Cryoprotective Agents Research Articles

Creation of a novel lipid-trehalose derivative showing positive interaction with the cell membrane and verification of its cytoprotective effect during cryopreservation

Cryopreservation is important for enabling long-term cell preservation. However, physical damage due to ice crystal formation and membrane permeation by dimethyl sulfoxide (DMSO) severely affects cryopreserved cell viability. To ensure cell survival and functional maintenance after cryopreservation, it is important to protect the cell membrane, the most vulnerable cell component, from freeze-thaw damage. This study aimed to create a glycolipid derivative having a positive interaction with the cell membrane and cytoprotective effects. As a result, we synthesized a novel trehalose derivative, oleyl-trehalose (Oleyl-Treh), composed of trehalose and oleyl groups. Its use led to increased viable cell counts when used with DMSO in a non-cytotoxic concentration range (1.6nM-16μM). Oleyl-Treh significantly improved viability and liver-specific functions of hepatocytes after cryopreservation, including albumin secretion, ethoxyresorufin-O-deethylase activity (an indicator of cytochrome P450 family 1 subfamily A member 1 activity), and ammonia metabolism. Oleyl-Treh could localize trehalose to the cell membrane; furthermore, the oleyl group affected cell membrane fluidity and exerted cryoprotective effects. This novel cryoprotective agent, which shows a positive interaction with the cell membrane, provides a unique approach toward cell protection during cryopreservation.

Cryopreservation of porcine urethral tissue: Storage at −20°C preserves the mechanical, failure and geometrical properties

Cryopreservation is required to preserve the native properties of tissue for prolonged periods of time. In this study, we evaluate the impact that 4 different cryopreservation protocols have on porcine urethral tissue, to identify a protocol that best preserves the native properties of the tissue. The cryopreservation protocols include storage in cryoprotective agents at −20 °C and −80 °C with a slow, gradual, and fast reduction in temperature. To evaluate the effects of cryopreservation, the tissue is mechanically characterised in uniaxial tension and the mechanical properties, failure mechanics, and tissue dimensions are compared fresh and following cryopreservation.The mechanical response of the tissue is altered following cryopreservation, yet the elastic modulus from the high stress, linear region of the Cauchy stress – stretch curves is unaffected by the freezing process. To further investigate the change in mechanical response following cryopreservation, the stretch at different tensile stress values was evaluated, which revealed that storage at −20 °C is the only protocol that does not significantly alter the mechanical properties of the tissue compared to the fresh samples. Conversely, the ultimate tensile strength and the stretch at failure were relatively unaffected by the freezing process, regardless of the cryopreservation protocol. However, there were alterations to the tissue dimensions following cryopreservation that were significantly different from the fresh samples for the tissue stored at −80 °C.Therefore, any study intent on preserving the mechanical, failure, and geometric properties of urethral tissue during cryopreservation should do so by freezing samples at −20 °C, as storage at −80 °C is shown here to significantly alter the tissue properties.

Effects of Porcine Immature Oocyte Vitrification on Actin Microfilament Distribution and Chromatin Integrity During Early Embryo Development in vitro.

Vitrification is mainly used to cryopreserve female gametes. This technique allows maintaining cell viability, functionality, and developmental potential at low temperatures into liquid nitrogen at −196°C. For this, the addition of cryoprotectant agents, which are substances that provide cell protection during cooling and warming, is required. However, they have been reported to be toxic, reducing oocyte viability, maturation, fertilization, and embryo development, possibly by altering cell cytoskeleton structure and chromatin. Previous studies have evaluated the effects of vitrification in the germinal vesicle, metaphase II oocytes, zygotes, and blastocysts, but the knowledge of its impact on their further embryo development is limited. Other studies have evaluated the role of actin microfilaments and chromatin, based on the fertilization and embryo development rates obtained, but not the direct evaluation of these structures in embryos produced from vitrified immature oocytes. Therefore, this study was designed to evaluate how the vitrification of porcine immature oocytes affects early embryo development by the evaluation of actin microfilament distribution and chromatin integrity. Results demonstrate that the damage generated by the vitrification of immature oocytes affects viability, maturation, and the distribution of actin microfilaments and chromatin integrity, observed in early embryos. Therefore, it is suggested that vitrification could affect oocyte repair mechanisms in those structures, being one of the mechanisms that explain the low embryo development rates after vitrification.

