Universidad de Buenos...
L-carnitine (LC) plays an important role in lipid catabolism and protects cells from damage caused by reactive oxygen species (ROS) due to its antioxidant activity. The \nobjective of this work was to evaluate if the supplementation of LC to the in vitro maturation medium (IVM) decreases the content...
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| Formato: | Tesis doctoral acceptedVersion |
| Lenguaje: | Español |
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Universidad de Buenos Aires. Facultad de Ciencias Veterinarias
2023
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| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_7378 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_7378.dir/7378.PDF |
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I28-R145-HWA_7378 |
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dspace |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-145 |
| collection |
Repositorio Digital de la Universidad de Buenos Aires (UBA) |
| language |
Español |
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spa |
| topic |
ovocitos criotolerancia L-Carlitine oocytes pigs cryotolerance embryonic development Porcinos desarrollo embrionario L-Carlitina Ciencias Veterinarias |
| spellingShingle |
ovocitos criotolerancia L-Carlitine oocytes pigs cryotolerance embryonic development Porcinos desarrollo embrionario L-Carlitina Ciencias Veterinarias Cruzans, Paula Romina Universidad de Buenos... |
| topic_facet |
ovocitos criotolerancia L-Carlitine oocytes pigs cryotolerance embryonic development Porcinos desarrollo embrionario L-Carlitina Ciencias Veterinarias |
| description |
L-carnitine (LC) plays an important role in lipid catabolism and protects cells from damage caused by reactive oxygen species (ROS) due to its antioxidant activity. The \nobjective of this work was to evaluate if the supplementation of LC to the in vitro maturation medium (IVM) decreases the content of intracellular lipids, decreases the \nintracellular level of ROS, favours mitochondrial function and increases the concentration \nof total glutathione (GSH) improving oocyte maturation, embryo quality and cryo-tolerance to vitrification of in vitro matured porcine oocytes. The cumulus-oocyte \ncomplexes were obtained by follicular aspiration of ovaries from sacrificed sows and matured in vitro for 44 h without LC (control) or with different concentrations of LC (0.6,\n1.25 or 2.5 mg/mL) (Sigma-Aldrich) in supplemented TCM-199. In vitro fertilization was performed with fresh boar semen for four h in 100 µL-drop of MCT 199 with caffeine, BSA, sodium lactate and pyruvate (20 denuded oocytes per drop, 1x106 spermatozoa/mL). Presumptive zygotes were washed and cultured at NCSU 23 at 39°C, \n7 % O2, 5 % CO2, and humidity. The percentage of cleavage was recorded on day two, and the percentage of blastocyst on day seven. In order to establish the best \nconcentration of LC to use to vitrify mature porcine oocytes, different determinations \nwere made: percentage of nuclear maturation, intracellular lipid content, intracellular level of ROS, mitochondrial function, apoptosis in cumulus cells (CC), total GSH concentration, post-IVF parameters and embryonic development. The concentration of 0.6 mg/mL of LC significantly decreased the intracellular lipid content, the intracellular \nlevel of ROS, and the mitochondrial function without affecting the percentage of nuclear \nmaturation or the total GSH concentration of the oocytes compared to the control. LC \nduring IVM affected the CC viability of mature porcine oocytes. The concentration of 2.5 mg/mL of LC significantly decreased the nuclear maturation of the oocytes to the control. \nNo significant differences between LC treatments and control were observed in post-IVF parameters (percentage of penetrated and monospermic oocytes, pronucleus formation, \nand efficiency). However, adding 0.6 mg/mL of LC during IVM decreased cleavage rates \nwithout modifying blastocyst rates, cell number, or blastocyst apoptosis compared with \nthe control. Hatched blastocysts were observed upon maturing LC oocytes but were not in control. Based on these previous results, the 0.6 mg/mL concentration of LC was selected to be used during IVM prior to the vitrification of mature oocytes with a new device (alternative vitrification support). LC during IVM decreased the number of dead oocytes after vitrification and tempering compared with the control. However, there were no significant differences in the percentages of early and late apoptosis, the intracellular ROS level and the oocytes' mitochondrial function in vitrified and tempered ripe, either using LC or not during IVM. Vitrification negatively affected porcine oocytes matured or not with LC with a low percentage of morphologically normal MII at 2 and 4 h post-tempering, without normal IVF (assessed by post-IVF parameters), with a low percentage of segmentation in control and null in the treatment with LC and without blastocysts in either of the two treatments. This is the first work in porcine species where the effect of LC is evaluated during IVM prior to the vitrification of mature oocytes. \nTreatment with 0.6 mg/mL of LC improved vitrified oocytes viability. However, neither the antioxidant function of LC nor the decrease in intracellular lipid content of oocytes was sufficient to reverse the adverse effects of cryopreservation of mature porcine oocytes. \nWork should continue on improvements during the vitrification and tempering of mature oocytes for this technique to be viable in porcine species. |
