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Targeted development and optimization of small-molecule ice recrystallization inhibitors (IRIs) for the cryopreservation of biological systems. 有针对性地开发和优化用于生物系统低温保存的小分子冰重结晶抑制剂(IRIs)。
IF 1 4区 生物学 Q3 BIOLOGY Pub Date : 2024-03-01
L E McMunn, E M Walsh, R N Ben

Despite the routine use of cryopreservation for the storage of biological materials, its outcomes are often sub-optimal (including reduced post-thaw viability, recovery, and functionality) due to the damage caused by uncontrolled ice growth. Traditional cryoprotective agents (CPAs), including dimethyl sulfoxide (DMSO), fail to prevent damage caused by ice growth and concerns over CPA cytotoxicity have fostered an increased interest in developing improved CPAs and cryoprotection strategies. The inhibition of ice recrystallization by natural antifreeze (glyco)proteins [AF(G)Ps] to improve cryopreservation outcomes has been examined; however, the ice binding properties of these substances and their challenging large-scale production make them poor CPA candidates. Therefore, the development and deployment of biocompatible, small-molecule ice recrystallization inhibitors (IRIs) for use as CPAs is a worthwhile objective. Extensive structure-activity relationship studies on AF(G)Ps revealed that simple carbohydrate derivatives could inhibit ice recrystallization. It was later discovered that this activity could be fine-tuned by delicately balancing the molecule's hydrophobicity and hydrophilicity. Current generation small-molecule IRIs have been meticulously designed to avoid binding to the surface of ice and subsequent biological testing (for both cytotoxicity and cryopreservation efficacy) has demonstrated significant improvements to the cryopreservation outcomes of several cell types. However, an individualized cell-specific approach for the simultaneous assessment of multiple cryopreservation outcomes is necessary to realize the full potential of IRIs as CPAs. This article provides a detailed overview of the development of small-molecule carbohydrate-based IRIs and highlights the crucial cell-specific biological considerations that must be taken into account when assessing cryopreservation outcomes. https://doi.org/10.54680/fr24210110112.

尽管生物材料的冷冻保存已成为常规储存方法,但由于不受控制的冰生长所造成的损害,其结果往往并不理想(包括解冻后的存活率、恢复能力和功能性降低)。包括二甲基亚砜(DMSO)在内的传统冷冻保护剂(CPAs)无法防止冰生长造成的损害,而对 CPA 细胞毒性的担忧又促使人们对开发改良型 CPAs 和冷冻保护策略的兴趣与日俱增。天然抗冻(糖)蛋白[AF(G)Ps]可抑制冰的再结晶,从而改善低温保存效果,但这些物质的冰结合特性及其大规模生产的挑战性使其成为不理想的 CPA 候选物质。因此,开发和应用生物相容性小分子冰重结晶抑制剂(IRIs)作为 CPAs 是一个值得实现的目标。对 AF(G)Ps 的广泛结构-活性关系研究发现,简单的碳水化合物衍生物可以抑制冰的再结晶。后来人们发现,这种活性可以通过微妙地平衡分子的疏水性和亲水性来进行微调。新一代小分子 IRI 经过精心设计,可避免与冰表面结合,随后进行的生物测试(细胞毒性和低温保存效果)表明,几种细胞类型的低温保存效果显著改善。然而,要充分发挥 IRIs 作为 CPA 的潜力,还需要一种针对特定细胞的个性化方法来同时评估多种低温保存结果。本文详细概述了基于碳水化合物的小分子 IRIs 的发展情况,并强调了在评估冷冻结果时必须考虑的关键细胞特异性生物学因素。https://doi.org/10.54680/fr24210110112。
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引用次数: 0
Testing of arctic insect hemolymph as a secondary agent in applied cryopreservation. 测试北极昆虫血淋巴作为应用冷冻保存的辅助剂。
IF 1 4区 生物学 Q3 BIOLOGY Pub Date : 2024-03-01
N G Li

Background: Cold hardiness of insects from extremely cold regions is based on a principle of natural cryoprotection, which is associated with physiological mechanisms provided by cryoprotectants.

