INFORMATION TO USERS
This was produced from a copy of a document sent to us for microfilming. While the most advanced technological means to photograph and reproduce this document have been used, the quality is heavily dependent upon the quality of the material submitted.
The following explanation of techniques is provided to help you understand markings or notations which may appear on this reproduction.
1.The sign or “target" for pages apparently lacking from the document photographed is “ Missing Page(s)”. If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting through an image and duplicating adjacent pages to assure you of complete continuity.
2. When an image on the film is obliterated with a round black mark it is an indication that the film inspector noticed either blurred copy because of movement during exposure, or duplicate copy. Unless we meant to delete copyrighted materials that should not have been filmed, you will find a good image of the page in the adjacent frame. If copyrighted materials were deleted you will find a target note listing the pages in the adjacent frame.
3. When a map, drawing or chart, etc., is part of the material being photo graphed the photographer has followed a definite method in “sectioning” the material. It is customary to begin filming at the upper left hand corner of a large sheet and to continue from left to right in equal sections with small overlaps. If necessary, sectioning is continued again—beginning below the first row and continuing on until complete.
4. For any illustrations that cannot be reproduced satisfactorily by xerography, photographic prints can be purchased at additional cost and tipped into your xerographic copy. Requests can be made to our Dissertations Customer Services Department.
5. Some pages in any document may have indistinct print. In all cases we have filmed the best available copy.
University Microfilms International 300 N. ZEEB RD„ ANN ARBOR, Ml 48106 8129102
Sto ck , Ro bert H ow ard
CHONDRODYSTROPHY IN BROILER CHICKS FED MANGANESE, BIOTIN AND CHOLINE CHLORIDE DEFICIENT DIETS
The Ohio State University P h .D . 1981
University Microfilms International 300 N . Zeeb Road, Ann Arbor, M I 48106 PLEASE NOTE:
In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V .
1. Glossy photographs or pages______
2. Colored illustrations, paper or print_____
3. Photographs with dark background
4. Illustrations are poor copy ______
5. Pages with black marks, not original copy 1
6. Print shows through as there is text on both sides of page______
7. Indistinct, broken or small print on several pages ______
8. Print exceeds margin requirements _____
9. Tightly bound copy with print lost in spine ______
10. Computer printout pages with indistinct print ______
11. Page(s)______lacking when material received, and not available from school or author.
12. Page(s)______seem to be missing in numbering only as text follows.
13. Two pages numbered______. Text follows.
14. Curling and wrinkled pages______
15. Other______
University Microfilms International CHONDRODYSTROPHY IN BROILER CHICKS FED MANGANESE, BIOTIN
AND CHOLINE CHLORIDE DEFICIENT DIETS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of-The Ohio State University
By
Robert Howard Stock, B.S., M.S.
*****
The Ohio State University
1981
Approved By
Reading Committee:
Dr. J. D. Latshaw Dr. E. C. Naber Dr. K. E. Nestor J Adviser Dr. J. A. Negulesco Department of Poultry Science The author would like to dedicate this dissertation to his parents, whose support and encouragement have helped make this goal a reality.
ii ACKNOWLEDGMENTS
The author wishes to express his thanks and appreciation to his adviser, Dr. J. D. Latshaw, for his friendship and guidance, which was so essential in the completion of this dissertation.
Gratitude is also expressed to Mr. R. E. Whitmoyer, Electron
Microscopist, for his technical advise and assistance in the completion of the electron microscopy work.
A special thanks is expressed to all the faculty and staff of The Poultry Science Department for their technical help and friendship. VITA
September 27, 1953 ...... Born - Canton, Ohio
1975 ...... B.S. in Biology, Case Western Reserve University, Cleveland, Ohio
1977-198 1 ...... Research Associate, Department of Poultry Science, The Ohio State University, Columbus, Ohio
1978 ...... M.S. in Poultry Nutrition, The Ohio State University, Columbus, Ohio
1978-1981 ...... Pre-Doctoral Traineeship, Department of Poultry Science, The Ohio State University, Columbus, Ohio
PUBLICATIONS
"The Effects of Manganese, Biotin and Choline on Cartilage Hexosamine and Hydroxyproline Content as Related to Perosis." M. S. Thesis, The Ohio State University, Columbus, Ohio, 1978.
"The Effects of Manganese, Biotin and Choline on Hexosamine and Hydroxyproline Content as Related to Leg Weakness," 1981. Poultry Science, 60: 1012-1016.
FIELDS OF STUDY
Major Field: Poultry Nutrition
Studies in Nutrition. Professors Edward C. Naber and Steven D. Clarke
Studies in Biochemistry. Professors George P. Royer and Elizabeth L. Gross
Studies in Physiology. Professors Harold S. Weiss and Jim A. Grossie
iv TABLE OF CONTENTS Page DEDICATION...... ii
ACKNOWLEDGMENTS...... i i i
VITA ...... iv
LIST OF TABLES...... v ii
LIST OF FIGURES...... Vi 1 i
LITERATURE REVIEW...... 1
Introduction ...... 1 Leg Weakness Nomenclature...... 2
Rickets ...... 3 Focal Osteodystrophy ...... 3 Twisted Leg...... 4 Chondrodystrophy...... 4 Tibial Dyschondroplasia...... 5 Spondylolisthesis...... 5 Osteoporosis...... 6
The Normal Avian Bone...... 6 Cartilage...... 8 Collagen...... 8 Proteoglycan ...... 11 Nutritional Deficiencies and their Interrelationships with Leg Problems...... 13
Manganese...... 14 Biotin . 18 Choline...... 19
Research Introduction ...... 22
MATERIALS AND METHODS...... 24
Experiment 1 ...... 24
Animals, Environment and D iet...... 24
Broiler Breeders...... 24 Chicks ...... 24
Measurement of Tarsometatarsus Length and Incidence of Leg Weakness ...... 28
v Experiment I I ...... 30
Electron Microscopy ...... 30
Experiment I I I ...... 30
Collagen S o lu b ility...... 30 Hydroxyproline Determination ...... 32
Experiment IV ...... 32
Autoradiography...... 32
Animals, Environment and Diets 32 Autoradiographic Procedures... 34
Statistical Methods ...... 34
RESULTS...... 35
Electron Microscopy ...... 40 Collagen S o lu b ility...... 48 Autoradiography...... 48
DISCUSSION...... 53
CONCLUSIONS...... 58
REFERENCES...... 60
v i LIST OF TABLES
Table Page
1 Composition of Basal Diet Fed to Broiler Breeders 25
2 Broiler Breeder Treatment Groups for Experiment I 26
3 Assignment of Chick Dietary Treatments ...... 27
4 Composition of Basal Chick D iet ...... 29
5 Audiographic Treatment Groups...... 33
6 Percent Egg Production by Week of Broiler Breeders on Various Diets...... 36
7 The Effect of Broiler Breeder Diet on the Three Week Weight of their Offspring...... 37
8 The Effect of Broiler Breeder Diet on the Tarsometatarsus Length of their Offspring...... 38
9 The Effect of Broiler Breeder Diet on the Incidence of Leg Weakness in their Offspring 39
10 Effect of Diet on Epiphyseal Cartilage Collagen S olubility...... 49
v i i LIST OF FIGURES
Figure Page
1 Epiphyseal Cartilage from a Chick Fed a Control Diet (X 22,500)...... 41
2 Epiphyseal Cartilage of a Chick Fed the Control Diet (X 6,750) ...... 42
3 Epiphyseal Cartilage of a Chick Fed a Manganese Deficient Diet (X 22,500) ...... 43
4 Epiphyseal Cartilage of a Chick Fed a Biotin Deficient Diet (X 22,500) ...... 44
5 Epiphyseal Cartilage of a Chick Fed a Biotin Deficient Diet (X 4,500) ...... 45
6 Epiphyseal Cartilage of a Chick Fed a Choline Chloride Deficient Diet (X 15,000) ...... 46
7 Epiphyseal Cartilage of a Chick Fed a Choline Chloride Deficient Diet (X 6,750) ...... 47
8 Autoradiography of Extracellular Matrix from a Chick Fed the Control Diet and Dosed with (1,2 14c) Choline Chloride (X 400) ...... 50
9 Autoradiography of Extracellular Matrix from a Chick Fed Choline Chloride Deficient Diet Diet and Dosed with (1,2 14C) Choline Chloride (X 400)...... 51
10 Autoradiography of Extracellular Matrix from a Chick Fed the Control Diet and Not Dosed with (1,2 14C) Choline Chloride (X 400)...... 52 LITERATURE REVIEW
Introduction
Leg weakness in broilers is a problem of considerable economic importance today. Putting a dollar value on the problem, however, is d iffic u lt, due to several variable factors. One is that the severity of the problem varies from flock to flock for no apparent reason. In addition, economic losses due to leg problems are due mainly to down grading of the carcasses and parts during processing.
The loss due to downgrading is affected by individual inspec tors. One inspector might downgrade broilers with misshapen legs, causing a large dollar loss. A different inspector might not downgrade for the same condition, thus causing l i t t l e loss.
I t appears that the selection of birds for faster growth rates may be, in part, responsible for the increased incidence of leg weak ness. I t is usually the faster-growing, larger birds which are most affected. Serfontein and Payne [1934], along with Sheridan, et a l.
[1978], have shown that the incidence of tib ia l dyschondroplasia, twisted leg and slipped tendon could be increased by selective breeding.
Many other factors, including environment, disease and nutrition, influence the incidence and type of leg weakness. A higher incidence of leg weakness is generally found in broilers reared in cages than those reared on li t t e r . The type of floor in the cage is also important, with those on wire floors having more leg problems than those on plastic mats [Naye and Simons, 1978]. Slinger, et a l.
[1955] found that turkeys raised on deep li t t e r had a lower incidence and severity of leg problems than those which were porch reared. The higher mechanical stress on the legs of the porch reared birds was theorized to be the main factor involved with the problem.
Nairn [1973] described bacterial osteomyelitis and synovitis as being common causes of leg weakness in turkeys. The organisms are also a problem in broilers. The level of manganese, biotin, choline, niacin, zinc, folic acid, riboflavin, pantothenic acid, vitamin E and phosphorus in the diet have all been shown to have some effect upon the incidence of leg weakness in broilers and turkeys [Scott, 1950; Jones, et a l., 1962; Briggs, 1946; Hunt and McGinnis, 1959; Scott, 1953].
