Comparison of canine sperm quality under different temperature storage
Autor(a) principal: | |
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Data de Publicação: | 2014 |
Tipo de documento: | Dissertação |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10348/2871 |
Resumo: | Successful gamete cryopreservation is essential to preserve the genetic pool of several species. The canine semen is known to possess a certain resilience to chilling procedures, which has a limited interest for long term gamete preservation; however, when frozen, canine sperm suffers an important decrease in quality. This feature determined the development of different extenders aiming the protection of the cell and to increase the success of canine sperm freezing/thawed procedures. In parallel, development of new techniques allowing a more objective evaluation of putative sperm fertility were also developed. However, those methods do not explain why some stud males are “good freezers” while other are “bad” freezers, when both were selected at start as adequate for freezing. Further, no clear definition exists on the cut-off values for specific seminal parameters to distinguish between a fertile and infertile stud, although the analysis of semen is regularly performed to assess the male fertility. In this study we plan to evaluate cold treatment associated changes on sperm cells by using two different approaches. For that, semen was collected from 8 dogs frequently used as semen donors. Freshly ejaculated samples were assess (Control group) before its use for chilling (Treatment A) or freezing (Treatment B), using routine procedures. For treatment A, three different times were considered: at 1.5h, at 4h and at 24h of chilling (respectively times 1, 2 and 3). Sperm characteristics were assessed by the convention methods, CASA and hypoosmotic test (HOST) for motility and movement parameters, velocity fractions, cell morphology and membrane integrity. Additionally, a molecular approach was tried by the study of heat sock protein 70 (HSP70) immunoexpression in the spermatozoa. HSP70 has protective effects over cells under conditions of thermal or proteotoxic stress. In the study presented here, the more conservative tests confirmed that chilling is not an aggressive procedure for dog semen in comparison to freezing, provided that the samples have a minimum acceptable quality at start. In frozen/thawed samples it was found a decrease in sperm motility and an increase in the percentage of static cells, along with a loss of the sperm membrane integrity. In parallel, a decrease in HSP intensity of immunostaining and a dislocation of the immunoreaction towards the flagellum were observed in the frozen samples (treatment B), while for treatment A no significant changes were found in the pattern of HSP immunoexpression. The use of molecular marker may reprosent an increase value in the study of the mechanisms underlaying the cenine sperm sensitivity to cryopreservation. |
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Comparison of canine sperm quality under different temperature storageSémenAnálise do sémenMarcadores molecularesCriopreservaçãoSuccessful gamete cryopreservation is essential to preserve the genetic pool of several species. The canine semen is known to possess a certain resilience to chilling procedures, which has a limited interest for long term gamete preservation; however, when frozen, canine sperm suffers an important decrease in quality. This feature determined the development of different extenders aiming the protection of the cell and to increase the success of canine sperm freezing/thawed procedures. In parallel, development of new techniques allowing a more objective evaluation of putative sperm fertility were also developed. However, those methods do not explain why some stud males are “good freezers” while other are “bad” freezers, when both were selected at start as adequate for freezing. Further, no clear definition exists on the cut-off values for specific seminal parameters to distinguish between a fertile and infertile stud, although the analysis of semen is regularly performed to assess the male fertility. In this study we plan to evaluate cold treatment associated changes on sperm cells by using two different approaches. For that, semen was collected from 8 dogs frequently used as semen donors. Freshly ejaculated samples were assess (Control group) before its use for chilling (Treatment A) or freezing (Treatment B), using routine procedures. For treatment A, three different times were considered: at 1.5h, at 4h and at 24h of chilling (respectively times 1, 2 and 3). Sperm characteristics were assessed by the convention methods, CASA and hypoosmotic test (HOST) for motility and movement parameters, velocity fractions, cell morphology and membrane integrity. Additionally, a molecular approach was tried by the study of heat sock protein 70 (HSP70) immunoexpression in the spermatozoa. HSP70 has protective effects over cells under conditions of thermal or proteotoxic stress. In the study presented here, the more conservative tests confirmed that chilling is not an aggressive procedure for dog semen in comparison to freezing, provided that the samples have a minimum acceptable quality at start. In frozen/thawed samples it was found a decrease in sperm motility and an increase in the percentage of static cells, along with a loss of the sperm membrane integrity. In parallel, a decrease in HSP intensity of immunostaining and a dislocation of the immunoreaction towards the flagellum were observed in the frozen samples (treatment B), while for treatment A no significant changes were found in the pattern of HSP immunoexpression. The use of molecular marker may reprosent an increase value in the study of the mechanisms underlaying the cenine sperm sensitivity to cryopreservation.A criopreservação de gâmetas é essencial à conservação do pool genético das espécies. O sémen canino é conhecido por ter uma capacidade de resistência à refrigeração aceitável, mas quando congelado, perde potencial fecundante. Esta sensibilidade do sémen canino à congelação incentivou o desenvolvimento de vários diluidores que potencializassem o sucesso da técnica, tendo sido neste processo uma parte integrante o desenvolvimento de métodos relativamente objectivos para avaliação do potencial fecundante do sémen ou dose seminal. No entanto, estes métodos não conseguem explicar os motivos subjacentes à perda de fertilidade observada nem porque alguns machos se apresentam como “bons congeladores” e outros como “maus congeladores” quando todos eles foram seleccionados como adequados para congelar. E apesar de a análise de sémen ser rotineiramente usada para avaliar a fertilidade do macho, não está claramente definido que valores dentro dos parâmetros de