Objective: Since arctic cold-hardy insects are producers of highly effective cryoprotectants, in this study, the hemolymph of Aporia crataegi L. and Upis ceramboides L. from an extremely cold area (Yakutia) was tested as a secondary component of cryoprotective agents (CPA) for cryopreservation (-80 degree C) of human peripheral blood lymphocytes and skin fibroblasts.

Materials and methods: Lymphocytes and skin fibroblasts were treated with various combinations of DMSO and hemolymph extract and step-wise cooled to -80 degree C. Post-cryopreservation cell viability was assessed by vital staining and morphological appearance.

Results: Viability was higher when cells were frozen with a mixture containing DMSO and Upis ceramboides hemolymph compared to the cells frozen in DMSO, while cells frozen with DMSO and Aporia crataegi hemolymph did not survive. The fact that hemolymph of not every cold-resistant insect can be used as a secondary agent along with DMSO indicates that only a unique combination of hemolymph components and its compatibility with cells might result in a positive effect.

Conclusion: Although the use of insect hemolymph as a complementary agent in applied cryopreservation is a problem in terms of practical application, such studies could initiate new trends in the search for the most successful hemolymph-like cryoprotectant systems. https://doi.org/10.54680/fr24210110712.

背景:极寒地区昆虫的耐寒性是基于自然低温保护的原理,这与低温保护剂提供的生理机制有关:由于北极耐寒昆虫是高效低温保护剂的生产者,本研究将极寒地区(雅库特)的 Aporia crataegi L. 和 Upis ceramboides L. 的血淋巴作为低温保护剂(CPA)的次要成分进行测试,以低温保存(-80 摄氏度)人类外周血淋巴细胞和皮肤成纤维细胞:用二甲基亚砜和血淋巴提取物的不同组合处理淋巴细胞和皮肤成纤维细胞,然后逐级冷却至零下 80 摄氏度:结果:与用二甲基亚砜冷冻的细胞相比,用含有二甲基亚砜和Upis ceramboides血淋巴的混合物冷冻的细胞存活率更高,而用二甲基亚砜和Aporia crataegi血淋巴冷冻的细胞则无法存活。并非所有耐寒昆虫的血淋巴都能与二甲基亚砜一起用作辅助制剂,这表明只有血淋巴成分的独特组合及其与细胞的相容性才可能产生积极的效果:尽管将昆虫血淋巴用作应用低温保存的辅助剂在实际应用方面存在问题,但此类研究可能会在寻找最成功的血淋巴低温保护剂系统方面引发新的趋势。https://doi.org/10.54680/fr24210110712。
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引用次数: 0
The kinetics of goat sperm is negatively affected after freezing in an extender including zinc oxide nanoparticles. 山羊精子在包括纳米氧化锌颗粒在内的扩展剂中冷冻后,其动力学会受到负面影响。
IF 1 4区 生物学 Q3 BIOLOGY Pub Date : 2024-03-01
L C Pereira Arruda, G de Oliveira Alves Pinto, G F Carneiro, M M Pessoa Guerra

Background: Nanotechnology can benefit livestock industries, especially through postharvest semen manipulation. Zinc oxide nanoparticles (Np-ZnO) are potentially an example.

Objective: To investigate how the addition of zinc oxide nanoparticles (Np-ZnO) affected the characteristics of post-thawed goat semen.

Materials and methods: Seminal pools from four Saanen bucks were used. Semen was diluted in Tris-egg yolk extender, supplemented with Np-ZnO (0, 50, 100 or 200 ug/mL), frozen and stored in liquid nitrogen (-196 degree C), and thawed in a water bath (37 degree C / 30 s). Semen samples were evaluated for sperm kinetics by computer-assisted sperm analysis (CASA), and assessed for other functional properties by epifluorescence microscopy, such as plasma membrane integrity (PMi), acrosomal membrane integrity (ACi) and mitochondrial membrane potential (MMP).

