Avaliação da qualidade folicular pós-criopreservação testando: um método alternativo de congelamento lento e hipotaurina no meio de transporte, de tecido gonadal de gatas

Abstract

Esta pesquisa teve por justificativa ampliar os estudos acerca da criopreservação de tecido ovariano de gatas domésticas, juntamente com o uso de antioxidante no meio de transporte, podendo servir de modelo experimental para felinos selvagens. Objetivou-se validar um método de congelamento lento sem a utilização de máquina (Capítulo 2). E comparar os efeitos da criopreservação sobre diferentes métodos vitrificação e congelamento lento, com e sem a adição de antioxidante hipotaurina no meio de transporte, em diferentes tempos de exposição, dos ovários (Capítulo 3). Foram utilizados os ovários de 320 gatas domésticas, submetidas à ovariectomia eletiva. O tecido ovariano foi dissecado e cortado em fragmentos de tamanhos pré-determinados 7 mm × 3 mm × 3 mm (63 mm3). Os fragmentos cortados foram divididos aleatoriamente sendo que metade foi direcionado para a avaliação a fresco e outra metade para criopreservação pelos métodos lento e rápido. Para o congelamento lento utilizou-se meio de cultivo MEM (earle) sem glutamina, com bicarbonato de sódio e antibiótico acrescido de dimetil sulfóxido. A vitrificação foi realizada empregando dimetil sulfóxido (DMSO) e solução de DAP 123 (acetamida 1M, DMSO 2M e propilenoglicol 3M). Fez-se análise dos fragmentos teciduais por histomorfologia, imuno-histoquímica utilizando a caspase-3 clivada. Os resultados adquiridos demonstraram a eficiência do protocolo de congelação testado, de maneira que não houve diferença entre o congelamento testado e o automatizado (p<0,05). Para hipotaurina no meio de transporte notou-se sua eficiência quando utilizada na refrigeração por 24 horas e criopreservação pelo método vitrificação (p<0,05). Assim, concluímos que o método alternativo de congelamento lento é eficiente e a hipotaurina ajuda a preservar a viabilidade folicular quando há a necessidade de refrigeração e vitrificação do fragmento ovariano.

This research had as justification to expand the studies about the cryopreservation of ovarian tissue of domestic cats, together with the use of antioxidant in the means of transport, being able to serve as an experimental model for wild cats. The objective was to validate a slow freezing method without using a machine (Chapter 2). And to compare the effects of cryopreservation on different methods of vitrification and slow freezing, with and without the addition of antioxidant hypotaurin in the transport medium, at different exposure times, of the ovaries (Chapter 3). The ovaries of 320 domestic cats, submitted to elective ovariectomy, were used. The ovarian tissue was dissected and cut into fragments of predetermined sizes 7 mm × 3 mm × 3 mm (63 mm3). The cut fragments were divided randomly, half of which was directed to fresh evaluation and the other half to cryopreservation by slow and fast methods. For slow freezing, MEM culture medium (earle) without glutamine, sodium bicarbonate and antibiotic plus dimethyl sulfoxide was used. Vitrification was performed using dimethyl sulfoxide (DMSO) and DAP 123 solution (1M acetamide, 2M DMSO and 3M propylene glycol). Tissue fragments were analyzed by histomorphology, immunohistochemistry using cleaved caspase-3. The ovaries of 320 domestic cats, submitted to elective ovariectomy, were used. The ovarian tissue was dissected and cut into fragments of predetermined sizes 7 mm × 3 mm × 3 mm (63 mm3). The cut fragments were divided randomly, half of which was directed to fresh evaluation and the other half to cryopreservation by slow and fast methods. For slow freezing, MEM culture medium (earle) without glutamine, sodium bicarbonate and antibiotic plus dimethyl sulfoxide was used. Vitrification was performed using dimethyl sulfoxide (DMSO) and DAP 123 solution (1M acetamide, 2M DMSO and 3M propylene glycol). Tissue fragments were analyzed by histomorphology, immunohistochemistry using cleaved caspase-3. The acquired results demonstrated the efficiency of the tested freezing protocol, so that there was no difference between the tested and the automated freezing (p <0.05). For hypotaurin in the transport medium, its efficiency was noted when used in refrigeration for 24 hours and cryopreservation by the vitrification method (p <0.05). Thus, we conclude that the alternative method of slow freezing is efficient and the hypotaurin helps to preserve follicular viability when there is a need for refrigeration and vitrification of the ovarian fragment.