Optimization of a sperm cryopreservation protocol for spotted wolffish (Anarhichas minor Olafsen, 1772)
Autor(a) principal: | |
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Data de Publicação: | 2018 |
Tipo de documento: | Dissertação |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10400.1/12583 |
Resumo: | Spotted wolffish is a potential candidate for cold water aquaculture, presenting high growth rates in captivity, good fillet yielding and a valuable and tasty meat. Reproduction in captivity is dependent on in vitro fertilization, however, low sperm volume with relatively low cell concentration and the lack of gametes synchronization (simultaneous availability of mature eggs and sperm) represent a challenge for the aquaculture industry. In this context, it is crucial the development of protocols, such as sperm cryopreservation, that allow sperm storage and maximization of its use. Sperm was diluted in a solution described by Kime and Tveiten (2002), and four sequential experiments were conducted. First, three different cryoprotectants (DMSO; 1, 2-propanediol; and methanol) at different concentrations (5, 10, and 20%) were tested for their toxicity. Second, sperm was cryopreserved in 0.5ml straws, at different distances from the liquid nitrogen (1.5, 2.5, 4.5, and 7.5cm) that correspond to different freezing rates. Afterwards, two different thawing rates (1min at 5ºC; and 25s at 10ºC) were tested. Finally, the fertilization capacity of cryopreserved sperm was tested against fresh, and cryopreserved and then washed sperm. The ratio of eggs with normal cell divisions, abnormal divisions or undeveloped were counted at the two-cell division stage. The trial on cryoprotectants toxicity revealed no differences between the control samples and cryoprotectants at concentration up to DMSO 10%, 1, 2-propanediol 10%, and methanol 20%, thus, these were chosen to use on the freezing trial. Motility values after freezing decreased for all the cryoprotectants, however, methanol revealed to have the lowest protective capacity and DMSO the highest throughout all the freezing rates, while propanediol presented the best results when freeze at 4.5 cm from liquid nitrogen. The thawing trial was carried using only DMSO 10% and 1, 2-propanediol 10%, revealing slight differences between the two temperatures. However, the best results were obtained using DMSO 10%. For the fertilization trial we selected the aforementioned cryoprotectant and thawing at 5ºC for 1min. Cryopreserved sperm showed lower fertilization capacity at a concentration of 5x104 spz/egg compared with fresh sperm. Nevertheless, at a concentration of 5x105 spz/egg, similar fertilizations rates to the fresh sperm were obtained. There was no improvement in the fertilization rates after washing the sperm to remove the cryoprotectant. An over dosage of DMSO revealed minor effects in abnormal divisions and lower number of developed eggs. To cryopreserve spotted wolffish sperm it is recommended that sperm is diluted on the extender described by Kime and Tveiten (2002), containing 10% DMSO, loaded on 0.5ml straws, freeze at a height of 4.5 or 7.5cm from liquid nitrogen for 10min and thawed for 1min at 5ºC. To obtain fertilization ratios equivalent to those obtained with fresh sperm, in vitro fertilization should be performed with a concentration of 5x105 cryopreserved sperm cells per egg. DMSO did not affect the eggs in the tested conditions during the contact time of 4 hours. |
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Optimization of a sperm cryopreservation protocol for spotted wolffish (Anarhichas minor Olafsen, 1772)Peixe-lobo pintadoAnarhichas minorSémenCriopreservaçãoMotilidadeDomínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e TecnologiasSpotted wolffish is a potential candidate for cold water aquaculture, presenting high growth rates in captivity, good fillet yielding and a valuable and tasty meat. Reproduction in captivity is dependent on in vitro fertilization, however, low sperm volume with relatively low cell concentration and the lack of gametes synchronization (simultaneous availability of mature eggs and sperm) represent a challenge for the aquaculture industry. In this context, it is crucial the development of protocols, such as sperm cryopreservation, that