Use of platelet-rich plasma on in vitro maturation during bovine embryo production

One of the crucial aspects to be considered for successful in vitro production (IVP) of embryos is the composition of the various media used throughout the stages of this reproductive biotechnology. The cell culture media employed should fulfill the metabolic requirements of both gametes during oocy...

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Autores:
Baia de Souza, Eduardo
Baia de Souza, Eduardo
Dubeibe, Diego
Dubeibe, Diego
Ramos, Anelise
Ramos, Anelise
Homobono, Bruno Porpino
Porpino Homobono, Bruno
Azevedo Ramos, Priscilla do Carmo de
de Azevedo Ramos, Priscilla do Carmo
Brito, Vanessa
Brito, Vanessa
Santos da Cruz, Gabriela
Nogueira da Costa de Almeida, Nathalia
Costa, Nathalia
Silva Cordeiro, Marcela da
Damasceno Santos, Simone do Socorro
Tipo de recurso:
Article of investigation
Fecha de publicación:
2024
Institución:
Universidad de Ciencias Aplicadas y Ambientales U.D.C.A
Repositorio:
Repositorio Institucional UDCA
Idioma:
eng
OAI Identifier:
oai:repository.udca.edu.co:11158/5777
Acceso en línea:
https://repository.udca.edu.co/handle/11158/5777
https://doi.org/10.1590/1984-3143-ar2023-0107
Palabra clave:
590 - Animales::591 - Temas específicos en historia natural de los animales
Plasma Rico en Plaquetas
Embriones animales
Bovinos
In vitro
Rights
openAccess
License
https://creativecommons.org/licenses/by-nc-sa/4.0/legalcode.es
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dc.title.none.fl_str_mv Use of platelet-rich plasma on in vitro maturation during bovine embryo production
title Use of platelet-rich plasma on in vitro maturation during bovine embryo production
spellingShingle Use of platelet-rich plasma on in vitro maturation during bovine embryo production
590 - Animales::591 - Temas específicos en historia natural de los animales
Plasma Rico en Plaquetas
Embriones animales
Bovinos
In vitro
title_short Use of platelet-rich plasma on in vitro maturation during bovine embryo production
title_full Use of platelet-rich plasma on in vitro maturation during bovine embryo production
title_fullStr Use of platelet-rich plasma on in vitro maturation during bovine embryo production
title_full_unstemmed Use of platelet-rich plasma on in vitro maturation during bovine embryo production
title_sort Use of platelet-rich plasma on in vitro maturation during bovine embryo production
dc.creator.fl_str_mv Baia de Souza, Eduardo
Baia de Souza, Eduardo
Dubeibe, Diego
Dubeibe, Diego
Ramos, Anelise
Ramos, Anelise
Homobono, Bruno Porpino
Porpino Homobono, Bruno
Azevedo Ramos, Priscilla do Carmo de
de Azevedo Ramos, Priscilla do Carmo
Brito, Vanessa
Brito, Vanessa
Santos da Cruz, Gabriela
Nogueira da Costa de Almeida, Nathalia
Costa, Nathalia
Silva Cordeiro, Marcela da
Damasceno Santos, Simone do Socorro
dc.contributor.author.none.fl_str_mv Baia de Souza, Eduardo
Baia de Souza, Eduardo
Dubeibe, Diego
Dubeibe, Diego
Ramos, Anelise
Ramos, Anelise
Homobono, Bruno Porpino
Porpino Homobono, Bruno
Azevedo Ramos, Priscilla do Carmo de
de Azevedo Ramos, Priscilla do Carmo
Brito, Vanessa
Brito, Vanessa
Santos da Cruz, Gabriela
Nogueira da Costa de Almeida, Nathalia
Costa, Nathalia
Silva Cordeiro, Marcela da
Damasceno Santos, Simone do Socorro
dc.subject.ddc.none.fl_str_mv 590 - Animales::591 - Temas específicos en historia natural de los animales
topic 590 - Animales::591 - Temas específicos en historia natural de los animales
Plasma Rico en Plaquetas
Embriones animales
Bovinos
In vitro
dc.subject.decs.none.fl_str_mv Plasma Rico en Plaquetas
dc.subject.agrovoc.none.fl_str_mv Embriones animales
Bovinos
In vitro
