Trehalose in glycerol-free freezing extender enhances post-thaw survival of boar spermatozoa

Cryopreservation of boar semen is still considered suboptimal due to lower fertility as compared with fresh samples when glycerol, a permeating cryoprotectant, is used. Trehalose is a non-permeable cryoprotectant and nonreducing disaccharide known to stabilize proteins and biologic membranes. The aim of this study was to evaluate the cryosurvival and in vitro penetrability of boar spermatozoa when glycerol was replaced with trehalose in a freezing extender. Ejaculated Berkshire semen samples were diluted in egg yolk-based freezing extender containing glycerol (100 mM) or trehalose (0, 50, 100, 150, 200 and 250 mM) and cryopreserved using a straw freezing procedure. Thawed samples were analyzed for motility, viability, mitochondrial membrane potential (MMP), and acrosome integrity. In experiment 2, penetrability of spermatozoa cryopreserved with 100 mM glycerol or trehalose was examined. Replacement of cryoprotectant glycerol (100 mM) with trehalose had no effect on sperm viability, but replacing it with 100 mM trehalose improved motility, MMP and acrosome integrity significantly. Sperm motility and MMP were considerably higher in 100 mM trehalose, whereas the acrosome integrity was substantially higher in 100–250 mM trehalose. The in vitro penetration rate was also significantly higher in spermatozoa cryopreserved with trehalose (61.3%) than in those cryopreserved with glycerol (43.6%). In conclusion, 100 mM non-permeable trehalose can be used to replace glycerol, a permeating cryoprotectant, for maintenance of better post-thaw quality of boar spermatozoa.     

Scouting the application of sourdough to frozen dough bread technology

The use of sourdough, even in combination with cryoprotectant (skim milk, sucrose and trehalose), conventional additives (guar gum, diacetyl tartaric acid esters of monoglycerides, ascorbic acid), honey or fructose and glucose, in frozen dough technology was investigated. After frozen storage, the leavening performance of doughs, and the hardness and texture of breads were compared to those of an unfrozen dough, and to those of a conventional frozen dough. All frozen doughs showed a longer fermentation time and a lower volume increase, with respect to unfrozen dough. When sourdough was combined with cryoprotectant, honey or both, the leavening performance improved compared to the use of sourdough alone. Compared to the conventional frozen dough, higher leavening performance was reached combining sourdough with cryoprotectant alone or together with honey. Sourdough combined with honey, fructose and glucose, honey and cryoprotectant, or conventional additives decreased bread hardness compared to the unfrozen dough bread and to the conventional frozen dough bread. Independently from the use of sourdough, conventional additives allowed to reach a specific volume not significantly different from that of unfrozen dough bread, and breads containing honey were characterized by low values of hardness and by high values of red index.     

不同稀释剂对猪冷冻精液的影响

手握法采集12月龄大约克种公猪精液,在37 ℃下离心,弃去精清,收集浓缩段富含精子部分,用7种不同的稀释液进行稀释,应用液氮熏蒸法制作颗粒冻精。结果表明,在7种稀释液中,7号稀释液 优于其他6种稀释液(p<0.05);与其他的冷冻保护剂相比,甘油的冷冻保护性能较好(p<0.01),其适宜浓度为2~4 ml·l-1;干解冻（40~45 ℃）效果优于湿解冻,解冻后精子活率达0.46~0.49（p<0.05）;稀释液中添加安钠咖可有效地延长精子的冻后存活时间(p<0.01)。     

牛胚胎玻璃化冷冻技术研究进展

胚胎冷冻保存是保存和繁殖遗传优势动物的主要工具,是胚胎移植产业的重要组成部分。目前广泛使用的冷冻方法主要有慢速冷冻和玻璃化冷冻2种。由于玻璃化冷冻具有成本低、效率高、操作简单等优点,玻璃化冷冻法越来越受到人们的重视。经过多年研究,研究人员已经在玻璃化冷冻方法、冷冻液等方面取得了大量进展,玻璃化冷冻法已经开始进入商业化应用。本文综述了牛胚胎冷冻保存技术的研究进展,旨在为相关从业人员提供一定借鉴和参考。     