allow sperm storage and maximization of its use. Sperm was diluted in a solution described by Kime and Tveiten (2002), and four sequential experiments were conducted. First, three different cryoprotectants (DMSO; 1, 2-propanediol; and methanol) at different concentrations (5, 10, and 20%) were tested for their toxicity. Second, sperm was cryopreserved in 0.5ml straws, at different distances from the liquid nitrogen (1.5, 2.5, 4.5, and 7.5cm) that correspond to different freezing rates. Afterwards, two different thawing rates (1min at 5ºC; and 25s at 10ºC) were tested. Finally, the fertilization capacity of cryopreserved sperm was tested against fresh, and cryopreserved and then washed sperm. The ratio of eggs with normal cell divisions, abnormal divisions or undeveloped were counted at the two-cell division stage. The trial on cryoprotectants toxicity revealed no differences between the control samples and cryoprotectants at concentration up to DMSO 10%, 1, 2-propanediol 10%, and methanol 20%, thus, these were chosen to use on the freezing trial. Motility values after freezing decreased for all the cryoprotectants, however, methanol revealed to have the lowest protective capacity and DMSO the highest throughout all the freezing rates, while propanediol presented the best results when freeze at 4.5 cm from liquid nitrogen. The thawing trial was carried using only DMSO 10% and 1, 2-propanediol 10%, revealing slight differences between the two temperatures. However, the best results were obtained using DMSO 10%. For the fertilization trial we selected the aforementioned cryoprotectant and thawing at 5ºC for 1min. Cryopreserved sperm showed lower fertilization capacity at a concentration of 5x104 spz/egg compared with fresh sperm. Nevertheless, at a concentration of 5x105 spz/egg, similar fertilizations rates to the fresh sperm were obtained. There was no improvement in the fertilization rates after washing the sperm to remove the cryoprotectant. An over dosage of DMSO revealed minor effects in abnormal divisions and lower number of developed eggs. To cryopreserve spotted wolffish sperm it is recommended that sperm is diluted on the extender described by Kime and Tveiten (2002), containing 10% DMSO, loaded on 0.5ml straws, freeze at a height of 4.5 or 7.5cm from liquid nitrogen for 10min and thawed for 1min at 5ºC. To obtain fertilization ratios equivalent to those obtained with fresh sperm, in vitro fertilization should be performed with a concentration of 5x105 cryopreserved sperm cells per egg. DMSO did not affect the eggs in the tested conditions during the contact time of 4 hours.O peixe-lobo pintado (Anarhichas minor Olafsen, 1772), é um potencial candidato para a diversificação da aquacultura em águas frias. Esta espécie apresenta uma taxa de crescimento elevada em cativeiro, bom rendimento de carcaça, e uma carne bastante valorizada pelo seu sabor. A sua reprodução em cativeiro está dependente da fertilização in vitro, no entanto, o baixo volume de sémen com relativa baixa concentração de células, associado à frequente falta de sincronização entre a produção de sémen por parte dos machos e a libertação ovos por parte das fêmeas representa um desafio para as empresas de aquacultura. Tendo estes factos em consideração, o desenvolvimento de protocolos de criopreservação, que permitem o armazenamento de sémen por tempo indeterminado e permitem também a maximização do seu uso e a melhor gestão de stock. As amostras de sémen de peixe-lobo pintado foram recolhidas usando a técnica de stripping, ao longo da época reprodutiva decorrida entre novembro e janeiro. A qualidade do sémen foi avaliada através dos parâmetros de mobilidade recolhidos pelo sistema CASA SCA 6.2. O sémen foi diluído na solução descrita por Kime e Tveiten (2012) que foi baseada nas análises ao plasma seminal desta espécie e é composta por 154 mM NaCl, 4,55 mM CaCl2, 2,37 mM MgSO4, 4,83 mM KHCO3, e 1 mM de glucose. Foram feitas quatro experiências sequenciais para examinar a resposta das células espermáticas a diferentes tratamentos. Primeiro, foi testada toxicidade de três crioprotectores diferentes, (DMSO, 1, 2-propanediol e metanol) a concentrações distintas (5, 10 e 20%). De seguida, o sémen foi congelado em palhinhas de criopreservação com um volume de 0,5ml, colocadas em caixas