Conduction Cooling and Plasmonic Heating Dramatically Increase Droplet Vitrification Volumes for Cell Cryopreservation.

Droplet vitrification has emerged as a promising ice‐free cryopreservation approach to provide a supply chain for off‐the‐shelf cell products in cell therapy and regenerative medicine applications. Translation of this approach requires the use of low concentration (i.e., low toxicity) permeable cryoprotectant agents (CPA) and high post cryopreservation viability (>90%), thereby demanding fast cooling and warming rates. Unfortunately, with traditional approaches using convective heat transfer, the droplet volumes that can be successfully vitrified and rewarmed are impractically small (i.e., 180 picoliter) for <2.5 m permeable CPA. Herein, a novel approach to achieve 90–95% viability in micro‐liter size droplets with 2 m permeable CPA, is presented. Droplets with plasmonic gold nanorods (GNRs) are printed onto a cryogenic copper substrate for improved cooling rates via conduction, while plasmonic laser heating yields >400‐fold improvement in warming rates over traditional convective approach. High viability cryopreservation is then demonstrated in a model cell line (human dermal fibroblasts) and an important regenerative medicine cell line (human umbilical cord blood stem cells). This approach opens a new paradigm for cryopreservation and rewarming of dramatically larger volume droplets at lower CPA concentration for cell therapy and other regenerative medicine applications.

The Effect of Cryoprotective Agents on Proteins of the Erythrocyte Membrane-Cytoskeleton Complex

The Effect of Cryoprotective Agents on Proteins of the Erythrocyte Membrane-Cytoskeleton Complex

Cryopreservation of Canine Erythrocytes Using Dimethyl Sulfoxide, Polyethylene Glycol and Sucrose

Cryoprotective properties of combined media of permeable (dimethyl sulfoxide) and impermeable (polyethylene glycol with m. w. 1500) cryoprotective agents during rapid cooling in liquid nitrogen of canine erythrocytes using saline and sucrose-saline media have been investigated. It was found that the use of combined solutions of cryoprotective agents based on polyethylene glycol with m.w. 1500 (15%) and dimethyl sulfoxide (2.5–10%) in saline was not quite effective for cryopreservation of canine erythrocytes. Reducing the salt concentration and adding cell-impermeable sucrose to the cryopreservation medium increase the preservation of erythrocytes after warming. The best cryoprotective properties for canine erythrocytes were demonstrated by 10% dimethyl sulfoxide based on sucrose-saline medium, with high preservation of cells after freeze-warming, mechanical and osmotic stability of warmed erythrocytes. This indicates the possibility of a long-term storage and use of cryopreserved canine erythrocytes for transfusions.

Computational screening of cryoprotective agents for regenerative medical products using quantum chemistry and molecular dynamics simulations

Cryoprotective agents (CPAs) are essential for the cryopreservation of cells. Thus far, dimethyl sulfoxide (DMSO) has been widely used as a CPA; however, DMSO is known to be toxic to cells. The damaged cells by the toxicity can present abnormal conditions, and should not be used for regenerative medical products because the cells/products are implanted directly into human bodies. With the aim of searching for an alternative CPA to DMSO, this work presents a computational screening of CPA candidate compounds using quantum chemistry and molecular dynamics (MD) simulations. Forty compounds were evaluated in regard to the solvation free energy and partition coefficient by quantum chemistry simulation and the root mean square deviation (RMSD) of a phospholipid bilayer which composes a cell membrane by MD simulation. The solvation free energy, partition coefficient, and RMSD were defined as indicators of osmoregulatory ability, affinity with a cell membrane, and ability to spread a cell membrane, respectively. The quantum chemistry simulation elucidated that the six compounds of trimethylglycine, formamide, urea, thiourea, diethylene glycol, and dulcitol were better than DMSO in either or both of the physical properties considered. This finding is based on the inherent physical property and is thus case-independent. Further characterization with the MD simulation suggested that formamide, thiourea, and urea should be the first candidates to investigate, although the result was valid only in the simulated condition. This work serves as the first step of multi-faceted computational evaluation of multiple compounds in the search for an effective CPA compound after DMSO.

