Physiological Studies of Turkey Tibial Dyschondroplasia

Physiological Studies of Turkey Tibial Dyschondroplasia N. C. RATH, G. R. BAYYARI, J. M. BALOG, and W. E. HUFF USDA, Agricultural Research Service, Poultry Production and Product Safety Research, University of Arkansas, Fayetteville, Arkansas 72701

1994 Poultry Science 73:416-424

1992). Genetic predisposition as well as susceptibility of these fast-growing breeds Avian tibial dyschondroplasia (TD) is of poultry towards a variety of nutritional characterized by a residual plug of car- and environmental factors have been imtilaginous tissue present in the proximal plicated as the cause of TD (Leach and aspects of tibiotarsal bone of fast-growing Nesheim, 1965; Siller, 1970; Walser et al, broiler chickens and turkeys (Leach and 1982; Edwards and Veltman, 1983). Nesheim, 1965; Riddell, 1981; Julian, 1982). A variety of experimental and physioThis cartilage fails to calcify and is not logical studies of TD have been conducted replaced by an endochondral bone. The in the past to understand the etiology of abnormal presence of this cartilage has the disease (Huff, 1980; Riddell, 1981; been associated with lameness, deformity, Edwards and Veltman, 1983; Vargas et al, osteomyelitis, and other bone disorders 1983; Lawler et al, 1988; Wu et al, 1993). (Riddell, 1981; Julian, 1982; Lynch et al, Dyschondroplastic tissues in general have exhibited lower metabolic profiles when compared to control cartilages (Lilburn and Leach, 1980; Freedman et al, 1985; Received for publication September 1, 1993. Gay et al, 1985; Farquharson et al, 1992). Accepted for publication November 12, 1993. INTRODUCTION

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ABSTRACT Comparative differences between tibial dyschondroplastic (TD) and age-matched control turkey epiphyseal cartilages were studied using cellular, metabolic, and extracellular matrix characteristics. Alkaline phosphatase and aryl sulfatase activities were measured as variables of calcification and cartilage degradation, respectively. There was a decrease in the activities of both enzymes in TD tissues. An increase in tissue phosphate concentrations was noted in the TD tissue whereas neither tissue calcium nor serum calcium and phosphorus concentrations were affected. Profiles of noncollagenous and collagenous proteins from normal and TD-affected tissues were compared following in vitro biotinylation of epiphyseal cartilage followed by a sequential extraction using 4 M guanidine HC1 and pepsin digestion, respectively. Electrophoretically separated proteins from both extracts were analyzed on Western blots and compared for any prominent differences between normal and TD cartilages. Biotinylation enhanced the detectibility of extracted proteins. There were, however, no major differences in the patterns of noncollagenous or collagenous proteins between the two groups of tissues. Tibial dyschondroplastic lesions further exhibited a large number of dead chondrocytes, which increased with severity of lesion. There appears to be no significant difference in the pattern of extracellular-matrix-associated proteins. However, enzyme and metabolic activities of TD-affected cartilages were significantly reduced, and this may be due to premature death of chondrocytes in the process of development. (Key words: tibial dyschondroplasia, alkaline phosphatase, aryl sulfatase, extracellular matrix, chondrocytes)

TURKEY TIBIAL DYSCHONDROPLASIA AND CARTILAGE PHYSIOLOGY

MATERIALS AND METHODS

Nicholas turkeys at various ages obtained from local commercial farms were killed by carbon dioxide asphyxiation. Following a sagittal cut in the proximal aspects of the tibiotarsal bone, birds were scored for the presence and severity of TD by visual examination. The severities of the lesions were assigned mild (< .5 cm), moderate (.5 to 1 cm), and severe (> 1 cm) with lesions extending deep into the tibia (Huff, 1980). Epiphyseal cartilages from birds with severe lesions were compared with age-matched normal epiphyseal tissue. For all biochemical studies, equal numbers of individual growth plate cartilages with severe TD lesions and agematched normal control growth plate were used. Cell isolation studies were done with tissues obtained from mild and various regions of severe TD lesions (Hargest et ah, 1985) and compared with tissue of normal epiphyseal cartilage. For analyses of calcium and phosphorus, blood was obtained before euthanasia by wing vein using a heparinized syringe. Sera from these turkeys were analyzed for calcium by atomic absorption spectrophotometry,1 and phosphorus was

measured using a clinical chemistry analyzer2 according to the manufacturer's suggested procedures. Metabolic Profiles