Results: For total motility (TM), the group treated with 200 ug/mL Np-ZnO was superior to the control. In straight-line velocity (VSL), the control was better than the group containing 200 ug/mL of Np-ZnO. For average path velocity (VAP), the control was higher than with 100 ug/mL Np-ZnO. For linearity (LIN), the control was higher than with 200 μg/mL Np-ZnO. In straightness (STR), the control and 100 μg/mL Np-ZnO were higher than with 200 ug/mL Np-ZnO. In wobble (WOB), the control was better than the 50 μg/mL Np-ZnO treatment. In PMi, ACi and MMP no significant differences were found.

Conclusion: The addition of Np-ZnO (200 ug/mL) to the goat semen freezing extender improved the total motility of cells, whilst negatively affecting sperm kinetics. https://doi.org/10.54680/fr24210110512.

背景:纳米技术可造福畜牧业,特别是通过采精后的处理。纳米氧化锌(Np-ZnO)就是一个潜在的例子:研究添加纳米氧化锌(Np-ZnO)如何影响解冻后山羊精液的特性:材料:使用四只萨能公羊的精液池。精液用三聚氰胺蛋黄扩展剂稀释,添加 Np-ZnO(0、50、100 或 200 微克/毫升),冷冻并储存在液氮中(-196 摄氏度),然后在水浴中解冻(37 摄氏度/30 秒)。精液样本通过计算机辅助精子分析(CASA)评估精子动力学,并通过外荧光显微镜评估其他功能特性,如质膜完整性(PMi)、顶体膜完整性(ACi)和线粒体膜电位(MMP):在总运动能力(TM)方面,用 200 微克/毫升 Np-ZnO 处理的组优于对照组。在直线速度(VSL)方面,对照组优于含有 200 微克/毫升 Np-ZnO 的组。在平均路径速度(VAP)方面,对照组高于含有 100 微克/毫升 Np-ZnO 的组别。在线性度(LIN)方面,对照组高于含有 200 微克/毫升 Np-ZnO 的组。在直线度(STR)方面,对照组和 100 μg/mL Np-ZnO 均高于 200 微克/毫升 Np-ZnO。在摇摆(WOB)方面,对照组优于 50 微克/毫升 Np-ZnO 处理组。在 PMi、ACi 和 MMP 中没有发现明显的差异:https://doi.org/10.54680/fr24210110512。
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引用次数: 0
A novel vitrified cryopreservation approach with stem cell-laden hydrogel microcapsules. 使用干细胞水凝胶微囊的新型玻璃化冷冻保存方法。
IF 1 4区 生物学 Q3 BIOLOGY Pub Date : 2024-03-01
T Song, B Liu

Background: Stem cell-laden hydrogel microcapsules construction is important for a wide application in tissue engineering and cell-based medicine, such as building an ideal immune barrier. Challenges are emerging for effectively storing such microcapsules by cryopreservation, and a large proportion of research has been on the cryopreservation of single cells encapsulated into microcapsules without a core-shell structure.

Objective: To achieve the effective cryopreservation of stem cell-laden hydrogel microcapsules with a core-shell structure.

Materials and methods: A novel core-shell alginate hydrogel encapsulation method was used to produce mesenchymal stem cell-laden microcapsules by microfluidic technique.

Results: This microcapsule could inhibit ice formation to achieve vitreous cryopreservation with a low concentration (2 M) of penetrating cryoprotectants.

Conclusion: Cell laden hydrogel microcapsules may have the potential to be the basis of a new strategy of cell cryopreservation and applications. https://doi.org/10.54680/fr24210110212.

