Browsing by Subject "Bovine"

Now showing 1 - 7 of 7
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    Caracterización de exosomas producidos por células oviductales in vivo e in vitro, en la especie bovina
    (Facultad de Veterinaria y el Servicio de Publicaciones de la Universidad de Murcia, 2023) Toledo Guardiola, Santa María; Matás Parra, Carmen; Rueda Gomariz, Almudena
    Las vesículas extracelulares (VEs), exosomas y micro vesículas son un tipo de estructuras heterogéneas pre-sentes en la mayoría de los fluidos orgánicos incluyendo el fluido oviductal. Las VEs contienen varios compuestos derivados de la célula original, como proteínas, lípidos, ARNm, miARN y ADN. Las VEs en el oviducto son producidas por las células epiteliales y entre sus funciones se encuentran: la interacción con los espermatozoides, mantener la viabilidad de éstos, participar en la maduración de los ovocitos y en el proceso de fecundación. Durante la fecundación in vitro y con el fin de mejorarla imitando las condiciones in vivo, numerosos investigadores han utilizado cultivos de células del epitelio oviductal bovino (CEOB) con notables mejoras. Estas células producen, entre otros componentes VEs, por ello, en este trabajo hemos planteado un estudio com-parativo de VEs presentes en el fluido oviductal (FO) bovino recogido en momentos próximos a la ovulación (in vivo) y de aquellas VEs producidas en cultivos de CEOB a los 7 días de cultivo (in vitro) comparando el tamaño, la distribución de la población y la concentración de proteína en ambos tipos. Las VEs se identificaron mediante microscopía electrónica, su tamaño mediante dispersión de luz láser y la concentración de proteínas mediante el método Bradford. Los resultados mostraron que el tamaño de las VEs fue similar entre ambos grupos experimentales. Por otro lado, sí que se observaron diferencias en cuanto a la concentración de proteínas. Las VEs obtenidas in vivocontenían mayor cantidad de proteína en su cargo que en las VEs obtenidas in vitro.En cuanto a identificación de las VEs mediante microscopía electrónica de transmisión, solo pudieron ser observadas aquellas obtenidas in vivo. Este hecho podría deberse al lugar de dónde han sido recogidas, al mét-odo de cultivo de células epiteliales oviductales bovinas o la escasez en su producción.
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    Comparison of chromaffin cells from several animal sources for their use as an in vitro model to study the mechanism of organophosphorous toxicity
    (Elsevier, 2006-04-28) Romero, D.; Quesada, E.; Sogorb, M. A.; Vilanova, E.; Carrera, V.; García Fernández, Antonio Juan; Ciencias Sociosanitarias
    It had been observed that the chromaffin cells of bovine adrenal medulla contain high levels of neuropathy target esterase (NTE), the esterase whose inhibition and aging is associated with induction of the organophosphorous induced delayed neuropathy. In this study, total esterase and NTE activities, and their inhibition kinetics by OPs are characterized in adrenal medulla of several species in order to find the best source for chromaffin cells. Total esterase activity in membrane fraction of bovine, equine, porcine, ovine and caprine were 6100±840, 4200±270, 5000±120, 28800±3000, and 10800±2400 mU/g tissue, respectively (mean±S.D., n = 3–4). NTE represented around 70%, 24%, 58%, 10% and 24% of the total esterases in the same tissues, respectively. It was deduced that NTE represents between 69% and 89% of the “B-activity” (activity resistant to 40 M paraoxon) in the membrane fraction of all species. The mipafox I50 calculated for 30-min inhibition of NTE at 37 ◦C ranged between 7.4 and 12 M. These values are in the range of that for brain NTE in hen (the usual model for testing OP delayed neurotoxicity). Considering that bovine adrenal medulla contains high NTE activity, that it represents a high proportion of total activity, it is easier to dissect than adrenal medulla from equine, caprine or ovine, and is more readily available than species cited previously, and that its inhibitory properties are similar to the classical hen brain model, it is deduced that bovine adrenal medulla is the most appropriate source of chromaffin cells to study OP toxicity, with porcine as the second alternative. The kinetic properties of chromaffin cell cultures from bovine and porcine were in accordance with their properties in homogenate and subcellular fractions, and they displayed an appropriate stability and viability of the primary culture to be used in in vitro toxicological studies for both mechanistic and testing purposes.
