Efeito do resfriamento e da associação de crioprotetores, penetrantes, não penetrante e ácido ascórbico na qualidade de folículos pré-antrais bovinos vitrificados
Autor(a) principal: | |
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Data de Publicação: | 2012 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | LOCUS Repositório Institucional da UFV |
Texto Completo: | http://locus.ufv.br/handle/123456789/5744 |
Resumo: | The present study aimed to evaluate the cooling effects and association of penetrant, non-penetrant cryoprotectors and ascorbic acid on the quality of vitrified bovine preantral follicles (FOPAs). Two experiments were conducted. In experiment I, the morphology of FOPAs after cooling followed by vitrification was studied. Ovaries were collected (n=10) from five crossbred heifers aged of 14 to 16 months. At the laboratory 22 ovarian fragments were taken from the cortical region and distributed as followed: two fragments for fresh control (zero hour) submitted to histological analysis and 20 fragments cooled at 4 ºC for four and 24 hours in TCM-199+HEPES+Antibiotics medium. From the 20 fragments cooled, four were fixed as control 4 and 24 hours (two piece each) and the remaining 16 fragments were distributed in four vitrification treatments for each cooling time, respectively: Treatments V4a and V24a: TCM-199 + dimethyl sulfoxide (DMSO) 1.5M + Ethylene glycol (EG) 1.5M; Treatments V4b and V24b: TCM-199 + DMSO 1.5M + EG 1.5M + sucrose (SUC) 0.5M; Treatments V4c and V24c: TCM-199 + DMSO 1.5M + EG 1.5M + Ascorbic Acid (AA) 0.1 mM/L and treatments V4d and V24d: TCM-199 + DMSO 1.5M + EG 1.5M + SUC 0.5M + AA 0.1mM/L. After being vitrified, fragments were stored in liquid nitrogen for three days. Fragments were then heated in solution of decreasing SUC and fixed for histology. In experiment II, the viability of FOPAs after cooling followed by vitrification by Trypan Blue staining (AT) was studied. Bovine ovaries (n = 10) were collected, fragmented and distributed as described in the first experiment, subsequently submitted to mechanical follicular isolation for analysis of viability. The morphology variable was evaluated by SNK test and viability was analyzed by Chi-square Test at 5% probability or the Fisher Exact Test when the number of repetitions was less than 30 follicles. There was no difference in percentage of morphologically normal follicles between fresh control group and control group cooled for 4 h (99.3% and 96.0% respectively; P>0.05), nevertheless, there was reduction of FOPAs morphological integrity of control cooled for 24 h (86%; P<0.05). After vitrification, there was reduction (P<0.05) of normal morphology in all treatments when compared with cooled control groups. On the other hand, the viability analysis showed that V4c treatment were not significantly different when compared with control 24h treatment and, together V24c treatment, showed higher capacity of morphological preservation of FOPAs than other treatments (P<0.05), indicating that AA can reduce the toxic and osmotic damages caused by cryopreservation procedure. In conclusion, FOPAs included in the ovarian bovine tissue conserve the morphology efficiently when cooled to 4 °C for 4 hours in TCM-199 + HEPES + Antibiotics and, the association of permeable cryoprotectants DMSO and EG with the antioxidant AA improves survival rates and maintain the morphologic integrity during the vitrification. |