A novel approach for improving yeast viability and baking quality of frozen dough by adding biogenic ice nucleators from Erwinia herbicola

Freezing deteriorates the baking quality of frozen bread dough by causing lethal injury to yeast cells and depolymerization to the gluten network. To investigate the potential of biogenic ice nucleators in frozen food applications, the effect of extracellular ice nucleators (ECINs) from Erwinia herbicola on the baking quality of frozen dough upon three freeze/thaw cycles were investigated. With addition of ECINs to the activity of 2.4 × 10⁶ units per gram of dough, hardening of bread crumb caused by three freeze/thaw cycles was alleviated by about 50% compared to the control. Additionally, the bread from frozen dough with added ECINs showed 50% larger specific volume compared to the control. The mechanism of cryoprotective effects from ECINs was possibly that ECINs helped in preserving the viability of yeast cells during freeze/thaw cycles. ECINs were able to improve the viability of log-phase and stationary-phase yeast cells in suspensions by about 100 and 10 fold, respectively, and viability of yeast in the frozen dough by 17%. This study revealed the potential of ECINs as a cryoprotectant for applications in the food and biotechnology industries.     

Cryopreservation of marine microalgae and potential toxicity of cryoprotectants to the primary steps of the aquacultural food chain

Cryopreservation, a technique of high potential for culture collections, might offer a solution for reliable supply of microalgae in aquaculture units. Marine microalgae used in aquaculture were cryopreserved under 4, −20 and −80 °C using common cryoprotectants (methanol, dimethylsulfoxide (DMSO), propylene glycol and polyvinylpyrrolidone (PVP)) with promising results for Chlorella minutissima, Chlorella stigmatophora, Isochrysis galbana and Dunaliella tertiolecta. As cryoprotectants usually are toxic above certain concentrations and exposure time, and assuming that low amounts of cryoprotectants will remain in regenerated cultures, an experimental scheme was employed to explore the lower limits of safety for these algae and their primary consumers in hatchery food chains. Results showed that methanol was well tolerated by C. stigmatophora and D. tertiolecta up to a concentration of 1.6% (v/v) while I. galbana could not survive in culture at any concentration and C. minutissima exhibited some 30% of the control's yield at 0.2%. DMSO was highly tolerated up to 1.0% by all strains with the Chlorella strains surviving well up to 2%. Propylene glycol was not only tolerated up to 8% by Dunaliella but induced mixotrophic growth as well, while for Isochrysis it was lethal at any concentration. Among zooplanktonic consumers, brine shrimp Artemia nauplii could tolerate very high concentrations of the tested cryoprotectants, the rotifer Brachionus plicatilis was found sensitive to low amounts of PVP, while the nauplii of the shrimp Penaeus japonicus and the crab Eriocheir sinensis were in general very sensitive to all cryoprotectants and in several cases to much lower amounts than 1%. However, as long as the residues of cryoprotectants are kept below 1% in the regenerated cultures, there will be no problem with the animal consumers.     

Equilibrium vitrification of mouse embryos at various developmental stages using low concentrations of cryoprotectants

We previously developed a new vitrification method (equilibrium vitrification) by which two-cell mouse embryos can be vitrified in liquid nitrogen in a highly dehydrated/concentrated state using low concentrations of cryoprotectants. In the present study, we examined whether this method is effective for mouse embryos at multiple developmental stages. Four-cell embryos, eight-cell embryos, morulae, and blastocysts were vitrified with EDFS10/10a, 10% (v/v) ethylene glycol and 10% (v/v) DMSO in FSa solution. The FSa solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus 0.5 M sucrose. The state of dehydration/concentration was assessed by examining the survival of vitrified embryos after storage at –80°C. When four-cell embryos and eight-cell embryos were vitrified with EDFS10/10a in liquid nitrogen and then stored at –80°C, the survival rate was high, even after 28 days, with relatively high developmental ability. On the other hand, the survival of morulae and blastocysts vitrified in liquid nitrogen and stored at –80°C for four days was low. Therefore, morulae and blastocysts cannot be vitrified in a highly dehydrated/concentrated state using the same method as with two-cell embryos. However, when blastocysts were shrunken artificially before vitrification, survival was high after storage at –80°C for four days with high developmental ability. In conclusion, the equilibrium vitrification method using low concentrations of cryoprotectants, which is effective for two-cell mouse embryos, is also useful for embryos at multiple stages. This method enables the convenient transportation of vitrified embryos using dry ice.     