de criopreservação, a diferentes alturas em relação à superfície do azoto líquido (1,5, 2,5, 4,5 e 7,5 cm) para que fossem obtidas taxas de congelação distintas. De seguida, foram testadas duas taxas de descongelação (1 min a 5ºC, e 25 s a 10ºC). Finalmente, a capacidade de fertilização do sémen criopreservado foi testada contra a mesma do sémen fresco, e sémen criopreservado e posteriormente “lavado” para remover o crioprotector e reduzir a possível toxicidade inerente ao mesmo devido ao longo tempo de quatro horas de contacto entre os ovos e o sémen. O rácio de ovos desenvolvidos com divisões normais, divisões anormais, e não-desenvolvidos foi obtido no estado de duas divisões celulares, 20 a 24 horas após o início da fertilização. A experiência sobre a toxicidade dos crioprotectores não revelou diferenças significativas entre o controlo com sémen fresco e o sémen com os crioprotectores DMSO e 1, 2-propanediol até uma concentração de 10%, e metanol até 20%, por essa razão, estes foram os tratamentos escolhidos para proceder às seguintes experiências. Os valores de motilidade para o sémen criopreservado decresceram em relação ao controlo, para todos os tratamentos, no entanto, o sémen criopreservado utilizando metanol revelou uma motilidade bastante baixa, enquanto que a preservação feita com DMSO apresentou os valores mais elevados. O ensaio sobre a descongelação procedeu apenas utilizando os tratamentos com DMSO 10% e 1, 2-propanediol 10%. Foram reveladas diferenças mínimas entre as duas temperaturas de descongelação, no entanto, considerando os crioprotectores, o tratamento com DMSO apresentou os melhores valores de motilidade. O teste à capacidade de fertilização foi feito utilizando apenas este crioprotector, e uma descongelação por 1 minuto a 5ºC. O esperma criopreservado apresentou uma capacidade de fertilização mais baixa em relação ao controlo à concentração de 5x104 spz/ovo, no entanto, utilizando uma concentração de 5x105 spz/ovo foram obtidas taxas de fertilização similares às obtidas utlizando sémen fresco. Não foi observada nenhuma mais valia em relação à remoção do crioprotector por lavagem do sémen. A sobredosagem de DMSO revelou alguns efeitos no número crescente de divisões anormais, e na redução no número de ovos desenvolvidos. Para criopreservar sémen de peixe-lobo pintado é recomendado que o sémen seja diluído no extender descrito por Kime e Tveiten (2012), contendo DMSO a 10%, sendo de seguida inserido em palhinhas de criopreservação com a capacidade de 0,5 ml e congelado a 4,5 ou 7,5 cm da superfície do azoto líquido e descongelado a 5ºC por 1 minuto. De forma a serem obtidos os mesmo valores de fertilização equivalentes aos obtidos com sémen fresco, a fertilização in vitro deve ser feita utilizando uma concentração de 5x105 spz/ovo.Cabrita, ElsaSantos, José Beirão dosSapientiaSantana, João Paulo Ribeiro Proença2019-05-29T15:06:29Z2018-12-1320182018-12-13T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10400.1/12583TID:202243702enginfo: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:RCAAP2023-07-24T10:24:34Zoai:sapientia.ualg.pt:10400.1/12583Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T20:03:54.824989Repositório 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 |
Optimization of a sperm cryopreservation protocol for spotted wolffish (Anarhichas minor Olafsen, 1772) |
title |
Optimization of a sperm cryopreservation protocol for spotted wolffish (Anarhichas minor Olafsen, 1772) |
spellingShingle |
Optimization of a sperm cryopreservation protocol for spotted wolffish (Anarhichas minor Olafsen, 1772) Santana, João Paulo Ribeiro Proença Peixe-lobo pintado Anarhichas minor Sémen Criopreservação Motilidade Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
title_short |
Optimization of a sperm cryopreservation protocol for spotted wolffish (Anarhichas minor Olafsen, 1772) |
title_full |
Optimization of a sperm cryopreservation protocol for spotted wolffish (Anarhichas minor Olafsen, 1772) |
title_fullStr |
Optimization of a sperm cryopreservation protocol for spotted wolffish (Anarhichas minor Olafsen, 1772) |
title_full_unstemmed |
Optimization of a sperm cryopreservation protocol for spotted wolffish (Anarhichas minor Olafsen, 1772) |
title_sort |