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Triana, Erly Luisana Carrascalhttp://lattes.cnpq.br/2151557305473376Carvalho, Giovanni Ribeiro dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4723068Z6Costa, Eduardo Paulino dahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787237D6Torres, Ciro Alexandre Alveshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787213D4Espeschit, Claudio José BorelaGuimarães, José Domingoshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4782270U62015-03-26T13:55:12Z2013-04-162015-03-26T13:55:12Z2012-07-25TRIANA, Erly Luisana Carrascal. Cooling effects and association of penetrant, non-penetrant cryoprotectors and ascorbic acid on the quality of vitrified bovine preantral follicles. 2012. 69 f. Dissertação (Mestrado em Genética e Melhoramento de Animais Domésticos; Nutrição e Alimentação Animal; Pastagens e Forragicul) - Universidade Federal de Viçosa, Viçosa, 2012.http://locus.ufv.br/handle/123456789/5744The present study aimed to evaluate the cooling effects and association of penetrant, non-penetrant cryoprotectors and ascorbic acid on the quality of vitrified bovine preantral follicles (FOPAs). Two experiments were conducted. In experiment I, the morphology of FOPAs after cooling followed by vitrification was studied. Ovaries were collected (n=10) from five crossbred heifers aged of 14 to 16 months. At the laboratory 22 ovarian fragments were taken from the cortical region and distributed as followed: two fragments for fresh control (zero hour) submitted to histological analysis and 20 fragments cooled at 4 ºC for four and 24 hours in TCM-199+HEPES+Antibiotics medium. From the 20 fragments cooled, four were fixed as control 4 and 24 hours (two piece each) and the remaining 16 fragments were distributed in four vitrification treatments for each cooling time, respectively: Treatments V4a and V24a: TCM-199 + dimethyl sulfoxide (DMSO) 1.5M + Ethylene glycol (EG) 1.5M; Treatments V4b and V24b: TCM-199 + DMSO 1.5M + EG 1.5M + sucrose (SUC) 0.5M; Treatments V4c and V24c: TCM-199 + DMSO 1.5M + EG 1.5M + Ascorbic Acid (AA) 0.1 mM/L and treatments V4d and V24d: TCM-199 + DMSO 1.5M + EG 1.5M + SUC 0.5M + AA 0.1mM/L. After being vitrified, fragments were stored in liquid nitrogen for three days. Fragments were then heated in solution of decreasing SUC and fixed for histology. In experiment II, the viability of FOPAs after cooling followed by vitrification by Trypan Blue staining (AT) was studied. Bovine ovaries (n = 10) were collected, fragmented and distributed as described in the first experiment, subsequently submitted to mechanical follicular isolation for analysis of viability. The morphology variable was evaluated by SNK test and viability was analyzed by Chi-square Test at 5% probability or the Fisher Exact Test when the number of repetitions was less than 30 follicles. There was no difference in percentage of morphologically normal follicles between fresh control group and control group cooled for 4 h (99.3% and 96.0% respectively; P>0.05), nevertheless, there was reduction of FOPAs morphological integrity of control cooled for 24 h (86%; P<0.05). After vitrification, there was reduction (P<0.05) of normal morphology in all treatments when compared with cooled control groups. On the other hand, the viability analysis showed that V4c treatment were not significantly different when compared with control 24h treatment and, together V24c treatment, showed higher capacity of morphological preservation of FOPAs than other treatments (P<0.05), indicating that AA can reduce the toxic and osmotic damages caused by cryopreservation procedure. In conclusion, FOPAs included in the ovarian bovine tissue conserve the morphology efficiently when cooled to 4 °C for 4 hours in TCM-199 + HEPES + Antibiotics and, the association of permeable cryoprotectants DMSO and EG with the antioxidant AA improves survival rates and maintain the morphologic integrity during the vitrification.O presente trabalho teve por objetivo avaliar o efeito do resfriamento e da associação de crioprotetores penetrantes e não penetrante acrescido de ácido ascórbico na qualidade de folículos pré-antrais (FOPAs) bovinos vitrificados. Para isso, foram realizados dois experimentos. No experimento I, foi estudada a morfologia dos FOPAs após resfriamento seguido de vitrificação. Ovários (n=10) foram coletados de cinco novilhas mestiças de 14 a 16 meses de idade. No laboratório, 22 fragmentos ovarianos foram retirados da região cortical, os quais