Vitrificação de tecido ovariano de gatas domésticas: o tamanho do fragmento influencia a viabilidade pós descongelação?
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Data
2018-04-27
Autores
Gorricho, Camila Mario
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Universidade Estadual Paulista (Unesp)
Resumo
A criopreservação de ovário ou tecido ovariano permite a preservação do material genético de qualquer espécie animal que seja submetido à gonadectomia por indicação preventiva, terapêutica ou, até mesmo, por morte inesperada. O objetivo do presente trabalho foi avaliar se o tamanho do fragmento ovariano influencia ou não a resistência aos crioprotetores. Para tanto, os ovários foram colhidos de 34 gatas domésticas (várias raças, 1-5 anos de idade) por ovariectomia de rotina, transportados ao laboratório e depois seccionados em fragmentos de diferentes tamanhos (3 x 3 x 3mm, 5 x 3 x 3mm e 7 x 3 x 3mm) e destinados aleatoriamente aos grupos de controle (GC3, GC5 e GC7, respectivamente) ou vitrificados (GV3, GV5 e GV7, respectivamente). Os fragmentos vitrificados-aquecidos foram avaliados por histomorfologia e imunohistoquímica (para taxas de apoptose utilizando a caspase-3 clivada). A avaliação histológica demonstrou que 72,97% dos folículos presentes em GV3 e 72,58% nos fragmentos do grupo GV5 eram normais, enquanto que nos fragmentos do GV7 essa taxa foi de apenas 42,86%. A principal alteração morfológica foi o desprendimento das células epiteliais da membrana basal presentes em todos os grupos. Da mesma forma, a avaliação imunohistoquímica, utilizando a caspase 3, revelou uma pequena proporção de células apoptóticas nos fragmentos do grupo GV3 (53%), enquanto que no grupo GV7, 43,58% das células expressaram a caspase 3 clivada. Esses achados indicam que fragmentos seccionados em 3 x 3 x 3mm (27mm³) são mais adequados para a perfusão do crioprotetor, sem causar danos celular após o descongelamento.
Cryopreservation of ovary or ovarian tissue allows preservation of genetic material of any animal species that is submitted to gonadectomy for preventive, therapeutic or even by unexpected death. The aim of the present study was to investigate whether or not the size of the ovarian fragment influence its resistance to cryostorage. For that purpose, ovaries were collected from 34 queens (various breeds, age 1-5 y) by routine ovariectomy, transported to the laboratory and then sectioned in different sizes (3 x 3 x 3 mm, 5 x 3 x 3 mm and 7 x 3 x 3 mm) and randomly assigned to a control (GC3, GC5 and GC7, respectively) or vitrified (GV3, GV5 and GV7, respectively) groups. Vitrified-warmed fragments were evaluated by histomorphology and immunohistochemistry (for apoptotic rates by using cleaved caspase-3). Histological examination reveal that 72.97% of the follicles in GV3 and 72.58% in GV5 were normal while only 42.86% of the follicles in GV7. The main morphological alteration presented in all groups was a detachment of the epithelial cells by basement membrane. Similarly, immunohistochemistry evaluation using caspase 3 revealed a small proportion of apoptotic cells in GV3 (53%) while in GV7 43.58% of the cells expressed cleaved caspase-3. These findings indicate that fragments sectioned in 3 x 3 x 3 mm (27mm3) seems more adequate for perfusion of the cryoprotectant, causing less damage to the cell after vitrification-warming.
Cryopreservation of ovary or ovarian tissue allows preservation of genetic material of any animal species that is submitted to gonadectomy for preventive, therapeutic or even by unexpected death. The aim of the present study was to investigate whether or not the size of the ovarian fragment influence its resistance to cryostorage. For that purpose, ovaries were collected from 34 queens (various breeds, age 1-5 y) by routine ovariectomy, transported to the laboratory and then sectioned in different sizes (3 x 3 x 3 mm, 5 x 3 x 3 mm and 7 x 3 x 3 mm) and randomly assigned to a control (GC3, GC5 and GC7, respectively) or vitrified (GV3, GV5 and GV7, respectively) groups. Vitrified-warmed fragments were evaluated by histomorphology and immunohistochemistry (for apoptotic rates by using cleaved caspase-3). Histological examination reveal that 72.97% of the follicles in GV3 and 72.58% in GV5 were normal while only 42.86% of the follicles in GV7. The main morphological alteration presented in all groups was a detachment of the epithelial cells by basement membrane. Similarly, immunohistochemistry evaluation using caspase 3 revealed a small proportion of apoptotic cells in GV3 (53%) while in GV7 43.58% of the cells expressed cleaved caspase-3. These findings indicate that fragments sectioned in 3 x 3 x 3 mm (27mm3) seems more adequate for perfusion of the cryoprotectant, causing less damage to the cell after vitrification-warming.
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Palavras-chave
criopreservação, felinos, histologia, imunohistoquímica, ovário, cryopreservation, felids, histology, immunohistochemistry, ovary