Vitrification of porcine immature oocytes: Association of equilibration manners with warming procedures,and permeating cryoprotectants effects under two temperatures

The aim of this study was to evaluate the association of equilibration manners with warming procedures, and the different permeating cryoprotectants (pCPAs) effects under two temperatures, in terms of survival, maturation and subsequent parthenogenetic development of porcine immature oocytes after Cryotop vitrification. In Experiment 1, oocytes were equilibrated by exposure to 5% (v/v) ethylene glycol (EG) for 10 min (EM1) or stepwise to 7.5% (v/v) and 15% (v/v) EG for 2.5 min respectively (EM2). Warming procedures were performed in 1.0 M sucrose for 1 min, then in 0.5 and 0.25 M sucrose for 2.5 min respectively (WP1), or in 0.5, 0.25 and 0.125 M sucrose each step for 2 min (WP2), or in 0.25, 0.125 and 0.063 M sucrose each step for 2 min (WP3). After 2 h of warming, the survival rate of oocytes treated by EM1 and WP1 was significantly higher (P < 0.05) than that of the other groups. Moreover, a similar proportion of survival and nuclear maturation in all vitrified groups was obtained after completion of the IVM. No significant difference in blastocyst development was observed among vitrified groups except the group treated by EM2 and WP3. In Experiment 2, oocytes were vitrified by using EG alone, EG combined with dimethyl sulphoxide (EG + DMSO) or propylene glycol (EG + PROH) as pCPAs under 25 °C and 39 °C. The percentages of cryosurvival and nuclear maturation were similar in all vitrified groups. Under 25 °C, the embryo development and total cell numbers of blastocysts were not significantly different among EG, EG + DMSO and EG + PROH groups. However, the application of EG + PROH at 39 °C resulted in significantly decreased both cleavage and blastocyst formation rates. In conclusion, our data showed that equilibration manner and warming procedure affect the cryosurvival of porcine immature oocytes, and the combination of pCPAs cannot give a better cryopreservation outcome whether 25 °C or 39 °C. Notably, the Cryotop vitrification accompanied by our modified strategy for porcine immature oocytes could achieve high survival and respectable blastocyst production.     

Improved cryopreservation yield of pancreatic islets using combination of lower dose permeable cryoprotective agents

Islet transplantation has been shown to restore normoglycemia in animal models and for type 1 diabetic patients in clinical trials. One method of storing islets intended for transplantation is via cryobanking at very low temperatures (−196 °C). Cryobanking islets without the use of cryoprotecting agents (CPAs) contributes to cellular shear stress and cell death. Although current CPA protocols vary, high concentrations of these agents are toxic to islets cells. This study tested the effects of the permeating CPA dimethyl sulfoxide (Me₂SO) with the addition of ethylene glycol (EG), both at reduced concentrations, on rat and human islet cell yield, viability, and glucose stimulated insulin release (GSIR). To test this, islets were treated using three combinations of CPAs (2M ME₂SO, 1M ME₂SO + 1M EG, and 1M ME₂SO + 0.5M EG). Next, fresh islets, 2M ME₂SO islets, and 1M ME₂SO + 0.5M EG isolated rat islets were transplanted into streptozotocin-induced (STZ) diabetic mice. Our data showed that cryopreservation with a reduced concentration of ME₂SO (1M ME₂SO + multimolar EG) achieved a higher percent yield and viability when compared to the current standard 2M ME₂SO treatment for both rat and human islets. Furthermore, STZ-induced diabetic mice achieved normoglycemia after transplantation with 1000 islet equivalents (IE), an average 12 days sooner, with islets cryopreserved with reduced-concentration (ME₂SO + 0.5M EG), compared to islets preserved with 2M ME₂SO. In conclusion, reduced concentration of penetrating CPAs during islet cryopreservation increases islet yield and viability in vitro and reduces delay before normoglycemia in diabetic mice.     