Growth plate cartilages from normal and TD lesions were obtained from nine individual turkeys of 7 and 8 wk of age in each group, weighed, homogenized in a buffer containing .15 M NaCl and 3 mM NaHC0 3 , pH 7.2 according to Reddi and Huggins (1972), and centrifuged at 10,000 x g. Alkaline phosphatase and aryl sulfatase activities in the supernatant were determined according to earlier procedures with minor modifications (Reddi and Huggins, 1972; Rath et at, 1981). Alkaline phosphatase was assayed at pH 9.5 using pnitrophenyl phosphate as substrate. Aryl sulfatase was assayed at pH 5.5 using pnitro catechol sulfate as substrate. The activities of the enzymes were expressed as micromoles of substrate utilized (units) at 37 C in 30 min per gram of tissue. The residues following enzyme extraction were further treated with 2 mL of .5 M HC1 to release mineral-bound calcium and phosphorus by stirring at room temperature for 16 h, when the supernatant was recovered by centrifugation as above. Total calcium in the supernatant was determined by atomic absorption spectrophotometry as above and total phosphate concentration was determined colorimetrically (Chen et ah, 1956). The results were evaluated statistically using an analysis of variance program (SAS Institute, 1985). Biotin Labeling and Extraction of Tissues

Noncollagenous and collagenous protein extracted from normal and TD-affected epiphyseal cartilages were compared to assess differences in the extracellularmatrix-associated proteins that could be attributed to the development of TD. In view of sensitivity offered by biotinylated proteins to subsequent detection on Western blot, the cartilage pieces were biotinylated in vitro prior to extraction as follows. Approximately 300 mg of minced Model 1100B, Perkin-Elmer, Norwalk, CT 06859. epiphyseal cartilage obtained from 7- and 2 Express Plus, Ciba Corning, Medfield, MA 02052. 11-wk-old normal and TD-affected turkeys

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Nevertheless, the exact mechanisms leading to this disorder remain poorly understood. In turkeys, TD may lead to osteomyelitis, osteochondrosis, and femoral head necrosis (Julian, 1982). The percentage of turkeys afflicted with TD may reach as high as 80% or more around 10 to 15 wk of age, which also coincides with an increase in osteomyelitis (Walser et at, 1982). However, fewer experimental studies have been conducted on turkey TD. In the present study, we have compared metabolic, extracellular matrix, and cellular profiles of cartilage tissue obtained from TD lesions of tibia as well as age-matched normal epiphyseal growth plate of turkeys to understand the physiological basis of this defect.

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3

Pierce Chemical Co., Rockford, IL 61105. "ICN, Irvine, CA 92713. 5 BioRad, Hercules, CA 94547. 6 Boehringer-Mannheim, Indianapolis, IN 46250. 7 Zymed Laboratories, Palo Alto, CA 94080.

Sodium Dodecyl Sulfate Gel Electrophoresis and Western Blotting Aliquots of SDS-solubilized extracts from both groups of tissues were electrophoretically separated under reducing conditions in 10% polyacrylamide gels using the Laemmli buffer system (Laemmli, 1970). Prestained and biotinylated molecular weight marker proteins were obtained from BioRad Laboratories. 5 The proteins were then electroblotted to polyvinylidene difluoride (PVDF) membrane using a transblot apparatus 5 according to Tawbin et al. (1979). The membrane was blocked for 1 h using 5% nonfat dry milk in Tris-buffered saline (500 mM NaCl, 20 mM Tris, p H 7.4) containing .2% Tween-20 (TBST). The biotinylated proteins were visualized after incubating the membrane with streptavidin-alkaline phosphatase conjugate 6 in TBST solution for 1 h, washed three times, and subsequently developed with 5-bromo4-chloro-3-indolyl phosphate/Nitroblue tetrazolium 7 substrate. Comparative patterns of noncollagenous (GuHCl extract) and collagenous proteins (pepsin extract) from normal and TD tissues were visually evaluated based on their migratory patterns using reference preparations of Type II and Type X collagens (Bashey et al, 1989; Miller and Rhodes, 1982). In some studies, gels were stained with Coomassie blue and identical preparations were used for Western blotting. Cell Isolation Chondrocytes were isolated according to a previously described procedure (Rath et al, 1988) using collagenase digestion of normal and TD-affected epiphyseal cartilage obtained from turkeys at 12 wk of age. Normal epiphyseal cartilages were carefully scrapped using a 5-mm micro dissecting curette. The TD tissues were obtained under semi-sterile conditions following a longitudinal split of growth plate. In case of mild lesion, cartilage tissues were obtained from the indented area of growth plate, whereas in severe TD the regions were approximately divided to proximal, medial and distal according to Hargest et al (1985). Three cartilage samples in each group were individually digested under

