Vitrification of zebrafish embryo blastomeres in microvolumes

Cryopreservation of fish embryos may play an important role in biodiversity preservation and in aquaculture, but it is very difficult. In addition, the cryopreservation of fish embryo blastomeres makes conservation strategies feasible when they are used in germ-line chimaerism, including interspecific chimaerism. Fish embryo blastomere cryopreservation has been achieved by equilibrium procedures, but to our knowledge, no data on vitrification procedures are available. In the present work, zebrafish embryo blastomeres were successfully vitrified in microvolumes: a number of 0.25 microl drops, sufficient to contain all the blastomeres of an embryo at blastula stage (from 1000-cell stage to oblong stage), were placed over a 2.5 cm loop of nylon filament. In this procedure, where intracellular cryoprotectant permeation is not required, blastomeres were exposed to cryoprotectants for a maximum of 25 sec prior vitrification. The assayed cryoprotectants (ethylene glycol, propylene glycol, dimethyl sulphoxide, glycerol and methanol) are all frequently used in fish embryo and blastomere cryopreservation. Methanol was finally rejected because of the excessive concentration required for the vitrification (15M). All other cryoprotectants were prepared (individually) at 5 M in Hanks' buffered salt solution (sigma) plus 20% FBS (vitrification solutions: vs). After direct thawing in Hanks' buffered salt solution plus 20% FBS, acceptable survival rates were obtained with ethylene glycol: 82.8%, propylene glycol: 87.7%, dimethyl sulphoxide: 93.4%, and glycerol: 73.9% (p < 0.05). Dimethyl sulphoxide showed the highest blastomere survival rate and allowed the rescue of as much as 20% of the total blastomeres from each zebrafish blastula embryo.     

In vitro and in vivo survival of mouse blastocysts after repeated vitrification with the open pulled straw (OPS) method

This study addresses the in vitro and in vivo survival of mouse embryos repeatedly vitrified by the OPS-technique of Vajta et al. (24). Of 225 vitrified blastocysts, after warming one third was cultured in vitro, the other two-thirds were re-vitrified. After these were warmed, the second third was put to culture. With the remaining third the procedure was repeated. Of embryos vitrified once, 97% developed to expanded blastocysts and 81% proceeded to hatching. Corresponding values for embryos re-vitrified once were 93% and 72%, respectively (P > 0.05). After another re-vitrification, expansion and hatching rates were reduced to 76% and 35%, respectively (P < 0.01). Of 10 recipients provided with 10 embryos each that had been vitrified once, 80% remained pregnant with 5.5 +/- 0.3 fetuses. Corresponding values for re-vitrified embryos were 80% and 5.0 +/- 0.3. Of all embryos transferred, 44% became vital fetuses after a single vitrification, compared to 40% after revitrification.     

The incidence of mitotic abnormalities in cryopreserved eight-cell early and compacted mouse embryos

The effects of slow freezing-rapid thawing on viability and chromosomal complement of eight-cell early and eight-cell compacted mouse embryos were investigated. The abnormalities connected with damage to the mitotic apparatus, and with chromosome damage were investigated in cryopreserved embryos by cytological analysis. The embryos were preserved using 1M glycerol as cryoprotectant and a slow cooling regime to -30 degrees C before transfer to liquid nitrogen. The proportion of mitotic abnormalities in compacted embryos was significantly higher (13.1%) than in early embryos (5.9%) and unfrozen control embryos (3.9%) in these studies performed after thawing. This was reflected after cryopreservation by a reduced viability of the embryos as judged by culture to the hatching blastocyst stage--compacted 8-cell embryos (71.8+/-4.7%) versus controls (78.4+/-5.9%) and cryopreserved early-stage 8-cell embryos (86.1+/-4.0%)--p<0.05 in each case. However, aneuploidy rates were low in all groups in both fresh and cryopreserved embryos (around 3%).     