Ethylene glycol and glycerol loading and unloading in porcine meniscal tissue

The development of a long-term storage method for meniscus, a complex tissue of the knee prone to injury, would improve the procedure and outcomes of meniscus transplantation. Cryopreservation uses cryoprotective agents (CPAs) including ethylene glycol (EG) and glycerol to preserve a variety of live tissues, and understanding of the CPA permeation kinetics will be critical in designing a vitrification protocol for meniscus.The purpose of this preliminary study was to understand the loading and unloading behaviours of EG and glycerol in meniscus by observing their efflux. For the main experiment, lateral and medial porcine menisci were incubated with CPA for 24 h at three temperatures (i.e., 4, 22, and 37 °C). Then, the menisci were immersed in 25 ml of X-VIVO™10 and CPA efflux was recorded by monitoring the molality of two consecutive washout solutions at different time points. In a subsequent experiment, menisci were incubated in the CPA solutions for 48 h at 22 °C, and the results were compared to those obtained at 22 °C in the main experiment.Results showed a rapid efflux of CPA from meniscus at the beginning of each wash. With increasing temperature, the amount of CPA efflux (and hence loading) increased. Using 24 h incubation, EG loaded the menisci more completely than glycerol. But after 48 h of incubation, both EG and glycerol achieved approximately the same degree of meniscus loading.This study provides preliminary data that will facilitate future design of experiments aimed at development of meniscus permeation studies.     

Osmotic and cryoprotective effects of a mixture of DMSO and ethylene glycol on rabbit morulae

Comparisons were made of the osmotic and cryoprotective effects on rabbit embryos preserved by vitrification with 2 solutions and by conventional freezing. Embryos obtained from rabbits killed 70 to 72 h after mating were used in the study (n = 948). Initially, toxicity of the 3 cryoprotectants was studied in fresh (unfrozen) embryos (n = 135). Subsequently, embryos placed in ethylene glycol (EG, 40% v/v; n = 88) and ethylene glycol with dimethyl sulfoxide (EG+DMSO, 20% v/v each, respectively; n = 344) were loaded into straws and plunged directly into liquid nitrogen. Embryos placed in 1.5 M DMSO and 20% heat inactivated rabbit serum were subjected to conventional freezing in a programmable freezer (control group, n = 363). The osmotic effect was estimated by measuring the changes in the embryonic and interzonal volume (crossectional area) and in the thickness of the mucin coat (n = 18). Cryoprotective effectivity was determined by development to the blastocyst stage in vitro, or birth of normal pups after transfer into synchronized recipients. Osmotic effects of cryoprotective solutions on embryonic and interzonal volume and mucin coat thickness were variable and overall not significant. Survival rate of cryopreserved embryos in vitro and development to blastocysts, was worst in the EG-treated embryos. Survival rate at birth was higher in vitrified vs frozen embryos. We conclude that rabbit morulae can be vitrified successfully in EG+DMSO medium.     

Effect of the cryoprotectant concentration on the in vitro embryo development and cell proliferation of OPS-vitrified porcine blastocysts

Our objective was to study the effect of the concentration of ethylene glycol (EG) and dimethyl sulfoxide (Me₂SO) during vitrification on the development of porcine blastocysts. Vitrification was performed with 0.4 M sucrose and either a Me₂SO and EG mixture (15%, 16% and 17% v/v of each) or EG alone (40% v/v), using superfine open pulled straws. Fresh and vitrified blastocysts were cultured for 48 h and the survival and hatching rates were evaluated. Some vitrified and fresh embryos were processed for Hoechst 33342 staining and proliferation cell nuclear antigen (PCNA) inmunolocalization to determine the proliferation index. The survival rate was similar for fresh and vitrified blastocysts, except for blastocysts vitrified using 15% of cryoprotectants, which displayed lower (P < 0.05) survival than fresh blastocysts. Vitrified and fresh blastocysts had a similar cell proliferation index (range: 75.8 ± 3.2 to 83.7 ± 3). When only hatched blastocysts among groups were compared, the proliferation rate decreased (P < 0.05) after vitrification with 17% of EG–Me₂SO. In conclusion, the concentration of EG–Me₂SO could be decreased to 16% in the vitrification medium with no reduction of the in vitro developmental ability of the blastocysts. In addition, a 40% EG-based medium can be used for vitrification with similar results to those achieved with a medium containing 16% EG–Me₂SO.     