description One of the crucial aspects to be considered for successful in vitro production (IVP) of embryos is the composition of the various media used throughout the stages of this reproductive biotechnology. The cell culture media employed should fulfill the metabolic requirements of both gametes during oocyte maturation and sperm development, as well as the embryo during its initial cell divisions. Most IVP protocols incorporate blood serum into the media composition as a source of hormones, proteins, growth factors, and nutrients. Numerous studies have suggested Platelet-Rich Plasma (PRP) as a substitute for fetal sera in cell culture, particularly for stem cells. Therefore, the objective of this study is to assess the potential use of PRP as a replacement for fetal bovine serum (FBS) during oocyte maturation for in vitro production of bovine embryos. During in vitro maturation (IVM), cumulus-oocyte complexes (COCs) were allocated into the following experimental groups: Group G1 (IVM medium with 5% PRP); Group G2 (MIV medium with 5% PRP and 5% SFB); Group G3 (MIV medium with 5% SFB); and Group G4 (MIV medium without either PRP or SFB). Subsequently, the cumulus-oocyte complexes were fertilized with semen from a single bull, and the resulting zygotes were cultured for seven days. Cleavage and blastocyst formation rates were assessed on days 2 and 7 of embryonic development, respectively. The quality of matured COCs was also evaluated by analyzing the gene expression of HSP70, an important protein associated with cellular stress. The results demonstrated that there were no significant differences among the experimental groups in terms of embryo production rates, both in the initial cleavage stages and blastocyst formation (except for the G4 group, which exhibited a lower blastocyst formation rate on D7, as expected). This indicates that PRP could be a cost-effective alternative to SFB in the IVP of embryos.
publishDate 2024
dc.date.accessioned.none.fl_str_mv 2024-05-29T13:50:44Z
dc.date.available.none.fl_str_mv 2024
2024-05-29T13:50:44Z
dc.date.issued.none.fl_str_mv 2024
dc.type.none.fl_str_mv Artículo de revista
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dc.identifier.citation.none.fl_str_mv De Souza, E. B., Marin, D. D., Ramos, A. S., Homobono, B. P., De Azevedo Ramos, P. D. C., De Brito, V. C., Da Cruz, G. S., Da Costa, N. N., Da Silva Cordeiro, M., & Santos, S. D. S. D. (2024). Use of platelet-rich plasma on in vitro maturation during bovine embryo production. Animal Reproduction, 21(1). https://doi.org/10.1590/1984-3143-ar2023-0107
dc.identifier.issn.none.fl_str_mv 18069614
dc.identifier.uri.none.fl_str_mv https://repository.udca.edu.co/handle/11158/5777
dc.identifier.doi.none.fl_str_mv https://doi.org/10.1590/1984-3143-ar2023-0107
identifier_str_mv De Souza, E. B., Marin, D. D., Ramos, A. S., Homobono, B. P., De Azevedo Ramos, P. D. C., De Brito, V. C., Da Cruz, G. S., Da Costa, N. N., Da Silva Cordeiro, M., & Santos, S. D. S. D. (2024). Use of platelet-rich plasma on in vitro maturation during bovine embryo production. Animal Reproduction, 21(1). https://doi.org/10.1590/1984-3143-ar2023-0107
18069614
url https://repository.udca.edu.co/handle/11158/5777
https://doi.org/10.1590/1984-3143-ar2023-0107
dc.language.iso.none.fl_str_mv eng
language eng
dc.relation.indexed.none.fl_str_mv LaReferencia
dc.relation.citationedition.none.fl_str_mv (2024) Artículo número e20230107
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dc.relation.citationissue.none.fl_str_mv 1
dc.relation.citationstartpage.none.fl_str_mv 1
dc.relation.citationvolume.none.fl_str_mv 21
dc.relation.ispartofjournal.none.fl_str_mv Animal Reproduction
