真鲷（Pagrus major）胚胎超低温损伤机理研究

IOCAS-IR > 海洋环流与波动重点实验室

	真鲷（Pagrus major）胚胎超低温损伤机理研究
其他题名	Studies on cryoinjury mechanism in red seabream（Pagrus major） embryo cryopreservation
	张莲蕾
学位类型	博士
	2009-06-01
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	真鲷胚胎冷冻保存冷冻损伤机理抗冻剂冰晶形成
摘要	鱼类胚胎由于其自身结构特征：体积大、含水量高、多室结构等，迄今超低温保存尚未成功。超低温保存过程中所造成的冷冻损伤是制约鱼类胚胎超低温保存成功与否的关键，具体表现为渗透压影响、抗冻剂毒性、冰晶损伤等。系统研究并阐明鱼类胚胎冷冻损伤机理，是成功建立鱼类胚胎超低温保存技术的基础。本论文主要针对胚胎对渗透压的耐受性、抗冻剂对胚胎的渗透性、降温速率对胚胎内外冰晶形成温度的影响等冷冻损伤机理进行了系统研究，主要研究结果如下： 1.通过检测胚胎在不同浓度人工海水（0%、25%、50%、75%、1×、2×、3×、4×，渗透压范围0~3740 mOsm/kg）中的孵化率，确定了真鲷不同发育时期胚胎对渗透压的耐受范围，以及心跳期胚胎浸泡不同时间对渗透压的耐受范围。结果显示：①真鲷2-4细胞期、原肠期、10-14体节期胚胎、心跳期和出膜前期胚胎孵化率>50%时渗透压的范围依次为：919~1391 mOsm/kg、919~1391 mOsm/kg、462 ~1391 mOsm/kg、232~1878 mOsm/kg和692~1391 mOsm/kg，表明心跳期胚胎对渗透压变化的耐受范围最广；②在不同浓度人工海水中分别浸泡10 min、30 min、1 h、5 h和10 h后，真鲷胚胎孵化率无显著变化的渗透压范围分别为0~2804 mOsm/kg、0~1878 mOsm/kg、232~1391 mOsm/kg、232~1391 mOsm/kg和919~1391 mOsm/kg；结果表明心跳期胚胎对渗透压的耐受范围随浸泡时间的延长而减小。 2.采用毛细管电泳技术检测胚胎内部DMSO的浓度，并且分析了胚胎孵化率和胚胎内部DMSO的浓度随浸泡时间变化与外部抗冻剂的关系。结果表明胚胎孵化率随胚胎外部抗冻剂溶液浓度和浸泡时间的增加而降低；胚胎内部DMSO浓度随胚胎外部抗冻剂溶液浓度和浸泡时间的增加而增加。对胚胎孵化率（y1）随抗冻剂溶液浓度（x）的变化进行一元三次多项式回归，当浸泡时间分别为10 min、30 min和60 min时，回归方程依次为：y1 = -2832.7x3 + 575.01x2 - 37.011x + 99.641（R2 = 0.9722）；y1 = 30288x3 - 16322x2 + 2077.3x + 27.603（R2 = 0.9876）；y1 = 16052x3 - 5985.2x2 - 32.696x + 119.6（R2 = 0.9124）。对胚胎内部DMSO浓度（y2）随抗冻剂溶液浓度（x）的变化进行回归，当浸泡时间分别为10 min、30 min和60 min时，回归方程依次为：y2 = 0.2584e6.7294x（R2 = 0.9876）；y2 = 0.2521e10.964x（R2 = 0.9644）；y2 = 0.4054e10.95x（R2 = 0.8954）。 3. 利用低温显微镜观察了不同降温速率（20、40、60、80、100、120℃/min）对胚胎内外冰晶形成温度的影响。胚胎外部冰晶形成温度（TEIF）随降温速率的增加显著下降，在降温速率大于80℃/min之后，TEIF随降温速率增加而降低的幅度减小；胚胎内部冰晶形成温度（TIIF）在降温速率小于80℃/min 时随降温速率的升高而降低，在降温速率大于80℃/min 时随降温速率的升高而升高；胚胎内外冰晶形成温度差值（TEIF - TIIF）在降温速率小于80℃/min时随降温速率的升高而增大，在降温速率大于80℃/min时随降温速率的升高而减小。 4. 在低温显微镜下观察了真鲷胚胎低温保存中有复活胚胎记录的保存方法在冷冻解冻过程中的冰晶形成过程，结果表明：①在冷冻过程中，玻璃化法冷冻的胚胎的内部冰晶形成温度（-53.70，-64.33℃）显著低于程序降温法（-17.51，-21.40℃）；而且在玻璃化法冷冻的胚胎内部冰晶形成温度高于外部冰晶后形成（-70.30℃），程序降温法中则相反，胚胎内部冰晶形成温度显著低于外部冰晶形成温度（-4.93，-5.00℃）；玻璃化法中，40%PG冷冻的胚胎外部溶液出现玻璃化现象，其他组均未出现；②在解冻过程中，各组均出现重结晶现象；解冻后，玻璃化法的胚胎完整率（62.82%）远高于程序降温法（9.21%）。
其他摘要	Successful fish embryo cryopreservation has not been achieved due to its large volume, large content of water, complex multi-compartmental system, etc. During the process of cooling and thawing, embryos are subjected to a series of cryoinjuries such as cold shock, ice formation, cryoprotectant toxicity and osmotic effect. Cryoinjury is one of the most important obstacles for cryopreservation. Understanding the mechanism of cryoinjury is the basis for establishing fish embryo cryopreservation method. This study investigated the red seabream embryo tolerance for osmotic pressure, penetrability to cryoprotectant, and the extra-and intra-cellular ice formation temperatures at different cooling rates. The detailed results are as follows: 1. We tested the hatching rates of embryos immersing in artificial sea water (ASW) with different concentrations (0%, 25%, 50%, 75%, 1×, 2×, 3×, 4×. osmotic pressure: 0~3740 mOsm/kg). The optimal osmotic pressure ranges for embryos in different stages, and for heart-beaten embryos in different exposure time were obtained, respectively. The result revealed that 1) For 2-4 cells embryo, gastrulae, 10-14 somite embryo, heart-beaten stage embryo and embryo before hatching, when the osmotic pressure of artificial sea water 919~1391 mOsm/kg, 919~1391 mOsm/kg, 462 ~1391 mOsm/kg, 232~1878 mOsm/kg and 692~1391 mOsm/kg, respectively, the hatching rate were high than 50%. And heart-beaten stage embryo showed the best tolerance for osmotic pressure. 