Impact of methoxyacetic acid on mouse Leydig cell gene expression

Background  

Methoxyacetic acid (MAA) is the active metabolite of the widely used industrial chemical ethylene glycol monomethyl ether, which is associated with various developmental and reproductive toxicities, including neural toxicity, blood and immune disorders, limb degeneration and testicular toxicity. Testicular toxicity is caused by degeneration of germ cells in association with changes in gene expression in both germ cells and Sertoli cells of the testis. This study investigates the impact of MAA on gene expression in testicular Leydig cells, which play a critical role in germ cell survival and male reproductive function.     

Hypothermic preconditioning of endothelial cells attenuates cold-induced injury by a ferritin-dependent process

Hypothermia for myocardial protection or storage of vascular grafts may damage the endothelium and impair vascular function upon reperfusion/rewarming. Catalytic iron pools and oxidative stress are important mediators of cold-induced endothelial injury. Because endothelial cells are highly adaptive, we hypothesized that hypothermic preconditioning (HPC) protects cells at 0°C by a heme oxygenase-1 (HO-1) and ferritin-dependent mechanism. Storage of human coronary artery endothelial cells at 0°C caused the release of lactate dehydrogenase, increases in bleomycin-detectible iron (BDI), and increases in the ratio of oxidized/reduced glutathione, signifying oxidative stress. Hypoxia increased injury at 0°C but did not increase BDI or oxidative stress further. HPC at 25°C for 15–72 h attenuated these changes by an amount achievable by pretreating cells with 10–20 μM deferoxamine, an iron chelator, and protected cell viability. Treating cells with hemin chloride at 37°C transiently increased intracellular heme, HO-1, BDI, and ferritin. Elevated heme/iron sensitized cells to 0°C but ferritin was protective. HPC increased iron maximally after 2 h at 25°C and ferritin levels peaked after 15 h. HO-1 was not induced. When HPC-mediated increases in ferritin were blocked by deferoxamine, protection at 0°C was diminished. We conclude that HPC-mediated endothelial protection from hypothermic injury is an iron- and ferritin-dependent process.     

Combination of intracellular cryoprotectants preserves the structure and the cells proliferative capacity potential of adult collared peccary testicular tissue subjected to solid surface vitrification

The objective was to evaluate different permeating cryoprotectants to vitrify testicular tissue biopsies from adult collared peccaries. Five pairs of testicles were dissected into fragments (9 mm³) that were allocated to non-vitrified (control) and vitrified groups using a solid-surface method following exposure to different cryoprotectants (3.0 M dimethyl sulfoxide (DMSO), 3.0 M ethylene glycol (EG) or 1.5 M DMSO + 1.5 M EG). After warming, samples were evaluated for histomorphology, ultrastructure, viability, and proliferative capacity potential. The appropriate conservation of the ultrastructural organization of the seminiferous tubule in terms of lumen presence and cell junctions was only observed at the use of DMSO/EG combination. Regardless of the cryoprotectant, the vitrification effectively preserved cell nuclear visualization and condensation similarly as observed at the non-vitrified group. Moreover, DMSO/EG combination provided a better preservation of basal membranes of seminiferous tubules than DMSO (P < 0.05). The occurrence of cell swelling was more evident in the use of DMSO than EG (P < 0.05), but both isolate cryoprotectants were similar to the DMSO/EG combination. Only the DMSO/EG combination maintained the proliferative capacity potential for spermatogonia (3.69 NORs/cell) and Sertoli cell (3.19 NORs/cell) similar to controls (3.46 and 3.31 NORS/cell, respectively). Moreover, ~40% cell viability was found after vitrification independent of cryoprotectant. In conclusion, DMSO/EG in combination is better than DMSO or EG alone for SSV of testicular tissue biopsies from adult collared peccaries.     

Effects of four cryoprotectants in combination with two vehicle solutions on cultured vascular endothelial cells.

