Cultivo in vitro de células somáticas derivadas de tecido auricular de catetos (Pecari tajacu Linnaeus, 1758) em meio com diferentes suplementações
Ano de defesa: | 2016 |
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Autor(a) principal: | |
Orientador(a): | |
Banca de defesa: | , |
Tipo de documento: | Dissertação |
Tipo de acesso: | Acesso aberto |
Idioma: | por |
Instituição de defesa: |
Universidade Federal Rural do Semi-Árido
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Programa de Pós-Graduação: |
Programa de Pós-Graduação em Ciência Animal
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Departamento: |
Não Informado pela instituição
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País: |
Brasil
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Palavras-chave em Português: | |
Área do conhecimento CNPq: | |
Link de acesso: | https://repositorio.ufersa.edu.br/handle/tede/566 |
Resumo: | The maintenance of metabolic activities during the in vitro culture of somatic cells of wild animals, especially collared peccary (Pecari tajacu), is an interesting step in the conservation of these cells for use in nuclear transfer (cloning). In this context, it is necessary to optimize the in vitro culture conditions of somatic cell by establishment of some appropriate supplementations to the media, in order to ensure the maximum preservation of the viable cell characteristics. Therefore, this study aimed to optimize the composition of the culture means of somatic cell derived from ear tissue of collared peccaries, evaluating two concentrations of fetal bovine serum (E1: FBS; 10% vs. 20%) and epidermal growth factor (E2: EGF, 5 ng/mL vs. 10 ng/mL). Thus, tissue fragments from 18 adult animals were submitted to primary culture and subcultures for 40 days until the fourth passage and the resulting cells were analyzed for morphology, adhesion, subconfluence, proliferative activity for developing growth curve for seven days and determining the population doubling time (PDT), viability by trypan blue and functional/metabolic activity by assay 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Moreover, in the E1, comparisons as cell adhesion were performed with cells cultured in the presence of bovine serum albumin (BSA, 0.5% and 1.0%). All data were analyzed by ANOVA followed by post hoc test. In the E1, no difference (P>0.05) was observed between the concentrations of FBS for the number of adhered [SFB10: 39/39 vs. SB20: 35/39] and subconfluent fragments [SFB10: 39/39 vs. SB20: 35/39], day all adhered [SFB10: 3.5 vs. SFB20: 3.0], with growth [SFB10: 7.4 vs. SFB20: 7.2] and subconfluent samples [SFB10: 11.8 vs. SFB20: 11.8]. However, significant values were observed in cells cultured in the presence of 20% FBS for viability [SFB10: 85.6% vs. SFB20: 98.2%], PDT [SFB10: 155.4 h vs.77.25 h] and MTT assay [SFB10: 0.57-0.57 vs. SFB20: 0.82-0.99 (D5-D7)]. Additionally, comparisons of supplementation of BSA and FBS confirm the potential FBS cell adhesion. Thus, 20% FBS was used in the following experiment. In the evaluation of the presence of EGF in culture, no difference was observed in the evaluated parameters for the number of attached [EGF0: 31/31 vs. EGF5: 31/31 vs. EGF10: 31/31] and subconfluent fragments [EGF0: 31/31 vs. EGF5: 31/31 vs. EGF10: 31/31] day all adhered [EGF0: 4.9 vs. EGF5: 7.0 vs. EGF10: 3.5] growth [EGF0: 7.2 vs. EGF5: 8.2 vs. EGF10: 7.9] and subconfluent samples [0 EGF: 12.6 vs. EGF5: 16.6 vs. EGF10:12.6], viability [EGF0: 84.3% vs. EGF5: 88.8% vs. EGF10: 87.0%], PDT [EGF0: 69.6 h vs. EGF5: 64.8 h vs. EGF10: 65.3 h] and MTT assay [EGF0: 1.26-1.38 vs. EGF5: 1.06-1.14 vs. EGF10: 1.13-1.16 (D5-D7)]. In all experiments, the growth curves showed clear log and lag phases of development. In conclusion, 20% FBS is suitable for the recovery of somatic cells in vitro; however, EGF does not improve the quality of growing these cells |
