Publication: Avances en la aplicación de marcadores de salud y bienestar en saliva en el ganado vacuno de leche
Authors
Vallejo Mateo, Pedro
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Escuelas::Escuela Internacional de Doctorado
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Lamy, Elsa ; Contreras Aguilar, María Dolores ; Franco Martínez, Lorena
Publisher
Universidad de Murcia
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DOI
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info:eu-repo/semantics/doctoralThesis
Description
Abstract
El estudio de las enfermedades relacionadas con el periparto en el vacuno lechero, principalmente metritis, cetosis o mamitis, cada día tiene más importancia en la industria de la producción lechera. Esto se debe a que muchas de las pérdidas económicas que se producen en las explotaciones ganaderas son debido a procesos patológicos que ocurre en esta fase, debido a una serie de cambios metabólicos y hormonales que predisponen a la aparición de dichas enfermedades. Estos cambios se relacionan con una disminución de la inmunidad, con aumento de la inflamación y con el estrés oxidativo.
El uso de fluidos diagnósticos diferentes a la sangre para evaluar el estado de salud animal está siendo cada vez más estudiado, utilizando muestras que se puedan tomar de forma no invasiva ni dolorosa. Este es el caso de la saliva, en la que recientemente se han validado métodos automatizados en el vacuno de leche para la evaluación de biomarcadores relacionados con el estrés, inmunidad, estado oxidativo, y del metabolismo general y daño hepático, muscular y renal. Sin embargo, actualmente estos biomarcadores en saliva de vaca no han sido evaluados en conjunto como biomarcadores de diagnóstico y monitorización en enfermedades relevantes en el vacuno, como la metritis, ni tampoco se conoce el efecto de la presencia de alimento en saliva en los resultados de los marcadores evaluados en este fluido.
Los objetivos de esta tesis doctoral fueron: 1) valorar las interferencias que se puede producir en la medición de analitos en saliva del ganado vacuno lechero debidas a la presencia de alimento ; y 2) evaluar si el uso de saliva puede ser de interés para el diagnóstico de metritis en el ganado vacuno lechero mediante la evaluación de 2.1) un perfil bioquímica salival mediante técnicas de análisis rutinarias y 2.2) posibles diferencias en el perfil proteico salivar entre vacas sanas y vacas con metritis, utilizando un enfoque proteómico. Para el primer objetivo se evaluó un perfil analítico mediante ensayos automatizados en saliva de vacas lecheras con diferentes alimentos añadidos in vitro como heno, pasto y premezcla, y se compararon con los resultados obtenidos de salivas tras lavar la boca a las vacas. Para el segundo objetivo, se evaluaron los posibles cambios analíticos en saliva de vacas lecheras sanas y con metritis, comparando los resultados salivares en conjunto con los obtenidos por hematología, bioquímica sérica y parámetros clínicos de la exploración. Para la consecución del tercer objetivo, se realizó un estudio proteómico con técnicas de alta sensibilidad para identificar los cambios en el proteoma que se producen en la saliva y suero en vacas con metritis, al comparar el perfil proteico con el observado en animales sanos.
En conclusión se determinó que: 1) la contaminación de la saliva con alimento puede producir cambios en los resultados de los analitos salivales relacionados con el bienestar y el estado de salud, lo que podría influir en la interpretación clínica; 2) el perfil bioquímico salivar automatizado muestra cambios en marcadores de estrés, inflamación, sistema inmune y metabolismo general entre vacas sanas y vacas con metritis; y 3) existen cambios significativos en la expresión de proteínas entre vacas lecheras sanas y con metritis, relacionadas con el sistema inmunológico, la inflamación, el metabolismo de lípidos y la homeostasis, el estrés oxidativo, la reparación del ADN, la señalización celular, y en diversas funciones estructurales y mucosas. En el caso de la calprotectina, una validación clínica posterior confirmó concentraciones más altas en saliva de vacas con metritis, pudiendo ser un nuevo biomarcador en esta especie.