foram distribuídos: dois para controle fresco (zero hora), sendo imediatamente fixados para análise histológica, e 20 fragmentos resfriados à 4 ºC por quatro e 24 horas em meio TCM-199+HEPES e antibióticos. Dos 20 fragmentos resfriados, quatro foram fixados como controle 4 e 24 horas (dois fragmentos cada) e os 16 restantes foram distribuídos em quatro tratamentos de vitrificação para cada tempo de resfriamento, respectivamente: Tratamentos V4a e V24a: TCM-199 + Etilenoglicol (EG) 1,5M + Dimetilsulfoxido (DMSO) 1,5M; Tratamentos V4b e V24b: TCM-199 + DMSO 1,5M + EG 1,5M + sacarose (SAC) 0,5M; Tratamentos V4c e V24c: TCM-199 + DMSO 1,5M + EG 1,5M + Ácido Ascórbico (AA) 0,1 mM/L e tratamentos V4d e V24d: TCM-199 + DMSO 1,5M + EG 1,5M + SAC 0,5M + AA 0,1mM/L. Após a vitrificação, os fragmentos permaneceram armazenados em Nitrogênio Líquido por três dias, e posteriormente aquecidos em soluções decrescentes de SAC e fixados para histologia clássica. No experimento II, foi estudada a viabilidade dos FOPAs após resfriamento seguido de vitrificação pela coloração Azul de Trypan (AT). Ovários bovinos (n=10) foram coletados, fragmentados e distribuídos nos quatros tratamentos conforme descrito no experimento I, os quais foram posteriormente submetidos ao isolamento folicular mecânico para análise da viabilidade. A variável morfologia foi avaliada pelo Teste SNK e a viabilidade foi analisada pelo teste Qui-quadrado com nível de significância de 5%, ou pelo Teste Exato de Fisher quando o número de repetições foi menor que 30 folículos. Não houve diferença na percentagem de folículos morfologicamente normais entre o grupo controle fresco e o grupo controle 4 h (99,3% e 96,0% respectivamente; P>0,05). Entretanto, houve redução da integridade morfológica dos FOPAs após 24 h de resfriamento (controle 24 h: 86%; P<0,05). Após a vitrificação, houve uma redução da integridade morfológica e viabilidade folicular em todos os tratamentos em relação aos controles resfriados (P<0,05). No entanto, o tratamento V4c manteve a viabilidade folicular semelhante ao controle 24 h (P > 0.05). Este mesmo tratamento (V4c) mostrou maior capacidade de preservação em relação aos demais tratamentos de vitrificação (P<0,05). Em conclusão, FOPAs inclusos no tecido ovariano bovino conservam a morfologia eficientemente quando são resfriados a 4 ºC por até quatro horas em meio TCM-199+HEPES+Antibióticos. Além disso, a associação dos agentes crioprotetores penetrantes DMSO / EG, com o agente antioxidante AA melhora as taxas de sobrevivência e mantêm a integridade morfológica durante a vitrificação.application/pdfporUniversidade Federal de ViçosaMestrado em ZootecniaUFVBRGenética e Melhoramento de Animais Domésticos; Nutrição e Alimentação Animal; Pastagens e ForragiculOvárioFolículos pré-antraisCriopreservaçãoResfriamentoOvaryPreantral folliclesCryopreservationCoolingCNPQ::CIENCIAS AGRARIAS::ZOOTECNIA::PRODUCAO ANIMALEfeito do resfriamento e da associação de crioprotetores, penetrantes, não penetrante e ácido ascórbico na qualidade de folículos pré-antrais bovinos vitrificadosCooling effects and association of penetrant, non-penetrant cryoprotectors and ascorbic acid on the quality of vitrified bovine preantral folliclesinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf1494903https://locus.ufv.br//bitstream/123456789/5744/1/texto%20completo.pdf868284081df988ac35cef0a3dbbd85c9MD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain120911https://locus.ufv.br//bitstream/123456789/5744/2/texto%20completo.pdf.txt57bb4f6331785ee94382272a40027291MD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3750https://locus.ufv.br//bitstream/123456789/5744/3/texto%20completo.pdf.jpg616f0180801e04e943e4f2278a93c2c9MD53123456789/57442016-04-10 23:15:55.603oai:locus.ufv.br:123456789/5744Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-04-11T02:15:55LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false |
dc.title.por.fl_str_mv |
