may account for the

anatomic conformation of various bones.

Techniques used in assessing phosphorus status. Modern animal

production practices have greatly enhanced the importance of optimum

bone development and strength at an early age, especially in regards

to prolonging the useful life of breeding animals. All phases of

livestock production have realized the need for quality bone











development. This is clearly seen where the push for maximum

production in beef, dairy, poultry and swine operations under

confinement situations has created need for situations where optimal

bone strength is important in relation to the performance of the

animal.

(1) Rib and tail biopsies. Withdrawal of minerals during

periods of inadequate intake does not take place equally from

different parts of the skeleton. The spongy bones, ribs, vertebrae

and sternum, which contain less inorganic salts, are the first to be

affected, together with the cancellous ends of the long bones. The

compact shafts of the long bones such as the humerus, femur and tibia

and of the small bones of the extremities are the last reserves to be

used. In each case, the essential change is a reduction in the total

mineral content of the bones, with little alteration in the

proportions of the minerals in the remaining ash (Underwood, 1981).

This is probably due in fact to the mechanism by which mineral is

lost from bone (i.e., resorption versus demineralization).

Resorption involves the removal of both bone matrix and mineral

matter, consequently reducing bone mass and ash, but not necessarily

bone ash percentage. Duncan (1958) stated that percentage ash in a

sample of bone gave no indication of the status of reserve and

suggested that comparison of the ash weight of representative whole

bones would be a better indication. Maynard et al. (1979) reported

that in mammals the bone is made of approximately 36% Ca, 17% P and

.8% Mg, based on dry fat-free bone. Benzie et al. (1959) reported

that the axial skeleton and ribs of the ewe were resorbed and











rehabilitated more readily than long bones, therefore suggesting

changes in nutrient intake is better detected from the axial skeleton

and ribs.

Most studies of the skeleton were conducted on animals

slaughtered specifically for that purpose, until the development by

Little (1972b) of a rib bone biopsy technique which enables serial

sampling of both cattle and sheep. This technique has many

advantages in studies where the monitoring of Ca and P in the

skeleton are primary goals--especially in terms of economics and the

ability to sample the same animal repeatedly.

Little (1972b) reported that expressing Ca and P content per

unit volume has greater sensitivity than per unit of fresh weight,

whereas expressing Ca and P on a moisture-free, fat-free basis was

not sensitive because changes in water and fat contents of bone were

not taken into account. In a 6 wk depletion study of yearling cattle

(diet contained .08% P and 8% CP, DMB), Little (1972b) revealed an

increase of 23.0 to 27.9% in moisture and 1.7 to 5.2% in fat of rib

bone biopsies taken at the initiation of depletion and the end of the

6 wk period, respectively. Rib bone biopsies also indicated a

decreased bone density (as measured by specific gravity), 1.68 vs

1.59 g-cm3 for pre- and postdepletion periods, respectively.

Little and Minson (1977) have suggested that the same rib should

be sampled in all animals in any one investigation due to variation

of P content among ribs within an animal. They suggest the use of

the 12th rib for best results. Also, Little and Ratcliff (1979)

reported that unbiased results may be obtained from samples of bone










taken from a rib sampled previously, 6 mo or more after the earlier

biopsy, and that it is advisable to obtain the first sample from a

site ventral to that proposed for the second sample. It has been

suggested by Little and Shaw (1979) that possible critical levels of

120 mg P-cm-3 bone from the 12th rib may indicate deficiency of P

while levels over 150 mg P*cm-3 suggest adequacy when using the rib

biopsy technique.

Cohen (1973a) correlated the P concentration in pasture samples,

blood, hair and bone. He found that there was a significant

correlation (r=.97) between P in pasture and in dry, fat-free ribs.

Correlation between P levels in pasture and in either hair or blood

were not significant.

Rib P levels may not always accurately reflect skeletal P

levels. As previously mentioned, Benzie et al. (1959) reported that

sheep bones high in cancellous tissue, such as ribs, were the first

to be depleted during P deficiency and to be repaired during P

adequacy. Hoey et al. (1982) and Gartner et al. (1982) reporting on

different aspects of the same study concluded that the use of

cortical rib biopsy P concentration to differentiate between P

deficient and P adequate animals is questionable. They found that

cattle fed a diet containing .09% (DMB) ceased to grow after 19 wk

although the lowest rib P levels in cortical rib biopsy samples

varied from 169-181 mg P*cm3 in P deficient animals and from

171-186 mg P-cm-3 in P adequate animals. Read et al. (1986b)

concluded that P content of bone samples expressed as mg P*cm-3

(fresh basis) was a reliable sensitive indicator of the P status of











grazing crossbred cows sampled at different stages of their

reproductive cycles.

Wilson and Hatfield (1974) developed a simple tail biopsy

procedure and reported it to be a sensitive criterion of response to

P source and level. Ash weight of caudal vertebrae responded

linearly over a 112 d feeding period to different levels of P

supplementation. These authors suggested that this technique in

combination with other procedures could increase the accuracy of

interpretation of data in regard to P nutrition.

(2) Mechanical properties of bone. Mechanical properties of

bone, such as breaking strength, have been used by nutritionists in

determining the bioavailability of minerals and establishing nutrient

requirements for many years (Haugh et al., 1971; Crenshaw et al.,

1981a). The response of bone to the variety of forces which it is

exposed to throughout the life of an animal is a function of the

mechanical properties of bone (Evans, 1973). Frankel and Burstein

(1970) stated that the structure of bone responds to and is derived

from its mechanical properties. Before discussing research involving

P and the mechanical properties of bovine bone, a review of some of

the basic principles of mechanics as they relate to the properties of

bone may be helpful. A better understanding of principles involved

in mechanical properties of bone would allow more accurate

conclusions to be made concerning the effect of nutrients on

mineralization and more accurate comparisons to be made among various

experiments (Crenshaw et al., 1981a).











Stress and strain are created when a force acts upon a body.

Strain is the change in length per unit length of a body when a force

is applied; whereas stress is the force applied per unit area of the

body. Bones are subjected continually to various forces acting upon

them. The magnitude, direction, duration and rate at which a force

is applied influences the responsiveness of the organ.

