Transporte e criopreservação de tecido testicular de gatos domésticos em meio contendo hipotaurina
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Data
2020-12-01
Autores
Senna, Izabella Pazzoto Alves
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Universidade Estadual Paulista (Unesp)
Resumo
Durante o processo de criopreservação de gametas ou tecidos, as células podem sofrer danos morfofuncionais causados pelo choque térmico ou estresse osmótico e oxidativo. Nesse contexto, propomos que a adição de antioxidantes nos meios durante este processo pode promover proteção das células contra a ação dos radicais livres. Assim, o objetivo deste estudo foi investigar os efeitos da suplementação de hipotaurina nos meios de transporte e congelação de tecido testicular de gatos domésticos. Para isso, testículos (n = 30) de quinze gatos foram transportados a aproximadamente 4ºC por 1 ou 6 horas e criopreservados em meios contendo diferentes concentrações de hipotaurina. Após os períodos de refrigeração e pós-descongelação os espermatozoides obtidos foram analisados quanto à integridade de membrana e DNA. Durante a refrigeração a integridade da membrana plasmática não foi afetada pelo tempo ou pelos diferentes meios de transporte (P > 0,05). Quando a hipotaurina foi adicionada apenas no meio de transporte e procedeu-se a criopreservação, a integridade da membrana plasmática dos espermatozoides testiculares somente se manteve no meio contendo 25 mM de hipotaurina (P = 0,08), nos demais houve diminuição significativa após a criopreservação quando o tecido foi refrigerado por 6 horas (P = 0,04 e P = 0,02, respectivamente). A integridade do DNA não foi afetada pelo tempo de refrigeração nem pelo meio de transporte. Quando analisada a influência da hipotaurina tanto no meio de transporte quanto no meio de criopreservação, observou-se diminuição na porcentagem de espermatozoides com membrana e DNA íntegros quando utilizado crioprotetor com concentrações mais altas de antioxidante. Estas lesões foram possivelmente causadas por alteração da osmolaridade ou ainda pelo efeito redutor devido ao excesso de antioxidante. Pode-se concluir que a hipotaurina deve estar presente no meio de transporte de testículos de gatos domésticos, uma vez que a adição deste antioxidante, melhora as características dos espermatozoides recuperados pós-descongelação. Mais estudos são necessários para verificar as possíveis causas de prejuízo na qualidade espermática quando esse antioxidante é adicionado no meio de criopreservação.
During the cryopreservation process, the thermal shock or osmotic and oxidative stress can cause morphofunctional damage to gamete or tissue cells. In this context, the addition of antioxidants to the medium is proposed as a method for promoting cell protection against the action of free radicals. This study aims to investigate whether hypotaurine supplemented to the medium allows for longer transport times while reducing the damage caused to the testicular parenchyma of cats by the refrigeration and cryopreservation processes. For this, testicles (n = 30) from fifteen cats were transported at about 4ºC for 1 or 6 hours and cryopreserved in medium containing different concentrations of hypotaurine. In the post-refrigeration and post-thaw periods, the sperm samples were analyzed to determine membrane and DNA integrity. During refrigeration, the plasma membrane integrity was not affected by transportation time or the different transportation means (P> 0.05). After adding hypotaurine only in the transport medium and accomplished cryopreservation, the integrity of the plasma membrane of testicular sperm was maintained only in the medium with 25 mM of hypotaurine (P = 0.08), whereas plasma membrane integrity decreased significantly in the other two treatments, MTh0 and MTh10, after cryopreservation (P = 0.04 and P = 0.02, respectively) and after being refrigerated for 6 hours. DNA integrity was not affected by the refrigeration time or transportation means. However, when adding hypotaurine in both the transportation medium and the cryopreservation medium, the percentage of sperm with intact membrane and DNA decreased when the added cryoprotectant had higher antioxidant concentrations. The observed damage possibly resulted from either the changing osmolarity or the reduction effect due to an excess of antioxidants. The results show that hypotaurine must be present in the transportation medium used to transfer the testicles of domestic cats, since the added antioxidant protects the testicular tissue, improving the characteristics of sperm recovered after thawing. However, further studies are needed to determine the possible causes of impaired sperm quality when this antioxidant is added to the cryopreservation medium as well.
During the cryopreservation process, the thermal shock or osmotic and oxidative stress can cause morphofunctional damage to gamete or tissue cells. In this context, the addition of antioxidants to the medium is proposed as a method for promoting cell protection against the action of free radicals. This study aims to investigate whether hypotaurine supplemented to the medium allows for longer transport times while reducing the damage caused to the testicular parenchyma of cats by the refrigeration and cryopreservation processes. For this, testicles (n = 30) from fifteen cats were transported at about 4ºC for 1 or 6 hours and cryopreserved in medium containing different concentrations of hypotaurine. In the post-refrigeration and post-thaw periods, the sperm samples were analyzed to determine membrane and DNA integrity. During refrigeration, the plasma membrane integrity was not affected by transportation time or the different transportation means (P> 0.05). After adding hypotaurine only in the transport medium and accomplished cryopreservation, the integrity of the plasma membrane of testicular sperm was maintained only in the medium with 25 mM of hypotaurine (P = 0.08), whereas plasma membrane integrity decreased significantly in the other two treatments, MTh0 and MTh10, after cryopreservation (P = 0.04 and P = 0.02, respectively) and after being refrigerated for 6 hours. DNA integrity was not affected by the refrigeration time or transportation means. However, when adding hypotaurine in both the transportation medium and the cryopreservation medium, the percentage of sperm with intact membrane and DNA decreased when the added cryoprotectant had higher antioxidant concentrations. The observed damage possibly resulted from either the changing osmolarity or the reduction effect due to an excess of antioxidants. The results show that hypotaurine must be present in the transportation medium used to transfer the testicles of domestic cats, since the added antioxidant protects the testicular tissue, improving the characteristics of sperm recovered after thawing. However, further studies are needed to determine the possible causes of impaired sperm quality when this antioxidant is added to the cryopreservation medium as well.
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Palavras-chave
Antioxidante, Espermatozoide testicular, Integridade de DNA, Integridade de membrana, Antioxidant, Testicular sperm, DNA integrity, Membrane integrity