Research on the problem of leg weakness is complicated by the interactions of genetics, environment, nutrition and disease. Thus, when comparing the findings of different researchers, these factors must be taken into consideration. The outcome of the research can also be affected by the way the birds are handled. Rough handling increases the mechanical stress on the legs of the birds and this seems to increase the incidence of leg weakness.
Leg Weakness Nomenclature
Early research in the area tended to include a ll forms of leg problems under the term "perosis." This practice has further confused an already complex research area. To avoid this, the terminology set forth by Wise [1975] and Nairn and Watson [1972] w ill be followed. The leg weaknesses of most concern in nutritional studies are the osteo-
dystrophic ones. Osteodystrophy refers to a basic defect in bone
formation with no concern as to where the lesion occurs [Nairn and
Watson, 1972]. Although there are many forms of osteodystrophy, those of major concern are rickets, focal osteodystrophy, twisted leg, chondrodystrophy, tib ia l dyschondroplasia, spondylolisthesis and osteoporosis [Wise, 1975].
Rickets
Rickets is characterized by soft, long bones and beaks, with the long bones bending rather than breaking. Affected birds exhibit poor feathering and a reluctance to move. Poor mineralization is the main pathological feature and is due to a deficiency of calcium, phos phorus, vitamin Dg or a calcium-phosphorus imbalance. Rickets is not a common problem now and when observed, i t is usually due to an error in feed formulation, mixing, or the in ab ility of the bird to u tilize the vitamin D present in the diet.
Focal Osteodystrophy
The presence of a residual necrotic cartilage tongue is usually indicative of focal osteodystrophy. Involvement is usually bilateral and affected birds have a "wobbling gait" with an enlargement and out ward bowing of the hock jo in t. Although the lesions are usually found in the proximal end of the tib ia , they may sometimes be observed at the proximal end of the tarsometatarsus. This condition was fir s t described in turkeys. Focal osteodystrophy is also found in many different genetic lines of chickens [Hemsley, 1970]. The pathogenecity of the condition is unknown.
Twisted Leg
Twisted leg is a condition affecting all poultry. The in ci dence of the disorder in any given flock is usually very low and it is usually unilateral. The hock jo int becomes severely deformed, and the tibiotarsal bone exhibits a lateral twisting and bending. There are no chondrodystrophic changes and although the etiology of the problem is unknown, i t has been theorized that the problem is due mainly to traumatic damage [Wise, 1975; Nairn and Watson, 1972].
Chondrodystrophy
Chondrodystrophy is a generalized disorder of the epiphyseal plate long bones. Mineralization and appositional growth of the bones are unaffected. In severe cases the hock jo in t becomes so deformed that the gastrocnemius tendon slips o ff the intercondyloid grooves of the tarsometatarsus. This happening gives rise to the term "slipped tendon," which has often been used interchangeably with the term
"perosis."
Many nutritional deficiencies, including manganese, biotin, choline, nicotinic acid, folic acid, zinc, pyridoxine and vitamin E can lead to chondrodystrophy [Scott, 1950; Hunt and McGinnis, 1959;
Wilgus, et a l., 1936; Jukes, 1940; Gries and Scott, 1972; O'Dell and
Savage, 1957]. Wolback and Hegsted [1953] showed that bone mineraliza tion was apparently normal and suggested that the problem may be due to an abnormal epiphyseal cartilage matrix proliferation and growth.
Studies by Leach [1968] and Westmoreland and Hoekstra [1969] indicated
that the problem is that the cells within the epiphyseal are dis oriented. Thus, the pathology appears to be one of matrix formation, since cell proliferation is apparently not affected, as was suggested by Wolback and Hegsted [1953].
The most prevalent form of chondrodystrophy is seen today in
"turkey syndrome 65" (T.S. 65) [Wise, et a l., 1974]. The histopathology of "turkey syndrome 65" is identical to that found in chondrodystrophy due to a choline, nicotinic acid or zinc deficiency. The quality of the starter ration seems to play some role in the flock incidence according to Wise, et a l. [1974].
Tibia! Dyschondroplasia
Tibial dyschondroplasia was fir s t reported by Leach and Nesheim
[1965] in chicks fed a purified diet. The condition is characterized by an uncalcified plug of avascular hypertrophic cartilage usually in the proximal tibiotarsus. While tib ia l dyschondroplasia is a fa irly common defect, i t rarely results in clinical symptoms of lameness.
Leach and Nesheim [1972] suggest that the problem is an inherited defect whose expression is under nutritional control. I t also seems to be more of a problem in the larger and faster growing strains of birds.
Spondylolisthesis
Kinky back, or spondylolisthesis, involves a basic defect in the sixth thorasic vertebra. The vertebra rotates and twists, resulting in damage to the spinal cord, which causes a bilateral leg weakness. I t appears that the problem is under a strong genetic influence [Nairn and Watson, 1972].
Osteoporosis
In osteoporosis, birds exhibit a bilateral leg weakness, are reluctant to move and s it back on their hocks. While l i t t l e is known of the etiology of the problem, it is known that it primarily affects caged laying birds and is due to a withdrawal of mineral from the skeleton [Riddell, et al., 1968].
The Normal Avian Bone
Bone is a very important tissue, both anatomically and physio logically, in birds. Not only do bones provide structural support for the body, but they also provide a reservoir of ions. This is of particular importance in meeting the increased demands for ions brought about by egg production. About 99 percent of the total body calcium and 80 percent of the phosphorus are present in the skeletal system
[Bell and Freeman, 1971].
The primary constituent of bone is calcium phosphate, with over half of this compound being present as hydroxyapatite crystals. The calcium phosphate is deposited both within and between the collagen fib rils of bone and is permeated by living cells. Three main types of cells are associated with bone: osteoblasts, osteoclasts and osteo- cytes. Osteoblasts are usually found at the surface of new bone forma tion (endosteuus and periosteuus) and are responsible for the deposition of new bone. Bone reabsorption is carried out primarily by osteoclasts which are large multinucleated cells. Osteocytes, in turn, are embedded in the bone and function to maintain the bone.
Under pathological conditions, or due to various irrita n ts , osteo clasts may be involved in reabsorption of deep lying bone minerals
[Bell and Freeman, 1971; Ackerman, et al., 1976].
Bone growth in chicks, as in mammals, involves three major processes. First is the growth of the epiphyseal cartilage and its replacement by bone. This is followed by the appositional growth and remodeling of the bone [Wolback and Hegsted, 1952], While bone growth in chicks is similar to that in mammals, there are some basic d if ferences which need to be pointed out.
In chicks, once the shell of the bone is formed, blood vessles perforate and excavate the enclosed cartilage, but unlike mammals, i t is not replaced by trabecular bone. The only site of ossification in the shi+t is in the periphery of the cartilage. The growth at the ends of the long bones is s tric tly cartilagenous in chicks. Sim ilarly, in chicks the zone of cartilage proliferation of the epiphysis has a greater height than those seen in mammals. In addition, the cells of the epiphyseal plates are very fla t and tig htly packed. Vascular in filtra tio n in mammals is preceded by a more complex degeneration of the bone cells than is found in the chick. The pattern of vascular infiltration is also considerably different. In the chick*, the tunneling is more widely and uniformly spaced. The uniform spacing found in chicks would indicate that there is some determining factor controlling vascular in filtra tio n . However, no morphological evidence of this has been found yet. Another major difference is that in chicks some of the cartilage cells remain in columns and penetrate the metaphysis from their origin in the epiphyseal cartilage. The manner in which these cartilage cells are eventually replaced by bone is not yet known [Wolback and Hegsted, 1952; Bloom and Fawcett, 1966].
Cartilage
Cartilage consists of chondrocytes, chondroblasts, chondro- clasts and an extracellular matrix which is composed primarily of col lagen and a small amount of proteoglycan [Ackerman, et al., 1976].
The proteoglycan is attached to the collagen fib rils via chondroitin sulfate which it contains. The chondroitin sulfate is covalently bonded to the protein core of the proteoglycan and firmly attached to the collagen. Current knowledge debates whether the chondroitin sul fate is covalently attached to the collagen or not [Leach, 1972]. The fibrous network formed by the proteoglycan-collagen interaction forms many pockets which may be the site of apatite crystal formation.
Another theory is that the pockets are not the site of apatite crystal formation, but that they serve to align the apatite crystals with the collagen fib rils . Either way, i t is apparent that the integrity of the proteoglycan-collagen network is essential for proper bone forma tion.
Collagen
Collagen is an abundant structural protein with several unique characteristics. I t contains high levels of glycine and is the only 9
protein which contains significant amounts of hydroxyproline. Thus,
hydroxyproline determinations are commonly used as an indication of
collagen content. Collagen also contains very little cystine,
methionine, phenylalanine, tyrosine, tryptophane, and histidine.
Three polypeptide chains are arranged in a trip le helix to form col-
lagen [Gould, 1968].
Collagen molecules line up in an overlapping fashion forming
fibrils with a pattern of alternating light and dark segments. The
gaps between adjacent molecules form holes which may be the site of
initial mineralization. The biosynthesis of collagen is a complex
process which is not completely understood. I t is known that col
lagen is synthesized in precursor form. Bone collagen contains three
polypeptide chains, two of the chains are identical and are called
al chains, with the remaining chain being called a2. Both sets of
chains have precursors, proal for al and proa2 for a2 [Martin, et al.,
1975]. The proa chains have been shown to contain both amino terminal
and carboxy terminal groups which are specific to the precursor
[Monson, et al., 1975]. The proa chains are assembled into procollagen
linked by disulfide bonds. Conversion to collagen proceeds in a step wise fashion, beginning with the removal of the amino terminal groups.
