减毒猪霍乱沙门氏菌C500抗冻保护剂的优化研究

本试验旨在优化减毒猪霍乱沙门氏菌C500抗冻保护剂，提高工程菌C500的存活率。通过单因素试验考察了4种类型的抗冻保护剂（渗透性、半渗透性、非渗透性保护剂及抗氧化剂）对工程菌C500冷冻保存前后存活率的影响，各保护剂设置不同的浓度并与工程菌C500等体积混合，于液氮中保存2周后进行平板计数以计算其成活率，初步筛选出最有利于菌种保存的渗透性保护剂、半渗透性保护剂、非渗透性保护剂和抗氧化剂及其最佳保护浓度，最后将筛选出的4种保护剂进行四因素三水平的正交试验以得出最佳的组合配方。单因素试验的结果表明，渗透性保护剂中乙二醇、半渗透性保护剂中蔗糖、非渗透性保护剂中聚乙烯吡络烷酮（PVP）及抗氧化剂中甘氨酸对工程菌C500的保护作用最佳，最佳保存浓度分别为15%、6%、6%和0.25%；正交试验结果表明，各因素均对工程菌C500的存活率有显著影响，筛选出的最佳组合配方为17%乙二醇、9.0%蔗糖、6.0% PVP、0.35%甘氨酸，最佳组合在-80 ℃的保存效果最佳，此温度下保存6周存活率可达88.34%，其次为液氮组、-20 ℃组及4 ℃组。这表明本试验得到的保护剂配方极大地提高了工程菌C500的抗冻能力，使其存活率大大提高。     

小鼠肾细胞冻存过程中甘油浓度的优化

目的:探讨在冻存小鼠肾细胞过程中,通过优化冷冻保护剂中甘油浓度的加入量,以达到小鼠肾细胞的最佳冻存效果。方法:将小鼠肾细胞制备成单细胞悬液,然后加入不同浓度的冷冻保护剂甘油,冻存细胞,比较细胞冻存效果,计算细胞成活率。四个组的甘油加入量分别为5%、10%、15%、20%。结果:在小鼠肾细胞冻存过程中,当冷冻保护剂中甘油的浓度达到10%时,冻存效果最佳,细胞成活率达到89.57%。     

Effect of cryoprotectants and thawing temperatures on chicken sperm quality

There is need for standardization of freezing–thawing protocol for rooster semen to minimize variability among results. Therefore, we aimed to compare effect of four different permeating cryoprotectants and two thawing temperatures (37 vs. 5°C) on sperm post‐thaw motility and to analyse combined effect of the best permeating cryoprotectant (P‐CPA) with one of four non‐permeating cryoprotectants (N‐CPA) on post‐thaw quality of rooster semen evaluated in vitro. Pooled semen from Ross PM3 rooster heavy line was diluted in Kobidil extender and frozen in cryoprotectant solution containing 6% dimethylacetamide, 7.5% dimethylformamide, 9% N‐methylacetamide or 8% ethylene glycol (EG) in liquid nitrogen vapours. To determine the best thawing rate, straws were thawed either at 37 or 5°C. Furthermore, samples were frozen in the presence of the best N‐CPA either with 0.75 mol/L ficoll, 0.2 mol/L sucrose, 0.2 mol/L trehalose or 0.05 mol/L glycine. Sperm motility, membrane destabilization and viability were analysed to compare different freezing–thawing conditions. In addition, morphology and ultrastructure analysis were performed to compare fresh and frozen‐thawed sperm quality. Our results indicate that the combination of EG and the thawing at 5°C improves (p ≤ .05) sperm post‐thaw motility. Moreover, ficoll addition to EG‐based freezing extender provided additional beneficial effect (p ≤ .05) on progressive movement and apoptosis incidence. Further work should evaluate different N‐CPA concentrations to improve freezing protocol. In addition, fertility evaluation and testing on different chicken lines are needed in order to contribute to animal genetic resources bank.     