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Pereira, Alexsandra FernandesSilva, Alexandre Rodrigueshttp://lattes.cnpq.br/1959482950237684Silva, Alexandre Rodrigueshttp://lattes.cnpq.br/1959482950237684Freitas, Vicente José de Figueirêdohttp://lattes.cnpq.br/812347831520530804353236369http://lattes.cnpq.br/2828706813975444http://lattes.cnpq.br/8114638410593492Santos, Magda Lorena Turbano dos2017-03-07T12:56:11Z2016-03-30SANTOS, Magda Lorena Turbano dos. Cultivo in vitro de células somáticas derivadas de tecido auricular de catetos (Pecari tajacu Linnaeus, 1758) em meio com diferentes suplementações. 2016. 74 f. Dissertação (Mestrado) - Curso de Pós-graduação em Ciência Animal, Universidade Federal Rural do Semi-Árido, Mossoró, 2016.https://repositorio.ufersa.edu.br/handle/tede/566The maintenance of metabolic activities during the in vitro culture of somatic cells of wild animals, especially collared peccary (Pecari tajacu), is an interesting step in the conservation of these cells for use in nuclear transfer (cloning). In this context, it is necessary to optimize the in vitro culture conditions of somatic cell by establishment of some appropriate supplementations to the media, in order to ensure the maximum preservation of the viable cell characteristics. Therefore, this study aimed to optimize the composition of the culture means of somatic cell derived from ear tissue of collared peccaries, evaluating two concentrations of fetal bovine serum (E1: FBS; 10% vs. 20%) and epidermal growth factor (E2: EGF, 5 ng/mL vs. 10 ng/mL). Thus, tissue fragments from 18 adult animals were submitted to primary culture and subcultures for 40 days until the fourth passage and the resulting cells were analyzed for morphology, adhesion, subconfluence, proliferative activity for developing growth curve for seven days and determining the population doubling time (PDT), viability by trypan blue and functional/metabolic activity by assay 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Moreover, in the E1, comparisons as cell adhesion were performed with cells cultured in the presence of bovine serum albumin (BSA, 0.5% and 1.0%). All data were analyzed by ANOVA followed by post hoc test. In the E1, no difference (P>0.05) was observed between the concentrations of FBS for the number of adhered [SFB10: 39/39 vs. SB20: 35/39] and subconfluent fragments [SFB10: 39/39 vs. SB20: 35/39], day all adhered [SFB10: 3.5 vs. SFB20: 3.0], with growth [SFB10: 7.4 vs. SFB20: 7.2] and subconfluent samples [SFB10: 11.8 vs. SFB20: 11.8]. However, significant values were observed in cells cultured in the presence of 20% FBS for viability [SFB10: 85.6% vs. SFB20: 98.2%], PDT [SFB10: 155.4 h vs.77.25 h] and MTT assay [SFB10: 0.57-0.57 vs. SFB20: 0.82-0.99 (D5-D7)]. Additionally, comparisons of supplementation of BSA and FBS confirm the potential FBS cell adhesion. Thus, 20% FBS was used in the following experiment. In the evaluation of the presence of EGF in culture, no difference was observed in the evaluated parameters for