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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Biomechanical and biochemical study of a standardized wound healing model.

Standardized protocols were developed for use in a detailed investigation into the biomechanical and biochemical properties of a dermal wound healing model in the rat. The use of a rapid freezing method at -80 degrees C minimized the detrimental effects of freezing on the biomechanical properties of the tissue and also allowed for convenient inter-laboratory collaboration to be performed. The methodology described allowed for the simultaneous and reproducible measurement of tensile strength, collagen cross-linking and proteolytic enzyme activity. Increases in the tensile properties of the tissue with time were consistent with an active process of remodelling process as indicated by changes in the cross-link and enzyme profiles. Initially the granulation tissue was comparatively rich in the keto-imine cross-link hydroxylysino-keto-norleucine, which was later replaced by the aldimine cross-link dehydro-hydroxy-lysinonorleucine. The mature cross-link histidino-hydroxy-lysinonorleucine was not observed within the granulation tissue at any stage and was also absent in aged control skin. A peak of matrix metalloproteinase-9 activity was observed at early timepoints (48 hr) and then decreased rapidly to normal levels and is consistent with an acute inflammatory response. In contrast matrix metalloproteinase-2 activity peaked later (3 days) and then decreased gradually, consistent with its role as one of the predominant enzymes involved in the remodelling process. The results described validate the animal model used and emphasize its potential for use in combined biomechanical and biochemical studies of acute wound healing.

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.

Clarification of immunoblots on polyvinylidene difluoride (PVDF) membranes for transmission densitometry.

The opacity of polyvinylidene difluoride (PVDF) transfer membrane can be reduced by infiltration with liquid. A mixture of 9:1 v/v ethylene glycol/glycerol gives complete transparency; clarification by this mixture is stable due to the low volatility of the components and is reversed by washing in water without loss of band intensity. The mixture is easy to apply to the membrane and being water soluble, non-corrosive and relatively non-toxic, is convenient to use. Clarification and densitometric analysis of stained immunoblots of PVDF membranes is at least as effective as that previously described for nitrocellulose, but the greater ease of use of the new mixture makes PVDF the membrane of choice for transmission densitometry. Using a blot onto PVDF of monoclonal IgG probed with anti-mouse antibody, laser transmission densitometry revealed that the stained peak area was related to loading in a linear fashion over a 60-fold range on a single blot.

Immunohistological characterisation of retinoblastoma and related ocular tissue.

The immunohistological reactivity of six retinoblastomas was investigated by means of 18 monoclonal antibodies and compared with that of adult and fetal retina. The antigenic profiles were found to be characteristic for each cell type studied and indicated that a panel of monoclonal antibodies could achieve a specific immunolocalisation not afforded by any single antibody. Immunohistological comparison between retinoblastoma and adult and fetal retinal cells provided evidence of the histogenesis of the tumour. The data suggest that the tumour arises from an early multipotential cell, which retains the capacity to develop differentiation characteristics associated with inner or outer retinal cell types, resulting in a heterogeneous tumour cell population. A cell with such differentiation potential predominates in the retina prior to the primitive neuroepithelial layer division at eight weeks' gestation.

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 continuous elongation technique for severe Dupuytren's disease. A biochemical mechanism.

Continuous extension of Dupuytren's contracture prior to fasciectomy results in a softening of the tissue, allowing straightening of the fingers. The observed change in cross-link profile indicates an increase in newly synthesised collagen due to increased turnover. This was confirmed by demonstration of the increases in levels of the degradative enzymes, the neutral metalloproteinases, collagenase and gelatinase and the acidic cathepsins B and L. Both types of enzyme effectively depolymerize the collagen fibres, albeit by different mechanisms, leading initially to loss of tensile strength and ultimately to solubilization. We suggest that the increase in enzyme activity is generated by tension on the fibroblasts of this metabolically active tissue produced during the continuous extension of the retracted fingers. The weakening of the fibres by degradation and the increase in newly synthesized collagen provide an explanation for the extension of the tissue without trauma.

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.

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.

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.