Sequential cleavage of the carboxy terminal groups produce trip le
stranded intermediate molecules. A final cleavage produces collagen and a trip le stranded carboxy residue [Fessler, et al., 1975; Morris, et al., 1975; Davidson, et al., 1975]. Normally, the conversion to
collagen is rapid, but there are some inherited defects in man and
cattle where the conversion is incomplete due to a decreased peptidase activity [Martin, et al., 1975]. 10
There are several pathologic conditions which lead to changes
in collagen metabolism. Most changes in collagen metabolism are
later expressed as defects in connective tissue or bone. The effects of la th y ritic agents on collagen metabolism have probably been the most extensively studied. Experimental lathyrism is characterized by gross deformities of the connective tissue and a loss of tensile strength. The extractable collagen from la th y ritic chicks has normal molecular dimensions, helical conformation and s ta b ility to heat de- naturation. I t does vary in two ways from normal collagen. The
la th y ritic collagen fib rils are temperature sensitive, that is , they go back into solution upon cooling. In addition, the lathyritic collagen exhibits a decrease in intramolecular cross-linkage [Gross,
1963; Martin, et al., 1961], Tanzer and Gross [1964], using radio isotope incorporation studies (with uniformly labeled L-proline and tritiated L-proline 3,4 H), showed lathyrism did not signifi cantly interfere with collagen synthesis or fib ril formation. Their results also indicated that the extractable collagen from la th y ritic chicks was newly synthesized. Work by Smiley, et a l. [1962] opposed this view and suggested that while collagen synthesis is not affected, fib r il formation is inhibited. Their results indicate that the increased extractable collagen is newly synthesized and does not arise from previously insoluble collagen. Smith and Shuster [1962] further support the idea that the increased soluble collagen is newly synthe sized. They also showed that collagen synthesis and breakdown were accelerated by la th y ritic agents. From this they postulated that the in a b ility of newly synthesized collagen to form fib rils may work via a feedback mechanism to increase collagen synthesis. 11
Osteogenesis imperfecta is an inherited condition in mammals characterized by skeletal fragility, poor teeth and hypermobility of the joints. The basic mechanism was shown by Fujii and Tanzer [1977] to involve a defect in collagen metabolism. Their results indicate that the amino acid composition of the collagen was not affected but that there was a much higher proportion of reduced aldehydes and crosslinkages. This indicates a delayed maturity of crosslinking in collagen of osteogenesis imperfecta which could be responsible for the increased fra g ility of collagen during bone formation. The opposite situation occurs in osteopetrotic bone where there is an increase in the crosslinks. Although osteopetrosis is generally a genetic dis order, i t can be produced in birds with an avian leukosis. Lack of reabsorption characterizes the disorder [Banes, et al., 1978]. Since the collagen crosslinks are responsible for stabilizing the matrix, i t seems plausible that the increased crosslinking may be responsible for the lack of bone resorption [Banes, et al., 1978].
Proteoglycans
The integrity of the proteoglycan-collagen network is essential for normal bone formation. Proteoglycans are complexes formed from glycosaminoglycan groups and a protein backbone. Glycosaminoglycans, in turn, are composed of repeating dimers which contain an amino sugar in their makeup [Eisenstein, et al., 1971]. Some common glycosamino glycans are keratan sulfate, hyaluronic acid, dermatan sulfate and chondroitin sulfate. As an example, chondroitin sulfate is a disaccharide composed of repeating units of D-glucuronic acid and ■
N-acetyl galactosamine [Vaughn, 1975; Stedman's Dictionary, 1966]. 12
Proteoglycan aggregates ave composed of proteoglycan subunits which are non-covalently bonded together. Rosenberg, et a l. [1975] found that there was no difference in the size of the proteoglycan subunits in large or small aggregates and that the size of the aggre gate is primarily determined by the length of the filamentous back bone. The basic proteoglycan subunit is one which cannot be further divided without breaking the covalent bonds.
In addition to being necessary for proper matrix formation,
Baylink, et a l. [1972] theorize that loss of proteoglycans is somehow involved with the beginning of mineralization. Their research showed a loss of proteoglycans occurring at the mineralization front.
The fir s t evidence tying problems in proteoglycan synthesis to their effect on bone growth was provided by Leach [1962]. This inves tigator demonstrated that a manganese deficiency which increased leg weakness also caused a decrease in glycosaminoglycan content of epiphyseal cartilage. Further work by Leach and Munster [1962] showed that a direct relationship existed between the level of manganese in the diet and the glycosaminoglycan content of the epiphyseal cartilage.
The glycosaminoglycan content of other organic matrices was also affected by the level of manganese in the diet. The glycosaminoglycan content was, however, not affected by the level of dietary choline.
In an attempt to delineate the effects of a manganese deficiency on glycosaminoglycan content, Tasi and Everson [1967] employed guinea pigs in their experiments. Their data indicated that manganese is not involved in the metabolism of a specific glycosaminoglycan but, rather, that i t is essential for the preliminary synthesis which is common to 13 all glycosaminoglyeans. I t appears that the problem leading to skeletal abnormalities ir. manganese deficient guinea pigs is in the metabolism of the cartilage matrix. Leach and Bush [1977] found that cartilage from turkeys with inherited chondrodystrophy contained one- half the normal amount of glycosaminoglycans. Sim ilarly, a decreased amount of glycosaminoglycans is seen in analysis of osteoarthritic cartilage [Bollet and Nance, 1966].
Brachymorphic mice which are homozygous for the genetic defect have shortened long bones. Orkin, et a l . [1977] showed that the epiphyseal growth plage in brachymorphic mice was decreased in size.
In addition, the extracellular matrix did not respond to stains which were specific for sulfated glycosaminoglycans, suggesting a defect in the proteoglycan fraction of the cartilage matrix. In later work,
Orkin, et a l. [1976], using radioisotopes, showed that the proteogly cans from brachymorphic mice were, in fact, undersulfated and that the entire growth plate was affected. Autoradiographic studies by Greene, et a l. [1978] confirmed this finding and pointed out that the epiphy seal matrix granules were reduced in both size and number.
Nutritional Deficiencies and their
Interrelationship with
Leg Problems
As pointed out e a rlie r, many different nutritional deficiencies can lead to leg abnormalities. Titus and Ginn [1931] found that rice bran contains some factor which prevents leg abnormalities, while
Heller and Penquite [1936] discovered a heat stable factor in water soluble extract of bran which prevented chondrodystrophy. In the same
year, Sherwood and Fraps [1936] showed that the ash of wheat gray
shorts also prevented chondrodystrophy in birds. The fir s t major
breakthrough was made by Wilgus, et a l. [1937], who found that wheat
germ prevented chondrodystrophy, and on further experimentation,
demonstrated that manganese almost completely eliminated the disorder.
They also found that the chondrodystrophy preventing capabilities of
feeds were directly proportional to their manganese content.
In addition to manganese, many other substances have been shown
through the years to serve as anti-chondrodystrophic agents. Jukes
[1940] showed that choline was essential for the prevention of
chondrodystrophy. A biotin deficiency was reported by McElroy to
cause chondrodystrophy [McElroy and Jukes, 1940]. Daniel, et a l.
[1946] reported that folic acid was necessary to alleviate the car
tilage abnormality. More recently, Scott [1953] indicated that the
effectiveness of dried brewer's yeast in preventing chondrodystrophy
was due to its high level of niacin and antioxidants which protect
vitamin E. He concluded that high levels of both niacin and vitamin
E were required for complete protection against chondrodystrophy.
Manganese
Manganese was fir s t shown to be an essential nutrient by
Kemmer, et a l. [1931]. Their work indicated that supplementing man
ganese to an a ll milk diet improves the growth and reproduction of mice. Manganese has two major metabolic roles: i t is essential for
chondroitin sulfate formation, a component of proteoglycans which is 15 essential for normal bone formation, and two, manganese is also a com ponent of some metalloenzymes and plays a role in carbohydrate metabolism.
A manganese deficiency will produce different symptoms in different animals. The general symptoms include decreased growth, slightly reduced bone mineralization, defective bones and decreased reproductive performance [Scott, et a l., 1969].
As the absorption of dietary manganese is poor, and the avail a b ility of manganese from plant source is rather lim ited, i t has become a routine practice to supplement it to poultry rations [Heller and Penquite, 1937].
Wilgus, et a l. [1937] were the fir s t investigators able to show that manganese prevented chondrodystrophy and that the protection cereals gave against chondrodystrophy was due .to their manganese con tent. The findings of Wilgus, et a l . [1937] were confirmed by Heller and Penquite [1937], who found that 0.02 percent of the diet as MnCO^ would prevent chondrodystrophy. Lyons, et a l. [1938] demonstrated that intraperitoneal injections of MnSO^ were effective in preventing chondrodystrophy, but zinc, aluminum or iron injections were ineffec tive. Oral administration of MnSO^ was also found to be effective in some instances, but it afforded little protection against chondrodys trophy when diets were high in calcium and phosphorus [Lyons, et a l .,
1938]. The effect of manganese was found to be enhanced by the addi tion of rice bran, which may indicate that i t contains some labile substances which may help to prevent chondrodystrophy [Weise, et a l.,
1938]. Gallup, et a l. [1939] found that increasing the manganese content of the diet to 50ppm with M nClg'^O , MnSO^MHgO, KMnO^,
MnCOg* or MnO^ reduced the incidence of chondrodystrophy. Additional manganese, up to lOOOppm, was found to be both non-toxic and non- effective in preventing the chondrodystrophy that occurred early.
Early developing chondrodystrophy is probably due to a deficiency or problem occurring during embryonic development. Differences in the manganese requirements of different breeds and strains were also evident.
Work by Gallup, et a l. [1938] and Caskey, et a l. [1939] showed that the bones of manganese deficient chicks were thicker and shorter than those of normal chicks. Although there was a slight reduction in the bone ash content of the manganese deficient chicks, calcifica tion appeared to be normal. This was supported by the radioactive isotope work of Parker, et a l. [1956] which indicated that the manga nese content of the diet does not significantly change the amount of 45 32 calcium or phosphorus deposition or their location in the tibiae.
Wolback and Hegsted [1953] found distinct changes in the histological appearance of the epiphyseal cartilage of bones from manganese and choline deficient chicks. They suggest that the effects of chondro dystrophy may be due to problems in the epiphyseal cartilage matrix formation or cell proliferation and growth. Leach and Munster [1962] showed that a manganese deficient diet decreased growth rate, increased both the incidence and severity of chondrodystrophy and decreased the hexosamine content of the epiphyseal cartilage (which is commonly used as an indication of glycosaminoglycan content). The amount of glycosaminoglycan present in the epiphyseal cartilage was found to be 17 directly related to the manganese content of the diet [Leach and
Muenster, 1962]. Neither food intake nor choline content of the diet appeared to have any influence on the glycosaminoglycan content of the epiphyseal cartilage.
