A multidisciplinary approach to the reduction in lameness and improvement in dairy cow welfare in the European Union

Deliverables

A series of guidelines are in preparation. These are based on the analysis of data collected during the assessment of husbandry systems in GB, NL, D and PL, which led to the identification of specific lameness risk factors. The guidelines are intended as an aid in the implementation of 'Best Practice' dairy husbandry systems designed to reduce the incidence of lameness.

Modern technology makes it possible to record electronic data during claw trimming. Using this technology it is possible to link the data on claw health with basic cow data such as registration number, date of birth, pedigree and milk yield data and then integrate the new data. The recording of claw health is achieved using a hand held pocket PC. Software, developed as part of the Framework 5 LAMECOW project, allows the pocket PC to be used independently when hoof trimming is in progress to record claw condition, lesions, disease and treatments administered. The programme is designed to take the user through a logical sequence of data recording. Firstly, it allows recording of farm data such as housing, herd size. The second stage is to call up individual cow data, already in memory from a previous visit or, for new cows, entered on site. The third stage is the recording of the cleanliness of each animal and an assessment of its lameness in one of three categories, sound, slightly lame or lame. The next stage is to select the foot to be examined. A diagnosis is then made of the lesion(s)/disease(s) observed and this is can be recorded on a diagram of the foot showing the medial and lateral claws, each divided into 6 zones, and the interdigital cleft. A total of 17 different claw conditions can be recorded. The farmer can be given a printed version of the diagnoses made for each cow during the trimming session. The pocket PC can subsequently be linked via the internet to download animal biodata and milk production data from external data servers by FTP connection or an MSN link. In this mode the biodata for cows already stored on the pocket PC can be automatically updated each time the system is linked to the appropriate external data server via the internet. Alternatively the claw heath data recorded on the pocket PC can be downloaded to a suitable server where for it can be analysed in conjunction with other animal biodata and performance records. We have created a recording programme that is easily transported and simple to use. If linked to a central server, farmers could have the opportunity to access the data on each of his animals. In addition, the pocket PC has the potential to be a powerful research tool for the collection of data at different geographical locations and subsequent transmission to a central server for data analysis.

The biomechanic forces at work within the bovine claw, important factors in the development of lameness in the cow, are impossible to measure directly. We developed computer models that simulated these forces and their effects in the normal claw form and in three diseased claw forms, a flat claw, a contracted claw and a laminitis-type claw. Measurements of important mechanical characteristics including elasticity modulus values were used to construct the model. Elasticity modulus values in the dorsal region of the claw were higher than in the bulbar region and the sole. This could point to a key biomechanic role that the claw has to play in motion. As it lands on its more elastic palmar/plantar region, the claw assumes a shock absorbing function. As the claw rolls onto the toe and pushes off, the stiffer lateral and dorsal wall allows for a more efficient force transfer. The correlation of the modulus of elasticity to dry matter levels was proven in the physiological claw, but could not be shown in the material of the pathological claw forms. These results suggest that the obvious deterioration in the horn quality of claws affected by chronic laminitis (grooves, loss of shine, deformation, and discolouration) also led to the significant reduction of the modulus of elasticity, with the moisture content having little effect. The computer models of the claw were based on so-called finite elements, a technique used often in engineering and architecture. These models were used to evaluate the way normal and pathological forms of bovine feet are loaded when standing on different flooring systems. The finite element model of the normal bovine claw under a load of 756 N (a value representing a normal cow's weight on one foot) showed only minimal deformation, most of which took place at the inner wall. Highest stresses were evident in the proximal inner wall, the outer edge of the weight-bearing surface and under the heels. The claw-floor contact image showed a pressure distribution resembling the distal rim of the claw wall. On a hard surface, the maximum stresses were three times higher than those on the soft surface. Analyses of the laminitic claw model showed altered stress and strain distributions compared to the sound claw model, with higher burdens on the dorsal margin, the solear surface and the inner wall. The flat dorsal wall angle of the flat claw shifted the major part of the loading to the back part of the claw capsule in the finite element model, leaving the dorsal aspect of the claw showing less stress and strain. The finite element model of the contracted claw indicated mechanical changes especially in the distal outer wall, where the turned-in (contracted) claw wall compresses the underlying soft and hard tissue structures. High stresses were predominately seen in the areas where most horn and claw defects are found in clinical research. While previous simulations could only calculate stress and strain in the horn capsule, the new full foot model included bone and connective tissue. Deformation seen in the model calculation was seen primarily in the softer tissues of the foot. High levels of strain in the region distal to the tuberculum flexorum correspond to the typical location of sole ulcers in diseased claws. The possibility of being able to visualise deformation and material stress in the bovine claw on hard and soft surfaces and of using physiological and pathological claw forms, has opened up new perspectives in the study of claw biomechanics. Testing and virtual validation of claw trimming techniques as well as the testing of precisely-defined flooring designs such as slatted or grooved flooring with or without rubber mats could be attempted with this model in the future.