the number of attached [EGF0: 31/31 vs. EGF5: 31/31 vs. EGF10: 31/31] and subconfluent fragments [EGF0: 31/31 vs. EGF5: 31/31 vs. EGF10: 31/31] day all adhered [EGF0: 4.9 vs. EGF5: 7.0 vs. EGF10: 3.5] growth [EGF0: 7.2 vs. EGF5: 8.2 vs. EGF10: 7.9] and subconfluent samples [0 EGF: 12.6 vs. EGF5: 16.6 vs. EGF10:12.6], viability [EGF0: 84.3% vs. EGF5: 88.8% vs. EGF10: 87.0%], PDT [EGF0: 69.6 h vs. EGF5: 64.8 h vs. EGF10: 65.3 h] and MTT assay [EGF0: 1.26-1.38 vs. EGF5: 1.06-1.14 vs. EGF10: 1.13-1.16 (D5-D7)]. In all experiments, the growth curves showed clear log and lag phases of development. In conclusion, 20% FBS is suitable for the recovery of somatic cells in vitro; however, EGF does not improve the quality of growing these cellsA manutenção das atividades metabólicas durante o cultivo in vitro de células somáticas de animais silvestres, especialmente catetos (Pecari tajacu), representa uma etapa interessante na conservação dessas células para aplicação na transferência nuclear (clonagem). Nesse contexto, faz-se necessário a otimização das condições de cultivo in vitro de células somáticas pelo estabelecimento de algumas suplementações adequadas aos meios, visando garantir a máxima preservação das características celulares viáveis. Portanto, o presente trabalho teve como objetivo otimizar a composição do meio de cultivo de células somáticas derivadas de tecido auricular de catetos, avaliando duas concentrações de soro fetal bovino (E1: SFB; 10% vs. 20%) e fator de crescimento epidermal (E2: EGF; 5 ng/mL vs. 10 ng/mL). Para tanto, fragmentos teciduais de 18 animais adultos foram submetidos ao cultivo primário e subcultivos por 40 dias, até a quarta passagem e as células resultantes foram analisadas quanto à morfologia, aderência, subconfluência, atividade proliferativa pela elaboração de curva de crescimento por sete dias e determinação do tempo de duplicação da população (PDT), viabilidade por azul de tripano e atividade funcional/metabólica pelo ensaio de brometo de 3-[4,5-dimetil-tiazol-2-il]-2,5-difeniltetrazólio (MTT). Além disso, no E1, comparações quanto à adesão celular foram realizadas com células cultivadas na presença de albumina sérica bovina (BSA, 0,5% e 1,0%). Todos os dados foram analisados por ANOVA seguido por teste post-hoc. No E1, nenhuma diferença (P>0,05) foi observada entre as concentrações de SFB para o número de fragmentos aderidos [SFB10: 39/39 vs. SB20: 35/39] e subconfluentes [SFB10: 39/39 vs. SB20: 35/39], dia de todas as amostras aderidas [SFB10: 3,5 vs. SFB20: 3,0], com crescimento [SFB10: 7,4 vs. SFB20: 7,2] e subconfluentes [SFB10: 11,8 vs. SFB20: 11,8]. Contudo, valores significativos foram observados em células cultivadas na presença de SFB a 20% quanto à viabilidade [SFB10: 85,6% vs. SFB20: 98,2%], PDT [SFB10: 155,4 h vs.77,2 h] e ensaio de MTT [SFB10: 0,57-0,57 vs. SFB20: 0,82-0,99 (D5-D7)]. Adicionalmente, comparações da suplementação do BSA e SFB confirmaram o potencial de adesão celular do soro. Assim, SFB a 20% foi empregado no experimento seguinte. Já na avaliação da presença de EGF no cultivo, nenhuma diferença foi observada nos parâmetros avaliados para o número de fragmentos aderidos [EGF0: 31/31 vs. EGF5: 31/31 vs. EGF10: 31/31] e subconfluentes [EGF0: 31/31 vs. EGF5: 31/31 vs. EGF10: 31/31], dia de todas as amostras aderidas [EGF0: 4,9 vs. EGF5: 7,0 vs. EGF10: 3,5], em crescimento [EGF0: 7,2 vs. EGF5: 8,2 vs. EGF10: 7,9] e subconfluentes [EGF0: 12,6 vs. EGF5: 16,6 vs. EGF10: 