Behavior of materials, when subjected to mechanical loading, may

be described in terms of four basic rheologic properties: 1)

elasticity, 2) plasticity, 3) viscosity and 4) strength. In most

materials, including biological materials like bone, these properties

exist in combination.

Bone can be tested as either a structure or material. When

tested as a structure, a force is applied until the bone fails. The

breaking load (BL), which is a structural test, represents the force

at which any further increase in force results in rupture. A

material property of bone, stress, results from an applied load. The

maximum stress the bone material can withstand is termed breaking

strength (BS). Crenshaw et al. (1981a, b) and Lawrence (1986) have

pointed out a major problem in the literature in that BL and BS,

which are different, are mistakingly used interchangeably by numerous

researchers.

Breaking load, the force required until failure of the bone, is

dependent on both the size of the bone and quality of the material

present. Breaking strength, however, takes into account not only the

force required to cause failure, but the area and geometrical shape

of area over which the force is applied (Crenshaw et al., 1981a).











Therefore, in order to calculate the BS, the area moment of inertia

(I) of the bone must be determined. Breaking strength allows

comparisons between anatomically similar bones which differ in size.

Equations for calculating I from simple measurements of an

object have been derived for geometrical configuration of known

shapes (circles, triangles, rectangles, etc.). However, bones are

irregular in shape, making it difficult to determine the area and

geometry for calculation of I. Some researchers have dealt with this

by machining small uniform sections of known geometrical shape. When

bone is mechanically tested in this manner, its material properties

are examined. If the whole bone is tested, its structural (material

and geometrical) properties are tested. Evans (1973) has stated that

this type of testing is more representative of forces acting on bone

in the live animal.

Lawrence (1986), using the SCADS computer program (Piotrowski

and Kellman, 1973), determined the actual area and I of 2 cm thick

equine third metacarpal (McIII) midshaft cross-sections irregardless

of their geometry and found that elliptical estimates very closely

approximated both the actual area and I of these sections when

compared to circular estimates of these parameters. Values reported

were 7.40, 3.55 and 3.66 cm4 for the circular, elliptical and SCADS

I's, respectively. The use of the circular I overestimated the

actual I by approximately 100%. Since I is used in calculating BS,

erroneous values may be obtained if an appropriate estimate of I is

not used.











Strength of materials can be determined by numerous types of

tests. The most commonly used to evaluate the mechanical properties

of bone by nutritionists is a flexure test (Baker and Haugh, 1979;

Crenshaw et al., 1981a; Lawrence, 1986). In a flexure (bending)

test, the bone is rested on two fulcra, one at each end of the bone,

and a force is applied from above. A flexure test involves both

compressive and tensile forces (Figure 2). Compressive forces tend

to "push" an object together, while tensile forces tend to "pull" it

apart. When a force is applied to bone in a flexure test,

compressive forces are exerted on the top fibers while tensile forces

are exerted on the bottom fibers. The neutral axis is the plane of

material in the center which is neither in compression nor tension.

In a flexure test, force is plotted against deformation, which

produces a force-deformation curve (Figure 3). Elastic deformation

occurs in the initial linear phase of this curve. In this area, when

the applied force is removed, the bone will return to its original

shape, with no damage, as no permanent damage is done to the bone.

However, the linear phase, the amount of force applied to the bone is

sufficient to result in permanent damage to the bone. This is the

region of plastic deformation (i.e., removal of the applied force

will not result in return of the bone to its original shape). These

theological properties of bone give it its viscoelastic properties.

Traits that describe the mechanical properties of bone as

determined by the flexure test include BS, BL, I, modulus of

elasticity (E) and strain. Equations and variables used in the

calculation of these parameters are presented in the material and












FORCE


NEUTRAL
AXIS




















NEUTRAL
AXIS


FORCE


-L -


Diagram illustrating 3-point flexure test of bovine third
metacarpal: a) pretest and b) during testing with force
applied to the posterior surface. Neutral axis is
represented by dashed line, C is distance from extreme
outer fiber to neutral axis, L is the length between the
roller supports, F.


Figure 2.



















ELASTIC LIMIT


ELASTIC
REGION


PLASTIC
REGION
L BREAK
POINT


DEFORMATION, Cm


Force-deformation curve resulting from a flexure test.


Figure 3.











methods section. Breaking strength, BL, I and strain have been

previously discussed. Modulus of elasticity (E) (or Young's modulus)

expresses stiffness under normal stress in the linear elastic region

(Figure 3). It is the ratio of stress to strain. It is essential

that deformation rates be included with results of flexure tests. As

loading rate is increased, long bones have increased capacity to

absorb energy (McElhaney, 1966), thus resulting in increased BS.

Young's modulus is also dependent upon the rate of deformation and

increases with increasing rates of deformation (Sedlin and Hirsch,

1966). Frost (1973) reported that bone may demonstrate nearly twice

the stiffness at high speed loading rates compared to low rates.

Very few animal nutritionists have reported loading rates in

presentation of their results.

When assessing results of mechanical properties of bone derived

from flexure tests, nutritionists must consider the nature of

material being tested (i.e., nature of preparation, wet or dry;

cancellous vs cortical bone). It was noted long ago that marked

differences exist between bone in its wet and dry states. Dry bone

has a slightly higher modulus of elasticity, is stiffer under elastic

deformation and also has a higher ultimate stress--but it is quite

brittle. In contrast, wet bone is quite ductile and allows

considerable plastic behavior (bending past elastic limit) before

fracture (i.e., dry bones are more nearly elastic and bend less upon

testing than wet bones). As little as 10 min exposure in air and

bone begins to show increased strength (Sedlin and Hirsch, 1966).

Crenshaw et al. (1981a) have reported that nutritionists can use











either dry or wet bones (if treated uniformly) when examining the

response of nutrient quality of the diet; however, wet bones would

more closely represent bones as they exist in the animal.

Mechanical properties of bone also vary greatly with the age of

the animal. Recent work with equine McIIIs (Bynum et al., 1971;

El Shorafa et al., 1979; Glade et al., 1986; Lawrence, 1986) have

shown that BS increases rapidly from day of birth until 5-7 yr of

age, then declines. Lawrence (1986) also found dramatic increases in

E of equine McIIIs in the first year of life, reaching a maximum

value at 4 yr of age (from .98 to 3.6 GPa). From 4 to 27 yr the

stiffness did not change significantly.