Toxicity of zinc oxide nanoparticles to zebrafish embryo: a physicochemical study of toxicity mechanism

The biological impact of engineered nanomaterials released into the aquatic environment is a major concern. In this work, the properties of ZnO nanoparticles (nano-ZnO, 30 nm) were characterized in a water suspension (E3 medium), and a zebrafish 96-h post fertilization (hpf) embryo–larval test was performed to assess the toxicity of nano-ZnO suspension. Nano-ZnO was found to readily form aggregates with different sizes; small aggregates (142.4–517.7 nm) were still suspended in E3 medium, but large aggregates (>1 μm) quickly deposited on the bottom of 24-well plates; nano-ZnO was partially dissolved to Zn species (Zn_(dis)) in E3 medium. In the nano-ZnO suspension, small aggregates, Zn_(dis), and large aggregates might jointly exert influence on the development of zebrafish embryos. The embryo toxicity test revealed that nano-ZnO killed zebrafish embryos (50 and 100 mg/L), retarded the embryo hatching (1–25 mg/L), reduced the body length of larvae, and caused tail malformation after the 96 hpf exposure. Zn_(dis) only partially contributed to the toxicity of nano-ZnO. This research highlights the need to further investigate the ecotoxicity of nano-ZnO in the water environment.     

Ultra rapid freezing and vitrification of human embryos derived from abnormally fertilised zygotes

The present study was undertaken to compare the developmental capacity of human embryos derived from abnormally fertilised zygotes (1 PN, > 3 PN; 16-18 hours after ICSI) cryopreserved using two techniques: ultra rapid freezing and vitrification. At 2-4 cell stage, (48 hours after ICSI), these abnormally fertilised embryos were then distributed in three groups: a) embryos that were cryopreserved by ultra rapid freezing (URF Group), b) embryos cryopreserved by vitrification (V Group) and c) embryos that were not cryopreserved (Control group). Survival rates and embryo development after 24 hours of in vitro culture (72 hours after ICSI) were compared. 42 embryos were cryopreserved by ultra rapid freezing in 0.5 mL straws, using a mixture of dimethyl sulphoxide (3M) and sucrose (0.25M) in a base solution consisting of IVF medium plus 20 percent (v/v) of Human Serum Albumin (HSA), and 24 embryos were vitrified in 0.25 ml straws, using a two step protocol with an equilibration solution consisting of 10 percent ethylene glycol (1.79 M) and 10 percent dimethyl sulphoxide (1.41 M) in a base solution of modified phosphate buffered saline (PBS) with 20 percent of HSA and a vitrification solution consisting of 20 percent ethylene glycol (3.58 M), 20 percent dimethyl sulphoxide (2.82 M) and 0.5 M sucrose in base solution. The recovery rate after thawing/warming was lower for the vitrification group (75 percent V; 83 percent URF). The number of embryos with less than 50 percent of intact blastomeres after cryopreservation was significantly higher for the URF group (0 percent V; 34 percent URF). After in vitro culture, the rate of embryos not cryopreserved (Control group) that developed in vitro (72 hours after ICSI) was the highest (86 percent), followed by group V (50 percent), while group URF was the lowest (13 percent). These differences were statistically significant. This straw method of vitrification is successful and safe.     

Increasing storage capability of pacu (Piaractus mesopotamicus) embryos by chilling: development of a useful methodology for hatcheries management

Cryopreservation of fish gametes has been studied extensively in the last few decades, but the successful cryopreservation of fish embryos remains elusive. However, recent studies using short-term chilling techniques have shown that it is possible to store embryos at low temperatures with no significant loss in viability. Information on cryopreservation of Neotropical freshwater fish embryos has so far been very limited in the literature. In the present study, chilling protocols for storage of pacu embryos at -8°C for up to 24 h were studied using different concentrations of sucrose in methanol. Embryos tolerated the subzero temperature for up to 6 h with no adverse effects (P > 0.05). After 12 h chilling, hatching rate of 64.0 +/- 3.5 percent was recorded. Low temperature storage of pacu embryos by chilling is detailed here for the first time. Further studies are needed to extend the storage time and to improve the hatching rate.     

Influences of ultrasound on hatching of the fish eggs

The effects of ultrasound on the hatching of the goldfish(Carassiusauratus auratus)and loach(Paramisgurnus dabryanus(sauvage))cytulas werestudied.The ultrasound with certain frequencies can accelerate the developmentof cytulas,induce the embryos to break membrane earlier,increase the survivalrate of larvas,and increase 50% hatching rate of loach cytulas at lower temper-ature.     