背景:干细胞水凝胶微胶囊的构建对于组织工程和细胞医学的广泛应用非常重要,例如构建理想的免疫屏障。通过低温冷冻有效保存此类微胶囊的挑战正在出现,而大部分研究都是针对封装在无核壳结构微胶囊中的单个细胞的低温冷冻:材料与方法:采用一种新型的核壳海藻酸盐水凝胶封装方法,通过微流控技术制备出含有间充质干细胞的微胶囊:结果:这种微胶囊可以抑制冰的形成,从而在低浓度(2 M)渗透性低温保护剂的作用下实现玻璃体低温保存:细胞载体水凝胶微胶囊有可能成为细胞冷冻保存和应用新策略的基础。https://doi.org/10.54680/fr24210110212。
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引用次数: 0
Optimizing semen cryopreservation in Calomys laucha: a step forward in rodent reproductive research. 优化 Calomys laucha 的精液冷冻保存:啮齿动物生殖研究的一大进步。
IF 1 4区 生物学 Q3 BIOLOGY Pub Date : 2024-03-01
C D Corcini, J Vilela, N Gatti, B Mion, N Castro, R T Franca, A S Varela Junior

Background: Examining semen cryopreservation in Calomys laucha offers valuable insights for reproductive research and species conservation.

Objective: To determine the most effective sugar for the cryopreservation of C. laucha semen.

Materials and methods: Using 36 epididymides from C. laucha, semen samples were diluted in a 3% skimmed milk medium supplemented with one of four sugars (glucose, fructose, lactose, or sucrose) at a concentration of 0.3 M. These mixtures underwent a conditioning phase at 37 degree C for 10 min, cooled to -80 degree C for another 10 min, and were subsequently stored in liquid nitrogen.

Results: Upon thawing, samples treated with lactose and glucose solutions show superior sperm motility, achieving 8.2% and 10.0% respectively, in contrast to the fructose (2.0%) and sucrose (4.1%) mixtures. Furthermore, samples preserved in glucose registered the highest sperm penetration rates, reaching 44.9%.

Conclusion: Our findings suggest that a cryopreservation medium containing 0.3 M glucose can contribute to the safeguarding C. laucha rodent semen. https://doi.org/10.54680/fr24210110612.

背景:研究Calomys laucha的精液冷冻保存为生殖研究和物种保护提供了有价值的见解:目的:确定冷冻保存乌贼精液最有效的糖:精液样本在 3% 脱脂奶培养基中稀释,培养基中添加浓度为 0.3 M 的四种糖(葡萄糖、果糖、乳糖或蔗糖)之一:解冻后,经乳糖和葡萄糖溶液处理的样本精子活力更强,分别达到 8.2% 和 10.0%,与果糖(2.0%)和蔗糖(4.1%)混合物形成鲜明对比。此外,在葡萄糖中保存的样本精子穿透率最高,达到 44.9%:https://doi.org/10.54680/fr24210110612。
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引用次数: 0
RNA-Seq analysis reveals the mechanism in response to cold stress of peach cv 'Dingjiaba'. RNA-Seq 分析揭示了桃品种 "丁家坝 "对冷胁迫的响应机制。
IF 1 4区 生物学 Q3 BIOLOGY Pub Date : 2024-03-01
X Li, H Wang, X Wu

Background: 'Dingjiaba' is an important Prunus persica cultivar (cv) mainly grown in the Hexi corridor in northwest China, which has an inherited strong cold tolerance.

Objective: To compare the transcriptome and physiology data of leaves of cvs 'Dingjiaba' (D) and 'Kanoiwa' (K) following cold treatment at different time periods, in order to gain new insights into the mechanisms of cold adaptation in 'Dingjiaba'.

Materials and methods: We analyzed the transcriptomic and physiological data of leaves of D and K cvs exposed to 0 h (D0/K0), 2 h (D2/K2), 6 h (D6/K6) and 12 h (D12/K12) cold stress.

