BACKGROUND & AIMS: -2

背景与目标： -2

BACKGROUND & AIMS: In our centre, embryos are judged to have survived cryopreservation if at least half of the initial number of blastomeres remain intact. Therefore both fully intact and partially damaged embryos are transferred. The aim of this study was to investigate the viability of partially damaged human embryos after cryopreservation. We retrospectively analysed the implantation and in-vivo development of embryos which were either fully intact or had lost some blastomeres after cryopreservation. Oocytes were collected following stimulation with the gonadotrophin-releasing hormone (GnRH)-agonist Buserelin and human menopausal gonadotrophin. Supernumerary multicellular embryos with not more than 20% of their volume filled with anucleate fragments were frozen on day 2 or day 3 of the cycle using a slow cooling procedure with dimethylsulphoxide as the cryoprotectant. Following slow thawing, 431 fully intact embryos were transferred in 314 embryo transfer procedures and 488 partially damaged embryos were transferred in 327 such procedures. The percentage of gestational sacs with fetal heartbeat obtained after transfer of fully intact embryos was almost three times higher than that after transfer of partially damaged embryos (11.4 versus 3.5%). Forty-five children (birth rate 10% per embryo transfer) were born after transfer of fully intact embryos and 14 after transfer of embryos from which some blastomeres had been lost following cryopreservation. In conclusion, although children have been delivered after transfer of partially damaged embryos, the aim of a cryopreservation programme must be to obtain fully intact embryos after thawing.

背景与目标： 在我们的中心，如果至少一半的初始卵裂球数量保持完整，则判定胚胎可以在冷冻保存中存活。因此，完全完整和部分受损的胚胎都被转移了。这项研究的目的是研究冷冻保存后部分受损的人类胚胎的生存能力。我们回顾性分析了冷冻保存后完全完整或失去一些卵裂球的胚胎的植入和体内发育。用促性腺激素释放激素 (GnRH) 激动剂Buserelin和人类更年期促性腺激素刺激后收集卵母细胞。在循环的第2天或第3天，使用以二甲基亚砜为冷冻保护剂的缓慢冷却程序，将其体积不超过20% 的多细胞胚胎冷冻。缓慢解冻后，在314胚胎移植程序中转移431完全完整的胚胎，在327这样的程序中转移488部分受损的胚胎。在转移完全完整的胚胎后获得的具有胎儿心跳的妊娠囊的百分比几乎是转移部分受损的胚胎后的三倍 (11.4对3.5%)。45名儿童 (每次胚胎移植的出生率10%) 在完全完整的胚胎移植后出生，14名在冷冻保存后丢失了一些卵裂球的胚胎移植后出生。总之，尽管儿童是在转移了部分受损的胚胎后分娩的，但冷冻保存计划的目的必须是在解冻后获得完整的胚胎。

BACKGROUND & AIMS: BACKGROUND:Vitrification is a prospective technology in ovarian tissue cryopreservation, but it is still in an initial stage. This study was conducted to investigate a modified vitrification protocol for human ovarian tissue, which can be used as an alternative to preserve fertility for young women with cancer who have to undergo cytotoxic therapy and sterilization. METHODS:Ovarian tissue samples were collected from 15 patients and randomly allocated to groups of fresh, vitrification, and conventional slow freezing. A modified carrierless vitrification method was applied. The proportion of morphologically intact follicles in fresh ovarian tissues was compared with that in warmed/thawed tissues. The initial growth of the follicles and the concentrations of estradiol and progesterone were detected to determine the viability and endocrine function of the cryopreserved tissues. RESULTS:The proportion of morphologically intact primordial follicles in the fresh group (97.6%) was significantly higher than that in the other two groups (vitrification group 80.3% and slow-freezing group 72.6%, P < 0.001). In both the vitrification and slow-freezing groups, estradiol and progesterone were secreted continuously during 2-week culture in vitro, the proportion of primary follicles were both significantly increased compared to the fresh group. No statistically significant differences existed between the two groups after cryopreservation in the proportion of both primordial and primary follicles, and the concentrations of estradiol and progesterone (P > 0.05). CONCLUSION:The modified vitrification method for cryopreservation of human ovarian tissues is effective, simple, and inexpensive.

