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This is to certify that the

thesis entitled

EFFICACY 0F 2S-HYDROXYCHOLECALCIFEROL
ON THE PREVENTION OF
TIBIAL DYSCHONDROPLASIA IN ROSS BROILER CHICKS

presented by
MARTIN F. LEDWABA

has been accepted towards fulfillment
of the requirements for

MASTER dggree in ANIMAL SCIENCE

 

 

{at fl/w

, A
Major professor

Date 455%;

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

LIBRARY
Michigan State
University

 

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

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6/01 c:/CIRC/DatoDue.p65«p.15

 

EFFICACY OF 25-HYDROXYCHOLECALCIFEROL ON THE PREVENTION OF
TIBIAL DYCHONDROPLASIA IN ROSS BROILER CHICKS

By

Martin F. Ledwaba

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Animal Science

2002

ABSTRACT

EFFICACY OF 25-HYDROXYCHOLECALCIFEROL ON THE PREVENTION OF
TIBIAL DYCHONDROPLASIA IN ROSS BROILER CHICKS

By

Martin F. Ledwaba

Six experiments were conducted to study the effects of 25-hydroxycholecalciferol
(ZS-(OH) D3) on growth performance, incidence and severity of tibial dyschondroplasia
(TD) and phytate phosphorus retention in Male Ross x Ross broilers grown in battery
brooders. Experiment 1 was a 2 x 3 factorial design with 2 levels of ultraviolet (UV) light
(no UV-light and UV-light) and 3 concentrations of 25-(OH)D3. In Experiment 2 chicks
were fed a TD-inducing diet and in Experiment 3-6 a normal broiler starter diet. In
Experiments 2-6 all chicks received no UV-light and their diets were supplemented with
various levels of 25-(OH)D3 ranging from O, 10, 18, 36, 40, 54, 70, 72 or 90ug/kg
depending on the experiment. Experiment 6 included a grower phase, that consisted of
birds transferred from the starter phase, in which a grower diet was fed supplemented
with 25-(OH)D3. The UV-light, 25-(OH)D3 or the combination of both improved growth
performance, phosphorus utilization and reduced rickets, severity and incidence of TD.
Supplementation with 25-(OH)D3 does not seem to improve performance in normal
starter or grower broiler diets. Severity and incidence of TD decreased linearly only in
Experiments 2 and 3 with 25-(OH)D3 supplementation. From the data we conclude that
the low TD incidence observed with 25-(OH)D3 supplementation is partly due to Ross x
Ross strain. Lower levels of 25-(OH)D3 can increase phytate phosphorus in broiler starter

diets and 40 pg/kg can improve phytate phosphorus retention in grower diets.

I dedicate this thesis to my grandfather France Ledwaba who I never got to meet before
he passed and who never had the opportunity to learn how to read and write while he was

alive.

iii

ACKNOWLEDGEMENTS

I would like to thank all the people that were helpful in this project which include
my advisor, committee members, faculty, staff, graduate assistants, farm manager and
family:

Dr. Kevin Roberson- thank you for giving me the opportunity to do a Masters Degree.
Dr. Richard Balander- thank you for the inspiration and encouragement.

Dr. Michael Orth and Dr. Richard Fulton - thank you for your support and guidance.
Angelo Napolitano- thank you for all the help and positive encouragement.

Dr. Gretchen Hill and Dr. Allen - thank you for letting me use your lab.

Jane Link, Dana Dvoracek-Driksna, Dave Maine, Ying“Jacky”Yun, and Dewey
Longuski- thank you for all the help in the lab.

A Special thanks to Dr. James E. Jay “Tabo”. -without your encouragement, guidance,
and support I could not have completed this project.

Finally, I would like to thank my parents who are the foundation of my determination and

force. I love you with all my heart.

iv

TABLE OF CONTENTS

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

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

LIST OF ABBREVIATIONS ............................................................................................ xi

INTRODUCTION .............................................................................................................. 1
CHAPTER 1

LITERATURE REVIEW ................................................................................................... 3

The Vitamin D System and 25-(OH)D3 ............................................................... 3

The Effect of Vitamin D on the Intestine .......................................................... 10

Calcium Absorption ..................................................................... 10

Phosphorus absorption ................................................................. 11

The Effect of Vitamin D on the Kidney ............................................................ 13

The Effect of Vitamin D in Bone ....................................................................... l4

Tibial Dyschondroplasia .................................................................................... 19

25-Hydroxycholecalciferol Supplementation .................................................... 32

REFERENCES ................................................................................................................. 38
CHAPTER 2

Materials and Methods ....................................................................................... 47

General Procedure ........................................................................ 47

Bone ............................................................................................. 50

Feed .............................................................................................. 5 l

Excreta ......................................................................................... 52

Blood ............................................................................................ 52

Statistical Analysis ....................................................................... 53

Results ................................................................................................................ 54

Experiment 1 ................................................................................. 54

Experiment 2 ................................................................................. 55

Experiment 3 ................................................................................. 56

Experiment 4 ................................................................................. 57

Experiment 5 ................................................................................. 58

Experiment 6 ................................................................................. 59

Starter ......................................................................... 59

Grower ....................................................................... 59

Discussion .......................................................................................................... 60

CHAPTER 3
SUMMARY, CONCLUSION AND IMPLICATION ..................................................... 77

REFERENCES ............................................................................................................... 1 10

vi

LIST OF TABLES

 

 

 

Table 1. Composition of the experimental diets 48
Table 2. Summary of Experiments 79
Table 3. Summary of Experiments 80
Table 4. Summary of results (compared to control) 81

 

Table 5. Effects of ultraviolet light and 25-hydroxycholecalciferol on performance,
serum calcium and phosphorus, and phytate phosphorus retention in 16-day
Ross X Ross broiler chicks fed a Vitamin D3 deficient diet, Experiment 1 ....... 96

Table 7. Effects of 25-hydroxycholecalciferol on performance, serum calcium and
phosphorus, and phytate phosphorus retention in 17 -day Ross X Arbor Acres
broiler chicks fed 3 Vitamin D3 deficient diet, Experiment 2. 98

 

Table 8. Effects of 25-hydroxycholecalciferol on incidence and severity of tibial
dyschondroplasia and percentage bone ash in 17-day Ross X Arbor Acres
broiler chicks fed a Vitamin D3 deficient diet, Experiment 2. 99

 

Table 9. Effects of 25-hydroxycholecalciferol on performance, serum calcium and
phosphorus, and phytate phosphorus retention in 20-day Ross X Ross broiler
chicks fed a normal broiler starter diet, Experiment 3. 100

 

Table 10. Effects of 25-hydroxycholecalciferol on incidence and severity of tibial
dyschondroplasia and percentage bone ash in 20-day Ross X Ross broiler
chicks fed a normal broiler starter diet, Experiment 3. 101

 

Table 11. Effects of 25-hydroxycholecalciferol on performance, serum calcium and
phosphorus, and phytate phosphorus retention in 20-day Ross X Ross broiler
chicks fed a normal broiler starter diet, Experiment 4. 102

 

Table 12. Effects of 25-hydroxycholecalciferol on incidence and severity of tibial
dyschondroplasia and percentage bone ash in 20-day Ross X Ross broiler
chicks fed a normal broiler starter diet, Experiment 4. 103

 

Table 13. Effects of 25-hydroxycholecalciferol on performance, serum calcium and
phosphorus, and phytate phosphorus retention in 20-day Ross X Ross broiler
chicks fed a normal broiler starter diet, Experiment 5. 104

 

vii

Table 14. Effects of ZS-hydroxycholecalciferol on incidence and severity of tibial
dyschondroplasia and percentage bone ash in 20-day Ross X Ross broiler
chicks fed a normal broiler starter diet, Experiment 5. 105

 

Table 15. Effects of 25-hydroxycholecalciferol on performance, serum calcium and
phosphorus, and phytate phosphorus retention in 17-day Ross X Ross broiler
chicks fed a normal broiler starter diet, Experiment 6. 106

 

Table 16. Effects of 25-hydroxycholecalciferol on incidence and severity of tibial
dyschondroplasia and percentage bone ash in 17-day Ross X Ross broiler
chicks fed a normal broiler starter diet, Experiment 6. 107

 

Table 17. Effects of 25-hydroxycholecalciferol on performance, serum calcium and
phosphorus, phytate phosphorus retention and incidence and severity of leg-
weakness in 35-day Ross X Ross broiler chicks fed a normal broiler grower
diet, Experiment 6. 108

 

Table 18. Effects of 25-hydroxycholecalciferol on incidence and severity of tibial
dyschondroplasia and percentage bone ash in 35-day Ross X Ross broiler
chicks fed a normal broiler grower diet, Experiment 6. 109

 

viii

LIST OF FIGURES

Figure 1. Diagram of Vitamin D3 production from 7-dehydroxycholecalciferol by
ultraviolet light 5

 

Figure 2. Diagram of Vitamin D metabolism. 7

 

Figure. 4. Effects of dietary 25-(OH)D3 with or without UV-light on 16-day body
weight. (Experiment 1). 82

 

Figure 5. Effects of dietary 25-(OH)D3 with or without UV-light on rickets incidence
at 16 days of age (Experiment 1). 83

 

Figure 6. Effects of dietary 25-(OH)D3 with or without UV-light on TD incidence at
16 days of age (Experiment 1). 84

 

Figure 7. Effects of dietary 25-(OH)D3 with or without UV-light on TD severity
(N3%) at 16 days of age (Experiment 1). 8S

 

Figure 8. Effects of dietary 25-(OH)D3 on TD incidence at 17 days of age
(EXPeriment 2) 86

 

Figure 9. Effects of dietary 25-(OH)D3 on TD severity (%N3) at 17 days of age
(Experiment 2)- 87

 

Figure 10. Effects of dietary 25-(OH)D3 on % phytate phosphorus (PP) retention at
17 days of age (Experiment 2). 88

 

Figure 11. Effects of dietary 25-(OH)D3 on TD incidence at 20 days of age
(Experiment 3). 89

 

Figure 12. Effects of dietary 25-(OH)D3 on TD severity (%N3) at 20 days of age
(Experiment 3)- 90

 

Figure 13. Effects of dietary 25-(OH)D3 on % phytate phosphorus retention (PP) at
20 days of age (Experiment 3). 91

 

Figure 14. Effects of dietary 25-(OH)D3 on TD incidence at 20 days of age
(Experiment 4)- 92

 

Figure 16. Effects of dietary 25-(OH)D3 on TD incidence at 20 days of age
(Experiment 5)- 93

 

ix

Figure 17. Effects of dietary 25-(OH)D3 on TD incidence at 17 days of age
(Experiment 6, Phase I). - - 94

 

Figure 18. Effects of dietary 25-(OH)D3 on % phytate phosphorus retention at 35
days of age (Experiment 6, Phase II). 95

 

LIST OF ABBREVIATIONS

ATP- Adenosine triphosphate
BMP- Bone morphogenetic protein
CaBP- Calcium binding protein
DBP- vitamin D binding protein
ECF- Extracellular fluid

HTD- High TD incidence

LTD- Low TD incidence

NpP- Non-phytate phosphorus

Pi- Inorganic phosphorus

PTH- Parathyroid hormone

TD- Tibial dyschondroplasia
TGFB- Transforming growth factor-beta
VBP- Vitamin D binding protein

VDR- Vitamin D receptor

xi

INTRODUCTION

Improved growth rates and feed efficiency have been the primary concerns for
broiler breeders over the past years. Geneticists have selected for these economic traits
and in turn nutritionists have made it possible to provide the necessary combination of
nutrients to optimize those traits. As the meat-type commercial poultry industry grew, so
have the birds used for production. As a result, metabolic parameters of the young
growing birds have come under heavy pressure causing muscle growth and skeletal
development to be out of synchrony. Skeletal deformities and leg weakness have become
one of the major problems to the poultry industry from the standpoint of economics and
welfare. Skeletal problems likely cause the broiler industry close to 120 million and the
turkey industry 40 million dollars in losses every year (Sullivan, 1994).

Tibial dyschondrOplasia (TD) is a common skeletal abnormality found in young
rapidly growing meat-type poultry (ducks, broiler chickens and turkeys) and is influenced
by nutrition as well as genetics (Riddel, 1981; Edwards, 1984). Tibial Dyschondroplasia
is a bone abnormality arising from a growth disorder in endochondral long bone and
lesions associated with TD were first observed by Leach and Nesheim (1965). In TD,
ossification of the hypertrophic region of the growth plate does not occur. The cartilage
which is responsible for long bone growth in young birds is not replaced by bone, but is
instead retained, leaving an island of degenerative cartilage cells that are arrested in early
hypertrophy (Hargest et al., 1985). The soft cartilage that remains in a TD growth plate
may lead to distress to the bird and as a result they are reluctant to walk. Reluctance in

walking often results in a change in feeding behavior and body weight gain is negatively

affected. As a result, the number of culls increases when birds become weak and are not
reaching target weights (Morris, 1993). Squatting or sitting can increase breast blisters,
the part of the bird that holds the most value in the poultry meat market. In addition, the
bone is more prone to deformities and breakage especially during processing which can
lead to downgrading of the carcass or condemnation (Burton et al., 1981).

Dietary supplementation with various Vitamin D3 metabolites to low calcium
diets as well as to diets adequate in calcium can reduce the incidence and severity of TD
(Edwards, 1989, 1990; Rennie et al., 1993, 1995.). The vitamin D3 metabolite that is most
commonly used as a feed additive and is available commercially today is 25-
hydroxycholecalciferol (25-(OH)D3). Feeding 25-(OH)D3 provides growth stimulation in
addition to TD prevention and the metabolite can improve phosphorus utilization in
broiler chicks. Phosphorus pollution in the soil and water has been blamed partially on
the poultry industry due to run-off from fields in which manure/litter has been used as
fertilizer. Few studies have been published on the effectiveness of 25-(OH)D3 on the
digestibility of phosphorus (Applegate et al., 2000; Angel et al., 2001).

Previous studies on requirements for 25-(OH)D3 in the diet of broiler chicks are _
mainly based on weight gain and feed efficiency (McNutt and Haussler, 1973; Cantor and
Bacon, 1978; Yarger et al 1996). However, the inconsistent results in recent studies with
25-(OH)D3 on TD incidence and severity suggest that the requirement for 25-(OH)D3
might be substantially lower than recommended. The objective of this study is to provide
more information on the effectiveness of 25-(OH)D3 in reducing TD incidence and
severity and improving phosphorus utilization without compromising performance

specifically when a marginal calcium diet is fed (85%).

CHAPTER 1

LITERATURE REVIEW

The Vitamin D System and 25-(OH)D3

Vitamin D is the collective name given to a family of compounds or related
sterols that exhibit anti-rachitic activity (curing or preventing rickets). The metabolite,
25-hydroxycholecalciferol, is one of the compounds that belongs to the vitamin D family.
Vitamin D is characterized as a fat-soluble vitamin and was first named and discovered
by McCullom in 1922 (McDowell, 1989). Before its discovery, vitamin D activity was
linked to vitamin A. This was true until the findings that cod-liver oil, which was known
to exhibit strong vitamin A and anti-rachitic activity, still preserved its anti-rachitic
activity after all vitamin A had been destroyed in the oil.

A more traditional cure for rickets, which was known long before the discovery of
the nutritional cure from vitamin D, is the healing property of UV-light. However, the
mutual relationship between vitamin D and UV-light in healing rickets was recognized
after experiments showed that UV-light exposure increased production of vitamin D in
livers of animals (Goldblatt and Soames, 1923) and stimulated vitamin D activity in
certain foods (Steenbock and Black, 1924). Identification of the vitamin D form, vitamin
D3, occurred in 1931 and the precursor to vitamin D3 (cholecalciferol), 7—
dehydrocholesterol, was first isolated from the skin in pigs in 1937 (Deluca, 1982). It was
not until the 1960’s that studies showed Vitamin D3 (the parent metabolite) could be
metabolized to different metabolites and that at least one of these metabolites functioned
as an active hormone (Deluca, 1979). The first metabolite of cholecalciferol to be

identified and isolated was 25-hydroxycholecalciferol (Blunt et al., 1968).

The vitamin D molecule including all its metabolites, is a secosteroid and its
structure consist of 4 rings of a cyclopentanoperhydrophenanthrene system in which one
ring, the B-ring, has undergone fission and is opened, distinguishing it from classical
steroids. The structure also consists of differing side chains from one secosteroid to
another. (See figure l-diagram of structure.) The most prominent secosteroids or forms of
vitamin D are ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3). These two
different forms of vitamin D are derived from pre-cursors known as ergosterol and 7-
dehydrocholesterol, respectively. Only after irradiation by UV-light, are these pre-cursors
characterized as a form of vitamin D (Norman, 1990). Ergocalciferol can be
manufactured synthetically from plant sterols. Vitamin D2 cannot be efficiently
metabolized by birds and is therefore not considered a functional metabolite in this
species (Valinetse and Bauman, 1981). In contrast, 7 -dehydrocholesterol that is derived
from cholesterol occurs naturally and is found in animal tissue. It can be synthesized in
the liver during normal steroid production then transported to the skin by a transporter
protein (Soares, 1984). Moreover, 7-dehydrocholesterol can be produced directly from
cholesterol in the epithelial cells of the skin, as well as in the intestinal wall (Klasing,
1998). The vitamin D precursor, 7-dehydrocholesterol, is more common in the skin of the
legs and feet of chickens than in the body (Koch and Koch, 1941). The oil of the preen-
gland (uropygial gland) also contains some amounts of this pro-vitamin. During preening
it may be spread onto the feathers, and is subsequently converted into vitamin D after
which ingestion may occur, but in a very inefficient way (Taylor and Dacke, 1984). The

conjugated double bond system (double bonds five to seven) in the B-ring of the

Figure 1. Diagram of Vitamin D3 production from 7-
dehydroxycholecalciferol by ultraviolet light

 

7 -dehydrocholesterol ergosterol

hy hV

 

Cholecalciferol Ergocalciferol
(Vitamin D2)
(Vitamin D3)

Adapted from Syed N asrat Imam (2001).

provitamin structure allows absorption of UV-photons, preferably of wavelength 285-315
nm, transforming 7-dehydrocholesterol to the pre-vitamin D3 (Norman, 1990). The final
and complete cholecalciferol is only formed after 2-3 days of thermal isomerization
which involves a series of transformations of its structure with rotations of the A—n'ng
(McDowell, 1989).

The cholecalciferol that is formed in the skin is absorbed into the blood and
transported by a binding protein (Gc globulin; group—specific component), which is
mostly a gamma globulin in birds, but may be an alpha and beta globulin as well in some
avian species (Dacke, 2000). The binding protein protects the vitamin from being
inactivated by oxidation. Cholecalciferol has little metabolic activity and is highly
hydrophobic. It can be stored in only limited amounts, especially in birds, mostly in
adipose tissue from where it can be released slowly during vitamin D deficiency
(Hurwitz, 1989). Cholecalciferol in circulation is rapidly carried to the liver and
metabolized into 25-(OH)D3. Although, the liver is the major site of the conversion, it
may also take place in small amounts in the kidney and intestine (Tucker et al., 1973).
During the transformation of cholecalciferol into 25-(OH)D3, a hepatic enzyme, 25-
hydroxylase, hydroxylates the 25th carbon of the side-chain (see figure 2.). The
hydroxylation primarily takes place in the rnicrosome, apparently because the
microsomal enzyme has a low Km or a high affinity for its substrate, but it can also occur
in the mitochondria especially during excessive levels of cholecalciferol. The 25-
hydroxylase requires N ADPH, molecular oxygen, cytochrome P450, and flavoprotein
(Deluca, 1979). The enzyme is one of the P450 hydroxylases and its activity does not

seem to be regulated by 25-(OH)D3 or 1,25-dihydroxycholecalciferol (1,25(OH)2D3). The

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25-hydroxylase may be under limited control and an increase or decrease in dietary
cholecalciferol or UV-light exposure results in a linear increase/decrease in plasma
concentration levels of 25-(OH)D3, respectively (Soares et al., 1995). This correlation has
made circulating 25-(OH)D3 an appropriate index for assessing vitamin D status in birds.
The 25-(OH)D3 metabolite is the major circulating metabolite, serving as the major
storage form of all the vitamin D metabolites. In comparison to cholecalciferol, the 25-
(OH)D3 metabolite is absorbed more efficiently when these metabolites are given orally,
66.5% vs. 83.6%, respectively (Bar et al., 1980). The hydroxylation to 25-(OH)D3
improves water solubility (Blunt et al., 1968) and the storage site where majority of 25-
(OH)D3 can be found, is in the blood. The half-life of 25-(OH)D3 in blood is about 3
weeks (Holick, 1990).