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were washed with 50 mM NaHC0 3 / pH 8.5, over 1 h at 4 C. Two individual samples were used in each group of experiment. The tissues were then incubated in a solution containing 2 m g / m L each of sulfo-NHS biotin and NHS-LC-biotin 3 in the same buffer over ice with a low speed stirring for 2 h. Finally, the tissues were washed three times with 20 mL of ice cold phosphatebuffered saline, p H 7.4, and extracted by stirring with 2 mL of 4 M guanidine HC1 (GuHCl)/20 mM Na-acetate solution, pH 6.5, containing protease inhibitors (1 mM phenylmethylsulfonylfluoride, 5 mM Nethyl maleimide, 5 mM benzamidine, and 10 U / m L aprotinin) for 48 h at 4 C. The extraction scheme described by Ronziere et al. (1990) was used with minor modifications. The supernatant was removed by centrifugation at 10,000 x g for 30 min and the process was repeated with additional 1 mL of guanidine HC1 solution for 8 h. Pooled guanidine extract was dialyzed against distilled and deionized water over 2 d at 4 C with successive changes and lyophilized. The residues from guanidine extraction were washed four times using distilled water and rinsed with 2 mL of .5 M acetic acid. To each tissue sample, 2 mL of pepsin solution (1 m g / m L in .5 M acetic acid) was added and stirred at 4 C for 24 h to extract pepsin-soluble collagen. The pepsin digests were collected by centrifugation at 10,000 x g for 15 min, and the supernatants were neutralized by the addition of Tris HC1 and dialyzed exhaustively against distilled deionized water. The dialyzed products of GuHCl and pepsin extracts were lyophilized. Equal amounts of lyophilized products by weight were dissolved in 2% SDS and used for polyacrylamide gel electrophoresis. Pepsin and hyaluronidase digestion of lyophilized guanidine extracted samples were done for 24 h at 4 C using pancreatic pepsin as above and testicular hyaluronidase 4 (Ronziere et al, 1990).

TURKEY TIBIAL DYSCHONDROPLASIA AND CARTILAGE PHYSIOLOGY

RESULTS Metabolic Profiles The serum concentrations of calcium and inorganic phosphate measured from 8 to 15 wk did not show any difference between normal and the turkeys with severe TD (x ± SD, Ca; normal, 9.86 ± .75 m g / d L , n = 17; TD, 10.27 ± .36 m g / d L , n = 17; inorganic phosphate; normal, 5.30 ± .40 m g / d L , n = 17; TD, 4.88 ± .05 mg/dL, n = 17). A significant decrease in alkaline phosphatase and aryl sulphatase activities were noted in TD lesions. There was no change in acidreleasable tissue calcium concentration between normal and TD cartilages; however, the concentration of inorganic phosphate exhibited a fivefold increase (Table 1).