A decrease in the hexosamine content of the epiphyseal c a r til age also decreased its chondroitin sulfate content, which is important for the maintenance of rig id ity in connective tissues. Leach [1977] examined the problem further by trying to answer the question as to whether the decreased chondroitin sulfate was due to a decrease in its synthesis or an increase in its catabolism. No evidence was ob tained by Leach which would indicate that catabolism was increased.
This, along with the finding that chicks on diets supplemented with manganese converted more glucosamine to galactosamine than did defi cient chicks, indicates that manganese plays an important role in chondroitin sulfate synthesis. Further work by Leach showed that man ganese appears to be of importance in two enzyme systems needed for proteoglycan synthesis. Manganese is required for the polymerase system which is involved in polysaccharide chain elongation and the galactosyl transferase system which incorporates galactose into the galactose-galactose-xylose trisaccharide which serves as the linkage between the polysaccharide chain and the protein. Although the precise role of manganese in these systems is not known, i t appears that manganese is not as tig h tly bound to these complexes as i t is in metalloenzymes [Leach, 1971]. In addition to these functions, lack of manganese leads to a decreased width of the epiphyseal plate as well as the metaphysis. The growth plate of manganese deficient chicks- 18 shows a disarrangement of the cells and a reduction in the extracell ular matrix without affecting cell proliferation. These changes appear specific for a manganese deficiency, since none of the other known causes of chondrodystrophy showed the severe reduction in the extracellular matrix [Leach, 1968].
Biotin
In 1932, a new factor co-enzyme R was discovered, which was necessary for respiration in legume nodule bacteria. Then Kogl and
Tonnis extracted a substance they called "biotin" from the yolks of eggs which was necessary for the growth of yeast. At the same time,
Gyorgy found a protective factor, vitamin H, which gave protection against the effect of raw egg whites. The work of Gyorgy, et a l.
[1940] indicated that co-enzyme R, biotin and vitamin H were one and the same compound.
The main known function of biotin is to participate as a co factor in carboxylation reactions. Biotin is known to be a cofactor for five enzyme systems; acetly CoA carboxylase, B-methyl crotonyl CoA carboxylase, methylmalonyl-oxaloacetic transcarboxylase, and propionyl
CoA carboxylase. A biotin deficiency usually leads to decreased growth, decreased feed efficiency, dermatitis and chondrodystrophy.
Biotin occurs naturally in many feedstuffs either bound and unavailable or free. Some good sources of biotin are liver, yeast, egg yolks and peanuts. The biotin requirements of animals vary con siderably, depending on the amount of intestinal synthesis taking place and the amount of coprophagy going on. In some cases, intestinal 19 synthesis of biotin may be sufficient to meet the animal's entire requirement. Other factors, however, affect biotin's a v ailab ility; avidin, a constituent of raw egg whites, binds to biotin, making it unavailable. Biotin is also subject to oxidative rancidity and des truction by certain intestinal micro-organisms [Scott, et al., 1969].
In comparison to manganese and choline, very l i t t l e research has been done on biotin and its effect on chondrodystrophy. McElroy and Jukes [1940] found that the anti-egg white factor (biotin) is formed in the rumen of cattle. One of the symptoms they observed with the egg white syndrome was chondrodystrophy. I t was found that this chondrodystrophy was not prevented by choline.
Jukes and Bird [1942] showed that a biotin deficiency produces both dermatitis and chondrodystrophy. At the same time, Patrick, ej: a l. [1942] found that a biotin deficiency caused chondrodystrophy, dermatitis, increased mortality and a decreased growth rate in chicks and poults. They also found that the incidence of chondrodystrophy did not necessarily correspond to the incidence of dermatitis.
Choiine
Best and Huntsman [1932] showed that choline was the active ingredient of lecithin and that it was choline that was responsible for the effectiveness of lecithin in preventing fatty liv e r. Choline,
"beta-hydroxyethyl trimethyl ammonium hydroxide," serves the following functions [Rechcigal, 1978]:
1. It is a structural part of lecithin and sphingomyelin. 20
2. I t is important as acetylcholine in the trans mission of nerve impulses between parasympa thetic nerves.
3. I t furnishes the labile methyl groups for the formation of methionine and creatine.
The general signs of a choline deficiency are decreased growth rate, fatty liv e r, hemorrhaghic kidneys and chondrodystrophy in chickens. Some animals can synthesize sufficient choline to meet their requirements i f they have an adequate source of methyl groups in their diet. The young chick, however, can not synthesize the compound in sufficient amounts in order to meet its daily requirement. The a b ility of the chick to synthesize choline increases with age, and i t is difficult to produce a choline deficiency in birds at eight weeks of age. Some feedstuffs which are good sources of choline are liv e r, fish meal, yeast, d is tille rs solubles and soybean meal [Scott, et a l .,
1969].
Jukes [1940] found that choline was essential for the preven tion of chondrodystrophy. He found that only 0.1 percent of the diet as choline was needed to achieve adequate growth, but that 0.2 percent choline was needed to prevent chondrodystrophy. This finding is in agreement with la te r work of Hegsted, et a l. [1941], who showed that
0.1 percent of the diet was adequate for growth and less than 0.1 per cent was required for the prevention of chondrodystrophy. The d if ference in the choline requirements for the prevention of chondrodys trophy are coupled to other dietetic factors. As an example, Young, et al. [1955] found that folic acid is required to ward against chondrodystrophy, but that i t has no preventive capabilities in the absence of choline. Similarly, the amount of folic acid required for
adequate growth is tripled in the absence of choline.
Cumming and Tribe [1956], working with the interrelationship
of diet and environment, found that the incidence of chondrodystrophy
increased in animals raised on wire floors and that this incidence
could be greatly reduced by increasing the amount of dietary choline.
The beneficial effects of choline are not limited. There is a point
beyond which adding additional choline does not decrease the incidence of chondrodystrophy. Other dietary substances have been tested to
check their interrelationships with choline. Leach, et al. [1962]
found that diethylstilbestrol improved growth and decreased leg abnormalities on a choline deficient diet. When diethylstilbestrol was used with a diet adequate in choline, a negative response was obtained. Paired feeding was used to show that the decreased leg abnormalities were not due to increased feed intake with d ie th y ls til bestrol supplementation [Leach, et a l ., 1962]. Diethylstilbestrol was also found to have a slight effect on a zinc deficiency, but no effect on a manganese deficiency. Baker, et a l. [1970] showed that varying the dietary levels of gylcine in the diet had no effect on either the incidence or severity of chondrodystrophy due to a choline deficiency.
Jukes [1940] found that arsenocholine, monoethyl choline and diethyl choline were as good as choline for the prevention of chondro dystrophy. Arsenochol ine and monoethyl choline were found to promote growth but not as well as choline. Betaine and betaine aldehyde, while they did increase growth, were ineffective in the prevention of chondrodystrophy unless they were fed in conjunction with the precursors of choline [Jukes and Olson, 1954; Jukes and Welch, 1942]. 22
Research Introduction
A high v a ria b ility in the incidence of chondrodystrophy from one experiment to another has made research on the problem d iffic u lt.
Even using the same diet, environmental conditions and obtaining the chicks from the same flock does not reduce this variab ility. Thus, i t was postulated that the nutritional status of the broiler breeders may be playing a role.
To investigate this, Experiment I was set up to determine the effects of additional supplementation of the broiler breeder diets on the following parameters:
1. Percent egg production
2. The three week weight of their chicks on various levels of biotin and choline chloride
3. The tarsometatarsus length of the chicks
4. The incidence of chondrodystrophy in the chicks
In an attempt to further delineate what is happening in the epiphyseal cartilage extracellular matrix, Experiment I I , an electron microscopy study, was set up with the following objectives:
1. To compare the histopathology of epiphyseal car tilage from manganese, biotin and choline chloride deficient chicks to control chicks.
2. To examine the histology of the extracellular matrix in an attempt to localize the lesion and correlate what is seen with the biochemical func tions of manganese, biotin and choline chloride.
Previous work [Stock, 1978] has shown that the collagen con tent of epiphyseal cartilage was not decreased with a manganese, biotin or choline chloride deficiency. While these deficiencies did not alter the collagen content of epiphyseal cartilage, they may have altered the metabolism of collagen. An example of altered collagen metabolism is found in la th y ritic chicks where there is an increase in the amount of soluble collagen [Martin, et a l., 1961]. Thus, a collagen solu b ility study was set up in Experiment I I I to determine the effects of manganese, biotin and choline chloride deficiencies on it.
Experiment IV was set up to see where choline chloride was being deposited in the cartilage. Choline chloride has generally been considered to be important as a structural component of phospholipid. 14 Thus, it was theorized that the use of (1,2 C) choline chloride and autoradiographic techniques might confirm this. In addition, i t was hoped that the autoradiography might localize where the choline chloride was being deposited. MATERIALS AND METHODS
Experiment I
Animals, Environment and Diet
Broiler Breeders
Cobb Silver 100 Plus broiler breeders which were forty-six weeks old were used in the present experiment. The breeders were randomly divided into four groups of f if t y hens and five cocks each and placed in floor pens. The birds were maintained on the restricted feeding program which is recommended by Cobb. They received sixteen pounds of feed the fir s t week of the experiment and 17.5 pounds the following three weeks per fifty-five birds. The birds were fed a basal broiler breeder diet (Table l),and i t was supplemented as shown in Table 2. Eggs were collected and saved after the birds were fed the diets for two weeks. The eggs were washed, candled and set in a
Robbins incubator.