sémen distinguem um macho fértil de um infértil. Neste trabalho propusemo-nos a avaliar as alterações associadas ao tratamento térmico pelo frio por dois tipos de metodologia. Assim foram obtidas amostras de sémen de 8 animais dadores regulares de sémen. As amostras foram analisadas a fresco (grupo controlo) e depois processadas de forma rotineira para refrigeração (Tratamento A) e congelação (tratamento B). No grupo de tratamento A foram ainda considerados 3 tempos (tempos 1, 2 e 3, respectivamente às 1.5h, 4h e 24h de refrigeração). O estudo comparativo do potencial fecundante foi realizado por intermédio de métodos convencionais, por CASA e pelo teste hipoosmótico(HOST), para a motilidade e parâmetros de movimento, características de velocidade e integridade funcional da membrana. Esta avaliação foi complementada pelo estudo de imunoexpressão de HSP70 (proteína de shock térmico 70), uma molécula com efeitos protectores sobre a célula em situações de stress térmico e proteotóxico, No trabalho agora apresentado, os testes mais conservadores permitiram confirmar que a refrigeração não é uma técnica muito agressiva para o espermatozóide canino, comparativamente à congelação. Associada a esta última foi observado uma perda na motilidade e na velocidade além de um aumento do nº de células com alteração na função de membrana. Em simultâneo, foi observado um decréscimo na intensidade de marcação para a HSP70, associada a uma deslocação da marcação para a cauda do espermatozóide. A utilização de marcadores moleculares pode revelar-se útil na identificação de mecanismos biológicos subjacentes à sobrevivência do espermatozóide à congelação.2014-01-20T10:49:06Z2014-01-20T00:00:00Z2014-01-20info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10348/2871engBorges, Paulo Alexandre Paulosinfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-02-02T13:00:20Zoai:repositorio.utad.pt:10348/2871Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T02:07:08.993313Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Comparison of canine sperm quality under different temperature storage |
title |
Comparison of canine sperm quality under different temperature storage |
spellingShingle |
Comparison of canine sperm quality under different temperature storage Borges, Paulo Alexandre Paulos Sémen Análise do sémen Marcadores moleculares Criopreservação |
title_short |
Comparison of canine sperm quality under different temperature storage |
title_full |
Comparison of canine sperm quality under different temperature storage |
title_fullStr |
Comparison of canine sperm quality under different temperature storage |
title_full_unstemmed |
Comparison of canine sperm quality under different temperature storage |
title_sort |
Comparison of canine sperm quality under different temperature storage |
author |
Borges, Paulo Alexandre Paulos |
author_facet |
Borges, Paulo Alexandre Paulos |
author_role |
author |
dc.contributor.author.fl_str_mv |
Borges, Paulo Alexandre Paulos |
dc.subject.por.fl_str_mv |
Sémen Análise do sémen Marcadores moleculares Criopreservação |
topic |
Sémen Análise do sémen Marcadores moleculares Criopreservação |
description |
Successful gamete cryopreservation is essential to preserve the genetic pool of several species. The canine semen is known to possess a certain resilience to chilling procedures, which has a limited interest for long term gamete preservation; however, when frozen, canine sperm suffers an important decrease in quality. This feature determined the development of different extenders aiming the protection of the cell and to increase the success of canine sperm freezing/thawed procedures. In parallel, development of new techniques allowing a more objective evaluation of putative sperm fertility were also developed. However, those methods do not explain why some stud males are “good freezers” while other are “bad” freezers, when both were selected at start as adequate for freezing. Further, no clear definition exists on the cut-off values for specific seminal parameters to distinguish between a fertile and infertile stud, although the analysis of semen is regularly performed to assess the male fertility. In this study we plan to evaluate cold treatment associated changes on sperm cells by using two different approaches. For that, semen was collected from 8 dogs frequently used as semen donors. Freshly ejaculated samples were assess (Control group) before its use for chilling (Treatment A) or freezing (Treatment B), using routine procedures. For treatment A, three different times were considered: at 1.5h, at 4h and at 24h of chilling (respectively times 1, 2 and 3). Sperm characteristics were assessed by the convention methods, CASA and hypoosmotic test (HOST) for motility and movement parameters, velocity fractions, cell morphology and membrane integrity. Additionally, a molecular approach was tried by the study of heat sock protein 70 (HSP70) immunoexpression in the spermatozoa. HSP70 has protective effects over cells under conditions of thermal or proteotoxic stress. In the study presented here, the more conservative tests confirmed that chilling is not an aggressive procedure for dog semen in comparison to freezing, provided that the samples have a minimum acceptable quality at start. In frozen/thawed samples it was found a decrease in sperm motility and an increase in the percentage of static cells, along with a loss of the sperm membrane integrity. In parallel, a decrease in HSP intensity of immunostaining and a dislocation of the immunoreaction towards the flagellum were observed in the frozen samples (treatment B), while for treatment A no significant changes were found in the pattern of HSP immunoexpression. The use of molecular marker may reprosent an increase value in the study of the mechanisms underlaying the cenine sperm sensitivity to cryopreservation. |
publishDate |
2014 |
dc.date.none.fl_str_mv |
2014-01-20T10:49:06Z 2014-01-20T00:00:00Z 2014-01-20 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
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publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10348/2871 |
url |
http://hdl.handle.net/10348/2871 |
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eng |
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eng |
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info:eu-repo/semantics/openAccess |
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openAccess |
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application/pdf |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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RCAAP |
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RCAAP |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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1799137160242135040 |