Light-based monitoring devices to assess range use by laying hens.

Access to an outdoor range has many potential benefits for laying hens but range use can be poor due to factors only partly understood. Techniques to monitor individual range use within commercial flocks are crucial to increase our understanding of these factors. Direct observation of individual range use is difficult and time-consuming, and automatic monitoring currently relies on equipment that is difficult to use in an on-farm setting without itself influencing range use. We evaluated the performance of a novel small, light and readily portable light-based monitoring system by validating its output against direct observations. Six commercial houses (2000 hens/house) and their adjacent ranges were used, three of which were equipped with more structures on the range than the others (to determine whether cover would influence monitoring accuracy). In each house, 14 hens were equipped with light monitoring devices for 5 discrete monitoring cycles of 7 to 8 consecutive days (at 20, 26, 32, 36 and 41 weeks of age). Light levels were determined each minute: if the reading on the hen-mounted device exceeded indoor light levels, the hen was classified as outside. Focal hens were observed directly for 5 min/hen per week. Accuracy (% of samples where monitoring and direct observations were in agreement) was high both for ranges with more and with fewer structures, although slightly better for the latter (92% v. 96% ± 1 SEM, F1,19 = 5.2, P = 0.034). Furthermore, accuracy increased over time (89%, 94%, 95%, 98% ± 1 SEM for observations at 26, 32, 36 and 41 weeks, respectively, F3,19 = 3.2, P = 0.047), probably due to progressively reduced indoor light levels resulting from partial closing of ventilation openings to sustain indoor temperature. Light-based monitoring was sufficiently accurate to indicate a tendency for a greater percentage of monitored time spent outside when more range structures were provided (more: 67%, fewer: 56%, SEM: 4, $\chi_1^2 = 2.9$, P = 0.089). Furthermore, clear and relatively consistent individual differences were detected. Individuals that were caught outside at the start of the experiment ranged more throughout its duration (caught outside: 72%, caught inside 51%, SEM: 4, $\chi_1^2 = 10.0$, P = 0.002), and individual range use was correlated between monitoring cycles (for adjacent monitoring cycles: $r_s^2 = 0.5-0.7$, P < 0.0001). This emphasizes the importance of studying range use on an individual level. In conclusion, our light-based monitoring system can assess individual range use accurately (although accuracy was affected by house characteristics to some extent) and was used to show that both cover availability and individual characteristics affected range use.

Mechanical comparison of monofilament nylon leader and orthopaedic wire for median sternotomy closure.

OBJECTIVES: To compare the mechanical properties of monofilament nylon leader and orthopaedic wire for median sternotomy closure in the dog. MATERIALS AND METHODS: Median sternotomy was performed in 14 canine cadaver sternums with the manubrium intact. The sternotomy was closed with either 80 lb monofilament nylon leader or 20G orthopaedic stainless steel wire in a peristernal figure of 8 pattern. Constructs were loaded in a servohydraulic material testing machine. Load at yield, maximum load, stiffness, displacement and mode of failure were compared between constructs subjected to a single cycle load to failure. RESULTS: No significant differences were found in load at yield, maximum load, stiffness or displacement between the monofilament nylon leader and the stainless steel wire constructs. No implant failure was evident in the stainless steel wire constructs. Four of the monofilament nylon leader constructs failed by pulling of the nylon through the crimp. CLINICAL SIGNIFICANCE: Monofilament nylon leader is mechanically comparable to stainless steel wire and potentially a suitable alternative for closure of median sternotomy in the dog.

The effects of long (C20/22) and short (C18) chain omega-3 fatty acids on keel bone fractures, bone biomechanics, behavior, and egg production in free-range laying hens.

