Survivability and developmental competences of domestic cat (Felis catus) oocytes after Cryotech method vitrification

The aim of this study was to evaluate the applicability of the Cryotech technique for the vitrification of domestic cat (Felis catus) oocytes, as a model for other feline species threatened with extinction. This technique, in which oocytes are stored in a minimal volume of medium, is already widely used in human assisted reproductive technology. In the first part of this study, a viability test (EtBr/FDA) was used to evaluate the toxicity of the vitrification media (solutions). After IVM, oocytes were placed in vitrification and warming solutions according to the manufacturer's procedure, with or without exposure to liquid nitrogen. The solutions and the vitrification procedure each caused a reduction in oocyte viability, with survival rates of 71.4% in oocytes exposed to the Cryotech media (without cooling in liquid nitrogen), and 62% in oocytes that were vitrified. In the second part of the experiment, parthenogenetic activation was used to evaluate the developmental potential of oocytes previously vitrified using the Cryotech method. After warming, the oocytes were activated using a combination of 0.7 µM ionomycin in TCM 199 medium (5 min) followed by 2 mM 6-DMAP in TCM 199 supplemented with 10% FBS (3 hr), then cultured and evaluated every 24 hr for parthenogenetic cleavage. In the experimental group, 23/50 (46%) cleaved embryos were obtained. Domestic cat oocytes, vitrified by the Cryotech method, are characterized by high survival rates. However, it is necessary to improve the technique to increase the developmental competence of embryos obtained from vitrified oocytes.     

Droplet-vitrification of apical shoot tips of Rubus fruticosus L. and Prunus cerasifera Ehrh.

Apical shoot tips excised from in vitro plantlets of blackberry (Rubus fruticosus L. ‘?a?anska Bestrna’) and cherry plum (Prunus cerasifera Ehrh.) were tested for recovery after cryopreservation using the droplet-vitrification technique. Following treatment for 30 min with a loading solution comprising 1.9 M glycerol and 0.5 M sucrose, explants were dehydrated with a highly concentrated cryoprotectant solution, so called vitrification solution. Shoot tips were dehydrated for 10, 20 and 30 min at room temperature with a solution derived from the original PVS2 solution (containing 37.5% (w/v) glycerol, 15% (w/v) dimethylsulfoxide, 15% (w/v) ethylene glycol and 22.5% (w/v) sucrose) and for 60, 90 and 120 min using the PVS3 solution (containing 50% (w/v) glycerol and 50% (w/v) sucrose). Explants were cooled by direct immersion in LN in 10 μl droplets of vitrification solution placed on aluminium foil strips. Rewarming was done by direct plunging of foil strips in a preheated (37 °C) unloading solution (0.8 M sucrose) for 30 s, after which an equal volume of unloading solution (at room temperature) was added for further incubation for 30 min. As for regrowth of blackberry, PVS3 proved more effective than the modified PVS2, but the difference was significant (P < 0.05) only for the shortest treatment duration. The duration of PVS3 treatment had no significant effect on regrowth of cryopreserved shoot tips (45.8–70%). By contrast, a 30-min treatment with modified PVS2 solution resulted in a significant increase in regeneration percentage (30%), as compared with a 10-min treatment with the same solution (5%). Cherry plum shoot tips were very sensitive to both vitrification solutions and growth recovery of cryopreserved samples was generally lower (5–20%) than that of blackberry explants. No significant influence of PVS treatment (both type of solution and treatment duration) on regrowth of cryopreserved shoot tips was observed with cherry plum shoot tips. Experiments performed in France and in Serbia produced similar results, thereby showing the robustness and reproducibility of the protocols developed.     

苹果砧木试管苗发生玻璃化的因素及预防

对苹果砧术试管苗继代培养过程中形成玻璃苗的可能因素，如瓶内湿度、琼脂浓度、细胞分裂素浓度进行比较试验，观察玻璃苗的形成及变化情况．结果表明：瓶内湿度过大，琼脂浓度略低，细胞分裂素浓度过高，都易引起玻璃化现象，而改变这些因素，可以有效地预防和缓解玻璃苗的发生．     

Cell Ultrastructure of Kiwifruit (Actinidia chinensis) Shoot Tips During Cryopreservation