Results: Low temperature stress caused membrane damage and led to increased rate of electrolyte leakage and increased MDA content. Cold stress induced the accumulation of soluble sugars, soluble proteins and proline in leaves of both cvs, with a lower increase in K compared to D. Transcriptome analysis identified 4,631, 5,069, 5,662 and 3,886 differentially expressed genes (DEGs) between D0 and K0, D2 and K2, D6 and K6 and D12 and K12, respectively. The differentially expressed genes significantly enriched in metabolic pathways and biosynthesis of secondary metabolites. We further validated the reliability of sequencing data of the RNA-Seq with Real-Time Quantitative PCR, which suggested that the expression trend of the RNA-Seq were same as RT-PCR.

Conclusions: These results provide novel insights into a series of molecular mechanisms underlying physiological metabolism and defense. https://doi.org/10.54680/fr24210110312.

背景:'丁家坝'是一个重要的柿树栽培品种(cv),主要生长在中国西北部的河西走廊,具有遗传性强的耐寒性:目的:比较 "丁家坝"(D)和 "香乃岩"(K)在不同时期经冷处理后叶片的转录组和生理数据,以期对 "丁家坝 "的冷适应机制有新的认识:我们分析了暴露于 0 小时(D0/K0)、2 小时(D2/K2)、6 小时(D6/K6)和 12 小时(D12/K12)低温胁迫的 D 和 K 品种叶片的转录组和生理数据:低温胁迫造成膜损伤,导致电解质渗漏率增加和 MDA 含量增加。转录组分析在 D0 和 K0、D2 和 K2、D6 和 K6 以及 D12 和 K12 之间分别发现了 4 631、5 069、5 662 和 3 886 个差异表达基因(DEGs)。差异表达基因明显富集于代谢途径和次生代谢物的生物合成。我们进一步用实时定量 PCR 验证了 RNA-Seq 测序数据的可靠性,结果表明 RNA-Seq 的表达趋势与 RT-PCR 相同:https://doi.org/10.54680/fr24210110312。
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引用次数: 0
Viscoelastic properties of decellularized and freeze-dried human dermis between i909c and 40cC. i909c 和 40cC 之间脱细胞和冻干人体真皮的粘弹性能。
IF 1 4区 生物学 Q3 BIOLOGY Pub Date : 2024-01-01
Q P Jia, S L Huang, Y T Tang, W Q Sun

Background: Human donor skin is processed to make the acellular dermis matrix (ADM) for tissue repair and regeneration. There is no data on the viscoelastic properties of ADM at room and subzero temperatures.

Objective: The work evaluated the temperature dependence of viscoelastic properties of freeze-dried ADM.

Materials and methods: Donor skin was de-epidermized, de-cellularized and freeze-dried with trehalose as the lyo-protectant. Glass transition of freeze-dried ADM was measured by differential scanning calorimeter (DSC), and viscoelastic properties were examined by dynamic mechanical analyzer (DMA).

Results: At the low moisture range (1.4 +/- 0.5%), the glass transition temperature (Tg) of freeze-dried ADM was 90 degree C to 100 degree C. As the moisture content increased, the Tg decreased steadily. At the high moisture range (10.8 +/- 2.9%), the Tg was 40 degree C to 60 degree C. There were large donor-to-donor variations in viscoelastic properties of freeze-dried ADM as demonstrated by the changes in storage modulus (G'), loss modulus (G") and damping factor tan delta (G"/G'). However, the trends of the temperature dependence for G', G" and tan delta were similar among all 8 donors. For each donor, changes in G' and G" were relatively small between -90 degree C and 40 degree C, and G' was at least one order of magnitude greater than G". Two viscoelastic relaxations were observed in freeze-dried ADM, one at -20 degree C and the other at -60 degree C respectively.

Conclusion: Freeze-dried ADM was protected in the glassy carbohydrate matrix. DMA observed two viscoelastic relaxations (i.e., alpha process at -20 degree C and beta process at -60 degree C). Overall changes in G' and G'' of freeze-dried ADM were relatively small within one order of magnitude between -90 degree C and 40 degree C. https://doi.org/10.54680/fr24110110612.