Keel fractures in the laying hen are the most critical animal welfare issue facing the egg production industry, particularly with the increased use of extensive systems in response to the 2012 EU directive banning conventional battery cages. The current study is aimed at assessing the effects of 2 omega-3 (n3) enhanced diets on bone health, production endpoints, and behavior in free-range laying hens. Data was collected from 2 experiments over 2 laying cycles, each of which compared a (n3) supplemented diet with a control diet. Experiment 1 employed a diet supplemented with a 60:40 fish oil-linseed mixture (n3:n6 to 1.35) compared with a control diet (n3:n6 to 0.11), whereas the n3 diet in Experiment 2 was supplemented with a 40:60 fish oil-linseed (n3:n6 to 0.77) compared to the control diet (n3:n6 to 0.11). The n3 enhanced diet of Experiment 1 had a higher n3:n6 ratio, and a greater proportion of n3 in the long chain (C20/22) form (0.41 LC:SC) than that of Experiment 2 (0.12 LC:SC). Although dietary treatment was successful in reducing the frequency of fractures by approximately 27% in Experiment 2, data from Experiment 1 indicated the diet actually induced a greater likelihood of fracture (odds ratio: 1.2) and had substantial production detriment. Reduced keel breakage during Experiment 2 could be related to changes in bone health as n3-supplemented birds demonstrated greater load at failure of the keel, and tibiae and humeri that were more flexible. These results support previous findings that n3-supplemented diets can reduce fracture likely by increasing bone strength, and that this can be achieved without detriment to production. However, our findings suggest diets with excessive quantities of n3, or very high levels of C20/22, may experience health and production detriments. Further research is needed to optimize the quantity and type of n3 in terms of bone health and production variables and investigate the potential associated mechanisms.

Do intervertebral discs degenerate before they herniate, or after?

The belief that an intervertebral disc must degenerate before it can herniate has clinical and medicolegal significance, but lacks scientific validity. We hypothesised that tissue changes in herniated discs differ from those in discs that degenerate without herniation. Tissues were obtained at surgery from 21 herniated discs and 11 non-herniated discs of similar degeneration as assessed by the Pfirrmann grade. Thin sections were graded histologically, and certain features were quantified using immunofluorescence combined with confocal microscopy and image analysis. Herniated and degenerated tissues were compared separately for each tissue type: nucleus, inner annulus and outer annulus. Herniated tissues showed significantly greater proteoglycan loss (outer annulus), neovascularisation (annulus), innervation (annulus), cellularity/inflammation (annulus) and expression of matrix-degrading enzymes (inner annulus) than degenerated discs. No significant differences were seen in the nucleus tissue from herniated and degenerated discs. Degenerative changes start in the nucleus, so it seems unlikely that advanced degeneration caused herniation in 21 of these 32 discs. On the contrary, specific changes in the annulus can be interpreted as the consequences of herniation, when disruption allows local swelling, proteoglycan loss, and the ingrowth of blood vessels, nerves and inflammatory cells. In conclusion, it should not be assumed that degenerative changes always precede disc herniation. Cite this article: Bone Joint J 2013;95-B:1127-33.

Impact of a mixed chain length omega-3 fatty acid diet on production variables in commercial free-range laying hens.

1. Previous studies found beneficial effects on the prevalence of keel fractures and bone health in laying hens receiving a short chain ω3 fatty acid diet. However, subsequent work using a mixed short and long chain ω3 diet (MCD) found little benefit to bone health and substantial production losses. 2. The current study examined commercial use of a mixed chain length ration in multiple farms and flocks and identified a detrimental effect on productivity including: mortality, number of poor quality eggs, hen daily average, feed consumption, and increased egg weight. 3. Production data were acquired from 11 flocks housed on 5 farms and modelled using MlwiN, a statistical package developed for analysis of hierarchical data. Production responses were individually modelled with duration of exposure to the diet, age of introduction, flock size and farm, included as prediction variables. 4. Analysis indicated birds receiving the MCD manifested dramatic increases in mortality, poor quality eggs, mean egg weight, feed consumption and decreased production in comparison to control flocks receiving a standard ration. Increased exposure time appeared to increase the magnitude of the diet effect. 5. Although no data were collected to explain the potential mechanisms of this diet, the authors discuss several possible scenarios including excessive long chain ω3 content overwhelming eicosanoid regulatory capacity and changes in behaviour patterns.

Mechanical comparison of median sternotomy closure in dogs using polydioxanone and wire sutures.

OBJECTIVES: To compare the mechanical properties of two different materials for canine median sternotomy closure. STUDY DESIGN: In vitro biomechanical study. SAMPLE POPULATION: Twelve canine cadaveric entire sternum and portion of associated ribs and musculature. METHODS: Median sternotomy leaving the manubrium intact was closed using polydioxanone and stainless steel wire in a figure of eight pattern. Constructs were loaded in a servohydraulic material testing system and displacement was compared at loads of 125, 150, 200, 250, 300, 350 and 400 N, and at failure. RESULTS: Displacement at loads up to 400 N and failure did not show any statistical difference between the polydioxanone and the stainless steel wire. However, the loading forces to create failure of the construct were superior for the stainless steel. Polydioxanone and stainless steel wire had the same response to distractive forces for loads applied up to 400 N. CLINICAL SIGNIFICANCE: This study cannot recommend the use of polydioxanone over wires but suggests that polydioxanone can potentially be an alternative for primary median sternotomy closure in selected patients.