12,6], viabilidade [EGF0: 84,3% vs. EGF5: 88,8% vs. EGF10: 87,0%], PDT [EGF0: 69,6 h vs. EGF5: 64,8 h vs. EGF10: 65,3 h] e ensaio de MTT [EGF0: 1,26-1,38 vs. EGF5: 1,06-1,14 vs. EGF10: 1,13-1,16 (D5-D7)]. Em todos os experimentos, as curvas de crescimento apresentaram nítidas fases log e lag de desenvolvimento. Em conclusão, o SFB a 20% é adequado para a recuperação de células somáticas in vitro; contudo, o EGF não melhora a qualidade do cultivo dessas célulasCoordenação de Aperfeiçoamento de Pessoal de Nível Superior2017-03-07application/pdfhttp://repositorio.ufersa.edu.br/retrieve/1275/MagdaLTS_DISSERT.pdf.jpgporUniversidade Federal Rural do Semi-ÁridoPrograma de Pós-Graduação em Ciência AnimalUFERSABrasilCC-BY-SAinfo:eu-repo/semantics/openAccessConservaçãoAnimais silvestresTecido somáticoFonte proteicaFator mitóticoConservationWild animalsSomatic tissueProtein sourceMitotic factorCNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIACultivo in vitro de células somáticas derivadas de tecido auricular de catetos (Pecari tajacu Linnaeus, 1758) em meio com diferentes suplementaçõesIn vitro culture of somatic cells derived from ear tissue of collared peccary (Pecari tajacu Linnaeus, 1758) in medium with different supplementsinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisreponame:Biblioteca Digital de Teses e Dissertações da UFERSAinstname:Universidade Federal Rural do Semi-Árido (UFERSA)instacron:UFERSAORIGINALMagdaLTS_DISSERT.pdfMagdaLTS_DISSERT.pdfapplication/pdf1454861https://repositorio.ufersa.edu.br//bitstream/tede/566/1/MagdaLTS_DISSERT.pdf4612469437c8dd924c24af03a052758dMD51LICENSElicense.txtlicense.txttext/plain; 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dc.title.por.fl_str_mv |
Cultivo in vitro de células somáticas derivadas de tecido auricular de catetos (Pecari tajacu Linnaeus, 1758) em meio com diferentes suplementações |
dc.title.alternative.por.fl_str_mv |
In vitro culture of somatic cells derived from ear tissue of collared peccary (Pecari tajacu Linnaeus, 1758) in medium with different supplements |
title |
Cultivo in vitro de células somáticas derivadas de tecido auricular de catetos (Pecari tajacu Linnaeus, 1758) em meio com diferentes suplementações |
spellingShingle |
Cultivo in vitro de células somáticas derivadas de tecido auricular de catetos (Pecari tajacu Linnaeus, 1758) em meio com diferentes suplementações Santos, Magda Lorena Turbano dos Conservação Animais silvestres Tecido somático Fonte proteica Fator mitótico Conservation Wild animals Somatic tissue Protein source Mitotic factor CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA |
title_short |
Cultivo in vitro de células somáticas derivadas de tecido auricular de catetos (Pecari tajacu Linnaeus, 1758) em meio com diferentes suplementações |
title_full |
Cultivo in vitro de células somáticas derivadas de tecido auricular de catetos (Pecari tajacu Linnaeus, 1758) em meio com diferentes suplementações |
title_fullStr |
Cultivo in vitro de células somáticas derivadas de tecido auricular de catetos (Pecari tajacu Linnaeus, 1758) em meio com diferentes suplementações |
title_full_unstemmed |
Cultivo in vitro de células somáticas derivadas de tecido auricular de catetos (Pecari tajacu Linnaeus, 1758) em meio com diferentes suplementações |
title_sort |
Cultivo in vitro de células somáticas derivadas de tecido auricular de catetos (Pecari tajacu Linnaeus, 1758) em meio com diferentes suplementações |
author |
Santos, Magda Lorena Turbano dos |