2) For heart-beaten stage embryos, after immersing in ASW with different concentrations for 10 min, 30 min, 1 h, 5 h and 10 h, the osmotic pressure range , 0~2804 mOsm/kg, 0~1878 mOsm/kg, 232~1391 mOsm/kg, 232~1391 mOsm/kg and 919~1391 mOsm/kg, respectively, had no significant influence on embryo hatching rate. The result reveals that the heart-beaten embryo’s tolerance of osmotic pressure decreased with the increase of exposure time. 2. We tested the concentration of DMSO inside the embryo using capillary electrophoresis, and analyzed the relationship between inner-cellular DMSO concentration and outer cryoprotectent concentration. The results indicated that the hatching rate (y1) decreased with the increase of outer cryoprotectent concentration (x). When the exposure time were 10 min, 30 min and 60 min, the regression equation were y1 = -2832.7x3 + 575.01x2 - 37.011x + 99.641 (R2 = 0.9722), y1 = 30288x3 - 16322x2 + 2077.3x + 27.603 (R2 = 0.9876), y1 = 16052x3 - 5985.2x2 - 32.696x + 119.6 (R2 = 0.9124), respectively. The inner-cellular DMSO concentration (y2) increased with the increase of outer cryoprotectent concentration (x). When the exposure time were 10 min, 30 min and 60 min, the regression equation were, y2 = 0.2584e6.7294x (R2 = 0.9876); y2 = 0.2521e10.964x (R2 = 0.9644); y2 = 0.4054e10.95x (R2 = 0.8954), respectively. 3. We examined the effect of cooling rate (20, 40, 60, 80, 100 and 120 oC/min) on the temperature of extra-and inner- cellular ice formation (TEIF and TIIF) under cryomicroscope. TEIF decreased with the increase of cooling rate, and TEIF decreased slightly when the cooling rate was more than 80℃/min. TIIF decreased with the increase of cooling rate (<80 oC/min), and increased with the increase of cooling rate (>80 oC/min). While TEIF - TIIF increased with the increase of cooling rate (<80 oC/min), and decreased with the increase of cooling rate (>80 oC/min). 4. We observed the ice formation of four embryos cryopreservation methods under cryomicroscope. The results showed that ①In the cooling process, both TEIFs and TIIFs in programmed cooling groups were significantly higher than those in vitrification groups. And in vitrification groups, TIIF values were higher than those of TEIF, indicating intra-cellular ice formed earlier than extra-cellular ice, which was opposite to programmed cooling groups. In 40%PG vitrification group, the solutions outside the embryos vitrified, while other groups didn’t. ② In the cooling process, recrystallization had also occurred within the embryos in all four groups. After thawing, the percentages of embryo with intact morphology in vitrification groups (62.82%) were significantly higher than that in programmed cooling groups (9.21%).
页数	78
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/699
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	张莲蕾. 真鲷（Pagrus major）胚胎超低温损伤机理研究[D]. 海洋研究所. 中国科学院海洋研究所,2009.

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