Estabelecimento de protocolos para a criopreservação de tecido testicular de catetos (pecari tajacu linnaeus, 1758)
Autor(a) principal: | |
---|---|
Data de Publicação: | 2019 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Repositório Digital da Universidade Federal Rural do Semi-Árido (RDU) |
Texto Completo: | https://repositorio.ufersa.edu.br/handle/prefix/5397 |
Resumo: | Cryopreservation of male gonadal tissue can be used in the conservation of genetic material, in order to form a germplasm bank allowing the maintenance of genetic variability in prepubertal and adult animals. Therefore, the aim was to establish an efficient protocol for cryopreservation of testicular tissue of collared peccaries. Therefore, the study was divided into two experiments, using 10 adult animals, 5 for each experiment, from the UFERSA Center for Wild Animals Multiplication. In experiment I, five pairs of testicles were fragmented (9 mm3) and allocated to non-vitrified (control) and vitrified solid-surface (SSV) groups following exposure to different cryoprotectants (3.0 M dimethyl sulfoxide (DMSO), 3.0 M ethylene glycol (EG) or 1.5 M DMSO/1.5 M EG). After warming, samples were evaluated for histomorphology, ultrastructure, viability, and proliferative capacity potential. The appropriate conservation of the ultrastructural organization of the seminiferous tubule in terms of lumen presence and cell junctions was only observed at the use of DMSO/EG combination. Regardless of the cryoprotectant, the vitrification effectively preserved cell nuclear visualization and condensation similarly as observed at the non-vitrified group. Moreover, DMSO/EG combination provided a better preservation of basal membranes of seminiferous tubules than DMSO (P < 0.05). Only the DMSO/EG combination maintained the proliferative capacity potential for spermatogonia (3.69 Nucleolus Organizing Regions - NORs/cell) and Sertoli cell (3.19 NORs/cell) similar to controls (3.46 and 3.31 NORS/cell, respectively). In conclusion, DMSO/EG in combination is better than DMSO or EG alone for SSV of testicular tissue biopsies from adult collared peccaries. In experiment II, five pairs of testicles were also fragmented (3 mm3) and allocated to non-cryopreserved (control) and cryopreserved groups using three cryopreservation methods: slow freezing (SF), cryotube vitrification (CV) and solid surface vitrification (SSV), and then exposed to different combinations of cryoprotectants (1.5 M DMSO/1.5 M EG, 1.5 M DMSO/1.5 M glycerol - G, and 1.5 M G/1.5 M EG). Non-cryopreserved and cryopreserved samples were evaluated for histomorphology, viability, proliferative potential and DNA fragmentation. Only in the use of DMSO/EG during SF and CV, it was possible to preserve DNA integrity in a similar way to control (P> 0.05). In addition, it was observed that, regardless the cryopreservation method, the DMSO/EG and DMSO/G combinations were able to preserve viability (P> 0.05). All treatments maintained the proliferative capacity potential for spermatogonia in a similar manner; however, G/EG in the SF and SSV methods impaired the proliferative potential of Sertoli cells (P> 0.05). Finally, the SF protocol using the DMSO/EG or DMSO/G combinations were better at preventing edema than G/EG for SF and DMSO/ G for SSV (P <0.05). In conclusion, we suggest the use of a DMSO/EG combination associated with slow freezing or vitrification in cryotubes for cryopreservation of testicular tissue of adult peccaries. |
id |
UFER_6fcd0ab801518d97f214dae45578e927 |
---|---|
oai_identifier_str |
oai:repositorio.ufersa.edu.br:prefix/5397 |
network_acronym_str |
UFER |
network_name_str |
Repositório Digital da Universidade Federal Rural do Semi-Árido (RDU) |
repository_id_str |
|
spelling |
