显微注射抗冻剂对牙鲆(Paralichthys olivaceus)胚胎毒性及冷敏感性的影响

摘要 鱼类胚胎冷冻保存经过多年研究，尽管有所突破，但目前仍未达到应用水平，其主要原因是由于鱼类胚胎体积大、卵膜通透性差等特点，导致传统的平衡法，无法使抗冻剂足够、有效的进入胚胎内部起到保护胚胎的作用。而在鱼类胚胎冷冻保存中利用显微注射技术，可直接将抗冻剂注射到鱼类胚胎卵黄囊内，这一定程度上弥补了普通平衡法的不足，提高抗冻剂对胚胎的保护程度。本研究主要对抗冻剂的选择、抗冻剂对牙鲆胚胎的注射剂量、注射浓度、以及注射后牙鲆胚胎的冷敏感性进行了比较分析。结果表明：1、牙鲆胚胎较适宜的显微注射剂量为750pl。2、几种抗冻剂注射牙鲆胚胎后的毒性大小相对排列为PVP>蔗糖，DMSO > MeOH > PG，其中注射PG获得的胚胎成活率和孵化率最高，注射PVP获得的胚胎成活率和孵化率最低，而PG与MeOH组成混合抗冻剂PM毒性更低获得的胚胎成活率和孵化率更高。而任何一种抗冻剂均随着浓度的增加其毒性随之增加。3、另外，在冷敏感实验中，显微注射6M的PM的胚胎胚胎，应用程序化法处理，以2℃/min的速率降至-20℃，平衡10min后解冻处理，发现注射PM的胚胎低温处理后成活率为25.07±1.57%，而6M的PM五步平衡法同步处理的对照组胚胎成活率为20.88±2.84%，说明显微注射的胚胎冷敏感性一定降低。

Study on toxicity and chilling sensitivity of flounder Paralichthys olivaceus embryos microinjected cryoprotants

In cryopreservation of fishes embryos, due to the characteristics of fishes embryos, such as big physical volume, the bad permeability of vitelline membrane, etc, traditional techniques for the incorporation of cryoprotectants have failed to protect all embryo compartments. But the method microinjection could incorporate cryoprotectants into the yolk sac of fish's embryos and could offset the shortage of traditional methods to some extent and raise the protection degree of eryoprotectants to embryos. In the present study, several factors relating to cryopreservation of flounder Paralichthys olivaceus embryos by vitrification were studied: cryoprotectants, microinjection volumes of cryoprotectants, microinjection concentration of cryoprotectants, and the cooling sensitivity of embryos after being microinjected. The results showed that 1, the becoming microinjcction volume was 750pl. 2, the toxicity of five single-agent cryoprotectants sequenced as follows PVP>Sucrose, DMSO>MeOH>PG. Among these cryoprotectants, the embryos injected with PG got the highest survival rate and hatching rate, with PVP got the lowest, however, there was a mixture of PG and MeOH (PM), its toxicity was much lower than PG and the embryos injected with it got higher survival rate and hatching rate than with PG. The toxicity of cryoprotectans increased as the concentration of its increased.3, the survival rate of embryos microinjected with 6mol/L PM was (25.07 ± 1.57)% after being coolled in-20 ℃ for 10 minutes by programmed cooling method, whenas ,the survival rate of controls dealt with five steps balance method was (20.88±2.84)%, which indicated that the chilling sensitivity of embryos microinjected decreased.