背景:人类供体皮肤经处理后可制成用于组织修复和再生的无细胞真皮基质(ADM)。目前还没有关于 ADM 在室温和零度以下的粘弹性能的数据:这项工作评估了冻干 ADM 粘弹性能的温度依赖性:对供体皮肤进行去表皮、去细胞和冷冻干燥处理,并使用妥尔糖作为低温保护剂。用差示扫描量热仪(DSC)测量冻干 ADM 的玻璃化转变,用动态机械分析仪(DMA)检测其粘弹性:在低水分范围(1.4 +/- 0.5%)内,冻干 ADM 的玻璃化转变温度(Tg)为 90 摄氏度至 100 摄氏度。冻干 ADM 的储藏模量(G')、损耗模量(G")和阻尼系数 tan delta(G"/G')的变化表明,供体与供体之间的粘弹性有很大差异。不过,所有 8 种供体的 G'、G "和 tan delta 随温度变化的趋势相似。对于每种供体,G'和 G "在-90 摄氏度和 40 摄氏度之间的变化相对较小,G'比 G "至少大一个数量级。在冷冻干燥的 ADM 中观察到两种粘弹性松弛,一种是在-20 摄氏度时,另一种是在-60 摄氏度时:结论:冻干 ADM 在玻璃状碳水化合物基质中受到保护。DMA 观察到两种粘弹性松弛(即 -20 摄氏度时的α过程和 -60 摄氏度时的β过程)。在-90 摄氏度和 40 摄氏度之间,冻干 ADM 的 G' 和 G'' 的总体变化在一个数量级内相对较小。https://doi.org/10.54680/fr24110110612。
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引用次数: 0
Supplementation of extender with chamuangone-enriched Garcinia cowa leaf extract improves quality parameters and longevity of cold-stored cat semen. 在扩增剂中添加富含辣椒素的加西牛肝菌叶提取物,可提高冷藏猫精液的质量指标和寿命。
IF 1 4区 生物学 Q3 BIOLOGY Pub Date : 2024-01-01
S Panyaboriban, N Kupthammasan, K Madsri, N Mukem, S Tarngwiriyakul, P Khirilak, P Panichayupakaranant, M Wittayarat

Background: Semen preservation by cooling is less expensive, simpler and results in less sperm damage than freezing does. However, spermatozoa can only be preserved for a short period due to the excessive formation of reactive oxygen species (ROS). Although several antioxidants can protect sperms from ROS damage during storage at low temperatures, the use of natural antioxidants derived from plants would be a better alternative.

Objective: To assess the effects of chamuangone, which can reduce oxidation reactions in cells, on cat semen quality after preservation at 4 degree C for 15 days.

Materials and methods: Epididymal sperm samples were collected before being diluted with tris-citric-fructose-egg yolk (TCFE) extender containing different concentrations of chamuangone (0, 50, 100, 150 and 200 ug/mL) and preserved at 4 degree C. Semen samples were evaluated before chilling and then every 3 days after chilling for up to 15 days. Each sample was assessed for sperm motility, viability, DNA integrity, plasma membrane integrity and percentage of spermatozoa with intact acrosomes.

Results: A significantly higher sperm motility was observed in the group supplemented with 100 ug/mL chamuangone compared to the control after 6 days of storage. However, the chamuangone concentration at 200 ug/mL did not significantly increase the sperm motility when compared to the control for the entire storage period.

Conclusion: 100 µg/mL chamuangone can improve sperm characteristics during 15 days of preservation at 4 degree C, keeping sperm alive (49.3 ± 5.2%) and moving (7.1 ± 2.4%). These results can be used for the development of breeding programs using technologically advanced reproductive procedures in domestic and wild cats. https://doi.org/10.54680/fr24110110212.