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spelling Baia de Souza, EduardoBaia de Souza, Eduardovirtual::43-1Dubeibe, DiegoDubeibe, Diegovirtual::44-1Ramos, AneliseRamos, Anelisevirtual::45-1Homobono, Bruno PorpinoPorpino Homobono, Brunovirtual::46-1Azevedo Ramos, Priscilla do Carmo dede Azevedo Ramos, Priscilla do Carmovirtual::47-1Brito, VanessaBrito, Vanessavirtual::48-1Santos da Cruz, GabrielaNogueira da Costa de Almeida, NathaliaCosta, Nathaliavirtual::49-1Silva Cordeiro, Marcela daDamasceno Santos, Simone do Socorro2024-05-29T13:50:44Z20242024-05-29T13:50:44Z2024De Souza, E. B., Marin, D. D., Ramos, A. S., Homobono, B. P., De Azevedo Ramos, P. D. C., De Brito, V. C., Da Cruz, G. S., Da Costa, N. N., Da Silva Cordeiro, M., & Santos, S. D. S. D. (2024). Use of platelet-rich plasma on in vitro maturation during bovine embryo production. Animal Reproduction, 21(1). https://doi.org/10.1590/1984-3143-ar2023-010718069614https://repository.udca.edu.co/handle/11158/5777https://doi.org/10.1590/1984-3143-ar2023-0107One of the crucial aspects to be considered for successful in vitro production (IVP) of embryos is the composition of the various media used throughout the stages of this reproductive biotechnology. The cell culture media employed should fulfill the metabolic requirements of both gametes during oocyte maturation and sperm development, as well as the embryo during its initial cell divisions. Most IVP protocols incorporate blood serum into the media composition as a source of hormones, proteins, growth factors, and nutrients. Numerous studies have suggested Platelet-Rich Plasma (PRP) as a substitute for fetal sera in cell culture, particularly for stem cells. Therefore, the objective of this study is to assess the potential use of PRP as a replacement for fetal bovine serum (FBS) during oocyte maturation for in vitro production of bovine embryos. During in vitro maturation (IVM), cumulus-oocyte complexes (COCs) were allocated into the following experimental groups: Group G1 (IVM medium with 5% PRP); Group G2 (MIV medium with 5% PRP and 5% SFB); Group G3 (MIV medium with 5% SFB); and Group G4 (MIV medium without either PRP or SFB). Subsequently, the cumulus-oocyte complexes were fertilized with semen from a single bull, and the resulting zygotes were cultured for seven days. Cleavage and blastocyst formation rates were assessed on days 2 and 7 of embryonic development, respectively. The quality of matured COCs was also evaluated by analyzing the gene expression of HSP70, an important protein associated with cellular stress. The results demonstrated that there were no significant differences among the experimental groups in terms of embryo production rates, both in the initial cleavage stages and blastocyst formation (except for the G4 group, which exhibited a lower blastocyst formation rate on D7, as expected). This indicates that PRP could be a cost-effective alternative to SFB in the IVP of embryos.Incluye referencias bibliográficasapplication/pdfenghttps://creativecommons.org/licenses/by-nc-sa/4.0/legalcode.eshttps://creativecommons.org/licenses/by-nc-sa/4.0/info:eu-repo/semantics/openAccessAtribución-NoComercial-CompartirIgual 4.0 Internacional (CC BY-NC-SA 4.0)http://purl.org/coar/access_right/c_abf2https://www.scielo.br/j/ar/a/Fv5ppH9nyzGFVzj9mCjP8FC/?lang=en590 - Animales::591 - Temas específicos en historia natural de los animalesPlasma Rico en PlaquetasEmbriones animalesBovinosIn vitroUse of platelet-rich plasma on in vitro maturation during bovine embryo productionArtículo de revistahttp://purl.org/coar/resource_type/c_2df8fbb1info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionTexthttp://purl.org/redcol/resource_type/ARThttp://purl.org/coar/version/c_970fb48d4fbd8a85LaReferencia(2024) Artículo número e2023010781121Animal 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jecuta públicamente en forma digital la Obra o cualquier Obra Derivada u Obra Colectiva, Usted debe mantener intacta toda la información de derecho de autor de la Obra y proporcionar, de forma razonable según el medio o manera que Usted esté utilizando: (i) el nombre del Autor Original si está provisto (o seudónimo, si fuere aplicable), y/o (ii) el nombre de la parte o las partes que el Autor Original y/o el Licenciante hubieren designado para la atribución (v.g., un instituto patrocinador, editorial, publicación) en