In circulation, 25-(OH)D3 is mostly bound to the vitamin D binding
protein(VBP). The binding affinity of 25-(OH)D3 towards the VBP is higher than most
metabolites, especially 1,25(OH)2D3 and cholecalciferol (Soares et al., 1995). Vitamin D
binding protein transports 25-(OH)D3 to the kidney where it is further metabolized. In
the kidney a second hydroxylation occurs at the 1 carbon position, and the enzyme
responsible for converting 25-(OH)D3 into 1,25(OH)2D3 is called 25-(OI-DD3-l-0t-
hydroxylase. This particular enzyme is found in the mitochondria of the proximal tubules
of the kidney. This enzyme is a mixed function oxidase and requires 3 proteins; P450-
cytochrome, iron-sulfur protein (renal ferrodoxin) and flavoprotein (Deluca, 1979).
Similarly to 25-hydroxylase, NADPH fuels this particular system. The l-a-hydroxylase
is under very strict control and is found in very low levels under normal conditions.

However, during vitamin D deficiency when hypocalcemia sets in, the parathyroid

glandsecretes parathyroid hormone (PTH), which in turn stimulates the activity of 1-0t-
hydroxylase. Hypophosphotemia also regulates this enzyme, but not through PTH. In
addition, the enzyme reaction is product stimulated, its activity being modulated by
1,25(OH)2D3 itself via a powerful feedback mechanism. The metabolite 1,25(OH)2D3 is
the metabolically active form of vitamin D and acts as a hormone in various tissues
(Soares, 1984). Moreover, 1,25(OH)2D3 is more rapid than 25-(OH)D3 in inducing the
classical effects of vitamin D (DeLuca, 1973) and this might explain why 1,25(OH)2D3
has a relatively short half-life of 4 to 6 hours in the blood (Groff and Gropper, 2000a).
During periods of low circulating levels of 1,25(OH)2D3, the activity is increased.
Whereas, when circulating levels of 1,25(OH)2D3 are in excess, activity is suppressed
(Henry and Norman, 1984). During the latter circumstances, another hydroxylation is
taking place, primarily in the kidney. This is also a mitochondrial enzyme, but this step
involves 24-hydroxylation. The 1,25(OH)2D3-24-hydroxylase has the ability to convert
25-(OH)D3 to 24,25(OH)2D3 thereby preventing the formation of 1,25(OH)2D3. The 24-
hydroxylase enzyme can also be expressed at target tissue sites of 1,25(OH)2D3. The
enzyme is very important in reducing the life span of 1,25(OH)2D3 initiating catabolism .
of the metabolite. The 24,25(OH)2D3 metabolite is rapidly excreted in chickens and
therefore believed to serve as an excretory route for excess 25-(OH)D3 (Holick et al.,
1976). The metabolite, 24,25(OH)2D3, also appears to play an important role in normal
bone formation (Omoy et al., 1978). It has been demonstrated that 24,25(OH)2D3 has
biological activity in the chondrocytes of the growth plate (Corvol etal., 1978; Suda et
al., 1985). Atkin et a1. ( 1985) suggested that 24,25(OH)2D3 promotes cartilage

maturation, differentiation and bone mineralization.

 

Many other hydroxylations can take place resulting in many different vitamin D
metabolites, of which 35 or more have been isolated and chemically identified. However,
the primary metabolites that support normal mineralization in bone and/or calcium and

phosphorus homeostasis are 1,25(OH)2D3, 24,25(OH)2D3 and 25-(OH)D3.

The Effect of Vitamin D on the Intestine

Calcium Absorption

In order for bone to function properly the bone matrix must mineralize. Several
minerals, but primarily calcium and phosphorus need to be in the blood to enter the bone
fluid. Calcium (Ca) is in the form of Ca 2+ or amorphous calcium phosphate [Ca3(P04)2]
in solution. This non-crystalline Ca will precipitate out as salts on the surface of the bone
and eventually be converted into hydroxyapatite [Calo(PO4)6(OH)6] (crystalline calcium)
to complete the ossification process. Digestion and absorption of dietary calcium
accounts for a large part of the calcium influx into the blood. This is especially true in
young birds, which are still growing and have limited calcium stores in the skeleton.
Calcium absorption takes place primarily in the small intestine particularly from the
duodenum and jejenum. (Larbier and Leclercq, 1992). Calcium is absorbed in the
intestine by two main mechanisms. One occurs by a passive, unregulated, also referred to
as non-saturable mechanism, that takes place in between the cells or enterocytes by
diffusion. This pathway is often used when calcium intake is elevated (Bronner, 1990).
The other component involves the active energy dependent or saturable mechanism and is
active when calcium levels are low in the blood. In this case the transport of Ca is across

an enterocyte and is a protein-facilitated process, which is mediated by calcium binding

10

protein (CaBP or calbindin). Calcium binding protein serves as a protein carrier across
the basolateral membrane and is regulated by the vitamin D metabolite 1,25-OH2D3
(Groff and Gropper, 2000b). When vitamin D deficient chicks were administered a dose
of 1,25-OH2D3, the nuclear receptors of the vitamin D dependent CaBP (calbindin-ngK)
in the chick intestine were saturated with the hormone (Theofan et al., 1986). As a result,
calbindin-ngK-mRNA as well as CaBP levels were significantly increased.

When interacting with an intracellular nuclear vitamin D receptor protein (VDR)
of its target tissue, in this case the intestine, l,25(OI-I)2D3 can form a VDR-1,25(OH)2D3
complex. This complex can influence gene transcription by induction or suppression of
promoter regions containing a VDR response element. Hence, 1,25(OH)2D3 can improve
calcium absorption by interacting with the DNA of the enterocyte and promoting the
synthesis of CaBP as mentioned (Groff and Gropper, 2000b). It is also believed that
1,25(OH)2D3 can stimulate Ca absorption directly by inducing changes in the basolateral
membrane, a process known as transcaltachia (Nemere and Anthony, 1990). Although,
fractional calcium absorption by the gut is fairly poor (about 30-50% of ingested
calcium) 1,25(OH)2D3 has the potential to increase calcium absorption up to 75%

(Aumaud, 1990).

Phosphorus absorption

The active metabolite, 1,25(OH)2D3, not only plays a significant role in the
calcium absorption process, but also in phosphorus absorption. Phosphorus can be
absorbed from the jejenum in the small intestine. As with calcium, absorption of

phosphorus can occur via two mechanisms; the passive unregulated mechanism which is

11

a diffusion process and/or the ATP-regulated process which is coupled to a transport
system involving sodium (Larbier and Leclercq, 1992). The hormone, 1,25(OH)2D3, is
believed to accelerate this sodium dependent transport system (Klasing, 1998). The
metabolite, 1,25(OH)2D3, interacts with a receptor in the basolateral membrane of the
duodenal loop which in turn triggers lysosomes present there. These lysosomal vesicles
are believed to transport phosphate in a similar fashion in which calcium is transported
during transcaltachia (Nemere, 1996). Also, other vitamin D metabolites such as 24,
25(OH)2D3 as well as 25-(OH)D3 may have specific receptors (Nemere, 1994). Vitamin
D, via its active metabolite, appears to also improve phosphorus absorption by increasing
the activity of alkaline phosphatase in the brushborder membrane (Groff and Gropper,
2000a). Alkaline phosphatase (zinc-dependent enzyme) helps in the hydrolysis of organic
phosphates (Bikle et al., 1979). However, its role in phosphate transport across the
membrane is unclear (Bikle et al., 1979). Davies (1970) reported a small, but gradual
increase in the activity of alkaline phosphatase when phosphorus deficient diets (0.16%
P, 1.0% Ca) were supplemented with 750 ICU (18.7 jig/kg) or 187.5 ug/kg of
cholecalciferol (Davies et al., 1970). However, when phosphorus was adequate (0.48%
available P) in the basal diet, vitamin D3 supplementation did not increase the activity of
alkaline phosphatase. The stimulation of alkaline phosphatase by vitamin D is minimal
and it is difficult to explain the fact that alkaline phosphatase is mostly found in the
duodenum where the pH is low (acidic), but phosphate absorption is highest in jejenum

where the pH is high (alkaline) (Harrison and Harrison, 1961).

12

The Effect of Vitamin D on the Kidney

Understanding the ability of vitamin D to act in the kidney, as this tissue serves a
major role in maintaining the phosphorus and calcium balance in the body is crucial. The
kidney will excrete more than 80% of the phosphorus that is absorbed by the body. In the
event of a phosphorus deficiency, reabsorption of phosphorus can help the body maintain
a positive balance, especially since reducing renal excretion by as little as 50% is
equivalent to increasing dietary intake of phosphorus by three times (Aumaud, 1990).
Calcitrol (1,25(OH)2D3) may stimulate reabsorption of phosphorus in the distal renal
tubule. Showing that 1,25(OH)2D3 directly stimulates renal reabsorption of phosphate has
been difficult, because of the secondary effect caused by vitamin D in viva i.e. increased
serum calcium and serum phosphate as well as low levels of PTH (Maxwell and
Kleeman, 1994b). In other words, when the body experiences low levels of phosphorus,
there will be increased levels of ionized calcium causing PTH levels to decrease. This
low level of PTH signals the removal of the PTH-block on renal phosphate reabsorption,
leading to increased phosphate retention (Deluca, 1979). At the same time 1,25(OH)2D3
increases due to the low phosphorus levels. There is confusion as to whether
1,25(OH)2D3 stimulates the kidney directly or indirectly by lowering PTH and hence the
removal of the PTH-block on kidney reabsorption of phosphate. Liang et al. (1982) has
shown that isolated renal chick cells have increased phosphate uptake during
administration of 1,25(OH)2D3 in vitro. Also, in humans during failure of renal tubular
reabsorption of phosphate due to disease there are corresponding low circulating levels of
1,25(OH)3D3 (Bell, 1985). Furthermore, during prolonged absence of vitamin D, the

hypophosphotemia (low plasma phosphorus) that often follows is partly due to the

13

increased clearance of phosphate as a result of reduced renal reabsorption of organic
phosphate (McDowell, 1989). Nevertheless, the reduction in P excretion when vitamin D
is administered could also be attributed to increased P deposition into the skeleton, an
event that is also stimulated by vitamin D (Maxwell and Kleeman, 1994b).

Sutton & Dirks (1978) showed that 1,25(OH)2D3 improves calcium reabsorption
by the kidney and vitamin D dependent CaBP (calbindin-D28K) is also present in this
tissue (Christakos et al., 1979; Roth et al., 1982). However, the majority of calcium
(98%) that filters through the kidney is reabsorbed without the influence of 1,25(OH)2D3
or PTH. The remaining 2% of the filtered load that is reabsorbed is believed to actually
be stimulated by PTH and 1,25(OH)2D3, both acting in concert, and not solely by
1,25(OH)2D3 (Deluca, 1982). However, mineral reabsorption by the kidney can regulate
plasma concentrations of calcium in the event of short term rises or falls in intestinal
absorption and bone losses of calcium. When there is an increase in calcium absorption
there must be a corresponding change in calcium excretion to maintain a steady state, this
is often accomplished by a decrease in reabsorption by the kidney (Maxwell and
Kleeman, 1994a). In order for bone mineralization to take place, one of the challenges is
to maintain the mineral constituents of the extracellular fluid. This task is partly

controlled by vitamin D stimulated tubular reabsorption of the kidney (Deluca, 1982).

The Effect of Vitamin D in Bone
In bone 1,25(OH)2D3 may have various actions that are indirectly involved in
mineralization, new bone formation, as well as bone resorption. Bone, which is

comprised primarily of osteoblasts, osteoclasts and chondrocytes, actively responds to

14

endocrine hormones such as 1,25(OH)2D3, Osteoblasts (bone cells) serve several
functions, one of major importance is that of bone matrix synthesis. The bone matrix is
made up largely of collagen I and many various bone proteins that are involved in the
mineralization process. The capability of 1,25(OH)2D3 to stimulate osteoblasts to
synthesize osteocalcin, a localized vitamin K-dependent protein that acts as a chemo-
attractant and recruits more osteoblasts into the bone matrix during matrix breakdown,
enhances new bone formation. Osteocalcin is a calcium binding protein that can bind to
hydroxyapatite and become part of the bone matrix prior to mineralization. Some of the
osteocalcin is also released into the blood, and can serve as an indicator of osteoblast
metabolism (Maxwell and Kleeman, 1994a).

In addition, alkaline phosphatase hydrolyses organic phosphate esters and
pyrophosphate, which are known to inhibit the mineralization process. Osteoblasts are
believed to express alkaline phosphatase and are in fact very rich in this enzyme.
Osteoblasts can also produce growth factors such as transforming growth factor-B (T GF-
B), a potent enhancer of bone formation and inhibitor of osteoclasts. Thus, 1,25(OH)2D3
serves an important role in regulating the expression of genes that produce other localized
transcription factors or bone morphogenetic proteins (BMPs), such as TGF-B, which
induce differentiation of chondrocytes and osteoblasts (Maxwell and Kleeman, 1994a).

During long bone growth, as the shape of the bone changes or is altered, a process
known as modeling is actively occurring. Modeling occurs through resorption and
formation of bone at different sites. Resorption does not necessarily follow formation so
the two processes are not coupled. If bone modeling does not take place in a normal

fashion it can result in different skeletal abnormalities in young growing poultry

15

(Watkins, 1999). Bone resorption during modeling is carried out by osteoclasts. These are
multinucleated cells derived from hemapoetic stem cells. The differentiation from stem
cell to osteoclast is induced by 1,25(OH)2D3 (Maxwell and Kleeman, 1994a). The
principal effect of 1,25 (OH)2D3 on osteoclasts in this particular case is to increase the
number of mature osteoclasts. Once the mature osteoclasts are formed, vitamin D
receptors (VDRs) in these cells become absent and no further receptor binding of
1,25(OI-I)2D3 occurs (Merke et al., 1986). Suda et al. (1992) found receptors for
1,25(OH)2D3 in osteoblasts, but not in osteoclasts. The active metabolite, 1,25(OH)2D3,
can increase the activity of mature osteoclasts, but this is an indirect effect. This indirect
effect on mature osteoclasts is mediated by 1,25(OH)2D3 via its action on osteoblasts. In
other words, 1,25(OH)2D3 stimulates the osteoblasts to produce various molecules that
recruit osteoclasts to activate bone resorption.

Endochondral ossification involves the conversion of cartilage into bone. The
initial formation of cartilage occurs in three steps. First, mesenchymal cells proliferate.
Second, once these cells begin to accumulate they produce extracellular matrix proteins,
which induce condensation of these dividing cells. In the third step there is differentiation
of the mesenchymal cells into chondrocytes. The chondrocytes secrete the cartilage
extracellular matrix. When a long bone in the young chick grows, the center of the shaft
turns into bone first. Cartilage is replaced by bone progressively outward towards the
end of the shaft. At the distal end of the shaft there is a region which is always producing
new cartilage as long as the bone grows. This front or region is referred to as the
epiphyseal growth plate (see figure 3.). During the mineralization process chondrocytes

are replaced by osteoblasts. Before this can take place, the new cartilage being formed in

16

the epiphyseal growth plate must undergo three phases. In a normal growth plate,
chondrocytes also pass through three layers: 1) In the zone of resting cells, the
chondrocytes have not yet reached full maturity. However, these are committed cells that
are actively proliferating and can be characterized by their spherical shape; 2) In the zone
of proliferating cells, the cartilage cells have differentiated into mature chondrocytes
which are still actively proliferating, but are now depositing cartilage and therefore
increasing the length of the bone. These cells have a flattened appearance and are
arranged in a columnar fashion; 3) In this zone of hypertrophic cells, chondrocytes have
once more differentiated into spherical cells, increasing in size mainly by fluid entering
the cytoplasm. These cells have become large (interstitial growth) and are actively
depositing calcium in the extracellular matrix. Chondrocyte proliferation in this region
would normally be limited by apoptosis or programmed cell death. The hypertrophic
chondrocytes are more vulnerable to the invasion of capillaries from the periosteum. This
vascularization of the hypertrophic region allows blood to bring in more calcium and
osteoblasts, which secrete bone matrix onto which the calcium can precipitate for
calcification. This process is crucial for bone mineralization to take place, hence
replacement of cartilage by bone (Hargest et al., 1985).

Certain vitamin D metabolites, especially 1,25(OH)2D3 have a direct effect on
these chondrocytes. The hormone, 1,25(OH)2D3, stimulates differentiation of
chondrocytes, promoting the normal development and maturation of cartilage.
Farquharson et al. (1993) showed that 1,25(OH)2D3 promotes proliferation as well as
differentiation of chondrocytes in vivo and in vitro. Studies have shown that 1,25(OH)2D3

and 24, 25(OH)2D3 can be produced locally by chondrocytes in the growth plate (Suda et

17

 

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18

al., 1985). In a different study, Farquharson et al. (1995) isolated chondrocytes, which
were cultured and then examined to see if chondrocytes had the ability to metabolize
exogenous 25(0ng The chondrocytes converted 25-(OH)D3 to 24,25-(OH)2D3 but not
to 1,25(OH)2D3. However, there is controversy over which is the main metabolite
participating in Chondrocyte differentiation and whether the metabolites produced
endogenously (locally) or by renal synthesis are the one’s that influence Chondrocyte
differentiation (Farquharson et al., 1995). Chondrocyte differentiation is also known to be
stimulated by TGF—B, which is believed to be regulated by 1,25(OH)2D3.

Schwartz et al. (1988) showed that 1,25(OH)2D3 as well as 24,25-(OH)2D3 could
directly and non-genomically (no protein synthesis involved) stimulate the activity of
alkaline phosphatase of isolated rat Chondrocyte membranes (Schwartz et al., 1988).
Alkaline phosphatase is believed to be produced by matrix vesicles that are present in the
cartilage matrix. Matrix vesicles are described by Wuthier (1988) as a lipid enclosed
microenvironment produced by chondrocytes, that contain enzymes and ion-transport
proteins for phosphorus and calcium (Ca2+), important in making apatite. Also,
1,25(OH)2D3 stimulates calbindin synthesis by chondrocytes which leads to calcium
accumulation and cartilage calcification. The vitamin D dependent CaBP has also been
detected in bone tissue of chicks (Christakos and Norman, 1978) and in growth plate

cartilage of chick tibias (Zhou et al., 1986).

Tibial Dyschondroplasia
TD is a non-infectious metabolic bone disease that is especially prominent in fast

growing strains of meat -type poultry. Leach and Nesheim (1965) first described the

19

characteristics of the abnormality in 1965. The authors observed an abnormal cartilage
formation in chicks that had been fed a purified basal diet (98 ICU/kg cholecalciferol).
They described it as a mass of opaque cartilage irregular in size situated below the
epiphyseal growth plate in the proximal end of the tibiotarsus and tarsometatarsus. Siller
(1970) noticed a similar or identical cartilage abnormality in chicks of 5 weeks of age.
After histological examination of the growth cartilage in the proximal region of the tibia
of broilers, the investigator observed that there was a thickening of the hypertrophic
cartilage layer and that this zone extended distally into the zone where normal
ossification would take place. The chondrocytes of the mass of the abnormal cartilage
appeared shrunken and degenerative which extended into the region of diaphysis
replacing the trabecular bone that would normally form there. The investigator added that
the metaphyseal areas were less vascularized by blood vessels. He named this bone
disease Tibial DyschondrOplasia. Similar results were observed by Itakura and Goto
(1973) in heavy strain broiler chicks and by Wise and Nott (1975) in meat type ducks.
Several studies on commercial broiler chicks confirmed that the characteristics of TD,
especially the failure of vascularization of the excessive mature cartilage (pre-
hypertrophic chondrocytes) (Riddell, 1975b; Riddell et al., 1971). Poulos et a]. (1978)
found that pre-hypertrophic chondrocytes in TD lesions did not reach full maturity or
hypertrophy. Hargest et al. (1985) actually saw a 40% reduction in cell size of
chondrocytes in the hypertrophic region of TD lesions. Furthermore, this group detected
necrotic cells adjacent to the early hypertrophic cells of the growth plate. Some apoptotic
cells were found occasionally in less severe lesions, but were totally absent in severe

lesions indicating that chondrocytes have the capacity to undergo normal physiological

20

death if conditions are favorable. Leach and Lilbum (1980) observed that chondrocytes
of TD lesions had reduced oxidative capacity compared to normal tissue. Similar results
were obtained by Hargest et al. (1985), who saw signs of energy depletion and low
calcium and phosphorus in chondrocytes of the hypertrophic region. Hargest et al. (1985)
further demonstrated that growth plate cartilage in TD lesions have very low calbindin
and alkaline phosphatase concentrations when compared to normal growth plates. The
low or absent levels of calbindin in TD lesions may explain the lack of intracellular
calcium deposits in the bones affected by TD (Xu et al., 1991). Many biochemical
studies of the tibial growth plate were conducted in the 1990’s in an attempt to
understand the mechanism of TD. However, the exact mechanism and primary factor
involved in the development of TD remains unclear. Orth et al. (1991) hypothesized that
the increased collagen crosslinks observed in the TD lesion inhibited enzymes such as
collagenases which are important in the degradation of matrix, allowing the cells to
expand (undergo hypertrophy), and aid in vascularization for metaphyseal blood vessels
to invade.

