Padronização de uma técnica de congelamento de sêmen em cães Standartization of a technique for canine semen freezing

Many researches have been developed to improve the canine semen cryopreservation technique. The aims of the present work were to determine the freezing extenders adequacy to freeze canine semen using a computadorized system. Two thaw temperature was evaluated as well. Five dogs were used and three ejaculates/animal were collected. The extenders tested were Tris-citric-yolk (G1), lactose-yolk (G2) and INRA 82 (G3), with 5% glycerol. A computadorized freezing system was used and cooling rate was -0,5oC/min, from 25-29oC to 5oC, the equilibration period was at 5oC/1h followed by freezing the samples at a rate of -20oC/min, from 5oC to -120oC. Thawing temperatures were: A) 37oC/30s and B) 52oC/10s followed by 37oC/30s. Evaluation post thaw attributes were spermatic motility and morphology, HOST and sperm membrane integrity by a fluorescence test using CFDA/PI. Motility and sperm morphology were better preserved with the extenders Tris-citric yolk and lactose-yolk than with INRA 82 (p 0.05) evaluations. Thawed temperature of 52oC provoked more acrosome abnormality than when 37oC was used (p<0.05). In conclusions, Tris- citric and Lactose-yolk were more efficient than INRA 82 to preserve the spermatozoa viability post-thaw and thawing at 37oC/30s showed to be more suitable when the cooling and freezing curve were -0,5oC/min -20oC/min, respectively.