Chicks
Broiler chicks hatched from eggs produced by broiler breeders fed the four experimental diets were divided into groups of ten. The chicks from each hen diet were randomly divided within their group and placed into a battery brooder. Two replicates of ten birds each were assigned to the different treatments, as shown in Table 3. Three birds
24 TABLE 1
COMPOSITION OF BASAL DIET FED TO BROILER BREEDERS
Ingredient Percent of Diet
Corn, Ground Yellow 66.0
Soybean Meal (44% Protein) 14.0
Meat and Bone Scrap (50% Protein) 5.0
Alfalfa Meal (17% Protein) 3.0
Limestone 9.0
Dicalcium Phosphate 0.5
Vitamin and Mineral Premix1 2.5
Vitamin and mineral premix is formulated to supply the following per kg of diet: 2,214 I.U. vitamin A, 1,050 I.C.U. vitamin D, 4.7 mg Riboflavin, 3.9 mg Panothenic acid, 3.8 ug B] 2 , 1.08 g MHA, 125 mg BHT, 5 g NaC l, 54 mg Mn (MN0), 65 mg Zn (ZnO). TABLE 2
BROILER BREEDER TREATMENT GROUPS FOR EXPERIMENT I
Group Treatment
I Basal Hen Diet
II Basal Hen Diet + 1300 mg/kg Choline Chloride
III Basal Hen Diet + 0.15 mg/kg Biotin
IV Basal Hen Diet + Vitamin Mix^
Vitamin mix was formulated to supply the fo l lowing per kg of diet: 4,000 I.U. vitamin B, 500 I.C.U. vitamin D3 , 3.8 Riboflavin, 10 mg Pantothenic acid, 33.0 ug Cobalamin, 0.35 mg Folacin, 4.5 mg Pyridoxine, 16 mg Niacin. 0.8 mg Thiamin, 0.15 mg Biotin, 130G mg Choline Chloride. TABLE 3
ASSIGNMENT OF CHICK DIETARY TREATMENTS
Hen Diets Basal + Basal + Basal Choline Basal^ + A ll B iotin Chloride Vitamins Chick Diets Chick Diets Chick Diets Chick Diets 2 Basal * + 0.05 mg Biotin/kg Basal + 385 mg Choline Basal^ + 0.05 mg Biotin/kg Basal^ + 0.05 mg B iotin/kg 0.10 770 Chloride/kg 0.10 0.10 0.15 1155 0.15 0.15 0.20 0.20 0.25 0.25 0.30 0.30
Basal2 + 385 mg Choline Basal2 + 385 mg Choline 770 Chloride/kg 770 Chloride/kg 1155 1155 1540 1925 2310
^Provided 1925 mg Choline Chloride/kg Diet 2 Provided 0.11 mg Biotin/kg Diet 28
from each replicate were selected at random and wing banded for tarso-
metatarsus measurements. The chicks had ad libitum access to feed
and water. The chicks were fed the basal diet (Table 4) supplemented
as shown in Table 3. The brooder temperature was maintained at 35°C
the fir s t week, with the temperature being decreased 3°C a week. The
birds received sixteen hours of light per day for the entire time.
At three weeks of age, tarso-metatarsus lengths were measured, the
birds were scored for incidence of leg weakness and samples were taken
for electron microscopy.
Measurement of Tarsometatarsus Length
and Incidence of Leg Weakness
The tarso-metatarsus length was measured weekly to the nearest
tenth o f a centimeter. The foot of the bird was placed on a fla t
surface andthe tibiotarsus was held at a 90° angle to the tarso- metatarsus. The length was measured from the surface to the top of
the hock jo in t.
Scoring for leg weakness was done on a subjective basis. Birds
which exhibited a slight bowing or deformation of the hock, and exhib
ited a reluctance to move, were scored as having a leg weakness prob
lem only i f both legs were affected. All birds which showed extreme
distortion of a leg and had impaired locomotion were scored as having
a leg weakness problem even i f only one leg was involved. TABLE 4
COMPOSITION OF BASAL CHICK DIET
Percent Ingredient Of Diet
Corn, Ground Yellow 40.0
Soybean Meal (44% Protein) 15.0
Isolated Soybean Protein (87% Protein) 14.7
Sucrose 16.8
Corn Oil 4.4
Salt 0.5
MHA 0.5
Dicalcium Phosphate 1.81
Limestone 1.86
Cellulose 3.82
Vitamin and Mineral Premix^ 0.61
Vitamin and mineral premix is formu lated to provide the following per kg of diet: 3,000 I.U . Vitamin A, 600 I.C.U. vitamin D3 , 30 mg vitamin E, 1.52 mg vitamin K, 1.8 mg Thiamin, 6.1 mg Riboflavin, 39.9 mg Niacin, 1.2 mg Pyridoxine, 15.2 mg Panto thenic acid, 1.8 mg Folacin, 17.6 ug B12, 250 mg Inositol, 44 mg Zn (ZnO), and 200 mg FeS04 30
Experiment I I
Electron Microscopy
The proximal tarso-metatarsus was quickly exposed after the bird was killed and cubes of the epiphyseal cartilage approximately
2 mm on an edge were removed and placed in fixative 3 percent gluteraldehyde, 2 percent paraformaldehyde and 1.5 percent acrolein in a 0.1M collidine buffer at a pH of 7.3. Once in fixative, the samples were kept under refrigeration until processed. The samples were washed with 0 . 1M collidine buffer and post fixed in it with 2 percent osmium textroxide. They were then washed with 1 percent uranyl acetate and stained overnight in i t . The samples were dehy drated in increasing concentrations of ethanol and embedded in Spurr plastic. The samples were then sectioned on a Sowall MT-28 ultra microtome, placed on grids and post stained with 0.5 percent uranyl acetate and 0.1 percent lead c itra te . They were viewed and photo graphed with a Philips 201 electron microscope.
Experiment I I I
Collagen Solubility
Day-old Cobb broiler chicks were used. The chicks were ran domly dividend into twelve groups of ten and placed into a battery brooder. The chicks were fed a basal diet (Table 4) supplemented as follows:
Group I received the basal diet plus 1925 mg choline chloride and 145 mg M^O per kg of diet. In Group I I the basal diet was 31 supplemented with 0.11 mg biotin and 145 mg M^O per kg of diet. Group
III received 0.11 mg biotin and 1925 mg choline chloride per kg of basal diet. In Group IV, 1925 mg of choline chloride, 0.11 mg biotin and 145 mg M^O per kg of diet was added to the basal diet.
Three replicate groups of ten birds each were assigned to each treatment. The chicks had ad libitum access to both feed and water.
The brooder temperature was maintained at 35° the fir s t week and the temperature was reduced 3°C per week. At three weeks of age three birds from each group were killed and the epiphyseal cartilage from the proximal tarso-metatarsus was removed. Collagen solubility was determined by following the procedures of Naber, et a l. [1965]. The cartilage was homogenized in a V irtis 45 homogenizer for four minutes at 40,000 RPM in a chloroform:methanol (2:1) solution. The homo- genate was vacuum filtered and washed with several volumes of chloro form: methanol . I t was then freeze-dried and weighed. The freeze- dried cartilage was sonicated for one minute at fifty watts in 10 ml of .14M NaCl, and the container was kept in an ice bath throughout the sonication. The samples were then placed in a shaker bath at 4°C for twenty-four hours. They were then centrifuged at 100,000g for one hour in a ultracentrifuge (Spinco). The supernatant was collected and the residue was resuspended in 10 ml of 1M NaCl. The above men tioned process was then repeated. This supernatant was removed and the residue was resuspended in 10 ml of 50mM citrate-phosphate buffer at pH 3.5. The above process was again repeated and the supernatant was removed. The remaining residue was freeze-dried. Hydroxyproline content was then determined for the various fractions. 32
Hydroxyproline Determination
Hydroxyproline was determined by the method of Newman and
Logan [1950], as modified by Martin and Axelrod [1953], with a few
minor exceptions. Approximately 5 mg of the freeze-dried cartilage
residue was placed in a screw top test tube, along with 2 ml of 6N
HC1. One ml aliquots of the supernatant extracts of the cartilage
were pipeted, along with 1 ml of 12N HC1, into screw top test tubes.
The samples were then autoclaved for twenty-four hours under fifteen
pounds of pressure. They were then brought to neutrality and a
final volume of 20 ml. A 1 ml aliquot of this was used for deter mining hydroxyproline content. The samples were read on spectrometer
at a wavelength of 550 nm in tubes with a pathlength of 1.6 cm.
Experiment IV
Autoradiography
Animals, Environment and Diets
Day-old Cobb broiler chicks were weighed, wing banded and
divided"at random into two groups of fifteen chicks each. Each group was placed in a metabolism pen. The chicks had ad libitum access to
feed and water. They were provided sixteen hours of light throughout the experiment. The temperature was maintained at 35°C the fir s t week
and was decreased 3° per week. The chicks were fed the basal diet
(Table 4) supplemented as shown in Table 5. Starting on day sixteen,
the chicks received a daily dose of (1,2 C ^) choline chloride (Table
5) by direct placement into the crop. On day twenty-one, the birds TABLE 5
AUTORADIOGRAPHIC TREATMENT GROUPS
Treatment Dose u Ci/Day Group Basal Chick (1,2 Cl4) Choline Diet Plus Chloride
I 0.11 mg B iotin 5 145 mg M|\j02 and 1925 mg Choline 1 Chloride per kg of Diet
II 0.11 mg Biotin and 145 mg Mn02 per kg of Diet 1
III 0.11 mg Biotin, 145 mg Mn 02> and 1925 mg Choline 3 Chloride per kg of Diet
IV 0.11 mg Biotin, and 145 mg Mn 02 per kg of Diet 3 34 were killed and samples of the epiphyseal cartilage were taken as for electron microscopy. After the samples were embedded in plastic 2 u thick, samples were taken and affixed to slides.
Autoradiographic Procedures
The basic techniques of Stein and Yanishevsky [1979], along with some of the techniques of Rogers (1979], were followed. After the sample was affixed to the slides, the slides were dipped in Kodak
NTB 2 nuclear emulsion. The emulsion was kept in a water bath at 38°C while the slides were being dipped in i t . The slides were then placed horizontally in a test tube rack tilte d at a 45° angle to dry (2 hours) before being placed in a slide box, along with a packet of d rie rite . The box was made light tig ht with tape and then placed inside another lig h t tight box. The slides were exposed at 5°C for two weeks. They were then developed in Kodak D19 developer for two minutes, followed by a th irty second rinse in a 1 percent acetic acid stop bath. Finally, they were fixed for five minutes and rinsed under tap water for twenty minutes and allowed to dry. All the above work was done in a dark room at 5°C, using a dark red number two safe-light.
Statistical Methods
All the statistical analyses (one way analysis of variance and two way analysis of variance) were conducted by the author. The statistical tables used were taken from S tatistical Methods by Snedecor and Cochran [1967]. RESULTS
Experiment I was set up in order to demonstrate the effect of the broiler breeder diet (Table 1) on the incidence of chondrodys trophy on the offspring of these birds. The four treatments had no effect on egg production (Table 6 ). This was expected since the basal diet was considered to be an adequate diet for broiler breeders in production.