Several researchers have studied the relationship between stress

and percentage of ash. El Shorafa et al. (1979) showed a positive

correlation (r=.58) between BS and ash. Vose and Kubala (1959)

reported that small increases in ash dramatically increased the BS in

human femurs. These authors fit an exponential curve to their data

as they felt a linear curve would not make "biological" sense (i.e.,

linearity would have predicted that a femur with a cortical ash of

60.8% would have a BS of zero). Currey (1969) later explained the

lack of a linear relationship between BS and ash and the failure of

bone to completely mineralize in that mineralization beyond an

optimum value would decrease resilience of the bone and reduce its

ability to resist dynamic loading. Currey (1969) suggested that the

relation between bone mineral and collagen in bone matrix is

responsible for the correlations of breaking strength and ash.

Mineral is used to stiffen bone, but at low values, mineral crystals










disrupt the collagen molecules, when a load is applied. Currey

(1969) also reported that, as ash values reach 63 to 68%, the

organization of the mineral-collagen matrix produces the optimum

strength. Currey (1969) also reported a linear relationship between

E and ash content of bone. Schryver (1978) has also reported that

differences in breaking strength in horses were positively related to

specific gravity.

There are few reports in the literature that pertain to the

mechanical properties of bovine bone in bending with respect to

mineral nutrition (i.e., that follow the testing procedures

previously described), whereas thorough investigations with equine

(Schryver, 1978; Lawrence, 1986) and swine (Nimmo et al., 1980;

Crenshaw et al., 1981b) have been reported.

Early studies by Becker and Neal (1930) reported data on bone

strength of 15 mature cattle on low Ca and low P rations. Values in

McIIIs ranged from 710 Kg in a Florida native cow receiving no

supplemental Ca or P to 1581 Kg in a Guernsey cow receiving a bone-

meal supplement. "Strength" values were also reported for third

metatarsals, ribs, humeri and femora. These authors concluded that

physical and chemical analyses of bone have a definite place beside

mineral balance determinations in long term investigations of mineral

requirements. Raulerson (1964) reported "breaking strengths" of

right McIII in rams fed either .06 or .67% P (DM) of 71.9 vs

108.3 Kg, respectively. Method of testing was not reported.

Deficient rams also had smaller, lighter bones than controls. More

recently, Magomedov (1978) has reported that Black Pied heifers fed











either 20% above, 20% below or at recommended P intakes had McIII

breaking strength values of 167, 175 and 171 MPa, respectively.

Mechanical properties of bone show differential responses to

varying nutrient levels. Miller et al. (1962), working with baby

pigs, indicated that bone stress reaches a maximum before BL in

femurs. These workers fed increasing levels of Ca (.4, .8, 1.2 or

1.6%) and reported that BS increased with increasing Ca up to .8% Ca,

while BL continued to increase up to 1.6% Ca. These data indicate

that the .8% Ca level was adequate for optimum bone mineralization

but more total mass of bone was deposited at higher Ca levels.

Critical consideration must be given to these mechanical traits when

assessing requirements for various classes of livestock.

(3) Noninvasive techniques. The Ca and P status at the

skeleton of an animal can be simply determined by ash analysis of the

bones. This method, however, necessitates either surgery or

slaughter and thus may not be the preferred method of choice to the

investigator.

Bone composition has been shown to be stable except when animals

are subject to mineral deficiency or imbalance (Field et al., 1975),

compared to flexibility of bone size and density. Therefore, any

method from which all these parameters could be measured

nondestructively would be valuable.

The accurate measurement of bone mineral content (BMC) is of

interest to both clinicians and scientists. In vivo measurement of

BMC is useful in evaluating sequential changes in bone density and

other bone parameters. The quantification of bone mineral kinetics











is valuable in assessing skeletal changes that may be associated with

normal skeletal development, gerontologic demineralization,

restricted physical activity, nutritional influences, altered gravity

exposure, radiation exposure, or endocrine and renal imbalancing

conditions with secondary bone effects.

Three noninvasive techniques, radiographic photometry, photon

absorptiometry and ultrasound, have received considerable attention

and study in domestic animals over the last two decades for their

ability to predict BMC and/or bone strength (Lawrence, 1986).

a) Radiographic photometry. Quantifying the mineral content of

the skeleton has been puzzling researchers for centuries. Certain

pathologic and histologic methods were used to measure skeletal mass;

in vitro bone density measurements have been reported in the 19th

century, but in vivo techniques have been developed in this century

(Stoliker et al., 1976). The advent of radiology as a separate

discipline, due to Roentgen's discovery of the x-ray in 1895, altered

the approach to the problem.

Attempts to correlate actual bone mineral content with a

radiograph were made as early as 1920 (Griffiths and Zimmerman,

1978). The technique for evaluation of BMC is based on the principle

that the attenuation of x-radiation as it passes through bone and,

therefore, the amount of light passing through a radiograph is

related to the mineral content of the bone. Ardran (1951) and

Lachman (1955), however, indicated that the use of conventional

radiography in assessing BMC may require a 30 to 60% loss of bone

mineral before a rarefaction could be visually detected. Excised











bone or stepwedges were later (1950s) placed on radiographs for

comparative purposes. A reference standard must be exposed

simultaneously with the bone in order to make direct

photodensitometric comparison between x-rays. Several materials have

been used as reference standards. These have included bone

preparations, ivory, dipotassium hydrogen phosphate and aluminum

(Al).

Omnell (1957) stated x-rays are absorbed by Al to the same

degree as bone mineral; therefore, results can be expressed in mm of

Al or BMC. Pridie (1967) reported that standards made of materials

other than bone produce different absorption curves which may vary

with the composition of the standard material and energy level of the

x-rays. Regardless, Al stepwedges have been used successfully

(Meakim et al., 1981; Landau et al., 1985; Lawrence, 1986).

Photodensitometric BMC determinations of defleshed chicken bones

had coefficients of variation from .88 to 1.22%, similar to the

values reported by Meakim et al. (1981). In vivo precision errors as

low as 1% and as high as 15% have been reported (Lawrence, 1986).