背景与目标：

BACKGROUND & AIMS: :In the present work, taurine and hypotaurine were evaluated as potential additives to improve European sea bass (Dicentrarchus labrax) sperm quality after cryopreservation. For cryopreservation, three different extenders were used: control extender (NAM), supplemented with 1mM taurine or supplemented with 1mM hypotaurine, all of them containing 10% Me₂SO as cryoprotectant. To evaluate sperm quality of fresh and thawed sperm, motility (CASA: computer assisted sperm analysis), viability (SYBR Green/propidium iodide), lipid peroxidation (malondialdehyde level), protein oxidation (carbonyl content), glutathione peroxidase, glutathione reductase and superoxide dismutase activities and DNA fragmentation (comet assay) were quantified. The result demonstrated that 1 mM hypotaurine supplemented extender increased total motility (30.1 ± 3.2%), and that 1 mM taurine extender produced higher velocity (18.1 ± 2.6 μm/s) and linearity (46.0 ± 4.8%) than the control extender (21.8 ± 3.2%, 15.5 ± 1.3 μm/s, 41.8 ± 2.4%, respectively). Cell viability, lipid peroxidation and protein oxidation were not statistically different between treatments. Similar results were obtained for glutathione peroxidase and superoxide dismutase activities. Only glutathione reductase showed differential activity before and after freezing, increasing its activity in thawed sperm. Regarding the comet assay results, taurine and hypotaurine significantly reduced DNA fragmentation (52.8 ± 0.9% and 51.8 ± 0.9%, respectively) in comparison to the control (55.7 ± 0.8%). In conclusion, for European sea bass sperm cryopreservation, extenders supplemented with 1 mM taurine and 1 mM hypotaurine improved some parameters of sperm quality after thawing, resulting in better motility and lower DNA damage than the control, two very important factors related to fertilization success.

背景与目标： : 在目前的工作中，牛磺酸和次牛磺酸被评估为潜在的添加剂，可在冷冻保存后提高欧洲鲈鱼 (Dicentrarchus labrax) 的精子质量。对于冷冻保存，使用三种不同的增量剂: 补充1mm牛磺酸或补充1mm次牛磺酸的对照增量剂 (NAM)，所有这些都含有10% me2 so作为冷冻保护剂。为了评估新鲜和解冻的精子质量，运动能力 (CASA: 计算机辅助精子分析)，活力 (SYBR Green/碘化丙啶)，脂质过氧化 (丙二醛水平)，蛋白质氧化 (羰基含量)，谷胱甘肽过氧化物酶，定量了谷胱甘肽还原酶和超氧化物歧化酶活性和DNA片段化 (彗星试验)。结果表明，添加1毫米的次牛磺酸增量剂可提高总运动性 (30.1 ± 3.2%)，而1毫米的牛磺酸增量剂产生的速度 (18.1 ± 2.6 μ m/s) 和线性度 (46.0 ± 4.8%) 高于对照增量剂 (21.8 ± 3.2%，15.5 ± 1.3 μ m/s，分别为41.8 ± 2.4%)。处理之间的细胞活力，脂质过氧化和蛋白质氧化无统计学差异。谷胱甘肽过氧化物酶和超氧化物歧化酶活性获得了相似的结果。冷冻前后只有谷胱甘肽还原酶表现出不同的活性，从而增加了其在解冻精子中的活性。关于彗星测定结果，与对照 (55.7 ± 0.8%) 相比，牛磺酸和次牛磺酸显着减少了DNA片段化 (分别为52.8 ± 0.9% 和51.8 ± 0.9%)。综上所述，对于欧洲鲈鱼精子冷冻保存，补充1毫米牛磺酸和1毫米次牛磺酸的增量剂可改善解冻后精子质量的某些参数，从而比对照组具有更好的运动性和更低的DNA损伤，这两个非常重要的因素与受精成功有关。

BACKGROUND & AIMS: :Although currently investigational, ovarian tissue cryopreservation and oocyte cryopreservation hold promise for future female fertility preservation, particularly following aggressive chemotherapy and/or radiotherapy treatment protocols.