The changes in the cell ultrastructure of in vitro cultured shoot tips from dwarf genotype of kiwifruit (Actinidia chinensis Ganmi 5) during cryopreservation were investigated. Shoot tips were preserved in liquid nitrogen using vitrification, and the cell ultrastructure was examined using transmission electron microscopy (TEM). The regular ultrastructure of the cell wall, cell membrane and nucleus of shoot tips could be damaged during the freezing and thawing associated with preservation using liquid nitrogen. The cell plasmolysis was increased and freezing tolerance was improved after preculturing and dehydrating in a preservation and vitrification solution (PVS2 ) (30% glycerol (Gly)+ 15% ethylene glycol (EG)+ 15% dimethylsulfoxide (DMSO) + 0.4 mol L-1 sucrose). The structure of some cells with low degree of injury and reversible damage was similar to that of the control and they could undergo normal cell division and differentiation. Besides, they could recover automatically and regenerate after their reculture.     

牛胚胎玻璃化超快速冷冻一步法移植试验

应用 30 %乙二醇 0 .3mol/ L 蔗糖 - m- PBS液 (VS1)、30 %乙二醇 0 .3m ol/ L 蔗糖 5 %葡聚糖 (T- 5 0 0 ) - m-PBS液 (VS2 )、30 %乙二醇 0 .3mol/ L蔗糖 10 %葡聚糖 (T- 5 0 0 ) - m- PBS液 (VS3)玻璃化超快速冷冻奶牛胚胎 ,一步法移植。结果显示 ,VS1、VS2、VS3组玻璃化超快速冷冻奶牛胚胎解冻后的形态正常率分别为 10 0 % (2 0 / 2 0 )、95 %(19/ 2 0 )和 5 5 .6 % (5 / 9) ;培养存活率分别为 70 % (14 / 2 0 )、75 % (15 / 2 0 )和 2 2 .2 % (2 / 9) ;囊胚孵化率分别为 0、2 0 % (4/2 0 )和 0。VS1、VS2组玻璃化冷冻奶牛胚胎解冻后的形态正常率极显著地高于 VS3组 (P<0 .0 1) ;VS1、VS2组玻璃化冷冻奶牛胚胎解冻后的培养存活率分别显著 (P<0 .0 5 )和极显著 (P<0 .0 1)地高于 VS3组。 VS1组冷冻的胚胎经一步法移植了 5头 (1枚 /头 ) ,未获得妊娠 (0 / 5 ) ;VS2组冷冻的胚胎用一步法移植了 10头 (1枚 /头 ) ,结果获得 2头妊娠(2 / 10 ) ,并产下 2头正常犊牛。     

木本植物试管苗玻璃化成因与控制研究进展

玻璃化是非受伤胁迫条件下试管苗的一种生理病变,试管苗的玻璃化,严重影响了微体快速繁殖的效率.综述国内外研究发现,木本植物试管苗玻璃化的形成,与培养基中糖（碳源）浓度、琼脂种类及浓度、离子（主要为Cu2）含量、细胞分裂素、乙烯及生长素等因子有关;过氧化物酶-IAA氧化酶体系对乙烯释放的控制,苯丙氨酸解氨酶和酸性过氧化物酶活性降低对木质化过程进行的抑制以及纤维素、木质素的缺乏,细胞壁压的降低等,均使得细胞过分吸水,从而导致木本植物玻璃化的发生.国内外试管苗玻璃化的控制技术主要有：增加固体培养基中琼脂浓度和蔗糖含量;降低培养基中Cl-、NH4＋的浓度及细胞分裂素含量,或向培养基中加入间苯三酚或根皮苷;材料需经低温处理并降低环境中的相对湿度.     

驱蚊香草的组织培养技术

利用驱蚊香草茎段进行组织培养，研究了添加在培养基中不同激素组成、浓度、培养条件等因素对不定芽诱导、继代增殖、防止玻璃化以及生根的影响．结果表明：适于驱蚊香草不定芽诱导的培养基为MS＋6-BA1．5mg／L＋NAA0．05mg／L，适于继代增值的培养基为MS＋6-BA1．5mg／L＋NAA0．05mg／L．适于生根的培养基为配方为1／2MS＋IBA0．5mg／L＋NAA0．1mg／L；用透气设施的封口材料明显地降低驱蚊香草的玻璃化；基质培养基生根优于琼脂培养基.     