teoglycans using Western blot analysis was significantly enhanced as compared with identical preparations that were stained with Coomassie blue (Figure 1A and B). The guanidine hydrochloride-extractable proteins were highly susceptible to pepsin digestion, whereas the hyaluronidase was able to partially eliminate some components of the extract, suggesting that the major constituents of the guanidine extract consisted of noncollagenous proteins and proteoglycans (Figure 2). Comparative patterns of biotinylatedstructural proteins and proteoglycans in the guanidine extract as well as in pepsin extract are shown in Figures 3 and 4. The guanidine-extractable materials from agematched normal and TD tissues had comparable profiles. There were, nevertheless, age-related differences in staining intensity and presence of additional bands both in normal and TD-affected growth plate. Minor differences were noted between normal and TD cartilages with respect to some high molecular weight proteins. Similarly, in pepsin extracts of cartilage matrix, Types II and X were identified by their electrophoretic migration pattern (Figure 4); however there were many additional bands present. As in noncollagenous protein profiles, the comparative patterns varied between 7 and 11 wk in terms of intensity and appearance of certain bands; nonetheless, within the same age group the band patterns were comparable between normal and TD cartilages (Figure 4). Cell Morphology

Composition Profiles of Biotinylated Matrices Guanidine hydrochloride and pepsin extracts of biotinylated cartilages, obtained from 7- and 11-wk-old turkeys, were subjected to polyacrylamide gel electrophoresis and Western blot analysis using alkaline phosphatase-labeled streptavidin as the probe. The sensitivity of detection of guanidine-extracted proteins and pro-

8

Cellox, Hopkins, MN 55343. ^HyClone, Logan, UT 84321. 10 Sigma Chemical Co., St. Louis, MO 63178-9916.

The viability as measured by trypan blue exclusion of chondrocytes was determined in three individual samples in each group obtained from turkeys of 12 wk of age (Table 2). The cells from mild lesions had comparable viability to chondrocytes derived from normal epiphyseal cartilage. However, cells from severe lesions had significantly (P < .05) less viable cells than normal and mild TD lesions. Cells isolated from proximal aspects of severe TD lesions had a higher number of viable cells as compared with medial or distal part of the lesion. A steady decrease in the population of living chondrocytes was noted as the lesions progressed from proximal to distal

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identical conditions in Dulbecco's modified Eagle's medium^ (DME with 2.5 mM glutamine, penicillin-streptomycin, 50 /*g/mL of ascorbic acid, and 10% fetal bovine serum?) containing 2 m g / m L of collagenase D for 16 h to release the chondrocytes. The percentage of viable cells were enumerated using the trypan blue dye exclusion method. In some experiments, chondrocytes derived from normal epiphyseal cartilage as well as mild and severe lesions were plated at a density of 2x 10 4 cells per well in 24-well plates and observed u p to 2 wk. The gross morphology of the cells in culture were examined microscopically on subsequent days. All chemicals were obtained from Sigma 10 except where noted.

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RATH ET Ah. TABLE 1. Biochemical profiles of normal and tibial dyschondroplastic (TD) tissues 1

Tissue

Calcium

Phosphate

Alkaline phosphatase

Aryl sulfatase

Normal Severe TD

.63 + .40 1.06 + .37

.11 + .04b .53 ± .61*

68.21 ± 19.9P 14.71 ± 10.0b

.91 ± .24a .22 ± .15b

a b

' Values within a column with no common superscript differ significantly (P < .05). Walues represent the x + SD of nine individual tissue samples taken from turkeys at 7 and 8 wk of age.

DISCUSSION The present study was performed to investigate physiological differences between normal and TD cartilages derived from turkey growth plates. Alkaline phosphatase and aryl sulfatase activities were measured as the metabolic variables of calcification (Robinson, 1923; Poole, 1992) and matrix degradation (Dorey and Bick, 1977a,b; Rath et al, 1981), respectively. Both enzymes exhibited significantly lower activities, indicating failure of both cartilage calcification and matrix degradation. These results are in agreement with several studies in the past, which have demonstrated lower activities of many

-200kDa

FIGURE 1. Western blot profiles of guanidine HC1 extracted proteins from 11-wk-old turkey growth plate cartilage using Coomassie blue staining in polyacrylamide gel (A) and an identical preparation following transfer to polyvinylidene difluoride membrane that was probed with streptavidin-alkaline phosphatase (B). N = normal, TD = tibial dyschondroplasia.