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    Efecto de las secreciones de organoides de endometrio humano sobre el cultivo in vitro de embriones bovinos
    (Facultad de Veterinaria y el Servicio de Publicaciones de la Universidad de Murcia, 2023) Masegosa Domínguez, Alicia María; Navarro-Serna, Sergio; Pérez-García, Vicente; Coy, Pilar; Juan, Ángela; Romero Aguirregomezcorta, Jon
    Durante los últimos 50 años, la fertilidad de las vacas lecheras de alta producción ha disminuido debido a la intensidad de la selección genética. Para superar este obstáculo, se ha recurrido a las técnicas de reproducción asistida. Sin embargo, la producción de embriones in vitro es un proceso relativamente ineficiente, con tasas máximas de blastocistos obtenidos de alrededor del 35%. Para solventar este problema, se planteó la hipótesis de añadir al medio de cultivo secreciones de organoides de endometrio, así, los embriones producidos in vitroestarían expuestos a un ambiente más fisiológico y el rendimiento de la técnica aumentaría. Para ello, se llevó a cabo la recolección de ovocitos bovinos procedentes de un matadero local, y se procedió a su maduración in vitro, fecundación in vitro y cultivo embrionario in vitro. El medio de cultivo embrionario se suplementó al 1% (v/v) con i) secreciones de organoides de endometrio humano (ya que no existe disponibilidad de secreciones bovinas), ii) medio de cultivo Boretto, y ii) gotas de matrigel con medio de cultivo Boretto, como controles de procedimiento. Además, se incluyó un grupo control sin ningún suplemento. Se evaluó el desarrollo embriona-rio a los días 2, 7 y 8 de cultivo. El porcentaje de blastocistos a día 7 y a día 8 post inseminación fue significa-tivamente mayor (P<0.05) cuando se añadió el medio de cultivo Boretto. Además, este tratamiento incrementó la cinética de desarrollo de los embriones a día 7 post inseminación, y la calidad de los blastocistos evaluada por el número total de células, con un 85.8% más que el control. Aunque las secreciones de endometrio humano no han dado el resultado esperado, sería necesario comprobar si las secreciones de organoides específicas de endometrio bovino, a distintas concentraciones, podrían incrementar el rendimiento de la producción in vitro de embriones bovinos.
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    Embryonic development of the bovine pineal gland (Bos taurus) during prenatal life (30 to 135 days of gestation)
    (Murcia : F. Hernández, 2005) Regodón, S.; Roncero, V.
    The ontogenesis of the pineal gland of 30 bovine embryos (Bos taurus) has been analysed from 30 until 135 days of gestation by means of optical microscopy and immunohistochemical techniques. For this study, the specimens were grouped into three stages in accordance with the most relevant histological characteristics: Stage 1 (30 to 64 days of prenatal development); Stage 2 (70 to 90 days) and Stage 3 (106 to 135 days). In the cow, it is from 30 days of gestation that the first glandular outline becomes differentiated from the diencephalic ependyma of the third ventricle. This differentiation includes the phenomena of proliferation and multiplication of the ependymal cells that form the epithelium of the pineal outline in development. At 82 days of intrauterine life, in the interior of the pineal parenchyma, we witnessed some incipient pseudoglandular structures that at 135 days were well differentiated. The pineal parenchyma displays a cytology made up of two cellular types of structurally distinct characteristics: pinealoblasts and interstitial cells. Both cellular types begin differentiation at 70 days of embryonic development, the pinealoblasts being greater in number than the interstitial cells. The glandular stroma is formed from the capsular, trabecular and the perivascular connective tissue, filling the interparenchymal space. A dense network of capillaries, which drive across the trabecular connective tissue towards the central glandular zone where their density increases and their calibre is reduced, complete the glandular structure. GFAP positive cells were observed in the embryonic pineal parenchyma in stage 3. At 135 days of gestation, NPY positive fibers entered the pineal gland through the pineal capsule occupying a perivascular localization. Morphological studies of this nature are vital for future use as parameters, indicative of the functional activity of the bovine pineal gland during embryonic development.
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    Respuesta a la estimulación ovárica mediante FSH (folltropin®) y rendimiento de opu en vacas adultas obtenidas por diferentes técnicas de reproducción asistida
    (Universidad de Murcia. Servicio de publicaciones, 2022-11-15) Morera Jiménez, Alba; Velasco García, Elena; Ruiz López, Salvador; Romero Aguirregomezcorta, Jon; Anatomía y Anatomía Patológica Comparadas
    Los objetivos de este trabajo han sido realizar un seguimiento ecográfico de los ovarios bovinos para comprobar la respuesta folicular de vacas adultas nacidas por diferentes técnicas de reproducción asistida (TRA), tras aplicar un protocolo de estimulación ovárica con FSH (Folltropin®); y determinar la eficacia reproductiva de los animales estudiados, analizando la cantidad y calidad de los ovocitos obtenidos por aspiración folicular (Ovum Pick-Up, OPU) antes y después del protocolo de estimulación empleado. La experiencia se ha llevado a cabo en las instalaciones del santuario bovino de la Granja Docente Veterinaria de la Universidad de Murcia. Se han utilizado 4 hembras bovinas obtenidas por distintas TRA: inseminación artificial (IA), transferencia de embriones (TE) producidos por TRA con ovocitos cultivados in vitro en medios convencionales y por TE obtenidos por TRA con ovocitos cultivados con fluidos naturales (folicular y oviductal).Los animales fueron estimulados durante 5 semanas mediante un protocolo que consistía en la aplicación de GnRH y posteriormente, FSH (Folltropin®, 350 UI/animal) durante 3 días en dosis decrecientes. Una vez a la semana, al finalizar el protocolo de estimulación ovárica, se realizaba una ecografía de los ovarios y una aspiración folicular ecoguiada (OPU) para comprobar la respuesta al protocolo de estimulación y determinar el número de folículos y de ovocitos obtenidos tras la OPU y su calidad ovocitaria. Previamente al periodo de estimulación con FSH, las vacas fueron ecografiadas y sometidas a OPU 3 semanas sin estimulación ovárica como grupo control.A lo largo del trabajo realizado, se ha comprobado la respuesta al protocolo de estimulación ovárica ensa-yado, y se ha determinado la eficacia reproductiva de los animales, según el número de folículos aspirados y la cantidad y calidad de los ovocitos obtenidos por OPU en cada una de ellas.
