Efeito da inibição da fosfolipase C pelo U73122 sobre o sêmen ovino
| Autor(a) principal: | |
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| Data de Publicação: | 2016 |
| Tipo de documento: | Dissertação |
| Idioma: | por |
| Título da fonte: | Biblioteca Digital de Teses e Dissertações da UFRRJ |
| Texto Completo: | https://rima.ufrrj.br/jspui/handle/20.500.14407/14219 |
Resumo: | O objetivo do presente trabalho foi avaliar o efeito do U73122 sobre a congelabilidade do sêmen ovino, uma vez que este reagente pode evitar a capacitação prematura e reação acrossomal induzidas durante o processo de criopreservação. Para isso, ejaculados de quatro carneiros da raça Santa Inês foram obtidos com auxílio de vagina artificial. No ensaio I, espermatozoides de quatro ejaculados, in natura, foram submetidos à incubação de 4h a 37°C, suplementados ou não com as concentrações de 10, 20 ou 30μM do U73122, na presença ou ausência do glicerol. Parâmetros cinéticos foram avaliados ao decorrer da incubação, a fim de verificar o comportamento dos espermatozoides pelo uso do reagente, e selecionar doses a serem utilizadas durante a criopreservação. Dezesseis ejaculados foram utilizados no ensaio II, os quais foram criopreservados com diluentes Botubov® fração única, acrescidos ou não do reagente U73122 nas concentrações de 10 e 20μM. As amostras descongeladas foram avaliadas quanto à cinética espermática, pelo sistema “Computer Assisted Sperm Analysis” (CASA), e quanto à integridade de membranas pela associação de sondas fluorescentes iodeto de propídeo, PSA-FITC e JC-1. Adicionalmente, foram realizadas avaliações da capacitação espermática e reação acrossomal com o uso da sonda fluorescente hidrocloreto de clortetraciclina (CTC) antes e após as amostras de sêmen criopreservadas serem submetidas à indução da capacitação in vitro durante 4h de incubação a 37°C. Todos os resultados foram submetidos à análise de variância e ao teste de diferença mínima significativa (DMS) de Tukey ao nível de 5% de probabilidade. O tratamento dos espermatozoides in natura com diferentes concentrações do U73122, durante incubação de 4h a 37°C, revelou um efeito dose e tempo-dependente sobre a utilização deste reagente em espermatozoides. Além disso, a presença do glicerol potencializou o efeito tóxico do U73122 sobre as células. Nos espermatozoides criopreservados, o U73122 não afetou nenhum dos parâmetros cinéticos avaliados pelo sistema CASA, nem o número dos espermatozoides com as membranas plasmáticas, acrossomais e mitocondriais íntegras (PIAIC) ou lesadas (PLALS). As avaliações realizadas imediatamente após o descongelamento mostraram que a utilização de 10 ou 20μM do inibidor reduziu o percentual de espermatozoides capacitados e com acrossoma reagido em relação ao grupo controle (p<0,05). Após a indução da capacitação in vitro, houve uma redução (p<0,05) do número de espermatozoides não capacitados em todos os tratamentos sugerindo um efeito reversível do U73122 sobre os processos de capacitação espermática e reação acrossomal. Conclui-se que o uso de 10 μM do reagente U73122 não altera a cinética dos espermatozoides in natura, e que, concentrações de 10 ou 20 μM deste reagente podem ser utilizadas em diluentes para criopreservação, uma vez que previnem a capacitação prematura e reação acrossomal induzidas durante este processo, sem alterar a cinética e a integridade das membranas espermáticas. |