Antioxidant effect of Elamipretide on bull's sperm cells during freezing/thawing process.

Spermatozoa are subjected to drastic changes in temperature, ice crystal formation, and diverse types of stresses (chemical, physical, osmotic, and oxidative) during the cryopreservation process, which severely compromise sperm quality and fertility. In this study, we aimed to investigate the protective role of Elamipretide in the cryopreservation of bull's spermatozoa. The study included 36 healthy Simmental bulls with an average age of 2±0.5years housed individually in pens. Two ejaculates were collected from each bull using an artificial vagina at 7 a.m. Subsequently, the semen was extended with animal protein-free commercial BIOXcell® extender (IMV Technologies) to a final concentration of 160×106 spermatozoa/ml, and rated in terms of motile sperm percentage, progressive motility, viability, and morphological abnormality of spermatozoa. Semen samples that showed more than 60% motility and 60% viability, were selected for the experiment. The fresh semen was then divided into five equal fractions. The first fraction was left for the control group (without Elamipretide), to the next were added in succession 0.1; 1; 5; and 10μM of Elamipretide TFA (Trifluoroacetic) (MedChemExpress). After that semen was subjected to freezing and thawing. Next semen was assessed for motility, viability, mitochondrial membrane potential and acrosome integrity, and antioxidant activity (SOD, CAT, MDA). It has been shown that a concentration of 5 and 10μM proved to be the most effective in terms of tested parameters of the quality of sperm cells subjected to cryopreservation. In conclusion, addition of the Elamipretide to the cryopreservation extender significantly improved frozen-thawed sperm cells quality and their function. The results of this study indicate that Elamipretide can be used as a cryoprotective agent to protect cells against the devastating effects of oxidative stress and increasing sperm survival after cryopreservation.

Evaluation of the effectiveness of a new cryopreservation system based on a two-compartment vial for the cryopreservation of cell therapy products

Background aimsSuccessful cell cryopreservation and banking remain a major challenge for the manufacture of cell therapy products, particularly in relation to providing a hermetic, sterile cryovial that ensures optimal viability and stability post-thaw while minimizing exposure to toxic cryoprotective agents, typically dimethyl sulfoxide (Me2SO). MethodsIn the present study, the authors evaluated the effectiveness and functionality of Limbo technology (Cellulis S.L., Santoña, Spain). This system provides a hermetic vial with two compartments (one for adding cells with the cryoprotective agent solution and the other for the diluent solution) and an automated defrosting device. Limbo technology (Cellulis S.L.) allows reduction of the final amount of Me2SO, sidestepping washing and dilution steps and favoring standardization. The study was performed in several Good Manufacturing Practice laboratories manufacturing diverse cell therapy products (human mesenchymal stromal cells, hematopoietic progenitor cells, leukapheresis products, fibroblasts and induced pluripotent stem cells). Laboratories compared Limbo technology (Cellulis S.L.) with their standard cryopreservation procedure, analyzing cell recovery, viability, phenotype and functionality. ResultsLimbo technology (Cellulis S.L.) maintained the viability and functionality of most of the cell products and preserved sterility while reducing the final concentration of Me2SO. ConclusionsResults showed that use of Limbo technology (Cellulis S.L.) offers an overall safe alternative for cell banking and direct infusion of cryopreserved cell products into patients.

Cryopiece, a novel carrier with faster cooling rate, high recovery rate and retrieval rate, for individual sperm cryopreservation.