Cohn (1981) reported that predicting ash weight by radiographic

photometry had a standard error of the estimate of 6%.

Soft tissue covering introduces much of the error in bone

density estimations (Meema et al., 1964). To avoid errors associated

with soft tissue, researchers have attempted to choose sites where

soft tissue covering is minimal. Most radiographic determinations of

BMC have been limited to the cortical area of the long bones, in

which, as Meema et al. (1964), stated that cortical bone exists in a










virtually pure form (i.e., little or no cancellous bone). This

makes the distal metacarpal a likely candidate for study in horses

and cattle. Also, since cortical bone comprises 80% of the skeleton,

information on cortical bone should reflect overall skeletal status

(Meema and Meema, 1981).

Morphometric measurements of cortical bone in third metacarpals

can also be obtained with radiography (Meakim et al., 1981; Lawrence,

1986). This allows serial assessment of changes in cortical area of

these bones (as related to changes at the periosteal and endosteal

surfaces).

Meakim et al. (1981) and Lawrence (1986) correlated radiographic

bone aluminum equivalents (RBAE) as determined by radiographic

photometry and BMC as determined by ashing of equine McIIIs from

horses of varying breed and age. They found high correlations (from

.88 to .95) using this technique.

Landau et al. (1985) studied the relationship between Ca and P

retention in 2 wk old Fresian calves and metacarpal radiographic

photometry. They found it to be a sensitive technique and concluded

it was a reliable method for evaluating mineralization in the bovine.

Correlations of radiographic measurements and BMC have been good

despite imperfection in bone geometry and variation in the

composition of compact bone (Meema and Meema, 1981). Exton-Smith

et al. (1969) reported a high correlation (r=.85) between

radiographic cortical area and bone ash per unit length of 29 third

proximal phalanxes of human subjects. Low correlations were found

for both cortical thickness and cortical thickness/width ratio, which











is similar to the findings of Suttie et al. (1983) who reported poor

correlations between cortical thicKness/width ratio (or cortical bone

index) of McIII of 69 red deer stags from 15 to 75 mo of age and

mineral status, a technique developed by Barnett and Nordin (1960).

However, it should be noted that these researchers correlated whole

bone mineral instead of BMC at the point of radiographic

measurement. Little (1984) reported that this cortical index method

provided a sensitive criterion for assessing P status in cattle.

Cattle fed either high P or low P (dietary levels not provided) were

distinguished by a cortical bone index determined from radiographs,

.68 vs .60 for high P vs low P, respectively, (p<.05). Medullary

cavities tended to be greater in the low P group. Meakim et al.

(1981) compared circular and elliptical cross-sectional indices

determined from radiographs with BMC of equine McIIIs and reported

correlations of .88 and .95, respectively. Lawrence (1986) reported

a high correlation (r=.89) between BMC and radiographic circular

index calculated using equine McIIIs of varying ages and breeds.

b) Photon absorption. A specialized technique, photon

absorption, for determining bone mineral mass utilizing the

absorption and recording of radiant gamma energy was first reported

by Cameron and Sorenson in 1963. The technique is based on the same

principle as that of the roentgenographic method, namely that the

mass of bone mineral present is directly proportional to the amount

of photon energy absorbed by the bone. As the bone is scanned, the

number of photons reaching the scintillation detector is plotted in a

transmittance curve. The area of the curve is proportional to BMC











(Smith et al., 1972). Calibration of photon absorption units is

accomplished by reference standards that simulate bone and serve as a

reference guide for BMC calculations (Mazess, 1981).

Cameron and Sorenson (1963) indicated that this method was

distinguished from radiographic photometry procedures in that 1) the

transmission of the photon beam is measured directly by counting

techniques, by means of a scintillation detector system; 2) the

photon beam is monochromatic and highly collimated; and 3) the effect

of the tissues around the bone are accounted for (i.e., fat, muscle,

cartilage and other soft tissues appear similar to each other and to

the absorption pattern of water). These authors point out that these

factors abolish errors associated with radiographic methods,

including variation in x-ray films and subsequent development

techniques, soft tissue effects, uncertain absorption coefficients

and scattered radiation.

Either single or dual photon absorption units are available

presently. In single photon absorption, the radiation source

(usually 125I) is rigidly coupled to a NaI crystal scintillation

detector system. The detector and source simultaneously scan the

bone transverse to its longitudinal axis. Soft tissue effects are

accounted for by use of a tissue equivalence bag which has a similar

absorption pattern as soft tissue. Dual photon absorption utilizes

the emission of two separate photon energies which discriminate

between bone mineral and soft tissue, thus dual photon absorptiometry

provides the advantage of not having to surround the bone with a










tissue equivalent. Radionuclide sources used in dual photon

absorptiometry include 125/241Am.

Repeatability and accuracy of bone mineral mass estimations

using photon absorptiometry have been actively studied in many

species. Using parafin blocks containing homogeneously mixed CaCO3

in known amounts, Cameron and Sorenson (1963) reported accuracy of

mineral content estimations to 3%. These same authors reported a

reproducibility of within 3% when 137 human female subjects were

scanned. Smith et al. (1972) reported coefficients of variation of

2.5 and 2.7% for cortical and trabecular bone, respectively, on 21

repetitive BMC measurements of a normal human radius over a 2 wk

period. Coefficients of variation associated with direct photon

absorption from 2.2 to 6.6% for 10 BMC/width measures on six rats

have been reported (Sanchez et al., 1981). These authors also

reported correlations between BMC/width and whole femur ash weignt of

r=.93 and dry weight of r=.96. Excellent correlations between bone

mass as determined by photon absorption and bone ash (g.cm ) as

determined by ashing have been reported in poultry (r=.93) (Meyer

et al., 1968) and in canine and equine species (r=.99) (Wentworth

et al., 1971).

Photon absorption has been used as a tool to observe the effects

of dietary concentrations of Ca on BMC and to observe

remineralization of bone following the change from a Ca deficient to

a Ca repletion diet (Krook et al., 1971; Wentworth et al., 1971).