la información de los derechos de autor del Licenciante, términos de servicios o de otras formas razonables; el título de la Obra si está provisto; en la medida de lo razonablemente factible y, si está provisto, el Identificador Uniforme de Recursos (Uniform Resource Identifier) que el Licenciante especifica para ser asociado con la Obra, salvo que tal URI no se refiera a la nota sobre los derechos de autor o a la información sobre el licenciamiento de la Obra; y en el caso de una Obra Derivada, atribuir el crédito identificando el uso de la Obra en la Obra Derivada (v.g., "Traducción Francesa de la Obra del Autor Original," o "Guión Cinematográfico basado en la Obra original del Autor Original"). Tal crédito puede ser implementado de cualquier forma razonable; en el caso, sin embargo, de Obras Derivadas u Obras Colectivas, tal crédito aparecerá, como mínimo, donde aparece el crédito de cualquier otro autor comparable y de una manera, al menos, tan destacada como el crédito de otro autor comparable.</li>
      <li>
        Para evitar toda confusión, el Licenciante aclara que, cuando la obra es una composición musical:
        <ol type="i">
          <li>Regalías por interpretación y ejecución bajo licencias generales. El Licenciante se reserva el derecho exclusivo de autorizar la ejecución pública o la ejecución pública digital de la obra y de recolectar, sea individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, SAYCO), las regalías por la ejecución pública o por la ejecución pública digital de la obra (por ejemplo Webcast) licenciada bajo licencias generales, si la interpretación o ejecución de la obra está primordialmente orientada por o dirigida a la obtención de una ventaja comercial o una compensación monetaria privada.</li>
          <li>Regalías por Fonogramas. El Licenciante se reserva el derecho exclusivo de recolectar, individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, los consagrados por la SAYCO), una agencia de derechos musicales o algún agente designado, las regalías por cualquier fonograma que Usted cree a partir de la obra (“versión cover”) y distribuya, en los términos del régimen de derechos de autor, si la creación o distribución de esa versión cover está primordialmente destinada o dirigida a obtener una ventaja comercial o una compensación monetaria privada.</li>
        </ol>
      </li>
      <li>Gestión de Derechos de Autor sobre Interpretaciones y Ejecuciones Digitales (WebCasting). Para evitar toda confusión, el Licenciante aclara que, cuando la obra sea un fonograma, el Licenciante se reserva el derecho exclusivo de autorizar la ejecución pública digital de la obra (por ejemplo, webcast) y de recolectar, individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, ACINPRO), las regalías por la ejecución pública digital de la obra (por ejemplo, webcast), sujeta a las disposiciones aplicables del régimen de Derecho de Autor, si esta ejecución pública digital está primordialmente dirigida a obtener una ventaja comercial o una compensación monetaria privada.</li>
    </ol>
  </li>
  <br/>
  <li>
    Representaciones, Garantías y Limitaciones de Responsabilidad.
    <p>A MENOS QUE LAS PARTES LO ACORDARAN DE OTRA FORMA POR ESCRITO, EL LICENCIANTE OFRECE LA OBRA (EN EL ESTADO EN EL QUE SE ENCUENTRA) “TAL CUAL”, SIN BRINDAR GARANTÍAS DE CLASE ALGUNA RESPECTO DE LA OBRA, YA SEA EXPRESA, IMPLÍCITA, LEGAL O CUALQUIERA OTRA, INCLUYENDO, SIN LIMITARSE A ELLAS, GARANTÍAS DE TITULARIDAD, COMERCIABILIDAD, ADAPTABILIDAD O ADECUACIÓN A PROPÓSITO DETERMINADO, AUSENCIA DE INFRACCIÓN, DE AUSENCIA DE DEFECTOS LATENTES O DE OTRO TIPO, O LA PRESENCIA O AUSENCIA DE ERRORES, SEAN O NO DESCUBRIBLES (PUEDAN O NO SER ESTOS DESCUBIERTOS). ALGUNAS JURISDICCIONES NO PERMITEN LA EXCLUSIÓN DE GARANTÍAS IMPLÍCITAS, EN CUYO CASO ESTA EXCLUSIÓN PUEDE NO APLICARSE A USTED.</p>
  </li>
  <br/>
  <li>
    Limitación de responsabilidad.