The necessary first step in successful organ cryopreservation will be the maintenance of endothelial cell integrity during perfusion of high concentrations of cryoprotective agents (CPAs). In this report we compare the effects of incubation on cultured porcine endothelial cells at 10 degrees C for 1 h with the CPAs glycerol, dimethyl sulfoxide (Me2SO), ethanediol (EG), and propane-1,2-diol (PG) in the vehicle solutions RPS-2 (high potassium, high glucose) and HP-5NP (low potassium, high sodium), both with and without added colloids. Tritiated adenine uptake and acid phosphatase estimation of cell number were used as indicators of cell viability. HP-5NP was superior to RPS-2 except with Me2SO when the differences in viability were not significant. Adding Haemaccel to HP-5NP improved the results, but adding albumin to RPS-2 was of no significant benefit. Osmotic stress appeared to be the major problem with glycerols use. Beyond 3.0 M the toxicity of Me2SO increased dramatically but it could not be determined if this was osmotic or chemical toxicity. PG was remarkably well tolerated to 3.0 M but a sharp decrease in cell viability beyond this concentration suggests that PG may be most useful with mixtures of other CPAs. Overall, EG appeared to be the least toxic CPA and in the context of vascular preservation warrants further investigation.     

Tolerance of human embryonic stem cell derived islet progenitor cells to vitrification-relevant solutions

We have previously shown that human embryonic stem cell derived islet progenitors (hESC-IPs), encapsulated inside an immunoprotective device, mature in vivo and ameliorate diabetes in mice. The ability to cryopreserve hESC-IPs preloaded in these devices would enhance consistency and portability, but traditional ‘slow freezing’ methods did not work well for cells encapsulated in the device. Vitrification is an attractive alternative cryopreservation approach. To assess the tolerance of hESC-IPs to vitrification relevant conditions, we here are reporting cell survival following excursions in tonicity, exposure to fifteen 40% v/v combinations of 4 cryoprotectants, and varied methods for addition and elution. We find that 78% survival is achieved using a protocol in which cells are abruptly (in one step) exposed to a solution containing 10% v/v each dimethyl sulfoxide, propylene glycol, ethylene glycol, and glycerol on ice, and eluted step-wise with DPBS + 0.5 M sucrose at 37 °C. Importantly, the hESC-IPs also maintain expression of the critical islet progenitor markers PDX-1, NKX6.1, NGN3 and NEURO-D1. Thus, hESC-IPs exhibit robust tolerance to exposure to vitrification solutions in relevant conditions.     

Cryoprotective agent toxicity interactions in human articular chondrocytes

Background

Vitrification is a method of cryopreservation by which cells and tissues can be preserved at low temperatures using cryoprotective agents (CPAs) at high concentrations (typically ?6.0 M) to limit the harmful effects of ice crystals that can form during cooling processes. However, at these concentrations CPAs are significantly cytotoxic and an understanding of their toxicity characteristics and interactions is important. Therefore, single-CPA and multiple-CPA solutions were evaluated for their direct and indirect toxicities on chondrocytes.

Methods

Chondrocytes were isolated from human articular cartilage samples and exposed to various single-CPA and multiple-CPA solutions of five common CPAs (dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), glycerol (Gy) and formamide (Fm)) at both 6.0 and 8.1 M concentrations at 0 °C for 30 min. Chondrocyte survival was determined using a fluorescent cell membrane integrity assay. The data obtained was statistically analyzed and regression coefficients were used to represent the indirect toxicity effect which a specific combination of CPAs exerted on the final solution’s toxicity.

Results

Multiple-CPA solutions were significantly less toxic than single-CPA solutions (P < 0.01). The indirect toxicity effects between CPAs were quantifiable using regression analysis. Cell survival rates of approximately 40% were obtained with the four-CPA combination solution DMSO–EG–Gy–Fm. In the multiple-CPA combinations, PG demonstrated the greatest degree of toxicity and its presence within a combination solution negated any benefits of using multiple lower concentration CPAs.

Conclusions

Multiple-CPA solutions are less cytotoxic than single-CPA solutions of the same total concentration. PG was the most toxic CPA when used in combinations. The highest chondrocyte survival rates were obtained with the 6.0 M DMSO–EG–Gy–Fm combination solution.     