Estabelecimento de protocolos para a criopreservação de tecido testicular de catetos (pecari tajacu linnaeus, 1758)BiobancoGermoplasmaTestículoCélula germinativaBiobankGermplasmTesticleGerm cellCNPQ::CIENCIAS AGRARIAS::AGRONOMIACryopreservation of male gonadal tissue can be used in the conservation of genetic material, in order to form a germplasm bank allowing the maintenance of genetic variability in prepubertal and adult animals. Therefore, the aim was to establish an efficient protocol for cryopreservation of testicular tissue of collared peccaries. Therefore, the study was divided into two experiments, using 10 adult animals, 5 for each experiment, from the UFERSA Center for Wild Animals Multiplication. In experiment I, five pairs of testicles were fragmented (9 mm3) and allocated to non-vitrified (control) and vitrified solid-surface (SSV) groups following exposure to different cryoprotectants (3.0 M dimethyl sulfoxide (DMSO), 3.0 M ethylene glycol (EG) or 1.5 M DMSO/1.5 M EG). After warming, samples were evaluated for histomorphology, ultrastructure, viability, and proliferative capacity potential. The appropriate conservation of the ultrastructural organization of the seminiferous tubule in terms of lumen presence and cell junctions was only observed at the use of DMSO/EG combination. Regardless of the cryoprotectant, the vitrification effectively preserved cell nuclear visualization and condensation similarly as observed at the non-vitrified group. Moreover, DMSO/EG combination provided a better preservation of basal membranes of seminiferous tubules than DMSO (P < 0.05). Only the DMSO/EG combination maintained the proliferative capacity potential for spermatogonia (3.69 Nucleolus Organizing Regions - NORs/cell) and Sertoli cell (3.19 NORs/cell) similar to controls (3.46 and 3.31 NORS/cell, respectively). In conclusion, DMSO/EG in combination is better than DMSO or EG alone for SSV of testicular tissue biopsies from adult collared peccaries. In experiment II, five pairs of testicles were also fragmented (3 mm3) and allocated to non-cryopreserved (control) and cryopreserved groups using three cryopreservation methods: slow freezing (SF), cryotube vitrification (CV) and solid surface vitrification (SSV), and then exposed to different combinations of cryoprotectants (1.5 M DMSO/1.5 M EG, 1.5 M DMSO/1.5 M glycerol - G, and 1.5 M G/1.5 M EG). Non-cryopreserved and cryopreserved samples were evaluated for histomorphology, viability, proliferative potential and DNA fragmentation. Only in the use of DMSO/EG during SF and CV, it was possible to preserve DNA integrity in a similar way to control (P> 0.05). In addition, it was observed that, regardless the cryopreservation method, the DMSO/EG and DMSO/G combinations were able to preserve viability (P> 0.05). All treatments maintained the proliferative capacity potential for spermatogonia in a similar manner; however, G/EG in the SF and SSV methods impaired the proliferative potential of Sertoli cells (P> 0.05). Finally, the SF protocol using the DMSO/EG or DMSO/G combinations were better at preventing edema than G/EG for SF and DMSO/ G for SSV (P <0.05). In conclusion, we suggest the use of a DMSO/EG combination associated with slow freezing or vitrification in cryotubes for cryopreservation of testicular tissue of adult peccaries.A criopreservação de tecido gonadal masculino pode ser usada na conservação de material genético, no intuito da formação de um banco de germoplasma permitindo a manutenção da variabilidade genética em animais pré-puberes e adultos. Diante disso, objetivou-se estabelecer um protocolo eficiente de criopreservação de tecido testicular de catetos. Para tanto, o estudo foi dividido em dois experimentos, sendo utilizados 10 animais adultos, 5 em cada experimento, oriundos do Centro de Multiplicação de Animais Silvestres da UFERSA. No experimento I, cinco pares de