BackgroundCryopreservation of extremely few spermatozoa is still a major challenge for male fertility preservation. This study aims to evaluate the cooling rate, recovery rate, and retrieval rate, along with other parameters of spermatozoa that cryopreserved using Cryopiece, a novel carrier, for individual sperm cryopreservation.MethodsSemen samples from 60 fertile donors were collected, and each semen sample was screened for motile sperm and mixed with cryoprotective agent (CPA), and then frozen using Cryopiece, micro-straw, and mini-straws. The cooling rate, retrieval rate, and recovery rate, morphology, DNA fragmentation index (DFI) and mitochondrial membrane potential (MMP), were compared among the un-frozen sperm and the sperm cryopreserved using these carriers.ResultsCryopiece possessed the fastest cooling rate. After freeze-thaw, the average retrieval rate of sperm cryopreserved using Cryopiece was 96.25%, and the average recovery rate was 64.40%, which were higher than that of sperm cryopreserved using the other two carriers (71.42% and 54.30% for micro-straw, and 63.54% and 58.04% for mini-straw, respectively). There was no significant impact on DFI after sperm cryopreservation, and no significant difference in morphology between sperm cryopreserved using these carriers was observed. Though MMP of sperm changed significantly after cryopreservation, micro-straw maintained sperm MMP better than Cryopiece and mini-straw did, while no significant difference was observed in MMP between sperm cryopreserved using Cryopiece and mini-straw.ConclusionsCryopiece produced satisfying retrieval and recovery rates in sperm cryopreservation and should be an ideal carrier for cryopreservation of small number of sperm.

Cryopreservation of Cyanobacteria and Eukaryotic Microalgae Using Exopolysaccharide Extracted from a Glacier Bacterium.

Exopolysaccharide (EPS) has been known to be a good cryoprotective agent for bacteria, but it has not been tested for cyanobacteria and eukaryotic microalgae. In this study, we used EPS extracted from a glacier bacterium as a cryoprotective agent for the cryopreservation of three unicellular cyanobacteria and two eukaryotic microalgae. Different concentrations of EPS (10%, 15%, and 20%) were tested, and the highest concentration (20%) of EPS yielded the best growth recovery for the algal strains we tested. We also compared EPS with 5% dimethyl sulfoxide (DMSO) and 10% glycerol for the cryopreservation recovery. The growth recovery for the microalgal strains after nine months of cryopreservation was better than 5% DMSO, a well-known cryoprotectant for microalgae. A poor recovery was recorded for all the tested strains with 10% glycerol as a cryoprotective agent. The patterns of growth recovery for most of these strains were similar after 5 days, 15 days, and 9 months of cryopreservation. Unlike common cryopreservants such as DMSO or methanol, which are hazardous materials, EPS is safe to handle. We demonstrate that the EPS from a psychrotrophic bacterium helped in the long-term cryopreservation of cyanobacteria and microalgae, and it has the potential to be used as natural cryoprotective agent for other cells.

Investigating solution effects injury of human T lymphocytes and its prevention during interrupted slow cooling

Cooling rate is a critical parameter affecting the success of cell cryopreservation. Fast cooling can result in intracellular ice formation (IIF), while slow cooling can bring solution effects injury, both are detrimental to the cells. Whilst most of the studies have investigated how IIF affects cells, solution effects injury has received little attention. Here, we studied the solution effects injury of human T lymphocytes by cryomicroscopy and tested the osmoprotective ability of some frequently used cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol, trehalose, urea and l-proline. We further investigated the relationship between cell volume, latent heat and solution effects cell injury. We found that solution effects injury during interrupted slow cooling was caused by high concentration of the extracellular solution rather than eutectic formation and solutes precipitation. DMSO, glycerol and trehalose can protect cells from solution effects injury, while l-proline and urea cannot under the same condition. The cell volume and latent heat are not crucial for causing solution effects injury in cells. This work confirms that high osmotic pressure, rather than eutectic formation, leads to cell injury. It also suggests that cell volume and latent heat may not be a key factor for explaining solution effects injury and its prevention in the cryopreservation of human T lymphocytes.