背景:与冷冻相比,冷却法保存精液成本更低、更简单,对精子的损伤也更小。然而,由于活性氧(ROS)的过度形成,精子只能保存很短的时间。虽然有几种抗氧化剂可以保护精子在低温储存期间免受 ROS 的损害,但使用从植物中提取的天然抗氧化剂将是更好的选择:目的:评估可减少细胞氧化反应的香豆酮在 4 摄氏度下保存 15 天后对猫精液质量的影响:收集附睾精子样本,然后用含有不同浓度(0、50、100、150 和 200 微克/毫升)香豆素的三柠檬果糖蛋黄(TCFE)扩展剂稀释,并在 4 摄氏度下保存。每个样本都要进行精子活力、存活率、DNA完整性、质膜完整性和顶体完整精子百分比的评估:结果:与对照组相比,补充 100 微克/毫升香豆酮的组在储存 6 天后精子活力明显提高。结论:100 µg/mL chamuangone 可以改善精子在 4 摄氏度下保存 15 天的特性,保持精子存活(49.3 ± 5.2%)和运动(7.1 ± 2.4%)。https://doi.org/10.54680/fr24110110212。
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引用次数: 0
Dog sperm cryopreservation using cryovials and different dilution steps. 使用低温瓶和不同稀释步骤进行狗精子冷冻保存。
IF 1 4区 生物学 Q3 BIOLOGY Pub Date : 2024-01-01
S Ibrahim, N A Hamad Talha, J Cho, Y Jeon, I J Yu

Background: The conventional sperm freezing method for dog sperm is with straws and includes two-step dilution and a long equilibration time.

Objective: To develop a more efficient freezing method using cryovials.

Materials and methods: Three freezing protocols using cryovials (0.5 mL) were conducted with dog spermatozoa at 1 x 108 sperm/mL: Group 1 spermatozoa were cooled in cryovials and extender 1 (E1) and extender 2 (E1 +1 M glycerol) at 4 degree C for 50 min and then frozen over LN2 for 20 min; Group 2 sperm was cooled and frozen in cryovials with a mixture of E1 and E2 (1:1) in a deep freezer (-80 degree C) for 30 min; Group 3 sperm in cryovials and E1 were cooled at 4 degree C for 20 min, cooled for an additional 20 min after addition of E2 (E1:E2, 1:1), and then frozen using LN2/ vapour for 20 min. The control (Group 4) consisted of spermatozoa in straws being frozen using the conventional freezing method using two-step dilution. All groups were plunged and stored in LN2 after freezing and their functional performance and gene expression determined.

Results: Progressive motility and acrosome integrity were highest (P < 0.05) in Groups 2, 3 and 4 (only acrosome integrity). Viability in Group 3 was significantly better that in the other Groups, and the reactive oxygen species (ROS) level and phosphatidylserine (PS) translocation index were significantly lower in Group 2 than the other Groups. The expression of sperm mitochondria-associated cysteine-rich protein (SMCP) and anti-apoptotic B-cell lymphoma 2 (BCL2) genes was highest (P < 0.05) in Group 2 and the expression of pro-apoptotic Bcl2-associated X protein (BAX) was lowest (P < 0.05) in Group 4.

Conclusion: The sperm frozen using cryovials, one step dilution and the deep freezer (Group 2) proved to be a simple and suitable cryopreservation method for dog sperm. https://doi.org/10.54680/fr24110110312.

