Effect of the holding time at 15 °C prior to cryopreservation,the thawing rate and the post-thaw incubation temperature on the boar sperm quality after cryopreservation

The aim of the present study was to evaluate the effect of the holding time at 15 °C prior to cryopreservation (2, 4 and 8 h), thawing rate (37 °C for 20 s or 70 °C for 8 s) and post-thaw incubation temperature (15 °C or 37 °C) on the post-thaw boar sperm quality. These are important time periods in the freezing–thawing process which have been less studied. Sperm-rich ejaculate fractions from three healthy boars were collected once a week for five consecutive weeks and were cryopreserved with the lactose-egg yolk extender (LEY). Sperm quality was determined by assessing the motility, the acrosome status, and the sperm plasma membrane integrity at 30, 150 and 240 min of incubation. The results show that with the holding time at 15 °C prior to cryopreservation there was not a clear effect until at least 24 h of holding time. The thawing rate and the post-thaw incubation temperature, however, had a marked effect on sperm quality. When the samples were thawed at 70 °C for 8 s, the sperm viability, motility and some kinetic variables (VCL, VSL, VAP and ALH) were greater than with results observed when the samples were thawed at 37 °C for 20 s. In addition after thawing the sperm samples incubated at 15 °C had a sustained sperm quality for longer, up to 4 h post-thawing.     

Effects of thawing,refreezing and storage conditions of tissue samples and protein extracts on 2‐DE spot intensity

We report that reliable quantitative proteome analyses can be performed with tissue samples stored at ?80°C for up to 10 years. However, storing protein extracts at 4°C for 24 h and freezing protein extracts at ?80°C and thawing them significantly altered 41.6 and 17.5% of all spot intensities on 2‐DE gels, respectively. Fortunately, these storing effects did not impair the reliability of quantifying 2‐DE experiments. Nonetheless, the results show that freezing and storage conditions should be carefully controlled in proteomic experiments.     

Cryopreservation of protozoan parasites

Conventional methods for the propagation and preservation of parasites in vivo or in vitro have some limitations, including the need for labor, initial isolation and loss of strains, bacterial, and fungal contamination, and changes in the original biological and metabolic characteristics. All these disadvantages are considerably reduced by cryopreservation. In this study, we examined the effects of various freezing conditions on the survival of several protozoan parasites after cryopreservation. The viability of Entamoeba histolytica was improved by seeding (p < 0.05, chi2 test), while this was not so effective for Trichomonas vaginalis. Of six cryoprotectants examined, dimethyl sulfoxide (Me(2)SO), and glycerol showed the strongest cryoprotective effects. The optimum conditions for using Me(2)SO were a concentration of 10% with no equilibration, and those for glycerol were a concentration of 15% with equilibration for 2h. The optimum cooling rate depended on the parasite species. Trypanosoma brucei gambiense and Leishmania amazonensis were successfully cryopreserved over a wide range of cooling rates, whereas the survival rates of E. histolytica, T. vaginalis, Pentatrichomonas hominis, and Blastocystis hominis were remarkably decreased when frozen at improper rates. Unlike the cooling rate, exposure of the protozoans to a rapid thawing method produced better motility for all parasites.     

The effect of warming velocity on motility and acrosomal integrity of boar sperm as influenced by the rate of freezing and glycerol level

The effect of thawing velocities ranging from 10°C/min to 1.800°C/min on the motility and acrosomal integrity of boar spermatozoa frozen at 1°C/min (suboptimal), 5°C/min, and 30°C/min (optimal) rate was studied with the sperm suspended for freezing in diluent containing 2, 4, or 6% of glycerol (v/v). The influence of thawing on sperm survival depends on the rate at which the sperm had been frozen. In semen frozen at a suboptimal rate of 1°C/min, the percentage of motile sperm (FMP) initially fell to 3.5–4.0% when the thawing rose to 200°C/ min, but, with further increases in thawing rate, increased and reached peak values (10.3–11.0% FMP) after thawing at 1,800°C/min. The percentage of sperm with normal apical ridge (NAR) also increased moderately with thawing rate, but the degree of improvement decreased as the glycerol level was increased. In semen frozen at 1°C/min, acrosomal integrity (NAR) was best maintained in 2% glycerol, reaching 22.9% NAR after thawing at 1,800°C/min. In semen frozen at the optimal rate of 30°C/min, the increases in thawing rates above 200°C/min substantially improved motility. Motility was generally higher in semen protected by 4 or 6% glycerol, with the peak values of 44 or 46% FMP, respectively, after thawing at 1,200°C/min. The proportion of sperm with NAR also increased with thawing rate, but as in the case of suboptimally frozen sperm it was influenced negatively by the glycerol concentration. The peak value 53% NAR was recorded in semen protected by 2% glycerol, frozen at 30°C/min, and thawed at 1,200°C/min. In view of the inverse relationship between FMP and NAR, selection of optimal conditions from among the interacting variables, freezing rate, glycerol concentration, and thawing rate requires compromising between maximal FMP and maximal NAR. Accordingly, we have adopted as optimal a protocol with a thawing rate of 1,200°C/min, a freezing rate of 30°C/min and concentrations of 3% glycerol. © 1993 Wiley-Liss, Inc.     