背景与目标： : 尽管目前正在研究中，卵巢组织冷冻保存和卵母细胞冷冻保存有望在未来的女性生育能力保存中，尤其是在积极的化学疗法和/或放射疗法治疗方案之后。

BACKGROUND & AIMS: :This study was carried out to test how sperm cryopreservation affected nuclear DNA stability and whether progeny development was modified when eggs were fertilized with cryopreserved spermatozoa. The "comet assay" (alkaline single-cell gel electrophoresis assay) was adapted to trout spermatozoa to estimate DNA stability as measured by alkali-induced DNA strand break formation. Because trout eggs develop in water after fertilization (oviparous species) and that eggshell is easy to clear up after fixative treatment, progeny development was assessed from the blastodisc flattening stage of the embryos to the first feeding stage of the hatched fries by direct observation. All parameters under study were analyzed on each sperm and comparisons between parameters were made using paired data. Freeze-thawing of sperm slightly but significantly increased the percentage of nuclei showing altered DNA after comet assay. This increase was correlated to the decrease in fertilization rates of sperm, but the absolute percentage of altered nuclei was not predictive of the absolute fertilization ability of sperm. Assessment of progeny development showed that survival rate and abnormality rate obtained after fertilization with cryopreserved sperm were not different from those obtained with fresh sperm. It is concluded that trout sperm cryopreservation only slightly affected sperm DNA stability and that the use of cryopreserved spermatozoa did not impair offspring survival and quality.

背景与目标： : 进行这项研究是为了测试精子冷冻保存如何影响核DNA的稳定性，以及当用冷冻保存的精子使卵受精时是否改变了后代的发育。“彗星测定” (碱性单细胞凝胶电泳测定) 适用于鳟鱼精子，以估算通过碱诱导的DNA链断裂形成测得的DNA稳定性。由于鳟鱼卵在受精后 (卵生物种) 在水中发育，并且在固定处理后蛋壳易于清除，因此通过直接观察评估了从胚胎的胚盘扁平阶段到孵化的薯条的第一个饲养阶段的后代发育。对每个精子分析了所有研究参数，并使用配对数据进行了参数之间的比较。彗星分析后，精子的冻融略有增加，但显着增加了显示DNA改变的核百分比。这种增加与精子受精率的降低有关，但是细胞核改变的绝对百分比不能预测精子的绝对受精能力。对后代发育的评估表明，冷冻保存的精子受精后的存活率和异常率与新鲜精子受精后的存活率和异常率没有差异。结论是，鳟鱼精子冷冻保存仅轻微影响精子DNA的稳定性，并且使用冷冻保存的精子不会损害后代的存活率和质量。

BACKGROUND & AIMS: :Current therapy of childhood cancer makes long-term survival a realistic outcome for most patients. However, some treatment regimens entail a significant risk of infertility. No established method for preservation of female fertility is currently available. Ovarian cryopreservation is an experimental technology that is being offered with increasing frequency to women undergoing cancer therapy. It has not yet been reported in children and adolescent girls. The aim of this review is to stimulate discussion on the possibility of performing ovarian cryopreservation in pre-menarcheal girls in advance of therapies that may induce ovarian failure. We present a multi-disciplinary discussion of the risks and benefits associated with the procedure and propose guidelines for its implementation. We propose that all girls about to receive treatment that has a high risk for infertility be offered consultation about the possibility of ovarian cryopreservation.