A Comparative Study on DMSO-Induced Modulation of the Structural and Dynamical Properties of Model Bilayer Membranes

Modulating the structures and properties of biomembranes via permeation of small amphiphilic molecules is immensely important, having diverse applications in cell biology, biotechnology, and pharmaceuticals, because their physiochemical and biological interactions lead to new pathways for transdermal drug delivery and administration. In this work, we have elucidated the role of dimethyl sulfoxide (DMSO), broadly used as a penetration-enhancing agent and cryoprotective agent on model lipid membranes, using a combination of fluorescence microscopy and time-resolved fluorescence spectroscopy. Spatially resolved fluorescence lifetime imaging microscopy (FLIM) has been employed to unravel how the fluidity of the DMSO-induced bilayer regulates the structural alteration of the vesicles. Moreover, we have also shown that the dehydration effect of DMSO leads to weakening of the hydrogen bond between lipid headgroups and water molecules and results in faster solvation dynamics as demonstrated by femtosecond time-resolved fluorescence spectroscopy. It has been gleaned that the water dynamics becomes faster because bilayer rigidity decreases in the presence of DMSO, which is also supported by time-resolved rotational anisotropy measurements. The enhanced diffusivity and increased membrane fluidity in the presence of DMSO are further ratified at the single-molecule level through fluorescence correlation spectroscopy (FCS) measurements. Our results indicate that while the presence of DMSO significantly affects the 1,2-dimyristoyl-rac-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-rac-glycero-3-phosphatidylcholine (DPPC) bilayers, it has a weak effect on 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (DMPG) vesicles, which might explain the preferential interaction of DMSO with the positively charged choline group present in DMPC and DPPC vesicles. The experimental findings have also been further verified with molecular dynamics simulation studies. Moreover, it has been observed that DMSO is likely to have a differential effect on heterogeneous bilayer membranes depending on the structure and composition of their headgroups. Our results illuminate the importance of probing the lipid structure and composition of cellular membranes in determining the effects of cryoprotective agents.

Effect of diluent variation on cryopreservation of large yellow croaker Larimichthys crocea

The aim of this research was to investigate different factors, including cryoprotective agents (CPAs), diluents, dilution ratios, equilibrium times, freezing rates, and thawing methods to optimize cryopreservation protocols for large yellow croaker (Larimichthys crocea). The parameters evaluated were sperm motility, sperm activity index (SAI), survival rate, and DNA damage. Different types of CPAs, such as dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), methanol, and glycerol, were tested for sperm preservation. The highest motility, SAI, and survival rate were observed when EG was used. Different diluents such as Stein’s solution, Hank’s balanced salt solution, marine fish Ringer’s solution, artificial seminal plasma (ASP) of small yellow croaker, and Cortland solution were investigated. The highest post-thaw motility was observed upon using ASP as the diluent. Different concentrations of EG were then mixed with ASP to identify the optimal EG concentration. Experimental results showed that the motility (70.33 ± 1.20%), SAI (5), and survival rate (78.30 ± 0.42%) of post-thaw sperm were optimum when 10% EG and ASP were used as the CPA and diluent of cryopreservation, respectively. Post-thaw sperm motility was high at equilibration times below 150 s and at an optimum dilution ratio of 1:1 (sperm: CPA + diluent) and was not significantly different compared with fresh sperm motility. The freezing rate was found to be slow below −10°C/min. The thawing temperature of 45°C was identified as ideal. The percentage of tail DNA in post-thaw sperm at 10% EG and ASP was also investigated and was found to have more significant DNA damage than that in fresh sperm but significantly lower damage than that in post-thaw sperm at EG concentrations of 5%, 15%, and 20% (p &lt; 0.05). The cryopreservation protocols obtained in this study will be useful in large yellow croaker hatcheries.

Comprehensive survey of United States internet users' sentiments towards cryopreservation.