Henrikson et al. (1970) used photon absorption to study the effect of

dietary fluoride on bone of dogs. Jeffcott (1985) recently used










photon absorptiometry to assess bone quality (BMC) as influenced by

chronological age, partial weightlessness and forced walking and

trotting exercise in horses. Also, Holmberg et al. (1985) have

conducted long term studies using photon absorption in dairy cattle

to monitor bone mineral changes in coccygeal vertebrae during

different lactations and aid in studying parturient paresis.

In vivo bone mineral analysis by photon absorptiometry in

animals has been found to be a sensitive, reproducible, noninvasive

and available method, and with the advent of large animal scanners as

described by Jeffcott (1985) will become a powerful tool for serially

monitoring changes in bone mass of large animals.

c) Ultrasound. Sound waves travel through bone with a velocity

(m-s-1) that reflects the material and structural properties of bone

(Gerlanc et al., 1975). Pratt (1982) has termed this velocity the

"effective" velocity; whereas, Jeffcott and McCartney (1985) have

used the term "apparent" velocity, as the actual path the sound waves

take through the bone has not been revealed.

The "effective" or "apparent" velocity is determined by placing

transducers that transmit and receive sound waves from an ultrasonic

wave generator on opposing surfaces of the bone. The time it takes

for the propagation of a sound wave to travel from the transmitting

transducer through the bone to the receiving transducer (t) is

divided by the width of the bone being tested (d). This calculation

yields an estimate of velocity (v) of sound through the bone.

Lawrence et al. (1986), to better approximate the flight of the

ultrasonic beam through bone, compared the "effective" velocity with










that of velocity based on area indices at the point of

transmission. They reported the more accurate the estimate of area

the higher the correlation between ultrasound and BMC.

Using fresh rectangles of bovine cortical bone, Rich et al.

(1966) studied the relationship between ultrasound and chemical

properties of bone. They reported a correlation between mg Ca-cm-2

in bovine cortical bone and ultrasound velocity of r=.99. Bokman et

al. (1983) measured ultrasonic velocities at the midpoints of McIII

of eight Angus cows and eight Hereford heifers. They reported a

correlation of .63 between bone density and ultrasonic velocity.

Recently, Lawrence and Ott (1985) reported a correlation of .92 for

BMC and ultrasound in 25 pairs of McIII from equine cadavers of

varying breeds and ages. Jones (1984) has suggested that ultrasound

is a better indicator of BS than BMC.

Abendschein and Hyatt (1970) studied the relationship between E

in standardized specimens of human cortical bone and ultrasound. The

modulus of elasticity of bone has been studied in its relationship to

the functional capacity of bone and directly related to its breaking

strength. Abendschein and Hyatt (1972) proposed that ultrasonic

velocity is a function of E and the density of bone. These authors

found a linear relationship between E determined by testing guinea

pig femurs loaded in compression and the ultrasound velocity.

Abendschein and Hyatt (1972) further state that ultrasonic velocity

can accurately quantitate E and other physical properties in bone.

Glade et al. (1986) reported BS calculated from sound velocity

measurements in equine McIIIs were within 2% of BSs measured











mechanically (r=.91). Lawrence and Ott (1985) reported a correlation

of .82 between sound velocities through soft tissue-free equine

cadaver McIIIs and their measured BS. Jeffcott and McCartney (1985)

compared ultrasonic velocities with specific gravity of McIIIs

obtained from 18 normal equine cadavers. They reported an apparent

linear correlation but noted that much of the variation in velocity

was due to age.

Ultrasound appears to be both accurate and reproducible. Rabin

et al. (1983) reported on ultrasound data collected on 262 horses.

They reported their accuracy to be better than 1% and reproducability

to be better than 0.5%. Comparable results have been reported

(Greenfield et al., 1981).

None of the techniques previously discussed (i.e., radiographic

photometry and photon absorption) that estimate bone mineral density

consider the architectural framework in which the mineral fraction

resides. Breaking strength (and other physical properties of bone)

is a function of mineral lattice and collagen orientation and

organization (Carter and Spengler, 1978), which are not detected by

x-rays or gamma-rays, but which affect sound transmission in bone.

Therefore, assessment of structural integrity, quality, change and

development in long bones may be best monitored by ultrasound instead

of techniques which measure mineral parameters of bone only (Glade

et al., 1986).

Feces

Fecal samples are easily obtained, and, since P homeostasis in

ruminants is achieved to a great extent in the gastro-intestinal










tract by controlling the secretion and reabsorption of salivary P,

fecal losses should be related to intake or absorption (Clark et al.,

1973). Several reports in the literature suggest that the status of

P nutrition in ruminants may be assessed by fecal collection methods

(Moir, 1960; 1966; Belonje, 1978). It has been suggested that fecal

grab samples from both cattle and sheep may be useful in estimating

the P content of the pasture being grazed.

Problems with the use of a fecal grab sample may be encountered

if, for example, large differences in feed intake are seen. Total

fecal output may differ, resulting in a difference in total mass of P

excreted (g.d-), but with no apparent difference in % P in the

samples. Read et al. (1986a) indicated that fecal P concentration

determined from fecal grab samples was not sensitive as a P status

indicator due to differences previously mentioned.

Cohen (1974) reported that Angus steers fed carpet grass, with

or without alfalfa and supplemented with various amounts of sodium

orthophosphate, had P intakes related to total daily fecal P

excretion (r=.98). Cohen (1974) suggests that the P intake of cattle

may be estimated from regression equations of the intake of a

specific feed source on total daily P excretion, using different

equations for different feed sources in order to eliminate

differences in digestibility. Cohen (1974) also stated that

selective grazing by sheep and the existence of plant species in a

natural or mixed pasture with varying digestibilities, which may vary

with season or time of day, make fecal collection very unreliable for

assessing the P status of grazing animals.










Moir (1966) reported seasonal fluctuations in P and crude

protein (CP) in feces from cattle in four areas of Queensland, while

using these determinations as indicators of P and protein intakes.

Fecal CP levels did not exhibit the marked drop during the dry season

shown from two of the other areas, and a markedly increased ratio of

fecal protein to fecal P occurred over this period. The fecal P was

reduced to about half the wet season level. Moir (1966) predicted

pasture contents of P and Ca from fecal samples. Lebdosoekojo (1977)

using fecal grab samples found fecal P to be highly correlated with

pasture P (r=.84) in beef cattle grazing mainly Melinis minutiflora

at Carimagua, Colombia.