Efeito do resfriamento e da associação de crioprotetores, penetrantes, não penetrante e ácido ascórbico na qualidade de folículos pré-antrais bovinos vitrificados |
dc.title.alternative.eng.fl_str_mv |
Cooling effects and association of penetrant, non-penetrant cryoprotectors and ascorbic acid on the quality of vitrified bovine preantral follicles |
title |
Efeito do resfriamento e da associação de crioprotetores, penetrantes, não penetrante e ácido ascórbico na qualidade de folículos pré-antrais bovinos vitrificados |
spellingShingle |
Efeito do resfriamento e da associação de crioprotetores, penetrantes, não penetrante e ácido ascórbico na qualidade de folículos pré-antrais bovinos vitrificados Triana, Erly Luisana Carrascal Ovário Folículos pré-antrais Criopreservação Resfriamento Ovary Preantral follicles Cryopreservation Cooling CNPQ::CIENCIAS AGRARIAS::ZOOTECNIA::PRODUCAO ANIMAL |
title_short |
Efeito do resfriamento e da associação de crioprotetores, penetrantes, não penetrante e ácido ascórbico na qualidade de folículos pré-antrais bovinos vitrificados |
title_full |
Efeito do resfriamento e da associação de crioprotetores, penetrantes, não penetrante e ácido ascórbico na qualidade de folículos pré-antrais bovinos vitrificados |
title_fullStr |
Efeito do resfriamento e da associação de crioprotetores, penetrantes, não penetrante e ácido ascórbico na qualidade de folículos pré-antrais bovinos vitrificados |
title_full_unstemmed |
Efeito do resfriamento e da associação de crioprotetores, penetrantes, não penetrante e ácido ascórbico na qualidade de folículos pré-antrais bovinos vitrificados |
title_sort |
Efeito do resfriamento e da associação de crioprotetores, penetrantes, não penetrante e ácido ascórbico na qualidade de folículos pré-antrais bovinos vitrificados |
author |
Triana, Erly Luisana Carrascal |
author_facet |
Triana, Erly Luisana Carrascal |
author_role |
author |
dc.contributor.authorLattes.por.fl_str_mv |
http://lattes.cnpq.br/2151557305473376 |
dc.contributor.author.fl_str_mv |
Triana, Erly Luisana Carrascal |
dc.contributor.advisor-co1.fl_str_mv |
Carvalho, Giovanni Ribeiro de |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4723068Z6 |
dc.contributor.advisor-co2.fl_str_mv |
Costa, Eduardo Paulino da |
dc.contributor.advisor-co2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787237D6 |
dc.contributor.advisor1.fl_str_mv |
Torres, Ciro Alexandre Alves |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787213D4 |
dc.contributor.referee1.fl_str_mv |
Espeschit, Claudio José Borela |
dc.contributor.referee2.fl_str_mv |
Guimarães, José Domingos |
dc.contributor.referee2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4782270U6 |
contributor_str_mv |
Carvalho, Giovanni Ribeiro de Costa, Eduardo Paulino da Torres, Ciro Alexandre Alves Espeschit, Claudio José Borela Guimarães, José Domingos |
dc.subject.por.fl_str_mv |
Ovário Folículos pré-antrais Criopreservação Resfriamento |
topic |
Ovário Folículos pré-antrais Criopreservação Resfriamento Ovary Preantral follicles Cryopreservation Cooling CNPQ::CIENCIAS AGRARIAS::ZOOTECNIA::PRODUCAO ANIMAL |
dc.subject.eng.fl_str_mv |
Ovary Preantral follicles Cryopreservation Cooling |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS AGRARIAS::ZOOTECNIA::PRODUCAO ANIMAL |
description |
The present study aimed to evaluate the cooling effects and association of penetrant, non-penetrant cryoprotectors and ascorbic acid on the quality of vitrified bovine preantral follicles (FOPAs). Two experiments were conducted. In experiment I, the morphology of FOPAs after cooling followed by vitrification was studied. Ovaries were collected (n=10) from five crossbred heifers aged of 14 to 16 months. At the laboratory 22 ovarian fragments were taken from the cortical region and distributed as followed: two fragments for fresh control (zero hour) submitted to histological analysis and 20 fragments cooled at 4 ºC for four and 24 hours in TCM-199+HEPES+Antibiotics medium. From the 20 fragments