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。

BACKGROUND & AIMS: :Cryopreservation of coconut can be used as a strategy to back up the establishment of living collections which are expensive to maintain and are under constant threat from biotic and abiotic factors. Unfortunately, cryopreservation protocols still need to be developed that are capable of producing a sizeable number of field-grown plants. Therefore, we report on the development of an improved cryopreservation protocol which can be used on a wide range of coconut cultivars. The cryopreservation of zygotic embryos and their recovery to soil-growing plants was achieved through the application of four optimised steps viz.: (i) rapid dehydration; (ii) rapid cooling; (iii) rapid warming and recovery in vitro and (iv) acclimatization and soil-supported growth. The thermal properties of water within the embryos were monitored using differential scanning calorimetry (DSC) in order to ensure that the freezable component was kept to a minimum. The feasibility of the protocol was assessed using the Malayan Yellow Dwarf (MYD) cultivar in Australia and then tested on a range of cultivars which were freshly harvested and studied in Indonesia. The most efficient protocol was one based on an 8-h rapid dehydration step followed by rapid cooling step. Best recovery percentages were obtained when a rapid warming step and an optimised in vitro culture step were used. Following this protocol, 20% (when cryopreserved 12 days after harvesting) and 40% (when cryopreserved at the time of harvest) of all MYD embryos cryopreserved could be returned to normal seedlings growing in soil. DSC showed that this protocol induced a drop in embryo fresh weight to 19% and significantly reduced the amount of water remaining that could produce ice crystals (0.1%). Of the 20 cultivars tested, 16 were found to produce between 10% and 40% normal seedlings while four cultivars generated between 0% and 10% normal seedlings after cryopreservation. This new protocol is applicable to a wide range of coconut cultivars and is useful for the routine cryopreservation of coconut genetic resources.

背景与目标： : 椰子的冷冻保存可用作支持建立生活收藏品的策略，这些收藏品的维护成本高昂，并且不断受到生物和非生物因素的威胁。不幸的是，仍然需要开发能够生产大量田间种植植物的冷冻保存方案。因此，我们报告了一种改进的冷冻保存方案的开发，该方案可用于各种椰子品种。通过应用四个优化步骤 (即 :( i) 快速脱水; (ii) 快速冷却; (iii) 体外快速升温和恢复，以及 (iv) 适应和土壤支持的生长，实现了合子胚的冷冻保存及其对土壤生长的恢复。使用差示扫描量热法 (DSC) 监测胚胎中水的热性能，以确保将可冻结的组分保持在最低水平。使用澳大利亚的马来亚黄矮星 (MYD) 品种评估了该方案的可行性，然后在印度尼西亚新鲜收获和研究的一系列品种上进行了测试。最有效的方案是基于8小时快速脱水步骤，然后是快速冷却步骤。当使用快速升温步骤和优化的体外培养步骤时，可获得最佳回收率。按照该方案，可以将冷冻保存的所有MYD胚胎的20% (在收获后12天冷冻保存时) 和40% (在收获时冷冻保存时) 返回到在土壤中生长的正常幼苗中。DSC显示，该方案诱导胚胎鲜重下降至19%，并显著减少了可产生冰晶的剩余水量 (0.1%)。在测试的20个品种中，发现有16个在低温保存后产生10% 至40% 个正常幼苗，而四个在0% 至10% 个正常幼苗之间产生。此新协议适用于广泛的椰子品种，对于椰子遗传资源的常规冷冻保存很有用。

BACKGROUND & AIMS: :Highly differentiated mature spermatozoa carry not only genetic but also epigenetic information that is to be transmitted to the embryo. DNA methylation is one epigenetic actor associated with sperm nucleus compaction, gene silencing, and prepatterning of embryonic gene expression. Therefore, the stability of this mark toward reproductive biotechnologies is a major issue in animal production. The present work explored the impact of hormonal induction of spermiation and sperm cryopreservation in two cyprinids, the goldfish (Carassius auratus) and the zebrafish (Danio rerio), using LUminometric Methylation Assay (LUMA). We showed that while goldfish hormonal treatment did increase sperm production, it did not alter global DNA methylation of spermatozoa. Different sperm samples repeatedly collected from the same males for 2 months also showed the same global DNA methylation level. Similarly, global DNA methylation was not affected after cryopreservation of goldfish spermatozoa with methanol, whereas less efficient cryoprotectants (dimethylsulfoxide and 1,2-propanediol) decreased DNA methylation. In contrast, cryopreservation of zebrafish spermatozoa with methanol induced a slight, but significant, increase in global DNA methylation. In the less compact nuclei, that is, goldfish fin somatic cells, cryopreservation did not change global DNA methylation regardless of the choice of cryoprotectant. To conclude, global DNA methylation is a robust parameter with respect to biotechnologies such as hormonal induction of spermiation and sperm cryopreservation, but it can be altered when the best sperm manipulation conditions are not met.