Saliva

Salivary P concentration of P deficient animals was reported to

be approximately 50% that of animals supplemented with P (Clark,

1953). However, it has been suggested that salivary P concentration

is not a reliable indicator of P status due to variation in P

concentration caused by varying amounts of saliva secreted daily by

cattle (Anon., 1976, cited in Loxton et al., 1983). This conclusion

is consistent with the work of Perge et al. (1983) who found wide

variation in salivary P concentration as affected by dietary supply

of Ca and P as well as time of sampling.
















CHAPTER THREE
MATERIALS AND METHODS


General Protocol

The objectives of this experiment were to determine the effect

of different phosphorus (P) concentrations on performance of growing

beef heifers and evaluate criteria for assessing P status in these

animals. The experiment consisted of a P depletion phase (270 d), an

adaptation period to the basal P supplementation diet (10 d) and a P

supplementation phase (ranging from 245 to 492 d in length; common

experimental endpoint was day 245 of supplementation phase for

nonpregnant and 3 wk postpartum for pregnant heifers). For the

entire duration of the experiment (525 to 772 d), all animals were

housed at the Animal Nutrition Laboratory, University of Florida,

Gainesville Experiment Station.

Depletion Phase

Fifteen weaned Angus heifers,1 7 to 8 mo of age, 1603 kg

initially were randomly assigned to three pens (5 animals-pen- ) and

housed under dry lot conditions on concrete floors in a covered

barn. All heifers were allowed ad libitum intake of the P depletion

diet2 (Table 2) and free access to water throughout the 270 d

depletion phase (October 16, 1982, to July 12, 1983). The P



1 Obtained from University of Florida AREC, Quincy, FL.

2 Prepared at Seminole Stores Incorporated, Ocala, FL.












Table 2. Composition of basal diets fed during phosphorus depletion
and supplementation phases.



Amounta

Ingredient Depletion Phase Supplementation Phaseb


Citrus pulp, % 30.0 35.0
Cottonseed hulls, % 30.0 17.5
Soybean hulls, % 20.0
Coastal Bermuda Hay,
ground, pelleted, % 15.5
Cardboard paper,
ground, % 10.5 11.0
Cane molasses, % 10.0 10.0
Animal fat, % 2.5
Urea, % 2.0 2.0
Mineral premix,c % 2.0 2.0
Vitamin A and D,d % + +
Total 100.0 100.0

Chemical composition
Calcium, % .60 .62
Phosphorus, % .10 .12
Magnesium, % .32 .34
Potassium, % 1.17 1.31
Sodium, % .28 .24
Zinc, mg/kg 37.60 39.40
Copper, mg/kg 13.80 14.90
Cobalt, mg/kg .13 .15
Molybdenum, mg/kg 4.30 4.70
Crude protein, % 10.83 11.91


a Dry-matter basis (DMB).

b MonofosTM (International Mineral and Chemical Corp., Mundelein, IL)
added to the basal diet at expense of cane molasses to achieve .20%
total P in supplemented diet.











Table 2--continued.



c Supplied mg per kg diet DM (compound, element): Sodium selenite
(Na2SeO) .21, .10; Nickel sulfate (NiSO4-6H20) 8.96, 2; Stannous
chloride (SnCl212H20) 3.80, 2; Chromium chloride (CrC1 6H20) .51,
.10; Ammonium vanadate (NH4VO ) .23, .10; Potassium iodate (KIO )
.17, .10; Sodium molydbate (Na2Mo04*2H20) 12.5, 5; Cobalt carbonate
(CoCO3) .22, .10; Cupric sulfate (CuS04*5H20) 24, 4; Manganese
oxide (MnO2) 15.8, 10; Zinc oxide (ZnO) 37.5, 30; Ferrous sulfate
(FeSO4*7H20) 49.7, 10; Sodium Chloride (NaC1) 2,700, 2,700; Calcium
carbonate (CaCO ) 450, 180; Potassium Chloride (KC1) 6,350, 3,300;
Dyna-Mate (18%K, 22%S, 11%Mg; International Mineral and Chemical
Corp., Mundelein, IL) 11,200 (2,000, 2,425, 1,223).

d Supplied per kg of diet: 2200 USP units Vitamin A palmitate and
440 USP units Vitamin D3.

e By laboratory analysis.

Average phosphorus (P) content of basal diet; P content varied from
.11 to .13% during course of supplementation phase, dry matter
basis.











depletion diet was formulated to be low in P yet provide adequate

energy, protein, and other minerals and vitamins to promote .5 kg-d-

gain. The mineral mix and vitamin premix used in the depletion diet

(as well as in the supplementation phase) were prepared at the Animal

Nutrition Laboratory. Calcium to P ratio of P depletion diet was

6:1. All animals were dewormed at initiation and midpoint of the

depletion phase. Animals were bled periodically by jugular vein

puncture using 15 gauge California Bleeding needles1 (Table 3)

according to methods described by Fick et al. (1979).

Supplementation Phase

Prior to the beginning of the P supplementation phase all

heifers were allowed a 10 d adaptation period to the basal diet.2 On

July 22, 1983, 14 heifers (16 to 17 mo of age; 2106 kg, initially)

were weighed, dewormed and randomly allotted (7 animals-group-) to

one of two dietary levels of P: 1) continuation of low P (LP) basal

diet containing .12% P (dry matter basis, DMB) or 2) high P (HP) diet

which consisted of the basal diet supplemented to provide .20% total

P in the diet dry matter (Table 2). The basal diet was formulated to

be low in P yet provide adequate energy, nitrogen, other minerals and

vitamins to promote .5 kg.d gain. Calcium to P ratios ranged from

3.1:1 in the HP diet to 5.2:1 in the LP diet.

Animals were allowed ad libitum intake of their respective diets

for the initial 210 d of the P supplementation phase (July 22, 1983,



1Jensen-Salsberg Laboratories, Inc., California Bleeding Needles,
Kansas City, MO.

2 Prepared at High Springs Milling, High Springs, FL.












Table 3. Bleeding dates and blood constituents sampled during
depletion phase.