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Arrais, Aline MatosMello, Marco Roberto Bourg de190.517.028-92http://lattes.cnpq.br/3560332978218414Dias, Ângelo José Burla641.157.986-53http://lattes.cnpq.br/9765923327693409Brandão, ZandonadiOliveira, Rodrigo Vasconcelos de134.435.077-19http://lattes.cnpq.br/77879901766028812023-12-22T02:57:37Z2023-12-22T02:57:37Z2016-08-22ARRAIS, Aline Matos. Efeito da inibição da fosfolipase C pelo U73122 sobre o sêmen ovino. 2016. 50 f. Dissertação (Programa de Pós-Graduação em Medicina Veterinária, Patologia e Ciências Clínicas) - Universidade Federal Rural do Rio de Janeiro, Seropédica, 2016.https://rima.ufrrj.br/jspui/handle/20.500.14407/14219O objetivo do presente trabalho foi avaliar o efeito do U73122 sobre a congelabilidade do sêmen ovino, uma vez que este reagente pode evitar a capacitação prematura e reação acrossomal induzidas durante o processo de criopreservação. Para isso, ejaculados de quatro carneiros da raça Santa Inês foram obtidos com auxílio de vagina artificial. No ensaio I, espermatozoides de quatro ejaculados, in natura, foram submetidos à incubação de 4h a 37°C, suplementados ou não com as concentrações de 10, 20 ou 30μM do U73122, na presença ou ausência do glicerol. Parâmetros cinéticos foram avaliados ao decorrer da incubação, a fim de verificar o comportamento dos espermatozoides pelo uso do reagente, e selecionar doses a serem utilizadas durante a criopreservação. Dezesseis ejaculados foram utilizados no ensaio II, os quais foram criopreservados com diluentes Botubov® fração única, acrescidos ou não do reagente U73122 nas concentrações de 10 e 20μM. As amostras descongeladas foram avaliadas quanto à cinética espermática, pelo sistema “Computer Assisted Sperm Analysis” (CASA), e quanto à integridade de membranas pela associação de sondas fluorescentes iodeto de propídeo, PSA-FITC e JC-1. Adicionalmente, foram realizadas avaliações da capacitação espermática e reação acrossomal com o uso da sonda fluorescente hidrocloreto de clortetraciclina (CTC) antes e após as amostras de sêmen criopreservadas serem submetidas à indução da capacitação in vitro durante 4h de incubação a 37°C. Todos os resultados foram submetidos à análise de variância e ao teste de diferença mínima significativa (DMS) de Tukey ao nível de 5% de probabilidade. O tratamento dos espermatozoides in natura com diferentes concentrações do U73122, durante incubação de 4h a 37°C, revelou um efeito dose e tempo-dependente sobre a utilização deste reagente em espermatozoides. Além disso, a presença do glicerol potencializou o efeito tóxico do U73122 sobre as células. Nos espermatozoides criopreservados, o U73122 não afetou nenhum dos parâmetros cinéticos avaliados pelo sistema CASA, nem o número dos espermatozoides com as membranas plasmáticas, acrossomais e mitocondriais íntegras (PIAIC) ou lesadas (PLALS). As avaliações realizadas imediatamente após o descongelamento mostraram que a utilização de 10 ou 20μM do inibidor reduziu o percentual de espermatozoides capacitados e com acrossoma reagido em relação ao grupo controle (p<0,05). Após a indução da capacitação in vitro, houve uma redução (p<0,05) do número de espermatozoides não capacitados em todos os tratamentos sugerindo um efeito reversível do U73122 sobre os processos de capacitação espermática e reação acrossomal. Conclui-se que o uso de 10 μM do reagente U73122 não altera a cinética dos espermatozoides in natura, e que, concentrações de 10 ou 20 μM deste reagente podem ser utilizadas em diluentes para criopreservação, uma vez que previnem a capacitação prematura e reação acrossomal induzidas durante este processo, sem alterar a cinética e a integridade das membranas espermáticas.