The study of peripartum-related diseases in dairy cattle, mainly metritis, ketosis, or mastitis, is becoming increasingly important in the dairy production industry. This is because many of the economic losses occurring on livestock farms are caused by pathological processes that take place during this phase, resulting from a series of metabolic and hormonal changes that predispose animals to the development of these diseases. These changes are associated with decreased immunity, increased inflammation, and oxidative stress. The use of diagnostic fluids other than blood to assess animal health status is being increasingly studied, utilising samples that can be collected in a non-invasive and painless manner. This is the case with saliva, for which automated methods have recently been validated in dairy cattle for evaluating biomarkers related to stress, immunity, oxidative status, general metabolism, and hepatic, muscular, and renal damage. However, at present, these biomarkers in cow saliva have not been evaluated together as diagnostic and monitoring markers in relevant bovine diseases, such as metritis, nor is it known how the presence of feed in saliva might affect the results of the markers assessed in this fluid. The objectives of this doctoral thesis were: 1) to assess the interferences that might occur in the measurement of analytes in saliva from dairy cattle due to the presence of feed; and 2) to evaluate whether the use of saliva may be of interest for the diagnosis of metritis in dairy cattle through the assessment of 2.1) a salivary biochemical profile using routine analytical techniques, and 2.2) possible differences in the salivary protein profile between healthy cows and cows with metritis, using a proteomic approach. For the first objective, an analytical profile was evaluated through automated assays in saliva from dairy cows with different feeds added in vitro, such as hay, grass, and premix, and these results were compared with those obtained from saliva after washing the cows’ mouths. For the second objective, potential analytical changes in the saliva of healthy and metritic dairy cows were evaluated, comparing the salivary results with those obtained by haematology, serum biochemistry, and clinical examination parameters. To achieve the third objective, a proteomic study was carried out using high-sensitivity techniques to identify changes in the proteome occurring in the saliva and serum of cows with metritis, by comparing the protein profile with that observed in healthy animals. In conclusion, it was determined that: 1) contamination of saliva with feed can cause changes in the results of salivary analytes related to welfare and health status, which could influence clinical interpretation; 2) the automated salivary biochemical profile shows changes in markers of stress, inflammation, immune system, and general metabolism between healthy cows and cows with metritis; and 3) there are significant changes in the expression of proteins between healthy and metritic dairy cows, related to the immune system, inflammation, lipid metabolism and homeostasis, oxidative stress, DNA repair, cell signaling, and various structural and mucosal functions. In the case of calprotectin, subsequent clinical validation confirmed higher concentrations in the saliva of cows with metritis, suggesting it may be a new biomarker in this species.
The study of peripartum-related diseases in dairy cattle, mainly metritis, ketosis, or mastitis, is becoming increasingly important in the dairy production industry. This is because many of the economic losses occurring on livestock farms are caused by pathological processes that take place during this phase, resulting from a series of metabolic and hormonal changes that predispose animals to the development of these diseases. These changes are associated with decreased immunity, increased inflammation, and oxidative stress. The use of diagnostic fluids other than blood to assess animal health status is being increasingly studied, utilising samples that can be collected in a non-invasive and painless manner. This is the case with saliva, for which automated methods have recently been validated in dairy cattle for evaluating biomarkers related to stress, immunity, oxidative status, general metabolism, and hepatic, muscular, and renal damage. However, at present, these biomarkers in cow saliva have not been evaluated together as diagnostic and monitoring markers in relevant bovine diseases, such as metritis, nor is it known how the presence of feed in saliva might affect the results of the markers assessed in this fluid. The objectives of this doctoral thesis were: 1) to assess the interferences that might occur in the measurement of analytes in saliva from dairy cattle due to the presence of feed; and 2) to evaluate whether the use of saliva may be of interest for the diagnosis of metritis in dairy cattle through the assessment of 2.1) a salivary biochemical profile using routine analytical techniques, and 2.2) possible differences in the salivary protein profile between healthy cows and cows with metritis, using a proteomic approach. For the first objective, an analytical profile was evaluated through automated assays in saliva from dairy cows with different feeds added in vitro, such as hay, grass, and premix, and these results were compared with those obtained from saliva after washing the cows’ mouths. For the second objective, potential analytical changes in the saliva of healthy and metritic dairy cows were evaluated, comparing the salivary results with those obtained by haematology, serum biochemistry, and clinical examination parameters. To achieve the third objective, a proteomic study was carried out using high-sensitivity techniques to identify changes in the proteome occurring in the saliva and serum of cows with metritis, by comparing the protein profile with that observed in healthy animals. In conclusion, it was determined that: 1) contamination of saliva with feed can cause changes in the results of salivary analytes related to welfare and health status, which could influence clinical interpretation; 2) the automated salivary biochemical profile shows changes in markers of stress, inflammation, immune system, and general metabolism between healthy cows and cows with metritis; and 3) there are significant changes in the expression of proteins between healthy and metritic dairy cows, related to the immune system, inflammation, lipid metabolism and homeostasis, oxidative stress, DNA repair, cell signaling, and various structural and mucosal functions. In the case of calprotectin, subsequent clinical validation confirmed higher concentrations in the saliva of cows with metritis, suggesting it may be a new biomarker in this species.
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