Sperm cells need hexoses as a substrate for their function, for both the maintenance of membrane homeostasis and the movement of the tail. These cells have a peculiar metabolism that has not yet been fully understood, but it is clear that they obtain energy from hexoses through glycolisis and/or oxidative phosphorylation. Spermatozoa are in contact with different external environments, beginning from the testicular and epididymal fluid, passing to the seminal plasma and finally to the female genital tract fluids; in addition, with the spread of reproductive biotechnologies, sperm cells are diluted and stored in various media, containing different energetic substrates. To utilize these energetic sources, sperm cells, as other eukaryotic cells, have a well-constructed protein system, that is mainly represented by the GLUT family proteins. These transporters have a membrane-spanning α-helix structure and work as an enzymatic pump that permit a fast gradient dependent passage of sugar molecules through the lipidic bilayer of sperm membrane. Many GLUTs have been studied in man, bull and rat spermatozoa; the presence of some GLUTs has been also demonstrated in boar and dog spermatozoa. The aims of the present study were - to determine the presence of GLUTs 1, 2, 3, 4 and 5 in boar, horse, dog and donkey spermatozoa and to describe their localization; - to study eventual changes in GLUTs location after capacitation and acrosome reaction in boar, stallion and dog spermatozoa; - to determine possible changes in GLUTs localization after capacitation induced by insulin and IGF stimulation in boar spermatozoa; - to evaluate changes in GLUTs localization after flow-cytometric sex sorting in boar sperm cells. GLUTs 1, 2, 3 and 5 presence and localization have been demonstrated in boar, stallion, dog and donkey spermatozoa by western blotting and immunofluorescence analysis; a relocation in GLUTs after capacitation has been observed only in dog sperm cells, while no changes have been observed in the other species examined. As for boar, the stimulation of the capacitation with insulin and IGF didn’t cause any change in GLUTs localization, as well as for the flow cytometric sorting procedure. In conclusion, this study confirms the presence of GLUTs 1, 2 ,3 and 5 in boar, dog, stallion and donkey spermatozoa, while GLUT 4 seems to be absent, as a confirmation of other studies. Only in dog sperm cells capacitating conditions induce a change in GLUTs distribution, even if the physiological role of these changes should be deepened. / Gli spermatozoi di mammifero necessitano di zuccheri per espletare le loro funzioni, come il mantenimento dell’omeostasi membranale ed il movimento. Queste cellule hanno un metabolismo particolare, che non è ancora stato del tutto compreso, anche se è chiaro che ottengano energia da esosi tramite il processo glicolitico e la fosforilazione ossidativa. Gli spermatozoi sono a contatto con ambienti esterni molto diversi: dai fluidi testicolare ed epididimale, per passare al plasma seminale ed infine alle secrezioni dell’apparato genitale femminile. Inoltre, con la diffusione delle biotecnologie riproduttive, il liquido seminale è diluito e conservato in svariati media contenenti diversi substrati energetici. Per sfruttare queste fonti energetiche gli spermatozoi, come le altre cellule eucariotiche, hanno un sistema proteico di membrana ben definito, rappresentato principalmente dalla famiglia dei GLUT. Queste proteine hanno una struttura transmembranale ad alfa elica e funzionano come una pompa enzimatica che permette un trasporto passivo veloce e secondo gradiente di concentrazione delle molecole di zucchero attraverso lo strato lipidico. Alcuni GLUT sono stati studiati negli spermatozoi di uomo, ratto e toro e la presenza di alcuni altri è stata dimostrata in cane e maiale. Gli scopi di questo studio sono stati: • determinare la presenza dei GLUT 1, 2, 3, 4 e 5 in spermatozoi di maiale, cane , stallone e asino e descrivere la loro localizzazione; • studiare eventuali cambi di localizzazione dovuti alla capacitazione o alla reazione acrosomiale in spermatozoi di maiale, cane e stallone; • valutare cambiamenti nella disposizione dei GLUT negli spermatozoi di maiale in seguito a capacitazione stimolata con insulina ed IGF; • valutare possibili cambiamenti di localizzazione dei GLUT a seguito del processo di “sessaggio” mediante citofluorimetro sorter. La presenza e la localizzazione dei GLUT 1, 2, 3 e 5 sono state dimostrate negli spermatozoi di maiale, asino, cavallo e cane mediante le tecniche di western blotting ed immunofluorescenza indiretta; una rilocalizzazione delle proteine dopo capacitazione è stata osservata solo negli spermatozoi di cane e nessun cambiamento è stato registrato nelle altre specie. Per quanto riguarda il maiale, non si sono rilevate rilocalizzazioni dei GLUT a seguito della capacitazione con stimolazione con insulina ed IGF e nemmeno a seguito del processo di “sex sorting”. Concludendo, questo studio conferma la presenza dei GLUT 1, 2, 3 e 5 negli spermatozoi di maiale, cane stallone e asino, mentre il GLUT 4 sembra essere assente, a conferma di alcuni studi precedenti. Solo negli spermatozoi di cane le condizioni capacitanti inducono un cambiamento nella distribuzione dei GLUT, anche se il ruolo fisiologico di questi cambiamenti deve essere ancora approfondito.