Effect of cooling rate and cryoprotectant on the cryosurvival of equine spermatozoa

Cryosurvival of cells is reduced if the cooling rate used is suboptimal. If cells cool too rapidly, intracellular water will freeze, causing intracellular ice crystals. However, if spermatozoa are cooled too slowly, excessive cellular dehydration occurs, causing irreversible damage to cellular compartments. In addition, cryoprotectants are added to the freezing diluent to protect cells from damage during cryopreservation. This study was conducted to determine the optimal cooling rate for stallion spermatozoa frozen in the presence of three different cryoprotectants. Spermatozoa were frozen in a skim milk, egg yolk diluent containing 4% glycerol, and ethylene glycol or dimethyl formamide at 10 different cooling rates ranging from 5°C/min to 50°C/min. The percentage of viable spermatozoa was higher for spermatozoa cooled at 10°C/min than at 50°C/min (P < .05). Spermatozoa frozen using glycerol as the cryoprotectant had higher percentages of motile and progressively motile spermatozoa compared with spermatozoa frozen using the other two cryoprotectants (P < .05). In conclusion, the cryosurvival of stallion spermatozoa is similar when cooling rates of 5°C/min to 45°C/min are used, and when 4% cryoprotectant is used, glycerol is a more effective cryoprotectant than ethylene glycol or dimethyl formamide.     

Comparative Study between Slow Freezing and Vitrification of Mouse Embryos Using Different Cryoprotectants

The objective of this study was to evaluate the effects of different cryoprotectants and different cryopreservation protocols on the development of mouse eight-cell embryos. Mouse eight-cell embryos were cryopreserved by using propylene glycerol (PROH), ethylene glycerol (EG), dimethyl sulfoxide (DMSO) or glycerol (G) as cryoprotectant with slow-freezing or Vit-Master vitrification protocol. After thawing, the survival rate, blastocyst formation rate and blastocyst hatching rate of the embryos were compared. When the mouse eight-cell embryos were cryopreserved by the slow-freezing, the survival rate, the blastocyst formation rate and the blastocyst hatching rate of the embryos with PROH were significantly higher than those of DMSO and G (p < 0.05, respectively), but not significantly different among those of DMSO, G and EG (p > 0.05, respectively), and not significantly different between those of PROH and EG (p > 0.05, respectively). When the mouse eight-cell embryos were cryopreserved by Vit-Master vitrification, the survival rate, the blastocyst formation rate and the blastocyst hatching rate of the embryos with EG were significantly higher than those of PROH, DMSO and G (p < 0.05, respectively). Yet, there were no significant differences among those of PROH, DMSO and G (p > 0.05, respectively). In conclusion, PROH was the optimal cryoprotectant for the cryopreservation of mouse eight-cell embryos by slow-freezing protocol. EG was the optimal cryoprotectant for the cryopresevation of mouse eight-cell embryos by Vit-Master vitrification protocol, which may be commonly used in clinical and laboratory practice.     

冷冻家猫附睾精液效果初探

为了筛选出适宜冷冻家猫精液的稀释液、冷冻保护剂和解冻温度,本试验采用家猫附睾精液,分别用2种不同的稀释液,3种不同的冷冻保护剂进行稀释、冷冻和解冻。结果显示,Ⅱ号稀释液冷冻效果优于Ⅰ号稀释液。用乙二醇作为冷冻保护剂,解冻后家猫精子活率达到52.7%±4.9%,畸形率为37.3%±4.2%,顶体完整率为52.4%±4.1%,各项指标均显著高于甘油组和二甲基亚砜组（P<0.05）;其中甘油组解冻后活率为35.5%±7.6%,顶体完整率为39.8%±4.4%,高于二甲基亚砜组的33.6%±7.3%和39.1%±4.1%,但两者之间差异不显著（P>0.05）。37 ℃水浴解冻后顶体完整率为36.4%±8.7%,显著高于30 ℃水浴解冻后的25.3%±6.7%（P<0.05）,但它们之间的活力,畸形率差异不显著,总体上37 ℃的解冻效果优于30 ℃。因此,乙二醇和稀释液Ⅱ配合使用冷冻家猫附睾精液效果较好,37 ℃为较优解冻温度。     