testículos foram fragmentados (9 mm3) e alocados em grupos não-vitrificados (controle) e vitrificados em superfície sólida (VSS), após a exposição a diferentes crioprotetores (dimetilsulfóxido - DMSO 3,0 M, etileno glicol - EG 3,0 M e a combinação 1,5 M DMSO/1,5 M EG). As amostras não-vitrificadas e vitrificadas foram avaliadas quanto à histomorfologia, ultraestrutura, viabilidade e potencial de capacidade proliferativa. A conservação adequada da organização ultraestrutural do túbulo seminífero em termos de presença de lúmen e junções celulares foi observada somente com o uso da combinação DMSO/EG. Independentemente do crioprotetor, a vitrificação conservou efetivamente a visualização e a condensação nuclear das células de maneira semelhante à observada no grupo não vitrificado. Além disso, a combinação DMSO/EG proporcionou uma melhor conservação das membranas basais dos túbulos seminíferos que o DMSO (P < 0,05). Somente a combinação DMSO/EG manteve o potencial de capacidade proliferativa para espermatogônia (3,69 Regiões Organizadoras de Nucléolos - NORs/célula) e célula de Sertoli (3,19 NORs/célula) semelhantes aos controles (3,46 e 3,31 NORS/célula, respectivamente). Portanto, DMSO/EG é melhor que o DMSO ou o EG sozinho para VSS de tecido testicular de cateto adulto. No experimento II, cinco pares de testículos foram fragmentados (3 mm3) e alocados a grupos não-criopreservados (controle) e criopreservados usando três métodos de criopreservação: congelação lenta (CL), vitrificação em criotubos (VC) e VSS, sendo então expostos a diferentes combinações de crioprotetores (1,5 M DMSO/1,5 M EG, 1,5 M DMSO/1,5 M glicerol – G, e 1,5 M G/1,5 M EG). As amostras, não-criopreservadas e criopreservadas foram avaliadas quanto à fragmentação de DNA, viabilidade, potencial de capacidade proliferativa e histomorfologia. Apenas no uso de DMSO/EG durante a CL e VC foi possível conservar a integridade do DNA de modo similar ao controle (P>0,05). Em adição, observou-se que, independentemente do método de criopreservação, as combinações DMSO/EG e DMSO/G foram capazes de conservar a viabilidade (P>0,05). Todos os tratamentos mantiveram o potencial de capacidade proliferativa para espermatogônia de modo similar; entretanto, apenas o G/EG, nos métodos de CL e VSS, foram inferiores ao controle para célula de Sertoli (P>0,05). Finalmente, o protocolo de CL usando as combinações DMSO/EG e DMSO/G foram melhores em evitar o aparecimento de edemas que G/EG – CL e DMSO/G – VSS (P<0.05). Assim, sugere-se a utilização da combinação DMSO/EG associada aos métodos de congelação lenta ou vitrificação para a criopreservação de tecido testicular de catetos adultos.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESUniversidade Federal Rural do Semi-ÁridoBrasilCentro de Ciências Agrárias - CCAUFERSAPrograma de Pós-Graduação em Ciência AnimalPereira, Alexsandra Fernandes91307198368http://lattes.cnpq.br/8114638410593492Silva, Alexandre Rodrigues70298254387http://lattes.cnpq.br/1959482950237684Pereira, Alexsandra Fernandes91307198368http://lattes.cnpq.br/8114638410593492Lima, Gabriela Liberalino01359058427http://lattes.cnpq.br/0329054086208548Moura, Carlos Eduardo Bezerra de03597959431http://lattes.cnpq.br/4717410137206021Silva, Lúcia Daniel Machado da65696972691http://lattes.cnpq.br/3610262531647532Silva, Andréia Maria da2020-08-31T01:53:35Z2020-01-272020-08-31T01:53:35Z2019-12-18info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisapplication/pdfCitação com autor incluído no texto: Silva (2020) Citação com autor não incluído no texto: (SILVA, 2020)https://repositorio.ufersa.edu.br/handle/prefix/5397porSILVA, Andréia Maria da. Estabelecimento de protocolos para a criopreservação de tecido testicular de catetos (pecari tajacu linnaeus, 1758). 2020. 