The three week weights of the birds are presented in Table 7.
No significant differences in three week weights were found due to either the various chick or hen diets. There was also no significant interaction between the chick and hen diets on three week weight. The tarsometatarsus lengths (Table 8 ) were not significantly affected by the hen diet, chick diet or their interaction.
The incidence of leg weakness in broiler chicks on the various levels of choline chloride was not significantly affected by the hen diets (Table 9). However, on the hen diets which were supplemented with choline chloride (Diet II) and with all vitamins (Diet IV), only
1155 mg of choline chloride per kg of chick diet was required to reduce the incidence of chondrodystrophy to a basal level while a higher level was required on the basal diet (Diet I).
The incidence of chondrodystrophy in the chicks fed graded levels of biotin (Table 9) was found to be significantly (P<0.05) affected by the hen diet. Both the hen diet supplemented with biotin
35 TABLE 6
PERCENT EGG PRODUCTION BY WEEK OF BROILER BREEDERS ON VARIOUS DIETS
Hen Diet I II III IV Week Basal + Basal + Basal + Basal Choiine All Biotin Chloride Vitami ns
1 47 52 52 43
2 50 60 53 41
3 47 56 55 53
4 57 63 63 59 TABLE 7
THE EFFECT OF BROILER BREEDER DIET ON THE THREE WEEK WEIGHT (g ) OF THEIR OFFSPRING1
Hen Diet Chick I II III IV Treatment Basal + Basal + Basal + Basal Choline All Biotin Chloride Vitamins
Added Biotin Diet mg/kg
0.05 469.9 ± 21.6 ------416.7 ± 53.9 492.0 ± 8.6 0.10 444.3 ± 27.9 -- 453.4 ± 4.7 449.1 ± 1.4
0.15 423.5 ± 2.7 ------444.5 ± 8.3 468.1 ± 13.0
Added Choline Chloride Diet mg/kg .
385 471.8 ± 53.7 475.5 ± 2.5 ------457.5 ± 77.3 770 427.1 ± 72.1 503.5 ± 29.8 -- 469.2 ± 26.4 1115 • 471.8 ± 40.9 490.9 ± 0.1 490.6 ± 19.9
^Threp week weight is given as weight ± standard deviation. TABLE 8
THE EFFECT OF BROILER BREEDER DIET ON THE TARSOMETATARSUS LENGTH OF THEIR OFFSPRING1
Hen Diet Chick I II III IV Treatment Basal + Basal + Basal + Basal Choiine All Biotin Chloride Vitamins
Added Biotin Diet mg/kg
0.05 6.8 ± 0.29 6.3 ± 0.52 6.7 ± 0.48 0.10 6.1 ± 0.38 -- 6.6 ± 0.39 6.6 ± 0.29 0.15 6.6 ± 0.32 --- 6.3 ± 0.50 6 .6 ± 0.29
Added Choline Chloride Diet mg/kg
385 6.8 ± 0.14 6.3 ± 0.56 --- 6.3 ± 0.16 770 6.5 ± 0.26 6.7 ± 0.44 -- 6.5 ± 0.35 1155 5.5 ± 0.15 6.7 ± 0.25 -- 6.5 ± 0.40
Harsometatarsus length is mean length (cm) ± standard deviation for six chicks. TABLE 9
THE EFFECT OF BROILER BREEDER DIET ON THE INCIDENCE OF LEG WEAKNESS IN THEIR OFFSPRING1
Hen Diet Chick I II III* IV* Treatment Basal + Basal + Basal + Basal Choii ne All Biotin Chloride Vitamins
Added Biotin Diet mg/kg 0.05 6.5 ± 2.1 --- 3.5 ± 0.7 5.0 ± 0.0 0.10 7.0 ± 1.4 -- 3.5 ± 0.7 3.5 ± 0.7 0.15 4.0 ± 1.4 --- 4.5 ± 0.7 3.5 ± 0.7
Added Choline Chloride Diet mg/kg 385 5.5 ± 0.7 5.0 ± 1.4 4.0 ± 0.0 770 5.5 ± 2.1 4.5 ± 0.7 -- 4.5 ± 2.1 1155 6.5 ± 0.7 2.5 ± 0.7 “ — 3.0 ± 1.4
^Incidence of leg weakness is expressed as mean number of birds affected ± standard deviation. 2 Hen diets III and IV significantly (P<0.05, two way analysis of variance) reduced the incidence of leg weakness for the chicks on the biotin deficient diets. 40
(Diet III) and the one supplemented with all vitamins (Diet IV) sig
nificantly decreased the incidence of chondrodystrophy in chicks fed
all levels of supplemental biotin.
Electron Microscopy
In an attempt to understand what is happening in the extra
cellu lar matrix of epiphyseal cartilage in biotin, manganese or choline
chloride deficient chicks, electron microscopy was used. Upon careful
examination of many micrographs i t was concluded that the structure
of the osteoblast was not affected by the various deficiencies. There
were, however, definite differences in the appearance of the extra
cellu lar matrix. Figures 1 and 2 are good examples of what a normal
extracellular matrix and osteoblast look like.
On a manganese deficient diet (Figure 3) the extracellular
matrix can be seen to contain less proteoglycan aggregates, and there
are more visible collagen fib rils present. In addition, the collagen
fib rils present are both larger in diameter and length than those of
the control. One other striking feature is the lack of the lacunae.
The lacunae are the clear areas surrounding the osteoblasts.
The results of a biotin deficiency (Figures 4 and 5) are very
similar to those seen with a manganese deficiency. The lacunae are missing and the collagen fib rils are longer and larger in diameter.
In addition, there seems to be a reduction in the number of large
proteoglycan aggregates present.
A choline chloride deficiency (Figures 6 and 7) produces quite
different effects from either a manganese or biotin deficiency. The 41
Figure 1. Epiphyseal cartilage from a chick fed a control diet. (X 22,500) L - lacuane, P - proteoglycan aggregate, C - coV lagen fib r il. 42
i 1 1
Figure 2. Epiphyseal cartilage of a chick fed the control diet. (X 6,750).
< 43
Figure 3. Epiphyseal cartilage of a chick fed a manganese deficient diet. (X 22,500). Figure 4. Epiphyseal cartilage of a chick fed a biotin defi cient diet. (X 22,500). 45
?jg$£a
8?fc ’ -.»
Figure 5. Epiphyseal cartilage of a chick fed a biotin d e fi cient diet. (X 4,500). 46
v W , "■.
Figure 6. Epiphyseal cartilage of a chick fed a choline chloride deficient diet. (X 15,000). 47
Figure 7. Epiphyseal cartilage of a chick fed a choline chloride deficient diet. (X 6,750). only sim ilarity is that the lacunae are missing in all three defi ciencies. The extracellular matrix appears much different with a choline chloride deficiency. The proteoglycan aggregates are not as
large as in the control, but appear to be more numerous. The collagen
fib rils are also smaller and shorter.
Collagen Solubility
Collagen solu bility was determined for chicks fed biotin, manganese, and choline chloride deficient diets. The results (Table
10) show that there is no significant difference in collagen solu b ility due to feeding the various diets.
Autoradiography
The autoradiographs showed that (1,2 ^ C ) choline chloride is incorporated into the extracellular matrix of epiphyseal cartilage of chicks fed a choline chloride adequate diet (Figure 8). However, no incorporation of the label is seen in the chicks on a choline chloride deficient diet (Figure 9). Figure 10 is the autoradiograph of a control chick which received no labeled choline chloride. As can be seen from Figure 10, the background level is low. TABLE 10
EFFECT OF DIET ON EPIPHYSEAL CARTILAGE COLLAGEN SOLUBILITY (ug/gm Tissue)!>2
Group I II III IV Biotin Choline Manganese Control Deficient Deficient Deficient
Extract I (.14 m NaCl) 85 ± 17 77 ± 4 72 ± 10 79 ± 23
Extract I I (1.0 m NaCl) 68 ± 21 65 ± 12 69 ± 4 44 ± 2
Extract III 50 mm Citrate- Phosphate Buffer 42 ± 22 43 ± 8 39 ± 10 32 ± 5
Resi due • 2340 ± 491 2245 ± 157 2432 ± 283 2492 ± 448
^Collagen solubility is expressed as mean ± standard deviation. 2 No significant difference in collagen solubility was found using one way analysis of variance. 50
Figure 8. Autoradiography of extracellular matrix from a chick fed the control diet and dosed with (1,2 14c) choline chloride. (X 400). 51
Figure 9. Autoradiography of extracellular matrix from a chick fed a choline chloride deficient diet and dosed with (1,2 choline chloride. (X 400). 52
. • & r ;*?%'. ; ; y...... :
Figure 10. Autoradiography of extracellular matrix from a chick fed the control diet and not dosed with (1,2 ^C ) choline chloride. (X 400). DISCUSSION
I t has been shown by Leach [1962] that chondrodystrophy in
chicks is due to a manganese deficiency related to a decrease in
glycosaminog!yean synthesis. The present data support these findings.
The collagen solubility study shows that a manganese deficiency has no effect on collagen solubility,and finds support in previous work
[Stock, 1978] indicating that the collagen content was normal under
these conditions. These findings indicate that the collagen portion of the extracellular matrix is normal and that the problem is, there fore, most lik e ly in the proteoglycan portion of the extracellular matrix. This is further supported by the electron microscopy study which shows a decrease in the proteoglycan content of the extracellu lar matrix of chicks fed a manganese deficient diet. Although i t is not known why the lacunae are missing in the cartilage of manganese deficient chicks, i t may be the result of a decreased proteoglycan content and the subsequent defect in the extracellular cartilage matrix formation.
While i t has been shown that a biotin deficiency will cause chondrodystrophy [McElroy and Jukes, 1940; Jukes and Bird, 1942;
Patrick, et a l ., 1942], very l i t t l e research has been conducted on its mode of action. Previous work [Stock, 1978] has shown that a biotin deficiency had no effect on the collagen content of the extracellular matrix. Although a biotin deficiency can decrease the glycosamino-
53 54 glycan content of the extracellular matrix, i t was not correlated with the incidence of chondrodystrophy [Stock, 1978]. As with a manganese deficiency, a biotin deficiency was shown to have no effect on col lagen solubility. I t appears that collagen metabolism is normal with a biotin deficiency.