背景与目标： : 目前儿童癌症的治疗使大多数患者的长期生存成为现实的结果。但是，某些治疗方案会带来不孕症的重大风险。目前尚无保留女性生育力的既定方法。卵巢冷冻保存是一种实验性技术，正在为接受癌症治疗的妇女提供越来越多的频率。尚未在儿童和少女中报告。这篇综述的目的是激发关于在可能导致卵巢衰竭的疗法之前对月经前的女孩进行卵巢冷冻保存的可能性的讨论。我们对与该程序相关的风险和收益进行了多学科讨论，并提出了实施指南。我们建议向所有即将接受不育高风险治疗的女孩提供有关卵巢冷冻保存可能性的咨询。

BACKGROUND & AIMS: :Vitrification is considered as the most promising method for long-term storage of tissues and organs. An effective way to reduce the accompanied cryoprotectant (CPA) toxicity, during CPA addition/removal, is to operate at low temperatures. The permeation process of CPA into/out of biomaterials is affected by the viscosity of CPA solution, especially at low temperatures. The objective of the present study is to measure the viscosity of the ternary solution, dimethyl sulfoxide (Me2SO)/water/sodium chloride (NaCl), at low temperatures and in a wide range of concentrations. A rotary viscometer coupled with a low temperature thermostat bath was used. The measurement was carried out at temperatures from -10 to -50°C. The highest mass fraction of Me2SO was 75% (w/w) and the lowest mass fraction of Me2SO was the value that kept the solution unfrozen at the measurement temperature. The concentration of NaCl was kept as a constant [0.85% (w/w), the normal salt content of extracellular fluids]. The Williams-Landel-Ferry (WLF) model was employed to fit the obtained viscosity data. As an example, the effect of solution viscosity on modeling the permeation of Me2SO into articular cartilage was qualitatively analyzed.

背景与目标： : 玻璃化被认为是组织和器官长期储存的最有前途的方法。在添加/去除CPA期间，降低伴随的冷冻保护剂 (CPA) 毒性的有效方法是在低温下运行。CPA进入/离开生物材料的渗透过程受CPA溶液粘度的影响，尤其是在低温下。本研究的目的是在低温和较宽的浓度范围内测量三元溶液二甲基亚砜 (Me2SO)/水/氯化钠 (NaCl) 的粘度。使用了旋转粘度计和低温恒温浴。测量是在-10至-50 °C的温度下进行的。Me2SO的最高质量分数是75% (w/w)，Me2SO的最低质量分数是使溶液在测量温度下保持解冻的值。NaCl的浓度保持恒定 [0.85% (w/w)，即细胞外液的正常盐含量]。采用Williams-Landel-Ferry (WLF) 模型拟合获得的粘度数据。例如，定性分析了溶液粘度对模拟Me2SO渗透到关节软骨中的影响。

BACKGROUND & AIMS: :This study investigated effects of hexoses, fetal calf serum (FCS), and phenazine ethosulfate (PES) during the culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. The basal, control medium was chemically defined (CDM) plus 0.5% fatty acid-free BSA. In vitro-produced bovine zygotes were cultured in CDM-1 with 0.5 mM glucose; after 60 hr, 8-cell embryos were cultured 4.5 days in CDM-2. The 8-cell embryos were randomly allocated to a 2 x 3 x 2 x 3 factorial experimental design with two energy substrates (2 mM glucose or fructose); three additives (0.3 microM PES, 10% FCS, and control); two cryopreservation methods using no animal products (conventional slow freezing or vitrification); and semen from three bulls with two replicates for each bull. A total of 1,107 blastocysts were produced. Fructose resulted in 13% more blastocysts per oocyte than glucose (37.2% vs. 32.9%), and per 8-cell embryo (51.3% vs. 45.3%; P < 0.01). No differences were found for additives (P > 0.1) control, FCS, or PES for blastocysts per oocyte or per 8-cell embryo. There was a significant interaction (P < 0.05) between additives and hexoses for blastocyst production; although trends were similar, the benefit of fructose compared to glucose was greater for controls than for FCS or PES. Culture of embryos with PES, which reduces cytoplasmic lipid content, improved cryotolerance of bovine embryos; post-cryopreservation survival of blastocysts averaged over vitrification and slow freezing (between which there was no difference) was 91.9%, 84.9%, and 60.2% of unfrozen controls (P < 0.01) for PES, control, and FCS groups, respectively.