We developed a novel organotypic long term perfusion cell culture system basing on our cell cultures of dermal fibroblasts and epidermal keratinocytes both isolated from the bovine claw. The model was developed using commercially available perfusion chambers. Because of some technical limitations we developed and optimised 2 new perfusion chambers allowing long term culture on bio-membranes as basement membrane equivalents. These chambers were developed by Dirk Hoffmann as part of his Dr med vet thesis and assembled by a private engineering group. Bovine keratinocytes and fibroblasts were obtained from bovine hooves of routinely slaughtered animals from the local abattoir. For the procedure of isolation of these cells see Nebel and Mülling (2002). The cells were cultivated with Dulbeccos modified Eagles medium (Biochrom, Berlin) supplemented with 10% foetal bovine serum (Sigma, Taufkirchen). First, fibroblasts were seeded onto the lower side of the Millicell® insert and allowed to grow for 4 days. Then the perfusion chamber was mounted by placing the fibroblast into the chamber and subsequently seeding epidermal keratinocytes onto the upper side of the Millicell® insert. The keratinocytes were allowed to adhere for 1 day. Then the cabmber was connected to the tube system with an attached Ismantec 8 cannel pump (Ismatec, Wertheim-Mondfeld) and a gas permeable media bag (Oligene, Berlin). We worked with different flow rates ranging from 0.035ml/min to 0.35ml/min. The system was run for up to 20 days. During this time it was possible to observe the cultivated cells under the light microscope at any time. The perfusion chamber of Dirk Hoffmann was handled similarly. The only difference was that the keratinocytes were allowed to adhere for 4 days. In addition, some Millicell® inserts were coated with Matrigel® basement membrane matrix (Becton Dickinson, Heidelberg) using the thin coating method (working instructions of Becton Dickinson) to permit the keratinocytes an improved adherence to the Millicell ® inserts. These Millicell® inserts were handled as described above. Using the Matrigel® thick gel method it was possible to grow cells within a three dimensional matrix. Fibroblasts were dispersed in Matrigel ® subsequently seeded onto the upper side of Millicell® inserts and grown for 4-5 days under standard cell culture conditions. Afterwards, keratinocytes were seeded onto the top of this Matrigel® and allowed to adhere for one day. Then the Millicell® inserts were placed into the perfusion chamber and connected to the fluid flow system. In our chambers different types of long term culture of fibroblasts and keratinocytes was established and carried out successfully. Keratinocytes showed a tissue specific differentiation and superficial cornification. Morphological examination and protein analysis demonstrated organ specific characteristics. The model is a valid tool for studies of dermo-epidermal cross talk and epidermal proliferation, differentiation and cell death. Experiments carried out with our culture model revealed the effects of selected cytokines and growth factors on cellular proliferation and differentiation. With our experimental work we generated initial evidence for the existence of a local paracrine regulatory mechanism in claw tissue potentially involved in the initiation of laminitis. This mechanism explains tissue alterations in damaged tissues as well as the reactive proliferation present in tissues from laminitic cows. IL-1 produced in the epidermis stimulates the production of KGF originating from fibroblasts. KGF is a potent stimulator of keratinocyte proliferation. Proliferation is also stimulated by GM-CSF, suggesting this an other factor in the development of laminitis. TNF-a depresses cellular proliferation and differentiation of keratinocytes, as does IL-1-a. In case of damage of dermal fibroblasts in disease IL- 1 may not cause stimulation but inhibition of keratinocyte proliferation.

The colour atlas provides photographs with a written description of the common lesions associated with lameness in dairy cattle. - Current status Complete. - Key innovative features An agreed description and nomenculature for common lesions associated with lameness in dairy cows. - Dissemination It may be used to benefit society and science. The atlas can be used as a whole without charge by anyone interested in recording causes of lameness or lesions on dairy cow feet and limbs. It is not to be sold. The photographs belong to members of the Lamecow team and are not to be sold or used separately from the atlas without permission. - Benefits The atlas can be used to assist training in lesion recognition. This will assist farmers and advisors to know the types of lesions in a dairy herd and consequently assist in development of a lameness reduction programme.