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorFAPERJ - Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de JaneiroThe aim of this study was to evaluate the effect of U73122 on freezing ability of ram semen, since this reagent could prevent premature capacitation and acrosome reaction induced during the cryopreservation process. For this, ejaculates from 4 Santa Ines rams were obtained with the aid of artificial vagina and were used in two experiments. In experiment I, in natura sperm from 4 ejaculated were subjected to incubation at 37°C during 4h, supplemented or not with concentrations of 10, 20 or 30μM of U73122 in the presence or absence of glycerol. Kinetic parameters were evaluated during the incubation, in order to verify the behavior of sperm by the use of the reagent, and selecting doses to be used during cryopreservation. Sixteen ejaculates were used in the experiment II, which were cryopreserved with Botubov® extender only fraction increased or not U73122 reagent in concentrations of 10 and 20μM. Thawed samples were evaluated for sperm kinetics using the "Computer Assisted Sperm Analysis (CASA) system, and for membranes integrity by the association of fluorescent probes propidium iodide, PSA-FITC and JC-1. In addition, capacitation and acrosome reaction were evaluated using fluorescent probe hydrochloride chlortetracycline (CTC) before and after the cryopreserved semen samples undergoing induction in vitro capacitation for 4h of incubation at 37°C. The data were submitted to analysis of variance and least significant difference test (DMS) of Tukey at 5% probability. The treatment of in nature spermatozoa with different concentrations of U73122 for 4h incubation at 37°C, revealed a dose-effect and time dependent on the use of this reagent in spermatozoa. Furthermore, the presence of glycerol potentiated the toxic effect of U73122 on the cells. In cryopreserved sperm, the U73122 did not affect any of the kinetic parameters evaluated by CASA system, nor the number of sperm with the plasma membrane, acrosomal and mitochondrial you merge (PIAIC) or injured (PLALS). The evaluations carried out immediately after thawing showed that the use of 10 or 20μM of inhibitor reduced the percentage of capacitated sperm and acrosome reacted in the control group (p>0.05). After induction of in vitro capacitation, there was a decrease (p<0.05) in the number of uncapacitated sperm in all treatments suggesting a reversible effect of U73122 on sperm capacitation process and acrosome reaction. In conclusion, that the use of 10 uM of U73122 reagent does not alter the kinetics of sperm in natura and, 10 or 20 uM concentrations of this reagent can be used in diluents for cryopreservation, since they prevent premature capacitation and acrosome reaction induced during this process, without altering the kinetics and the integrity of the sperm membrane.application/pdfporUniversidade Federal Rural do Rio de JaneiroPrograma de Pós-Graduação em Medicina Veterinária (Patologia e Ciências Clínicas)UFRRJBrasilInstituto de VeterináriaespermatozoidescongelamentocarneirospermfreezingramMedicina VeterináriaEfeito da inibição da fosfolipase C pelo U73122 sobre o sêmen ovinoEffect of U73122 inhibitor on freezing ability of ram semeninfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisABDEL-LATIF, A. T. A. A. Biochemical and functional interactions between the inosito l 1,4, 5 - trisphosphate - calcium and cyclic amp signalling systems in smooth muscle. 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S.; SCHAFER- SOMI, S. Involvement of cholesterol , calcium and progesterone in the induction of capacitation and acrosome reaction of mammalian spermatozoa. Animal Reproduction Science, v. 102, p. 181–193, 2007. 39 WOLKERS, W. F.; WALKER, J. M. Cryopreservation and Freeze-Drying Protocols. Hannover: [s.n.]