不同冷冻保护剂对羔羊睾丸组织冷冻效果的影响

为探讨非渗透性冷冻保护剂与渗透性冷冻保护剂配伍对羔羊睾丸组织冷冻效果的影响,以φ=10%DMSO、φ=10%EG、φ=10%DMSO+5g/L BSA和φ=10%EG+5g/L BSA为冷冻保护剂,测定冷冻前后羔羊睾丸组织的细胞活率、曲细精管完整率、睾酮浓度、抑制素质量浓度以及碱性磷酸酶(AKP)和酸性磷酸酶(ACP)活性。结果表明,φ=10%DMSO+5g/L BSA组的细胞活率、曲细精管完整率、睾酮浓度、抑制素的质量浓度及AKP和ACP活性均显著高于其他处理组,但显著低于对照组(新鲜组织);φ=10%EG+5g/L BSA组与φ=10%DMSO组的AKP和ACP活性差异不显著,但均显著高于φ=10%EG组。说明φ=10%DMSO中添加5g/L的BSA能有效改善羔羊睾丸组织的冷冻效果。     

长双歧杆菌BBMN68冷冻保护剂筛选与优化

为提高长双歧杆菌BBMN68的冷冻保藏效果,以冷冻存活率和菌株活力为评价指标,分别评价了4个类型的18种冷冻保护剂.结果表明,脱脂乳、海藻糖、果糖、甘油、维生素C、L-Glu能对BBMN68有效保护.采用二次回归正交设计,对筛选得到的6种保护剂进行复配,得到复配保护剂的最优配方(质量分数)为:甘油3%、海藻糖5%、果糖5%、脱脂乳8%、维生素C 0.05%、L-Glu 0.05%.在复配保护剂的作用下,-80℃保藏期间BBMN68菌数稳定,活菌对数值始终保持在10.5lg(cfu/mL);冷冻浓缩物作为直投发酵剂应用于酸奶中,在21d内菌数变化不大,始终保持在107cfu/mL以上.     

冷冻保护剂对犊牛睾丸组织标志性酶活性的保护作用

为了探讨犊牛睾丸组织适宜的冷冻保存体系,设计以甘油、DMSO、乙二醇、海藻糖、BSA和睾丸液作为主要成分的6种冷冻保护液,以碱性磷酸酶(AKP)、酸性磷酸酶(ACP)、乳酸脱氢酶(LDH)和β-D葡萄糖苷酶(β-D-Glucosidase)的酶活性作为测定指标,以新鲜犊牛睾丸组织的4种酶活性为空白对照,使用6种冷冻保护液将睾丸组织于液氮中冷冻保存7d,然后37℃水浴解冻并无菌培养24h,再制备成φ=10%的睾丸组织匀浆液并测定4种酶活性,最后分析6种冷冻保护液对犊牛睾丸组织标志性酶活性的冷冻保护效果。结果表明,含ρ=0.015g/mL牛血清白蛋白(BSA)的冷冻保护液冷冻7d后,睾丸组织AKP、ACP、LDH和β-D葡萄糖苷酶的活性分别为599.99、129.11、1 891.53和489.98U/g,均显著优于其他组(P0.05);含ρ=0.04g/mL海藻糖的冷冻保护液冷冻7d后,AKP、ACP、LDH和β-D葡萄糖苷酶活性分别为454.88、114.63、1 862.79和457.8U/g,其保护效果次于0.015g/mL BSA组,但均显著优于其他组(P0.05);经DMSO、乙二醇和睾丸液冷冻7d后,4种标志性酶的活性均低于0.015g/mL BSA组和0.04g/mL海藻糖组,甘油组冷冻保护液的效果最差。因此,含ρ=0.015g/mL BSA和ρ=0.04g/mL海藻糖的冷冻保护液能够较好地保护犊牛睾丸组织的酶活性,是犊牛睾丸组织适宜的冷冻保护剂。