背景与目标： : 高度分化的成熟精子不仅携带遗传信息，还携带待传递至胚胎的表观遗传信息。DNA甲基化是与精子核压实，基因沉默和胚胎基因表达预形成相关的表观遗传因素之一。因此，该标志对生殖生物技术的稳定性是动物生产中的主要问题。本工作使用光度甲基化测定法 (LUMA) 探讨了激素诱导精子和精子冷冻保存对两种鲤鱼，金鱼 (Carassius auratus) 和斑马鱼 (Danio rerio) 的影响。我们表明，尽管金鱼激素治疗确实增加了精子的产生，但它并未改变精子的整体DNA甲基化。从同一男性重复收集2个月的不同精子样本也显示出相同的全球DNA甲基化水平。同样，用甲醇冷冻保存金鱼精子后，整体DNA甲基化不受影响，而效率较低的冷冻保护剂 (二甲基亚砜和1,2-丙二醇) 降低了DNA甲基化。相反，用甲醇冷冻保存斑马鱼精子会导致整体DNA甲基化略有但显着增加。在不紧密的细胞核中，即金鱼鳍体细胞，无论选择冷冻保护剂，冷冻保存都不会改变整体DNA甲基化。总而言之，对于生物技术 (例如激素诱导精子和精子冷冻保存) 而言，全球DNA甲基化是一个可靠的参数，但是当不满足最佳的精子操作条件时，可以改变它。

BACKGROUND & AIMS: :The USDA-ARS National Plant Germplasm System (NPGS) maintains more than 200 Allium sativum (garlic) accessions at the Western Regional Plant Introduction Station in Pullman, WA. All accessions must be grown out in the field annually since garlic plants from these accessions do not reliably produce seeds and bulbs do not store well. Shoot tips excised from garlic cloves can be successfully cryopreserved using either Plant Vitrification Solution 2 (PVS2; 15 percent v/v DMSO, 15 percent v/v ethylene glycol, 30 percent v/w glycerol, 0.4 M sucrose) or Plant Vitrification Solution 3 (PVS3; 50 percent v/w sucrose, 50 percent v/w glycerol). We compared regrowth of shoot tips representing diverse garlic germplasm after exposure to either PVS2 or PVS3 during the cryopreservation procedure. At the USDA-ARS National Center for Genetic Resources Preservation, a component of the NPGS, we consider accessions successfully preserved if a minimum of 40 percent of explants exhibit regrowth after liquid nitrogen exposure and at least 60 viable shoot tips remain in long-term storage. Ten of twelve diverse garlic accessions were successfully cryopreserved using either PVS2 or PVS3 as cryoprotectants. Five genotypes had the best post liquid nitrogen regrowth after exposure to PVS2, four genotypes had the best regrowth after exposure to PVS3, and three genotypes performed equally well using either cryoprotectant solution. This project is part of an ongoing program to cryopreserve accessions of NPGS clonal crop collections.

背景与目标： : usda-ars国家植物种质系统 (NPGS) 在华盛顿州普尔曼市的西部地区植物引种站保留了200多种葱属 (大蒜)。所有种质必须每年在田间种植，因为这些种质的大蒜植物不能可靠地产生种子，并且鳞茎不能很好地储存。可以使用植物玻璃化溶液2 (PVS2; 15% v/v DMSO，15% v/v乙二醇，30% v/w甘油，0.4 M蔗糖) 或植物玻璃化溶液3 (PVS3; 50% v/w蔗糖，50% v/w甘油)。我们比较了在冷冻保存过程中暴露于PVS2或PVS3后代表不同大蒜种质的茎尖的再生长。在npg的组成部分usda-ars国家遗传资源保存中心，如果至少40% 的外植体在液氮暴露后表现出再生，并且至少有60个可行的茎尖在长期储存中，我们认为种质成功保存。使用PVS2或PVS3作为冷冻保护剂成功冷冻保存了十二种不同大蒜中的十种。暴露于PVS2后，五种基因型的液氮再生长最佳，四种基因型的暴露于PVS3后再生长最佳，使用任一冷冻保护剂溶液，三种基因型的表现均相同。该项目是正在进行的一项计划的一部分，该计划旨在冷冻保存NPGS克隆作物集合的加入。