Optimization of a sperm cryopreservation protocol for spotted wolffish (Anarhichas minor Olafsen, 1772) |
author |
Santana, João Paulo Ribeiro Proença |
author_facet |
Santana, João Paulo Ribeiro Proença |
author_role |
author |
dc.contributor.none.fl_str_mv |
Cabrita, Elsa Santos, José Beirão dos Sapientia |
dc.contributor.author.fl_str_mv |
Santana, João Paulo Ribeiro Proença |
dc.subject.por.fl_str_mv |
Peixe-lobo pintado Anarhichas minor Sémen Criopreservação Motilidade Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
topic |
Peixe-lobo pintado Anarhichas minor Sémen Criopreservação Motilidade Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
description |
Spotted wolffish is a potential candidate for cold water aquaculture, presenting high growth rates in captivity, good fillet yielding and a valuable and tasty meat. Reproduction in captivity is dependent on in vitro fertilization, however, low sperm volume with relatively low cell concentration and the lack of gametes synchronization (simultaneous availability of mature eggs and sperm) represent a challenge for the aquaculture industry. In this context, it is crucial the development of protocols, such as sperm cryopreservation, that allow sperm storage and maximization of its use. Sperm was diluted in a solution described by Kime and Tveiten (2002), and four sequential experiments were conducted. First, three different cryoprotectants (DMSO; 1, 2-propanediol; and methanol) at different concentrations (5, 10, and 20%) were tested for their toxicity. Second, sperm was cryopreserved in 0.5ml straws, at different distances from the liquid nitrogen (1.5, 2.5, 4.5, and 7.5cm) that correspond to different freezing rates. Afterwards, two different thawing rates (1min at 5ºC; and 25s at 10ºC) were tested. Finally, the fertilization capacity of cryopreserved sperm was tested against fresh, and cryopreserved and then washed sperm. The ratio of eggs with normal cell divisions, abnormal divisions or undeveloped were counted at the two-cell division stage. The trial on cryoprotectants toxicity revealed no differences between the control samples and cryoprotectants at concentration up to DMSO 10%, 1, 2-propanediol 10%, and methanol 20%, thus, these were chosen to use on the freezing trial. Motility values after freezing decreased for all the cryoprotectants, however, methanol revealed to have the lowest protective capacity and DMSO the highest throughout all the freezing rates, while propanediol presented the best results when freeze at 4.5 cm from liquid nitrogen. The thawing trial was carried using only DMSO 10% and 1, 2-propanediol 10%, revealing slight differences between the two temperatures. However, the best results were obtained using DMSO 10%. For the fertilization trial we selected the aforementioned cryoprotectant and thawing at 5ºC for 1min. Cryopreserved sperm showed lower fertilization capacity at a concentration of 5x104 spz/egg compared with fresh sperm. Nevertheless, at a concentration of 5x105 spz/egg, similar fertilizations rates to the fresh sperm were obtained. There was no improvement in the fertilization rates after washing the sperm to remove the cryoprotectant. An over dosage of DMSO revealed minor effects in abnormal divisions and lower number of developed eggs. To cryopreserve spotted wolffish sperm it is recommended that sperm is diluted on the extender described by Kime and Tveiten (2002), containing 10% DMSO, loaded on 0.5ml straws, freeze at a height of 4.5 or 7.5cm from liquid nitrogen for 10min and thawed for 1min at 5ºC. To obtain fertilization ratios equivalent to those obtained with fresh sperm, in vitro fertilization should be performed with a concentration of 5x105 cryopreserved sperm cells per egg. DMSO did not affect the eggs in the tested conditions during the contact time of 4 hours. |
publishDate |
2018 |
dc.date.none.fl_str_mv |
2018-12-13 2018 2018-12-13T00:00:00Z 2019-05-29T15:06:29Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/masterThesis |
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masterThesis |
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publishedVersion |
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http://hdl.handle.net/10400.1/12583 TID:202243702 |
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eng |
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