Medical cryopreservation is the speculative practice of using low temperatures and medical-grade cryoprotective agents to halt the decay of a recently-deceased person’s brain and body for the prospect of future resuscitation and restoration of function. We conducted a survey of 1,487 internet users in the United States to measure familiarity with, interest in, beliefs about, and attitudes towards cryopreservation. The majority of respondents (75%) had previously heard of the topic. Respondents tended to underestimate the cost of cryopreservation and number of previous cases but overestimate the number of providers. While many respondents expressed interest in signing up (20%) or had actively researched the topic (21%), a much smaller fraction have decided to be cryopreserved (6%). This level of interest is much greater than the number of previous preservation cases would indicate. We found that respondents’ attitudes towards death significantly correlated with their general sentiments towards the topic, with those expressing a desire for longer life or to see the future being more interested and positively inclined. Fear of death was not associated with interest in cryopreservation. Negative sentiments towards cryopreservation were less common than respondents perceived. For example, 14% of respondents believed that “most people” think cryopreservation should be illegal, but only 4% of respondents actually did. Many respondents (42%) were pessimistic regarding the likelihood of cryopreservation being successful, but the mean estimate of time until revival of cryopreserved bodies would be possible was 82 years.

Study of Intracellular Ice Structure during Cryopreservation Process using Polyvinyl Pyrrolidone as Cryoprotectant Agent

Study of Intracellular Ice Structure during Cryopreservation Process using Polyvinyl Pyrrolidone as Cryoprotectant Agent

The effect of docetaxel on survival, fertilization rate and apoptosis-related genes mRNA expression in mouse metaphase II oocytes following vitrification.

Docetaxel is beneficial in oocyte cryopreservation. The effect of docetaxel, on the survival, fertilization rate and mRNA expression of apoptosis-related genes of vitrified mature oocytes was investigated. Mature oocytes were divided into eight experimental groups, including I) control, II) docetaxel, III) docetaxel + cryoprotectant agent 1 (CPA1), IV) docetaxel + CPA2, V) docetaxel + vitrification 1 (Vit1), VI) docetaxel + Vit2, VII) Vit1, and VIII) Vit2. The survival and fertilization rates, and the mRNA expression level of Bcl-xl, Bax and caspase-3 as apoptosis-related genes were evaluated. The survival rates in Vit1, and Vit2 groups were significantly lower than in the control group (P<0.05). The fertilization rates in docetaxel + Vit1, docetaxel + Vit2, Vit1, and Vit2 were significantly lower than the control, docetaxel, and related groups using docetaxel and CPAs. Bax expression was significantly increased in groups which oocytes vitrified. Also, its expression in the Vit2 group increased significantly in comparison to the docetaxel + Vit2 group. The expression of the Bcl-xl gene was downregulated in docetaxel + CPA2, docetaxel + Vit2 and Vit2 compared to docetaxel group. The Bax/Bcl-xl ratio significantly increased in docetaxel + CPA2, docetaxel + Vit1, docetaxel + Vit2, Vit1 and Vit2 groups compared to control, docetaxel and the docetaxel + CPA1 group. Caspase-3 expression significantly increased in all six groups in comparison to the control, and docetaxel groups. Its expression significantly increased in the Vit1 and Vit2 groups in comparison with docetaxel + Vit1, and docetaxel + Vit2, respectively. Docetaxel ameliorates the damages to oocytes during vitrification by altering the expression of apoptosis-related genes and its effects are dependent on the vitrification solution used in cryopreservation of oocytes.