Cryopreservation of Sheep Primordial Follicles

The aim of this study was to evaluate the efficiency of 1 M dimethylsulphoxide (DMSO), ethylene glycol (EG), propylene glycol (PROH) and glycerol (GLY) to cryopreserve primordial follicles. The first evaluation was performed soon after cryopreservation and the second evaluation after 4 days of in vitro culture, using the cryoprotectants that allowed the higher results (higher follicular survival rate) after cryopreservation. The results after follicular isolation (control) and cryopreservation using 1 M DMSO, EG, PROH and GLY showed that the mean number (+/- SEM) of live follicles per millilitre was 3204 (100%) +/- 319.27, 2798 (87%) +/- 239.14, 2492 (78%) +/- 345.8, 448 (14%) +/- 46.3 and 208 (7%) +/- 75.26, respectively. Higher follicular survival was reported when DMSO and EG were used. Control follicles and follicles cryopreserved with these two cryoprotectants were cultured and the percentage of follicular survival was 55% (control), 42% (EG) and 34% (DMSO). Similar results were found between control and follicles cryopreserved with EG. In conclusion, 1 M EG is the most effective cryoprotectant to preserve primordial follicles isolated from ovaries of sheep.     

不同冷冻保护剂和冷冻方法对小鼠耳皮肤成纤维细胞冻存效果的影响

通过比较不同冷冻保存方法和冷冻保护剂对小鼠耳皮肤成纤维细胞冷冻-解冻复苏后细胞存活率、48h贴壁率和细胞生长曲线的影响,筛选适宜的小鼠耳皮肤成纤维细胞冷冻保存方法和冷冻保护剂。结果表明,以100mL/L二甲基亚砜(DMSO)作为冷冻保护剂进行小鼠耳皮肤成纤维细胞冷冻保存时,方法3处理组解冻复苏后细胞存活率和培养48h细胞贴壁率均高于方法2处理组(P>0.05),并分别极显著高于方法1和方法4。采用方法3进行冷冻保存时,以100mL/L DMSO作为冷冻剂的细胞贴壁率显著高于100mL/L甘油(GL)组(P<0.05),且冷冻解冻后的细胞呈现正常的分裂增殖生长模式。因此,宜选择100mL/L胎牛血清(FBS)+100mL/L DMSO+DMEM作为冷冻保护液,采用方法3进行小鼠耳皮肤成纤维细胞的冷冻保存。     

Simple prediction of the survival of follicles in cryopreserved human ovarian tissue

This study examines the possible predictive value of the LIVE/DEAD fluorescence viability assay for evaluation of survival of cryopreserved human ovarian tissue. Ovarian tissue from ten patients was examined by LIVE/DEAD viability staining before and after cryopreservation and after freezing in a -20 C freezer (negative control). After cryopreservation with a slow freezing protocol and cryoprotectant the LIVE/DEAD assay showed 86% viable follicles (an intact oocyte and at least more than 50% of the granulosa cells alive), whereas after freezing at -20 C the survival rate was 67%. The healthy follicular loss after cryopreservation was 4%, whereas with freezing at -20 C, it was 25%. Although this assay overestimates the survival rate of cryopreserved primordial follicles, if the LIVE/DEAD assay yields greater than approximately 85% viable follicles, it can be assumed that the follicles in the cryopreserved tissue have maintained their developmental potential and that the tissue is suitable for retransplantation.     

Functional Activity of Frozen Thawed Chinchilla lanigera Spermatozoa Cryopreserved with Glycerol or Ethylene Glycol

The cryopreservation of spermatozoa constitutes a valuable tool for the captive breeding management of valuable and/or threatened species. Chinchilla lanigera is a species almost extinct in the wild, and the domestic counterpart has one of the most valuable pelts in the world. The objectives of this study were to: (i) compare the functional activity of post‐thawed chinchilla spermatozoa cryopreserved at ?196°C either with glycerol (G) or ethylene glycol (EG) as cryoprotectants (1 m final concentration) and (ii) investigate the effects of incubating the gametes for 4 h in the presence or in the absence of the cryoprotectants; evaluations were performed taking into account motility, viability, response to hypo‐osmotic shock and acrosome integrity of the cells. Parameters reflecting post‐thaw (0 h) sperm functional activity were significantly lower than those of freshly ejaculated gametes. When comparing the cryoprotectant efficiency of G vs EG, neither cryoprotectant agent offered appreciable advantages. After 4 h of incubation, in the presence or absence of the cryoprotectant agent, a rapid and significant decrease was found in all functional parameters and remained at ～ 20–30% motile, viable and viable acrosome intact cells. Viability was significantly lower when the cryoprotectant was removed from the media (possibly due to the centrifugation process). With respect to the maintenance of sperm membrane integrity, only ～ 10% of cells showed membrane resistance to hypo‐osmotic conditions after the 4 h incubation period. These results constitute new insights for cryopreservation protocols and the development of assisted reproductive techniques in this species.     