Date Constituenta


December 20, 1982 (66)b S

February 16, 1983 (124)c S,P

April 13, 1983 (180) S,P,W

May 25, 1983 (222) S,P,W

July 6, 1983 (264) S,P


a S = serum, P = plasma, W = whole blood.

b Day of P depletion phase.

c Heifer 232 died February 14, 1983, of causes not related to
experimental diets.









to February 16, 1984). Throughout this period, feed intake was

recorded daily for each group. Feed offered was reduced to 6.8

kg.head-l day-1 (as-fed) until May 10, 1984, at which time it was

further reduced to 4.5 kg-head-l*day-1 (as-fed). Feed intake was

restricted in an attempt to limit body condition in order to minimize

potential calving difficulties. Feed offered was periodically sampled

for chemical analyses. Heifers were provided free-access to water

throughout the supplementation phase. Heifers were weighed at 2 wk

intervals at 0800 h prior to feeding.

Ovarian activity of heifers was determined monthly by rectal

palpation from October 14, 1983, to March 16, 1984 (6 palpations).

Heifers were observed each morning (0730 h) for at least 30 min for

signs of sexual activity in both P depletion and supplementation

phases. Heifers were exposed twice daily (0730 and 1700 h) for a 30-

min period to a bull from October 28 to November 23, 1983. From

November 24, 1983, until removal of the bull (February 26, 1984), all

heifers were placed with the bull overnight (i.e., from 1700 to

0730 h) in a small dirt dry lot which had been cleared of all

vegetation.1 The bull had access to the heifers for a total of

120 d. After the final palpation (March 16, 1984) two heifers from

both LP and HP groups were diagnosed as nonpregnant2 and were

slaughtered on March 22, 1984, and samples recovered (Table 4).


1 Monsanto Corporation, RoundUpTm, St. Louis, MO.

2 Upon examination of the reproductive tracts of these heifers
obtained at slaughter, heifer No. 172 of the LP group was found to
be pregnant with a 55 to 60 d concepts. All other tracts were
found to possess ovaries with follicles or CL present except heifer
No. 278 of the HP group. This heifer was diagnosed as having a
prepubertal uterus at this time.







78





Table 4. Samples obtained at slaughter of
experimental animals.



1. Third metacarpus (right and left)
2. Tail coccygeall vertebrae V, VI and VII)
3. 12th rib (right and left)
4. Liver (rignt lobule)
5. Kidney
6. Heart
7. Longissimus dorsi muscle
8. Rumen contents with fluid component
9. Abomasal contents with fluid componenta
10. Reproductive tractsb


a Not collected from animals slaughtered on
3-22-84.

Only collected from animals slaughtered on
3-22-84. Tracts were visually examined
for any abnormalities that may have been
associated with the nonpregnant status of
the heifers.










The remaining pregnant heifers (5 per group; 10 total) were

maintained until 3 wk postpartum at which time they were slaughtered

and samples obtained (Table 4). Calving occurred between August 11,

1984 (birth of first calf) and November 3, 1984 (birth of last

calf). Calves were weighed at day of birth (DOB) and 3 wk of age.

Blood samples were obtained from both heifers and calves at birth and

3 wk postpartum. Colostrum samples were taken from heifers within

the first 6 h after birth of calves. Milk samples were collected

3 wk postpartum. Heifers that lost calves due to death were milked

out by hand twice daily (0800 and 1700 h) until 3 wk postpartum.

Heifers were periodically sampled throughout the P

supplementation phase (Table 5). Animal stress and excitement were

avoided or minimized during and prior to tissue sample collection.

Sample collection was performed during the early morning (from 0730

to 0800 h).

Blood samples were obtained by jugular vein puncture as

described in the depletion phase. Blood plasma was obtained using

lithium citrate (20% w/v; .1 ml-10 ml blood-1) in the collection

tubes. Blood serum was obtained using serum separation tubes.1

Whole blood was collected in plastic tubes prepared as for plasma and

frozen (-15 C) for subsequent analysis. Plasma and serum were

separated by centrifugation (2500 rpm for 20 min) 20 min after

collection and frozen (-15 C) until further analysis.



1 Becton-Dickinson Co., Vacutainer Serum Separation Tubes (SST),
Rutherford, NJ.













Table 5. Sample collection during P supplementation phase.


Datea


Samples Collectedb


October 8, 1983 (79)c P, S, W

November 18, 1983 (120) P, S, W, BB, LB, H, F

January 22, 1984 (186) P, S, W

March 15, 1984 (238) P, S, W, H, F, Sv

May 10, 1984d (294) P, S, W, BB, H, F, Sv, RF


a Does not include serum, plasma, whole blood and colostrum/milk
samples collected at parturition and 3 wk postpartum from heifers
and calves.

P = plasma, S = serum, W = whole blood, BB = rib bone biopsy, LB =
liver biopsy, H = hair, F = fecal grab sample, Sv = Saliva, RF =
rumen fluid.

c Day of P supplementation phase.

d This collection included only 5 heifers-group- (10 total) as four
heifers were slaughtered on 3-22-83.










Rib cortical bone biopsy samples were obtained using a modified

version of the surgical procedure described by Little (1972b).

Samples were obtained from the 12th rib of all animals (left side on

d 120; right side on d 294 of the P supplementation phase) using a

1.4 cm diameter trephine. Trephine depth was set to allow both

medial and lateral aspects of the intercostal appendage to be

collected. These samples were wrapped in .9% saline soaked gauze and

frozen (-15 C) until further analysis.

Liver samples were taken in vivo using the liver biopsy

technique described by Fick et al. (1979). These samples were frozen

(-15 C) immediately after collection.

Hair samples were obtained by clipping at 30 cm2 area on the

left flank of the animal using electric clippers. Hair was stored in

plastic bags until further analysis.

Fecal grab samples were obtained by using plastic rectal

palpation gloves while animals were restrained in a squeeze chute.

Approximately 100 g of feces were collected and placed in plastic

bags and frozen (-15 C) until subsequent analysis.

Salivary samples were obtained using a modification of a

technique described by Murphy and Connell (1970). Approximately 3 to

10 ml of saliva per heifer was collected using a suction device.

Saliva was transferred into plastic tubes and centrifuged (3000 rpm

for 15 min) to remove food particles and soil, then frozen (-15 C)

until further analysis.