29 The effect of vitrification at the immature stage on DNA methylation in porcine oocytes and its relevance to subsequent embryo development

Oocyte vitrification is an important approach for invitro gene banking of female germplasm; however, in pigs, it hampers embryo development. In cattle, vitrification at the MII stage was reported to alter epigenetic status in oocytes and even in subsequently developing embryos (Chen et al. 2016 Theriogenology 86, 868-878). The present study investigated the effect of vitrification at the immature stage of porcine oocytes on DNA methylation status and its relevance to subsequent embryo development. Immature cumulus–oocyte complexes were vitrified in microdrops and warmed (vitrified group) or treated with cryoprotectant agents (17.5% ethylene glycol + 17.5% propylene glycol, CPA group) by our method (Appeltant et al. 2018 Cryobiology 85, 87-94). Then they were subjected to IVM, parthenogenetic activation (PA), and embryo culture. From each batch, a group of oocytes was processed without treatment (control group). Oocyte survival and polar body extrusion were recorded after IVM. Cleavage and blastocyst developmental rates were recorded on Day 2 and 6 of culture, respectively (Day 0=PA). In each replication, DNA methylation was assayed in representative oocytes at the MII stage after IVM and in embryos at the 2- to 4-cell stage on Day 2 by immunostaining with 5-methylcytosine (5mC). Relative fluorescent intensity of 5mC in the chromatin was compared among groups. The experiment was replicated 3 times. Data were analysed by ANOVA. After IVM, there was no significant difference among the control, CPA, and vitrified groups in terms of the percentage of live oocytes (99.3, 96.4, and 94.0%, respectively) or polar body extrusion (88.6, 86.9, and 79.6%, respectively). After PA of oocytes with a polar body, there was no difference between the control and CPA groups in the percentage of cleavage (84.1 and 80.7%, respectively) or blastocyst development of cleaved embryos (63.3 and 79.3%, respectively). However, in the vitrified group, cleavage and blastocyst development rates (46.6 and 33.5%, respectively) were reduced (P&amp;lt;0.05) compared with the other groups. The 5mC fluorescence in the DNA of oocytes at the MII stage in the CPA and vitrified groups were similar and significantly lower than that in the control group (0.88±0.02, 0.87±0.001, and 1.0±0.02, respectively) but higher than that in the negative control processed without primary antibody (0.33±0.02). In the embryos at the 2- to 4-cell stage, 5mC fluorescence was not significantly different among the control, CPA, and vitrified groups (1.0±0.1, 0.99±0.1, and 0.96±0.1, respectively) but was significantly higher than that of the negative control (0.36±0.04). In conclusion, CPA treatment reduced DNA methylation levels in oocytes. However, it was restored during early embryo development and did not affect blastocyst development. The results suggest that reduced DNA methylation in vitrified oocytes is caused by CPA but it may not be responsible for their reduced ability to develop to blastocysts.

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.

Comparison of different cryoprotective agents on swamp buffalo semen cryopreservation

Present study aimed to determine the effectivenessof dimethylformamide (DMF)as cryoprotective agent (CPA) by determining its optimum concentration inTris Eggyolk (TEY) extender on the quality of post-thaw swamp buffalo semen. Only ejaculates having &gt; 60% sperm motility were divided into eight s tubes. Four tubes were diluted in TEY with 4, 5, 6, 7% of glycerol and four other tubes were diluted in TEY with 4, 5, 6, 7% of DMF. Diluted semen then packed into ministraw (0.25 mL), equilibrated at 4°C for 4 hours and freezed at 8 cm above liquid nitrogen vapour for 10 minutes and then stored at liquid nitrogencontainer. After 24 hours of storage, straws were thawed at 37°C for 30 seconds. The result showed that sperm motility after equilibration tend to be the same at all four glycerol concentrations, whereas the decrease in motility was highest in extenders with a DMF concentration of 7% (P &lt;0.05). Post-thaw sperm motility and recovery rate in TEY extender containing 5% DMF or 7% glycerol were highest (P&lt;0.05) than other glycerol or DMF concentration. Sperm viability and membrane integrity did not differ among TEY containing 6% or 7% glycerol, both were higher (P&lt;0.05) than 4% or 5% glycerol. Viability and membrane integrity of sperm in 5% DMF was superior (P&lt;0.05) to 4%, 6% or 7%. This research concluded that 5% DMF or 7% glycerol was considered to be the most suitable CPAs for swamp buffalo semen cryopreservation.