. WU, H. et al. Sperm Factor Induces Intracellular Free Calcium Oscillations by Stimulating the Phosphoinositide Pathway 1. Biology of Reproduction, v. 64, p. 1338–1349, 2001. YANAGIMACHI, R. Mammalian Sperm Acrosome Reaction : Where Does It Begin Before Fertilization ? Biology of Reproduction, v. 85, p. 4–5, 2011. YÁNIZ, J. LUIS et al. Effect of diluent composition on the dynamics of sperm DNA fragmentation and other sperm quality parameters in ram during incubation at 37 ◦ C. Small Ruminant Research, v. 129, p. 92–96, 2015. ZHANG, D. et al. Strontium promotes calcium oscillations in mouse meiotic oocytes and early embryos through InsP3 receptors , and requires activation of phospholipase and the synergistic action of InsP3. Human Reproduction, v. 20, n. 11, p. 3053–3061, 2005.https://tede.ufrrj.br/retrieve/54934/2016%20-%20Aline%20Matos%20Arrais.pdf.jpghttps://tede.ufrrj.br/jspui/handle/jspui/2920Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2019-09-25T17:50:30Z No. of bitstreams: 1 2016 - Aline Matos Arrais.pdf: 1298839 bytes, checksum: 34142cbe4d5bf5d300fb857a95dfdd34 (MD5)Made available in DSpace on 2019-09-25T17:50:30Z (GMT). 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| dc.title.por.fl_str_mv |
Efeito da inibição da fosfolipase C pelo U73122 sobre o sêmen ovino |
| dc.title.alternative.eng.fl_str_mv |
Effect of U73122 inhibitor on freezing ability of ram semen |
| title |
Efeito da inibição da fosfolipase C pelo U73122 sobre o sêmen ovino |
| spellingShingle |
Efeito da inibição da fosfolipase C pelo U73122 sobre o sêmen ovino Arrais, Aline Matos espermatozoides congelamento carneiro sperm freezing ram Medicina Veterinária |
| title_short |
Efeito da inibição da fosfolipase C pelo U73122 sobre o sêmen ovino |
| title_full |
Efeito da inibição da fosfolipase C pelo U73122 sobre o sêmen ovino |
| title_fullStr |
Efeito da inibição da fosfolipase C pelo U73122 sobre o sêmen ovino |
| title_full_unstemmed |
Efeito da inibição da fosfolipase C pelo U73122 sobre o sêmen ovino |
| title_sort |
Efeito da inibição da fosfolipase C pelo U73122 sobre o sêmen ovino |
| author |
Arrais, Aline Matos |
| author_facet |
Arrais, Aline Matos |
| author_role |
author |
| dc.contributor.author.fl_str_mv |
Arrais, Aline Matos |
| dc.contributor.advisor1.fl_str_mv |
Mello, Marco Roberto Bourg de |
| dc.contributor.advisor1ID.fl_str_mv |
190.517.028-92 |
| dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/3560332978218414 |
| dc.contributor.advisor-co1.fl_str_mv |
Dias, Ângelo José Burla |
| dc.contributor.advisor-co1ID.fl_str_mv |
641.157.986-53 |
| dc.contributor.advisor-co1Lattes.fl_str_mv |
http://lattes.cnpq.br/9765923327693409 |
| dc.contributor.referee1.fl_str_mv |
Brandão, Zandonadi |
| dc.contributor.referee2.fl_str_mv |
Oliveira, Rodrigo Vasconcelos de |
| dc.contributor.authorID.fl_str_mv |
134.435.077-19 |
| dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/7787990176602881 |
| contributor_str_mv |
Mello, Marco Roberto Bourg de Dias, Ângelo José Burla Brandão, Zandonadi Oliveira, Rodrigo Vasconcelos de |
| dc.subject.por.fl_str_mv |
espermatozoides congelamento carneiro |
| topic |
espermatozoides congelamento carneiro sperm freezing ram Medicina Veterinária |
| dc.subject.eng.fl_str_mv |
sperm freezing ram |
| dc.subject.cnpq.fl_str_mv |
Medicina Veterinária |
| description |