118 f. Tese (Doutorado em Ciência Animal), Universidade Federal Rural do Semi-Árido, Mossoró, 2019.CC-BY-SAinfo:eu-repo/semantics/openAccessreponame:Repositório Digital da Universidade Federal Rural do Semi-Árido (RDU)instname:Universidade Federal Rural do Semi-Árido (UFERSA)instacron:UFERSA2023-10-30T20:27:42Zoai:repositorio.ufersa.edu.br:prefix/5397Repositório Institucionalhttps://repositorio.ufersa.edu.br/PUBhttps://repositorio.ufersa.edu.br/server/oai/requestrepositorio@ufersa.edu.br || admrepositorio@ufersa.edu.bropendoar:2023-10-30T20:27:42Repositório Digital da Universidade Federal Rural do Semi-Árido (RDU) - Universidade Federal Rural do Semi-Árido (UFERSA)false |
dc.title.none.fl_str_mv |
Estabelecimento de protocolos para a criopreservação de tecido testicular de catetos (pecari tajacu linnaeus, 1758) |
title |
Estabelecimento de protocolos para a criopreservação de tecido testicular de catetos (pecari tajacu linnaeus, 1758) |
spellingShingle |
Estabelecimento de protocolos para a criopreservação de tecido testicular de catetos (pecari tajacu linnaeus, 1758) Silva, Andréia Maria da Biobanco Germoplasma Testículo Célula germinativa Biobank Germplasm Testicle Germ cell CNPQ::CIENCIAS AGRARIAS::AGRONOMIA |
title_short |
Estabelecimento de protocolos para a criopreservação de tecido testicular de catetos (pecari tajacu linnaeus, 1758) |
title_full |
Estabelecimento de protocolos para a criopreservação de tecido testicular de catetos (pecari tajacu linnaeus, 1758) |
title_fullStr |
Estabelecimento de protocolos para a criopreservação de tecido testicular de catetos (pecari tajacu linnaeus, 1758) |
title_full_unstemmed |
Estabelecimento de protocolos para a criopreservação de tecido testicular de catetos (pecari tajacu linnaeus, 1758) |
title_sort |
Estabelecimento de protocolos para a criopreservação de tecido testicular de catetos (pecari tajacu linnaeus, 1758) |
author |
Silva, Andréia Maria da |
author_facet |
Silva, Andréia Maria da |
author_role |
author |
dc.contributor.none.fl_str_mv |
Pereira, Alexsandra Fernandes 91307198368 http://lattes.cnpq.br/8114638410593492 Silva, Alexandre Rodrigues 70298254387 http://lattes.cnpq.br/1959482950237684 Pereira, Alexsandra Fernandes 91307198368 http://lattes.cnpq.br/8114638410593492 Lima, Gabriela Liberalino 01359058427 http://lattes.cnpq.br/0329054086208548 Moura, Carlos Eduardo Bezerra de 03597959431 http://lattes.cnpq.br/4717410137206021 Silva, Lúcia Daniel Machado da 65696972691 http://lattes.cnpq.br/3610262531647532 |
dc.contributor.author.fl_str_mv |
Silva, Andréia Maria da |
dc.subject.por.fl_str_mv |
Biobanco Germoplasma Testículo Célula germinativa Biobank Germplasm Testicle Germ cell CNPQ::CIENCIAS AGRARIAS::AGRONOMIA |
topic |
Biobanco Germoplasma Testículo Célula germinativa Biobank Germplasm Testicle Germ cell CNPQ::CIENCIAS AGRARIAS::AGRONOMIA |
description |
Cryopreservation of male gonadal tissue can be used in the conservation of genetic material, in order to form a germplasm bank allowing the maintenance of genetic variability in prepubertal and adult animals. Therefore, the aim was to establish an efficient protocol for cryopreservation of testicular tissue of collared peccaries. Therefore, the study was divided into two experiments, using 10 adult animals, 5 for each experiment, from the UFERSA Center for Wild Animals Multiplication. In experiment I, five pairs of testicles were fragmented (9 mm3) and allocated to non-vitrified (control) and vitrified solid-surface (SSV) groups following exposure to different cryoprotectants (3.0 M dimethyl sulfoxide (DMSO), 3.0 M ethylene glycol (EG) or 1.5 M DMSO/1.5 M EG). After warming, samples were evaluated for histomorphology, ultrastructure, viability, and proliferative capacity potential. The appropriate conservation of the ultrastructural organization of the seminiferous tubule in terms of lumen presence and