猪GV期卵母细胞玻璃化冷冻保存技术研究

旨在探讨提高猪GV期卵母细胞冷冻保存效率的可能途径。将EDS、EFS40和ES冷冻保护液用作卵母细胞冻前、冻后程序的处理,但不冷冻,比较3种冷冻保护剂对猪GV期卵母细胞的毒性作用;采用ES液作玻璃化液,比较常规细管法、OPS法和电镜铜网法3种冷冻载体对猪GV期卵母细胞的冷冻效果;并以ES液作玻璃化液,OPS管为冷冻载体,将猪GV期卵母细胞分5组,即对照组、CB+离心处理组、直接冷冻组、CB+冷冻组、CB+离心+冷冻组进行对比处理,比较各处理卵母细胞的冻后存活率与发育率。结果表明,在不同冷冻保护液中,以ES液组合作为玻璃化冷冻液时的毒性作用最低,与对照组间无明显差异(P>0.05);在3种冷冻载体中,用OPS法冷冻猪GV期卵母细胞,所获冻后存活率明显高于电镜铜网法和细管法(65.4%对45.0%和38.6%,P<0.05),并可获得最佳的冻后成熟率(43.3%);猪GV期卵母细胞单纯经细胞松弛素B和离心极化处理,不会严重影响卵母细胞的存活,但卵裂率显著低于对照组(39.0%对52.1%,P<0.05);细胞松弛素B处理或细胞松弛素B+离心极化处理后再进行玻璃化冷冻,并不能提高卵母细胞的冻后存活率与发育率。采用OPS法直接进行GV期卵母细胞的玻璃化冷冻,可获得7.8%的冻后卵裂率,并能获得桑椹胚发育,是一种有效的猪卵母细胞冷冻保存技术。     

Good Thermally Conducting Material Supports Follicle Morphologies of Porcine Ovaries Cryopreserved with Ultrarapid Vitrification

Effects of supporting materials during vitrification procedure on the morphologies of preantral follicles of pig ovaries were assessed. Ovarian cortical sections of prepubertal pigs were randomly allocated to 5 groups. The sections were vitrified ultrarapidly with 5 different vitrification devices. The sections were put on 4 fine needles (Cryosupport), on a thin copper plate, or on a carbon graphite sheet or were sandwiched between copper plates or between carbon graphite sheets before cooling. The cooling and warming rates with the graphite sheets were significantly higher than those with the copper plates (P<0.05). A total of 3,064 follicles were analyzed following HE staining after vitrification with 5 different devices. The morphologies follicles vitrified on the Cryosupport or on the graphite sheet were well preserved compared with those vitrified on the copper plate or between copper plates (P<0.01). The morphologies of follicles vitrified between copper plates were mostly damaged (P<0.05). Taken together, good thermally conducting material supports follicle morphologies of ovaries cryopreserved with ultrarapid vitrification.     

Vitrification procedure decreases inositol 1,4,5‐trisphophate receptor expression,resulting in low fertility of pig oocytes

Although cryopreservation of mammalian oocytes is an important technology, it is well known that unfertilized oocytes, especially in pigs, are highly sensitive to low temperature and that cryopreserved oocytes show low fertility and developmental ability. The aim of the present study was to clarify why porcine in vitro matured (IVM) oocytes at the metaphase II (MII) stage showed low fertility and developmental ability after vitrification. In vitro matured cumulus oocyte complexes (COCs) were vitrified with Cryotop and then evaluated for fertility through in vitro fertilization (IVF). Although sperm‐penetrated oocytes were observed to some extent (30–40%), the rate of pronuclear formation was low (9%) and none of them progressed to the two‐cell stage. The results suggest that activation ability of cryopreserved oocytes was decreased by vitrification. We examined the localization and expression level of the type 1 inositol 1,4,5 trisphosphate receptor (IP₃R1), the channel responsible for Ca²⁺ release during IVF in porcine oocytes. Localization of IP₃R1 close to the plasma membrane and total expression level of IP₃R1 protein were both decreased by vitrification. In conclusion, our present study indicates that vitrified‐warmed porcine COCs showed a high survival rate but low fertility after IVF. This low fertility seems to be due to the decrease in IP₃R1 by the vitrification procedure.