Regulation of osteoarthritis by omega-3 (n-3) polyunsaturated fatty acids in a naturally occurring model of disease.

OBJECTIVE: To examine effects of high omega-3 (n-3) polyunsaturated fatty acid (PUFA) diets on development of osteoarthritis (OA) in a spontaneous guinea pig model, and to further characterise pathogenesis in this model. Modern diets low in n-3 PUFAs have been linked with increases in inflammatory disorders, possibly including OA. However, n-3 is also thought to increases bone density, which is a possible contributing factor in OA. Therefore we aim to determine the net influence of n-3 in disease development. METHOD: OA-prone Dunkin-Hartley (DH) Guinea pigs were compared with OA-resistant Bristol Strain-2s (BS2) each fed a standard or an n-3 diet from 10 to 30 weeks (10/group). We examined cartilage and subchondral bone pathology by histology, and biochemistry, including collagen cross-links, matrix metalloproteinases (MMPs), alkaline phosphatase, glycosaminoglycan (GAG), and denatured type II collagen. RESULTS: Dietary n-3 reduced disease in OA-prone animals. Most cartilage parameters were modified by n-3 diet towards those seen in the non-pathological BS2 strain - significantly active MMP-2, lysyl-pyridinoline and total collagen cross-links - the only exception being pro MMP-9 which was lower in the BS2, yet increased with n-3. GAG content was higher and denatured type II lower in the n-3 group. Subchondral bone parameters in the DH n-3 group also changed towards those seen in the non-pathological strain, significantly calcium:phosphate ratios and epiphyseal bone density. CONCLUSION: Dietary n-3 PUFA reduced OA in the prone strain, and most disease markers were modified towards those of the non-OA strain, though not all significantly so. Omega-3 did not increase markers of pathology in either strain.

High-intensity exercise induces structural, compositional and metabolic changes in cuboidal bones--findings from an equine athlete model.

Fatigue fracture of cuboidal bones occurs in the human foot as well as the equine carpus. The racehorse provides a naturally-occurring model to study the effects of high-intensity exercise on the morphology and metabolism of cuboidal bones. We studied both the mineral and the collagenous matrix of the third (C(3)) and radial (C(r)) carpal bones of raced and non-raced Thoroughbred (TB) horses. We hypothesised that racehorses would show increases in the mineral component of these bones and post-translational modifications of the collagenous matrix alongside changes in markers of collagen remodelling and bone formation. C(3) and C(r) carpal bones were retrieved from raced TB horses (n=14) and non-raced TB horses (n=11). Standardised proximal-distal sections were taken from each bone and these were sliced transversely to study the proximal-distal differences in bone metabolism from the subchondral plate through to trabecular bone. Histomorphometry and bone mineral density measurements were performed in parallel with biochemical analyses including total collagen, collagen synthesis and cross-links, matrix metalloproteinases-2 and 9 and their inhibitors, calcium and phosphate, and bone alkaline phosphatase. The results of this study show that, while there is a net increase in bone formation in the racehorses, there is additionally an increase in bone collagen synthesis and remodelling, particularly within the trabecular regions of the bone. The increase in bone density would lead to greater stiffness, particularly in the cortical bone, and failure of this 'stiffer' cortical bone may result from its lack of support from the rapidly remodelling and structurally weakened underlying trabecular bone.

Use of matrix metalloproteinases 2 and 9 and white blood cell counts in monitoring the treatment and predicting the survival of horses with septic arthritis.

Thirty-nine samples of synovial fluid were collected from the joints of 32 horses with suspected septic arthritis and 39 samples were collected from horses euthanased for non-orthopaedic conditions. The white blood cell counts (WBCC) were determined and the pro and active forms of matrix metalloproteinases (MMPs) 2 and 9 were measured by gelatin zymography and image analysis in each sample. The initial measurements of the ratio of proMMP9:proMMp2 and WBCC were good prognostic indicators of the survival of the horses. There was no significant relationship between the interval between the injury and the horse being referred for treatment and either the WBCC or the levels of MMP2 and MMP9 initially, and no evidence that this interval significantly affected the chances of the horses surviving.

Effects of housing, parturition and diet change on the biochemistry and biomechanics of the support structures of the hoof of dairy heifers.