Variations in vascular resistance of isolated rat hearts during normothermic and hypothermic experiments

The flexibility of an automated, modified Langendorff perfusion column is illustrated by a series of experiments on isolated rat hearts under the following conditions: normothermia, hypothermia, the addition of a cryoprotective agent - ethylene glycol and cooling to −13°C and −22°C. Successful normothermic perfusions of up to 14 h were achieved. Hypothermia prevented a rise in vascular resistance with time and improved the electrocardiograms. Ethylene glycol was administered during the cooling period to −22°C at temperatures below 23°C. It was removed upon warming and before toxic effects were visible, thus leading to good recovery. By controlling the speed of the perfusate's peristaltic pump, the perfusion pressure was not allowed to exceed a pre-set level. A constant and standard vascular resistance at any selected perfusion temperature, normal heart rate and electrocardiograms were the criteria for normality.     

不同防冻剂对兔胚胎干细胞慢速冷冻保存的影响

干细胞冷冻保存是干细胞研究和临床应用中的必需技术。为提高兔胚胎干细胞在慢速冻存过程中的保存效果,比较了二甲基亚砜(DMSO)和乙二醇(ethylene glycol,EG)对兔胚胎干细胞冷冻保护效果。对冷冻复苏后的细胞进行台盼蓝染色,并研究其胚胎干细胞分子特性,结果表明DMSO比EG具有更好的冷冻保护效果。再在以10%DMSO为基础的防冻液中添加膜稳定剂海藻糖(trehalose)或谷氨酰胺(glutamine),细胞冷冻复苏后结果显示,谷氨酰胺对兔胚胎干细胞有明显的冷冻保护作用,使细胞存活率从71%提高到83.7%。当谷氨酰胺浓度为0、5、10、20、40mmol/L分别加入防冻液中后,20mmol/L的谷氨酰胺具有最佳的冷冻保护效果。以上结果得出兔胚胎干细胞慢速冷冻的防冻液改进配方为:在胚胎干细胞培养液中添加10%DMSO 20mmol/L谷氨酰胺。     

A study on cryoprotectant solution suitable for vitrification of rat two-cell stage embryos

The present study was performed to develop a suitable cryoprotectant solution for cryopreservation of rat two-cell stage embryos. First, we examined the cell permeability of several cryoprotectants; propylene glycol had the fastest permeability compared to dimethyl sulfoxide, ethylene glycol, and glycerol. Embryos were then exposed to a solution containing propylene glycol to evaluate its effects on fetal development. As the development was similar to that of fresh embryos, P10 (10% v/v propylene glycol in PB1) was used as a pretreatment solution. Next, the effects of the vitrification solution components (sucrose, propylene glycol, ethylene glycol, and Percoll) were examined by observing the vitrification status; 10% v/v propylene glycol, 30% v/v ethylene glycol, 0.3 mol sucrose, and 20% v/v Percoll in PB1 (PEPeS) was the minimum essential concentration for effective vitrification without the formation of ice crystals or freeze fractures.     

Cryopreservation of a marine microalga, Nannochloropsis oculata (Eustigmatophyceae)

The cryopreservation of algae could prevent genetic drift and minimize labor costs compared to the current method of maintenance and subculturing. Clear, simple protocols for cryopreservation of marine microalga, Nannochloropsis oculata were developed and cryoprotectant choice and concentration optimized. The viability of the microalga was assessed directly after thawing, and algal concentration was measured after 2-30 days of growth. Five cryoprotectants (dimethyl sulphoxide, Me2SO; ethylene glycol, EG; glycerol, Gly; methanol, MeOH; and propylene glycol, PG) at five concentrations (10, 20, 30, 40, and 50%; v/v) were evaluated to determine the toxicity of various cryoprotectants to N. oculata. The toxicity of cryoprotectant (Me2SO, EG, MeOH, and PG) was observed only at higher concentrations of CPAs: > 20% for EG, > 30% for Me2SO and methanol, and > 40% for PG. Direct freezing of algae in liquid nitrogen resulted in a severe loss of viability and a modified cryopreservation protocol proved to be more appropriate for the preservation of N. oculata. Cryopreservation protocols developed and tested in the present study might be applied to cryopreserving other strains, or species, in this genus.     

