Qualidade das células espermáticas criopreservadas obtidas de tecido testicular de gatos domésticos: influência do tamanho dos fragmentos e avaliação dos crioprotetores
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Data
2017-11-06
Autores
Macente, Beatrice Ingrid
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Universidade Estadual Paulista (Unesp)
Resumo
Esta pesquisa teve por justificativa ampliar os estudos acerca da criopreservação de tecido gonadal e células espermáticas de gatos domésticos, podendo servir de modelo experimental para felinos selvagens. Objetivou-se comparar os efeitos da criopreservação sobre diferentes tamanhos de fragmentos testiculares, empregando dois crioprotetores. Utilizaram-se os testículos de 31 gatos domésticos, submetidos à orquiectomia eletiva. Os testículos foram pesados e apenas os que apresentarem pesos entre 1 e 2 g foram empregados. O tecido testicular foi dissecado e cortado em fragmentos de tamanhos pré-determinados (0,3cm3 e 0,5cm3). Uma amostra foi direcionada para as avaliações a fresco: integridade de membrana e do DNA; histopatologia; incidência de apoptoses por imuno-histoquímica; e índice de peroxidação lipídica - TBARS. A criopreservação foi feita em meio Tris-gema Equex STM suplementado com 3% de crioprotetores (glicerol e propanediol) através da técnica congelação rápida. Após a descongelação, foram realizados os mesmos testes iniciais. Os primeiros resultados obtidos com os 12 gatos iniciais apontaram na avaliação histomorfológica e pelo teste de lipoperoxidação lipídica que o glicerol foi mais eficiente que o propanediol na criopreservação de fragmentos testiculares de gatos domésticos de 0,5cm3 (Capítulo 2). No experimento seguinte, avaliaram-se os fragmentos testiculares de outros 31 gatos domésticos, seccionados nos mesmos tamanhos e criopreservados nas mesmas condições do experimento anterior, empregando-se 3% glicerol ou 3% propanediol no meio diluente Tris-gema Equex STM, utilizando a técnica de congelamento rápido, seguindo das avaliações pelos testes para dano ao DNA dos espermatozoides por meio da acridina laranja; e análise dos fragmentos teciduais semi-quantitativamente por histomorfologia e imuno-histoquímica. A avaliação imuno-histoquímica foi mais eficiente em verificar os danos às células tubulares seminíferas, sendo possível observar a marcação diferenciada da caspase presente no citoplasma e núcleo das células espermáticas, bem como na cabeça dos espermatozoides. Por meio da caspase foram verificado que os fragmentos frescos já apresentavam danos consideráveis as células teciduais e que não diferiram dos achados para os fragmentos criopreservados, demonstrando a eficiência do protocolo de congelação adotado, sem a observação de superioridade entre os dois tamanhos de fragmentos ou entre os crioprotetores. Contudo, a avaliação apenas do espermatozoides tanto pela técnica de acridina laranja, como pela imuno-histoquímica, pode-se verificar a inferioridade do propanediol em relação ao glicerol para fragmentos de 0,5 cm³ (Capítulo 3). Novas pesquisas que possam complementar os testes realizados neste estudo, como o cultivo dos fragmentos, a fertilização in vitro por meio de ICSI, ou ainda, o uso dos fragmentos para xenotrasnplantes poderiam avaliar com maior acurácia os achados das pesquisas reportadas nesta tese.