No description available.

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Il lavoro eseguito comprende 3 differenti studi inerenti l’approfondimento di diversi aspetti della fisiopatologia della prostata, in particolare l’aspetto clinico, biochimico ed ormonale. Per quanto riguarda l’aspetto clinico è stato eseguito uno studio ecografico sull’accrescimento e sviluppo della ghiandola, a partire dai 4 giorni di vita all’anno di età del cane. L’aspetto biochimico dell’organo è stato indagato nel secondo studio, analizzando l’eventuali differenze nel profilo proteico e nel profilo dello zinco, valutato con spettrofotometria ad assorbimento atomico, su campioni di plasma seminale di cani sani o cani affetti da iperplasia prostatica benigna sintomatica od asintomatica. Lo studio è stato eseguito allo scopo di valutare se l’approccio biochimico ai problemi prostatici possa essere d’aiuto nella diagnosi della più comune patologia della prostata, l’iperplasia prostatica benigna. Il terzo studio inerente l’aspetto ormonale della prostata, completa lo studio precedente, analizzando le eventuali differenze ormonali (Testosterone, Estrogeni e Progesterone) riscontrabili fra due gruppi di cani: i cani sani e quelli affetti da iperplasia prostatica benigna.

General aim of the study is equine welfare, particularly concerning different husbandry methodic and inter-specific relational factors. Specific aim is the evaluation of possible mutual (to humans and to equines) benefits and the analysis of critical factors/strength points, of human-horse relationship within Therapeutic Riding context (TR). The peculiarities of human-horse relationship (compared to the bond with “Pet”) are analyzed, concerning their socio-anthropological, psychological, psycho-dynamic distinctive characteristics. 8 European representative therapeutic riding centers (TRC) were therefore selected (on the basis of their different animals’ husbandry criteria, and of the different rehabilitative methodologies adopted). TRC were investigated through 2 different questionnaires, specifically settled to access objective/subjective animal welfare parameters; the quality of human-horse relationship; technicians’ emotional experienced. 3 Centers were further selected, and behavioral (145 hours of behavioral recording) and physiological parameters (heart rate and heart rate variability) were evaluated, aimed to access equine welfare and horses’ adaptive responses/coping (towards general environment and towards TR job). Moreover a specific “handling-task” was ideated and experimented, aimed to measure the quality of TR technicians-horses relationship. We did therefore evaluate both the individual horses’ responses and the possible differences among Centers. Data collected highlight the lack of univocal standardized methodic, concerning the general animals’ management and the specific methodologies (aimed to improve animal welfare and to empower TR efficacy). Some positive and some critical aspects were detected concerning TR personnel-horse relationship. Another experimental approach did evaluate the efficacy (concerning the mutual benefits’ empowerment) of an “ethologically-fitted” TR intervention, aimed to educate children to and through the relationship with horses. Our data evidenced that the improvement of human horse relationship, through structured educational programs for TR personnel might have important consequences both to human and equine welfare.

