超低温冷冻对中华绒螯蟹胚胎形态结构的影响

Effects of cryopreservation on exterior shape and interior structure of Chinese mitten crab (Eriocheir sinensis) embryos

为了解超低温冷冻对胚胎形态结构的影响,以中华绒螯蟹为研究对象,运用石蜡切片、扫描电镜和透射电镜观察细胞分裂期、原肠期和原溞状幼体期胚胎,分别用玻璃化液处理和经超低温冷冻后外部形态和内部结构的变化。结果发现:(1)显微观察表明,细胞分裂期胚胎在玻璃化液中用二步平衡法处理后吸水膨胀明显,三步平衡后胚胎形态无明显变化,超低温冷冻后,卵黄物质从细胞中溢出,细胞破损严重;原肠期胚胎在玻璃化液中用二步平衡法处理后,外部形态与鲜胚无明显差异,经过冷冻后,所有胚胎内部变成粉红色,胚体由原来的透明变成不透明状,细胞膜边缘模糊似绒毛状;(2)扫描电镜观察,玻璃化液处理后的所有原肠期胚胎表面褶皱呈沟壑状,形成一层网状结构;透射电镜观察,处理后胚胎细胞内出现白色团块,细胞边缘变得粗糙有突起,细胞内冰腔清晰可见,空泡形成,80%以上线粒体解体,细胞破裂明显;(3)组织切片观察,原肠期胚胎细胞外面的膜脱落破损,胚层内有大小不一的冰腔,细胞内出现明显的空泡。原溞状幼体期胚胎经过玻璃化液处理后,外部形态与鲜胚间无明显区别,但经过超低温冷冻后,95%以上胚胎组织呈弥散状,部分卵黄物质碎裂成颗粒状,胚胎的细胞膜脱落,胚层内出现大量冰腔和空泡,90%胚胎表面皱缩凹陷,但仍有10%的胚胎表面保持光滑完整,表明原溞状幼体期胚胎是适合进行冷冻保存的时期。

In order to deeply understand the effects of cryopreservation on the configuration of embryos, we chose the Eriocheir sinensis as the object, and observed the exterior shapes and interior structure of different developmental stages embryos after treatment with cryoprotectants and cryopreservation by means of microscope, slice and electron microscopy. Results showed that： （1） Cleavage embryos swelled clearly after treatment with vitrifying solutions by two-step methods, exterior shape of the embryos had no significant difference compared to the control group by three-step equilibration methods, some yolk over- flowed from the inner cells and cells were damaged seriously after cryopreservation. （2） Exterior shape of gastrula stage embryos was similar to the control group after treatment with two-step equilibration methods, surface of gastrula stage embryo treated with cryoprotectants crinkled and shaped reticulation structure by scanning electron microscopy （SEM）, white gobbet appeared in cells of embryos and fringe of cell was coarse and protuberant by transmission electron microscopy （TEM）. After cryopreservation, color of em- bryos was pink, cell became opaque, the brim of cell membrane was blurry and shaped floss, segmental yolk was disintegrated into granule, the surface of embryos was sunken and wrinkled. （3） Cell membrane of embryos broke off, ice holes of different size appeared inside embryos, visible vacuoles appeared inside cells by slice observation. Ice holes in cells were very clear and some vacuoles appeared, about 80% mitochondria dissolved, breach of cells was very obvious by TEM. Exterior shape of the original zoea stage embryos had no difference compared to that of the control group after treatment with cryoprotectants by microscope and SEM. After cryopreservation, above 95% tissues shaped dispersion, partial yolk was dis- integrated into granule, cell membrane of embryos broke off, ice holes appeared inside embryos. Surface of 90% embryos was sunken and wrinkled, but the cuticle of 10% embryos retained slick and intact by SEM. The results implied that the original zoea stage of embryos was the appropriate stage for cryopreservation.