The Training Package has been designed in English prototype form to be accessed as a CD/DVD package designed to inform the dairy sector from individual stockpeople and farmers to the various business, commercial and academic support activites, such as veterinarians, feed manufacturers and suppliers, farm building designers, research scientist and educationalists about the nature, causes and prevention of lameness in dairy cows. The main sections of the Training Package are as follows Introduction; The European Dairy Industry; The Bovine Foot; Disease Library; Risk Factors (intrinsic and extrinsic); Treatment; Prevention and Elimination, Weigh Bearing and Locomotion; Fundamental Knowledge for Hoof Trimmers; Claw Trimming; Project Research. The Training Package will be used as one of the vehicles to disseminate the findings of the project and will be used as an educational and support tool in disseminating information to the European dairy and associated sectors about nature, causes and prevention of lameness in dairy cows. To achieve optimum benefit from the work that has gone into developing the protype Training Package, a further stage of development is required to take the product to the full market readiness. This will require the involvement of professional media companies.

A quantity of data (epectronic and biological) from farms and experimental studies have been collected and stored as part of this project. These data are a valuable resource for future research and interested parties may contact the relevant holder of the data.

An isolated haemoperfused distal cow limb model for experimental studies on the pathogenesis of bovine laminitis The actual knowledge on the pathomechanisms of bovine laminitis is still incomplete despite of progressively increasing research activities. Isolated perfused distal limbs are a promising model to study the biological mechanisms that lie behind the laminitis syndrome. For ethical and financial reasons experiments using this model are a preferable alternative to animal experiments. We developed an isolated haemoperfused distal cow limb model basing on an existing porcine limb model used already for studying the effects of pharmaceuticals. This model is described in a Standard Operating Procedure (SOP) enabling the use of the model for experimental studies by other groups. The isolated limbs were obtained from routinely slaughtered cows and subsequently perfused in the laboratory of a local biotech company (Vitro-tec Entwicklungsgesellschaft mbH, Berlin). A standard perfusion apparatus enables isolated limb perfusion for up to 5 hours under close to physiological conditions. Perfusion pressure and flow were calculated and adjusted basing on values available for horses and pigs. The comparison of the different initial experiments showed that 190 ml/min is an optimal value for the flow of perfusate. The oxygen saturation was adjusted to 100 % and the pH preset between 7.35 and 7.45. Glucose was added as nutrient. The weight increase of the limb and the perfusion pressure served as vitality indicators. The threshold for an acceptable weight gain of the distal limb was 10 % and for the upper and lower values of perfusion pressure 150 mmHg and 30 mmHg respectively. In addition, systematic light- and electron microscopic examination of tissue samples from all segments of the claws was carried out to evaluate tissue vitality according to a list of standardised morphological criteria. A thermographic camera was used to detect "cold spots as an indicator for ischemic areas. A complete electrolyte solution added with washed erythrocytes (haematocrit 8 - 10 %) turned out to be optimal for perfusion and was used as standard perfusate. The model was challenged in a series of 52 experiments. Thereby the effects of candidate bioactive molecules relevant for the pathogenesis of laminitis on perfusion pressure and resulting tissue alterations were tested. The modern hypotheses on the pathogenesis of laminitis attribute a central role to disturbances in microcirculation and direct damage to the vascular endothelium in the initial phase of the development of laminitis. These disturbances and alterations are caused by toxins, vaso-active substances of metabolic by-products. Therefore, in the perfusion experiments of this work the effects of some of these substances most frequently discussed in relation to laminitis have been tested. The claw tissue was perfused under oxygen deficiency and reduced flow rates. Furthermore the model was challenged by endotoxin, lactate and histamine to detect whether these substances produce changes in perfusion or structure of the vascular system or cause tissue alterations relevant for the development of laminitis. It was possible to show that an oxygen deficiency between 15 30 % for 4 hours and agents like histamine and E. coli endotoxin provoke changes in perfusion parameters and alterations in tissue structure. The following changes were detected: An increase in pressure and organ resistance, alterations of vascular permeability, formation of oedema and cell damage. These alterations perfectly fit in the actual hypotheses on the pathogenesis of laminitis. The limited period of 5 hours however does not permit final conclusions whether this condition finally would lead to clinical laminitis. The model introduced here is interesting to investigations on a variety of specific questions in Veterinary Medicine. It is attractive in particular for further research into equine and bovine laminitis. Furthermore it is also attractive for research in human medicine with possible applications in Pharmacology and Toxicology. In combination with modern analytical methods such as PCR techniques the reactions of cells to the challenging factors could be detected on the molecular level. The combination of the described novel ex vivo model with real-time PCR for quantitative analysis of effects on the cellular protein expression is recommended for future investigations using this model.