Gli spermatozoi di suino sottoposti alla procedura di sessaggio mediante citofluorimetria presentano una serie di modificazioni morfo-funzionali che compromettono nel tempo la loro sopravvivenza e la capacità fecondante. Questi spermatozoi, inoltre, a causa della sensibilità ai danni indotti dalla crioconservazione, vengono solitamente conservati allo stato liquido a 15-17°C, con conseguente ulteriore peggioramento nel tempo della qualità delle cellule spermatiche sessate. Lo scopo della ricerca è stato quello di valutare le modificazioni di alcune caratteristiche morfo-funzionali degli spermatozoi in seguito a sex-sorting e conseguente conservazione. Successivamente si è cercato di migliorare i parametri qualitativi del seme sessato mediante l’aggiunta di sostanze antiossidanti e la messa a punto di una nuova metodica di conservazione. I risultati ottenuti hanno evidenziato che la procedura di sessaggio e la conseguente conservazione per 24-26 ore a 15°C hanno indotto un peggioramento significativo delle caratteristiche morfo-funzionali (vitalità, integrità acrosomiale, quantità e distribuzione dell’Hsp70, capacità fecondante). Mentre l’azione degli antiossidanti non si è rivelata efficace nel miglioramento della qualità degli spermatozoi durante le fasi di colorazione e passaggio attraverso il citofluorimetro, l’azione congiunta del plasma seminale e degli antiossidanti superossido-dismutasi ed epigallocatechina-3-gallato ha indotto un miglioramento significativo della vitalità degli spermatozoi. Per la conservazione del seme di suino è stata testata la tecnica di incapsulazione in membrane di alginato di bario che permette, durante l’inseminazione artificiale, un rilascio graduale degli spermatozoi e l’utilizzo di un quantitativo inferiore di materiale seminale. L’applicazione di tale tecnica per la conservazione degli spermatozoi di suino sessati non sembra provocare un calo significativo della vitalità, dell’integrità acrosomiale e dell’efficienza totale di fecondazione rispetto al seme sortato e conservato diluito suggerendo futuri studi in vivo. Una migliore conoscenza dei danni indotti da queste tecnologie e la loro minimizzazione potrà stimolare in futuro l’utilizzo su vasta scala del seme sessato nel suino. / Boar spermatozoa submitted to the sorting procedure show several morpho-functional modifications effective in compromising their survival and fertilization ability. Moreover ,boar spermatozoa, because of their susceptibility to damages induced by cryopreservation, are usually stored at 15-17°C after the sorting procedure; however, also the conservation at liquid state implies the worsening of semen quality. The aims of this research were: 1) to evaluate morpho-functional characteristics of sperm cells submitted to sex-sorting and consequent storage; 2) to try to improve the quality of sorted semen by the addition of antioxidants; 2) to set up a new storage method. Our results evidence a decreased quality of boar sorted-stored spermatozoa in term of: viability, acrosome integrity, amount and localization of Hsp70, fertilizing ability. During the staining step and the passage through the cytofluorimeter, antioxidants were not effective in improving sperm cells morpho-functional characteristics, while the addition of superoxide dismutase or epigallocatechin-3-gallate associated with seminal plasma induced an increase of viability of sorted boar spermatozoa stored 24 h at 15°C. Some researchers have utilized encapsulation in barium alginate membrane to store boar sperm cells. This technique allows a constant release of spermatozoa in sow reproductive system, avoiding the double/triple intervention of insemination and reducing the number of spermatozoa/insemination. The application of this technique in order to store boar sperm cells after sorting did not induce any impairment of sperm morpho-functional characteristics (viability, acrosome integrity, total efficiency of insemination) compared to sorted spermatozoa stored at liquid state, thus demonstrating the possibility to use this method to improve the reproductive performance of boar sorted semen.

