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钝吻黄盖鲽(Pseudopleuronectes yokohamae)是鲆鲽鱼类中重要的天然捕捞和养殖对象,但由于生态环境变化、人工捕捞过度等因素,导致其种质资源数量降低,进行精子冷冻保存技术研究,对其种质资源保存具有重要意义。本研究以性成熟的雄性钝吻黄盖鲽为实验材料,对精子稀释液种类及成分、抗冻剂种类、激活精子海水盐度和冷冻精液授精实验等进行筛选,实验数据利用SPSS 19.0软件进行单因素方差分析和Student-Newman-Keuls分析。结果表明,利用MFs-3(8 g/L Na Cl+0.65 g/L KCl+15 g/L Glucose)稀释液分别与20%1,2丙二醇(1,2-propylene glycol,PG)和20%乙二醇(ethylene glycol,EG)抗冻剂作为精子抗冻保存液时,冷冻效果最好,其中PG组对应的精子活力、快速运动时间和寿命分别为(95.26±0.39)%、(46.00±1.00)s和(124.33±4.04)s,EG组则为(95.15±0.41)%、(45.67±0.58)s和(124.00±3.00)s。利用盐度为10~50的人工配制的海水激活解冻后的精子,发现当盐度为30时,精子活力高达(95.07±0.69)%,与对照组相比不存在显著性差异。将PG组和EG组冷冻保存的精子解冻后分别与其卵进行授精实验,PG组受精率和孵化率为(80.08±0.68)%和(77.44±1.76)%,EG组则为(80.17±0.45)%和(77.92±1.33)%,与鲜精相比无显著性差异。利用计算机辅助精子分析系统(computer-aided sperm analysis,CASA)测定MFs-3+20%PG和MFs-3+20%EG冷冻保存的钝吻黄盖鲽精子的各项运动参数,结果显示,二者的曲线运动速度(curvilinear velocity,VCL)、直线运动速度(straight line velocity,VSL)、平均鞭打频率(beat cross frequency,BCF)、运动的直线性(linearity,LIN)和运动的前向性(straightness,STR)的数值差异性不显著。本研究利用MFs-3+20%PG和MFs-3+20%EG成功冷冻了钝吻黄盖鲽的精子,为钝吻黄盖鲽精子冷冻保存技术、人工杂交育种繁殖及种质资源库的建立奠定了基础。
Pseudopleuronectes yokohamae is an important natural fishing and breeding object of flounder, but due to the change of ecological environment and over-fishing, the quantity of its germplasm resources is reduced, the sperm cryopreservation technology is studied, Its germplasm resources preservation is of great significance. In this study, sexual maturity of male blunt nose yellow capillaris as experimental material, the type and composition of sperm diluent, antifreeze species, activated sperm salinity and frozen sperm insemination experiments were screened, the experimental data using SPSS 19.0 software One-way ANOVA and Student-Newman-Keuls analysis. The results showed that with the dilutions of MFs-3 (8 g / L Na Cl + 0.65 g / L KCl + 15 g / L Glucose) and 20% 1,2-propylene glycol The best freezing effect was obtained when ethylene glycol (EG) cryoprotectant was used as cryopreservation solution for sperm, in which the corresponding sperm motility, rapid exercise time and life span were (95.26 ± 0.39)% and (46.00 ± 1.00) s and (124.33 ± 4.04) s, while the EG group was (95.15 ± 0.41)%, (45.67 ± 0.58) s and (124.00 ± 3.00) s respectively. The results showed that when the salinity was 30, the sperm motility was as high as (95.07 ± 0.69)%, which was not significantly different from the control group. The fertilized and frozen eggs of PG and EG were (80.08 ± 0.68)% and (77.44 ± 1.76)%, respectively, compared with EG (80.17 ± 0.45) % And (77.92 ± 1.33)%, no significant difference compared with fresh fine. The movement parameters of Cryptomeria baicalensis sperm cryopreserved by MFs-3 + 20% PG and MFs-3 + 20% EG were determined by computer-aided sperm analysis (CASA) The curvilinear velocity (VCL), straight line velocity (VSL), beat cross frequency (BCF), linearity (LIN) and forward movement Straightness, STR) numerical differences were not significant. In this study, we successfully frozen the sperm of Amorpha fruticosa with MFs-3 + 20% PG and MFs-3 + 20% EG. The results showed that Cryptomeria baicalensis sperm cryopreservation, artificial breeding and germplasm bank Establishment laid the foundation.