This research had the justification to broaden the studies about the cryopreservation of gonadal tissue and sperm cells of domestic cats, and could serve as an experimental model for wild cats. The objective of this study was to compare the effects of cryopreservation on different sizes of testicular fragments using two cryoprotectants. The testicles of 31 domestic cats submitted to elective orchiectomy were used. The testes were weighed and only those weighing between 1 and 2 g were used. The testicular tissue was dissected and cut into fragments of predetermined sizes (0.3cm3 and 0.5cm3). A sample was directed to fresh evaluations: membrane integrity and DNA; histopathology; incidence of apoptosis by immunohistochemistry; and lipid peroxidation index - TBARS. Cryopreservation was done on Tris-egg yolk Equex STM medium supplemented with 3% cryoprotectants (glycerol and propanediol) by the rapid freezing technique. After thawing, the same initial tests were performed. The first results obtained with the 12 initial cats showed that glycerol was more efficient than propanediol in the cryopreservation of testicular fragments of domestic cats of 0.5 cm3 (Chapter 2). In the following experiment, the testicular fragments from 31 other domestic cats, sectioned in the same sizes and cryopreserved under the same conditions as in the previous experiment, were evaluated using 3% glycerol or 3% propanediol in the Tris- egg yolk Equex STM medium, using rapid freezing technique, followed by evaluations for DNA damage of the spermatozoa by acridine orange; and analysis of the tissue fragments semi-quantitatively by histomorphology and immunohistochemistry. The immunohistochemical evaluation was more efficient in verifying the damage to the seminiferous tubular cells, being possible to observe the differentiated marking of the caspase present in the cytoplasm and nucleus of the sperm cells, as well as in the spermatozoa head. By caspase, it was verified that the fresh fragments already presented considerable damage to the tissue cells and did not differ from the findings for the cryopreserved fragments, demonstrating the efficiency of the adopted freezing protocol, without the observation of superiority between the two sizes of fragments or between the cryoprotectants. However, the evaluation of spermatozoa by both acridine orange and immunohistochemistry, the inferiority of propanediol to glycerol can be verified for fragments of 0.5 cm³ (Chapter 3). New research that could complement the tests performed in this study, such as the culture of fragments, in vitro fertilization using ICSI, or the use of fragments for xenotransplantation could more accurately assess the findings of the research reported in this thesis.
This research had the justification to broaden the studies about the cryopreservation of gonadal tissue and sperm cells of domestic cats, and could serve as an experimental model for wild cats. The objective of this study was to compare the effects of cryopreservation on different sizes of testicular fragments using two cryoprotectants. The testicles of 31 domestic cats submitted to elective orchiectomy were used. The testes were weighed and only those weighing between 1 and 2 g were used. The testicular tissue was dissected and cut into fragments of predetermined sizes (0.3cm3 and 0.5cm3). A sample was directed to fresh evaluations: membrane integrity and DNA; histopathology; incidence of apoptosis by immunohistochemistry; and lipid peroxidation index - TBARS. Cryopreservation was done on Tris-egg yolk Equex STM medium supplemented with 3% cryoprotectants (glycerol and propanediol) by the rapid freezing technique. After thawing, the same initial tests were performed. The first results obtained with the 12 initial cats showed that glycerol was more efficient than propanediol in the cryopreservation of testicular fragments of domestic cats of 0.5 cm3 (Chapter 2). In the following experiment, the testicular fragments from 31 other domestic cats, sectioned in the same sizes and cryopreserved under the same conditions as in the previous experiment, were evaluated using 3% glycerol or 3% propanediol in the Tris- egg yolk Equex STM medium, using rapid freezing technique, followed by evaluations for DNA damage of the spermatozoa by acridine orange; and analysis of the tissue fragments semi-quantitatively by histomorphology and immunohistochemistry. The immunohistochemical evaluation was more efficient in verifying the damage to the seminiferous tubular cells, being possible to observe the differentiated marking of the caspase present in the cytoplasm and nucleus of the sperm cells, as well as in the spermatozoa head. By caspase, it was verified that the fresh fragments already presented considerable damage to the tissue cells and did not differ from the findings for the cryopreserved fragments, demonstrating the efficiency of the adopted freezing protocol, without the observation of superiority between the two sizes of fragments or between the cryoprotectants. However, the evaluation of spermatozoa by both acridine orange and immunohistochemistry, the inferiority of propanediol to glycerol can be verified for fragments of 0.5 cm³ (Chapter 3). New research that could complement the tests performed in this study, such as the culture of fragments, in vitro fertilization using ICSI, or the use of fragments for xenotransplantation could more accurately assess the findings of the research reported in this thesis.
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Palavras-chave
Felino, Criopreservação, Crioprotetor, Feline, Cryopreservation, Cryoprotector