Ohyama et al. (1997) suggested that TD development also involved a defect in
apoptosis (programmed cell death). The signals that trigger the transition from
hypertrophy to apoptosis are still under investigation. Nevertheless, there is
overwhelming evidence and a general agreement amongst most investigators that the
cartilage plug that accumulates in the TD lesion is a consequence of an interruption in the
timing and sequence of the episodes that takes place during long bone growth. The
inability of chondrocytes to undergo hypertrophy only permits them to reach a

transitional state with no apoptosis, leaving a poorly mineralized and inadequately

21

vascularized cartilage in the metaphysis. The cartilage accumulation is due to a lack of
degradation rather than an increase in the rate of production of chondrocytes.

After Leach and Neisheim’s discovery in 1965, more reports of cartilage
abnormality were reported in the 1970’s from England to Australia, Canada, Japan, South
Africa and the USA. Although Leach and Neisheim (1965) did not notice any signs of leg
weakness or lameness in their chicks with TD lesions at 2—4 weeks of age, the majority of
these reports associated this defect with lameness and leg deformities of meat-type
poultry causing a decline in commercial value especially after processing. Siller (1970)
might not have identified the cartilage abnormality had he not observed signs of unusual
posture, squatting, lameness and reluctance to walk in broilers at 5 weeks. Riddell (1971)
observed the lesion in broilers of commercial flocks only after having performed
radiographic studies at 9 weeks. They had initially seen clinical signs such as reluctance
to move, stilted gate, marked bowing of the tibia and even birds in a crippled state.
Radiography that was repeated on these chickens at 17 weeks of age showed partial
resorption of the abnormal cartilage and by 21 weeks it was completely gone. In 1972 at
a Japanese poultry farm, inside barns that had no windows, rapidly growing heavy breeds
showed clinical signs of TD at 35 days of age. Signs included leg deformity and unusual
gait (Itakura and Goto, 1973). Since the 1970’s, the growth rate of birds has increased
almost two-fold and the clinical signs of TD may not always be detected before
processing. However, clinical signs such as lameness, severe leg weakness, squatting
(sitting on hooks), abnormal posture and reluctance to move are often associated with TD
and are still occasionally seen in commercial flocks. Bowing of the tibial head has also

been attributed to TD (Riddell, 1975b), but this may also occur due to excessive weight.

22

If lesions persist this may lead to fractures and weight loss (Poulos et al., 1978). Prasad
(1972) observed a 25% weight loss in afflicted birds compared to normal birds. However,
birds that are afflicted with a cartilage abnormality usually have similar body weights
compared to birds without the cartilage abnormality. Under commercial conditions, birds
that have a severe cartilage abnormality and as a result have difficulty in moving, often
do not reach the feeders. Hence, the result is reduced feed efficiency and body weight.

More often TD lesions are subclinical and such lesions may only be seen after
closer examination of the growth plate either by the naked eye, histopathology, or by
using radiography or a lixiscope. Thorp et al. (1991) argued that naked eye assessment is
an inadequate means of identifying TD. The authors noticed that the lesions that
demonstrated the classical characteristics of TD, as examined by the naked eye, actually
did not exhibit the proper cellular morphology native to TD after histopathologic
examination. Nevertheless, many of the early studies have been assessed by the naked
eye and are often still performed this way. Tibial Dyschondroplasia lesions may first
appear at 14 days of age (Leach and Nesheim, 1965; Riddell, 1975a; Poulos et al., 1978;
Kling, 1985). In more recent studies, (Edwards, 1985) detected TD lesions in birds as
early as 7 days of age while Elliot et al. (1992) observed TD lesions in birds at 6 days of
age. The TD condition is reversible and spontaneous resorption of the TD lesion can
occur after 6 weeks of age (Poulos et al., 1978).

The incidence of TD in broiler chicks varies tremendously from flock to flock.
Different frequencies have been reported since the 70’s. Siller (1970) stated that the
incidence of clinical characteristics of TD in the fowl were in the range of 1-2 % as

suggested by breeders in England. After having visited several commercial poultry farms

23

in the USA in 1971, Prasad et al. (1972) reported about 30-40% incidence of clinical
signs of TD. Itakura and Goto (1973) reported approximately 1-5% incidence at a
Japanese poultry farm of about 28,000 broiler chickens reared in windowless houses.
However, subclinical cases of TD might have existed in these flocks, but were not
included in those numbers. Hemsley (1970) described the incidence of subclinical TD to
‘ be greater than 7 % in broiler chickens in Australia. Similar results were observed in
Western Canada (Riddell et al., 1971). When 200 birds in Australia were randomly
selected from each of 3 different processing plants and examined for TD, incidences of
35%, 27% and 14% were reported (Burton et al., 1981). Consideration to the difference
was given due to the different weight and age of birds from one plant to another. The
weight and age at which the birds are being examined will inevitably influence the
incidence of TD, but genetic factors also play a major role. Leach and Neisheim (1965)
actually increased the incidence of TD through genetic selection. The authors developed
a high incidence strain of chickens with 41 % TD incidence and a low incidence strain
with 16% TD. Since then other investigators have verified this and genetically
manipulated chicken’s predisposition to the development of TD (Riddell, 1976; Sheridan
et al., 1978).

The initial observation by Leach and Neishem (1965), that males are more
susceptible to TD than females has been observed by most studies involving tibial
dyschondroplasia. Friedman (1977) stated that the abnormality occurred exclusively in
males. Wise and Jennings (1972) observed the same thing in ducks as well as chickens.
However, Riddell et al. (1971) did not see any marked differences between females and

males. The reason for the high incidence of TD in males of meat-type poultry may be the

24

faster growth rate that exists in males compared to females. Overall, the issue of growth
rate and body weight gain was initially thought to be one of the primary factors in the
development of TD. Riddell (1971) noticed that endochondral bone growth took place the
fastest in the proximal tibiotarsus and proximal tarsometatarsus indicating that the fast
growth in meat type chickens could be a cause of TD (Riddell et al., 1971). Wise and
Jennings (1972) also discussed the possibility that TD was caused by a rapid early growth
rate by chicks and that this weight exerts excessive pressures on the growth plate
cartilage resulting in a failure of metaphyseal blood vessel penetration (Wise and
Jennings, 1972). Riddell (1975b) tested the hypothesis that TD might be caused by the
excessive weight by inducing unilateral lameness in broiler chicks. Tendons of the
tibiotarsal joint were severed on one leg whereby more weight was placed on the other
leg due to the resulting limp. Results showed that weight was not the primary factor in
the development in TD. A weight -bearing study conducted by Cook et al. (1980)
confumed that weight had no direct effect on TD incidence. In another experiment, oat
hulls were added to a commercial broiler starter diet to slow growth of chicks and in
which the growth plates were measured, then compared to chicks fed only the starter diet
(Riddell, 19753). There was only a small correlation between growth rate of the growth
plate and the development of TD. Riddell (1975a) suggested that growth rate is only a
contributing factor rather than a primary factor. However, Poulos et al. (1978) noticed
that when broilers were fed a high-energy diet, the birds had a higher growth rate and a
higher incidence of TD than birds fed a low energy diet which had lower growth rate and
a low incidence of TD. The author was not clear on whether the high incidence of TD

was due to higher growth rates or due to the calcium levels which appeared to be higher

25

in the high-energy diet after analysis, 1.63% Ca vs 1.43% Ca, respectively (Poulos et al.,
1978). The author believed that excessively high calcium levels might increase TD
incidence.

Nevertheless, studies where feed restriction has been implemented on commercial
broiler chicks (Huff, 1980; Edwards and Sorensen, 1987; (Roberson et al., 1993) the TD
incidence was significantly reduced. However, in the study by Lile et al. (1989) the
body weights of these fasted birds at 28 days of age were not less than the birds that were
fed ad libitum (control). This is in contrast with Edwards (1987) who saw a 15%
reduction in BW gain at 20 days of age and Roberson et al. (1991) who saw a 6%
reduction, both correlated with reduced TD incidence. Roberson et al. ( 1991) also noticed
a greater accumulation of Ca in the growth plates of feed deprived birds. The timing of
the deprivation seems to be important in reducing the incidence of TD, suggesting that
feed deprivation is most effective during the starter period between ages 4 to 20 days
(Edwards and Sorensen, 1987) or 14 through 21 (Roberson et al., 1991). Elliot et al.
(1994) reported that feed withdrawal improved bone mineralization and decreased TD
incidence as well as severity. The author also reported seeing significantly increased
plasma 25(OH)D3, which is positively correlated with dietary calcium and dietary
vitamin D unlike 1,25(OH)2D3, which is negatively correlated. Sufficient supply of
calcium in the diet will decrease plasma 1,25(OH)2D3. Feed withdrawal will do the same,
indicating that calcium utilization is enhanced under these conditions. This would explain
the improved bone mineralization and possibly Roberson’s (1991) observation of Ca
accumulation in growth plates of feed deprived chicks. Slowing the growth rate may

result in a very low incidence of TD and may even prevent it from occurring. However,

26

in the meat-type industry, this is undesirable as higher body weights give higher dressing
percentage and convert into more profit.

In addition to the amount of feed given, the composition of a diet fed to broilers
may contribute significantly to the development of TD. Several nutrients when deficient
or in excess have been shown to induce TD: an excess of methionine metabolites such as
homocysteine, cysteine and cystine, but not methionine by itself (Orth et al., 1992); an
excess of histidine (Andrews et al., 1989); an excess of chloride (Leach and Neisheim,
1965); an excess or deficiency of vitamin D including some of its metabolites (Edwards,
1984); an excess or deficiency of calcium and phosphorus (Edwards and Veltmann,
1983). The nutrients that make the most impact on TD development, when their levels are
increased or decreased individually or at the same time in the diet, are vitamin D, calcium
and phosphorus. When Leach and Neisheim first observed TD, the diet that was fed to
these chicks appeared to be adequate for all nutrients. However, when a diet composed of
corn and soybean meal was supplemented with minerals and vitamins and fed to broilers,
it prevented the occurrence of the cartilage abnormality. Despite the fact that they tested
the levels of individual vitamins and minerals in the diet, they were unsuccessful in
identifying a single nutritional factor affecting the occurrence of the cartilage
abnormality. Groth and Frey (1966) observed that the calcium and phosphorus in the
bone ash of chick tibias showed a relationship with the mineral content of the diet.

Leach and Neisheim (1965) realized that changing the mineral mixture of the diet
had an effect on the acid-base and/or anion/cation balance, which affected the TD
incidence. A high chloride (anion) diet increased TD incidence and sodium bicarbonate

(cation) decreased TD incidence. The possibility that an imbalance in calcium to

27

phosphorus ratio was the cause of TD or at least the cause of leg weakness was
considered (Poulos et al., 1978). Hedhammer (1974) had reported that high intakes of
calcium had resulted in osteochondrosis in dogs. The lack of studies on calcium and
phosphorus role in the incidence of TD prompted Edwards and Veltmann (1983) to
conduct studies in this area. These authors observed that when a basal diet (11001CU/kg
cholecalciferol) with high calcium levels was fed to broiler chicks, TD incidence
decreased. Whereas when phosphorus was increased, TD incidence increased. Low levels
of calcium and high levels of phosphorus increased TD incidence, showing 39% TD
incidence with 0.8% calcium and 0.75% phosphorus present in the basal diet. Phosphorus
seems to upset the acid balance in the similar way as high chloride diets. Lilbum et al.
(1989) also noticed a strong positive relationship between levels of available phosphorus
and TD and expanded on the acid balance theory. In another study (Edwards, 1984)
confirmed that an increased calcium: phosphorus ratio was related to a decrease in TD
incidence and that if calcium was increased in combination with a phosphorus increase,
thus maintaining a 2: 1 ratio, it would result in a decrease in TD incidence (13% TD
incidence with 1.1 calcium (Ca) and 0.55% available phosphorus (aP)). In other words, it
is the ratio of Ca: aP, rather than total calcium plus phosphorus in the diet that is of prime
importance in the expression of the TD lesion. Riddell (1987) also confirmed the
observation that an increased calcium: phosphorus ratio in the diet reduced TD incidence
and severity and that an increasing phosphorus level relative to calcium is the cause of
greater incidence of TD rather than low calcium levels (Riddell and Pass, 1987). The
author reported 27% TD incidence in 4 week old chicks fed a starter diet containing 1.4

% Ca and 0.75% aP compared to 70% TD incidence in chicks fed a diet containing 0.8%

28

Ca and 0.75% aP. Roberson et al. (1993) showed a significant decrease in TD incidence
and severity when the Ca : P ratio was kept at about 2:1. Although, this study exhibited
TD incidences higher than normal the authors attributed this to the fact that the battery
brooder had no fluorescent lights and likely the amount of cholecalciferol (400 ICU/kg)
was inadequate under such conditions.

The effects of cholecalciferol in the diet, presence of ultraviolet light or the
combination of the two and its relationship with calcium and phosphorus levels on the
development of TD are nutritional and environmental factors that have been studied
extensively. The relationships of these factors have been long known to be highly
important in growth performance and bone development of chicks. Most of the early
studies attempting to establish vitamin D requirements were focusing on its effect on
phosphorus utilization as well as its antirachitic effects since vitamin D deficiency was
linked to rickets and therefore the parameters measured were often weight gain, feed
efficiency and bone ash. Waldroup et al. (1963) conducted a series of experiments with
broiler chickens in battery cages without any UV-light and fed basal diets with various
Ca : P ratios and levels of cholecalciferol. This group noticed that when phosphorus was
low (0.48 %) and Ca: P ratio was 1.4 : 1 or 1.8 :1 the body weight and bone ash was
depressed compared to a 1 : 1 ratio. However, if phosphorus was increased, there was a
need for increased ratios in order to see an increase in bone ash and body weight. As
mentioned earlier, this finding was later found to pertain to TD incidence as well.
Waldroup et al. (1964) also noticed that increasing levels of vitamin D were beneficial in
significantly increasing both body weight and bone ash, suggesting that the optimum

level of vitamin D3 to achieve this improvement for broiler chicks was 826 ICU/kg

29

compared to NRC (1960) recommended levels of 2001CU/kg. Waldroup (1965)
concluded that if calcium levels were below 1.0%, cholecalciferol levels needed to be
higher than 200 ICU/kg.

With the discovery and isolation of other vitamin D metabolites in the late 1960’s,
several studies were conducted on chicks using some of these various metabolites.
McNutt & Haussler (1973) fed leghom cockerelles (male chicks) a vitamin D deficient
diet and supplemented the birds with cholecalciferol, 25-(OH)D3 or 1,25(OH)2D3. Results
showed that 25-(OH)D3 gave a better growth response than either cholecalciferol or
1,25(OH)2D3. The 25-(OH)D3 metabolite was also more effective at increasing percent
bone ash over the control diet than the other two metabolites, at least at lower levels of
supplementation. The results suggested that overall, 25-OHD3 seemed to be the. most
nutritionally effective metabolite. McNaughton et al. (1976) attempted to establish the
25-(OH)D3 requirement in broiler chicks and reported a significant increase in tibia ash
when 25-(OH)D3 was added to a diet low in phosphorus (0.45%), compared to when
cholecalciferol was added in similar amounts (198 ICU or 300 ICU/kg). However, no
differences in bodyweights were observed. Cantor and Bacon (1978) saw a significant ,
improvement in weight gain and feed: gain when feeding broilers chicks 25-(OH)D3
compared to similar levels of cholecalciferol (1.25, 2.50 or 5.0 rig/kg).

The prevention of TD in poultry had not been extensively studied in the 1970’s
although there were speculations on the involvement of vitamin D metabolism in the
development of TD. Prasad (1971) attempted to eliminate the abnormal cartilage
formation by adding dicalcium phosphate and/ or adding cholecalciferol to the drinking

water, but without success. Sunde (1975) conducted several experiments on commercial

30

male poults and looked at the effects of cholecalciferol and 25-(OH)D3 on leg disorders.
The author did not clearly indicate that the leg disorder studied was TD. However, he
referred to similar clinical signs often associated with the leg abnormality in broilers,
assuming it to be the same condition observed by Hemsley (1970). Differences in
incidence of the leg disorders in 3-4 week old poults supplemented with 10 rig/kg of 25-
OHD3 or 25 rig/kg of cholecalciferol were not great. However, Sunde (1975) stated that
the diet containing 25-OHD3 appeared to give lower leg disorder incidence, and increased
bone ash and body weights. The author speculated that genetically there was an impaired
ability to convert cholecaliferol to 25-(OH)D3 or 1,25(OH)2D3 by young birds. Saveur &
Mongin (1978) also proposed that vitamin D metabolism played a part in the
development of TD. These authors had noticed that the metabolic acidosis reduced the
kidney’s ability to convert 25-(OH)D3 to 1,25(OH)2D3 by 40% in vitro and in viva.
Metabolic acidosis impairs normal cartilage maturation. In addition, vitamin D deficiency
was clearly linked to rickets, and the similarity between TD and rickets seemed great at
that time. Edwards began to test the effects of vitamin D metabolites on the incidence and
severity of TD in the early 1980’s. Broiler chicks that had received 0.2 ml of either 24,
25(OH)2D3 or 1,25(OH)2D3 each day showed no response (Edwards, 1984). The initial
conclusion that there was an impaired conversion of 25-(OH)D3 to 1,25(OH)2D3, leading
to a deficiency of this active metabolite and leads to a higher TD incidence was disputed
at that time by this author.

Edwards (1989) conducted a study in which broiler chicks were fed a TD
inducing diet (0.75% Ca and 0.76% P) supplemented with cholecalciferol and/or 25-

(OH)D3 or 1,25(OH)2D3. The effects on bone ash and the development of TD on birds

31

that had received no vitamin D by 16 days of age were described as very dramatic despite
the fact that they had received UV-light. When a vitamin D deficient basal diet was
supplemented with either 1100 ICU/kg cholecalciferol, 10 jig/kg 25(OH)D3 or 10 rig/kg
1,25(OH)2D3, all significantly reduced the TD incidence from 69% to 42, 28 or 3%,
respectively in one experiment. Supplementation of 25(OI-I)D3 did not show any
evidence of being more effective in reducing incidence or severity of TD, increasing bone
ash, increasing body weight gain or improving feed efficiency than supplementing
cholecalciferol at these levels. The metabolite 1,25 (OI-I)2D3 significantly reduced TD
incidence over the other two metabolites. However, in another experiment,
supplementation of either vitamin D metabolite did not significantly improve any of the

measured parameters.

25-Hydroxycholecalciferol Supplementation

The availability and the cost of producing vitamin D metabolites in the 1970’s
and early 1980’s had hindered investigators from conducting more studies on the
potential of usage of 1,25(OH)2D3 or 25-(OH)D3 as a supplement in commercial poultry
diets. However, by the 1990’s it was clear that overall, 25-(OH)D3 and 1,25(OH)2D3 had
many potential benefits including reduction in TD incidence and severity. Along with the
rapid growth of the poultry industry, problems such as environmental contamination due
to excess phosphorus in the manure has increased tremendously. This has become a large
issue especially in agricultural states practicing intensive livestock production. Much of
this can be blamed on the organically bound phosphorus called phytate or phytic acid.