背景与目标： : 这项研究调查了牛胚胎培养过程中己糖，胎牛血清 (FCS) 和吩嗪硫酸酯 (PES) 对通过缓慢冷冻或玻璃化冷冻保存后胚泡发育和存活的影响。基础对照培养基是化学定义的 (CDM) 加上0.5% 不含脂肪酸的BSA。用0.5 mM葡萄糖在CDM-1中培养体外产生的牛受精卵; 60小时后，在CDM-2中培养8细胞胚胎4.5天。将8细胞胚胎随机分配到2x3x2x3析因实验设计中，该实验设计具有两种能量底物 (2毫米葡萄糖或果糖); 三种添加剂 (0.3 microM PES，10% FCS和对照); 两种不使用动物产品的冷冻保存方法 (常规的缓慢冷冻或玻璃化); 和来自三只公牛的精液，每只公牛有两个重复。总共产生了1,107个囊胚。果糖导致每个卵母细胞比葡萄糖 (37.2% 对32.9%) 和每个8细胞胚胎 (51.3% 对45.3%; P < 0.01) 多13% 个胚泡。对于每个卵母细胞或每个8细胞胚胎的囊胚，未发现添加剂 (P > 0.1) 对照，FCS或PES的差异。在用于囊胚生产的添加剂和己糖之间存在显着的相互作用 (P <0.05); 尽管趋势相似，但与葡萄糖相比，果糖的益处对于对照比FCS或PES更大。用PES培养胚胎，这降低了细胞质脂质含量，提高了牛胚胎的低温耐性; 91.9%，84.9%，未冷冻对照的胚泡在玻璃化和慢速冷冻 (两者之间没有差异) 后的平均冷冻保存存活率 (P < 0.01) 为PES，对照，和FCS组。

BACKGROUND & AIMS: KEY MESSAGE:After cryopreservation, the occurrence of apoptosis-like programmed cell death events induced by the accumulation of ROS reduces pollen viability. Cryopreservation, as a biotechnological means for long-term preservation of pollen, has been applied to many species. However, after cryopreservation, the viability of pollen significantly decreases via a mechanism that is not completely clear. In this study, the pollen of Paeonia lactiflora 'Zi Feng Chao Yang', which exhibits significantly reduced viability after liquid nitrogen (LN2) storage, was used to study the relationship among pollen viability, programmed cell death (PCD) and reactive oxygen species (ROS). The apoptosis rate was increased significantly in pollen with decreased viability after cryopreservation, and the changes in ROS generation and hydrogen peroxide (H2O2) were consistent with the apoptosis rate. Correlation analysis results showed that the apoptosis rate is positively correlated with ROS generation and H2O2 content. In addition, ascorbic acid (AsA), glutathione (GSH) and ascorbic acid reductase (APX) levels were significantly correlated with ROS and H2O2. After LN2 preservation for 8 months, the exogenous antioxidants AsA and GSH at appropriate concentrations significantly decreased H2O2 content, inhibited PCD indicator levels, and increased cryopreserved pollen viability. These observations suggest that PCD occurred in pollen during LN2 preservation for 1-8 months and was induced by the accumulation of ROS in pollen after cryopreservation, thus explaining the main reasons for the reduction in pollen viability after cryopreservation in LN2.

背景与目标： 关键信息: 冷冻保存后，ROS积累引起的凋亡样程序性细胞死亡事件的发生会降低花粉活力。低温保存作为一种长期保存花粉的生物技术手段，已应用于许多物种。但是，冷冻保存后，花粉的活力通过尚不完全清楚的机制显着降低。在这项研究中，使用了在液氮 (LN2) 储存后表现出显着降低的Paeonia lactactflora 'Zi Feng Chao Yang' 花粉，研究了花粉活力，程序性细胞死亡 (PCD) 和活性氧 (ROS) 之间的关系。)。冷冻保存后，花粉的凋亡率显着增加，活力降低，ROS生成和过氧化氢 (H2O2) 的变化与凋亡率一致。相关分析结果表明，细胞凋亡率与ROS生成和H2O2含量呈正相关。此外，抗坏血酸 (AsA)，谷胱甘肽 (GSH) 和抗坏血酸还原酶 (APX) 的水平与ROS和H2O2显着相关。LN2保存8个月后，适当浓度的外源抗氧化剂AsA和GSH显着降低了H2O2含量，抑制了PCD指示水平，并提高了冷冻保存的花粉活力。这些观察结果表明，PCD在LN2保存1-8个月期间发生在花粉中，并且是由冷冻保存后花粉中ROS的积累引起的，从而解释了LN2冷冻保存后花粉活力降低的主要原因。