The electron micrographs of biotin deficient chicks (Figures
4 and 5) are sim ilar to those of manganese deficient chicks (Figure
3). However, sincn. the glycosaminoglyean content is not diminished with a biotin deficiency, the decrease in the number of large proteo glycan aggregates must be due to some other cause. The main known function of biotin is in carboxylation reactions [Rechcigal, 1978].
Therefore, it can be postulated that biotin is required in some carbo xylation reaction which is essential for the formation of the large proteoglycan aggregates. One may assume that on a biotin deficiency, although providing for fewer large proteoglycan aggregates, that i t would not decrease the glycosaminoglycan content of the cartilage.
Sim ilarly, the lack of the lacunae may be a result of the decreased number of large proteoglycan aggregates and the subsequent defect in matrix formation.
Supplementation of the broiler breeder diets with additional biotin or one times the NRC requirement of a ll vitamins had no effect on egg production, three week weight or the tarsometatarsus length.
However, the incidence of chondrodystrophy in chicks fed graded levels of biotin was significantly decreased (P<0.05) by the supplementation of the broiler breeder diets. In addition, no difference in the in ci dence of chondrodystrophy was found between the chicks from the breeders which received supplemental biotin or those receiving one
times the requirement of all vitamins. I t appears that the beneficial
effect of the additional vitamins was probably due to their biotin
content.
It is established that eggs are a good source of biotin
[Rechcigal, 1978]. The present findings suggest that the level of
biotin in the egg and the subsequent biotin reserves of the chick are
under the influence of the dietary status of the hen. This offers one
plausable explanation for the wide variation in the incidence of
chondrodystrophy found between different experiments using biotin
deficient diets.
Chondrodystrophy is known to be caused by a choline chloride deficiency [Gries and Scott, 1972; Young, e ta l., 1955]. The etiology of the problem, however, remains unknown. Previous research showed
that neither the glycosaminoglycan or the collagen content of the extracellular matrix was altered with a choline chloride deficiency.
The results of the collagen solubility study indicate that i t was also unaffected by a choline chloride deficiency, suggesting that collagen metabolism is normal under the present experimental conditions.
The electron micrographs of choline chloride deficient chicks
(Figures 6 and 7) are quite different than those of biotin and man ganese deficient chicks. A choline chloride deficiency increases the number of proteoglycan aggregates although they are much smaller in size than those of the control. This indicates that the proteoglycan subunits are not aggregating properly. Choline is probably playing a structural role as lecithin in being required for the formation of the 56 large proteoglycan aggregates. The defect in the extracellular matrix, although different from that found with manganese and biotin defi ciencies, leads, nevertheless, to the disappearance of the lacunae.
In order to follow up on the possible structural role of choline chloride, autoradiographic studies were used. Our findings indicate that the labeled choline chloride was only incorporated into the extracellular matrix of connective tissue from chicks fed a diet which was adequate in choline chloride. No incorporation was found in chicks on a choline chloride deficient diet. This was unexpected, as i t was thought that the incorporation would be better on the choline chloride deficient diet, since there would be less pool dilution.
However, it appears that on a choline chloride deficient diet that the limited amount of choline is preferentially used elsewhere, probably as a methyl donor. Thus i t appears that choline is an important structural component of the extracellular matrix which is required for the proper aggregation of the proteoglycan subunits.
Supplementation of the broiler breeder diets with additional choline chloride or one times the NRC requirement of all vitamins had no effect on egg production, three week weight, tarsometatarsus length, or the incidence of chondrodystrophy. The lack of any effect on the tarsometatarsus length was unexpected, since earlier research with a similar diet [Stock, 1978] produced a noticeable shortening of the tarsometatarsus of chicks on choline chloride deficient diets. The lack of any effect in this experiment was most lik e ly due to the d if ference between the basal diets. In order to approximate practical conditions, 15 percent soybean meal was included in the basal ration of these experiments (Table 4). The added soybean meal improved the texture and the p a la tib ility of the diet. The three week weights of the chicks fed these diets were about 150 grams heavier than those of the chicks fed the previous diet [Stock, 1978]. I t appears that while the current diet approximates practical conditions, the effects were somewhat different from those seen on a more purified diet. CONCLUSIONS
The etiology of chondrodystrophy is far from being understood.
Research in the area is beginning to provide some insights into i t .
In addition, research has narrowed the problem down to being one of cartilage matrix formation. The results from these experiments indi cate that the problem is in proteoglycan formation and aggregation.
The results indicate that supplementing additional biotin in the breeder ration can reduce the incidence of chondrodystrophy in chicks fed a biotin deficient diet. I t suggests that in dealing with chondrodystrophy that we should also be concerned with the breeder diet in addition to the chick diet.
The electron microscopy studies provide evidence that all three deficiencies involve problems with proteoglycan formation and aggregation. The effect of a manganese and biotin deficiency appear very sim ilar; however, e arlier research showed that there is a decrease in the glycosaminoglycan content with a manganese deficiency but not with a biotin deficiency. Thus, while the decreased proteoglycan con tent on a manganese deficiency is due to decreased glycosaminoglycan synthesis, some other factor is responsible in the case of a biotin deficiency. A choline chloride deficiency presents another picture, showing an increased number of smaller proteoglycan aggregates. I t has been postulated that choline functions as a structural component, possible as a phospholipid. This is supported by autoradiographic work
58 59 which showed (1,2 ^4C) choline chloride was incorporated into the extracellular matrix, indicating it plays a.structural role. Addi tional research is required to substantiate these findings.
Previous work [Stock, 1978] showed collagen content was not affected by manganese, biotin or choline chloride deficiencies. These results confirm that collagen solubility is not involved and further support the hypothesis that proteoglycan formation and aggregation are the areas of concern with these three deficiencies. REFERENCES
Ackerman, L. V.; Spjut, H. J .; and Abell, M. R., ed., 1976. Bones and joints. The Williams & Wilkins Co., Baltimore.
Baker, D. H.; Allen, N. H.; Boomgardt, T.; and Graber, G., 1970. Failure of excess glycine to aggrauate choline deficiency perosis in the young chick. Poultry Sci 49: 1753-54.
Banes, A. J.; Bernstein, P. H.; Smith, R. E.; and Mechanic, G. L., 1978. Collagen biochemistry of osteopetrotic bone: I. Quantitative changes in bone collagen cross-links in virsus- induced avian osteopetrosis. Biochem and Biophy Res Comm 81: 1390-92.
Baylink, D.; Wergedal, J.; and Thompson, E., 1972. Loss of protein polysaccharides at sites where bone mineralization is in itiated . J Histochem Cytochem 20: 279-92.
B ell, D. J ., and Freeman, B. M., ed., 1971. Physiology and biochemistry of the domestic fowl. Academic Press, New York.
Best, C. W., and Huntsman, M. E., 1932. The effects of components of lecithin upon deposition of fat in the liver. J Physiol 75: 405-12.
Bloom, M. W., and Fawcett, D. W., 1966. A textbook of histology. W. B. Saunders Co., Philadelphia.
B ollet, A. J ., and Nance, J. L ., 1966. Biochemical findings in normal and osteoarthritic articular cartilage. II. Chonroitin sulfate concentration and chain length, water and ash content. J Clin Invest 45: 1170-77.
Briggs, G. M., 1946. Nicotinic acid deficiency in turkey poults and the occurrence of perosis. J of Nutr 31: 79-84.
Caskey, C. D.; Gallup, W. D.; and Norris, L. C., 1939. The need for manganese in the bone development of the chick. J Nutr 17: 407-17.
Cummings, R. B., and Tribe, D. E., 1956. The effect of floor type and the choline content of the diet upon the incidence of perosis in chickens. J Agric Sci 47: 103-06.
60 61
Daniel, L. J.; Farmer, F. A.; and Norris, L. C., 1946. Folic acid and perosis. J Biol Chem 349-50.
Davidson, J. M.; McEneany, L. S. G.; and Bornstein, P., 1975. Inter mediates in the limited proteolytic conversion of procollagen to collagen. Biochem 14: 5188-94.
Eisenstein, R.; Sorgente, N.; and Kuettner, K. E ., 1971. Organization of extracellular matrix in epiphyseal growth plate. Amer J Path 65: 515-34.
Fessler, L. I . ; Morris, N. P.; and Fessler, J. H., 1975. Procollagen: Biological scission of amino and carboxyl extension peptides. Proceedings, Natural Academy of Sciences, U.S.A. 72: 4905-09.
F u jii, K., and Tanzer, M. L., 1977. Osteogenesis imperfected: Bio chemical studies of bone collagen. Clinical Orthopedics and Research 124: 271-77,
Gallup, W. D., and Norris, L. C., 1938. The essentialness of manganese for the normal development of bone. Science 17: 18-19.
______, 1939. The amount of manganese required to prevent perosis in the chick. Poultry Sci 18: 76-82.
Gould, B. S., 1968. Treatise on collagen, Vol. II. Biology of collagen, Part A. Academic Press, New York.
Greene, R. M.; Brown, K. S.; and Pratt, R. M., 1978. Autoradiographic analysis of altered glycosaminoglycan synthesis in the epiphy seal cartilage of neonatal brachymorphic mice. Anat Rec 191: 19-30.
Gries, C. L., and Scott, M. L., 1972. The pathology of pyridoxine deficiency in chicks. J Nutr 102: 1259-68.
Gross, J ., 1963. An intermolecular defect of collagen in experimental 1 athyrism. Biochimican et Biophysica Acta 71: 250-52.
Hegsted, D. M.; M ills, R. C.; Elvehtem, C. A.; and Hart, E. B., 1941. Choline in the nutrition of chicks. J Biol Chem 138: 459.
Heller, U. S., and Penquite, R., 1936. Factors producing and preventing perosis. Poultry Sci 15: 424.
______, 1937. Factors producing and preventing perosis in chickens. Poultry Sci 16: 243-46.
Hemsley, L. A., 1970. A cartilage abnormality of broiler chickens. Vet Rec 86: 385. 62
Hunt, J. R., and McGinnis, J ., 1959. The prevention of a perosis- like condition in turkey poults. Poultry Sci 38: 612-19.