Monogastric animals, such as chicks, cannot efficiently utilize the phytate and as a result,

32

phosphorus excretion can be very high in the feces. Poultry diets are mainly composed of
cereal grains that often have high phytate content ranging from 60 to 80% of the total
phosphorus found in the plant (Simon et al., 1990). In addition, because of the importance
of P in energy metabolism and bone development it must be present in feed in sufficient
quantities to meet the bird’s requirement. This has traditionally been achieved by
supplementing inorganic phosphorus, which results in increased P excretion (Sebastian et
al., 1998). Simply reducing the levels of phosphorus in the diet will compromise the
requirements to achieve optimum performance and bone development, possibly leading
to bone abnormalities such as tibial dyschondroplasia (van der Klis and Versteegh, 1999).

For many years vitamin D has been known to have the property of improving
phytate phosphorus digestibility (Mellanby, 1950). When cholecalciferol was added to a
diet low in P and Ca and fed to broiler chicks, the hydrolysis of P—phytate was enhanced
and phosphorus utilization was improved up to 77% (Mohammed et al., 1991). Studies on
phytate retention with other metabolites have showed positive results on this criterium.
McNaughton and Murray (1990) indicated that when 25-(OH)D3 was added to the feed it
was more effective in utilizing P than when cholecalciferol was added in similar amounts.
Supplementation with 1,25(OH)2D3 has also proved to be effective in increasing
phosphorus retention significantly from 31 to 68% (Edwards, 1993).

Supplemention of high levels of cholecalciferol to poultry feeds has not been able
to yield the same positive results as lower levels of 25-(OH)D3 or 1,25(OH)2D3. The
relative activities amongst cholecalciferol, 25-(OH)D3 and 1,25(OH)2D3 have been
documented Their potency can be placed in the order of 1,25(OH)2D3 as being the most

potent, and then 25-(OH)D3 and cholecalciferol as the least potent. These studies show

33

that when compared to cholecalciferol, the 25-(OH)D3 metabolite tend to be in the range
of about 1 to 4 times as active (McNaughton et al., 1976; Sunde, 1975) while
1,25(OH)2D3 range from 2 to 13 times as active depending on the criteria measured
(Boris, 1977; Edwards, 1989; Haussler and Rasmussen, 1972; Myrtle and Norman,
1971). As far as reducing TD incidence, 1,25(OH)2D3 has been the most successful
metabolite, and the most efficient dosage level in TD prevention from a safety standpoint
appears to be as low as 5 rig/kg (Rennie et al., 1993). Although, 10 jig/kg of 1,25(OH)2D3
has been shown to be more consistent in reducing both genetically induced TD (Thorp et
al., 1993) and nutritionally induced TD (Edwards, 1989, 1990; Roberson and Edwards,
1994), this particular level is regarded as slightly toxic. Rennie (1993) saw tendencies of
hypercalcemia and some growth depression with higher levels (10 jig/kg) of
1,25(0H)2D3. In combination with high calcium diets broilers are especially sensitive to
1,25(OH)2D3 (Edwards et al., 1992; Rennie et al., 1995). Due to the potency of
1,25(OH)2D3 there are major safety concerns for using this metabolite as a dietary
supplement in poultry feeds. In addition 1,25(OH)2D3 is quite expensive to produce, a
tightly regulated hormone, and currently is not approved for use in commercial poultry
feeds. In contrast, 25-(OH)D3 is not considered a hormone and can be produced fairly
cheap today and is available commercially. Therefore, 25-(OH)D3 has been considered a
good alternative to 1,25(OH)2D3 as a vitamin D supplement in feed. Comparative studies
between 1,25(OH)2D3 and 25-(OI-I)D3 have demonstrated that 25-(OH)D3 can be fed up
to 690 rig/kg for long periods of time without adverse effects (Yarger et al., 1995).
Rennie and Whitehead (1996) fed up to 250 jig/kg of 25-(OH)D3 and observed a

reduction in TD incidence from 21% to 5% with no negative side effects. Full renal

34

tubular calcification was observed in chicks, but only after levels as high as 1000 pig/kg
25-(OH)D3 had been fed for 14 days. However, minor signs of renal tubular calcification
had been observed with as little as 100 rig/kg 25-(OH)D3. Supplementation of 25-(OH)D3
to poultry diets to reduce TD has often given inconsistent and inconclusive results.

The development in technology allowed for the synthesis of 25-(OH)D3 in
kilogram quantities in the 1990’s. Amoco Bioproducts Corporation began formulating
25-(OH)D3, into hydrogenated vegetable oil-based beadlets and mixing it into a premix
using ricehulls as a carrier (Yarger et al., 1995). The basal level recommended for 25-
(OH)D3 was in the range of 50 to 70 jig/kg. However, 69 jig/kg was ultimately chosen as
the appropriate level based on studies conducted by Yarger et al. (1995) who showed that
supplementing feed with 25-(OH)D3 at 69 jig/kg is adequate for maximal body weight
gain and feed efficiency when the basal diet contains no cholecalciferol. However, the
only study based on TD was conducted by Edwards ( 1989) who had observed that 10
ug/kg 25-(OH)D3 reduced TD incidence and severity in low calcium diets, but results
were inconsistent. Rennie and Whitehead (1996) later concluded that higher
concentrations than Edwards (1989) were needed to effectively reduce TD. The authors
noticed that 75 pig/kg significantly reduced TD incidence to 10% with diets lacking in
cholecalciferol, but adequate in calcium. In 1996, Mireles et al. ( 1996) fed broilers 68.9
jig/kg from day 0 for 21 days and reported a significant reduction in TD incidence.
Furthermore, TD severity progressively decreased when fed over a period of 49 days.
When Mitchell et al. (1997) fed broilers selected for low incidence of TD (LTD) several
different levels of 25-(OH)D3, the group demonstrated that supplementing with as low as

5 jig/kg 25—(OH)D3 to high calcium (1.0 % Ca) diets significantly reduced TD incidence

35

and severity. The highest supplementation level of 40 rig/kg 25-(OH)D3 in the study
showed that TD incidence was not significantly different from supplementing 5 rig/kg.
The author claimed that 10 rig/kg 25-(OH)D3 was adequate to obtain maximal bone ash
and decreased TD incidence in 16 day LTD line chickens, provided that the diet
contained a basal level of 1100 ICU/kg cholecalciferol (Mitchell et al., 1997).

At Auburn University, a study was conducted in the attempt to examine the
effectiveness of 25-(OH)D3 in reducing the incidence of TD in LTD line chicks selected
aggressively against TD for nine generations. This group of scientists reported lower TD
incidence and severity when the diet was supplemented at 68.9 rig/kg (Zhang et al.,
1997). Four week old male broilers exhibited about 6.5 % TD when fed a diet
supplemented with 68.9 rig/kg compared to 14.6 % TD incidence for birds fed only the
basal diet which contained sufficient cholecalciferol (2200 ICU/kg). This is in contrast
with Roberson (1999) who saw no reduction in TD severity or incidence in 17 day broiler
chicks, fed a diet sufficient in calcium (0.95%) supplemented with low to high levels of
25-(OI-I)D3 (23,46, 69, 92 or 250 rig/kg). There are arguments on whether the level of
68.9 jig/kg 25-(OH)D3 is the correct level to satisfy performance and TD incidence as
pre-existing patents have recommended levels below 68.9 jig/kg (Dudley— Cash, 1996).

The objective of this study was to study the effects of 25-hydroxycholcalciferol
on the incidence and severity of TD and phosphorus utilization in broiler chicks and to
estimate the level needed to reduce TD and improve phosphorus utilization when chicks
are fed a normal broiler diet. Based on the previous studies we hypothesized that dietary

supplementation of 25-(OH)D3 will reduce the incidence of TD in broilers at a lower

36

level than the manufacturer’s recommendations of 69 jig/kg regardless of whether the

birds are fed a normal broiler diet or TD inducing diet.

37

REFERENCES

Andrews, P., M. W. Orth, and M. E. Cook. 1989. Disulfirarn and histidine induces tibial
dyschondroplasia in broiler chickens. Poult. Sci. 68(Suppl. 1):62. (Abstr.).

Atkin, I., J. C. Pita, A. Omoy, A. Agundez, G. Castiglione, and D. S. Howell. 1985.
Effects of vitamin D metabolites on healing of low phosphate, vitamin D essential
for bone formation. Nature 276:517.

Aumaud, C. 1990. Calcium and phosphorus. Pages 212-223 in Present knowledge in
nutrition. L. B. Myrtle, ed. International Life Sciences Institute-Nutrition
Foundation.

Bar, A., M. Sharvit, D. Noff, S. Edelstein, and S. Hurwitz. 1980. Absorption and
excretion of cholecalciferol and of 25-hydroxycholecalciferol and metabolites in
birds. J. Nutr. 110:1930-1934.

Bell, N. H. 1985. Vitamin D-endocrine system. J. Clin. Invest. 76: 1-6.

Bikle, D., R. Morrissey, and D. Zolock. 1979. The mechanism of action of vitamin D in
the intestine. Am. J. Clin. Nutr. 32:2322-2338.

Blunt, J. W., H. F. DeLuca, and H. K. Schnoes. 1968. 25-hydroxycholecalciferol. A
biologically active metabolite of vitamin D3. Biochemistry 723317-3322.

Boris, A. 1977. Relative activities of some metabolites and analogs of cholecalciferol in
stimulation of tibia ash weight in chicks otherwise deprived of vitamin D. J. Nutr.
107:194-198.

Bronner, F. 1990. Intestinal calcium transport: The cellular pathway. Miner. Electrol.
Metab. 16:94-100.

Burton, R., A. Sheridan, and C. Howlett. 1981. The incidence and importance of tibial
dyschondroplasia to the commercial broiler on industry in Australia. Br. Poult.
Sci. 22:153—160.

38

Christakos, S., E. J. Friedlander, B. R. Frandsen, and A. W. Norman. 1979. Studies on the
mode of action of calciferol. Xiii. Development of a radioimmunoassay for
vitamin D-dependent chick intestinal calcium- binding protein and tissue
distribution. Endocrinol. 104:1495-1503.

Christakos, S., and A. W. Norman. 1978. Vitamin D3-induced calcium binding protein in
bone tissue. Science 202:70-71.

Corvol, T., M. F. Dumontier, M. Garabedian, and R. Rappaport. 1978. Vitamin D and
cartilage. H. Biological activity of 25-hydroxycholecalciferol and 24,25 and 1,25-
hydroxycholecalciferol on cultured growth plate chondrocytes. Endocrinol.

102: 1269.

Davies, M., G. Ritcey, and I. Motzok. 1970. Intestinal phytase and alkaline phosphatase
of chicks: Influence of dietary calcium, inorganic and phytate phosphorus and
vitamin D3. Poult. Sci. 49:1280-1286.

Deluca, H. 1979. The vitamin D system in the regulation of calcium and phosphorus
metabolism. Nutr. Rev. 37:161-193.

Deluca, H. 1982. Metabolism and molecular mechanism of action of vitamin D.
Biochem. Soc. Trans. 10:147-158.

DeLuca, H. F. 1973. Vitamin D metabolites in medicine and nutrition. Proc. Georg. Natl.
Conf., Georgia. 88-108.

Dudley- Cash, W. 1996. Heat return to poultry science meetings. Feedstuffs 68:11.

Edwards, H., Jr. 1985. Observations on several factors influencing the incidence of tibial
dyschondroplasia in broiler chicks. Poult. Sci. 64:2325—2334.

Edwards, H. M., Jr. 1984. Studies on the etiology of tibial dyschondroplasia in chickens.
J. Nutr. 114:1001-1013.

Edwards, H. M., Jr. 1989. The effect of dietary cholecalciferol, 25-
hydroxycholecalciferol and 1,25-dihydroxycholecalciferol on the development of
tibial dyschondroplasia in broiler chickens in the absence and presence of
disulfiram. J. Nutr. 119:647-652.

39

Edwards, H. M., Jr. 1990. Efficacy of several vitamin D compounds in the prevention of
tibial dyschondroplasia in broiler chickens. J. Nutr. 120:1054-1061.

Edwards, H. M., Jr. 1993. Dietary 1,25-dihydroxycholecalciferol supplementation
increases natural phytate phosphorus utilization in chickens. J. Nutr. 123:567-577.

Edwards, H. M., Jr., M. A. Elliot, and S. Sooncharemying. 1992. Effect of dietary
calcium on tibial dyschondroplasia. Interaction with light, cholecalciferol, 1,25-
dihydroxycholecalciferol, protein, and synthetic zeolite. Poult. Sci. 71:2041-2055.

Farquharson, C., J. Berry, E. Mawer, E. Seawright, and C. Whitehead. 1995. Regulators
of Chondrocyte differentiation in tibial dyschondroplasia: An in vivo and in vitro
study. Bone 17:279-286.

Farquharson, C., C. Whitehead, J. Rennie, and N. Loveridge. 1993. In vivo effect of 1,25-
dihydroxycholecalciferol on the proliferation and differentiation of avian
chondrocytes. J. Bone Miner. Res. 8:1081-1088.

Goldblatt, H., and K. Soames. 1923. Biochem. J. 17:298-326.

Groff, J., and S. Gropper. 2000a. Fat soluble vitamins. Pages 316-370 in Advanced
nutrition and human metabolism. Marshall, P, Belmont, CA.

Groff, J ., and S. Gropper. 2000b. Macrominerals. Pages 373-385 in Advanced nutrition
and human metabolism. Marshall, P, Belmont, CA.

Hargest, T., R. Leach, Jr., and C. Gay. 1985. I. Ultrastructure. Am. J. Pathol. 1192175-
198.

Harrison, H., and H. Harrison. 1961. Intestinal transport of phosphate: Action of vitamin
D, calcium and potassium. Am. J. Physiol. 201: 1007.

Haussler, M., and H. Rasmussen. 1972. The metabolism of vitamin D3 in the chick. J.
Biol. Chem. 247:2328-2335.

Hemsley, L. 1970. A cartilage abnormality of broiler chickens. Vet. Rec. 86:385.

40

Henry, H., and A. Norman. 1984. Vitamin D: Metabolism and biological actions. Ann.
Rev. Nutr. 4:493-520.

Holick, M. F. 1990. The use and interpretation of assays for vitamin D and its
metabolites. J. Nutr. 120:Suppl. 11:1464-1469.

Holick, M. F., L. A. Baxter, P. K. Schraufrogel, T. E. Tavela, and H. F. DeLuca. 1976.
Metabolism and biological activity of 24,25-dihydroxyvitamin D3 in the chick. J.
Biol. Chem. 251:397-402.

Hurwitz, S. 1989. Calcium homeostasis in birds. Vitamins and Hormones 45: 173-221.

Itakura, C., and M. Goto. 1973. Tibial dyschondroplasia in broiler chickens in Japan. Jpn.
J. Vet. Sci. 35:289-297.

Klasing, K. 1998. Minerals and vitamins. Pages 234-329 in Comparative avian nutrition.
Cab International.

Koch, E., and F. Koch. 1941. The provitamin D of the covering tissue of chickens. Poult.
Sci. 20:33-35.

Larbier, M., and B. Leclercq. 1992. Calcium & vitamins. Pages 109-126 in Nutrition and
feeding of poultry. Nottingham University Press.

Liang, C., J. Barnes, and L. Cheng. 1982. Effects of 1,25-(OH)2D3 administered in vivo
on phosphate uptake by isolated chick renal cells vitamin D. Am. J. Physiol. 242:
3 12-3 18.

Maxwell, M., and C. Kleeman. 1994a. Calcium metabolism. Pages 243-306 in Maxwell
and Kleeman's clinical disorders of fluid and electrolyte metabolism. R. Narins,
ed. McGraw-Hill Inc., New York.

Maxwell, M., and C. Kleeman. 1994b. Physiology of phosphorus metabolism. Pages 307-
371 in Maxwell and Kleeman's clinical disorders of fluid and electrolyte
metabolism. R. Narins, ed. McGraw-Hill Inc., New York.

McDowell, L. 1989. Vitamin D. Pages 55-92 in Vitamins in animal nutrition. Academic
Press Inc., San Diego.

41

McNaughton, J ., J. Elbert, and B. Dilworth. 1976. The Chick's requirement for 25-
hydroxycholecalciferol and cholecalciferol. Poult. Sci. 56:51 1-516.

McNaughton, J., and R. Murray. 1990. Effect of 25-hyroxycholecalciferol and vitamin D3
on phosphorus requirement of broilers. Poult. Sci. 69(Suppl. 1): 178 (Abstr).

McNutt, K., and M. Haussler. 1973. Nutritional effectiveness of 1,25-
dihydroxycholecalciferol in preventing rickets in chicks. J. Nutr. 103:681-689.

Mellanby, E. 1950. A story of nutrition research. In William and Wilkins, Baltimore,
Maryland.

Merke, J ., G. Klaus, U. Hugel, R. Waldherr and E. Ritz. 1986. No 1,25-dihydroxyvitarnin
D3 receptors on osteoclasts of calcium-deficient chicken despite demonstrable
receptors on circulating monocytes. J. Clin. Invest.77:312-314.

Mitchell, R. D., H. M. Edwards, Jr., and G. R. McDaniel. 1997. The effects of ultraviolet
light and cholecalciferol and its metabolites on the development of leg
abnormalities in chickens genetically selected for a high and low incidence of
tibial dyschondroplasia. Poult. Sci. 76:346-354.

Mohammed, A., M. J. Gibney, and T. G. Taylor. 1991. The effects of dietary levels of
inorganic phosphorus, calcium and cholecalciferol on the digestibility of phytate-
P by the chick. Br. J. Nutr. 66:251-259.

Myrtle, J., and A. Norman. 1971. Vitamin D: A cholecalciferol metabolite highly active
in promoting intestinal calcium transport. Science 171:79-82.

Nemere, I. 1994. Identification of a specific binding protein for lalpha, 25-
dihydroxyvitamin D3 in basal-lateral membranes of chick intestinal epithelium
and relationship to transcaltachia. J. Biol. Chem. 269:23750-23756.

Nemere, I. 1996. Apparent nonnuclear regulation of intestinal phosphate transport:
Effects of 1,25-dihydroxyvitamin D3. Endocrinol. 13722254-2261.

Nemere, 1., and W. Anthony. 1990. Transcaltachia, vesicular calcium transport, and
microtubule-associated calbindin-ngk: Emerging views of 1,25-
dihydroxyvitarrrin D3-mediated intestinal calcium transport. Miner. Electrol.
Metab. 16:106-114.

42

 

Norman, A. 1990. Vitamin D. Pages108-1l6 in Present knowledge in nutrition. L. B.
Myrtle, ed. International Life Sciences Institute-Nutrition Foundation.

Omoy, A., D. Goodwin, D. Noff, and S. Edelstein. 1978. 24,25-Dihydroxyvitamin D is a
metabolite of vitamin D essential for bone formation. Nature. 276:517.

Orth, M. W., Y. Bai, I. H. Zeytun, and M. E. Cook. 1992. Excess levels of cysteine and
homocysteine induce tibial dyschondroplasia in broiler chicks. J. Nutr. 122:482-
487.

Poulos, P., S. Reiland, K. Elwinger, and S. Olsson. 1978. Skeletal lesions in the broiler,
with special reference to dyschondroplasia. Acta. Radiol. Suppl. 358:229-306.

Prasad, S., W. Hairr, and J. Dallas. 1972. Observations of abnormal cartilage formation
associated with leg weakness in commercial broilers. Avian Dis. 16:457-461.

Rennie, J. S., H. A. McCormack, C. Farquharson, J. L. Berry, E. B. Mawer, and
C. C. Whitehead. 1995. Interaction between dietary l,25-dihydroxycholecalciferol
and calcium and effects of management on the occurrence of tibial
dyschondroplasia, leg abnormalities and performance in broiler chickens.
Br. Poult. Sci. 36:465-477.

Rennie, J. S., and C. C. Whitehead. 1996. Effectiveness of dietary 25- and 1-
hydroxycholecalciferol in combating tibial dyschondroplasia in broiler chickens.
Br. Poult. Sci. 37 2413-421.

Rennie, J. S., C. C. Whitehead, and B. H. Thorp. 1993. The effect of dietary 1,25-
dihydroxycholecalciferol in preventing tibial dyschondroplasia in broilers fed on
diets imbalanced in calcium and phosphorus. Br. J. Nutr. 69:809-816.