BACKGROUND & AIMS: :Cryogenic storage is considered to be the most convenient method to maintain phenotypic and genetic stability of organisms. A cryopreservation technique based on encapsulation-drying of in vitro-produced arbuscular mycorrhizal fungi has been developed at the Glomeromycota In Vitro Collection. In this study, we investigated fungal morphology (i.e., number and size of spores, number of branched absorbing structures (BAS), hyphal length, and number of anastomosis per hyphal length), activity of acid phosphatase and alkaline phosphatase in extraradical hyphae, and variation in amplified fragment length polymorphism (AFLP) profiles of in vitro-produced isolates of five Rhizophagus species maintained by cryopreservation for 6 months at -130 °C and compared to the same isolates preserved at 27 °C. Isolates were stable after 6 months cryopreservation. Comparing isolates, the number of BAS increased significantly in one isolate, and hyphal length decreased significantly in another isolate. No other morphological variable was impacted by the mode of preservation. Phosphatase activities in extraradical hyphae and AFLP profiles were not influenced by cryopreservation. These findings indicate that cryopreservation at -130 °C of encapsulated-dried and in vitro-produced Rhizophagus isolates (i.e., Rhizophagus irregularis, Rhizophagus fasciculatus, Rhizophagus diaphanous, and two undefined isolates) is a suitable alternative for their long-term preservation.

背景与目标： : 低温储存被认为是维持生物体表型和遗传稳定性最方便的方法。已在肾小球菌门体外收集处开发了一种基于体外产生的丛枝菌根真菌包封干燥的冷冻保存技术。在这项研究中，我们研究了真菌形态 (即孢子的数量和大小，分支吸收结构的数量 (BAS)，菌丝长度和每菌丝长度的吻合次数)，根茎外菌丝中酸性磷酸酶和碱性磷酸酶的活性，以及通过在-130 °C下冷冻保存6个月并与在27 °C下保存的相同分离物进行比较的五种根瘤菌的体外产生的分离株的扩增片段长度多态性 (AFLP) 谱的变化。低温保存6个月后分离株稳定。比较分离株，一种分离株的BAS数量显着增加，而另一种分离株的菌丝长度显着减少。保存方式没有影响其他形态变量。低温保存不会影响根皮外菌丝和AFLP谱中的磷酸酶活性。这些发现表明，在-130 °C下冷冻保存包囊干燥和体外产生的根瘤菌分离物 (即，不规则根瘤菌，束状根瘤菌，双生根瘤菌和两个未定义的分离物) 是长期保存的合适替代品。

BACKGROUND & AIMS: :Despite significant progress in cryopreservation of mammalian oocytes and embryos, many ofthe molecular and biochemical events that underlie this technology are poorly understood. In recent years, researchers have focused on obtaining viable oocytes that are developmentally competent. Even under the most favourable conditions, experimental approaches have achieved only limited success compared with fresh oocytes used in routine in vitro embryo production. Chilling injuries and toxic effects of the cryoprotectants are the major adverse consequences following cryoprocedures. To overcome these problems, different strategies have been developed for improving cryopreservation results. These strategies include reducing container volumes, increasing the thermal gradient, changing the cell surface/volume ratio, enhancing cryotolerance by supplementation with various additives or modifying the lipid composition of the oocyte membrane. In order to develop new strategies for reducing the various forms of stress associated with oocyte cryopreservation, it is fundamental to gain a better understanding of the major changes responsible for poor post-thaw survival. With this knowledge, we hope that oocyte cryostorage will become a fully reliable reproductive technique in the near future.