| author2 |
Lombardo, Daniel Marcelo |
| author_facet |
Lombardo, Daniel Marcelo Cruzans, Paula Romina |
| format |
Tesis doctoral Tesis doctoral acceptedVersion |
| author |
Cruzans, Paula Romina |
| author_sort |
Cruzans, Paula Romina |
| title |
Universidad de Buenos... |
| title_short |
Universidad de Buenos... |
| title_full |
Universidad de Buenos... |
| title_fullStr |
Universidad de Buenos... |
| title_full_unstemmed |
Universidad de Buenos... |
| title_sort |
universidad de buenos... |
| publisher |
Universidad de Buenos Aires. Facultad de Ciencias Veterinarias |
| publishDate |
2023 |
| url |
http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_7378 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_7378.dir/7378.PDF |
| work_keys_str_mv |
AT cruzanspaularomina universidaddebuenos AT cruzanspaularomina suplementaciondelcarnitinaenlamaduracioninvitrodeovocitosporcinosefectosobrelacriotoleranciaalavitrificacionylacompetenciaparaeldesarrolloembrionarioinvitro |
| _version_ |
1824356463826436096 |
| spelling |
I28-R145-HWA_73782024-08-27 Universidad de Buenos... L-carnitine (LC) plays an important role in lipid catabolism and protects cells from damage caused by reactive oxygen species (ROS) due to its antioxidant activity. The \nobjective of this work was to evaluate if the supplementation of LC to the in vitro maturation medium (IVM) decreases the content of intracellular lipids, decreases the \nintracellular level of ROS, favours mitochondrial function and increases the concentration \nof total glutathione (GSH) improving oocyte maturation, embryo quality and cryo-tolerance to vitrification of in vitro matured porcine oocytes. The cumulus-oocyte \ncomplexes were obtained by follicular aspiration of ovaries from sacrificed sows and matured in vitro for 44 h without LC (control) or with different concentrations of LC (0.6,\n1.25 or 2.5 mg/mL) (Sigma-Aldrich) in supplemented TCM-199. In vitro fertilization was performed with fresh boar semen for four h in 100 µL-drop of MCT 199 with caffeine, BSA, sodium lactate and pyruvate (20 denuded oocytes per drop, 1x106 spermatozoa/mL). Presumptive zygotes were washed and cultured at NCSU 23 at 39°C, \n7 % O2, 5 % CO2, and humidity. The percentage of cleavage was recorded on day two, and the percentage of blastocyst on day seven. In order to establish the best \nconcentration of LC to use to vitrify mature porcine oocytes, different determinations \nwere made: percentage of nuclear maturation, intracellular lipid content, intracellular level of ROS, mitochondrial function, apoptosis in cumulus cells (CC), total GSH concentration, post-IVF parameters and embryonic development. The concentration of 0.6 mg/mL of LC significantly decreased the intracellular lipid content, the intracellular \nlevel of ROS, and the mitochondrial function without affecting the percentage of nuclear \nmaturation or the total GSH concentration of the oocytes compared to the control. LC \nduring IVM affected the CC viability of mature porcine oocytes. The concentration of 2.5 mg/mL of LC significantly decreased the nuclear maturation of the oocytes to the control. \nNo significant differences between LC treatments and control were observed in post-IVF parameters (percentage of penetrated and monospermic oocytes, pronucleus formation, \nand efficiency). However, adding 0.6 mg/mL of LC during IVM decreased cleavage rates \nwithout modifying blastocyst rates, cell number, or blastocyst apoptosis compared with \nthe control. Hatched blastocysts were observed upon maturing LC oocytes but were not in control. Based on these previous results, the 0.6 mg/mL concentration of LC was selected to be used during IVM prior to the vitrification of mature oocytes with a new device (alternative vitrification support). LC during IVM decreased the number of dead oocytes after vitrification and tempering compared with the control. However, there were no significant differences in the percentages of early and late apoptosis, the intracellular ROS level and the oocytes' mitochondrial function in vitrified and tempered ripe, either using LC or not during IVM. Vitrification negatively affected porcine oocytes matured or not with LC with a low percentage of morphologically normal MII at 2 and 4 h post-tempering, without normal IVF (assessed by post-IVF parameters), with a low percentage of segmentation in control and null in the treatment with LC and without blastocysts in either of the two treatments. This is the first work in porcine species where the effect of LC is evaluated during IVM prior to the vitrification of mature oocytes. \nTreatment with 0.6 mg/mL of LC improved vitrified oocytes viability. However, neither the antioxidant function of LC nor the decrease in intracellular lipid content of oocytes was sufficient to reverse the adverse effects of cryopreservation of mature porcine oocytes. \nWork should continue on improvements during the vitrification and tempering of mature oocytes for this technique