猪精液冷冻保护剂研究进展

 冷冻保护剂作为精液冷冻稀释液中的添加剂,能防止和减少细胞在冷冻及复苏过程中造成的损伤,确保细胞复苏后有较高存活率。论文就国内外猪精液渗透性和非渗透性精液冷冻保护剂的作用机理、应用进展等研究进行了综述。     

牛精液冷冻保护剂研究进展

牛的人工授精技术是目前应用最为广泛的动物繁殖技术之一,对牛的遗传改良做出了巨大贡献。但是,在冷冻一解冻过程中仍然有大约40％～50％的精子失去活性,限制良种公牛种用性能的发挥。论文在分析精子冷冻保存原理的基础上,阐述了渗透性、非渗透性冷冻保护剂以及低密度脂蛋白对牛精子的冷冻保护作用,以期为开发新型冷冻保护剂的研究提供一定参考,进一步提升牛冷冻精液的质量。     

Cryopreservation of llama semen using a combination of permeable cryoprotectants

Semen cryopreservation is not available for massive use in South American Camelids (SACs) due to the lack of an efficient protocol and the low pregnancy rates obtained with artificial insemination (AI). The use of a single cryoprotectant (CP) is commonly used in SACs frozen semen. The objective of the study was to evaluate the combined cryoprotective capacity of two permeable CPs at different stages of the cryopreservation protocol in llama semen. Sixteen ejaculates from 4 llama males were analysed, and sperm quality was assayed in raw semen, at 5°C, after equilibration of samples with the CPs and when samples were thawed. The following CPs and combination were used: 6% glycerol (GL), 6% dimethylformamide (DMF) and the combination of both CPs: 3% GL and 3% DMF. A Kruskal–Wallis test and an experimental factorial design, considering one factor with four levels (raw semen, 6% GL, 6% DMF and GL/DMF), were used. Total sperm motility and live sperm with intact acrosomes remained unchanged after equilibration of samples (p > .05). A significant decrease in the percentage of functional membrane, motile and live sperm with intact acrosomes was observed when samples were thawed (GL, DMF and GL/DMF). Nevertheless, the cryopreservation protocols used preserved sperm DNA quality; thus, sperm chromatin condensation and DNA fragmentation were unaffected (p > .05) when GL, DMF and GL/DMF were used. To conclude, no superiority was found between the use of a single or a combination of permeable cryoprotectants to freeze llama semen.     

Optimized cryopreservation of mouse sperm based on fertilization rate

Although procedures for in vitro fertilization with cryopreserved sperm have been published there is a lack of data indicating that the cryoprotectant and cryopreservation procedures used for those procedures were optimal. To redress this, fertilization rate of eggs exposed to sperm in vitro was used as the outcome in the optimization of raffinose concentration in the cryoprotectant (raffinose in water), volume of cryoprotectant, and freezing conditions for C57BL/6J mouse sperm. Sperm were frozen in a cylindrical Dewar with an internal diameter and height of 14.0 cm and 36.0 cm respectively. The optimal concentration of raffinose was 23-24% (510-540 mOsm/kg). The optimal volume of cryoprotectant used to prepare the sperm suspension from a single mouse was 180-400 μl, and sperm proved most fertile when frozen 13-25 mm above liquid nitrogen. Raffinose in the fertilization medium did not inhibit fertilization. Fertilized eggs transferred to oviducts of recipient mice developed into viable offspring.     