Riddell, C. 1975a. Studies on the pathogenesis of tibial dyschondroplasia in chickens.
Ii. Growth rate of long bones. Avian Dis. 19:490-496.

Riddell, C. 1975b. Studies on the pathogenesis of tibial dyschondroplasia in chickens.
Iii. Effect of body weight. Avian Dis. 19:497-505.

Riddell, C. 1976. Selection of broiler chickens for a high and low incidence of tibial
dyschondroplasia with observations on spondylolisthesis and twisted legs
(perosis). Poult. Sci. 55:145-151.

43

Riddell, C., J. Howell, and M. Kaye. 1971. Tibial dyschondroplasia in broiler chickens in
Western Canada. Avian Dis. 15:515-565.

Riddell, C., and D. Pass. 1987. The influence of dietary calcium and phosphorus on tibial
dyschondroplasia in broiler chickens. Avian Dis. 31:771-775.

Roberson, K., C. Hill, and P. Ferket. 1993. Addative amelioration of tibial
dyschondroplasia in broilers by supplemental calcium or feed deprivation. Poult.
Sci. 72:798-805.

Roberson, K. D., and H. M. Edwards. 1994. Effects of ascorbic acid and 1,25-
dihydroxycholecalciferol on alkaline phosphatase and tibial dyschondroplasia in
broiler chickens. Br. Poult. Sci. 35:763-773.

Roth, J ., S. Bonner-Weir, A. W. Norman, and L. Orci. 1982. Irnmunocytochemistry of
vitamin D-dependent calcium binding protein in chick pancreas: Exclusive
localization. Endocrinol. 110:2216—2218.

Schwartz, Z., D. Schlader, L. Swain, and B. Boyan. 1988. Direct effects of 1,25-
dihydroxyvitarnin D3 and 24,25-dihydroxyvitamin D3 on growth zone and resting
zone chondrocyte membrane alkaline phosphotase and phospholipase-A2 specific
activities. Endocrinol. 123:2878-2884.

Sheridan, A., C. Howlett, and R. Burton. 1978. The inheritance of tibial dyschondroplasia
in broilers. Br. Poult. Sci. 19:491-499.

Siller, W. 1970. Tibial dyschondroplasia in the fowl. J. Pathol. 101:39-46.

Soares, J. 1984. Calcium metabolism and its control- A review. Poult. Sci. 63:2075—2083.

Soares, J. H., Jr., J. M. Kerr, and R. W. Gray. 1995. 25-hydroxycholecalciferol in poultry
nutrition. Poult. Sci. 74:1919-1934.

Steenbock, H., and A. Black. 1924. J. Biol. Chem. 61:405-422.

Suda, S., N. Takahashi, T. Shinki, N. Horiuchi, A. Yamaguchi, S. Yoshiki, S. Enomoto,
and T. Suda. 1985. 1 alpha, 25-dihydroxyvitamin D3 receptors and their action in
embryonic chick chondrocytes. Calcif. Tiss. Int. 37:82-90.

Sullivan, T. W. 1994. Skeletal problems in poultry: Estimated annual cost and
descriptions. Poult. Sci. 73:879-882.

Sunde, M. 1975. What about 25-hydroxycholecalciferol on poultry? Proc. Dist. Feed Res.
Counc. 30:53-62.

Taylor, T., and C. Dacke. 1984. Calcium metabolism and its Regulation. Pages 125-170
in Physiology and biochemistry of the domestic fowl. B. M. Freeman, ed. vol. 5.
Academic Press, London.

Theofan, G., A. P. Nguyen, and A. W. Norman. 1986. Regulation of calbindin-ngk gene
expression by 1,25-dihydroxyvitamin D3 is correlated to receptor occupancy. J.
Biol. Chem. 261 : 16943-16947.

Thorp, B., C. Whitehead, and J. Rennie. 1991. Avian tibial dyschondroplasia: A
comparison of the incidence and severity as assessed by gross examination and
histopathology. Res. Vet. Sci. 51:48-54.

Thorp, B. H., B. Ducro, C. C. Whitehead, C. Farquharson, and P. Sorensen. 1993. Avian
tibial dyschondroplasia: The interaction of genetic selection and dietary 1,25-
dihydroxycholecalciferol. Avian Pathol. 22:31 1-324.

Tucker, G., 3rd, R. E. Gagnon, and M. R. Haussler. 1973. Vitamin D3 -25-hydroxylase:
Tissue occurrence and apparent lack of regulation. Arch. Biochem. Biophys. 155:
47-57.

Valinetse, M., and V. Bauman. 1981. Comparative antirachitic activity of vitamin D2 and
vitamin D3 in chicks. Appl. Biochem. Microb. 183: 168-175.

Van der Klis, J ., and H. Versteegh. 1999. Phosphorus nutrition of poultry. Pages 309-320
in Recent developments in poultry nutrition 2. J. Wiseman and P. C.
Gamsworthy, ed. Nottingham University Press.

Waldroup, P., C. Ammerman, and R. Harms. 1963. The relationship of phosphorus,
calcium, and vitamin D3 in the diet of broiler-type chicks. Poult. Sci. 42:982—989.

Watkins, B. 1999. Diet and legweakness in poultry. Pages 79-92 in Recent developments
in poultry nutrition 2. J. Wiseman and P. C. Gamsworthy, ed. Nottingham
University Press.

45

Wise, D., and A. Jennings. 1972. Dyschondroplasia in domestic poultry. Vet. Rec. 91:
285-286.

Yarger, J. G., C. L. Quarles, B. W. Hollis, and R. W. Gray. 1995. Safety of 25-
hydroxycholecalciferol as a source of cholecalciferol in poultry rations. Poult. Sci.
74: 1437-1446.

Zhang, X., G. Liu, G. R. McDaniel, and D. Roland. 1997. Responses of broiler lines
selected for tibial dyschondroplasia incidence to supplementary 25-
hydroxycholecalciferol. J. Appl. Poult. Res. 6:410-416.

Zhou, X. Y. et al. 1986. Bone vitamin D—dependent calcium-binding protein is localized
in chondrocytes of growth-plate cartilage. Calcif. Tiss. Int. 38:244-247.

CHAPTER 2

Materials and Methods

General Procedure

A total of 240 one-day-old male broiler chicks were used for each experiment.
Chicks were procured from a commercial hatchery and were of Ross X Ross strain except
for Experiment 2 in which Ross X Arbor Acres chicks were obtained. All research was
approved by the All-University Committee on Animal Use and Care of Michigan State
University in accordance with the Guide for the Care and Use of Agricultural Research
and Teaching Animal Care. Ten chicks per pen were assigned randomly to each of 24
pens in an electrically heated battery brooder1 housed in a room without windows. Room
temperature was maintained at approximately at 23°C. Incandescent lighting was
continuous in the room and fluorescent light2 (15 watt cool white bulb) was continuous in
the battery pens. Feed and water were provided ad libitum in all experiments. The
compositions of the com-soybean meal based diets are listed in Table 1. The 25-
hydroxycholecalciferol premix was donated by Monsanto3. Chromic oxide was used as
an external indicator at 0.10 % of the basal diet to determine phytate phosphorus
retention.

In Experiment 1, 12 pens randomly selected out of a total of 24 pens had plastic
filters4 designed to block ultraviolet radiation from fluorescent light to establish whether

the filters work under conditions of the experiment. Chicks were fed a basal diet that

 

lPetersime Incubator Co., Gettysburg, OH 54328.
ZOSRAM SYLVANIA, LTD, Danvers, MA 01923
3Monsanto Animal Nutrition, Naperville. IL 60563
4University Products, Inc., Holyoke, MA 01041

47

Table 1. Composition of the experimental diets

 

 

 

 

 

 

Starter diet Grower diet
Ingredients and analysis TD-inducin g Normal Normal
%
Ground yellow corn 56.61 51.00 61.13
Soybean meal (dehulled) 35.00 39.00 31.12
Soybean oil 5.00 6.07 4.32
Dicalcium phosphate 1.86 1.70 1.21
Limestone 0.28 0.98 1.06
Salt 0.45 0.45 0.40
DL-methionine 0.20 0.20 0.16
Trace nrineral mixz 0.25 0.25 0.25
Vitamin pre-mixl 0.25 0.25 0.25
Chromic oxide 0.10 0.10 0.10
Calculated Composition
Protein 21.6 23.0 20.0
ME, kcal/kg 3200 3200 3200
Calcium 0.65 0.85 0.76
Total Phosphorus 0.72 0.69 0.58
Nonphytate Phosphorus 0.48 0.45 0.35
Experiment
1 2 3 4 5 6 6
Analyzed Composition Starter Grower
Calcium ' 0.75 0.67 0.85 0.88 0.86 0.94 0.80
Total Phosphorus 0.78 0.73 0.70 0.71 0.67 0.70 0.62
Phytate Phosphorus 0.22 0.25 0.28 0.27 0.23 0.27 0.25

 

lVitamin premix provided per kilogram of diet: vitamin A (all-trans-retinyl acetate), 5500 IU; vitamin E
(all-rac-a-tocopheryl acetate), 11 IU; menadione (as menadione sodium bisulfite), 1.1 mg; riboflavin, 4.4
mg; Ca pantothenate, 10 mg; nicotinic acid, 44 mg; choline chloride, 600 mg; vitamin B 12, 0.1 mg; vitamin
B6, 3 mg; thiamin (as thiamin mononitrate), 2.2 mg; folic acid, 3 mg; biotin, 0.3 mg; and *cholecalciferol
1100 ICU (TD inducing diet) and 2200 ICU (normal diet).

2Mineral premix supplied per kilogram of diet: manganese, 120 mg; zinc, 100 mg; iron, 60 mg; copper, 10
mg; iodine, 2.1 mg; selenium, 0.1 mg.

48

contained no cholecalciferol and which was low in calcium and high in phosphorus
[0.65% calcium and 0.48% non-phytate phosphorus (in) by calculation], a diet known
to induce TD in broiler chicks (Edwards and Veltmann, 1983). The vitamin D3
metabolite, 25—hydroxycholecalciferol [25-(OH)D3] was added to the basal diet at 0, 10

or 70 rig/kg. The experiment was a 2 x 3 factorial design with 2 levels of ultraviolet light

(no light and light) and 3 concentrations of 25-(OH)D3 (0, 10 and 70 rig/kg). Four pens
were randomly assigned to each treatment group and the experiment lasted for 17 days.

Experiment 2 was designed to determine the effect of dietary supplementation of
25-(OH)D3 on tibial dyschondroplasia when a TD—inducing diet is fed. Ross X Arbor
Acres male chicks were used in this experiment. Fluorescent tubes in all of the 24 pens
were covered with plastic filters providing no source of ultraviolet light for any of the
chicks. The TD-inducin g diet fed in this experiment contained 1100 ICU/kg
cholecalciferol and 25-(OH)D3 was added at 0, 10, 40 or 70 jig/kg for 17 days. Each
treatment was fed to six pens of 10 chicks and was represented on each of the six decks
of the battery-brooder.

Experiment 3 was conducted to determine the effects of supplementing 25-
(OH)D3 on the development of TD when a normal broiler diet was fed to broiler chicks.
The experimental design was identical to Experiment 2 except the cockerels used in this
experiment were Ross X Ross and a normal broiler starter diet [0.85% calcium and
0.45% non-phytate P (in)] was fed containing 22001CU/kg cholecalciferol for 20 days.

Experiments 4 and 5 were conducted to further determine the effects of
supplementing a wider range of 25-(OH)D3 at even intervals on the development of TD

when a normal starter broiler diet containing 2200 ICU/kg was fed to broiler chicks. In

49

both of these experiments, 4 pens of 10 chicks each were randomly assigned each
treatment. Ross X Ross chicks were fed supplemental 25-(OH)D3 at 0, 18, 36, 54, 72 or
90ug/kg for 20 days.

Experiment 6 was conducted to determine if 25-(OH)D3 needed to be fed past the
starter period to prevent TD. Ross X Ross chicks were fed the starter diet described in
Experiments 3-5 for 17 days. Twelve pens/treatment were fed either 0 or 40 ug/kg 25-
(OH)D3. From 18 to 35 days, a grower diet formulated to contain 0.76% calcium, 0.35%
non-phytate phosphorus and 2200 ICU/kg cholecalciferol was fed to birds selected from
the starter period based upon proximity to the treatment body weight average. Pen-mates
from two starter pens per treatment were mixed in a grower pen at 7 birds/pen. There
were 4 pens/treatment for a total of 28 birds/treatment. Dietary treatments in Experiment
6 were: 1) 0 ug/kg 25-(OH)D3 from 1 to 35 days of age, 2) 40 ug/kg 25-(OH)D3 from 1 to
35 days of age, and 3) 40 ug/kg 25-(OH)D3 from 1-17 days and 0 ug/kg from 18-35 days

of age.

Bone

At the end of each experiment or phase, chicks were killed by cervical
dislocation. The right tibia of each bird was cut longitudinally at the proximal head of the
metaphysis to examine for the presence of TD. Tibias were scored for degree of severity
on a scale from 0 to 3, with score 3 representing the most severe case of TD (Edwards
and Veltmann, 1983). All the number 3 scores were calculated on a percent basis and a
TD severity index was calculated using two different scoring systems. Scoring index

number 1 defined the total TD score in each individual pen divided by the total number of

50

birds affected with TD per pen (Edwards, 1984). Scoring index number 2 defined the
total TD score in each individual pen divided by the number of birds in each pen whether
birds were affected with TD or not. The left tibia of each bird were collected and kept for
bone ash analysis according to the AOAC (1984) method. The tibiae were fat extracted
with ethanol and then ether before ashin g at 600°C overnight.

In order to examine one of the problems associated with TD, such as leg
weakness, all the birds were subjected to a water-bath leg weakness test at 34 days of age.
Birds were placed standing in a water-bath filled with 3 cm of water kept at 28°C. Each
bird was observed for 90 seconds and the time spent standing and sitting was recorded. A
scoring system for the severity of lameness was developed on a scale from 0-3 (score 3
representing the most severe case of lameness). Score 0 was assigned to birds standing
the entire 90 seconds; score 1 for birds making I attempt to sit, but standing for the
remainder until 90 seconds; score 2 for birds making 2 attempts to sit and standing for the
remainder until 90 seconds; score 3 for birds making 3 or more attempts to sit during 90
seconds or remain sitting for entire 90 seconds. The incidence of leg weakness and

number 3 scores were calculated on a percent basis.

Feed

Feed samples were first ground by a Wiley Mill5 using a 1mm screen then by a
1093 Cyclotech6 using a 0.5 mm screen. Samples were digested in a MARS 57
microwave. The feed was analyzed for Chromic oxide (Willams et al. 1962), calcium

(Clinical Application of Atomic Absorption Emission Spectroscopy, 1972) and

 

5Arthur H. Thomas Co., Philadelphia, PA.
°FOSS TECATOR, AB Hoganas, Sweden, S-2632
7Microwave Accelerated Reaction System, CBM Corporation, Mathews, NC 28106

51

phosphorus (Gomori, 1942) content using a UNICAM 989 atomic absorption
spectrophotometers. Phytate phosphorus content of the feed was determined by a DU
7400 spectrOphotometer9 as described by Latta and Eskin (1980). Feed consumption was
recorded throughout the trial and chicks were weighed once a week to calculate feed

efficiency (gain: feed).

Excreta

Excreta samples were obtained by collecting fresh excreta from each pen over a
24-hour period at the end of each experiment or phase. The excreta was dried at 50° C
and ground as previously cited for feed before digested in the microwave. Chronric oxide
was measured in the manure according to the previously cited method by flame atomic
absorption spectrOphotometrys. Excreta were analyzed for phytate phosphorus content by
the method described by Latta and Eskin (1980). Phytate phosphorus retention was
calculated using the Chromic oxide balance method as described by Edwards and Gillis

(1959).

Blood

Blood samples were obtained at 13 days of age via cardiac puncture from one
chick per pen. The blood was centrifuged for 15 minutes to extract serum and analyzed
for serum phosphorus (Goldenberg and Fernandez, 1966) and serum calcium (Moorhead

and Biggs, 1974) concentrations using a DU 7400 spectrophotometerg.

 

t"l'herme Electron Corporation, Franklin, MA 02038
’BECKMAN. Fullerton, CA

52

Statistical Analysis

Data was analyzed by Analysis of variance (ANOVA) and the SAS General
Linear Model Procedure. Experiment 1 was analyzed as a 2 x 3 factorial using 2-way
ANOVA in which main effects of light and 25-OH D3, and interaction between light and
25-(OH)D3 was incorporated into the model. Treatment means were separated by
Bonferroni’s multiple comparison test. To assess the fit of a regression model to the data,
a lack-of-fit test was performed. Data were subject to nonlinear regression when
appropriate. The breakpoint between the descending line and the plateau was determined
using the DUD method of the NLIN procedure (Robbins et al.1979) to estimate the

requirement for 25-(OH)D3 to minimize TD incidence and severe lesions.

53

Results

Experiment 1

Overall, exposure of birds to ultraviolet light resulted in higher 17-day body
weight (p<0.001), gain: feed (p=0.027), serum calcium (p=0.016), and bone ash
(p<0.001), and lower rickets and TD incidence (p<0.001), severity score #2 (p<0.001)
and severe lesions (p=0.002) than birds that were subjected to filtered fluorescent lights
(Tables 5 and 6). Phytate phosphorus retention (PP) increased (p=0.034) by the presence
of ultraviolet light. The effect was especially strong when the diet was deficient in
vitamin D increasing the phytate phosphorus retention from 32.9 to 56.4%. Serum
calcium concentration in birds fed the vitamin D deficient diet in pens with filtered lights
was extremely low. This response was not observed in any of the other groups where
serum calcium levels appeared normal and were influenced by other treatments in this
experiment. Serum phosphorus levels were significantly higher in the birds subjected to
filtered fluorescent lights with no cholecalciferol in the diet compared to any other
treatment group except for birds consuming 70 jig/kg 25-(OH)D3 and exposed to
ultraviolet light in this experiment.

The overall effects of addition of 25-(OH)D3 to the diet resulted in increased
bone ash and gain: feed (p<0.001) and decreased rickets, TD incidence, severity score # 1
and 2 (p<0.001) and number 3 scores (p=0.003) (Tables 5 and 6). Serum calcium, bone
ash and gain: feed only increased at 10ug/kg while body weight increased and TD
incidence decreased (p<0.001) with each level of 25-(OH)D3. However, TD severity
(severity score # 1 and 2, and number 3 scores) decreased (p<0.001) only at 70 jig/kg 25-

(OH)D3. Rickets was eliminated in chicks with the addition 70ug/kg 25-(OH)D3 to the

54

 

diet, but also at long/kg provided that ultraviolet light was present. The interaction g-‘
(p<0.001) is a significant decrease with ultraviolet light when no 25-(OH)D3 was added,
but no significant effects otherwise. Similarly, there were significant interaction effects
between addition of 25-(OH)D3 and ultraviolet light exposure for TD incidence, severity
score #2, number 3 scores and bone ash (p=0.031, p=0.028, p=0.02 and p<0.001) (Table
6). The highest supplementation level was needed to significantly decrease the TD
incidence and severity when chickens were deprived of ultraviolet light. However, when
the ultraviolet light was present 10 jig/kg was sufficient in significantly reducing TD
incidence and even eliminated severe lesions. Significant interactions were also observed
for body weight and gain: feed (p<0.001). The addition of 10 pig/kg 25-(OH)D3 to the
diet increased (p=0.013) phytate phosphorus retention. Further increasing the level of 25-
(OH)D3 to 70 rig/kg resulted in phytate phosphorus retention between 0 and 10 jig/kg
which was not different from either of the other two levels. However, there was an
ultraviolet light by 25-(OH)D3 interaction effect (p=0.021) similar to the rickets and bone

ash responses.