This study investigated effects of housing, diet and parturition on the biochemistry, biomechanics and pathology of feet of maiden, pregnant and lactating dairy heifers. Strength/laxity, laminar morphology, connective tissue (CT) biochemistry and sole lesions were assessed. Although no animals became clinically lame, severity of sole lesions was significantly greater in heifers housed in cubicles vs. straw yards, and in lactating/pregnant heifers vs. maidens. These effects were additive. Cubicle housing and parturition each increased CT metabolism (and were additive), and altered CT composition. Similarly, both impaired the biomechanical resilience of the hoof. There were no effects for diet. The results indicate that parturition/lactation causes non-inflammatory changes in CT that impair resilience of the feet to external stresses associated with poor housing. This "parturition effect" appears to be unrelated to change in diet and relatively brief, unless exacerbated by additional stresses of housing. Thus heifer lameness may be significantly reduced through short-term, low-cost improvements in husbandry around the time of calving.

Ultrastructural differences in cranial cruciate ligaments from dogs of two breeds with a differing predisposition to ligament degeneration and rupture.

Cranial (anterior) cruciate ligament (CCL) samples were obtained from dogs of the Labrador retriever (LR) and greyhound (GH) breeds, of which the former but not the latter is predisposed to CCL rupture. Electron microscopy revealed that the collagen fibril diameters of GHs were larger than those of LRs (P=0.03). Histological examination revealed a "fibrocartilaginous" appearance of CCLs in seven of eight GHs, and, to a lesser extent, in three of eight LRs. The formation of fibrocartilage is clearly not a disadvantage to the healthy racing GH, and cannot be regarded as a pathological degeneration in this breed. It is suggested that fibrocartilage is formed as a beneficial physiological adaptation to the compression of CCLs caused by tensile stress as a result of the tightening of two twisted bands. Fibrocartilage would appear to protect CCLs in the GH, but it may be indicative of a mild degenerative change, which may eventually lead to rupture in the LR.

Distal femoral intercondylar notch dimensions and their relationship to composition and metabolism of the canine anterior cruciate ligament.

OBJECTIVE: To determine the relationship between the dimensions of the distal femoral intercondylar notch (ICN) and the composition and metabolism of the anterior cruciate ligament (ACL) in three dog breeds with different relevant risks to ligament rupture and subsequent osteoarthritis (OA). DESIGN: ICN measurements were obtained from the femurs of Golden Retrievers (high risk), Labrador Retrievers (high risk) and Greyhounds (low risk). Femoral condyle width and height, ICN height and width indices, and notch shape index were measured using Vernier callipers in all dogs. Intact ACLs were obtained from the same dog breeds for a study of the impinged areas and were analysed for collagen content, collagen cross-links, and sulphated glycosaminoglycan (GAG) content, matrix metalloproteinase (MMP)-2 and the tissue inhibitors of metalloproteinases (TIMPs)-1 and -2. RESULTS: Femoral condyle width and height and ICN width indices were significantly greater in the low risk compared to the high risk breeds (P<0.01 for all parameters). In contrast, the pro (P=0.003) and active (P=0.007) forms of MMP-2 and sulphated GAGs (P=0.0002) were significantly greater in the impinged areas of the ACLs of the rupture predisposed breeds. CONCLUSIONS: Impingement by the ICN on the ACLs of the high risk breeds may result in increased collagen remodelling and increased sulphated GAG deposition, causing reduced structural integrity of the ligament. Altered ACL composition may predispose the ligament to increased laxity leading to joint degeneration and OA. This may have a comparative implication for pathogenesis of ACL rupture in humans.

Biomechanical and histopathological changes in the support structures of bovine hooves around the time of first calving.

Hooves were removed from heifers killed two weeks before calving (C-2), and four and twelve weeks post-calving (C+4 and C+12), and from age-matched maiden heifers. Segments were dissected from the anterior walls of lateral hind claws, to include horn, corium and bone, and the biomechanical properties were measured. There was reduced rigidity, both overall and at a physiologically appropriate displacement of 2 mm, and a greater displacement was required to reach initial and maximum support in the hooves of calving, compared with maiden, heifers. Histological examination revealed widened and distorted interdigitating laminae, progressing to C+12 in the hind claws, but with evidence of recovery in the front claws after C+4. Increased connective tissue strength from dorsal to ventral hoof segments was correlated with protein, proteoglycan, pro- and activated matrix metalloproteinase-2, and tissue inhibitors of metalloproteinases, and was inversely correlated with fat, water and collagen content. This implies that mechanical changes reflect alterations in the biochemistry of the connective tissue. This evidence supports the hypothesis that primary causal events associated with calving weaken the connective tissue of the hoof suspensory apparatus, leading to increased susceptibility to clinical lameness associated with sole ulcers and white line disease.