Cryopreservation of sperm from farmed Pacific abalone,Haliotis discus hannai

This study aimed to improve a sperm cryopreservation protocol for farmed Pacific abalone, Haliotis discus hannai. Dimethyl sulfoxide (Me₂SO), glycerol, ethylene glycol (EG), propylene glycol (PG), and methanol were chosen as cryoprotectants (CPAs). Four different equilibration time (5, 10, 30, and 60 min), and two types of equilibration temperature (4 °C and 20 °C) were selected at the present experiment. Most equilibration temperatures with each CPA showed significant differences among different equilibration time. Post-thaw sperm motility of five CPAs showed no significant difference at two equilibration temperature. Based on these results, 8% Me₂SO, 8% EG, 6% PG, 2% glycerol, and 2% methanol were chosen to determine optimal conditions for sperm cryopreservation of H. discus hannai. The highest post-thaw sperm motility (8% Me₂SO: 50.6%, 8% EG: 45.6%, 2% glycerol: 44.5%, 6% PG: 28.7%, 2% methanol: 25.4%) was achieved after exposing sperm to liquid nitrogen (LN₂) vapor for 10 min at 5 cm above the LN₂ surface and then submerging them in LN₂ for at least 2 h followed by thawing at 60 °C with seawater and recovering them at 20 °C with seawater. In this study, 8% Me₂SO and 2% glycerol were chosen to check post-thaw sperm quality to estimate percentages of plasma membrane integrity (PMI), mitochondrial potential analysis (MP), and acrosome integrity (AI) using fluorescent techniques. No significant difference in PMI, MP, and AI was found between sperm cryopreserved with 8% Me₂SO and those cryopreserved with 2% glycerol. The current study has demonstrated that 8% Me₂SO was optimal for sperm cryopreservation for H. discus hannai with 5 min of equilibration time, 5 cm of rack height and 60 °C of thawing temperature. The present research provides more effective cryopreservation methods for H. discus hannai sperm than previous studies.     

Cryopreservation of bovine in vitro produced embryos using ethylene glycol in controlled freezing or vitrification

In this study, the cryoprotectant ethylene glycol (EG) was tested for its ability to improve and facilitate the cryopreservation of in vitro produced (IVP) bovine embryos. Embryos were cryopreserved in EG solutions supplemented with either newborn calf serum (NBCS) or polyvinyl alcohol (PVA). To assess EG toxicity, the embryos were equilibrated in EG concentrations from 1.8 to 8.9 M at room temperature for 10 min and then cultured for 72 h on a cumulus cell monolayer. The hatching rate was highest for day 7 blastocysts frozen in 3.6 M EG (98%) and was not different from the control group (85%). The controlled freezing (0.3 degrees C/min to -35 degrees C) of expanded day 7 blastocysts resulted in a hatching rate of 81%, which was similar to that of the nonfrozen controls (76%). Differential staining revealed only very few degenerate blastomeres attributed to freezing and thawing. Upon direct nonsurgical transfer of day 7 expanded blastocysts frozen in 3.6 M EG, a pregnancy rate of 43% was achieved, while the pregnancy rate after transfer of other developmental stages was significantly lower (22% with expanded day 8 blastocysts). When bovine IVP embryos were incubated at room temperature in 7.2 M EG preceded by preequilibration in 3.6 M EG, the hatching rate of day 7 expanded blastocysts reached 93%. Upon vitrification of IVP day 7 and day 8 blastocysts and expanded blastocysts in 7.2 M EG, the latter showed a higher hatching rate (42%) than blastocysts (12%). Overall, PVA as supplement to the basic freezing solution instead of NBCS had deleterious effects on survival after controlled freezing or vitrification. The simple cryopreservation protocol employed in this study and the low toxicity of ethylene glycol highlight the usefulness of this approach for controlled freezing of IVP embryos. However, further experiments are needed to improve the pregnancy rate following embryo transfer and to enhance survival after vitrification.     