Experiment 2

Body weight gain, feed efficiency, and serum calcium and phosphorus were not
affected in this experiment (Table 7). Phytate phosphorus retention (PP) increased
linearly (p<0.001) with increasing dietary 25-(OH)D3 and was significantly higher when
40 pig/kg 25-(OH)D3 was fed compared to the control birds (Table 7). Phytate phosphorus
retention was almost doubled when 70 jig/kg 25-(OH)D3 was fed. Tibia bone ash

increased linearly from 41.8 to 43.9% (p<0.001) and birds receiving 25-(OH)D3 had

55

significantly higher percent bone ash than birds receiving no supplementation of 25-
(OH)D3 (Table 8). The incidence of TD, TD severity and number 3 scores decreased
linearly (p<0.001) by dietary 25-(OH)D3. The same parameters except for severity index
# 1 also were decreased quadratically (p<0.001). The control group had a higher
(p<0.001) TD incidence (73%) compared to birds that had been supplemented with
10ug/kg 25-(OH)D3 (26%). When supplementing at higher levels, (40 and 70 jig/kg of
25-(OH)D3), a further reduction in TD incidence (5% and 2%, respectively) was
observed. Incidence of number 3 scores were parallel to TD incidence with an initial
decrease (p<0.001) from 59% to 16% with supplementation of 10 rig/kg of 25-(OH)D3.
Similarly, treatment with 40 or 70 jig/kg of 25-(OH)D3 resulted in less severe lesions, but
were not different from each other. Addition of 70 jig/kg of 25-(OH)D3 to the TD-
inducing diet completely eliminated number 3 scores. Overall, severity was decreased
(p<0.001) at 40 rig/kg using severity index # l and at 10 rig/kg with severity index # 2
(p<0.001). Severity index 2 mirrored the results of number 3 score incidence. Analysis by
the non-linear regression (NLIN) procedure estimated the requirement of 25-(OH)D3 to

reduce TD incidence and severe lesions to be 14.8 rig/kg and 13.5 ug/kg, respectively.

Experiment 3

As in Experiment 2, body weight, feed efficiency and serum calcium and
phosphorus is not affected by supplementation of 25-(OH)D3 (Table 9). Supplementation
of 25-(OH)D3 had an effect on phytate phosphorus retention (p<0.001) (Table 9). Birds
fed 10 or 40ug/kg 25-(OH)D3 had significantly higher phytate phosphorus retention

(46.0% and 43.1%, respectively) compared to birds fed no 25-(OH)D3. However, 70

56

rig/kg significantly reduced phytate P retention to levels similar to controls resulting in a
quadratic response (p<0.001). There was no treatment effect on percent bone ash in this
experiment (Table 10). The incidence of TD was decreased linearly (p=0.003) by
increasing dietary 25-(OH)D3 (Table 10). The overall incidence of TD lesions was not as
high as in Experiment 2, as TD incidence was 25 % for the control group and
supplementing 10 ug/kg of 25-(OH)D3 did not significantly decrease TD incidence.
Addition of 70 jig/kg of 25-(OH)D3 was needed to significantly reduce TD incidence to
5%. However, the effect of feeding 40 ug/kg of 25-(OH)D3 on TD incidence was not
significantly different from 70 rig/kg of 25-(OH)D3. Severe TD lesions were decreased
linearly (p=0.004) with increasing 25-(OH)D3 (Table 10). Similar to Experiment 2, the
number 3 scores were eliminated when 70 [lg/kg 25-(OH)D3 was fed and TD severity
index # 2 was not different between feeding 40 or 70 jig/kg of 25-(OH)D3, but was
different for severity index # 1 (Table 10). According to the NLIN analysis, the estimated

level needed to minimize TD incidence in a normal broiler diet is 65.4 jig/kg.

Experiment 4

As in Experiment 3, body weight and gain: feed, serum calcium and phosphorus
and bone ash responses to the various levels of 25-(OH)D3 were not significant (Tables
11 and 12). Supplementation of 25-(OH)D3 increased phytate phosphorus retention at
p=0.083. The addition of 36 jig/kg 25-(OH)D3 caused an increase of phytate phosphorus
retention from 44.2 % to 50.2 % indicating higher phytate degradation at this level.
However, higher supplementation levels of 25-(OH)D3 were not significantly different

from control treatment. Supplementation of 25-(OH)D3 did not significantly affect the

57

incidence or severity of TD in this experiment (Table 12). In fact, TD incidence was
identical at levels of 0 and 72 jig/kg of 25-(OH)D3. Number 3 scores were actually absent
when 18 pig/kg 25(OH)D3 was fed, but overall no significant effects were observed for

TD severity due to low incidence in this experiment (Table 12).

Experiment 5

There was no difference in 20-day body weight, but there was a significant effect
on feed efficiency in this experiment (Table 13). The addition of 18 jig/kg 25-(OH)D3
had significantly lower gain: feed ratio than the addition of 54 rig/kg and higher levels of
25-(OH)D3, exhibiting a linear effect (p=0.030) due to this response. The level of 25-
(OH)D3 had no significant effect on serum concentration of either phosphorus or calcium.
In this experiment, phytate phosphorus retention was not affected by dietary 25-(OH)D3
and phytate phosphorus retention was higher than other experiments when a marginal

calcium diet was fed (Table 13). Incidence of TD and severity of birds fed 25-(OH)D3
were not significantly different from the control group (Tablel4). Although at 0 rig/kg
25-(OH)D3 TD incidence was similar to that observed in Experiment 3, there were no
significant effects from adding levels up to as high as 90 jig/kg. Dietary supplementation
of 25-(OH)D3 resulted in an increase in bone ash only at 36 rig/kg compared to the

negative control but not at higher levels.

58

Experiment 6
Starter

There were no improvements in body weight, feed efficiency, phytate phosphorus
retention or bone ash (1‘ ables 15 and 16) at 17-days of age by supplementing the diet with
40 jig/kg of 25-(OH)D3. The incidence of TD lesions was the same for the two groups,
exhibiting a TD incidence of 12% (Table 16). The severity of TD was too low to observe

a significant effect.

Grower

No significant differences were observed at 35-days of age for body weight or
gain: feed among broilers fed 0 rig/kg and previously fed 0 rig/kg (control), fed 40 jig/kg
for 5 weeks or 0 rig/kg fed after previously fed 40 ug/kg of 25-(OH)D3 (Table 17).
Supplementary 25-(OH)D3 in the grower diet increased (p=0.004) phytate phosphorus
retention compared to the control and the non-supplemented group previously fed 40
ug/kg 25-(OH)D3. No significant differences were observed for leg weakness (Table 17).
The removal or supplementation of 25-(OH)D3 in the grower diet did not influence the
incidence or severity of leg weakness of 35-day-old broilers. There were no responses
observed for TD incidence or severity in chicks at 35-days of age by the removal of 25-
(OH)D3 from the grower diet (Table 18). The TD incidence was totally absent for the
control and for the non-supplemented group previously fed 40 rig/kg. However, complete
resorption of the lesion had not occurred in the supplemented group and showed a TD
incidence and number 3 score of 7% for both parameters in birds fed 40 rig/kg the entire

experiment.

59

Discussion

The present study shows that ultraviolet light alone can effectively reduce rickets,
but not the development of TD in birds fed a vitamin deficient diet and that
supplementation of 25-(OH)D3 to broiler diets in the absence or presence of fluorescent
lighting can reduce TD incidence and severity and improve phosphorus utilization in
broiler chicks. Experiment 1, due to the high rickets percentage and very low bone ash
responses, clearly shows that the filters were effective in blocking ultraviolet light. In the
commercial poultry industry, broilers are often raised in large windowless houses and
exposure to ultraviolet radiation is limited. Since the mid 1990’s, many poultry producers
have switched from incandescent lamps to fluorescent bulbs as the source of light in their
facilities (Lewis and Morris, 1998). However, fluorescent lamps in battery brooders have
been estimated to provide the equivalent of 20 to 40 rig/kg cholecalciferol (Edwards et
al., 1994). The cholecalciferol produced in the skin from ultraviolet light in Experiment 1
was equivalent to 10 rig/kg 25-(OH)D3 obtained orally. The efficiency of utilization of
cholecalciferol activity from ultraviolet light may be because the cholecalciferol does not
have to be absorbed first in the gut in association with fats before reaching the liver, but
can diffuse directly from the skin into the blood stream and be carried quickly to the liver
by vitamin D binding protein for 25-(OH)D3 production (Holick, 1981). Assuming that
the biological activity of 25-(OH)D3 is about 2.5 times that of cholecalciferol (Sunde,
1975; Soares et al., 1978), the addition of 10ug/kg 25-(OH)D3 to a basal diet containing
no cholecalciferol is approximately equivalent to the addition of 27.5 jig/kg (1100

ICU/kg), which has been reported to be adequate vitamin D activity for broiler chicks

60

(Edwards et al., 1994). Experiment 1 clearly showed that rickets can be significantly
reduced with the addition of 10 rig/kg 25-(OH)D3 and even eliminated provided that
ultraviolet light is available. Elliot and Edwards (1997 ) reported 3% and 0% rickets
incidence in birds fed a basal diet (0.90% calcium and 0.46% in) supplemented with
27 .5 rig/kg or 50 rig/kg cholecalciferol, respectively, in the presence of ultraviolet light.
In our study, when fluorescent lights were filtered, rickets was eliminated with 70 rig/kg
25-(OH)D3. Aburto et a1. (1998) showed that rickets could be eliminated with
supplementation of 40 jig/kg 25-(OH)D3 in absence of ultraviolet light when the diet
contained either low or high vitamin A levels.

Ultraviolet light has also been shown to be very effective in reducing TD
incidence and severity and in increasing bone ash as well as overall performance in
broiler chicks (Edwards et al., 1994). However, in Experiment 1 of our study the TD
incidence was not influenced by addition of 10 pig/kg 25-(OH)D3 unless ultraviolet light
was available which served as an additional source of vitamin D activity to the chick.
This was likely due to the growth response to ultraviolet light when vitamin D3 was
deficient. After adequate growth was similarly restored by 10 jig/kg 25-(OH)D3 when
ultraviolet light was blocked, a reduction in TD was only observed by additional 25-
(OH)D3 (70 rig/kg). However, growth was not reduced by dietary vitamin D3 deficiency
when ultraviolet light was present. Therefore, for TD incidence, a response to dietary 25-
(OH)D3 was observed at a lower level (10 rig/kg).

Since ultraviolet radiation was not a factor in Experiment 2, the only source of
vitamin D was the cholecalciferol from the basal diet. When 1100 ICU/kg cholecalciferol

is present in a diet with high phosphorus and low calcium (TD-inducing) such as in

61

Experiment 2, vitamin D3 appears to be inadequate in overriding the TD inducing effect.
The possible reason for this is less efficient absorption of cholecalciferol compared to 25-
(OH)D3 (Bar, 1980). The hydroxylation of cholecalciferol or conversion to 25-(OH)D3
makes the metabolite more polar which changes its absorptive characteristics and
increases its water solubility (Blunt et al., 1968). Absorption of cholecalciferol is energy-
dependent and is associated with fats while 25-(OH)D3 absorption involves passive
diffusion across the intestinal wall into the blood stream (Calabotta, 1997). In addition,
once in the bloodstream, cholecalciferol has a lesser affinity for vitamin D binding
protein (DBP) than 25-(OH)D3, which strongly affects its transport (Haddad and Walgate,
1976). The inefficiency of vitamin D activity in the TD-inducing diet is clearly evident
from the high TD incidence of 73% in Experiment 2. Other investigators also reported
this type of inefficiency. Edwards and Veltrnan (1983) had the highest TD incidence
when chicks were fed a diet high in phosphorus, low in calcium and containing 1100
ICU/kg cholecalciferol. During the early 1990’s Edwards conducted a series of
experiments with this TD-inducing diet which contained 1100 ICU/kg cholecalciferol.
The investigators reported TD incidences of 42% and 51% at 16—days of age with
ultraviolet present (Edwards, 1989), 54% at 16-days of age with ultraviolet present
(Edwards, 1990), 94% at 16-days of age in absence of ultraviolet light (Edwards et al.,
1992) and 52% at 21 days of age in absence of ultraviolet light (Edwards et al., 1995), in
broiler chicks.

The addition of 25-(OH)D3 to the TD-inducing diet containing 1100 ICU/kg
cholecalciferol in Experiment 2 confirmed that supplementing this vitamin D metabolite

could significantly reduce the development of TD at less than 70 rig/kg. Supplementation

62

of 10 ug/kg 25-(OH)D3 in Experiment 2 reduced TD incidence and severity by 64% and
72 %, respectively. When Edwards (1989) supplemented 10 jig/kg of 25-(OH)D3 to a
similar diet, the author obtained a 28 % TD incidence, which is comparable with
Experiment 2 (26% TD incidence). Although TD incidence and severity was reduced at
long/kg of 25-(OH)D3, this is still relatively high and may be unacceptable in the
commercial broiler industry. The conversion of cholecalciferol to 25-(OH)D3 may be
inefficient or the plasma pool of 25-(OH)D3 is too low for a sufficient amount of 1,25-
(OH)2D3 synthesis to impact target tissues at the receptor level. Yarger et al. (1995)
observed significantly lower serum 1,25-(OH)2D3 concentrations in relation to higher 25-
(OH)D3 concentrations. As dietary 25-(OH)D3 increased from 69 to 690 jig/kg the serum
1,25-(OH)2D3 decreased while serum 25-(OH)D3 concentrations increased linearly. In a
similar study, Mitchell et al. (1997a) reported that dietary 25-(OH)D3 levels of 20 to 40
jig/kg decreased plasma 1,25-(OI-I)2D3. The investigators also reported that plasma 25-
(OH)D3 increased linearly with increasing levels of dietary 25-(OH)D3. The effect was
especially strong with the high TD incidence (HTD) line of chicks (interaction p<0.05).
The incidence of TD decreased at 5 ug/kg in the low incidence (LTD) line chicks and
was about 20% with increasing dietary levels up to 40 ug/kg. These authors had
somewhat higher dietary concentrations of calcium (1.0 %) than the present trial. It is
possible that some or a majority of the 1,25-(OH)2D3 is unbound and is functioning at the
cellular level (vitamin D receptor). The technique used to measure plasma 1,25-(OH);D3
only measures the bound form, therefore lower concentrations than actual of 1,25-
(OH)2D3 that are bound to DBP are detected in the plasma in this case (Soares, 1995).

Meanwhile, plasma 25—(OH)D3 is usually bound to DBP and also has a greater affinity for

63

 

the protein including albumin than 1,25-(OH)3D3 (Vieth et al.,1990). This might explain 0.
the linear increase in plasma 25-(OH)D3 in Yarger et al. (1996) and Mitchell et al.
(1997a) when dietary 25-(OH)D3 was increased in the diet. Whatever the mechanism,
increasing supplementation levels to 40 or 70 ug/kg of 25-(OH)D3 in a TD-inducing diet
of Experiment 2 in our study further reduced the TD incidence to 5% and 2%,
respectively, without compromising performance. A higher plasma pool of 25-(OH)D3
due to the increased dietary level might have allowed for more free 1,25-(OH)2D3 at
target tissues and greater 1,25-(OH)3D3 synthesis due to the calcium deficient diet. The
net availabilty of 1,25-(OH)2D3 for TD prevention may be the greatest when 25-(OH)D3
is supplemented between 40 to 70 jig/kg to a calcium deficient diet.

In Experiment 3 the TD incidence and severity were also decreased with 25-
(OH)D3, which suggests that 25-(OH)D3 supplementation is effective in reducing TD in
both normal broiler diets as well as TD-inducing diets. In Experiment 3 the
cholecalciferol was increased to 2200 ICU/kg together with increased calcium
concentration (0.85%) and lower non-phytate phosphorus concentration (0.45%). This
basal diet alone (control), without 25-(OH)D3 added to the diet, resulted in one-third the
incidence of TD compared to Experiment 2. Supplementing 2200 ICU/kg cholecalciferol
in combination with higher calcium and lower phosphorus levels alone can reduce TD
incidence in Ross x Ross chicks. Roberson (1999) fed a normal broiler (0.95% Ca) diet
with vitamin D3 activity of >800 ICU/kg and reported low TD incidence (24%). This is
in contrast to a study conducted by Lofton and Soares (1986), in which TD incidences
were rather high at 28 days (40%) after feeding broiler chicks a normal com-soybean

meal diet containing 2000 ICU/kg cholecalciferol. Edwards et al. (1992) also reported

high TD incidence in l6-day old chicks fed a near adequate calcium (95%) diet
containing 2000 ICU/kg. It is also of interest to note that supplementing this normal
broiler diet in Experiment 3 with 10 jig/kg of 25-(OH)D3 did not significantly decrease
the incidence of the TD lesion or its severity, which is in contrast to Experiment 2. Leach
and Lilbum (1992) stated that some other nutritional factors that influence TD incidence
may be independent of factors affecting lesion severity and effects may not always be
parallel. Incidence and severity of TD in Experiment 3 was not significantly different
from the control group until 70 ug/kg 25-(OH)D3 was fed.

Experiments 4 and 5 demonstrated that supplementing 25-(OH)D3 as a source of
vitamin D activity to reduce TD lesions in modern broiler chicks is not always effective
in the Ross x Ross strain. The results showed that TD incidence was consistently down to
about 8% when 36 to 54 pig/kg are fed, but was not a statistically significant response
even when TD incidence was 22% in the basal group of Experiment 5. This is in contrast
to Experiment 3 in which there was a linear decrease in TD incidence and severity at
levels of 25-(OH)D3 up to 70 ug/kg. Based on Experiments 3, 4, and 5, feeding 70 rig/kg
of 25-(OH)D3 is not much different from feeding intermediate levels in the range of 18 to
54 rig/kg. Mitchell et al. (1997a) also had conflicting results with regards to incidence
when the two experiments were compared. In one of the experiments, they reported a
significant reduction in TD incidence in LTD line of chicks, from 44% to 10% after 25-
(OH)D3 supplementation levels of as low as 5 rig/kg. In another experiment, 5 ug/kg 25-
(OH)D3 only reduced TD incidence from 61% to 38% (LTD line) while supplementation
levels between 10 to 40 jig/kg resulted in half the TD incidence observed at 5 rig/kg.

Rennie and Whitehead (1996) reported 10% TD incidence in Ross broiler chicks at 3

65

weeks of age with dietary addition of 75 jig/kg 25-(OH)D3. Many of the earlier studies
have rarely reported TD incidences below 10% with any supplementation level of 25-
(OH)D3 when feeding a normal broiler diet. Although complete prevention has been
reported, and birds were fed very high levels, 250 jig/kg 25-(OH)D3 in combination with
250 jig/kg ascorbic acid for 3 weeks (Rennie and Whitehead, 1996). The only metabolite
of vitamin D that seem to be able to absolutely prevent the condition consistently is the
active metabolite, 1,25-(OH)2D3 (Sorenson, 1991; Rennie, 1993) or the synthetic analog
la-hydroxycholecalciferol (Edwards, 1990).

The TD incidence of the control groups in both experiments reported by Mitchell
et al. (1997a) were fairly high compared to our experiments which was very similar in
design even when a low TD line was used. The reason for this might possibly be the
strain of chicks used in the author’s studies. Some broiler strains are more susceptible to
TD than others. When a "I'D-inducing diet was fed to Peterson x Hubbard and Ross x
Arbor Acres chicks, the TD incidence and severity was higher, and bone ash lower, in the
Peterson x Hubbard for all three experiments (Elliot and Edwards, 1994). In another
experiment, Edwards (1984) reported some indication that Ross x Arbor Acres chicks
had significantly lower TD incidence compared to Peterson x Hubbard. The Ross strain
may have been selected against TD so aggressively over the years that it is fairly resistant
to the metabolic disorder, provided that diets are adequate in calcium, phosphorus and
cholecalciferol. Genetics plays a significant role in TD incidence and therefore the
potency of 25-(OH)D3 in reducing TD depends to a large extent on the broiler strain.

Chickens with increased incidence of TD had fewer intestinal vitamin D receptors

(VDR) than chickens with low incidence (Soares et al., 1990). Comparisons between

66

HTD and LTD lines developed by Auburn University shows higher bone ash in LTD line
chicks and plasma concentrations of 25-(OH)D3 and 1,25-(OH)2D3 lower or equal to
HTD line chicks.