cooled, four were fixed as control 4 and 24 hours (two piece each) and the remaining 16 fragments were distributed in four vitrification treatments for each cooling time, respectively: Treatments V4a and V24a: TCM-199 + dimethyl sulfoxide (DMSO) 1.5M + Ethylene glycol (EG) 1.5M; Treatments V4b and V24b: TCM-199 + DMSO 1.5M + EG 1.5M + sucrose (SUC) 0.5M; Treatments V4c and V24c: TCM-199 + DMSO 1.5M + EG 1.5M + Ascorbic Acid (AA) 0.1 mM/L and treatments V4d and V24d: TCM-199 + DMSO 1.5M + EG 1.5M + SUC 0.5M + AA 0.1mM/L. After being vitrified, fragments were stored in liquid nitrogen for three days. Fragments were then heated in solution of decreasing SUC and fixed for histology. In experiment II, the viability of FOPAs after cooling followed by vitrification by Trypan Blue staining (AT) was studied. Bovine ovaries (n = 10) were collected, fragmented and distributed as described in the first experiment, subsequently submitted to mechanical follicular isolation for analysis of viability. The morphology variable was evaluated by SNK test and viability was analyzed by Chi-square Test at 5% probability or the Fisher Exact Test when the number of repetitions was less than 30 follicles. There was no difference in percentage of morphologically normal follicles between fresh control group and control group cooled for 4 h (99.3% and 96.0% respectively; P>0.05), nevertheless, there was reduction of FOPAs morphological integrity of control cooled for 24 h (86%; P<0.05). After vitrification, there was reduction (P<0.05) of normal morphology in all treatments when compared with cooled control groups. On the other hand, the viability analysis showed that V4c treatment were not significantly different when compared with control 24h treatment and, together V24c treatment, showed higher capacity of morphological preservation of FOPAs than other treatments (P<0.05), indicating that AA can reduce the toxic and osmotic damages caused by cryopreservation procedure. In conclusion, FOPAs included in the ovarian bovine tissue conserve the morphology efficiently when cooled to 4 °C for 4 hours in TCM-199 + HEPES + Antibiotics and, the association of permeable cryoprotectants DMSO and EG with the antioxidant AA improves survival rates and maintain the morphologic integrity during the vitrification. |
publishDate |
2012 |
dc.date.issued.fl_str_mv |
2012-07-25 |
dc.date.available.fl_str_mv |
2013-04-16 2015-03-26T13:55:12Z |
dc.date.accessioned.fl_str_mv |
2015-03-26T13:55:12Z |
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 |
TRIANA, Erly Luisana Carrascal. Cooling effects and association of penetrant, non-penetrant cryoprotectors and ascorbic acid on the quality of vitrified bovine preantral follicles. 2012. 69 f. Dissertação (Mestrado em Genética e Melhoramento de Animais Domésticos; Nutrição e Alimentação Animal; Pastagens e Forragicul) - Universidade Federal de Viçosa, Viçosa, 2012. |
dc.identifier.uri.fl_str_mv |
http://locus.ufv.br/handle/123456789/5744 |
identifier_str_mv |
TRIANA, Erly Luisana Carrascal. Cooling effects and association of penetrant, non-penetrant cryoprotectors and ascorbic acid on the quality of vitrified bovine preantral follicles. 2012. 69 f. Dissertação (Mestrado em Genética e Melhoramento de Animais Domésticos; Nutrição e Alimentação Animal; Pastagens e Forragicul) - Universidade Federal de Viçosa, Viçosa, 2012. |
url |
http://locus.ufv.br/handle/123456789/5744 |
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Universidade Federal de Viçosa |
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Mestrado em Zootecnia |
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UFV |
dc.publisher.country.fl_str_mv |
BR |
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Genética e Melhoramento de Animais Domésticos; Nutrição e Alimentação Animal; Pastagens e Forragicul |
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Universidade Federal de Viçosa |
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