基于可见/近红外光谱和深度学习的早期鸭胚雌雄信息无损检测

胚蛋雌雄识别一直是家禽业发展的瓶颈问题,在禽肉生产过程中倾向于养殖雄性个体,而禽蛋生产产业倾向于养殖雌性家禽。若能在孵化过程中较早鉴别出种蛋的雌雄,不仅能够降低家禽孵化产业的成本,还能够提高禽蛋和禽肉生产行业的经济效益。该文以种鸭蛋为研究对象,为了在种鸭蛋孵化早期实现对种蛋的雌雄识别,构建了可见/近红外透射光谱信息采集系统,在200～1 100 nm的波长范围内采集了345枚孵化了0~8 d的种鸭蛋光谱数据。搭建了适用于种鸭蛋光谱信息的6层卷积神经网络（convolutional neural network, CNN）,其中包括输入层、3个卷积层、全连接层与输出分类层。卷积层可以提取光谱中的有效信息,全连接层通过对卷积层提取的局部特征进行整合供输出层分类决策。另外在卷积神经网络中引入局部响应归一化和dropout操作能够加快网络的收敛速度。利用该卷积神经网络构建鸭胚雌雄信息识别网络,通过对比与分析不同孵化天数的识别效果,发现孵化7d的识别效果最佳。随后将孵化7 d的种鸭蛋原始光谱数据进行噪声去除,选取500~900 nm波段用于后续的特征波长选取和建模。分别运用了竞争性自适应重加权算法(CARS)、连续投影算法( SPA)与遗传算法(GA)选择能够区分鸭胚性别的波长点,将选取的特征波长转换为二维的光谱信息矩阵,二维光谱信息矩阵保留了一维光谱的有效信息,同时极大地方便了与卷积神经网络的结合。利用二维光谱信息矩阵和卷积神经网络相结合,实现孵化早期阶段鸭胚的雌雄识别。经检验,基于 SPA算法和CNN网络建立的模型效果较佳,其中训练集、开发集及测试集的准确率分别为93.36%,93.12%和93.83%;基于GA算法和CNN网络建立的模型效果次之,训练集、开发集及测试集的准确率分别为90.87%,93.12%和86.42%;基于CARS算法和CNN网络建立的模型的训练集、开发集及测试集的准确率分别为84.65%,83.75%和77.78%。研究结果表明基于可见/近红外光谱技术和卷积神经网络可以实现孵化早期鸭胚胎雌雄的无损鉴别,为后续相关自动化检测装置的研发提供了技术支撑。     

Histone deacetyltransferase1 expression in mouse oocyte and their in vitro-fertilized embryo: effect of oocyte vitrification

This study was conducted to investigate the expression of Histone Deacetyltransferase1 (HDAC1) in mouse embryos derived from the vitrified-warmed oocytes. Firstly, the mouse oocytes at metaphaseII (MII) stage were randomly allocated into three groups: A untreated (control), B exposed to vitri?cation solution (VS) without being plunged into liquid nitrogen (toxicity), or C vitri?ed by open-pulled straw (OPS) method (vitri?cation). After warming, they were fertilized in vitro. Fresh oocytes were used as control. Expression of HDAC1 was then examined in MII mouse oocytes and embryos by immunofluorescence with anti-HDAC1 polyclonal antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG. Results showed that after in vitro fertilization (IVF), developmental rates to two-cell embryos (39%), 4-cell embryos (35%), morula (32%) and blastocysts (26%) in cryopreserved oocytes were all significantly lower than those of fresh oocytes (P < 0.01). In addition, HDAC1 expression in the vitrified group was significantly lower (P< 0.05) than that in the control and toxicity groups at all developmental stages except for the blastocyst. Moreover, the vitrified-warmed oocytes showed significantly lower (P < 0.05) HDAC1 expression compared with that of control and toxicity groups. In conclusion, HDAC1 was expressed both in oocytes and in their in vitro-fertilized embryos. This decreased expression of HDAC1 in mouse oocytes and the embryos due to the cryopreservation may have a negative impact on embryo development.     