Jones, M. L .; Deyoe, C. W.; Davies, R. E.; and Couch, J. R ., 1962. Effect of phosphorus on growth and hock disorder of turkeys 8-23 weeks of age. Poultry Sci 41: 1925-28.
Jukes, T. H., 1940. Effect of choline and other supplements on perosis. J Nutr 20: 445-58.
, 1940. Prevention of perosis by choline. J Biol Chem 134: 789-90.
Jukes, T. H., and Bird, F. H., 1942. Prevention of perosis by biotin. Soc Exp Biol Med 49: 231-32.
Jukes, T. H., and Welch, A. D., 1942. The effects of certain analogues of choline on perosis. J Biol Chem 146: 19-24.
Jukes, T. H., and Olson, J. T ., 1954. Choline-like nutritional effect of dimethyaminoethanol. J Biol Chem 157: 419-20.
Kemmer, A. R.; Elvehtem, C. A.; and Hart, E. B., 1931. Studies on the relation of manganese to the nutrition of the mouse. J Biol Chem 92: 623-30.
Leach, R. J ., J r., 1960. The effect of manganese, zinc, choline, and folic acid deficiencies on the composition of the epiphyseal cartilage. Unpublished Ph.D. dissertation, Cornell University, Ithaca, New York.
______, 1968. Effect of manganese upon epiphyseal growth plate in young chicks. Poultry Sci 47: 828-30.
______, 1971. Role of manganese in mucopolysaccharide metabolism. Federation Proceedings 30: 991-94.
Leach, R. M., J r., and Muenster, A. M., 1962. Studies on the role of manganese in bone formation. J Nutr 78: 51-56.
Leach, R. M., J r., and Nesheim, M. C., 1965. N utritional, genetic and morphological studies of an abnormal cartilage formation in young chicks. J Nutr 86: 236-44.
______, 1972. Further studies on tib ia l dyschondroplasia (c a r til age abnormality) in young chicks. J Nutr 102: 1673-80.
Leach, R. M., J r., and Buss, E. G., 1977. The effect of inherited chondrodystrophy on the hexosamine content of cartilage from turkey embroys. Poultry Sci 56: 1043-45. 63
Leach, R. M., J r.; Norris, L. C.; and Scott, M. L„, 1962. The effect of diethylstilbesterol and choline deficiency in the chick. Poultry Sci 41: 1828-32.
Lyons, M. W.; Insko, M., J r.; and Martin, J. H., 1938. The effect of intraperitoneal injections of manganese, zinc, aluminum and iron salts on the occurrence of slipped tendons in chicks. Poultry Sci 17: 12-16.
Martin, C. J ., and Axelrod, A. E., 1953. A modified method for the determination of hydroxyproline. Soc Exp Biol Med 83: 461-62.
Martin, G. R.; Gross, T .; Piez, K. A.; and Lewis, M. S., 1961. On the intramolecular cross-linking of collagen in lathyritic rats. Biochimi can et Biophysica Acta 53: 599-601.
Martin, G. R.; Byers, P. H.; and Piez, K. A., 1975. Procollagen advances in enzymology and related areas of molecular biology. 42: 167-91.
McElroy, L. W., and Jukes, T. H., 1940. Formation of the anti-egg white injury factor (biotin) in the rumen of the cow. Soc Exp Biol Med 45: 296-97.
Monson, J. M.; Click, E. M.; and Bornstein, P., 1975. Further charac terization of procollagen, purification and analysis of the proal chain of chick bone procollagen. Biochem 18: 4088-92.
Morris, N. P.; Fessler, L. I . ; Weinstock, A.; and Fessler, J. H., 1975. Procollagen assembly and secretion in embryonic chick bone. J Biol Chem 250: 5717-26.
Naber, E. C.; Scott, K.; and Johnson, R. M., 1965. Effect of calcium on the incorporation of proline into hydroxyproline of collagen fractions from normal and la th y ritic chick embryos. Poultry Sci 44: 1540-45.
Nairn, M. E., 1973. Bacterial osteomyelitis and synovitis of the turkey avian disease. Avian Disease 17: 504-17.
Nairn, M. E., and Watson, A. R. A., 1972. Leg weakness of poultry-- a clinical and pathological characterisation. Australian Vet J 48: 645-56.
Naye, U., and Simons, P. C. M., 1978. Twisted leg in broilers. British Poultry Sci 19: 549-57.
Newman, B. E., and Logan, M. A., 1950. The determination of hydroxy proline. J Biol Chem 184: 299-305. 64
O'Dell, B. L., and Savage, J. E., 1957. Symptoms of zincdeficiency in the chick. Federation Proceedings 16: 394.
Orkin, R. W.; Pratt, R. M.; and Martin, G. R., 1976. Undersulfated chondroitin sulfate in the cartilage matrix of brachymorphic mice. Dev Bio 50: 82-94.
Orkin, R. W.; Williams, B. R.; Cranley, R. E.; Poppke, D. C.; and Brown, K. S., 1977. Defects in the cartilaginous growth plates of brachymorphic mice. J Cell Bio 73: 287-99.
Parker, H. E.; Andrews, F. N.; Carrick, C. W.; Hauge, R. D.; and Hauge, S. M., 1955. Effect of manganese on bone formation studied with radioactive isotopes. Poultry Sci 34: 1154-58.
Patrick, H.; Bochuher, R. V.; Dutcher, R. A.; and Knandel, H. C., 1942. Poultry Sci 21: 476.
Rechcigal, M., Jr., ed., 1978. CRC handbook series in nutrition and food. Section E: Nutritional disorders. Volume I I . CRC Press, West Palm Beach.
Riddell, C.; Helmboldy, C. F.; Singsen, E. P.; and Matterson, L. D., 1968. Bone pathology of birds affected with cage layer fatigue. Avian Disease 12: 285-97.
Rogers, A. W., 1979. Practical autoradiography. Review 20 by Amersham Corporation. Garrod and Lofthouse, England.
Rosenberg, L.; Heilman, W.; and Kleinschmidt, A. K., 1975. Electron microscopic studies of proteoglycan aggregates from bovine articular cartilage. J Biol Cnem 250: 1877-83.
Scott, M. L., 1950. Studies on the enlarged hockdisorder (perosis) in turkeys. J Nutr 40: 611-24.
______, 1953. Prevention of the enlarged hock disorder in turkeys with niacin and vitamin E. Poultry Sci 32: 670-77.
Scott, M. L.; Nesheim, M. C.; and Young, R. J ., 1969. Nutrition of the chicken. M. L. Scott and Associates, Ithaca.
Serfontein, P. J ., and Payne, L. F., 1934. Inheritance of abnormal anatomical condition of the tib ia l metatarsal joints. Poultry Sci 13: 61-63.
Sheridan, A. K.; Howlett, C. R.; and Burton, R. N., 1978. The inheri tance of tib ia l dyschondroplasia in broilers. British Poultry Sci 19: 491-99. 65
Sherwood, R. M., and Fraps, S. L., 1936. Constituents of wheat gray shorts which prevent slipped tendon. Poultry Sci 16: 109- 1 1 .
Slinger, S. T.; Petter, W. F.; and Arthur, D., 1955. Effect of phosphorus, vitamin E and environment on growth and the incidence of perosis in turkeys. Poultry Sci 34: 935-41.
Smiley, J. D .; Yeager, H.; and Z iff, M., 1962. Collagen metabolism in osteolathyrism in chick embryos: Site of action of B-aminopropionitrile. J Exp Med 116: 45-54.
Smith, D. J ., and Shuster, R. C., 1962. Biochemistry of lathyrism. I . Collagen biosynthesis in normal and la th y ritic chick embryos. Arch Biochem and Biophy 98: 498-501.
Snedecor, G. W., and Cochran, W. G., 1967. Statistical methods. Iowa University Press, Ames.
Stedman's Medical Dictionary, 1966. Williams & Williams Co., Baltimore.
Stein, G. H., and Yanishevsky, R., 1979. Autoradiography in methods in enzymology. William Btakoby and Ira Pasitan, ed. Academic Press, New York 58: 279-92.
Stock, R. H., 1978. The effects of manganese, biotin and choline on cartilage hexosamine and hydroxyproline content as related to perosis. Unpublished Master's thesis, The Ohio State University, Columbus, Ohio.
Tanzer, M. L ., and Gross, J ., 1964. Collagen metabolism in the normal and la th y ritic chick. J Exp Med 119: 275-89.
Tasi,Huan-Chang Chow, and Everson, G. J ., 1967. Effect of manganese deficiency on the acid mucopolysaccharides in the cartilage of guinea pigs. J Nutr 91: 447-52.
Titus, H. W., and Ginn, W. M., 1931. Rice bran, a preventive of perosis (deforming leg weakness) in chickens. Science 74: 249-50.
Vaughn, T ., 1975. The physiology of bone. Oxford University Press, Oxford.
Weise, A. C.; Elvehtem, C. A.; Hart, E. B.; and Halphin, J. S., 1938. Studies on the prevention of perosis in the chick. Poultry Sci 17: 33-37.
Westmorland, N., and Hoekstra, W. G., 1969. Pathological defects in the epiphyseal cartilage of normal and zine-deficient chicks. J Nutr 98: 76-82. 66
Wilgus, H. S.; Norris, L. C.; and Hueser, 1936. The role of certain inorganic elements in the cause and prevention of perosis. Science 84: 252-53.
______, 1937. The role of manganese and certain other trace elements in the prevention of perosis. J Nutr 14: 155-67.
Wise, D. R., 1975. Skeletal abnormalities in table poultry. Areview. Avian Pathology 4: 1-10.
Wise, D. R.-, Boldero, M. K.; and Thornton, G. A., 1974. The pathology and etiology of turkey syndrome '65 (T.S. 65). Res Vet Sci 14: 194-200.
Wolback, S. B., and Hegsted, D. M., 1952. Endochondral bone growth in the chick. AMA Arch Path 54: 1-12.
______, 1953. Perosis: Epiphyseal cartilage in choline and manganese deficiencies in the chick. AMA Arch Path 56: 437-53.
Young, R. J.; Norris, L. C.; Heuser, S. F., 1955. The chick's requirement for fo lic acid in the utilizatio n of choline and its precursors betaine and methyaminoethanol. J Nutr 85: 353-62.