tesis por compendio de publicaciones / La agalaxia contagiosa caprina (AC) es una de las enfermedades con mayores repercusiones socioeconómicas del sector de los pequeños rumiantes. Los avances en el conocimiento de la epidemiología molecular y analítica de la enfermedad, así como los aspectos más relevantes que habrán de ser estudiados en un futuro, fueron abordados en una revisión por invitación en la revista The Veterinary Journal (Estudio 1). No obstante, esta Tesis Doctoral se ha centrado en estudiar las implicaciones epidemiológicas del semental caprino en la AC. Dentro de este ámbito de estudio, se ha evaluado la posible distribución sistémica de Mycoplasma spp. en sementales portadores auriculares asintomáticos mediante el estudio microbiológico y molecular (PCR) (Estudio 2). Por otra parte, ante el riesgo que suponía para los centros de inseminación la entrada de sementales infectados, se hizo necesario implantar estrategias de prevención y vigilancia en los mismos que contemplaran la identificación de portadores asintomáticos. Este modelo fue basado en el muestreo tanto de animales candidatos a entrar en los centros como de aquellos presentes en los mismos (Estudio 3). El alto valor genético de algunos sementales caprinos infectados, motivó el desarrollo de otra experiencia evaluando la eficacia del tratamiento antibiótico con marbofloxacina, uno de los antibióticos con mayor eficacia in vitro. Además, también fueron consideradas las posibles repercusiones que este tratamiento podría tener sobre la calidad espermática de los sementales afectados (Estudio 4). Finalmente, se evaluó la supervivencia de los principales agentes etiológicos de la AC en la dosis seminal. Previamente, fue necesario se desarrollar un modelo de contaminación experimental que permitiera obtener resultados válidos y extrapolables a las condiciones de campo (Artículo 5). Gracias a ello, se pudo posteriormente desarrollar un estudio experimental para evaluar la supervivencia de estas especies de micoplasmas en semen diluido y eyaculados, así como su repercusión sobre la calidad espermática (Estudio 6). OBJETIVOS PRIMERO: Realizar una revisión bibliográfica en donde se planteen estrategias de futuro en la investigación de la AC. SEGUNDO: Estudiar la distribución de Mycoplasma spp. en sementales caprinos portadores auriculares asintomáticos. TERCERO: Desarrollar un programa de prevención y vigilancia de AC en centros de inseminación artificial. CUARTO: Estudiar la eficacia del tratamiento sistémico de sementales portadores y sus repercusiones sobre la calidad espermática. QUINTO: Desarrollar un modelo experimental para la contaminación de semen caprino con Mycoplasma spp. SEXTO: Evaluar la viabilidad de Mycoplasma agalactiae (Ma) y Mycoplasma mycoides subsp. capri (Mmc) en semen diluido así como sus repercusiones en la calidad espermática. CONCLUSIONES PRIMERA Se confirma la distribución sistémica de Ma y Mmc en sementales caprinos portadores auriculares asintomáticos. SEGUNDA La presencia de Ma y Mmc en el sistema respiratorio, digestivo y urogenital de portadores asintomáticos puede suponer un factor de riesgo para la transmisión de la AC mediante las secreciones procedentes de estas localizaciones. TERCERA Se ha determinado por primera vez la presencia de Mmc en semen caprino de sementales asintomáticos infectados de forma natural. CUARTA El análisis combinado de hisopos de conducto auditivo externo y muestras de semen para el diagnóstico de micoplasmas asociados a la AC, resulta una herramienta útil para controlar la presencia de sementales caprinos portadores asintomáticos. QUINTA Además de afectar a la calidad espermática, el tratamiento sistémico con marbobloxacina resulta ineficaz para eliminar las infecciones por Mmc del conducto auditivo externo de sementales caprinos infectados de forma natural. SEXTA La supervivencia a dosis infectivas de Ma y Mmc en semen diluido durante al menos 150 minutos supone un factor de riesgo para su transmisión venérea. SEPTIMA En las condiciones estudiadas, el semen diluido disminuye la viabilidad de Ma mientras que el eyaculado la incrementa, sugiriendo que la monta natural puede incrementar las posibilidades de transmisión venérea de la agalaxia contagiosa caprina respecto al uso de la inseminación artificial. / Recent published studies on the contagious agalactia (CA) and especially the advances in the knowledge of their molecular