31 Dec 2010
The recent study were attempting to develop spermatogonial germ cellÂ transplantation as a tool to preserve and propagate male germ-plasm from endangeredÂ fish species, as well as to produce surrogate broodstock of commercially valuableÂ fish. Spermatogonia identification and testes dissociation were the first necessaryÂ steps to obtain highly amount and viable population of spermatogonia as donor cellsÂ for transplantation. Using giant gouramy testes as a model, spermatogonia wasÂ histological characterized and two methods of testes dissociations were comparedÂ (i.e. medium A contained 0.5% trypsin in PBS and medium B contained 0.5% trypsin andÂ DNase 10 IU/Î¼L in PBS complemented with CaCl2, Hepes and FCS). Optimal incubationÂ times (1, 2, 3, 4 and 5 hours) in dissociation medium were also determined. FreshlyÂ isolated testes of immature giant gouramy were minced in dissociation medium andÂ then incubated to get monodisperce cell suspension. Parameters observed wereÂ number and viability of spermatogonia (Ã¸ > 10 Î¼m). The viability was analyzed usingÂ trypan blue exclusion dye. The results showed that the average number ofÂ spermatogonia observed in medium B was higher than in medium A (P<0.05), meanwhileÂ the viability of spermatogonia between medium A and B were not significantly differentÂ (P>0.05). The viability of spermatogonia decreased by the increasing duration time ofÂ dissociation. The viability of spermatogonia started to decrease significantly in 2Â hours incubation time in medium A and 4 hours incubation time in medium B (P<0.05).Â In conclusion, application of dissociation medium B yielded higher number of viableÂ spermatogonia than dissociation medium A.
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