author_facet |
Santos, Magda Lorena Turbano dos |
author_role |
author |
dc.contributor.authorID.por.fl_str_mv |
04353236369 |
dc.contributor.authorLattes.por.fl_str_mv |
http://lattes.cnpq.br/2828706813975444 |
dc.contributor.advisorLattes.por.fl_str_mv |
http://lattes.cnpq.br/8114638410593492 |
dc.contributor.advisor1.fl_str_mv |
Pereira, Alexsandra Fernandes |
dc.contributor.advisor-co1.fl_str_mv |
Silva, Alexandre Rodrigues |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://lattes.cnpq.br/1959482950237684 |
dc.contributor.referee1.fl_str_mv |
Silva, Alexandre Rodrigues |
dc.contributor.referee1Lattes.fl_str_mv |
http://lattes.cnpq.br/1959482950237684 |
dc.contributor.referee2.fl_str_mv |
Freitas, Vicente José de Figueirêdo |
dc.contributor.referee2Lattes.fl_str_mv |
http://lattes.cnpq.br/8123478315205308 |
dc.contributor.author.fl_str_mv |
Santos, Magda Lorena Turbano dos |
contributor_str_mv |
Pereira, Alexsandra Fernandes Silva, Alexandre Rodrigues Silva, Alexandre Rodrigues Freitas, Vicente José de Figueirêdo |
dc.subject.por.fl_str_mv |
Conservação Animais silvestres Tecido somático Fonte proteica Fator mitótico Conservation Wild animals Somatic tissue Protein source Mitotic factor |
topic |
Conservação Animais silvestres Tecido somático Fonte proteica Fator mitótico Conservation Wild animals Somatic tissue Protein source Mitotic factor CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA |
description |
The maintenance of metabolic activities during the in vitro culture of somatic cells of wild animals, especially collared peccary (Pecari tajacu), is an interesting step in the conservation of these cells for use in nuclear transfer (cloning). In this context, it is necessary to optimize the in vitro culture conditions of somatic cell by establishment of some appropriate supplementations to the media, in order to ensure the maximum preservation of the viable cell characteristics. Therefore, this study aimed to optimize the composition of the culture means of somatic cell derived from ear tissue of collared peccaries, evaluating two concentrations of fetal bovine serum (E1: FBS; 10% vs. 20%) and epidermal growth factor (E2: EGF, 5 ng/mL vs. 10 ng/mL). Thus, tissue fragments from 18 adult animals were submitted to primary culture and subcultures for 40 days until the fourth passage and the resulting cells were analyzed for morphology, adhesion, subconfluence, proliferative activity for developing growth curve for seven days and determining the population doubling time (PDT), viability by trypan blue and functional/metabolic activity by assay 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Moreover, in the E1, comparisons as cell adhesion were performed with cells cultured in the presence of bovine serum albumin (BSA, 0.5% and 1.0%). All data were analyzed by ANOVA followed by post hoc test. In the E1, no difference (P>0.05) was observed between the concentrations of FBS for the number of adhered [SFB10: 39/39 vs. SB20: 35/39] and subconfluent fragments [SFB10: 39/39 vs. SB20: 35/39], day all adhered [SFB10: 3.5 vs. SFB20: 3.0], with growth [SFB10: 7.4 vs. SFB20: 7.2] and subconfluent samples [SFB10: 11.8 vs. SFB20: 11.8]. However, significant values were observed in cells cultured in the presence of 20% FBS for viability [SFB10: 85.6% vs. SFB20: 98.2%], PDT [SFB10: 155.4 h vs.77.25 h] and MTT assay [SFB10: 0.57-0.57 vs. SFB20: 0.82-0.99 (D5-D7)]. Additionally, comparisons of supplementation