epidemiology and analytical as well as the most relevant aspects that are to be studied in the future were discussed in the review by invitation asked us magazine The Veterinary Journal (Study 1). The doctoral thesis has mainly focused on studying the epidemiological implications of bucks on the CA. Within this field of study has evaluated first the distribution of the organism in ear asymptomatic carrier bucks in order to determine the existence of possible systemic infections. All these questions were developed in a second wok, by microbiological and molecular (PCR) study of naturally infected stallions (Study 2). Moreover was necessary to implement prevention strategies and monitoring them to contemplate the identification of asymptomatic carriers in insemination centers (Study 3). This model is based on sampling animals both candidates to enter the centers as those present in them. The high genetic value of some of the bucks infected, motivated the development of another experience evaluating the effectiveness of the use of antibiotic treatment with marbofloxacin for ear asymptomatic carriers bucks. In addition, were also considered the potential impact that this treatment might have on sperm quality of stallions affected (Study 4). http://www.google.com/url?source=transpromo&rs=rssf&q=//translate.google.com/community?source=all Finally, the survival of the major etiological agents of CA, Mycoplasma agalactiae (Ma) and Mycoplasma mycoides subsp capri (Mmc) was evaluated in the diluted semen. Previously it was necessary to develop a model of experimental contamination which to obtain valid and extrapolated to field conditions results (Article 5). This model allowed subsequently developing a experimental study to evaluate the survival of these species of mycoplasmas in diluted semen and ejaculate, and their impact on sperm quality (Study 6). OBJECTIVES FIRST: Perform a literature review where progress is exposed in the study of molecular and analytical epidemiology of contagious agalactia, control and approach future strategies in their research. SECOND: Study the distribution of Mycoplasma spp. in ear asymptomatic carriers bucks. THIRD: Develop a program of prevention and monitoring of caprine contagious agalactia in artificial insemination centers effective for detecting asymptomatic carrier bucks candidates to enter in the centers and present inside. FOURTH: To study the efficiency of systemic treatment with marbofloxacin to eliminate infection in naturally infected ear asymptomatic carriers bucks with Mycoplasma subsp. mycoides subsp. capri, evaluating its effect on sperm quality. FIFTH: Develop an experimental model for goat semen contamination with Mycoplasma spp., to evaluate its effect on the viability and motility sperm, as well as its impact on sperm quality under different conditions of carriage. http://www.google.com/url?source=transpromo&rs=rssf&q=//translate.google.com/community?source=all SIXTH: Evaluation of the Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri viability in diluted semen and their impact on sperm quality. CONCLUSIONS FIRST Systemic distribution of M. agalactiae and Mycoplasma mycoides subsp capri is confirmed in naturally infected ear asymptomatic carriers bucks. SECOND The presence of Ma and Mmc in the respiratory, digestive and urogenital system of asymptomatic carriers can be a risk factor for CA transmission by related secretions. THIRD Has been determined for the first time the presence of Mycoplasma mycoides subsp. capri in semen of asymptomatic bucks. FOURTH The combined of ear canal swabs and semen samples for the diagnosis of mycoplasma associated with CA is a useful tool to monitor the presence of asymptomatic carriers bucks in insemination centers. FIFTH Marbobloxacin systemic treatment was ineffective to eliminate Mycoplasma mycoides subsp. capri ear infections of naturally infected bucks while affecting sperm quality. SIXTH The survival as infective dose of Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri diluted semen for at least 150 minutes is a risk factor for venereal transmission. SEVENTH Under the conditions studied, the seminal dose decreases the viability of Mycoplasma agalactiae while ejaculated is a medium that favors their growth, suggesting that natural breeding can increase the possibilities of venereal transmission of caprine contagious agalactia regarding the use of artificial insemination.