FIGURE 2. Profiles of enzyme-digested guanidineextractable proteins from 7-wk-old normal growth plate cartilage transblotted to polyvinylidene difluoride membrane. 1) control; 2) hyaluronidase; 3) pepsin; and 4) molecular weight markers.

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end (P < .05). The deeper part of the severe lesion had almost no viable cells. Whereas the chondrocytes from normal epiphyseal cartilages or mild lesions exhibited hypertrophic morphology in 72 h in culture, the cells from severe lesions did not exhibit any change under the same conditions (Figure 5A and B). No necrotic changes or cellular lysis were evident in these cells even up to 2 wk in culture when the normal cells exhibited necrotic changes and contained many vacuoles. The chondrocyte cultures from the proximal area of lesions exhibited a mixture of hypertrophic and nonhypertrophic cells at 96 h (not shown).

TURKEY TIBIAL DYSCHONDROPLASIA AND CARTILAGE PHYSIOLOGY

MW

7 WK

11 W K

7WK

421

i , WK

200kDa H6.25kDa

WW 80kDa -

66.2kDa -

Type | |

*•* I

49.5kDa - I

45kDa -

TD

N

FIGURE 3. Comparative profiles of guanidineextractable proteins from 7- and 11-wk-old cartilage tissues. N = normal; TD = tibial dyschondroplasia; MW = molecular weight.

enzymes and biochemicals in the TD tissues as compared with normal and hypertrophic growth plate tissues (Lilburn and Leach, 1980; Freedman et al, 1985; Gay et al, 1985; Lawler et al, 1988; Farquharson et al, 1992). Acid-extractable tissue calcium concentrations did not show any change, although an increase in phosphate was noted. Serum calcium and phosphorus concentrations were unaffected in turkeys with severe TD, ruling out any possible imbalance in calcium and phosphate metabolism that could contribute to the development of TD. However, studies by Sanders and Edwards (1991)

TD

TD

TD

FIGURE 4. Comparative profiles of pepsinextracted collagenous proteins from 7- and 11-wk-old cartilage tissues. N = normal; TD = tibial dyschondroplasia.

have shown that dietary imbalance in calcium and phosphorus may not be a factor in TD in turkeys, unlike that of chickens (Edwards and Veltman, 1983). The importance of cell and extracellular matrix interactions during organ development and morphogenesis has been recognized (Reddi, 1984). Therefore, any aberration in extracellular matrix structure may likely cause developmental abnormality. In skeletal tissue development, a variety of collagenous and noncollagenous proteins such as the Type X collagen, proteoglycans, chondrocalcin, and osteopontin have been implicated in the control of cartilage

TABLE 2. Percentage of viable cells (trypan blue negative) obtained from growth plat es of normal and tibial dyschondroplasia (TD)-affected 12-wk-old turkeys 1 Epiphyseal cartilage

Total

Normal Mild TD Severe TD

84.7 ± 6.9a 77.6 ± 5.7* 16.3 + 2.0b

Proximal

Middle

Distal

Vinhlr rrll- ("',)

ab

37.2 ± 6.4

10.5 ± 3.7

Values within a column with no common superscript differ significantly (P < .05). 'Values represent the x ± SD of three samples of tissues.

.7 ± .7

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N

Type X

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RATH ET AL.

exclusive synthesis by chondrocytes undergoing hypertrophy (Schmid and Linsenmayer, 1987; Petit et al, 1992). There were no apparent differences between normal and TD cartilages derived from either 7- or 11-wk-old turkeys. Bashey et al. (1989) using a differential salt precipitation method observed a decrease in the levels of Type X collagen relative to other collagen Types in TD tissues, although it was neither altogether absent nor was there any qualitative difference in biochemical composition in comparison to those derived from normal hypertrophic growth plate. However, in a recent study using immunochemical localization of Type X collagen, Chen et al. (1993) did not notice any difference between normal and TD tissues of chickens. Because Type X collagen appears to be exclusively associated with hypertrophic cartilages (Schmid and Linsenmayer, 1987), its presence in TD cartilage may suggest the reminiscence of that developmental process. It may be argued that the pathogenesis of TD may not be solely due to the failure of developing chondrocytes to undergo hypertrophy but possibly related to the failure of some later stages of development such as chondrolysis and cartilage degradation.