of BSA and FBS confirm the potential FBS cell adhesion. Thus, 20% FBS was used in the following experiment. In the evaluation of the presence of EGF in culture, no difference was observed in the evaluated parameters for the number of attached [EGF0: 31/31 vs. EGF5: 31/31 vs. EGF10: 31/31] and subconfluent fragments [EGF0: 31/31 vs. EGF5: 31/31 vs. EGF10: 31/31] day all adhered [EGF0: 4.9 vs. EGF5: 7.0 vs. EGF10: 3.5] growth [EGF0: 7.2 vs. EGF5: 8.2 vs. EGF10: 7.9] and subconfluent samples [0 EGF: 12.6 vs. EGF5: 16.6 vs. EGF10:12.6], viability [EGF0: 84.3% vs. EGF5: 88.8% vs. EGF10: 87.0%], PDT [EGF0: 69.6 h vs. EGF5: 64.8 h vs. EGF10: 65.3 h] and MTT assay [EGF0: 1.26-1.38 vs. EGF5: 1.06-1.14 vs. EGF10: 1.13-1.16 (D5-D7)]. In all experiments, the growth curves showed clear log and lag phases of development. In conclusion, 20% FBS is suitable for the recovery of somatic cells in vitro; however, EGF does not improve the quality of growing these cells |
publishDate |
2016 |
dc.date.issued.fl_str_mv |
2016-03-30 |
dc.date.accessioned.fl_str_mv |
2017-03-07T12:56:11Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
SANTOS, Magda Lorena Turbano dos. Cultivo in vitro de células somáticas derivadas de tecido auricular de catetos (Pecari tajacu Linnaeus, 1758) em meio com diferentes suplementações. 2016. 74 f. Dissertação (Mestrado) - Curso de Pós-graduação em Ciência Animal, Universidade Federal Rural do Semi-Árido, Mossoró, 2016. |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufersa.edu.br/handle/tede/566 |
identifier_str_mv |
SANTOS, Magda Lorena Turbano dos. Cultivo in vitro de células somáticas derivadas de tecido auricular de catetos (Pecari tajacu Linnaeus, 1758) em meio com diferentes suplementações. 2016. 74 f. Dissertação (Mestrado) - Curso de Pós-graduação em Ciência Animal, Universidade Federal Rural do Semi-Árido, Mossoró, 2016. |
url |
https://repositorio.ufersa.edu.br/handle/tede/566 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.rights.driver.fl_str_mv |
CC-BY-SA info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
CC-BY-SA |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Federal Rural do Semi-Árido |
dc.publisher.program.fl_str_mv |
Programa de Pós-Graduação em Ciência Animal |
dc.publisher.initials.fl_str_mv |
UFERSA |
dc.publisher.country.fl_str_mv |
Brasil |
publisher.none.fl_str_mv |
Universidade Federal Rural do Semi-Árido |
dc.source.none.fl_str_mv |
reponame:Biblioteca Digital de Teses e Dissertações da UFERSA instname:Universidade Federal Rural do Semi-Árido (UFERSA) instacron:UFERSA |
instname_str |
Universidade Federal Rural do Semi-Árido (UFERSA) |
instacron_str |
UFERSA |
institution |
UFERSA |
reponame_str |
Biblioteca Digital de Teses e Dissertações da UFERSA |
collection |
Biblioteca Digital de Teses e Dissertações da UFERSA |
bitstream.url.fl_str_mv |
https://repositorio.ufersa.edu.br//bitstream/tede/566/1/MagdaLTS_DISSERT.pdf https://repositorio.ufersa.edu.br//bitstream/tede/566/2/license.txt https://repositorio.ufersa.edu.br//bitstream/tede/566/3/MagdaLTS_DISSERT.pdf.txt https://repositorio.ufersa.edu.br//bitstream/tede/566/4/MagdaLTS_DISSERT.pdf.jpg |
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MD5 MD5 MD5 MD5 |
repository.name.fl_str_mv |
Biblioteca Digital de Teses e Dissertações da UFERSA - Universidade Federal Rural do Semi-Árido (UFERSA) |
repository.mail.fl_str_mv |
direcaosisbi@ufersa.edu.br|| direcaosisbi@ufersa.edu.br |
_version_ |
1757095600575217664 |