When Rennie and Whitehead (1996) fed a wheat based commercial basal diet
without 25-(OH)D3 supplementation to Ross chicks, the incidence was fairly high (64%)
displaying a TD incidence commonly seen with Peterson parental lines. The reason for
the high incidence may be that the basal diet was not a com-soybean diet, but consisted
of wheat-soybean diet. The wheat fibers contain non-fermentable fibers that can increase
the bulk of intestinal contents, increasing the rate of passage and enhancing gut motility.
This reduces the time for calcium absorption. Furthermore, these non-ferrnentable fibers
stimulate proliferation of microbes, which in turn binds minerals such as calcium making
it less available (Groff, 2000). Traditionally, practical starter diets used in broiler studies
on TD have been com-soybean meal based. No published studies compare the
effectiveness of com-soybean meal and wheat-soybean meal diets in alleviating TD.
However, Edwards (1985) demonstrated that various soybean meals when fed to broilers
can produce different TD incidences in chicks depending on the year and source from
which the soybean meal was obtained. A soybean meal from one source consistently
produced a TD incidence of 34-69% while a soybean meal from another source produced
only 14-18%. Leach and Neisheim (1965) stated that com and soybean meal diets appear
to have some protective properties against TD. After feeding broiler chicks this type of
diet Leach and Neisheim (1965) found that it completely prevented the occurrence of the
cartilage abnormality. The investigators speculated that there might be an unidentified

nutritional factor affecting bone formation in corn and soybean meal diets.

67

From the results in Experiment 6, it is difficult to know whether 25-(OH)D3 has
any effect on TD incidence at all in the current Ross x Ross broiler chick when fed a
normal broiler diet. The speculation that intermediate levels within the range of 18 to 40
pig/kg 25-(OH)D3 based from the previous experiments were necessary to reduce TD
incidence and severity, can be disputed. The low TD incidence of 12% with the control
diet suggests that the chicks were fairly resistant to the development of the cartilage
abnormality. On the other hand, it is possible that a significant number of the birds that
were not examined at l7-days and used for phase 2 had the TD condition at the time and
were not accounted for in the TD incidence of phase 1. The lesion in these second phase
birds might have spontaneously resorbed by 35-days of age, hence the low incidence in
the second phase birds. The majority of the TD lesions of the control group of phase 1
might have been less severe TD scores and therefore more easily resorbed during the
grower phase. As birds mature, the vitamin D system becomes more efficient with
regards to the conversion of cholecalciferol or 25-(OH)D3 to 1,25-(OH)zD3 which is less
efficient in young, immature and rapidly growing broilers (Edwards, 1989; Edwards,
1990). It is difficult to see any response by 25-(OH)D3 on TD during the grower phase
since we do not know accurately the birds subclinical condition at 17-days of age.
However, no effect was observed on TD in the second phase of Experiment 6. This is in
contrast to Zhang et al. (1997) who saw a slight decrease in TD incidence of LTD line
birds when fed a grower diet supplemented with 68.9 and 344.5 jig/kg from 4 to 6 weeks
of age. Birds fed the control diet in Zhang et al. (1997) had a similar TD incidence at 6
weeks and at 4 weeks of 17.2% and 14.2%, respectively. The control birds in phase 2 of

Experiment 6 in our study had virtually no TD incidence at 5 weeks of age. This is in

68

contrast with Scheideler and Ferket (2000) who reported 17.5% TD incidence at 3 weeks
and 20% at 9 weeks in a flock of male Ross x Ross birds fed a normal starter and grower
broiler diet, respectively.

Environmental factors might also have played a role in the low TD incidence as
well as severity observed in the non-supplemented birds of Experiment 4, 5 and 6. Birds
that are exposed to stress during their growth period may have higher incidence of TD.
Stressed birds have greater susceptibility to infection as the immune system is
suppressed. Toxic stresses which result in malabsorption of nutrients such as the fat-
soluble vitamin D3 (fat-malabsorption) that are essential for bone development and
growth can increase TD incidence (Calabotta, 1997). Stress levels are more likely to be
higher under commercial conditions. The level of stress that birds experienced during the
experimental periods might not have been high enough to induce a strong effect on the
development of TD. The type of surface that broiler chicks are raised on also influences
the incidence of TD. In the commercial broiler industry, broiler chicks are commonly
raised on the floor of various litters depending on the farm and flocks often exhibit 15 to
30% TD incidence (Veltrnann and Jensen, 1980). In a study by Veltrnann and Jensen
(1980), four-week old broiler chicks raised on wood shavings had 39% TD incidence
versus 2% incidence in chicks raised on wire floors of battery brooders. In another study,
chicks raised on wire floors had 8% TD versus 26% in chicks raised on conventional
floor pens. Certain types of mycotoxins have been shown to induce TD up to 85-90% in
chicks if the compound is present in feed (W alser et al., 1980) but a major outbreak of
mycotoxins in the feed is fairly rare and probably only plays a small factor in increased

TD incidence in the modern broiler industry.

69

Phytate phosphorus retention was clearly improved by ultraviolet light in
Experiment 1 as vitamin D activity increased resulting in an increased degradation of
phytate phosphorus. The improved retention of phosphorus and possibly calcium
probably allowed an increased availability of these minerals contributing to the improved
bone ash and reduction of rickets incidence in Experiment 1. Cations such as calcium can
chelate to phosphate groups of phytic acid forming insoluble Ca-phytate complexes in the
intestine (Nelson and Kirby, 1987). When the phytate is hydrolyzed minerals such as
calcium that are part of the molecule are released. Shafey et al. (1990) suggested that
cholecalciferol stimulates this hydrolysis resulting in enhanced absorption of both
phosphorus and calcium.

The linear increase observed on phytate phosphorus retention by the increasing
levels of dietary 25-(OH)D3 in Experiment 2 confirmed that 25-(OH)D3 can improve
phosphorus availability in chicks. Angel et al. (2001) reported that 70 jig/kg 25-(OH)D3
had a significant sparing effect (0.035% P spared) on non-phytate phosphorus in Ross
chicks fed a diets containing graded levels of non-phytate phosphorus (0.24, 0.32 and
0.40%) and calcium at 0.80%. As with the TD inducing diet of Experiment 2, the
retention of phytate phosphorus was also improved in a normal broiler diet in Experiment
3. Recent studies have shown that supplementation of 25-(OH)D3 to broiler diets improve
phytate phosphorus utilization. Edwards (1996) reported that a diet containing 0.67% Ca
and 0.33% in supplemented with 5 jig/kg 25-(OH)D3 resulted in increased phytate
phosphorus retention from 53% to 74%. In our studies, the phytate phosphorus retention
was not as high as Edwards (1996) with supplementation of 25-(OH)D3. This difference

is probably due to the lower calcium levels used in the study by Edwards (1996). When

70

calcium was decreased from 1.0% to 0.5%, phosphorus utilization was increased by 15 %
(Mohammed et al., 1991).

When Hubbard X Peterson chicks were fed diets containing 2200 ICU/kg
cholecalciferol and 0.33% phytate phosphorus (0.51% total P) with various levels of Ca
(0.4 or 0.88%), supplementation of 210 rig/kg 25-(OH)D3 improved ileal hydrolysis of
phytate phosphorus from 42.9% to 64 % (Applegate et al., 2000). In our study, the
supplementation of 25-(OH)D3 improved phytate phosphorus retention slightly, but
significantly at 36 jig/kg in Experiment 4 from 44.2 to 50.2% but not in Experiment 5.
However, the phytate phosphorus retention overall in Experiment 5 (average 51.9%) was
generally higher than any of the experiments when a marginal calcium diet was fed.
Analyzed total phosphorus was slightly lower for Experiment 5 (0.67%). Higher P.-
(inorganic phosphorus) are able to inhibit the catalytic action of certain phytases
(W oodzinsky and Ullah, 1996). Perhaps 25-(OH)D3 facilitates the activity of phytase in
the hydrolysis of phytic acid indirectly by transporting P,- away from the intestinal
mucosa into the blood, hence decreasing the inhibitory effect of P,-. The phytate
phosphorus retention in Experiment 6 (average 41%) for the starter group was also lower
than Experiment 5, resulting in no improvement by 40 jig/kg 25-(OH)D3
supplementation. The basal diet had a higher analyzed calcium level of 0.94%, which
possibly decreased hydrolysis of phytate.

Low bone ash (25.2%) when birds were fed a diet with no added cholecalciferol
in Experiment 1 is in agreement with Edwards et al. (1994) who reported 29% under
similar conditions in 16-day old birds. Exposing birds to ultraviolet light gave almost an

identical response in bone ash (41.6%) compared to Edwards et al. (1994) (42.1%)

71

whereas Elliot and Edwards (1997) observed 37% at 16-days of age. Addition of 10
[lg/kg 25-(OH)D3 in our study resulted in similar bone ash (41.2%) produced by
ultraviolet light alone. Sunde (1976) reported that 10 rig/kg 25-(OH)D3 did not result in
maximum bone ash at 3 weeks in turkey poults (42.9%) but that it was near the maximum
obtained with 25 rig/kg of cholecalciferol (46.7%). Boris et al. (1977) indicated that 25-
(OH)D3 is twice as potent as cholecalciferol in maintaining maximum bone ash.

Experiment 2 resulted in 41.8 % bone ash with 1100 ICU/kg cholecalciferol in the
control diet which confirmed the findings of Boris et al. (1977). However, Edwards et
al.( 1996) stated that 1,250 ICU/kg is not adequate to produce maximum bone ash.
Experiments 4 and 5 had a fairly consistent bone ash percentage of about an average of
43—44% with 22001CU/kg cholecalciferol with or without 25-(OH)D3 supplementation.
However, Experiment 3 had the highest and most consistent bone ash with an average of
45%. Although maximum bone ash was obtained with 36 jig/kg 25-(OH)D3 in
Experiment 5, a study by Mitchell et al. (1997a) achieved this at 10 and 20 jig/kg 25-
(OH)D3. In Experiments 4 and 5 of the present study, 36 rig/kg 25-(OH)D3 appears to
give the best results overall when considering bone ash %, the virtual absence of severe .
TD lesions and consistent gain: feed response.

The extremely low 17-day body weight (254 g) observed for chicks consuming
the vitamin D deficient diet in pens with filtered lights of Experiment 1 was expected.
Normally, slower grth rates result in lower TD incidence and severity (Edwards, 1987;
Roberson et al., 1993). In a study by Edwards et al.(1994), 16-day body weight was about
350 g compared to about 400 g when adequate vitamin D activity was provided. Once

ultraviolet light was available to the chicks bodyweight tended to increase with 25-

72

(OH)D3 supplementation, but was not significantly different. Many of the earlier studies
(McNutt and Haussler, 1973; Cantor and Bacon, 1978: Yarger et al., 1995) suggested that
adding 25-(OH)D3 to the diet for broiler chicks improved body weight gain and feed
efficiency compared to cholecalciferol. However, there were no responses observed for
body weight gain with 25-(OH)D3 supplementation in any of our experiments after
Experiment 1. Body weights averaged between 400 to 500 grams for l7-day old birds
(Experiment 2 and 6) and 600 and 700 grams in 20-day old birds (Experiment 3, 4 and 5),
which is consistent with earlier studies conducted with birds of the Ross strain that were
of similar design (Rennie and Whitehead, 1996; Roberson, 1999). In Experiment 2 Ross
x Arbor Acre chicks were used, but the strain difference does not seem to have affected
body weight gain.

Feed efficiency was restored with ultraviolet light in Experiment 1 and
supplementation of 25—(OH)D3 up to 70 rig/kg in presence or absence of light did not
give any beneficial results. If the diet is adequate in cholecalciferol (1100 ICU/kg ) body
weight and feed efficiency will normally not respond to supplemental 25-(OH)D3
(Edwards, 1989). This was confirmed with all our experiments except for Experiment 5
which had significantly lower gain: feed at 36 ug/kg 25-(OH)D3 but a ratio was still an
efficiency considered normal (0.729) for 20-day old broilers.

In Experiment 1, the low body weights were also accompanied by abnormally low
serum calcium (4.52 mg/dl), which largely contributed to the poor mineralization
reflected in the low bone ash percentage. These chicks were severely deficient in vitamin
D and as a result calcium absorption was inevitably impaired. Edwards et al.(1994)

showed that chicks fed a diet with no cholecalciferol in the absence of ultraviolet light

73

also had serum calcium levels of 7.7 mg/dl compared to about 11.0 mg/dl in chicks given
ultraviolet light or adequate vitamin D3. Mitchell et al. (1997a) reported increased plasma
calcium with as low as 5 jig/kg cholecalciferol. Serum calcium and serum phosphorus
were fairly constant across all experiments and were not affected by 25-(OH)D3
supplementation. Calcium ion concentrations in the circulating blood is necessary for
blood clotting and inter-neural signal transmission and its metabolic role is vital to life
and therefore extracellular calcium (ECF [Ca2*]) must be sustained. The skeleton serves
as a calcium reserve from which ECF can aquire calcium during deficiency and excess
calcium is simply excreted in the feces (Heaney, 2002). This is why serum calcium will
not be heavily influenced by supplemental 25-(OH)D3. However, prolonged severe
calcium or vitamin D deficiency will lower calcium in the blood as was observed in
Experiment 1 and the result of this is reflected in the bone mass or ash content.

A possible explanation for the relatively higher serum phosphorus concentrations
in Experiment 1 could be that it came from the dissolution of amorphous calcium
phosphate, which is found in large amounts in young bone. The amorphous calcium
phosphate has lower calcium to phosphorus ratio than pure hydroxyapatite, the latter that
is found more commonly in mature animals. Moreover, the TD-inducing diet is high in
phosphorus relative to calcium, which possibly increased serum phosphorus. Although,
the phosphorus absorption is not as efficient in the vitamin D deficient status, a low
absorption of phosphorus will suppress PTH levels, signaling to the kidney to increase
phosphorus reabsorption and decrease phosphate excretion. Phosphorus levels in the
body are highly regulated by the renal reabsorption and excretion process, whereas

calcium levels rely primarily on PTH to stimulate 1,25-(OH)2D3 production from 25-

74

(OH)D3 and less on renal reabsorption and excretion (it is presumed that 99% of all
filtered calcium is automatically absorbed by the kidney (Deluca 1982)). The latter
metabolite, which is probably already present in very low concentrations in the pool due
to the vitamin D deficient status inevitably, limits 1,25-(OH)2D3 production and
subsequently its participation in improving intestinal absorption of either mineral.
Despite the large amount of new information that has been published on TD
during the past decade the etiology of TD is still not clear. The implications of feeding
different metabolites of cholecalciferol such as 25-(OH)D3 have been the most
identifiable solutions in combating the TD lesions. The responses observed with 25-
(OH)D3 on TD deve10pment in our study and earlier nutritional studies are variable.
Studies on other suggested beneficial effects of dietary 25-(OH)D3 such as growth
stimulation have also been inconsistent, but to a lesser extent, and appears to be affected
by age, strain and genetics. The role of 25-(OH)D3 in phosphorus utilization has also
received more attention in recent years and a relationship between 25-(OH)D3 and
phytate degradation has been established although the mechanism in achieving this is also
unclear. There is evidence that shows that 1,25-(OH)zD3 is a more superior metabolite in
preventing TD (Edwards, 1989, 1990; Rennie et al., 1993) and increasing phytate
phosphorus retention (Edwards, 1993) in broiler chicks. The advantage of 1,25-(OI-D3D3
is its ability to interact with receptors at target tissues which is due to its hydroxylation at
position 1 on its carbon skeleton. The 1,25-(OH)3D3 metabolite binds to the VDR with
high affinity and specificity. The 25-(OH)D3 metabolite is only able to compete for the

receptor when concentrations are three-fold that of 1,25-(OH)2D3 (Soares, 1995). The

75

potency of 1,25-(OH)2D3 allows for a very small margin of safety from toxicity which is

why the commercial use of 1,25-(OH)2D3 is tightly regulated.

76

CHAPTER 3

SUMMARY, CONCLUSION AND IMPLICATION

Skeletal deformities and leg weakness have become one of the major problems to
the poultry industry. Skeletal problems likely cause the broiler industry close to 120
million and the turkey industry 40 million dollars in losses every year (Sullivan, 1994).
Tibial dyschondrOplasia (TD) is a common skeletal abnormality found in young rapidly
growing meat-type poultry (ducks, broiler chickens and turkeys) and is influenced by
nutrition as well as genetics (Riddel, 1981; Edwards, 1984). Feeding 25-(OH)D3 provides
growth stimulation in addition to TD prevention and the metabolite can improve
phosphorus utilization in broiler chicks. The objective of this study was to study the
effects of 25-hydroxycholcalciferol on the incidence and severity of TD and phosphorus
utilization in broiler chicks and to estimate the level needed to reduce TD and improve
phosphorus utilization when chicks are fed a normal broiler diet. Based on the previous
studies we hypothesized that dietary supplementation of 25-(OH)D3 will reduce the
incidence of TD in broilers at a lower level than the manufacturer’s recommendations of
69 rig/kg regardless of whether the birds are fed a normal broiler diet or TD inducing
diet.

In conclusion, the low TD incidence observed with 25-(OH)D3 supplementation
in our studies is partly contributed by the type of broiler strain that was used. The dietary
calcium concentration of about 0.90% seems to be a threshold level for Ross x Ross
chicks in our studies, a point at which TD incidence will not be reduced with any level of

25-(OH)D3 supplementation. This threshold appears to be different in other meat-type

77

strains. The addition of 40 to 70 jig/kg clearly reduced and minimized TD when birds
were affected by a low or marginal calcium diet. Lower level of 25-(OH)D3 is much more
effective when calcium is deficient (TD-inducing). Supplementation with 25-(OH)D3
does not seem to improve performance in normal starter or grower broiler diets. Lower
levels of 25-(OH)D3 can increase phytate phosphorus in broiler starter diets. The
inclusion of 25-(OH)D3 in the grower diet appears beneficial as it improved phytate
phosphorus retention.

Supplementation of 25-(OH)D3 to normal starter and grower diets may have
important practical effects in the commercial poultry industry as it can increase
phosphorus utilization. The 25-(OH)D3 metabolite increases phytate phosphorus retention
in the gut of the birds. Excessive levels of phosphorus in the manure is of great
environmental concern in areas of intensive animal agriculture production which poultry
growers and integrators are faced with. The majority of the producers in the commercial
poultry industry who supplement 25-(OH)D3 in their operations remove it from the diets
in the grower phase (personal communication, Marty Allison, 2002). This is likely due to
a cost factor as the price per pound (lb) per ton of feed (69ug) of 25-(OH)D3 is $1.65.
The inclusion of 25-(OH)D3 in the grower diet may potentially reduce some of the
excessive phosphorus that is excreted in the manure. In addition, with regards to 25-
(OH)D3 supplementation and TD prevention, Ross birds fed 0.90-1.0% Ca do not appear
to need Hy—D to prevent TD, but may be important for bone health for companies

formulating at 0.85%.

78

Table 2. Summary of Experiments

 

 

 

 

Experiment 1
Group 1 Diet UV-Light 25-(OH)D3 Cholecalciferol
(ug) (ICU)
1 1 No 0 0
2 1 Yes 0 0
3 2 No 10 0
4 2 Yes 10 0
5 3 N o 70 0
6 3 Yes 70 0
Experiment 2
Group 1 Diet UV-Light 25-(OH)D3 Cholecalciferol
(ug) (ICU)
1 1 No 0 1 100
2 2 No 10 1 100
3 3 No 40 1 100
4 4 No 70 1 100
Experiment 3
Group 1 Diet UV-Light 25-(OH)D3 Cholecalciferol
(ug) (ICU)
1 1 No 0 2200
2 2 No 10 2200
3 3 N0 40 2200
4 4 No 70 2200
Experiment 4
Group 1 Diet UV-Light 25-(OH)D3 Cholecalciferol
(ug) (ICU)
1 1 No 0 2200
2 2 No 18 2200
3 3 No 36 2200
4 4 No 54 2200
5 5 No 72 2200
6 6 N 0 90 2200

79

Table 3. Summary of Experiments

 

 

 

Experiment 5
Group 1 Diet UV-Light 25-(OH)D3 Cholecalciferol
(ug) (ICU)
l 1 No 0 2200
2 2 No 18 2200
3 3 No 36 2200
4 4 No 54 2200
5 5 No 72 2200
6 6 No 90 2200
Experiment 6
Phase 1
Group 1 Diet UV-Light 25-(OH)D3 Cholecalciferol
Starter (ug) (ICU)
1 1 No 0 2200
2 No 40 2200
Experiment 6
Phase 2
Group 1 Diet UV-Light 25-(OH)D3 Cholecalciferol
Grower (ug) (ICU)
1 1 No 0 2200
2 No 40 2200
3 3 No 0 2200

80

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81

Figure. 4. Effects of dietary 25-(OH)D3 with or without UV-light on 16-day body

weight. (Experiment 1).