BACKGROUND & AIMS: OBJECTIVE:To evaluate sperm cryopreservation-induced injuries using sperm plasma membrane protein P34H and alpha-tubulin as two different subcellular compartment markers. DESIGN:Prospective experimental study. SETTING:Academic hospital research center and fertility clinic. PATIENT(S):Semen samples obtained from healthy donors attending the fertility clinic. Sperm samples were either directly processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot experiments (control group) or cryopreserved in liquid nitrogen for different periods of time before being analyzed. INTERVENTION(S):SDS-PAGE, Western blotting, and densitometric quantification of P34H and alpha-tubulin before and after cryopreservation. MAIN OUTCOME MEASURE(S):Changes in protein quantification between the different groups as a result of sperm cryopreservation. RESULT(S):In the 31 sperm samples processed for P34H evaluation, a 50% decrease is observed after sperm cryopreservation as compared to the control group. The alpha-tubulin immunoblotting of 41 sperm samples revealed a 200% increase in the protein detection in the group of cryopreserved sperm as compared to the control group. Contrary to the P34H detection, this change in alpha-tubulin immunodetected levels appears to be related to the cryopreservation period as it increases during storage. CONCLUSION(S):These findings indicate that cryopreservation of human semen induces damages at different cellular levels. Moreover, some cryoinjuries are immediate although others seem to take place over time stored in liquid nitrogen.

背景与目标：

BACKGROUND & AIMS: :Human mesenchymal stromal cells (MSCs) can differentiate into various cell types, which makes them attractive for regenerative medicine and tissue engineering. Encapsulation of MSCs in alginate microspheres (AMS) is a novel and promising approach of tissue engineering. Application and research of such cell-hydrogel systems require selection of adequate cryopreservation protocols. In this study we investigated the response of MSCs encapsulated in AMS to different cryopreservation protocols. Bone marrow MSCs either encapsulated in AMS and or as cells in suspension, were cryopreserved with 5% and 10% of dimethyl sulfoxide (ME₂SO) using conventional 2-step slow cooling (protocol 1). The viability and metabolism of MSCs in AMS following cryopreservation with 5% Me₂SO were lower than in the group cryopreserved with 10% Me₂SO. MSCs in suspension were more resistant to cryopreservation than cells in AMS when cryopreserved with 5% Me₂SO, although when using a concentration of 10% Me₂SO, no differences were detected. Comparisons of the viability and metabolic activity of MSC cryopreserved either in AMS or as cell suspensions with 10% ME₂SO using protocol 1 (2-step cooling), protocol 2 (3-step slow cooling with induced ice nucleation) or protocol 3 (rapid 1-step freezing), showed that the highest viabilities and metabolic rates were obtained following cryopreservation of MSCs in AMS by protocol 2 (with controlled ice nucleation). Cryopreservation with protocol 3 resulted in critical damage of the encapsulated MSCs. After cryopreservation by protocol 2, AMS encapsulated MSCs were capable of achieving multilineage differentiation directed towards osteogenic, adipogenic and chondrogenic lineages. The data obtained indicate that cryo-banking of AMS encapsulated MSCs is feasible for future regenerative medicine projects.

背景与目标： : 人间充质基质细胞 (MSCs) 可以分化为各种类型的细胞，这使它们对再生医学和组织工程具有吸引力。将MSCs封装在藻酸盐微球 (AMS) 中是一种新颖而有前途的组织工程方法。这种细胞-水凝胶系统的应用和研究需要选择足够的冷冻保存方案。在这项研究中，我们调查了封装在AMS中的MSCs对不同冷冻保存方案的反应。用常规的2步缓慢冷却 (方案1)，用5% 和10% 二甲基亚砜 (me 2 so) 冷冻保存在am中或作为细胞悬浮的骨髓msc。用5% me 2 so冷冻保存后的AMS中msc的活力和代谢低于用10% me 2 so冷冻保存的组。当用5% me2 so冷冻保存时，悬浮液中的msc比AMS中的细胞对冷冻保存的抗性更高，尽管当使用浓度为10% me2 so时，未检测到差异。比较在AMS或10% me 2的细胞悬浮液中冷冻保存的MSC的活力和代谢活性，因此使用方案1 (2步冷却)，方案2 (3步缓慢冷却诱导冰成核) 或方案3 (快速1步冷冻)，结果表明，通过方案2 (受控的冰成核) 将msc冷冻保存在AMS中后，可获得最高的存活率和代谢率。使用方案3进行冷冻保存会导致封装的msc严重受损。通过方案2冷冻保存后，AMS封装的msc能够实现针对成骨，成脂和软骨谱系的多系分化。获得的数据表明，AMS封装的msc的冷冻银行对于未来的再生医学项目是可行的。