It was further noted that the TD tissues contained a significant number of dead chondrocytes compared with normal epiphyseal cartilage. The percentage of dead chondrocytes increased with severity of lesion. In the distal part of the lesion almost all chondrocytes were dead relative to the proximal part. In culture, these cells did not exhibit any hypertrophic changes, in contrast to cells derived from normal epiphysis. Hargest et al. (1985) similarly observed the presence of autolytic and necrotic mass of cartilage cells in TD lesions of chickens. Consequently, it appears that premature death of chondrocytes in the process of hypertrophy probably leads to the arrest of all cellular events W *? * ' leading to decrease in metabolism, impairment of chondrolysis, and to the persistCTtV* . " " , ence of cartilage. However, the causes and the mechanism of such cell death remains FIGURE 5. Chondrocyte cultures at 96 h. A) normal unknown. epiphyseal cartilage; and B) tibial dyschondroplasic Several mechanisms have been sugcartilage with severe lesion. Magnification lOOx. Scale gested in the etiology of TD that range bar = 10 urn. *

" • ' , >

- ' * ;

-

%

•

~

' " * -

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hypertrophy, calcification, and subsequent bone development (Heingard and Paulsson, 1987; Schmid and Linsenmayer, 1987; Poole, 1992). It was therefore of interest to compare the extracts of cartilage from normal and TD growth plates to examine whether differences between the two tissues could be attributed to the pathogenesis of TD. In view of superior sensitivity offered by biotinylated proteins to detection on Western blots (Wilcheck and Bayer, 1988), the cartilage pieces were biotinylated before extraction to analyze the patterns of proteins and proteoglycans in extracts. Whereas biotinylation significantly improved the detection of macromolecules, particularly in guanidine extracts, only few differences were noted in the comparative profiles of normal and diseased tissues either in noncollagenous or collagenous proteins. Similar observations were made by Lowther et al. (1974), who did not find much difference between the proteoglycans derived from normal and TD cartilages of chickens in spite of a significant depression of its biosynthesis in affected tissues. Similarly, no major differences were noted in the electrophoretic patterns of pepsin-extracted collagenous proteins. There were several comparable bands of collagenous proteins in the extract with predominant presence of Type II collagen. The Type X collagen was of particular interest in the present study because of its

TURKEY TIBIAL DYSCHONDROPLASIA AND CARTILAGE PHYSIOLOGY

ACKNOWLEDGMENTS The technical assistance of Lee Manuel, Kathy McElyea, Dana Bassi, and Scott Zornes during all phases of this study is gratefully acknowledged. Thanks are due to Hari Reddi and Walter Bottje for critical reading of the manuscript.

REFERENCES Bashey, R. I., M. Leach, C. V. Gay, and S. A. Jimenez, 1989. Type X collagen in avian tibial dyschondroplasia. Lab. Invest. 60:106-112. Chen, P. S., Jr., T. Y. Toribara, and H. Warner, 1956. Micro determination of phosphorus. Anal. Chem. 28:1756-1758. Chen, Q., E. P. Gibney, R. M. Leach, and T. F. Linsenmayer, 1993. Chicken tibial dyschondroplasia: a limb mutant with two growth plates and possible defect in collagen crosslinking. Dev. Dynam. 196:54-61. Dorey, C. K., and K. L. Bick, 1977a. Ultrastructural analysis of glycosaminoglycan hydrolysis in the rat periodontal ligament. I. Evidence for macrophage involvement in bone remodeling. Calcif. Tissue Res. 24:135-141. Dorey, C. K., and K. L. Bick, 1977b. Ultrahistochemical analysis of glycosaminoglycan hydrolysis in the rat periodontal ligament. II. Arylsulfatase and bone resorption. Calcif. Tissue Res. 24: 143-149. Edwards, H. M., Jr., 1990. Efficacy of several vitamin

