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Marc Roger Jean Marie Henryhttp://lattes.cnpq.br/1408982969631585Maria Madalena Pessoa GuerraMariana Machado NevesMonique de Albuquerque LagaresSildivane Valcácia SilvaYamê Fabres Robaina Sancler da SilvaJanaina Torres Carreirahttp://lattes.cnpq.br/2255404091157254Beatriz Parzewski Neves2021-01-22T19:29:36Z2021-01-22T19:29:36Z2019-04-30http://hdl.handle.net/1843/34840https://orcid.org/0000-0003-2172-6567Este estudo teve como objetivo avaliar, em espermatozoides bubalinos, as diferenças estruturais e funcionais, que podem influenciar a sua resistência ao processo de congelamento-descongelamento. Um total de 90 ejaculados, coletados por meio de vagina artificial de 13 touros Murrah (Bubalus bubalis), foram avaliados, diluídos em meio Tris-ácido cítrico-frutose contendo 10% de lipoproteínas de baixa densidade, e submetidos ao congelamento-descongelamento. Durante o processo, os ejaculados foram avaliados por sistema computadorizado (CASA) em seis momentos: pré-resfriamento (PR), pré-congelamento (PC), cinco minutos pós-descongelamento (PD_T5) e a cada uma hora durante a incubação pós-descongelamento à 37 °C (PD_T60, PD_T120 e PD_T180). De acordo com a longevidade da motilidade total, obtida pela diferença entre as avaliações no PR e no PD_T180, foram formados dois grupos de acordo com a resistência espermática ao processo de congelamento-descongelamento: os ejaculados que perderam menos que 41,2% da motilidade total entre as duas avaliações compuseram o grupo de resistência espermática boa (RB, n=23), e aqueles que perderam mais que 65,1% compuseram o grupo de resistência espermática ruim (RR, n=22). Em cada ejaculado, foram avaliados por citometria de fluxo: integridade das membranas plasmática e acrossômica (MPA), estabilidade da membrana plasmática, potencial de membrana mitocondrial (HMP), produção de peróxido de hidrogênio intracelular (H2O2), produção de ânion superóxido (O2•-) e peroxidação lipídica (LPO), utilizando as sondas IP e FITC-PSA, YOPRO-1, MitoStatus Red, CM-H2DCFDA, MitoSOXTM Red e C11-BODYPY (Molecular Probes), respectivamente. Considerou-se como significativo p≤0,05. Os espermatozoides do grupo RR apresentaram motilidade progressiva e os parâmetros de cinética VCL, VSL, VAP, BCF e ALH inferiores, principalmente durante a incubação após o descongelamento. Também foram inferiores na MPA e na estabilidade da membrana plasmática, nos tempos PD_T5 e PD_T180. O metabolismo mitocondrial foi inferior nos espermatozoides do grupo RR, tanto na porcentagem de células com HMP, como na intensidade de HMP em células íntegras no PD_T5 e PD_T180, e em células lesadas, no PD_T5. No grupo RR, a produção de O2•- foi superior e a produção de H2O2 foi inferior, tanto no PD_T5 quanto no PD_T180. A LPO foi semelhante entre os dois grupos, em ambos os tempos avaliados (PD_T5 e PD_T180), mesmo após o desafio com ácido araquidônico, realizado no PD_T5. Por este estudo, sugere-se que a diferença da resistência espermática ao congelamento-descongelamento, entre os ejaculados de touros bubalinos, se relaciona com a sensibilidade da membrana plasmática e com o metabolismo mitocondrial, que resultam em alterações dos parâmetros de motilidade e cinética espermáticas, sem induzir danos oxidativos como a peroxidação lipídica.This study aimed to evaluate the structural and functional differences in buffalo sperm that could influence its resistance to the freezing-thawing process. A total of 