Body weight
(g)

 

 

 

 

 

 

 

 

 

450 - ,’
- -A- -Filter
400 " I
3' +UV-
I, light
350 7 '1
300 r ,'
250
200 . i
0 10 70
25(OH)D3 (uglkg)

82

 

Figure 5. Effects of dietary 25-(OH)D3 with or without UV-light on rickets incidence

at 16 days of age (Experiment 1).

Rickets

 

incidence (% ) 100 1
90 ~ \

 

  

 

 

 

- -k - Filter

+ UV-
light

 

 

 

 

  

 

25-(OI'DD3 (ug/kg)

83

Figure 6. Effects of dietary 25-(OH)D3 with or without UV-light on TD incidence at
16 days of age (Experiment 1).

TD incidence
(”M 70

 

 

~ - -k - Filter

 

 

 

 

 

 

0 i i
0 10 70

25(OH)D3 (Hg/kg)

84

Figure 7. Effects of dietary 25-(OH)D3 with or without UV-light on TD severity
(N3%) at 16 days of age (Experiment 1).

'ID severity
(% N3)
40

 

 

 

 

 

 

 

 

 

10

-10

 

254011le (“g/kg)

85

 

Figure 8. Effects of dietary 25-(OH)D3 on TD incidence at 17 days of age
(Experiment 2).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TD incidence
(‘70) .
90 - 73%|
a
I
70 - l
50 3
26%|
30 b
T
i 5%I
2%|
10 ' ‘ c
f . . . FT”
0 10 40 70
-10

25(OH)D3 (“g/kg)

86

Figure 9. Effects of dietary 25-(OH)D3 on TD severity (%N3) at 17 days of age
(Experiment 2).

 

 

 

 

 

 

 

 

 

 

 

TD severity
(% N3)
80 _
59% l
70 -
a
- T
60 .l.
50 -
40 I l
30 - 16%|
b
20 -I I 27 ’ ”‘1'
3 o
10 - * c Oil
c
0 I‘LL—I T
l I l I
0 10 40 70
-10
25(OH)D3 (ug/kg)

87

Figure 10. Effects of dietary 25-(OH)D3 on % phytate phosphorus (PP) retention at
17 days of age (Experiment 2).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

%
PP retention 90
80 ~
54.7%
70 - __I
46.47 a
60 - _.”_I
39.6% I 3., T
.I.
so I 30 90/ I be I
40 — ' a T
c .I.
so — I
m -
10 -
0 . . .
o 10 40 7o
-1 r
0 25-(0H)D. (mg/kg)

88

Figure 11. Effects of dietary 25-(OH)D3 on TD incidence at 20 days of age
(Experiment 3).

TD incidence

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(%l 70
60-
50 «
40- 25%, ‘
21%|
3
30 ‘ ‘l' a
_ I» I :1
20 . 1 i
ab
10 -
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0 . . . I
0 10 40 70
'10 25—-(0H)1)3_(ug/kg)'

89

Figure 12. Effects of dietary 25-(OH)D3 on TD severity (%N3) at 20 days of age
(Experiment 3).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TD severity
(% N3) 60
50 .
40 <
30 -
13%|
20 ~ a 8% I 8% |
I ah ab 07
10 ~ - l T T °
1 1 b
0 I I I ‘1':
0 10 40 70
-10
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90

Figure 13. Effects of dietary 25-(OH)D3 on % phytate phosphorus retention (PP) at
20 days of age (Experiment 3).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

% PP
retention 90
so -
70 -
46% <
60 ‘ __I 43.1% |
50 - 34.3% I a a
1' . 342%|
40 - b 1
r b
l
30 - I
20 - '
I
10 ~
0 I I T
0 10 4o 70
.10
25(OH)D3 (“g/kg)

91

Figure 14. Effects of dietary 25-(OH)D3 on TD incidence at 20 days of age
(Experiment 4).

TD incidence

 

(%) 60
50 —
40 '

30‘

zo-El‘fl

 

 

10 ‘ I I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0 18 36

 

 

 

'10 25-(0H)D3(ug/kg)

92

Figure 16. Effects of dietary 25-(OH)D3 on TD incidence at 20 days of age
(Experiment 5).

TD incidence
(% )

 

50-

40-

22% I .
30 - 18%

I 15%| ’
20- I 10%| 8%

I
10- ~ W! I

12%

 

 

 

+——‘: ~—-—« I

 

 

 

 

 

 

 

 

 

 

 

 

 

0 r . . I
0 18 36 54 72 9o

 

 

 

-10
25(OH)D3 (“g/kg)

93

Figure 17. Effects of dietary 25(OH)D3 on TD incidence at 17 days of age
(Experiment 6, Phase I).

TD incidence
(% )

 

50~

401

30*

12% 12%

L
|_

20‘

10‘

 

 

 

 

-10
25-(OH)D3 (ug/kg)

94

Figure 18. Effects of dietary 25-(OH)D3 on % phytate phosphorus retention at 35
days of age (Experiment 6, Phase II).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

% PP
t ti
re err on 80
70 a
60 - ' 47.1%I
43.0%I a 42.7%I
50 r b i b
i E
40 -
30 .
20 .
10 i
0 i i
0.0 40-40 40-0
-10

25(OH)D3 (ug/kg)

95

Table 5. Effects of ultraviolet light and 25-hydroxycholecalciferol on performance,
serum calcium and phosphorus, and phytate phosphorus retention in l6-day Ross X
Ross broiler chicks fed a Vitamin D3 deficient diet, Experiment 1

 

 

 

 

 

Serum
Treatments Concentrations
17-d Phytate P
25-(OH)D3 UV light BW Gainzfeed Calcium Phosphorus Retention
(Mg/kg) ( g ) -— mg/dl (%)
0 no 254c 0.545“ 4.52“ 9.07“ 32.9“
0 yes 469“ 0.680“ 8.23“ 7.88“ 56.4“
10 no 475“ 0.696“ 8.26“ 7.92“ 58.0“
10 yes 510“b 0.673“ 8.86“ 7.75“ 60.5“
70 no 562“ 0.711“ 8.00“ 7.61“ 53.2“
70 yes 526““ 0.713“ 7.94“ 8.56““ 51.6“
x 466 0.670 7.64 8.13 52.1

Pooled SE 19 0.019 0.93 0.86 3.1
Main effect means

25-(OH)D3

0 361c 0.613“ 6.37“ 8.47 44.6“
10 492“ 0.684“ 8.52“ 7.85 59.3“
70 544“ 0.712“ 6.97““ 7.14 52.4““
Light

No 430“ 0.650“ 6.26“ 7.56 48.0“
yes 502“ 0.689“ 8.30“ 8.09 56.2“
ANOVA

Source of Variation Probabilities

25-(OH)D3 <0.001 <0.001 0.095 0.319 0.013
Light <0.001 0.027 0.016 0.496 0.034
25-(OH)D3 x Light <0.001 <0.001 0.133 0.077 0.021
Regression of 25-(OH)
Linear 0.002 0.008 0.870 0.181 0.598
Quadratic 0.014 0.076 0.065 0.634 0.030

 

 

 

N Means in a column with no common superscript differ significantly (P<.05)

96

Table 6. Effects of ultraviolet light and 25-hydroxycholecalciferol on rickets
incidence, development of tibial dyschondroplasia and percentage bone ash in 16-
day Ross X Ross broiler chicks fed a Vitamin D3 deficient diet, Experiment 1.

 

 

 

 

 

 

 

 

Treatments Rickets Tibial Dyschondroplasia
Severity
Severity
25-(OH)D3 UV light RI Incidence Score 1 Score 2 N3 Bone ash
(Hg/kg) (%) (%) (%) (%)
0 no 93“ 58“ 2.19““ 1.28“ 23““ 25.2“1
0 yes 13“ 42“ 1.96“b 0.78“ 12“c 41.6°
10 no 12“ 56“ 2.39“ 1.33“ 30“ 41.2“c
10 yes 0“ 15“ 1.75““ 0.23c 0“ 42.5““
70 no 0b 15“ 1.38“c 0.20“ 0c 43.1“
70 yes 0“ 7“ 0.75c 0.07c 0c 42.4““
Y 20 32 1.74 0.65 11 39.3
Pooled SE 4 6 0.29 0.17 5 0.5
Main effects means
25-(OH)D3
0 53“ 50“ 2.07“ 1.03“ 18“ 33“
10 6“ 36“ 2.07“ 0.78“ 15“ 42“
70 0“ 11c 1.06“ 0.14“ 0“ 42“
Light
yes 35“ 43“ 1.98 0.94“ 18“ 36“
no 4“ 21“ 1.49 0.36“ 4“ 42“
ANOVA
Source of Variation df Probabilities
25-(OH)D3 2 <0.001 <0.001 <0.001 <0.001 0.003 <0.001
Light <0.001 <0.001 0.054 <0.001 0.002 <0.001
25-(OH)D3 x Light 2 <0.001 0.031 0.737 0.028 0.017 <0.001
Linear 0.016 <0.001 <0.001 <0.001 0.005 0.019
Quadratic 0.008 0.463 0.313 0.858 0.771 0.006

 

“1 Means in a column with no common superscript differ significantly (P<.05)

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REFERENCES

Aburto, A., and W. M. Britton. 1998. Effects of different levels of vitamins A and E on
the utilization of cholecalciferol by broiler chickens. Poult. Sci. 77:570-577.

Angel, R., A. S. Dhandu, T. J. Applegate, and M. Christman. 2001. Phosphorus sparing
effect of phytase, 25-hydroxycholecalciferol, and citric acid when fed to broiler
chicks. Poult. Sci. 80(Supp1. 1):134. (Abstr.)

Applegate, T. J., R. Angel, H. L. Classen, R. W. Newkirk, and D. D. Maenz. 2000. Effect
of dietary calcium concentration and 25-hydroxycholecalciferol on phytate
hydrolysis and intestinal phytase activity in broilers. Poult. Sci. 79(Suppl. 1):21.
(Abstr.)

Association of Official Analytical Chemists,l984. Official methods of analysis. Assn.
Offic. Anal. Chemists, Washington, DC.

Bar, A., M. Sharvit, D. Noff, S. Edelstein, and S. Hurwitz. 1980. Absorption and
excretion of cholecalciferol and of 25- hydroxycholecalciferol and metabolites in
birds. J. Nutr. 110:1930-1934.

Blunt, J. W., Y. Tanaka, and H. F. DeLuca. 1968. The biological activity of 25-
hydroxycholecalciferol, a metabolite of vitamin D3. Proc. Natl. Acad. Sci. USA
61:714-718.

Boris, A. 1977. Relative activities of some metabolites and analogs of cholecalciferol in
stimulation of tibia ash weight in chicks otherwise deprived of vitamin D. J. Nutr.
107: 194- 198.

Burton, R., A. Sheridan, and C. Howlett. 1981. The incidence and importance of tibial
dyschondroplasia to the commercial broiler on industry in Australia. Br. Poult.
Sci. 22:153-160.

Calabotta, D. F. 1997. Use of 25-(OI-DD3 may improve bird performance. Feedstuffs. 69:
1 1-14.

Cantor, A., and W. Bacon. 1978. Performance of caged broilers fed vitamin D3 and 25-
OH vitamin D3. Poult. Sci. 57:1123-1124.

110

Clinical Application of Atomic Absorption Emission Spectroscopy. 1972. The IL
Application Laboratory. Lexington, MA.

Deluca, H. 1982. Metabolism and molecular mechanism of action of vitamin D.
Biochem. Soc. Trans. 10:147-158.

Edwards, H. M. 1985. Effects of different soybean meal on the incidence of tibial
dyschondroplasia. J. Nutr. 115: 1005-1015.

Edwards, H. M. 1995. Efficacy of several vitamin D compounds in increasing phytate
phosphorus utilization in chickens. Poult. Sci. 74(Suppl. 1):107. (Abstr.)

Edwards, H. M. 1999. Responses to vitamin D3 metabolites tend to vary. Feedstuffs 71:
14-15.

Edwards, H. M., and M. B. Gillis. 1959. A Chromic oxide balance method for
determining phosphate availability. Poult. Sci. 38:569-574.

Edwards, H. M., Jr. 1984. Studies on the etiology of tibial dyschondroplasia in chickens.
J. Nutr. 114:1001-1013.

Edwards, H. M., Jr. 1989. The effect of dietary cholecalciferol, 25-
hydroxycholecalciferol and 1,25-dihydroxycholecalciferol on the development of
tibial dyschondroplasia in broiler chickens in the absence and presence of
disulfiram. J. Nutr. 119:647-652.

Edwards, H. M., Jr. 1990. Efficacy of several vitamin D compounds in the prevention of
tibial dyschondroplasia in broiler chickens. J. Nutr. 120:1054-1061.

Edwards, H. M., Jr., M. A. Elliot, S. Sooncharernying, and W. M. Britton. 1994.
Quantitative requirement for cholecalciferol in the absence of ultraviolet light.
Poult. Sci. 73:288-294.

Elliot, M. A., and H. M. Edwards, Jr. 1994. Effect of genetic strain, calcium, and feed
withdrawal on growth, tibial dyschondroplasia, plasma 1,25-
dihydroxycholecalciferol, and plasma 25-hydroxycholecalciferol in sixteen-day-
old chickens. Poult. Sci. 73:509-519.

111

Elliot, M. A., and H. M. Edwards, Jr. 1997. Effect of 1,25-dihydroxycholecalciferol,
cholecalciferol, and fluorescent lights on the development of tibial
dyschondroplasia and rickets in broiler chickens. Poult. Sci. 76:570-5 80.

Goldenberg, H., and A. Fernandez. 1966. Simplified method for the estimation of
inorganic phosphorus in body fluids. Clin. Chem. 12:871-882.

Gomori, G. 1942. A modification of the colorimetric phosphorus determination for use
with photoelectric colorimeter. J. Clin. Lab. Med. 27:955-960.

Groff, J ., and S. Gropper. 2000. Fat soluble vitamins. Pages 316-370 in Advanced
nutrition and human metabolism. Marshall, P, Belmont, CA.

Haddad, J. G., Jr., and J. Walgate. 1976. 25-hydroxyvitamin D transport in human
plasma. Isolation and partial characterization of calcifidiol-binding protein. J.
Biol. Chem. 251:4803-4809.

Heaney, R. P. 2002. Ethnicity, bone status, and the calcium requirement. Nutr. Res. 22:
153-178.

Holick, M. F. 1981. The cutaneous photosynthesis of previtamin D3: A unique
photoendocrine system. J. Invest. Dermatol. 77:51-58.

Latta, M., and M. Eskin. 1980. Phytate phosphorus determination. J. Agric. Food Chem.
28:1313—1315.

Leach, R., Jr., and M. Neisheim. 1965. Nutritional, genetic and morphological studies of
an abnormal cartilage formation in young chicks. J. Nutr. 86:236-244.

Leach, R. M., and M. Lilbum. 1992. Current knowledge on the etiology of tibial
dyschondroplasia in the avian species. Poult. Sci. Rev. 4:57-65.

Lewis, P. D., and M. T. R. 1998. Responses of domestic poultry to various light sources.
World's Poult. Sci. J. 54:7-25.

Lofton, J. T., and J. H. Soares, Jr. 1986. The effects of vitamin D3 on leg abnormalities in
broilers. Poult. Sci. 65:749-756.

112

 

McNutt, K., and M. Haussler. 1973. Nutritional effectiveness of 1,25-
dihydroxycholecalciferol in preventing rickets in chicks. J. Nutr. 103:681-689.

Mitchell, R. D., H. M. Edwards, Jr., and G. R. McDaniel. 1997. The effects of ultraviolet
light and cholecalciferol and its metabolites on the development of leg
abnormalities in chickens genetically-selected for a high and low incidence of
tibial dyschondroplasia. Poult. Sci. 76:346-354.

Mohammed, A., M. J. Gibney, and T. G. Taylor. 1991. The effects of dietary levels of
inorganic phosphorus, calcium and cholecalciferol on the digestibility of phytate-
P by the chick. Br. J. Nutr. 662251-259.

Moorehead, W. R., and H. G. Biggs. 1974. 2-arnino-2-methyl-1-propanol as the
alkalizing agent in an improved continuous-flow cresolphthalein complexone
procedure for calcium in serum. Clin. Chem. 20:1458-1460.

Nelson, T. S., and L. K. Kirby. 1987. The calcium binding properties of natural phytate in
chick diets. Nutr. Rep. Int. 35:949-956.

Rennie, J. S., and C. C. Whitehead. 1996. Effectiveness of dietary 25- and l-
hydroxycholecalciferol in combating tibial dyschondroplasia in broiler chickens.
Br. Poult. Sci. 37:413-421.

Rennie, J. S., C. C. Whitehead, and B. H. Thorp. 1993. The effect of dietary 1,25-
dihydroxycholecalciferol in preventing tibial dyschondroplasia in broilers fed on
diets imbalanced in calcium and phosphorus. Br. J. Nutr. 69:809-816.

Robbins, K. R., H. W. Norton, and D. H. Baker. 1979. Estimation of nutrient
requirements from growth data. J. Nutr. 109:1710-1714.

Roberson, K. D. 1999. 25-hydroxycholecalciferol fails to prevent tibial dyschondroplasia
in broiler chicks raised in battery brooders. J. Appl. Poult. Res. 8:54-61.

Scheideler, S. E., P. R. Ferket. 2000. Phytase in broiler rations. Effects on carcass yields
and incidence of tibial Dyschondroplasia. J. Appl. Poult. Res. 9:468-475.

113

Shafey, T. M., M. W. McDonald, and R. A. Pym. 1990. Effects of dietary calcium,
available phosphorus and vitamin D on growth rate, food utilisation, plasma and
bone constituents and calcium and phosphorus retention of commercial broiler
strains. Br. Poult. Sci. 31:587-602.

Soares, J. H., Jr., T. S. Shellum, and J. M. Kerr. 1990. Vitamin D receptor number and
affinity in tibial dyschondroplasia. J. Bone Min. Res. 5(Suppl. 2):Sl67. (Abstr): 5

Soares, J. H., Jr., M. R. Swerdel, and E. H. Bossard. 1978. Phosphorus availability. 1.
The effect of chick age and vitamin D metabolites on the availability of
phosphorus in defluorinated phosphate. Poult. Sci. 57:1305-1312.

Sorensen, P. 1991. The genetics of leg disorders. Pages 213-229 in Bone biology and
skeletal disorders in poultry. C. C. Whitehead, ed. 23rd Poultry Science
Symposium, Edinburgh, Scotland. Carfax publishing Co., Abingdon, England.

Sunde, M. 1975. What about 25-hydroxycholecalciferol on poultry? Proc. Dist. Feed Res.
Counc. 30: 53-62.

Veltmann, J. R., Jr., and L. S. Jensen. 1980. Variations in the incidence of tibial
dyschondroplasia associated with different environmental conditions. Avian Dis.
24: 625-630.

Vieth, R., K. McCarten, and K. H. Norwich. 1990. Role of 25-hydroxyvitamin D3 dose in
determining rat 1,25- dihydroxyvitamin D3 production. Am. J. Physiol. 258:
E780-789.

Walser, M. M., N. R. Allen, and C. T. Mirocha. 1980. Tibial dyscondroplasia induced by
toxins of F usarium roseum. Poult. Sci. 59:1669-1670 (Abstr).

Williams, C., D. David, and O. Iisma. 1962. The determination of chromic oxide in
faeces samples by atomic absorption spectrophotometry. J. Agric. Sci. 59:381-
385.

Woodzinsky, R. J., and A. H. J. Ullah. 1996. Phytase. Advan. Appl. Micro. 42:263-302.

Yarger, J. G., C. L. Quarles, B. W. Hollis, and R. W. Gray. 1995a. Safety of 25-
hydroxycholecalciferol as a source of cholecalciferol in poultry rations. Poult. Sci.
74:1437-1446.

114

Yarger, J. G., C. A. Saunders, J. L. McNaughton, C. L. Quarles, B. W. Hollis, and R. W.
Gray. 1995b. Comparison of dietary 25-hydroxycholecalciferol and
cholecalciferol in broiler chickens. Poult. Sci. 74:1159-1167.

Zhang, X., G. Liu, G. R. McDaniel, and D. Roland. 1997. Responses of broiler lines
selected for tibial dyschondroplasia incidence to supplementary 25-
hydroxycholecalciferol. J. Appl. Poult. Res. 6:410-416.

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