Malachite green induces cardiovascular defects in developing zebrafish (Danio rerio) embryos by blocking VEGFR-2 signaling

Malachite green (MG) is a triphenyl methane dye used in various fields that demonstrates high toxicity to bacteria and mammalian cells. When bud stage zebrafish embryos were treated with MG at 125, 150, and 175 ppb for 14 h, the development of trunk including intersomitic vessels was inhibited in MG-treated flk-1-GFP transgenic embyos. MG clearly induced whole growth retardation. MG induced severe cell death in trunk intersomite region of zebrafish embryos and in human vascular endothelial cells in a dose-dependent manner. MG inhibited heart rates and cardiac looping. MG attenuated whole blood formation and inhibited vascular endothelial growth factor (VEGF)-induced receptor (R)-2 phosphorylation in vascular endothelial cells. In conclusion, MG significantly alters the cardiovascular development causing growth retardation in zebrafish through the blocking VEGFR-2 activation in early cardiovascular development. It suggests that MG may be an environmental toxic agent with the potential to induce embryonic cardiovascular defects in vertebrates.     

Cryopreservation of Chirostoma jordani sperm,fish model for the conservation of the genus in Mexico

Genus Chirostoma belongs to Atherinopsidae family and it is an endemic species from the Mesa Central in Mexico. Abundance of its species have decreased and some ones have been placed on the threatened species list, because of overfishing, urbanization, industrialization, destruction, habitat fragmentation, pollution and exotic species introduction. Chirostoma jordani (Woolman, 1894) is a freshwater fish with biological, ecological, cultural, and commercial importance. It has a broad distribution in Lerma drainage, Durango and Mexico City. In this last locality, their populations, although small, still persist in Xochimilco Lake; it is necessary to implement biotechnologies for their conservation, because of these causes and their basic biology. The aim was to standardize a sperm cryopreservation protocol in C. jordani, to determine extender solution, cryoprotective agent type and concentration, equilibrium time, freezing and thawed rate to be applied in assisted reproduction and conservation of genus Chirostoma. Chirostoma jordani adult males were collected in Atlangatepec Dam, Tlaxcala State, Mexico, to fresh seminal evaluation and cryopreservation protocol standardization. Four cryoprotectants effect was evaluated: dimethylsulfoxide (DMSO), methanol (MeOH), ethylene glycol (EG), and glycerol (GL) at five concentrations: 2%, 6%, 10%, 14% and 16% v/v. Higher and lower DMSO and MeOH 10% and EG 14%, decreased post-thaw motility percentage. Both DMSO and MeOH 10% and EG 14% had the highest post-thaw motility percentages, 48.8 ± 1.5%, 54.5 ± 1.0% and 53.5 ± 1.0%, at 15, 10, and 5 min equilibrium times, respectively, thawed at 40°C. Chirostoma jordani sperm can be cryopreserved with both DMSO and MeOH 10%, and EG 14%. These ones can be used for assisted reproduction. GL was not efficient, since it presented a post-thaw motility percentage very low.     

Toxic effects of some alcohol and ethylene glycol derivatives on Cladosporium resinae.

Eleven commercially available alcohol and ethylene glycol derivatives were tested for their toxicity toward a problem organism in jet fuel, Cladosporium resinae. In the presence of glucose, 20% (vol/vol) ethylene glycol monomethyl ether prevented spore germination and mycelial growth, and 10% (vol/vol) 2-ethoxybutanol, 10% 2-isopropoxyethanol, 10% 3-methoxybutanol, 5% 2-butyloxyethanol, 5% ethylene glycol dibutyl ether, and 5% diethylene glycol monobutyl ether were found to have similar effects. In a biphasic kerosene-water system, 3-methoxybutanol, 2-butyloxyethanol, and diethylene glycol monobutyl ether were again found to be more toxic than ethylene glycol monomethyl ether. Considerable potassium efflux, protein leakage, and inhibition of endogenous respiration were observed in the presence of the more toxic compounds. 2-Butyloxyethanol also caused loss of sterols from cells.