背景与目标： : 尽管在哺乳动物卵母细胞和胚胎的冷冻保存方面取得了重大进展，但该技术背后的许多分子和生化事件却知之甚少。近年来，研究人员专注于获得具有发育能力的活卵母细胞。即使在最有利的条件下，与常规体外胚胎生产中使用的新鲜卵母细胞相比，实验方法也仅取得了有限的成功。冷冻保护剂的冷害和毒性作用是冷冻程序后的主要不利后果。为了克服这些问题，已经开发了不同的策略来改善冷冻保存结果。这些策略包括减少容器体积，增加热梯度，改变细胞表面/体积比，通过补充各种添加剂或改变卵母细胞膜的脂质组成来增强低温耐热性。为了开发新的策略来减少与卵母细胞冷冻保存相关的各种形式的压力，至关重要的是要更好地了解导致解冻后生存不良的主要变化。有了这些知识，我们希望卵母细胞冷冻储存将在不久的将来成为一种完全可靠的生殖技术。

BACKGROUND & AIMS: :Using vitrification and encapsulation-vitrification protocols, we successfully cryopreserved shoot apices from in-vitro plants of different Gentiana cultivars (lines). Although both protocols gave high survival percentages after storage in liquid nitrogen, the encapsulation-vitrification protocol had several distinct advantages over the vitrification protocol: (i) survival was higher under optimal conditions, (ii) the range of optimal exposure periods to the plant vitrification solution 2 (PVS2) was broader, and (iii) regrowth of cryopreserved shoot apices was apparently more vigorous and faster. Shoot apices from ten cultivars/lines of three Gentiana species (G. scabra, G. triflora, and G. pneumonanthe) were successfully cryopreserved using the two protocols with average survival of 49.0 percent and 73.7 percent for vitrification and encapsulation-vitrification, respectively. These results indicate that the two protocols optimized in the present study are promising for cryopreservation of a wide range of Gentiana genetic resources.

背景与目标： : 使用玻璃化和封装玻璃化方案，我们成功地从不同龙胆品种 (系) 的体外植物中冷冻保存了茎尖。尽管两种方案在液氮中储存后均具有较高的存活率，但封装-玻璃化方案与玻璃化方案相比具有几个明显的优势 :( i) 在最佳条件下的存活率更高，(ii) 植物玻璃化溶液2 (PVS2) 的最佳暴露时间范围更广，(iii) 冷冻保存的芽尖的再生速度显然更加旺盛和更快。使用这两种方案成功地冷冻保存了三种龙胆物种 (G. scabra，G. triflora和G. pneumonanthe) 的十个品种/品系的茎尖，平均存活49.0% 和73.7% 分别用于玻璃化和包封玻璃化。这些结果表明，在本研究中优化的两个方案有望用于广泛的龙胆遗传资源的冷冻保存。

BACKGROUND & AIMS: :The evaluation of the motility data obtained with a CASA system, applying a Two-Step Cluster analysis, identified in seabream sperm 3 different sperm subpopulations that correlated differently with embryo hatching rates. Hence, we designed an experiment to understand the effect of the application of different cryopreservation protocols in these sperm motility-based subpopulations. We analyzed Sparus aurata frozen/thawed semen motility 15, 30, 45 and 60s after activation, using CASA software. Different protocols were applied for cryopreservation: three different cryoprotectants (Dimethyl Sulfoxide (Me(2)SO), Ethylene Glycol (EG) and Propylene Glycol (PG)) each at two different concentrations and two packaging volumes (0.5ml straws, and 1.8ml cryovials) were tested. Different freezing rates were evaluated corresponding to 1, 2, 3, 4 and 8cm above the liquid nitrogen surface for the straws and 1, 2 and 4cm for the cryovials. Motility parameters rendered by CASA were treated with a Two-Step Cluster analysis. Three different subpopulations were obtained: SP1 - slow non-linear spermatozoa, SP2 - slow linear spermatozoa and SP3 - fast linear spermatozoa. We considered SP3 as the subpopulation of interest and focused further analyses on it. Generally, SP3 was the best represented subpopulation 15s after activation and was also the one showing a greater decrease in time, being the least represented after 60s. According to the applied univariate general linear model, samples frozen in straws with 5% Me(2)SO and in cryovials with 10% Me(2)SO at 2 and 1cm from the LN(2,) respectively, produced the best results (closer to the control). Clustering analysis allowed the detection of fish sperm subpopulations according to their motility pattern and showed that sperm composition in terms of subpopulations was differentially affected by the cryopreservation protocol depending on the cryoprotectant used, freezing rates and packaging systems.