Effect of the polyvinylpyrrolidone concentration of cryoprotectant on mouse embryo development and production of pups: 7.5% of PVP is beneficial for in vitro and in vivo development of frozen-thawed mouse embryos

In this study, we investigated the effect of polyvinylpyrrolidone (PVP) concentration on in vitro and in vivo development of 2 cell stage, vitrified ICR mouse embryos using a cryoprotectant consisting of ethylene glycol (EG) and sucrose. M2 was selected as the basic medium for vitrification and thawing. After equilibration with 4% (v/v) EG at 37 C for 15 min, the embryos were vitrified with 35% EG, 5, 6 or 7.5% (w/v) PVP and 0.4 M sucrose at 37 C for 30 sec. One week later, the cryotubes of cryopreserved embryos in liquid nitrogen were directly immersed into a 37 C water bath for 1 min and transferred serially into 300 mul of 0.5 or 0.3 M sucrose at room temperature for 5 min and M2 medium at 37 C for 10 min. The surviving embryos were cultured in KSOM (potassium simplex optimized medium) for 96-120 h in an atmosphere of 5% CO(2) in humidified air. Survival was evaluated by morphological appearance, including membrane integrity and presence of apoptotic blastomeres after thawing. For in vivo evaluation, blastocysts were transferred to the uteri of pseudopregnant mice. The survival rates of the 5 and 7.5% PVP concentration groups showed a significantly higher difference compared with that of the 6% PVP group (85.5 and 86.5 vs. 71.2%), respectively. Each pup in the of 5 and 6% groups was cannibalized immediately after parturition. A litter of live pups was obtained from only the 7.5% PVP groups. Our study indicated that supplementation of EG and sucrose cryoprotectant solution with 7.5% PVP is optimal for successful vitrification of 2-cell stage ICR mouse embryos.     

A Trial to Cryopreserve Immature Medaka (Oryzias latipes) Oocytes after Enhancing Their Permeability by Exogenous Expression of Aquaporin 3

Fish oocytes have not been cryopreserved successfully, probably because it is difficult to prevent intracellular ice from forming. Previously, we have shown in medaka that immature oocytes are more suitable for cryopreservation than mature oocytes or embryos, in terms of permeability. We have also shown in immature medaka oocytes that the exogenous expression of aquaporin 3 (AQP3), a water/cryoprotectant channel, promotes the movement of water and cryoprotectants through the plasma membrane. In the present study, we attempted to cryopreserve immature medaka oocytes expressing AQP3. We first examined effects of hypertonic stress and the chemical toxicity of cryoprotectants on the survival of the AQP3-expressing oocytes. Exposure to hypertonic solutions containing sucrose decreased the survival of oocytes, but the expression of AQP3 did not affect sensitivity to hypertonic stress. Also, AQP3 expression did not markedly increase sensitivity to the toxicity of cryoprotectants. Of the four cryoprotectants tested, propylene glycol was the least toxic. Using a propylene glycol-based solution, therefore, we tried to cryopreserve immature oocytes by vitrification. During cooling with liquid nitrogen, all intact oocytes became opaque, but many AQP3-expressing oocytes remained transparent. This indicates that the expression of AQP3 is effective in preventing intracellular ice from forming during cooling. During warming, however, all the AQP3-expressing oocytes became opaque, indicating that intracellular ice formed. Therefore, the dehydration and permeation by propylene glycol were still insufficient. Further studies are necessary to realize the cryopreservation of fish oocytes.     

不同冻存液对奶牛乳腺上皮细胞冻存质量的影响

试验旨在探究奶牛乳腺上皮细胞（bovine mammary epithelial cells,BMECs）最佳的冻存液以改善乳腺上皮细胞的冻存质量。BMECs传至第5代后分别加入以下10种不同配方的冻存液。A组：85%DMEM＋10%胎牛血清＋5%DMSO;B组：80%DMEM＋10%胎牛血清＋10%DMSO;C组：75%DMEM＋10%胎牛血清＋15%DMSO;D组：70%DMEM＋10%胎牛血清＋20%DMSO;E组：85%DMEM＋5%胎牛血清＋10%DMSO;F组：75%DMEM＋15%胎牛血清＋10%DMSO;G组：70%DMEM＋20%胎牛血清＋10%DMSO;H组：80%DMEM＋10%胎牛血清＋10%甘油;I组：70%DMEM＋10%胎牛血清＋20%甘油;J组：60%DMEM＋10%胎牛血清＋30%甘油,冻存前统一调整细胞密度到1×106个/mL冻存。分别对复苏后的细胞进行台盼蓝染色计算存活率和PI/Hoechst33258双染计算凋亡率。结果表明,BMECs经不同冻存剂冻存复苏后,细胞活力、形态学及凋亡率表现有所不同,其中B组和G组的活力和24h贴壁率较其他组高,二者的凋亡率较低,二者之间差异无显著性（P〉0.05）;传代后B组细胞的生长状况最好。     