The role of up-regulated serine proteases and matrix metalloproteinases in the pathogenesis of a murine model of colitis.

Proteinases are important at several phases of physiological and pathological inflammation, mediating cellular infiltration, cytokine activation, tissue damage, remodeling, and repair. However, little is known of their role in the pathogenesis of inflammatory bowel disease. The aim of this study was to assess the role of tissue proteases in a mouse model of colitis. Proteolytic activity was analyzed, using gel and in situ zymography, in colonic tissues from severe combined immunodeficient mice with colitis induced by transfer of CD4(+) T lymphocytes. Serine proteinase levels increased in colitic tissue, with major species of 23 kd, 30 kd, and 45 kd. Co-migration and inhibition studies indicated that the 23-kd proteinase was pancreatic trypsin and that the 30-kd species was neutrophil elastase. Matrix metalloproteinase (MMP)-9 expression, and MMP-2 and MMP-9 activation, was elevated in colitic tissues. Proteinase levels followed a decreasing concentration gradient from proximal to distal colon. Proteolysis was localized to infiltrating leukocytes in diseased severe combined immunodeficient mice. Transmural inflammation was associated with serine proteinase and MMP activity in overlying epithelium and with marked subepithelial proteolytic activity. The results demonstrate a clear elevation in the levels and activation of proteases in colitis, potentially contributing to disease progression through loss of epithelial barrier function.

Prognostic value of markers of collagen remodeling in venous ulcers.

A 25 patient study was conducted into the relationship between markers of collagen metabolism in venous ulcer exudates and healing status, and their prognostic value in predicting healing performance. Wounds were sampled on at least 5 occasions over 12 months, the frequencies of which were determined by the need for clinic attendance. Specimens were taken from several sites on each ulcer using sterile preweighed filters. Wound margins were traced and sites recorded for each collection. Sample sites were evaluated for severity as improving, static, or deteriorating according to subsequent wound progression. Specimens were analyzed for levels of proenzyme and active forms of matrix metalloproteinases 2 and 9, neutrophil elastase, and type I collagen C propeptide. There was an overall trend of greater expression of all markers with increasing severity of wound site, this being highly significant for pro-matrix metalloproteinase-9 (p = 0.006). For samples collected simultaneously from improving and deteriorating regions of the same wound, paired data analysis showed statistically significant differences for pro-matrix metalloproteinase-9 (p < 0.001), neutrophil elastase (p < 0.005) and activated matrix metalloproteinase-9 (p < 0.05). Taken overall, these data show the potential of markers of collagen biochemistry as predictors of repair in venous ulcers; in particular pro-matrix metalloproteinase-9 and neutrophil elastase were found to be accurate prognostic indicators of subsequent healing.

Mechanical stress in vitro induces increased expression of MMPs 2 and 9 in excised Dupuytren's disease tissue.

We have previously shown that the ability to mechanically extend Dupuytren's contractures in vivo by the Continuous Elongation Technique before surgery resulted in increased metalloproteinase activity. However, under these conditions it was not possible to show whether the response was proportional to the mechanical stimulus or was inflammatory cell mediated. Using an in vitro system of controlled extensions in which inflammatory involvement is absent, we have now shown that there is a clear correlation between the load applied to the tissue and the release of matrix metalloproteinase-2. The subsequent degradation of the collagen results in a loss of mechanical strength reported in the preceding paper.

Postsurgical wound progression monitored by temporal changes in the expression of matrix metalloproteinase-9.

It is important to monitor the early stages of postoperative wound repair in order to identify those problems associated with impaired healing. Many of the crucial cellular responses of early wound healing, such as inflammatory infiltration, angiogenesis and re-epithelialization, are made possible through the action of matrix metalloproteinases (MMPs). Expression of MMP-2 and MMP-9 is elevated in acute wounds, and still greater levels are found in chronic wounds, indicating that uncontrolled proteolysis is a characteristic of retarded healing. Therefore, comparative measurements of MMPs may be used to monitor the progression of early wound healing. To investigate this, wound fluids and sera were collected from mastectomy and colectomy patients throughout early stages of repair, and the temporal expression profile established. Wounds which were healing were expressed maximal levels of MMP-9 at 24 h, followed by a significant decline by 48 h. Persistent elevation of MMP-9 expression was associated with infected and chronic wounds, and was identified in postoperative wounds by the absence of the significant decline between 24 and 48 h. Measurement of MMP-9 in postoperative wound fluids, therefore, provides an early indicator of impaired healing, which may be evaluated non-invasively within 48 h of closure.