    <p>A MENOS QUE LO EXIJA EXPRESAMENTE LA LEY APLICABLE, EL LICENCIANTE NO SERÁ RESPONSABLE ANTE USTED POR DAÑO ALGUNO, SEA POR RESPONSABILIDAD EXTRACONTRACTUAL, PRECONTRACTUAL O CONTRACTUAL, OBJETIVA O SUBJETIVA, SE TRATE DE DAÑOS MORALES O PATRIMONIALES, DIRECTOS O INDIRECTOS, PREVISTOS O IMPREVISTOS PRODUCIDOS POR EL USO DE ESTA LICENCIA O DE LA OBRA, AUN CUANDO EL LICENCIANTE HAYA SIDO ADVERTIDO DE LA POSIBILIDAD DE DICHOS DAÑOS. ALGUNAS LEYES NO PERMITEN LA EXCLUSIÓN DE CIERTA RESPONSABILIDAD, EN CUYO CASO ESTA EXCLUSIÓN PUEDE NO APLICARSE A USTED.</p>
  </li>
  <br/>
  <li>
    Término.
    <ol type="a">
      <li>Esta Licencia y los derechos otorgados en virtud de ella terminarán automáticamente si Usted infringe alguna condición establecida en ella. Sin embargo, los individuos o entidades que han recibido Obras Derivadas o Colectivas de Usted de conformidad con esta Licencia, no verán terminadas sus licencias, siempre que estos individuos o entidades sigan cumpliendo íntegramente las condiciones de estas licencias. Las Secciones 1, 2, 5, 6, 7, y 8 subsistirán a cualquier terminación de esta Licencia.</li>
      <li>Sujeta a las condiciones y términos anteriores, la licencia otorgada aquí es perpetua (durante el período de vigencia de los derechos de autor de la obra). No obstante lo anterior, el Licenciante se reserva el derecho a publicar y/o estrenar la Obra bajo condiciones de licencia diferentes o a dejar de distribuirla en los términos de esta Licencia en cualquier momento; en el entendido, sin embargo, que esa elección no servirá para revocar esta licencia o que deba ser otorgada , bajo los términos de esta licencia), y esta licencia continuará en pleno vigor y efecto a menos que sea terminada como se expresa atrás. La Licencia revocada continuará siendo plenamente vigente y efectiva si no se le da término en las condiciones indicadas anteriormente.</li>
    </ol>
  </li>
  <br/>
  <li>
    Varios.
    <ol type="a">
      <li>Cada vez que Usted distribuya o ponga a disposición pública la Obra o una Obra Colectiva, el Licenciante ofrecerá al destinatario una licencia en los mismos términos y condiciones que la licencia otorgada a Usted bajo esta Licencia.</li>
      <li>Si alguna disposición de esta Licencia resulta invalidada o no exigible, según la legislación vigente, esto no afectará ni la validez ni la aplicabilidad del resto de condiciones de esta Licencia y, sin acción adicional por parte de los sujetos de este acuerdo, aquélla se entenderá reformada lo mínimo necesario para hacer que dicha disposición sea válida y exigible.</li>
      <li>Ningún término o disposición de esta Licencia se estimará renunciada y ninguna violación de ella será consentida a menos que esa renuncia o consentimiento sea otorgado por escrito y firmado por la parte que renuncie o consienta.</li>
      <li>Esta Licencia refleja el acuerdo pleno entre las partes respecto a la Obra aquí licenciada. No hay arreglos, acuerdos o declaraciones respecto a la Obra que no estén especificados en este documento. El Licenciante no se verá limitado por ninguna disposición adicional que pueda surgir en alguna comunicación emanada de Usted. Esta Licencia no puede ser modificada sin el consentimiento mutuo por escrito del Licenciante y Usted.</li>
    </ol>
  </li>
  <br/>
</ol>