cell junctions was only observed at the use of DMSO/EG combination. Regardless of the cryoprotectant, the vitrification effectively preserved cell nuclear visualization and condensation similarly as observed at the non-vitrified group. Moreover, DMSO/EG combination provided a better preservation of basal membranes of seminiferous tubules than DMSO (P < 0.05). Only the DMSO/EG combination maintained the proliferative capacity potential for spermatogonia (3.69 Nucleolus Organizing Regions - NORs/cell) and Sertoli cell (3.19 NORs/cell) similar to controls (3.46 and 3.31 NORS/cell, respectively). In conclusion, DMSO/EG in combination is better than DMSO or EG alone for SSV of testicular tissue biopsies from adult collared peccaries. In experiment II, five pairs of testicles were also fragmented (3 mm3) and allocated to non-cryopreserved (control) and cryopreserved groups using three cryopreservation methods: slow freezing (SF), cryotube vitrification (CV) and solid surface vitrification (SSV), and then exposed to different combinations of cryoprotectants (1.5 M DMSO/1.5 M EG, 1.5 M DMSO/1.5 M glycerol - G, and 1.5 M G/1.5 M EG). Non-cryopreserved and cryopreserved samples were evaluated for histomorphology, viability, proliferative potential and DNA fragmentation. Only in the use of DMSO/EG during SF and CV, it was possible to preserve DNA integrity in a similar way to control (P> 0.05). In addition, it was observed that, regardless the cryopreservation method, the DMSO/EG and DMSO/G combinations were able to preserve viability (P> 0.05). All treatments maintained the proliferative capacity potential for spermatogonia in a similar manner; however, G/EG in the SF and SSV methods impaired the proliferative potential of Sertoli cells (P> 0.05). Finally, the SF protocol using the DMSO/EG or DMSO/G combinations were better at preventing edema than G/EG for SF and DMSO/ G for SSV (P <0.05). In conclusion, we suggest the use of a DMSO/EG combination associated with slow freezing or vitrification in cryotubes for cryopreservation of testicular tissue of adult peccaries. |
publishDate |
2019 |
dc.date.none.fl_str_mv |
2019-12-18 2020-08-31T01:53:35Z 2020-01-27 2020-08-31T01:53:35Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
Citação com autor incluído no texto: Silva (2020) Citação com autor não incluído no texto: (SILVA, 2020) https://repositorio.ufersa.edu.br/handle/prefix/5397 |
identifier_str_mv |
Citação com autor incluído no texto: Silva (2020) Citação com autor não incluído no texto: (SILVA, 2020) |
url |
https://repositorio.ufersa.edu.br/handle/prefix/5397 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.relation.none.fl_str_mv |
SILVA, Andréia Maria da. Estabelecimento de protocolos para a criopreservação de tecido testicular de catetos (pecari tajacu linnaeus, 1758). 2020. 118 f. Tese (Doutorado em Ciência Animal), Universidade Federal Rural do Semi-Árido, Mossoró, 2019. |
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 Brasil Centro de Ciências Agrárias - CCA UFERSA Programa de Pós-Graduação em Ciência Animal |
publisher.none.fl_str_mv |
Universidade Federal Rural do Semi-Árido Brasil Centro de Ciências Agrárias - CCA UFERSA Programa de Pós-Graduação em Ciência Animal |
dc.source.none.fl_str_mv |
reponame:Repositório Digital da Universidade Federal Rural do Semi-Árido (RDU) 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 |
Repositório Digital da Universidade Federal Rural do Semi-Árido (RDU) |
collection |
Repositório Digital da Universidade Federal Rural do Semi-Árido (RDU) |
repository.name.fl_str_mv |
Repositório Digital da Universidade Federal Rural do Semi-Árido (RDU) - Universidade Federal Rural do Semi-Árido (UFERSA) |
repository.mail.fl_str_mv |
repositorio@ufersa.edu.br || admrepositorio@ufersa.edu.br |
_version_ |
1809747463385382912 |