Expression of gelatinases within the trabecular bone compartment of ovariectomized and parathyroidectomized adult female rats.

Ovariectomized (ovx) and parathyroidectomized (ptx) rat models of disturbed bone metabolism have been widely used in evaluating bone changes resulting from hormonal depletion, and are characterized by elevated and depressed bone turnover, respectively. We report here the expression of gelatinases extracted from native trabecular bone in these models. Nine-month-old female Sprague-Dawley rats were sacrificed after 3 weeks following ovx or 10 days post ptx to determine the influence of these procedures on the levels of proximal tibial bone tissue gelatinases. Identification and quantitation of these enzymes were performed via gelatin gel zymography of native tissue extracts and laser densitometry of developed gels, respectively. In the ptx model, a reduction in tissue levels of pro- and active-MMP-2 and a 45 kDa activated fragment was seen, whereas ovx exhibited significant increases in these enzymes. The MMPs are therefore clearly under the influence of factors known to modulate bone remodeling in vivo. The study of MMP levels directly extracted from bone using these experimental models may assist in developing management regimes for metabolic bone diseases through the use of drugs aimed at controlling turnover.

Increased expression of promatrix metalloproteinase-9 and neutrophil elastase in canine dilated cardiomyopathy.

OBJECTIVE: Canine dilated cardiomyopathy, commonly affecting Doberman pinschers, results in extracellular matrix remodelling within the myocardium. The aim of this study was to examine the proteolytic activity in myocardium from Doberman pinschers with dilated cardiomyopathy. METHODS: Samples of myocardium, obtained rapidly post mortem from the left ventricular free wall of Dobermans with dilated cardiomyopathy, clinically normal Dobermans and control dogs (non-Dobermans), were examined for proteolytic activity using substrate gel zymography. Gels were analysed by scanning densitometry. RESULTS: Promatrix metalloproteinase-9 activity was significantly increased in all Doberman myocardium when compared to controls. A significant increase in an enzyme, identified to be neutrophil elastase by inhibition of its activity by Elastatinal and Western blotting, was also detected in all Dobermans when compared to controls. CONCLUSIONS: The results indicate that promatrix metalloproteinase-9 and neutrophil elastase, both of which are implicated in inflammatory responses, are present in significantly elevated levels in Doberman dilated cardiomyopathy and are raised in clinically normal Dobermans. Both proteolytic enzymes degrade a wide variety of connective tissue components and thus the increased levels found may play an important role in the structural remodelling seen in the myocardium and subsequent heart failure. Increased proteolytic enzyme levels in clinically normal Dobermans may be indicative of the predisposition of the breed to dilated cardiomyopathy.

Chemistry of collagen cross-linking: biochemical changes in collagen during the partial mineralization of turkey leg tendon.

With age, the proximal sections of turkey leg tendons become calcified, and this phenomenon has led to their use as a model for collagen mineralization. Mineralizing turkey leg tendon was used in this study to characterize further the composition and cross-linking of collagen in calcified tissues. The cross-link profiles of mineralizing collagen are significantly different from those of other collagenous matrices with characteristically low amounts of hydroxylysyl-pyridinoline and the presence of lysyl-pyridinoline and pyrrolic cross-links. However, the presence of the immature cross-link precursors previously reported in calcifying tissues was not supported in the present study, and was found to be due to the decalcification procedure using EDTA. Analysis of tendons from young birds demonstrated differences in the cross-link profile which indicated a higher level of hydroxylation of specific triple-helical lysines involved in cross-linking of the proximal tendon. This may be related to later calcification, suggesting that this part of the tendon is predestined to be calcified. The minimal changes in lysyl hydroxylation in both regions of the tendon with age were in contrast with the large changes in the cross-link profile, indicating differential hydroxylation of the helical and telopeptide lysine residues. Changes with age in the collagen matrix, its turnover and thermal properties in both the proximal and distal sections of the tendon clearly demonstrate that a new and modified matrix is formed throughout the tendon, and that a different type of matrix is formed at each site.