Zebrafish embryos (Danio rerio) using microinjection

Low membrane permeability is one of the major obstacles to the successful cryopreservation of zebrafish embryos. The aim of the present study was to explore if this could be overcome by yolk modification with different cryoprotectants by micro-injection. Initial investigation of two cryoprotectants, methanol and sucrose, was undertaken to determine their suitability for micro-injection supplementation of the yolk mass. Intact zebrafish embryos at 50% epiboly stage were injected with Hanks' solution, 5.2 M methanol or 1.3 M sucrose yielding approximate final concentrations of 2.0 and 0.5 M of the cryoprotectants within the yolk sac respectively. After micro-manipulation, the embryos were cultured at 28 degree C for three days and their survival assessed at the hatching stage. All micro-manipulations performed in the present study resulted in a significant decrease in embryo survival (P < 0.05). Embryos micro-injected with methanol or sucrose were also subjected to a cooling procedure. They were placed in 3M methanol + 0.5 M sucrose at room temperature for 30 min and then cooled from 20 degree C to 0 degree C at 2 degree/min, from 0 degree C to -7.5 degree/min at 1 degree/min, seeded at -7.5 degree C and held for 10 min, before cooling at 0.3 degree/min to - 20 degree C or until full crystallization in all embryos. The processes of extra- and intracellular crystallization were studied by cryomicroscopy. The temperature of intracellular crystallization did not differ significantly between control and injected embryos. However, it was found that intracellular crystallization did not always happen instantly after extracellular crystallization.     

硅衬底GaN蓝色发光材料转移前后应力变化研究

利用外延片压焊和湿法腐蚀技术将硅衬底上生长的InGaN多量子阱发光二极管(LED)薄膜材料转移到了新衬底上. 研究结果表明, 转移后的LED薄膜中GaN层受到的张应力变小,InGaN层受的压应力变大. 去除转移后LED薄膜中过渡层后,GaN层受到的张应力变大,而铟镓氮层受到的压应力基本不变. 将转移后的薄膜做成垂直结构的LED芯片后,其光电性能明显改善. 关键词： GaN 发光二极管 硅衬底 应力     

基于卷积神经网络的蛋胚活性精准检测方法研究

孵化的蛋胚是生产禽流感疫苗的载体,蛋胚的活性检测是疫苗生产中的关键环节,通过光电容积脉搏法检测蛋胚活性是提高蛋胚活性检测准确率的关键.为了提高蛋胚活性检测效率和检测准确率,采用滑动功率谱方法(PSD)将蛋胚脉搏波可视化,基于卷积神经网络对蛋胚活性进行精准分类.实验结果显示,采用卷积神经网络对单个蛋胚信号的计算时间仅为1...     

Study on permeability of DMSO in embryos of red seabream (Pagrus major) by capillary electrophoresis

The objectives were to investigate the permeability of DMSO to red seabream (Pagrus major) embryos by capillary electrophoresis and the effects of DMSO concentrations (5 to 40 percent, volume basis) and immersion times (10, 30 and 60 min) on hatching rate and morphology. The results suggested the internal DMSO concentrations were positively related with the external concentrations and exposure times, while the hatching rate was negatively related. The hatching rate decreased drastically (less than 50 percent) after exposure in 35 percent, 20 percent and 15 percent DMSO for over 10, 30 and 60 min, respectively. In all groups, when hatching rate was greater 50 percent, the internal DMSO concentration was less than 2 percent, which was still insufficient for successful cryopreservation. Morphological changes indicated the chorion was permeable to the cryoprotectant. A sign of dehydration in yolk were observed, for a significant decrease in the maximal yolk sac diameter. However, further research was needed to investigate whether the DMSO permeated into the yolk.     

虫蛀玉米种子的空气耦合超声波检测

提出了一种基于空气耦合超声波技术的玉米种子虫蛀孔洞颗粒和完好颗粒分类识别方法·首先根据玉米颗粒的弹性模量、泊松比和密度等物理量计算出了玉米颗粒的声速,并根据检测精度需求设定了激励信号频率。然后采用MATLAB对采集的两类种子超声波信号数据进行分析处理,并分析了种子厚度和摆放方位对超声波响应特征的影响。最后建立了K近邻(KNN)、簇类独立软模式法(SIMCA)、Fisher线性判别(LDA)和决策树(DT)识别模型,并对模型性能进行了测试.结果表明;种子孔洞深度、胚部厚度和正反面方位不同,即超声波在种子表面的反射程度不同、在种子中传播声程不同,则起声波信号衰减程度不同,导致接收到信号的幅值不同,且样本点在主成分分析(PCA)特征空间的分布也不同。4种识别模型均可以实现对两类玉米的分类识别,其中KNN模型性能最佳,其对虫蛀孔洞颗粒和完好颗粒的正确识别率分别为98%100%,误差带为2%,0。此结果说明采用空气耦合超声波技术可以实现对玉米种子虫蛀孔洞颗粒的检测。      

Evaluation of a biological wastewater treatment system combining an OSA process with ultrasound for sludge reduction