90 ejaculates, collected by artificial vagina method from 13 Murrah buffalo bulls (Bubalus bubalis), were evaluated, diluted in Tris-citric acid-fructose containing 10% of low-density lipoproteins extender and submitted to the freeze-thaw protocol. Computer Assisted Sperm Analysis (CASA) was undertaken in six different moments during the process: pre-cooling (PC), pre-freezing (PF) and five, 60, 120 and 180 minutes after thawing under incubation at 37 °C (PT_T5, PT_T60, PT_T120 and PT_T180, respectively). Sperm resistance to freezing-thawing was calculated for each ejaculate by the difference between total motility at PC and that at PT_T180 moment. Two groups of divergent sperm freezing-thawing resistances were then created: RB group (n=23) was composed by the ejaculates that had lost less than 41,2% of total motility and RR group (n=22) was composed by the ejaculates that had lost more than 65,1% of total motility. Flow cytometry analyses were performed for each ejaculate using the respective fluorescence probe: plasmatic and acrosomal membrane integrity (MPA; IP and FITC-PSA), plasmatic membrane stability (YOPRO-1), mitochondrial membrane potential (HPM; MitoStatus Red), intracellular hydrogen peroxide production (H2O2; CM-H2DCFDA), superoxide anion production (O2•-; MitoSOXTM Red) e lipid peroxide (LPO; C11-BODYPY). For all analyses, p≤0,05 was considered as significant. Progressive motility and the kinematic parameters VCL, VSL, VAP, BCF and ALH were lower in RR sperm cells, mainly on the post-thawing incubation moments. MPA and plasmatic membrane stability values were also lower in RR group at the both post-thawing evaluation times, PT_T5 and PT_T180. Besides, mitochondrial metabolism was lower in RR spermatozoa as lower values of the percentage of cells with HMP and, HMP intensity in intact cells at PT_T5 and PT_T180 and in non-intact cells at PT_T5 were observed in that group. Superoxide anion production was found greater, whereas H2O2 production was lower in RR group at the two evaluation times during post-thawing incubation period (PT_T5 and PT_T180). LPO did not differ between groups at PT_T5 and at PT_T180, even after arachidonic acid challenge of sperm cells at PT_T5. This study suggests that differences in sperm freezing-thawing resistance observed between buffalo bull ejaculates are related to plasmatic membrane sensibility and mitochondrial metabolism that results in altered motility and kinematic parameters, but not in oxidative damages as lipid peroxidation.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorporUniversidade Federal de Minas GeraisPrograma de Pós-Graduação em Ciência AnimalUFMGBrasilVET - DEPARTAMENTO DE CLÍNICA E CIRURGIAPrograma Institucional de Internacionalização – CAPES - PrInthttp://creativecommons.org/licenses/by-nc-nd/3.0/pt/info:eu-repo/semantics/openAccessCriopreservação sêmenCitometria de fluxoCongelabilidadeBúfaloComparação de características estruturais e funcionais de espermatozoides bubalinos com maior ou menor sensibilidade à criopreservaçãoComparison of structural and functional characteristics of buffalo sperm with greater or lesser sensitivity to cryopreservationinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisreponame:Repositório Institucional da UFMGinstname:Universidade Federal de Minas Gerais (UFMG)instacron:UFMGORIGINALTese.Beatriz.Parzewski.Neves.pdfTese.Beatriz.Parzewski.Neves.pdfapplication/pdf2028006https://repositorio.ufmg.br/bitstream/1843/34840/1/Tese.Beatriz.Parzewski.Neves.pdf16882d8f7c883dbb1cf9ef074c1b7c87MD51CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8811https://repositorio.ufmg.br/bitstream/1843/34840/2/license_rdfcfd6801dba008cb6adbd9838b81582abMD52LICENSElicense.txtlicense.txttext/plain; charset=utf-82119https://repositorio.ufmg.br/bitstream/1843/34840/3/license.txt34badce4be7e31e3adb4575ae96af679MD531843/348402021-01-22 16:29:36.22oai:repositorio.ufmg.br:1843/34840TElDRU7Dh0EgREUgRElTVFJJQlVJw4fDg08gTsODTy1FWENMVVNJVkEgRE8gUkVQT1NJVMOTUklPIElOU1RJVFVDSU9OQUwgREEgVUZNRwoKQ29tIGEgYXByZXNlbnRhw6fDo28gZGVzdGEgbGljZW7Dp2EsIHZvY8OqIChvIGF1dG9yIChlcykgb3UgbyB0aXR1bGFyIGRvcyBkaXJlaXRvcyBkZSBhdXRvcikgY29uY2VkZSBhbyBSZXBvc2l0w7NyaW8gSW5zdGl0dWNpb25hbCBkYSBVRk1HIChSSS1VRk1HKSBvIGRpcmVpdG8gbsOjbyBleGNsdXNpdm8gZSBpcnJldm9nw6F2ZWwgZGUgcmVwcm9kdXppciBlL291IGRpc3RyaWJ1aXIgYSBzdWEgcHVibGljYcOnw6NvIChpbmNsdWluZG8gbyByZXN1bW8pIHBvciB0b2RvIG8gbXVuZG8gbm8gZm9ybWF0byBpbXByZXNzbyBlIGVsZXRyw7RuaWNvIGUgZW0gcXVhbHF1ZXIgbWVpbywgaW5jbHVpbmRvIG9zIGZvcm1hdG9zIMOhdWRpbyBvdSB2w61kZW8uCgpWb2PDqiBkZWNsYXJhIHF1ZSBjb25oZWNlIGEgcG9sw610aWNhIGRlIGNvcHlyaWdodCBkYSBlZGl0b3JhIGRvIHNldSBkb2N1bWVudG8gZSBxdWUgY29uaGVjZSBlIGFjZWl0YSBhcyBEaXJldHJpemVzIGRvIFJJLVVGTUcuCgpWb2PDqiBjb25jb3JkYSBxdWUgbyBSZXBvc2l0w7NyaW8gSW5zdGl0dWNpb25hbCBkYSBVRk1HIHBvZGUsIHNlbSBhbHRlcmFyIG8gY29udGXDumRvLCB0cmFuc3BvciBhIHN1YSBwdWJsaWNhw6fDo28gcGFyYSBxdWFscXVlciBtZWlvIG91IGZvcm1hdG8gcGFyYSBmaW5zIGRlIHByZXNlcnZhw6fDo28uCgpWb2PDqiB0YW1iw6ltIGNvbmNvcmRhIHF1ZSBvIFJlcG9zaXTDs3JpbyBJbnN0aXR1Y2lvbmFsIGRhIFVGTUcgcG9kZSBtYW50ZXIgbWFpcyBkZSB1bWEgY8OzcGlhIGRlIHN1YSBwdWJsaWNhw6fDo28gcGFyYSBmaW5zIGRlIHNlZ3VyYW7Dp2EsIGJhY2stdXAgZSBwcmVzZXJ2YcOnw6NvLgoKVm9jw6ogZGVjbGFyYSBxdWUgYSBzdWEgcHVibGljYcOnw6NvIMOpIG9yaWdpbmFsIGUgcXVlIHZvY8OqIHRlbSBvIHBvZGVyIGRlIGNvbmNlZGVyIG9zIGRpcmVpdG9zIGNvbnRpZG9zIG5lc3RhIGxpY2Vuw6dhLiBWb2PDqiB0YW1iw6ltIGRlY2xhcmEgcXVlIG8gZGVww7NzaXRvIGRlIHN1YSBwdWJsaWNhw6fDo28gbsOjbywgcXVlIHNlamEgZGUgc2V1IGNvbmhlY2ltZW50bywgaW5mcmluZ2UgZGlyZWl0b3MgYXV0b3JhaXMgZGUgbmluZ3XDqW0uCgpDYXNvIGEgc3VhIHB1YmxpY2HDp8OjbyBjb250ZW5oYSBtYXRlcmlhbCBxdWUgdm9jw6ogbsOjbyBwb3NzdWkgYSB0aXR1bGFyaWRhZGUgZG9zIGRpcmVpdG9zIGF1dG9yYWlzLCB2b2PDqiBkZWNsYXJhIHF1ZSBvYnRldmUgYSBwZXJtaXNzw6NvIGlycmVzdHJpdGEgZG8gZGV0ZW50b3IgZG9zIGRpcmVpdG9zIGF1dG9yYWlzIHBhcmEgY29uY2VkZXIgYW8gUmVwb3NpdMOzcmlvIEluc3RpdHVjaW9uYWwgZGEgVUZNRyBvcyBkaXJlaXRvcyBhcHJlc2VudGFkb3MgbmVzdGEgbGljZW7Dp2EsIGUgcXVlIGVzc2UgbWF0ZXJpYWwgZGUgcHJvcHJpZWRhZGUgZGUgdGVyY2Vpcm9zIGVzdMOhIGNsYXJhbWVudGUgaWRlbnRpZmljYWRvIGUgcmVjb25oZWNpZG8gbm8gdGV4dG8gb3Ugbm8gY29udGXDumRvIGRhIHB1YmxpY2HDp8OjbyBvcmEgZGVwb3NpdGFkYS4KCkNBU08gQSBQVUJMSUNBw4fDg08gT1JBIERFUE9TSVRBREEgVEVOSEEgU0lETyBSRVNVTFRBRE8gREUgVU0gUEFUUk9Dw41OSU8gT1UgQVBPSU8gREUgVU1BIEFHw4pOQ0lBIERFIEZPTUVOVE8gT1UgT1VUUk8gT1JHQU5JU01PLCBWT0PDiiBERUNMQVJBIFFVRSBSRVNQRUlUT1UgVE9ET1MgRSBRVUFJU1FVRVIgRElSRUlUT1MgREUgUkVWSVPDg08gQ09NTyBUQU1Cw4lNIEFTIERFTUFJUyBPQlJJR0HDh8OVRVMgRVhJR0lEQVMgUE9SIENPTlRSQVRPIE9VIEFDT1JETy4KCk8gUmVwb3NpdMOzcmlvIEluc3RpdHVjaW9uYWwgZGEgVUZNRyBzZSBjb21wcm9tZXRlIGEgaWRlbnRpZmljYXIgY2xhcmFtZW50ZSBvIHNldSBub21lKHMpIG91IG8ocykgbm9tZXMocykgZG8ocykgZGV0ZW50b3IoZXMpIGRvcyBkaXJlaXRvcyBhdXRvcmFpcyBkYSBwdWJsaWNhw6fDo28sIGUgbsOjbyBmYXLDoSBxdWFscXVlciBhbHRlcmHDp8OjbywgYWzDqW0gZGFxdWVsYXMgY29uY2VkaWRhcyBwb3IgZXN0YSBsaWNlbsOnYS4KCg==Repositório InstitucionalPUBhttps://repositorio.ufmg.br/oaiopendoar:2021-01-22T19:29:36Repositório Institucional da UFMG - Universidade Federal de Minas Gerais (UFMG)false
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