Key Innovative Features of Flooring Studies: The objective of the study was to increase the understanding of the influence of flooring systems on the wellbeing and performance of dairy cows. The studies were carried out at Swedish experimental and commercial herds. Walking comfort: A method was developed to analyze the pattern of foot prints of the walking cow (track way analysis) to estimate the animals walking comfort on different surfaces. The best walking comfort was achieved on sand ground followed by yielding rubber flooring, solid and slatted. Least good comfort was seen on slippery concrete slats. Lame cows impaired their trackways most on concrete and differences between healthy and lame cows were least on rubber flooring and sand, and largest on concrete slatted flooring. On solid floors with different surface design rubber flooring also proved to give the best walking comfort i.e. longer strides indicating better slip-resistance. Grooved concrete and mastic asphalt improved trackways slightly compared to smooth concrete. The friction coefficient reflected partly the results, which is reasonable as the steps will be more extended when the animal trusts the surface. However, the method used here does not reflect the risk for slipping in an acute situation with crowding and riding animals. Preference: In a preference study the majority of cows (about 70%) preferred to stand on rubber flooring rather than on concrete floors. Cows also showed a tendency to stand on extra soft rubber mats compared to soft rubber mats. The majority of cows (about 75 %) preferred to walk on rubber mats compared to slatted concrete and a slightly higher preference for solid rubber compared to slatted rubber was observed. Longtime exposure and claw pressure: The pressure distribution between and within claws was studied before and after long term exposure for different flooring systems using an electronical pressure plate. It was found that biomechanical parameters of the claw sole were mainly affected by differences in horn wear of the studied flooring systems. On a rougher flooring (mastic asphalt, comparable to new concrete flooring) an over-wear of the rear claws´ outer wall was seen, and this responded in a greater growth rate. A very low or negative net growth in these cows resulted in shorter claws and steeper claw angle and a convex sole shape. When rubber equipped feed stalls were used together with mastic asphalt, wear was reduced, net growth was positive and the remarkable loss of the concavity was reduced. Rubber mats on walking areas and feed places resulted in a lower growth and wear rate, an increased net growth, and resulted in a concave sole conformation similar to what is seen in grazed cattle. The growth and wear on aged, smooth concrete, slatted floor was similar to rubber flooring. No or little differences of the conformation of medial claws were observed between the different flooring systems. Generally, only a few associations between flooring system, claw conformation, lameness and lesions were found in the present study. This might be a result of low prevalence of claw lesions during the study time. Scrapers on top of a concrete slatted floor (comparing with slats without scrapers) and a solid rubber floor (comparing with solid mastic asphalt) decreased the risk of lameness. Feed-stalls decreased the risk of lameness in cows of second parity. Claws with sole haemorrhages were likely to be longer and having a lower angle and claws with white line haemorrhages were characterised by a higher concavity of the claw sole. Hygiene and behaviour was shown to be altered with flooring system. Scrapers on slatted flooring significantly improved the floor hygiene. Feed stalls improved the floor hygiene on the slatted flooring without scrapers. With the present housing design, management and feeding strategy, feed-stalls resulted in increased feeding-time, and decreased competition and displacement. Foot bath: A seven percentage copper-sulphate solution on a hoof mat applied twice daily the risk of heel-horn erosion and digital dermatitis, increased the probability of improvement of feet with lesions initially, and decreased the risk of new or aggravated lesions during treatment. On the other hand, the study provided no evidence of an effect of a foam bath containing peracetic acid during on heel-horn erosion, digital dermatitis or interdigital hyperplasia. Benefit: Soft, hygienic and non slippery flooring with a moderate wear of the claws is possible to establish with present flooring systems and thereby improve the dairy cows´ locomotion comfort, health and welfare. Well managed foot baths can reduce the occurrence of infectious claw diseases in an infected herd but optimizing hygiene by the management system is of most importance. Rubber flooring, solid or slatted, showed benefits in many aspects but a combination of different floor surfaces may be optimal to balance claw growth.

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