LN_2保存锥栗种子的生理生化特性研究

运用LN2 ( - 196℃ )保存手段 ,结合添加和不添加防冻剂预处理 ,通过对锥栗种子LN2 保存前后种子质量特性和胚酶活性的分析 ,对其长期保存的可行性进行研究 ,结果表明 :含水量是影响锥栗种子和离体胚LN2 保存的重要因素 ,LN2 保存应进行适度脱水。当无防冻剂预处理时 ,LN2 保存宜采用 15 %～ 2 0 %含水量区间 ,冷解冻方式采用MF MT。当有防冻剂预处理时 ,各种酶活性的保存要比无预处理的要好 ,LN2 保存宜采用2 0 %～ 2 5 %含水量区间 ,冷解冻方式采用MF ST。     

Effects of five cryoprotective agents on quality of sheep epididymal spermatozoa during pre-freezing

The objective of this study was to test five cryoprotective agents during ram epidydimal spermatozoa incubation (at 4 °C), of up to 3 h, typical equilibration time before the freezing step begins, in order to establish a starting point for future freezing and thawing protocols. The parameters analyzed were: progressive motility (PM), vitality, and the plasma membrane functional integrity by the hypoosmotic swelling (HOS) test. Testes and epididymides were collected immediately after death. The tail of both epididymides were isolated and spermatozoa were recovered constituting one sample (n=20). A Tes–Tris–Yolk extender was employed. The extender contained five alternative CPAs: Dimethylacetamide (DMA), Dimethyl sulfoxide (DMSO), Ethylene glycol (EG), Glycerol (GLY) and Propylene glycol (PG) at three final concentrations: 2.5%, 5.0% and 10.0%. Control groups consisted of samples mixed only with the extender, without any CPA. All sample groups were exposed to the CPAs for 1 h or 3 h at 4 °C. EG exposure yielded better responses in both PM and HOS test parameters compared to extender only and also the other CPAs. There was no difference among all the treatments regarding vitality. EG (with best results at 2.5%) is thus proposed as a good CPA (followed by DMA as an explorable alternative) for the implementation of forthcoming ram epididymal spermatozoa freezing protocols.     

Cryoprotectant microinjection toxicity and chilling sensitivity in gilthead seabream (Sparus aurata) embryos

Cryopreservation of fish embryos requires an optimal distribution of cryoprotectants inside all embryo compartments. Traditional techniques for the incorporation of cryoprotectants (CPAs) have failed to protect all fish compartments, especially the yolk sac which has been considered the principal point of embryo chilling sensitivity. In the present study, microinjection was used to incorporate cryoprotectants into the yolk sac of gilthead seabream (Sparus aurata) embryos at tail bud stage. The effect of microinjection viability, cryoprotectant toxicity and chilling resistance was evaluated through the hatching rate. Larval survival at first feeding was also determined in microinjection viability and cryoprotectant toxicity studies. Permeabilized seabream embryos were microinjected with 2.35 nl dimethyl sulfoxide (Me₂SO), methanol (MeOH), ethylene glycol (EG) (5 M, 10 M and pure) or sucrose (10% and 15%). In a second experiment, 29.5 nl and 154.0 nl of the highest concentration of each cryoprotectant were used in the same embryo stage. To test the effect of microinjected cryoprotectants on embryo chilling resistance, 29.5 nl of pure Me₂SO or 15% sucrose was microinjected into the yolk sac of tail bud stage embryos and then at a later stage, (tail-bud-free), were exposed to 3 M Me₂SO solution at − 10 °C for 30 min. Our results showed that microinjection technique did not affect the viability of tail bud stage embryos as is shown by the high hatching and survival rates. Hatching and larval survival rate at first feeding were not affected with any of the CPAs tested, showing percentages higher than 75% and 90%, respectively, when embryos were microinjected with a smaller quantity of cryoprotectant. Sucrose was the cryoprotectant better tolerated at higher concentration and volume. Cryoprotectant concentration inside the yolk higher than 1.18 M for Me₂SO, 1.5 M for EG and 2 M for methanol decreased the hatching rate. Microinjection allowed the delivery of high concentrations of CPAs into the yolk sac without deleterious effects on the embryo, but did not provide a significant level of protection for the whole embryo against chilling injury.     

畜禽精液冷冻损伤保护研究进展

精液冷冻过程中液体冰晶化造成的机械损伤和氧化自由基造成的氧化损伤是畜禽冷冻精液产业化的主要限制因素。在冻精中添加外源抗氧化剂维持氧化还原平衡状态以此减轻氧化损伤、通过添加抗冻剂和控制冷冻速率来减少冰晶产生和减轻机械损伤成为畜禽冻精技术研究的重点。本文从不同种类抗氧化剂的抗氧化机理及其在冻精中的应用现状、添加抗冻剂和控制冷冻速率等方面减少机械损伤进行综述,以期为畜禽精液冷冻技术提供理论和技术参考。     

冷冻保护剂在卵母细胞上的研究进展

系统综述了细胞松弛素B、紫杉醇、咖啡因和谷胱甘肽4种冷冻保护剂在增强卵母细胞抗冻能力方面的研究,并分析了Bcl-2和Bax2种凋亡基因的表达程度与卵母细胞凋亡之间的关系,