The thesis is set in three different parts, according to the relative experimental models. First, the domestic pig (Sus scrofa) is part of the study on reproductive biotechnologies: the transgenesis technique of Sperm Mediated Gene Transfer is widely studied starting from the quality of the semen, through the study of multiple uptakes of exogenous DNA and lastly used in the production of multi-transgenic blastocysts. Finally we managed to couple the transgenesis pipeline with sperm sorting and therefore produced transgenic embryos of predetermined sex. In the second part of the thesis the attention is on the fruit fly (Drosophila melanogaster) and on its derived cell line: the S2 cells. The in vitro and in vivo models are used to develop and validate an efficient way to knock down the myc gene. First an efficient in vitro protocol is described, than we demonstrate how the decrease in myc transcript remarkably affects the ribosome biogenesis through the study of Polysome gradients, rRNA content and qPCR. In vivo we identified two optimal drivers for the conditional silencing of myc, once the flies are fed with RU486: the first one is throughout the whole body (Tubulin), while the second is a head fat body driver (S32). With these results we present a very efficient model to study the role of myc in multiple aspects of translation. In the third and last part, the focus is on human derived lung fibroblasts (hLF-1), mouse tail fibroblasts and mouse tissues. We developed an efficient assay to quantify the total protein content of the nucleus on a single cell level via fluorescence. We coupled the protocol with classical immunofluorescence so to have at the same time general and particular information, demonstrating that during senescence nuclear proteins increase by 1.8 fold either in human cells, mouse cells and mouse tissues.

En la especie canina, la rodilla es la articulación que más frecuentemente presenta alteraciones, siendo las patologías de los ligamentos y meniscos las más comunes, asociadas a cambios degenerativos articulares secundarios. Para evaluar las estructuras que componen la rodilla y poder diagnosticar dichas alteraciones podemos emplear numerosas técnicas de diagnóstico por imagen como la artrografia, ecografía así como estudios de imagen por resonancia magnética (IRM). La artrografia de la rodilla, es una técnica fácil de realizar y económica, mientras que el resto de métodos son más sofisticados y necesitan una mayor inversión para llevarlos a cabo. Los estudios artrográficos se han empleado durante años en medicina humana, especialmente para el diagnóstico de lesiones en meniscos y ligamentos cruzados. Sin embargo, en veterinaria existe poca información sobre la técnica de la artrografia y anatomía radiográfica en la articulación de la rodilla porque comúnmente se emplea para el diagnóstico de las osteocondritis disecante de la articulación del hombro. Otro medio de diagnóstico de utilidad en el diagnóstico de anomalías en cartílagos, meniscos, músculos, tendones y ligamentos, así como de artropatías y neoplasias es la ecografía. La capacidad de los ultrasonidos para ofrecer imágenes de tejidos blandos intraarticulares permite obtener información que puede ser empleada de forma adicional a la imagen radiográfica. Por último, el estudio por imagen de resonancia magnética (IRM) es una de las técnicas más recientes de diagnóstico por imagen, aunque su empleo actual en medicina veterinaria es aún limitado. Debido a su capacidad de visualizar estructuras intraarticulares es la modalidad de imagen preferida para el estudio del cartílago articular, los meniscos y los ligamentos de las articulaciones sinoviales, además al contrario que la artrografia, la IRM es una técnica no invasiva y no utiliza radiación ionizante. Uno de los objetivos de este trabajo ha sido desarrollar una técnica de artrografía de la articulación de la rodilla canina, determinando la combinación de concentración y volumen de contraste, así como la efectividad de la artrografía para delimitar las superficies articulares y ligamentos de la articulación de la rodilla. Por otro lado hemos querido describir la anatomía ecográfica y por IRM normal de la rodilla canina y correlacionar de forma directa los cortes anatómicos con las imágenes ecográficas y de RM, para facilitar su interpretación. Para determinar la concentración y volumen de contraste del estudio astrográfico se realizó una experiencia previa en 3 animales en los que se combinaron diferentes volúmenes y concentraciones de iohexol, contraste iodado hidrosoluble no iónico de baja osmolaridad. La combinación elegida fue la de 0'4ml de iohexol por centímetro de grosor mediolateral de la articulación a una concentración de 300mgI/ml. El estudio artrográfico se llevó a cabo en 12 animales de tamaño mediano y grande, y los disparos se realizaron a los tiempos O, 3, 5, 10, 15, 20 Y 30 minutos tras la administración del contraste en proyecciones lateral y caudocraneal De los 12 artrogramas, 11 (91'6%) fueron clasificados como aceptables, resultando diagnósticos hasta el tiempo 10 en la proyección caudocraneal (CdCr) y hasta el 15 en la mediolateral (ML), posteriormente la absorción del contrastese incrementaba no pudiéndose discernir las estructuras articulares. El ligamento cruzado craneal, el receso suprarrotuliano y el cartílago articular de los cóndilos del fémur se clasificaron como obvios, claramente delineados y accesibles para la evaluación radiográfica, mientras que el resto de estructuras articulares fueron visualizadas y resultaron distinguibles y eventualmente accesibles para la evaluación radiográfica. No se encontraron diferencias significativas (p< 0'05) entre los dos grupos de animales / In dogs, the stifle is the most frequently injured joint, ligamentous and meniscal injuries being the most common, with its associated secondary degenerative changes. Diagnosis of stifle joint disorders is generally based on history of lameness, physical examination and radiography. Diagnostic imaging techniques Iike arthrography, ulttasonography and magnetic resonance imaging could be used to further evaluate these sttuctures. Knee arthrography is a cheap technique easy to perform, meanwhile all other are more sofisticated and expensive methods. Arthrographic studies have been used along the years in human beings, specially in the diagnosis of meniscal and cruciate ligaments injuries. Canine arthrography of this joint has received little attention in the veterinary literature. Ulttasound may probe to be useful in the diagnosis of cartilage abnormalities, meniscal tears, muscle, tendon and ligament abnormalities, arthropathies and neoplasia. The ability of ulttasound to image intra-articular soft tissues, allows it to provide additional information that can be used in conjunction with radiography. Magnetic resonance imaging (MRI) is a valuable diagnostic tool in humans, but is relatively new to veterinary medicine because is an expensive method and few veterinary centers currently have the equipment avaliable. MRl has become the preferred imaging modality for the evaluation of the articular cartiIage, menisci and ligaments of synovial joints. Unlike arthrography, MRl is noninterventional and does not require the use of ionizing radiation. The present study was done to evaluate the technique of stifle arthrography in the dog, to determine the volume and concentration of the contrast material and to determine the accurate evaluation of intraarticular structures and articular surfaces. We also wanted to describe the normal ultrasonographic appearance as well as the normal anatomy by MRl To conlude, we can say that arthrography performed with iohexol, does not provide a full anatomic detail of all structures of the stifle joint in dogs, although some articular structures as we said before could be perfectly accesible for radiographic evaluation. Is a gafe technique because no inflamatory changes in the synovial fluid 7 days following injection of iohexol was observed. Ultrasonography of the canine stifle could be a useful clinical application to noninvasively evaluate most of the intraarticular structures, meanwhile the increased soft tissue contrast resolution with MRI provides better detail of the soft tissue structures of the knee than conventional radiography and ultrasound and therefore is the best diagnostic imaging tool to evaluate injuries at the canine stifle joint. arthrographic procedures were clear, colorless and with citologic values remained within normal limits in all dogs.

The evaluation of chronic activity of the hypothalamic-pituitary-adrenal (HPA) axis is critical for determining the impact of chronic stressful situations. The potential use of hair glucocorticoids as a non-invasive, retrospective, biomarker of long term HPA activity is of great interest, and it is gaining acceptance in humans and animals. However, there are still no studies in literature examining hair cortisol concentration in pigs and corticosterone concentration in laboratory rodents. Therefore, we developed and validated, for the first time, a method for measuring hair glucocorticoids concentration in commercial sows and in Sprague-Dawley rats. Our preliminary data demonstrated: 1) a validated and specific washing protocol and extraction assay method with a good sensitivity in both species; 2) the effect of the reproductive phase, housing conditions and seasonality on hair cortisol concentration in sows; 3) similar hair corticosterone concentration in male and female rats; 4) elevated hair corticosterone concentration in response to chronic stress manipulations and chronic ACTH administration, demonstrating that hair provides a good direct index of HPA activity over long periods than other indirect parameters, such adrenal or thymus weight. From these results we believe that this new non-invasive tool needs to be applied to better characterize the overall impact in livestock animals and in laboratory rodents of chronic stressful situations that negatively affect animals welfare. Nevertheless, further studies are needed to improve this methodology and maybe to develop animal models for chronic stress of high interest and translational value in human medicine.