Efeitos de diferentes diluentes comerciais para congelação de sêmen canino
Carregando...
Data
2022-05-02
Autores
Rodrigues, Dalmyr Roza
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
O uso de sêmen congelado depende que sejam mantidos alguns atributos espermáticos para se obter sucesso na fertilização do oócito, e a adição de substâncias crioprotetoras promovem proteção ao espermatozoide durante a congelação. A criopreservação induz uma série de modificações físicas e bioquímicas nos espermatozoides que levam a alterações na célula espermática. Estas alterações estão associadas à perda da motilidade, ruptura da membrana plasmática e acrossomal, estresse oxidativo, entre outras, promovendo danos irreversíveis ao espermatozoide. Além de fatores relacionados a cadela, como momento da ovulação, oócitos imaturos, e local de inseminação, a utilização de sêmen canino criopreservado é prejudicada pela viabilidade espermática pós-descongelação, que em muitas vezes pode ser baixa em decorrência de danos tanto na estrutura quanto na função dos espermatozoides e podem ser atribuídos ao choque térmico durante a congelação. Esses danos podem ser diminuídos com o uso de diluentes adequados para a criopreservação. Assim, o objetivo deste estudo é avaliar a eficácia de três diluentes comerciais para congelação de sêmen canino, sendo 2 a base de gema de ovo (Gema1 e Gema2) e 1 a base de lecitina de soja (Lecitina), sobre a qualidade espermática pós-descongelação. O estudo foi dividido em 2 experimentos, no experimento 1 foram 10 animais utilizados. Após a colheita, cada ejaculado foi dividido em 3 alíquotas iguais, diluídos nos respectivos meios de congelação na concentração de 100x106 espermatozoides/mL, envazados em palhetas de 0,5mL, submetidos à congelação obedecendo os critérios de estabilização de acordo com os fabricantes, e armazenadas em nitrogênio líquido. No experimento 2, foram selecionados os dois melhores diluentes do experimento 1 para nova avaliação e comparação, aumentando o número de amostras diminuindo assim o erro amostral, então foram adicionados mais 5 animais aumentando o número de amostras em 50%. A descongelação foi realizada a 37ºC por 30 segundos. As amostras foram submetidas a análise computadorizada da cinética espermática pelo CASA e integridade das membranas e potencial mitocondrial por citometria de fluxo após descongelação. No experimento 1 os diluentes a base de gema de ovo apresentaram resultado significativamente superior em comparação ao meio a base de lecitina de soja, para os parâmetros de motilidade total, motilidade progressiva, espermatozoides com movimento rápido, integridade de membrana plasmática e acrossomal, estabilidade de membrana plasmática, e alto potencial mitocondrial. No experimento 2, os 2 melhores diluentes do experimento 1 foram os dois a base de gema de ovo, e houve diferença significativa entre os meios que apresentaram motilidade total de 59,9%±3,3 e 52,3%±2,2 para Gema1 e Gema2 respectivamente, na avaliação da membrana por citometria o meio Gema1 também apresentou melhores resultados que o meio Gema2, 54,5%±3,3 e 45,0%±4,0 para integridade de membrana plasmática e acrossomal, e 47,6%±4,5 e 37,3%±3,5 para estabilidade de membrana plasmática respectivamente. Em conclusão pode-se afirmar que a lecitina de soja não atingiu resultados satisfatórios para os parâmetros espermáticos avaliados comparados a gema de ovo, e entre os meios a base de gema de ovo o meio Gema1 apresentou resultados superiores para os parâmetros avaliados pós-descongelação para espécie canina.