O objetivo do presente trabalho foi avaliar o efeito do U73122 sobre a congelabilidade do sêmen ovino, uma vez que este reagente pode evitar a capacitação prematura e reação acrossomal induzidas durante o processo de criopreservação. Para isso, ejaculados de quatro carneiros da raça Santa Inês foram obtidos com auxílio de vagina artificial. No ensaio I, espermatozoides de quatro ejaculados, in natura, foram submetidos à incubação de 4h a 37°C, suplementados ou não com as concentrações de 10, 20 ou 30μM do U73122, na presença ou ausência do glicerol. Parâmetros cinéticos foram avaliados ao decorrer da incubação, a fim de verificar o comportamento dos espermatozoides pelo uso do reagente, e selecionar doses a serem utilizadas durante a criopreservação. Dezesseis ejaculados foram utilizados no ensaio II, os quais foram criopreservados com diluentes Botubov® fração única, acrescidos ou não do reagente U73122 nas concentrações de 10 e 20μM. As amostras descongeladas foram avaliadas quanto à cinética espermática, pelo sistema “Computer Assisted Sperm Analysis” (CASA), e quanto à integridade de membranas pela associação de sondas fluorescentes iodeto de propídeo, PSA-FITC e JC-1. Adicionalmente, foram realizadas avaliações da capacitação espermática e reação acrossomal com o uso da sonda fluorescente hidrocloreto de clortetraciclina (CTC) antes e após as amostras de sêmen criopreservadas serem submetidas à indução da capacitação in vitro durante 4h de incubação a 37°C. Todos os resultados foram submetidos à análise de variância e ao teste de diferença mínima significativa (DMS) de Tukey ao nível de 5% de probabilidade. O tratamento dos espermatozoides in natura com diferentes concentrações do U73122, durante incubação de 4h a 37°C, revelou um efeito dose e tempo-dependente sobre a utilização deste reagente em espermatozoides. Além disso, a presença do glicerol potencializou o efeito tóxico do U73122 sobre as células. Nos espermatozoides criopreservados, o U73122 não afetou nenhum dos parâmetros cinéticos avaliados pelo sistema CASA, nem o número dos espermatozoides com as membranas plasmáticas, acrossomais e mitocondriais íntegras (PIAIC) ou lesadas (PLALS). As avaliações realizadas imediatamente após o descongelamento mostraram que a utilização de 10 ou 20μM do inibidor reduziu o percentual de espermatozoides capacitados e com acrossoma reagido em relação ao grupo controle (p<0,05). Após a indução da capacitação in vitro, houve uma redução (p<0,05) do número de espermatozoides não capacitados em todos os tratamentos sugerindo um efeito reversível do U73122 sobre os processos de capacitação espermática e reação acrossomal. Conclui-se que o uso de 10 μM do reagente U73122 não altera a cinética dos espermatozoides in natura, e que, concentrações de 10 ou 20 μM deste reagente podem ser utilizadas em diluentes para criopreservação, uma vez que previnem a capacitação prematura e reação acrossomal induzidas durante este processo, sem alterar a cinética e a integridade das membranas espermáticas. |
| publishDate |
2016 |
| dc.date.issued.fl_str_mv |
2016-08-22 |
| dc.date.accessioned.fl_str_mv |
2023-12-22T02:57:37Z |
| dc.date.available.fl_str_mv |
2023-12-22T02:57:37Z |
| 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 |
ARRAIS, Aline Matos. Efeito da inibição da fosfolipase C pelo U73122 sobre o sêmen ovino. 2016. 50 f. Dissertação (Programa de Pós-Graduação em Medicina Veterinária, Patologia e Ciências Clínicas) - Universidade Federal Rural do Rio de Janeiro, Seropédica, 2016. |
| dc.identifier.uri.fl_str_mv |
https://rima.ufrrj.br/jspui/handle/20.500.14407/14219 |
| identifier_str_mv |
ARRAIS, Aline Matos. Efeito da inibição da fosfolipase C pelo U73122 sobre o sêmen ovino. 2016. 50 f. Dissertação (Programa de Pós-Graduação em Medicina Veterinária, Patologia e Ciências Clínicas) - Universidade Federal Rural do Rio de Janeiro, Seropédica, 2016. |
| url |
https://rima.ufrrj.br/jspui/handle/20.500.14407/14219 |
| dc.language.iso.fl_str_mv |
por |
| language |
por |
| dc.relation.references.por.fl_str_mv |
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