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    Single-cell DNA methylation sequencing reveals epigenetic alterations induced by bovine oocyte in vitro maturation
    (Brazilian College of Animal Reproduction, 2024-06-28) Abril-Parreño, Laura; Lopes, Jordana S.; Galvao, Antonio; Kelsey, Gavin; Coy, Pilar; Romero Aguirregomezcorta, Jon; Fisiología; Facultad de Veterinaria
    Oocyte in vitro maturation (IVM) is a key step for the in vitro production of bovine embryos. However, there is a concern that this process may induce suboptimal developmental competence of bovine oocytes including epigenetic alterations. Currently, epigenetic profile of bovine oocyte IVM is relatively limited and inconsistent, probably arising from differences in the detection approach and experimental design used between studies. The aim of this work was to determine whether DNA methylome alterations are present in bovine oocytes that were in vitro matured and to identify conserved biomarkers across species. To achieve this, the results of this study were compared to those obtained in a similar study with in vitro matured porcine oocytes (unpublished data). The study was performed on 18 in vitro matured oocytes recovered from 2-8 mm follicles of abattoir-derived bovine ovaries and 28 in vivo matured oocytes collected by ovum-pick-up. Transvaginal aspiration was performed at 96–98 h after GnRH administration. The analysis of DNA methylation was performed by single-cell whole-genome bisulphite sequencing. Then, differentially methylated regions (DMRs, FDR < 0.05, FC > 0.1) were determined using the R package limma. Results showed that global DNA methylation profiles differed between in vitro and in vivo groups. Individual oocytes were clustered using Uniform Manifold Approximation and Projection analysis, which showed a clear separation within the in vivo group according to breed and age. The analysis of DMR identified a lower number of hypermethylated and hypomethylated regions in the IVM group, which were more frequent in variably methylated regions (VMRs), promoters, transcripts and imprinted genes. No differences were found in methylation of CpG islands of genes previously related to large offspring syndrome between groups. Regarding the effect on genomic imprinting, methylation was lower for IVM oocytes in the imprinted gene CDKN1C and higher in the BEGAIN gene when compared to the in vivo group. In addition, the analysis of genes that have been previously predicted for their possible function in the imprinting process showed a number of differences between the in vivo and in vitro group. For example, we identified lower methylation in in vitro matured oocytes in CpG islands of 5 “candidate” genes (SEMA7A, ZNF575, ATP4B, PDGFA, COMP) while only one was hypermethylated in the coding region of the PLCL2 gene. Finally, we identified conserved differences in methylation related to IVM between bovine and porcine oocytes for 14, 8 and 3 genomic features in the transcripts, VMRs and promoters, respectively. The findings indicate that some of the epigenetic alterations are associated with suboptimal developmental competence of IVM oocytes. In conclusion, these results could help to improve this technique when employing in vitro production procedures in cattle.
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    Spontaneous and ART-induced large offspring syndrome: similarities and differences in DNA methylome
    (Taylor and Francis Group, 2022-05-03) Yahan Li; Sena Lopes, Jordana; Coy-Fuster, Pilar; Melissa Rivera, Rocío; Didáctica y Organización Escolar
    Large/abnormal offspring syndrome (LOS/AOS) is a congenital overgrowth syndrome reported in ruminants produced by assisted reproduction (ART-LOS) which exhibit global disruption of the epigenome and transcriptome. LOS/AOS shares phenotypes and epigenotypes with the human congenital overgrowth condition Beckwith-Wiedemann syndrome. We have reported that LOS occurs spontaneously (SLOS); however, to date, no study has been conducted to determine if SLOS has the same methylome epimutations as ART-LOS. In this study, we performed whole- genome bisulphite sequencing to examine global DNA methylation in bovine SLOS and ART-LOS tissues. We observed unique patterns of global distribution of differentially methylated regions (DMRs) over different genomic contexts, such as promoters, CpG Islands, shores and shelves, as well as at repetitive sequences. In addition, we included data from two previous LOS studies to identify shared vulnerable genomic loci in LOS. Overall, we identified 320 genomic loci in LOS that have alterations in DNA methylation when compared to controls. Specifically, there are 25 highly vulnerable loci that could potentially serve as molecular markers for the diagnosis of LOS, including at the promoters of DMRT2 and TBX18, at the imprinted gene bodies of IGF2R, PRDM8, and BLCAP/NNAT, and at multiple CpG Islands. We also observed tissue-specific DNA methylation patterns between muscle and blood, and conservation of ART-induced DNA methyla-tion changes between muscle and blood. We conclude that as ART-LOS, SLOS is an epigenetic condition. In addition, SLOS and ART-LOS share similarities in methylome epimutations.