Rumen fluid (in vivo) was obtained while animals were restrained

in a squeeze chute using a stainless steel Frick speculum (3.02 cm










inner diameter, 51 cm in length) which was placed in the mouth

through which a stomach tube (1.9 cm inner diameter, 3 m in length)

was passed into the rumen. Approximately 25 ml of rumen fluid was

collected. This was frozen (-15 C) in plastic bottles until further

analysis.

Colostrum and milk samples were collected as previously

described. Prior to collection, the udders of all cows were cleaned

to minimize possible mineral contamination. Approximately 100 ml was

collected and frozen (-15 C) in plastic bottles until further

analysis.

Samples collected at slaughter of all animals are shown in

Table 4. All soft tissue (including periosteum) was removed from the

right and left third metacarpals (McIII) following slaughter. When

removing surrounding tissues including fibrous periosteum, great care

was taken so that the mineral fraction would not be disrupted. While

removing surrounding soft tissues, McIII were always kept submerged

in .9% saline. When complete, McIII were wrapped in .9% saline

soaked gauze and frozen (-15 C) to maintain original moisture content

until further analysis. Metacarpals were prepared in this manner to

ensure that biomechanical tests would be performed on "wet" bones

which resemble more closely the bones as they exist in the animal

(Sedlin and Hirsch, 1966). Right and left 12th ribs from the

transverse lumbar process to the distal articulating cartilaginous

ends were removed and prepared in the same manner as McIII. Tails

(with hide removed) were also wrapped in .9% saline soaked gauze at

slaughter and frozen (-15 C) until further analysis.










Approximately 100 g of liver (right lobule), kidney (capsule

intact), heart and longissimus muscle were collected at slaughter

following procedures outlined by Fick et al. (1979).

Rumen and abomasal samples were collected by making a small

incision through the respective compartments and extruding material

from within into 150 ml plastic bottles and frozen (-15 C) until

subsequent analysis.



Noninvasive and Mechanical Analysis of Bone

Photon Absorption

A Lunar Radiation CorporationI analyzer was employed for dual

photon absorptiometry bone analysis which utilizes a highly

collimated radionuclide source emitting radiation at two separate

energies. As McIII were scanned, radiation was transmitted,

scattered or absorbed depending upon the amount of mineral present.

Dual photon absorptiometry is designed to account for soft tissue

effects by using gamma rays at two different energies. Metacarpals

were submerged in .9% saline which has an absorption pattern similar

to soft tissue. Whole McIII were scanned yielding information on

such parameters of interest as estimated bone mineral content (g) and

-2
bone mineral density (g-cm-2). These parameters can be estimated for

the entire bone or specific sections as the scan line is 4.5 mm in

width. Estimated bone mineral content and bone mineral density were



Lunar Radiation Corporation, Bone Mineral Analyzer Dual Photon
Rectilinear Scanner Model DP3, Madison, WI.










determined for whole McIII and two separate diaphysis sections. Five

scan lines were read (2.25 cm) at both the midpoint and 3 cm proximal

to the midpoint (approximately 1.125 cm on either side of these

points).

Radiographic Photometry

Anterioposterior (AP) radiographs were taken of all McIII with

an aluminum stepwedge exposed simultaneously as a reference

standard. Radiographs were obtained using mobile x-ray equipment2

and veterinary x-ray film3 in cassettes with compatible intensifying

screens. Voltage, amperage, exposure time, film focal distance, film

developing and processing were performed as described by Meakim

et al. (1981). To ensure uniformity, all McIII radiographs were shot

on a single day. Photometric scans of the stepwedge and bone (at the

midpoint and 3 cm proximal to the midpoint) were performed using a

densitometer coupled to a microcomputer5 and digitizer. Peak

lateral, medial and midpoint radiographic bone aluminum equivalents

(RBAE) were averaged and this value reported as RBAE. This provided

an estimate of bone mineral content.



SAtomic Products Corp., Atomic Lab Div. Center, Al Stepwedge
Penetrometer, Moriches, NY.

2 Universal X-Ray Products, Easymatic Super 325, Chicago, IL.

3 3M Corp., Animal Care Products, Standard Veterinary X-Ray Film,
St. Paul, MN.

Southern Micro Instruments Inc., Microcomp Digital Image Analysis
System, Atlanta, GA.

5 Zenith Data System Corporation, Model Z-150 PC, St. Joseph, MI.

6 Houston Instrument, Hipad Digitizer, Austin, TX.










Ultrasound

Sound travels through bone with a velocity that reflects the

material and structural properties of bone (Gerlanc et al., 1975).

High frequency ultrasound was used to evaluate BMC and bone strength

by measuring time elapsed and distance over which a standardized

pulse of sound transmitted mediolaterally through McIII traveled

(Figure 4). Ultrasound measurements were made at both the midpoint

and 3 cm proximal to the midpoint, identical to those of photon

absorption and radiographic photometry. An ultrasonic pulse

generator transmitted and received signals via matched KB Aerotech

1 MHz transducers mounted on an electronic digital caliper.2 For all

measurements a liberal quantity of coupling medium was used to ensure

good sound transmission. It was only possible to obtain consistent

transmission of sound over the region of the McIII shaft that had

reasonably parallel sides. The elapsed time between transmission and

reception of a signal (t, ps) was displayed on an oscilloscope.3 The

mediolateral diameter of McIII (d, mm) was measured simultaneously by

the electronic caliper. The "effective" ultrasonic velocity (U,

-1
m*s ) of sound pulses through the McIII was calculated using the

expression U = d/t.

Mechanical Testing

After all noninvasive tests were complete mechanical properties

of all bones (McIII and ribs) were determined by three-point bending



1 Panametrics, Pulser Receiver 5055 PR, Waltham, MA.

2 Fowler, Ultracal Electronic Calipers, Detroit, MI.

SBallantine Lab Incorporated, Model 1023AB, Boonton, NJ.




























































Diagram illustrating the use of ultrasound on bovine third
metacarpals. McIII = third metacarpal, T = transducer, DC
= digital caliper and OS = oscilloscope. Adapted from
Jeffcott and McCartney (1985).


Figure 4.