Comparison of Cryoprotectants in the Preservation of Theileria parva Sporozoites Using an In Vitro Infectivity Assay

An in vitro infectivity assay was used to examine five cryoprotectants for their suitability for preserving Theileria parva sporozoites. All five were capable of preserving T. parva sporozoites through freezing, the optimal concentrations being 7.5% for glycerol, 5% for dimethyl sulphoxide (DMSO), poly (vinylpyrrolidone) (PVP) and poly(ethylene glycol) (PEG), and 2.5% for hydroxyethyl starch (HES). When the five cryoprotectants were compared at their optimal concentrations, using a modification of the standard method of stabilate preparation, glycerol was significantly better than the others (p<0.05). Measurement of the effects of each cryoprotectant on the osmolality of the media revealed that glycerol and DMSO elevated the osmolality significantly (p<0.05). Resuscitation of glycerol-preserved sporozoites required the presence of glycerol in the diluent to maintain infectivity. Studies on the effects of equilibration time in glycerol on the infectivity of sporozoites showed that those frozen immediately after mixing (2 min) were as infective as those frozen after 60 min of equilibration.     

Effect of different penetrating and non‐penetrating cryoprotectants and media temperature on the cryosurvival of vitrified in vitro produced porcine blastocysts

The aim of this study was to determine the most efficient vitrification protocol for the cryopreservation of day 7 in vitro produced (IVP) porcine blastocysts. The post‐warm survival rate of blastocysts vitrified in control (17% dimethyl sulfoxide + 17% ethylene glycol [EG] + 0.4 mol/L sucrose) and commercial media did not differ, nor did the post‐warm survival rate of blastocysts vitrified in medium containing 1,2‐propandiol in place of EG. However, vitrifying embryos in EG alone decreased the cryosurvival rate (55.6% and 33.6%, respectively, p < .05). Furthermore, the post‐warm survival rates of blastocysts vitrified with either trehalose or sucrose as the non‐penetrating cryoprotectant did not differ. There was also no significant difference in post‐warm survival of blastocysts vitrified in control (38°C) media and room temperature (22°C) media with extended equilibration times, although when blastocysts were vitrified using control media at room temperature, the post‐warm survival rate increased (56.8%, 57.3%, 72.5%, respectively, p < .05). The findings show that most cryoprotectant combinations examined proved equally effective at supporting the post‐warm survival of IVP porcine blastocysts. The improved post‐warm survival rate of blastocysts vitrified using media held at room temperature suggests that the cryoprotectant toxicity exerted in 22°C media was reduced.     

Effects of cryopreservation on the meiotic spindle, cortical granule distribution and development of rabbit oocytes

Although much progress has been made in oocyte cryopreservation since 1971, live offspring have only been obtained in a few species and in rabbits. The aim of our study was to evaluate the effect of vitrification and slow freezing on the meiotic spindle, cortical granule (CG) distribution and their developmental competence. Oocytes were vitrified in 16.84% ethylene glycol, 12.86% formamide, 22.3% dimethyl sulphoxide, 7% PVP and 1% of synthetic ice blockers using Cryotop as device or slow freezing in 1.5 m PROH and 0.2 m sucrose in 0.25 ml sterile French mini straws. Meiotic spindle and CG distribution were assessed using a confocal laser-scanning microscope. To determine oocyte competence, in vitro development of oocytes from each cryopreservation procedure was assessed using parthenogenesis activation. Our data showed that oocytes were significantly affected by both cryopreservation procedures. In particular, meiotic spindle organization was dramatically altered after cryopreservation. Oocytes with peripheral CG distribution have a better chance of survival in cryopreservation after slow-freezing procedures compared to vitrification. In addition, slow freezing of oocytes led to higher cleavage and blastocyst rates compared to vitrification. Our data showed that, in rabbits, structural alterations are more evident in vitrified oocytes than in slow-frozen oocytes, probably as a consequence of sensitivity to high levels of cryoprotectants. Slow-freezing method is currently the recommended option for rabbit oocyte cryopreservation.