to be viable in porcine species. Fil: Cruzans, Paula Romina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Buenos Aires, Argentina Lombardo, Daniel Marcelo Cruzans, Paula Romina 2023-12-06 La L-carnitina (LC) juega un papel importante en el catabolismo de los lípidos y protege a las células del daño causado por las especies reactivas del oxígeno (ERO) debido a su actividad antioxidante. El objetivo de este trabajo fue evaluar si la suplementación de LC al medio de maduración in vitro (MIV) disminuye el contenido de lípidos intracelulares, disminuye el nivel intracelular de ERO, favorece la función mitocondrial y \naumenta la concentración de glutatión (GSH) total mejorando la maduración ovocitaria, la calidad embrionaria y la criotolerancia a la vitrificación de los ovocitos porcinos madurados in vitro. Los complejos cúmulo-ovocito fueron obtenidos por aspiración folicular de ovarios de cerdas sacrificadas y madurados in vitro por 44 h sin LC (control) \no con diferentes concentraciones de LC (0,6; 1,25 o 2,5 mg/mL) (Sigma-Aldrich) en TCM-199 suplementado. La fecundación in vitro se realizó con semen fresco de verraco durante 4 h en 100 µL-gota de TCM 199 con cafeína, BSA, lactato de sodio y piruvato (20 ovocitos denudados por gota, 1x106 espermatozoides/mL). Los presuntos cigotos \nse lavaron y cultivaron en NCSU 23 a 39°C, 7 % de O2, 5 % de CO2 y humedad. El porcentaje de segmentación se registró el día 2 y el porcentaje de blastocisto el día 7. \nA fin de establecer la mejor concentración de LC a utilizar para vitrificar los ovocitos porcinos maduros, se realizaron diferentes determinaciones: porcentaje de maduración \nnuclear, contenido de lípidos intracelulares, nivel intracelular de ERO, función \nmitocondrial, apoptosis en células del cumulus (CC), concentración de GSH total, \nparámetros post-FIV y desarrollo embrionario. La concentración de 0,6 mg/mL de LC disminuyó significativamente el contenido de lípidos intracelulares, el nivel intracelular de ERO y la función mitocondrial sin afectar el porcentaje de maduración nuclear ni la concentración de GSH total de los ovocitos comparado con el control. El uso de LC \ndurante la MIV afectó la viabilidad de las CC de los ovocitos porcinos maduros. La concentración de 2,5 mg/mL de LC disminuyó significativamente la maduración nuclear \nde los ovocitos respecto del control. No se observaron diferencias significativas en los parámetros post-FIV (porcentaje de ovocitos penetrados y monospérmicos, formación \nde pronúcleo y eficiencia) entre los tratamientos de LC y el control. Pero el agregado de \n0,6 mg/mL de LC durante la MIV disminuyó los porcentajes desegmentación sin modificar los porcentajes de blastocistos ni el número de células ni la apoptosis de los \nblastocistos comparado con el control. Se observaron blastocistos eclosionados al \nmadurar los ovocitos con LC, pero no así en el control. De acuerdo con estos resultados \nprevios, se seleccionó la concentración de 0,6 mg/mL de LC para ser utilizada durante la MIV previo a la vitrificación de los ovocitos maduros con un nuevo dispositivo (soporte \nalternativo de vitrificación). La utilización de LC durante la MIV disminuyo el número de ovocitos muertos luego de la vitrificación y atemperado comparado con el control, pero no hubo diferencias significativas en los porcentajes de apoptosis temprana y tardía, el \nnivel intracelular de ERO y la función mitocondrial de los ovocitos maduros vitrificados y \natemperados ya sea utilizándose o no LC durante la MIV. La vitrificación afectó \nnegativamente a los ovocitos porcinos madurados o no con LC con bajo porcentaje de \nMII morfológicamente normales a las 2 y 4 h post-atemperado, sin una FIV normal (evaluada por los parámetros post-FIV), con muy bajo porcentaje de segmentación en \nel control y nulo en el tratamiento con LC y sin blastocistos en ninguno de los dos tratamientos. Este es el primer trabajo en la especie porcina en donde se evalúa el efecto de la LC durante la MIV previa vitrificación de los ovocitos maduros. El tratamiento con 0,6 mg/mL de LC mejoró la viabilidad de los ovocitos vitrificados, pero, tanto la función antioxidante de la LC como la disminución del contenido de lípidos intracelulares de los ovocitos, no fue suficiente para revertir los efectos adversos de la criopreservación de \nlos ovocitos porcinos maduros. Se deberá seguir trabajando en mejoras durante la vitrificación y atemperado de los ovocitos maduros para que esta técnica sea viable en la especie porcina application/pdf ovocitos criotolerancia L-Carlitine oocytes pigs cryotolerance embryonic development spa Universidad de Buenos Aires. Facultad de Ciencias Veterinarias info:eu-repo/semantics/openAccess htpps://creativecommons.org/licenses/by-nend/2.5/ar/ Porcinos desarrollo embrionario L-Carlitina Ciencias Veterinarias Doctora de Universidad de Buenos Aires en Ciencias Veterinarias Suplementación de L-Carnitina en la maduración in vitro de ovocitos porcinos : efecto sobre la criotolerancia a la vitrificación y la competencia para el desarrollo embrionario in vitro info:eu-repo/semantics/doctoralThesis info:ar-repo/semantics/tesis doctoral info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_7378 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_7378.dir/7378.PDF |