背景:狗精子的传统冷冻方法是使用吸管,包括两步稀释和较长的平衡时间:材料与方法:使用低温瓶(0.5 mL)的三种冷冻方案:第 1 组精子在冷冻瓶和扩展剂 1(E1)及扩展剂 2(E1 + 1 M 甘油)中于 4 摄氏度下冷却 50 分钟,然后在 LN2 上冷冻 20 分钟;第 2 组精子在冷冻瓶和 E1 及 E2 混合液(1:1)在深冷冻箱(-80 摄氏度)中冷冻 30 分钟;第 3 组精子在冷冻瓶中与 E1 一起在 4 摄氏度下冷却 20 分钟,加入 E2(E1:E2,1:1)后再冷却 20 分钟,然后用 LN2/ 蒸汽冷冻 20 分钟。对照组(第 4 组)是用两步稀释法冷冻吸管中的精子。冷冻后,将所有组的精子放入 LN2 中保存,并测定其功能表现和基因表达:第 2 组、第 3 组和第 4 组(仅顶体完整性)的进行性运动和顶体完整性最高(P < 0.05)。第 3 组的活力明显优于其他组,第 2 组的活性氧(ROS)水平和磷脂酰丝氨酸(PS)转位指数明显低于其他组。第 2 组精子线粒体相关富半胱氨酸蛋白(SMCP)和抗凋亡 B 细胞淋巴瘤 2(BCL2)基因的表达量最高(P < 0.05),第 4 组促凋亡 Bcl2 相关 X 蛋白(BAX)的表达量最低(P < 0.05):结论:使用低温瓶、一步稀释法和深冷箱冷冻精子(第 2 组)被证明是一种简单且适合狗精子的冷冻保存方法。https://doi.org/10.54680/fr24110110312。
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引用次数: 0
Effects of stearic acid on the embryo cryopreservation in mouse. 硬脂酸对小鼠胚胎冷冻的影响
IF 1 4区 生物学 Q3 BIOLOGY Pub Date : 2024-01-01
T N Igonina, T A Rakhmanova, A N Omelchenko, K A Okotrub, E Yu Brusentsev, I N Rozhkova, S Ya Amstislavsky

Background: Intracellular lipids are sensitive to freezing. Lipidome modification is an important tool for studying the role of intracellular lipids in cryotolerance of mammalian oocytes and preimplantation embryos.

Objective: To study the effects of in vitro exposure of murine embryos to saturated stearic acid (SA) on the lipid content, embryo development and cryotolerance.

Materials and methods: In vivo derived mouse embryos were cultured with 100 uM SA for 48 h up to the morula/blastocyst stage. Some of the SA-treated embryos were chosen for the evaluation of their development competence and the change in the lipidome, and other embryos were either slowly frozen or rapidly vitrified.

Results: Nile red staining combined with confocal laser scanning microscopy revealed a decrease in the total amount of lipids in the SA-treated embryos. Raman measurements showed that the lipid unsaturation was lower in embryos after in vitro SA culture. The addition of SA did not affect the embryo development before cryopreservation, but negatively affected the results of slow freezing cryopreservation and vitrification.

Conclusion: In vitro SA exposure lowered the total amount of intracellular lipids and unsaturation in mouse embryos. The changes were accompanied with a significantly lower efficacy of embryo cryopreservation. https://doi.org/10.54680/fr24110110512.

背景:细胞内脂质对冷冻敏感。脂质体修饰是研究细胞内脂质在哺乳动物卵母细胞和植入前胚胎耐冷冻性中的作用的重要工具:研究小鼠胚胎体外暴露于饱和硬脂酸(SA)对脂质含量、胚胎发育和低温耐受性的影响:将体内衍生的小鼠胚胎用 100 uM SA 培养 48 小时,直至胚泡/囊胚期。选择部分经 SA 处理的胚胎评估其发育能力和脂质体的变化,其他胚胎则被缓慢冷冻或快速玻璃化:结果:尼罗河红染色结合共焦激光扫描显微镜显示,经 SA 处理的胚胎中的脂质总量有所减少。拉曼测量显示,体外 SA 培养后的胚胎脂质不饱和度较低。添加 SA 不会影响冷冻保存前的胚胎发育,但会对慢冻冷冻和玻璃化的结果产生负面影响:结论:体外 SA 暴露降低了小鼠胚胎细胞内脂类的总量和不饱和度。https://doi.org/10.54680/fr24110110512。
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Cryo letters
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