The use of frozen semen depends on maintaining some sperm attributes for successful fertilization of the oocyte, and the addition of cryoprotective substances promotes protection to the sperm during freezing. Cryopreservation induces a series of physical and biochemical changes in sperm that lead to changes in the sperm cell. These changes are associated with loss of motility, rupture of the plasma and acrosomal membrane, oxidative stress, among others, promoting irreversible damage to the sperm. In addition to factors related to the bitch, such as time of ovulation, immature oocytes, and site of insemination, the use of cryopreserved canine semen is impaired by post-thawing sperm viability, which can often be low due to damage to both structure and structure. in sperm function and can be attributed to heat shock during freezing. These damages can be reduced with the use of diluents suitable for cryopreservation. Thus, the objective of this study is to evaluate the effectiveness of three commercial extenders for canine semen freezing, 2 of which are egg yolk based (Gema1 and Gem2) and 1 are soy lecithin (Lecithin) on sperm quality post-thawing. The study was divided into 2 experiments, in experiment 1 10 animals were used. After collection, each ejaculate was divided into 3 equal aliquots, diluted in the respective freezing media at a concentration of 100x106 spermatozoa/mL, poured into 0.5mL straws, subjected to freezing according to the stabilization criteria according to the manufacturers, and stored in liquid nitrogen. In experiment 2, the two best diluents from experiment 1 were selected for further evaluation and comparison, increasing the number of samples, thus decreasing the sampling error, then 5 more animals were added, increasing the number of samples by 50%. Thawing was performed at 37°C for 30 seconds. The samples were submitted to computerized analysis of sperm kinetics by CASA and membrane integrity and mitochondrial potential by flow cytometry post-thawing. In experiment 1, the egg yolk-based extenders presented a significantly superior result compared to the soy lecithin-based medium, for the parameters of total motility, progressive motility, fast moving spermatozoa, plasma and acrosomal membrane integrity, stability of plasma membrane, and high mitochondrial potential. In experiment 2, the 2 best extenders of experiment 1 were both egg yolk-based, and there was a significant difference between the media that showed total motility of 59.9%±3.3 and 52.3%±2.2 for Gema1 and Gema2 respectively, in the evaluation of the membrane by cytometry, the Gema1 medium also presented better results than the Gema2 medium, 54.5%±3.3 and 45.0%±4.0 for plasma and acrosomal membrane integrity, and 47.6%±4.5 and 37.3%±3.5 for plasma membrane stability respectively. In conclusion, it can be stated that soy lecithin did not achieve satisfactory results for the evaluated sperm parameters compared to egg yolk, and among the egg yolk-based medium, the Gema1 medium showed superior results for the parameters evaluated after thawing for canine species.
The use of frozen semen depends on maintaining some sperm attributes for successful fertilization of the oocyte, and the addition of cryoprotective substances promotes protection to the sperm during freezing. Cryopreservation induces a series of physical and biochemical changes in sperm that lead to changes in the sperm cell. These changes are associated with loss of motility, rupture of the plasma and acrosomal membrane, oxidative stress, among others, promoting irreversible damage to the sperm. In addition to factors related to the bitch, such as time of ovulation, immature oocytes, and site of insemination, the use of cryopreserved canine semen is impaired by post-thawing sperm viability, which can often be low due to damage to both structure and structure. in sperm function and can be attributed to heat shock during freezing. These damages can be reduced with the use of diluents suitable for cryopreservation. Thus, the objective of this study is to evaluate the effectiveness of three commercial extenders for canine semen freezing, 2 of which are egg yolk based (Gema1 and Gem2) and 1 are soy lecithin (Lecithin) on sperm quality post-thawing. The study was divided into 2 experiments, in experiment 1 10 animals were used. After collection, each ejaculate was divided into 3 equal aliquots, diluted in the respective freezing media at a concentration of 100x106 spermatozoa/mL, poured into 0.5mL straws, subjected to freezing according to the stabilization criteria according to the manufacturers, and stored in liquid nitrogen. In experiment 2, the two best diluents from experiment 1 were selected for further evaluation and comparison, increasing the number of samples, thus decreasing the sampling error, then 5 more animals were added, increasing the number of samples by 50%. Thawing was performed at 37°C for 30 seconds. The samples were submitted to computerized analysis of sperm kinetics by CASA and membrane integrity and mitochondrial potential by flow cytometry post-thawing. In experiment 1, the egg yolk-based extenders presented a significantly superior result compared to the soy lecithin-based medium, for the parameters of total motility, progressive motility, fast moving spermatozoa, plasma and acrosomal membrane integrity, stability of plasma membrane, and high mitochondrial potential. In experiment 2, the 2 best extenders of experiment 1 were both egg yolk-based, and there was a significant difference between the media that showed total motility of 59.9%±3.3 and 52.3%±2.2 for Gema1 and Gema2 respectively, in the evaluation of the membrane by cytometry, the Gema1 medium also presented better results than the Gema2 medium, 54.5%±3.3 and 45.0%±4.0 for plasma and acrosomal membrane integrity, and 47.6%±4.5 and 37.3%±3.5 for plasma membrane stability respectively. In conclusion, it can be stated that soy lecithin did not achieve satisfactory results for the evaluated sperm parameters compared to egg yolk, and among the egg yolk-based medium, the Gema1 medium showed superior results for the parameters evaluated after thawing for canine species.
Descrição
Palavras-chave
Criopreservação, Espermatozoide, Cães, Gema de ovo, Lecitina de Soja, Inseminação artificial, Cryopreservation, Sperm, Dogs, Egg Yolk, Soy Lecithin, Artificial insemination