Sludge production is an undesirable by-product of biological wastewater treatment. The oxic-settling-anaerobic (OSA) process constitutes one of the most promising techniques for reducing the sludge produced at the treatment plant without negative consequences for its overall performance. In the present study, the OSA process is applied in combination with ultrasound treatment, a lysis technique, in a lab-scale wastewater treatment plant to assess whether sludge reduction is enhanced as a result of mechanical treatment. Reported sludge reductions of 45.72% and 78.56% were obtained for the two regimes of combined treatment tested in this study during two respective stages: UO1 and UO2. During the UO1 stage, the general performance and nutrient removal improved, obtaining 47.28% TN removal versus 21.95% in the conventional stage. However, the performance of the system was seriously damaged during the UO2 stage. Increases in dehydrogenase and protease activities were observed during both stages. The advantages of the combined process are not necessarily economic, but operational, as US treatment acts as contributing factor in the OSA process, inducing mechanisms that lead to sludge reduction in the OSA process and improving performance parameters.     

基于紫外‐可见透射光谱技术和极限学习机的早期鸡胚雌雄识别

为了对鸡种蛋胚胎进行雌雄识别,探究利用紫外-可见-近红外透射光谱进行鸡胚雌雄识别的可行性,搭建了鸡种蛋透射光谱检测系统,采用横向和竖向大头朝上2种放置方式获取210枚鸡种蛋孵化0~15 d的光谱,光谱范围为360~1 000 nm。构建极限学习机（ELM）鸡胚雌雄识别模型,通过比较不同放置方式和孵化天数下模型的识别准确率,发现竖向放置且孵化第7 d的识别效果最好;将竖向放置孵化第7 d的光谱初步分为紫外（360~380 nm）、可见光（380~780 nm）、近红外（780~1 000 nm）、紫外-可见光（360~780 nm）和全波段（360~1 000 nm）5个不同的波段范围来分析,预测集准确率分别为82.86%,77.14%,75.71%,84.29%和81.43%,筛选出360~780 nm的紫外-可见光波段为有效波段;在紫外-可见光（360~780 nm）波段,采用多元散射校正（MSC）去噪,并用竞争性自适应重加权采样算法（CARS）和连续投影算法（SPA）筛选特征波长降维,建立不经筛选特征波长、CARS筛选特征波长和SPA筛选特征波长的3种ELM模型。其中不经筛选特征波长的ELM模型识别效果最好,但输入变量最多,隐含层神经元为680且激活函数为sig时,预测集准确率为84.29%。SPA筛选特征波长的ELM模型识别效果次之,输入变量有9个,隐含层神经元为840且激活函数为hardlim时,预测集准确率为81.43%。CARS筛选特征波长的ELM模型识别效果最差,输入变量有27个,隐含层神经元为100且激活函数为sig时,预测集准确率为78.57%;用遗传算法（GA）优化ELM模型的权值变量和隐含层阈值,不经筛选特征波长建立的GA-ELM模型,预测集准确率为87.14%,SPA筛选特征波长建立的GA-ELM模型,预测集准确率为87.14%,CARS筛选特征波长建立的GA-ELM模型,预测集准确率为81.43%。紫外-可见光波段不经筛选特征波长的GA-ELM模型识别效果和经SPA筛选特征波长的GA-ELM模型相同,表明SPA筛选的特征波长变量能够有效反映360~780 nm波段的信息,SPA使用的变量数仅占紫外-可见光波段的2.14%,因此,雌雄识别最佳模型为紫外-可见光波段经SPA筛选特征波长的GA-ELM模型,预测集准确率为87.14%,其中,雌性识别率为88.57%,雄性识别率为85.71%,单个样本平均判别时间0.080 ms。结果表明紫外-可见透射光谱技术和ELM模型为孵化早期鸡胚蛋雌雄识别提供了一种可行方法。     

The changing biochemical composition and organisation of the murine oocyte and early embryo as revealed by Raman spectroscopic mapping

