Ninhaus-Silveira Alexandre, Foresti Fausto, de Azevedo Alexandre, Agostinho Cláudio Angelo, Veríssimo-Silveira Rosicleire
Departamento de Biologia e Zootecnia - Universidade Estadual Paulista/Ilha Solteira, Av. Brasil, 56 - Centro Postal Box 31, CEP: 15385 - 000 Ilha Solteira, São Paulo, Brasil.
Zygote. 2009 Feb;17(1):45-55. doi: 10.1017/S0967199408004991. Epub 2008 Nov 20.
SummaryWhile the freezing techniques of mammal embryos have been providing promising results, the cryopreservation of teleostean eggs and embryos have remained unsuccessful up to now. Therefore, this work aimed to develop a procedure of cryogenic preservation of embryos of Prochilodus lineatus and to observe, at both structural and ultrastructural levels, the morphological alterations that took place after the application of freezing/thawing techniques. The embryos at the morula stage could not tolerate exposure to the cryoprotectants ethylene glycol monomethyl ether, propylene glycol monomethyl ether, methanol, dimethyl sulphoxide and propylene glycol, presenting 100% of mortality. Embryos at the 4- to 6-somites stage tolerated exposure to propylene glycol and dimethyl sulphoxide, and the results revealed no significant differences (alpha = 0.05) regarding survival from both treatments. None of the freezing, thawing and hydration protocols was effective on preserving embryo viability. The ultrastructural analyses of frozen and thawed embryos showed that cells from ectoderm, somites, notochord and endoderm were structurally intact, with well preserved nuclei and mitochondria. The yolk globules were able to tolerate the freezing process, but the yolk syncytial layer was unorganized, displaying an electron-dense and compacted appearance, collapsed reticules, nuclei with modified chromatin and ruptures on the plasmatic membrane at the contact zone with endoderm. It might be concluded that the procedures tested for freezing were unable to avoid the formation of intracellular ice crystals, leading to drastic morphological modifications and making P. lineatus embryos unviable.
摘要
虽然哺乳动物胚胎的冷冻技术已取得了令人鼓舞的成果，但硬骨鱼卵和胚胎的冷冻保存至今仍未成功。因此，本研究旨在开发一种巴西真鲷胚胎的低温保存方法，并在结构和超微结构水平上观察冷冻/解冻技术应用后发生的形态学变化。桑椹胚阶段的胚胎不能耐受暴露于冷冻保护剂乙二醇单甲醚、丙二醇单甲醚、甲醇、二甲基亚砜和丙二醇中，死亡率达100%。4至6体节阶段的胚胎能耐受暴露于丙二醇和二甲基亚砜中，两种处理的存活率结果显示无显著差异（α = 0.05）。冷冻、解冻和水化方案均不能有效保存胚胎活力。对冷冻和解冻胚胎的超微结构分析表明，外胚层、体节、脊索和内胚层的细胞结构完整，细胞核和线粒体保存良好。卵黄球能够耐受冷冻过程，但卵黄合胞体层无组织，呈现电子致密和致密的外观、网状结构塌陷、细胞核染色质改变以及与内胚层接触区域的质膜破裂。可以得出结论，所测试的冷冻程序无法避免细胞内冰晶的形成，导致形态发生剧烈改变，使巴西真鲷胚胎无法存活。