D compounds in the prevention of tibial dyschondroplasia in broiler chickens. J. Nutr. 113:1568-1575. Edwards, H. M, Jr., and J. R. Veltman, 1983. The role of calcium and phosphorus in the aetiology of tibial dyschondroplasia in young chickens. J. Nutr. 113:1568-1575. Farquharson, C, C. Whitehead, S. Rennie, B. Thorp, and N. Loveridg, 1992. Cell proliferation and enzyme activities associated with development of avian dyschondroplasia: an in situ biochemical study. Bone 13:59-67. Freedman, B. D., C. V. Gay, and R. M. Leach, 1985. Avian tibial dyschondroplasia II. Biochemical changes. Am. J. Pathol. 119:191-198. Gay, C. V., R. E. Anderson, and R. M. Leach, 1985. Activities and distribution of alkaline phosphatase and carbonic anhydrase in tibial dyschondroplastic lesions and associated growth plate of chicks. Avian Dis. 19:812-821. Hargest, T. E., R. M. Leach, and C. V. Gay, 1985. Avian tibial dyschondroplasia. I. Ultrastructure. Am. J. Pathol. 119:175-190. Heingard, D., and M. Paulsson, 1987. Cartilage. Methods Enzymol. 145:336-363. Huff, W. E., 1980. Evaluation of tibial dyschondroplasia during aflatoxicosis and feed restriction in young broiler chickens. Poultry Sci. 59:991-995. Julian, R. J., 1982. Osteochondrosis, dyschondroplasia and osteomyelitis causing femoral head necrosis in turkeys. Avian Dis. 29:854-866. Laemmli, U. K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 227:680-685. Lawler, E. M., J. L. Shivers, and M. M. Walser, 1988. Acid phosphatase activity of chondroclasts from fusarium induced tibial dyschondroplastic cartilage. Avian Dis. 32:240-245. Leach, R. M., and M. C. Nesheim, 1965. Nutritional, genetic and morphological studies of abnormal cartilage formation in young chicks. J. Nutr. 86: 236-244. Lilburn, M. S., and R. M. Leach, 1980. Metabolism of abnormal cartilage cells associated with tibial dyschondroplasia. Poultry Sci. 59:1892-1896. Lowther, D. A., H. C. Robinson, J. W. Dolman, and J. W. Thomas, 1974. Cartilage matrix components in chicken with tibial dyschondroplasia. J. Nutr. 104:922-929. Lynch, M., B. H. Thorp, and C. C. Whitehead, 1992. Avian tibial dyschondroplasia as a cause of bone deformity. Avian Pathol. 21:275-285. Miller, E. J., and R. K. Rhodes, 1982. Preparation and characterization of the different types of collagen. Methods Enzymol. 82:33-64. Orth, M. W., D. A. Martinez, M. E. Cook, and A. C. Vailas, 1991. Nonreducible cross link formation in tibial dyschondroplastic growth plate cartilage from broiler chicks fed homocysteine. Biochem. Biophys. Res. Commun. 179: 1582-1586. Petit, B., A. M. Freyria, M. vander Rest, and D. Herbage, 1992. Cartilage collagens. Pages 33-84 in: Biological Regulation of Chondrocytes. M. Adolphe, ed. CRC Press, Boca Raton, FL. Poole, A. R., 1992. The growth plate: cellular physiology, cartilage assembly and mineraliza-

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from deficiency of essential nutritional factors to a variety of autocrine and paracrine factors (Edwards, 1990; Orth et al, 1991; Farquharson et ah, 1992). Some of these deficiencies may relate to the impairment of vascularization restricting the migration of cells of hematopoietic origins, which take part in cytokine production as well as cartilage resorption and remodeling (Tyler, 1991) that influence chondrocyte function, the absence of which may lead to chondrocyte death. On the other hand, persistent exposure to certain cytotoxic factors such as mycotoxins (Huff, 1980; Lawler et al, 1988) or pesticides (Vargas et al, 1983; Wu et al, 1993) may produce chondrocyte death by virtue of their chronic and cumulative effect on developing cartilage. Such cytotoxic effects may in turn depend upon extent of clearance of these compounds from synovial spaces. In conclusion, it appears that TD could be the result of premature death of cartilage cells at the time of hypertrophy, the causes of which are unknown.

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