Despite an exponential uptake in recent years of assisted reproductive techniques, such as in vitro fertilisation, much is still not fully understood about the biochemical modifications that take place during the development and maturation of the egg and embryo. As such, in order to improve the efficiency of these techniques, furthering our understanding of the processes that underpin oocyte and embryo development is necessary. Raman spectroscopic mapping as a technique enables the investigation of biochemical variation within intact cells without the need for labelling. Here, Raman maps of fixed immature and mature oocytes along with early stage embryos were collected using 785 nm excitation and a step size of 2 µm. The results were analysed using both univariate and multivariate techniques. It was found that significant macromolecular accumulation took place during oocyte maturation, while a decrease in total lipid content consistent with the formation of new cellular membranes is observed upon embryo cleavage. Furthermore, an observed asymmetrical localisation of macromolecules in the mature oocyte may indicate the existence of cytoplasmic polarisation, a phenomenon that has been observed in the eggs of lower organisms. As such, these results indicate that Raman spectroscopic mapping may present an alternative analytical tool for investigating the biochemistry of egg and embryo development. In particular, these results indicate that temporal Raman analysis may help to reveal the existence of cytoplasmic polarisation in the murine egg. Copyright © 2011 John Wiley & Sons, Ltd.     

Electrical properties of indium implanted silicon

Electrical conductivity and Hall effect were measured from 100° to 278°K as a function of layer removal to determine the indium ionization energy and the presence of compensating centers resulting from the implantation of indium into silicon. The implants were fully annealed to reduce the influence of radiation damage. For comparison, similar measurements were performed on silicon shallow diffused with indium. Differential analysis methods were used to compute carrier concentration, mobility, and resistivity for the stripped layers. In addition, Hall measurements were performed on silicon uniformly doped with indium. In all three cases an indium energy level of 160 meV was observed. Mobility plots versus temperature were also consistent. However, significant compensation effects were noticed in the implants.     

Shear thickening behavior of nanoparticle suspensions with carbon nanofillers

Quantum dots (QDs) have widely been used in biomedical and biotechnological applications. However, few studies focus on the assessing toxicity of QDs exposure in vivo. In this study, zebrafish embryos were treated with CdTe QDs (4 nm) during 4–96 h post-fertilization (hpf). Mortality, hatching rate, malformation, heart rate, and QDs uptake were detected. We also measured the larval behavior to analyze whether QDs had persistent effects on larvae locomotor activity at 144 hpf. The results showed that as the exposure dosages increased, the hatching rate and heart rate of zebrafish embryos were decreased, while the mortality increased. Exposure to QDs caused embryonic malformations, including head malformation, pericardial edema, yolk sac edema, bent spine, and yolk not depleted. QDs fluorescence was mainly localized in the intestines region. The larval behavior testing showed that the total swimming distance was decreased in a dose-dependent manner. The lowest dose (2.5 nM QDs) produced substantial hyperactivity while the higher doses groups (5, 10, and 20 nM QDs) elicited remarkably hypoactivity in dark periods. In summary, the data of this article indicated that QDs caused embryonic developmental toxicity, resulted in persistent effects on larval behavior.     

Cryoprotectants protect medaka (Oryzias latipes) embryos from chilling injury

This study was conducted to investigate the effect of six cryoprotectants (dimethyl sulfoxide (DMSO), glycerol (Gly), methanol (MeOH), ethylene glycol (EG), 1,2-propylene glycol (PG) and N,N-dimethylformamide (DMF) on the survival of medaka (Oryzias lapites) embryos at low temperatures (0 and -5C). Firstly, the embryos at 8 to 16-cell stages were exposed to different concentrations (1 to 4 mol per L) of DMSO, Gly, MeOH, EG, PG and DMF for 40min at 26C. After removal of the cryoprotectants (CPAs), the embryo survivals were assessed by their development into live fries following 9 day of culture. The results showed that the higher concentration of the CPA, the lower survival of the embryos; and that the toxicity of the six CPAs to medaka embryos is in the order of PG < MeOH = DMSO < Gly < EG < DMF (P < 0.05). Secondly, based on the results obtained above, embryos at 8 to 16-cell stages or other stages were exposed to 2 mol per L of PG, MeOH or DMSO for up to 180 min at 0C and up to 80 min at -5C respectively. The 8 to 16-cell embryos treated with MeOH at low temperatures showed highest survival. Thirdly, when embryos at different stages were treated with 2 mol per L of MeOH at -5C for 60 min, 16-somite stage embryos showed highest survival, followed by 4-somite, neurula, 50 percent epiboly, blastula, 32-cell and 8 to 16-cell embryos. These results demonstrated that PG had the lowest toxicity to medaka embryos among the six permeable CPAs at 26C, whereas MeOH showed highest cryoprotective efficiency under chilling conditions and chilling injury decreased gradually with the development of medaka embryos.