Schistosoma mansoni: Cryopreservation of schistosomula by two-step addition of ethanediol and rapid cooling

A simple, inexpensive, and highly effective technique for the Cryopreservation of schistosomula of Schistosoma mansoni is outlined by experiments designed to clarify the role of each of the steps involved. The technique consists of incubating schistosomula in 10% ($\text{v}\text{v}$) ethanediol for 10 min at 37 C followed by 5 min at 0 C and for a further 10 min in 35% ($\text{v}\text{v}$) ethanediol at 0 C. The schistosomular suspension is then aliquotted in 20-μl drops onto 40 × 5.5-mm glass slivers prepared from standard microscope coverslips, each drop being spread out to cover an area of approximately 15 × 4 mm. These glass slivers are then dropped directly into liquid nitrogen giving a cooling rate of approximately 5000 C min^?1. Survival is further improved if the schistosomula are at least 90 min old before Cryopreservation and if the frozen organisms are thawed in culture medium prewarmed to +42 C. Levels of survival obtained with this technique are consistently high: 44 to 60% as assessed by motility. From 400 ± 11 cryopreserved schistosomula injected intramuscularly into eight mice, a mean of 54.5 ± 16.3 adult worms were recovered representing an infection level of 13.7%, and which in turn represents 47.4% of the unfrozen control level.     

一种改进的丝状真菌DNA提取方法

以丝状真菌雅致枝霉(Thamnidium elegans)和深黄伞形霉(Umbelopsis isabellina)为材料, 使用优化的CTAB法提取基因组DNA。改进后的方法使用液氮冻融以及玻璃珠振荡的方法代替了传统的液氮研磨, 所需菌体量少, 而得到的基因组DNA比用传统的CTAB法得到的基因组DNA产率高、纯度好、且步骤简单, 适用于一次微量提取多个样品的基因组DNA。这种方法得到的基因组DNA可用于大部分分子生物学基本实验如PCR和DNA的酶切等。     

一种简单快速植物组织冰冻切片方法

比较不同冷冻方法对植物细胞超微结构的影响,结果表明:直接包埋法处理的植物细胞超微结构保存较好,而液氮冷冻处理的植物细胞内膜系统损伤严重.建立了一种直接包埋冷冻和适当回温相结合的方法,不仅可以制作出植物细胞基本结构保存完整的组织切片,而且避免了使用冰冻保护剂的弊端.其操作程序是:样品固定→冰冻与包埋→适当回温→快速切片→展片→染色.此法制作的切片可进行不同的染色和组织细胞化学测定,具有操作简便,易于推广的特点.     

Assessment of two thawing processes of cryopreserved human sperm in pellets

In this study, we evaluated the effects of the thawing methodology on sperm function after cryopreservation in pellets. We compared the use of two thawing procedures: method (1) maintaining pellet for 10 min in air at room temperature, then another 10-min period in air at 37 °C followed by dilution in a thawing medium; and method (2) immersing the pellets directly in thawing medium at 37 °C for 20 min. This procedure leads to a higher rate of temperature increase and a dilution of the glycerol present in the freezing medium. We analyzed the effect of the thawing procedure on sperm motility, viability, membrane lipid packing disorder, acrosome status, reactive oxygen species (ROS) level and sperm chromatin condensation. This study revealed a positive effect of the M2 thawing methodology on sperm parameters. The percentage of spermatozoa with fast-linear movement is increased (M1: 17.26% vs. M2: 28.05%, p < 0.01), with higher viability (M1: 37.81% vs. M2: 40.15%, p < 0.01) and less acrosome damage (M1: 40.44% vs. M2: 35.45%, p = 0.02). We also detected an increase in the percentage of viable spermatozoa with low membrane lipid disorder (M1: 31.36% vs. M2: 33.17%, p = 0.03) and a reduction in chromatin condensation (44.62 vs. 46.62 arbitrary units, p = 0.02). Further studies will be necessary to evaluate the possible clinical applications.     

Pig sperm membrane microdomains contain a highly glycosylated 15-25-kDa wheat germ agglutinin-binding protein

A highly glycosylated protein, which has unique, novel features in localization, structure, and potential function, is found in pig sperm, and named WGA-gp due to its high binding property with wheat germ agglutinin (WGA). WGA-gp is localized mainly in flagella and enriched in membrane microdomains or lipid rafts. It is not detected by ordinary protein staining methods due to a high content of both N- and O-glycans consisting of neutral monosaccharides. Interestingly, WGA-gp may be involved in intracellular Ca²⁺ regulation. Treatment of sperm with anti-WGA-gp antibody enhances the amplitude of Ca²⁺ oscillation without changing the basal intracellular Ca²⁺ concentrations. All these features of WGA-gp, except for different carbohydrate structures occupying most part of the molecules, are similar to those of flagellasialin in sea urchin sperm, which regulates the intracellular Ca²⁺ concentration. Presence of carbohydrate-enriched flagellar proteins involved in intracellular Ca²⁺ regulation may be a common feature among animal sperm.     

Effect of common cryoprotectants on critical warming rates and ice formation in aqueous solutions

Ice formation on warming is of comparable or greater importance to ice formation on cooling in determining survival of cryopreserved samples. Critical warming rates required for ice-free warming of vitrified aqueous solutions of glycerol, dimethyl sulfoxide, ethylene glycol, polyethylene glycol 200 and sucrose have been measured for warming rates of order 10–10⁴ K/s. Critical warming rates are typically one to three orders of magnitude larger than critical cooling rates. Warming rates vary strongly with cooling rates, perhaps due to the presence of small ice fractions in nominally vitrified samples. Critical warming and cooling rate data spanning orders of magnitude in rates provide rigorous tests of ice nucleation and growth models and their assumed input parameters. Current models with current best estimates for input parameters provide a reasonable account of critical warming rates for glycerol solutions at high concentrations/low rates, but overestimate both critical warming and cooling rates by orders of magnitude at lower concentrations and larger rates. In vitrification protocols, minimizing concentrations of potentially damaging cryoprotectants while minimizing ice formation will require ultrafast warming rates, as well as fast cooling rates to minimize the required warming rates.