背景与目标： : 使用CASA系统获得的运动性数据的评估，应用两步聚类分析，在seabrain精子中确定了3个与胚胎孵化率不同相关的不同精子亚群。因此，我们设计了一个实验来了解不同冷冻保存方案在这些基于精子运动的亚群中的应用效果。我们使用CASA软件分析了激活后15、30、45和60s的Sparus aurata冷冻/解冻的精液运动能力。应用不同的方法进行冷冻保存: 测试三种不同的冷冻保护剂 (二甲基亚砜 (Me(2)SO) 、乙二醇 (EG) 和丙二醇 (PG))，各自以两种不同的浓度和两种包装体积 (0.5毫升吸管和1.8毫升冷冻瓶)。评估不同的冷冻速率，对应于吸管的液氮表面上方的1、2、3、4和8厘米，以及冷冻瓶的1、2和4厘米。通过两步聚类分析处理CASA提供的运动参数。获得了三个不同的亚群: SP1-慢非线性精子，SP2-慢线性精子和SP3-快线性精子。我们认为SP3是感兴趣的亚群，并对其进行了进一步的分析。通常，SP3是激活后15s代表最好的亚群，也是时间减少幅度更大的亚群，在60s后代表最少。根据所应用的单变量一般线性模型，分别在5% Me(2)SO的吸管和10% Me(2)SO的冷冻瓶中冷冻的样品分别在LN(2，) 的2和1厘米产生最佳结果 (更接近对照)。聚类分析可以根据鱼类精子的运动模式检测其精子亚群，并表明，根据低温保存方案，根据所使用的冷冻保护剂，冷冻速率和包装系统，精子亚群的组成受到不同的影响。

BACKGROUND & AIMS: :Cryopreservation of sperm is an extremely important issue in the field of male infertility as freezing can have detrimental effects on a variety of sperm functions, some of them not accessible to the traditional semen quality analysis. In this study, chromatin structure variations in human spermatozoa in semen were studied with the sperm chromatin structure assay (SCSA), both before and after cryopreservation. Samples were divided into two aliquots: the first was analysed without further treatment, while the second was stored in liquid nitrogen at -196 degrees C using standard cryopreservation techniques. The fresh and thawed aliquots were also assessed by light and fluorescence microscopy (after Acridine Orange staining, AO), and computer-assisted semen analysis (CASA) of motility. Overall sperm quality was found to deteriorate after cryopreservation. When thawed spermatozoa were subjected to an extra swim-up round, a general improvement in nuclear maturity was seen in post-rise spermatozoa.

背景与目标： : 精子的冷冻保存是男性不育领域中一个极其重要的问题，因为冷冻会对各种精子功能产生不利影响，其中一些是传统精液质量分析无法实现的。在这项研究中，使用精子染色质结构测定法 (SCSA) 研究了冷冻保存前后精液中人精子的染色质结构变化。将样品分成两个等分试样: 第一个在没有进一步处理的情况下进行分析，而第二个在-196 ℃ 下使用标准冷冻保存技术储存在液氮中。还通过光镜和荧光显微镜 (a啶橙染色后，AO) 以及运动的计算机辅助精液分析 (CASA) 评估了新鲜和解冻的等分试样。冷冻保存后发现总体精子质量下降。当解冻的精子进行额外的游泳回合时，上升后的精子的核成熟度普遍改善。