Studies on cryoprotectant toxicity to zebrafish (Danio rerio) ovarian tissue fragments

Cryopreservation of ovarian tissue is a viable alternative to cryopreservation of oocytes and embryos in many species but it has not been studied in fish. Selection of cryoprotectant is an important step in designing cryopreservation protocols. In order to identify the optimum cryoprotectant (CPA) in a suitable concentration for zebrafish ovarian tissue cryopreservation, studies on toxicities of five commonly used cryoprotectants methanol, ethanol, dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) were carried out. Experiments were conducted on ovarian tissue fragments consisting of stage I and stage II ovarian follicles. Ovarian tissue fragments were incubated in 90% L-15 medium (pH 9) containing 1-4M cryoprotectants for 30min at 22°C. Three different tests were used to assess ovarian tissue fragment viability: trypan blue (TB) staining, fluorescein diacetate (FDA) combined with propidium iodide (PI) staining and adenosine 5′- triphosphate (ATP) assay. Results from these tests showed that ATP assay was more sensitive than FDA+PI or TB staining for assessing cryoprotectant toxicity to follicles in tissue fragments. Methanol and ethanol were the least toxic cryoprotectants tested. Cryoprotectant toxicity increased in the order of methanol/ethanol, DMSO, PG and EG. Ethanol was used for zebrafish ovarian tissue for the first time and the results showed that the effect of methanol and ethanol on ovarian tissue fragments were comparable. As methanol has been shown to be the most effective cryoprotectant for zebrafish ovarian follicles in our laboratory, the use of ethanol will also be considered in assisting future freezing protocol design. The present study also showed that stage II ovarian follicles are more sensitive to cryoprotectant treatment than stage I follicles in tissue fragments. The results obtained in this study provided useful information for ovarian tissue fragment cryopreservation protocol design in the future.     

Impact of cryoprotectants and cryopreservation on metabolic activity and cytoskeleton proteins of zebrafish (Danio rerio) ovarian fragments

Cryopreservation of reproductive cells and tissues of aquatic species offers many benefits to the field of conservation, aquaculture and biomedicine. Although cryopreservation of fish sperm has been successfully achieved, cryopreservation of embryos and oocytes remains unsuccessful. Several studies have been undertaken on cryopreservation of isolated fish ovarian follicles at different stages, although the protocols used lead to a compromised viability. The present study investigates the effect of cryoprotectants and cryopreservation on the viability of ovarian tissues of zebrafish (Danio rerio). The effect of permeating cryoprotectants (CPAs) methanol, dimethyl sulfoxide (DMSO), and ethylene glycol (EG) on ovarian tissues were investigated in a series of toxicity tests. Controlled slow cooling of ovarian tissues using 1M and 4M methanol was also carried out. Ovarian tissue viability was assessed by trypan blue (TB) and fluorescence diacetate (FDA)-propidium iodide (PI) tests. In addition, the effect of methanol exposure and cryopreservation on ovarian follicle ATP level, mitochondria, actin and tubulin distribution were also investigated. Results showed that cryoprotectant toxicity to ovarian fragments increased in the order of methanol, DMSO and EG. The results from controlled slow cooling showed that 1M methanol was more effective than 4M methanol although subsequent cryopreservation induced decreases in ATP levels. Immunocytochemistry and actin staining results showed impacts of cryopreservation on mitochondria and cytoskeleton proteins distribution.     

Cryopreservation of zebrafish (Danio rerio) oocytes by vitrification

Cryopreservation of fish oocytes is challenging because these oocytes have low membrane permeability to water and cryoprotectant and are highly chilling sensitive. Vitrification is considered to be a promising approach for their cryopreservation as it involves rapid freezing and thawing of the oocytes and therefore minimising the chilling injury. In the present study, vitrification properties and the toxicity of a range of vitrification solutions containing different concentrations of Me2SO, methanol, propylene glycol and ethylene glycol were investigated. Two different base media and vitrification methods were compared. The effect of different post-thaw dilution solutions together with incubation periods on oocyte viability were also investigated. Stage III zebrafish oocytes were equilibrated in increasing concentrations of cryoprotectants for 30 min in 3 steps. Oocytes were thawed rapidly in a water bath and cryoprotectants were removed in 4 steps. Oocyte viability was assessed using trypan blue staining. The results showed that vitrification solutions V3 and V4 in KCl buffer had low toxicity and vitrified well. The survivals of oocytes after stepwise dilution using solutions containing permeable cryoprotectants were significant higher than those diluted in 0.5M glucose, and the use of CVA65 vitrification system improved oocyte survival when compared with plastic straws after 30 min at 22 degrees C post-thawing. Cryopreservation of zebrafish oocytes by vitrification is reported here for the first time, although oocyte survivals after cryopreservation assessed by trypan blue staining were relatively high shortly after thawing, they became swollen and translucent after incubation in KCl buffer. Further studies are needed to optimise the post-thaw culturing conditions.     

Study on the mitochondrial activity and membrane potential after exposing later stage oocytes of two gorgonian corals (Junceella juncea and Junceella fragilis) to cryoprotectants

Coral reefs provide a valuable habitat for many economically valuable fish and invertebrates. However, they are in serious jeopardy, threatened by increasing over-exploitation, pollution, habitat destruction, disease and global climate change. Here, we examined the effect of cryoprotectant exposure on mitochondrial activity and membrane potential in coral oocytes in order to find suitable cryoprotectants towards their successful cryopreservation. According to the No Observed Effect Concentrations (NOECs), methanol was found to be the least toxic cryoprotectant whilst DMSO was the most toxic cryoprotectant. The results also demonstrated that there were no significant differences (p > 0.05) in ATP concentrations between Junceella juncea and Junceella fragilis after exposure to all concentrations of all cryoprotectants for 30 min. Using confocal microscopy, JC-1 (5,50,6,60-tetrachloro-1,10,3,30-tetraethyl-imidacarbocyanine iodide) staining indicated that the mitochondrial membrane potential of Junceella fragilis oocytes reduced after 1 M and 2 M methanol treatment and a loss of the mitochondrial distribution pattern and poor green fluorescence after 3M methanol treatment. Therefore, even oocytes that show no adverse effect of cryoprotectants on survival might suffer some more subtle impacts. The results obtained from this study will provide a basis for development of protocols to cryopreserve the oocytes of gorgonian corals.     

Vitrification of zebrafish embryo blastomeres in microvolumes

Cryopreservation of fish embryos may play an important role in biodiversity preservation and in aquaculture, but it is very difficult. In addition, the cryopreservation of fish embryo blastomeres makes conservation strategies feasible when they are used in germ-line chimaerism, including interspecific chimaerism. Fish embryo blastomere cryopreservation has been achieved by equilibrium procedures, but to our knowledge, no data on vitrification procedures are available. In the present work, zebrafish embryo blastomeres were successfully vitrified in microvolumes: a number of 0.25 microl drops, sufficient to contain all the blastomeres of an embryo at blastula stage (from 1000-cell stage to oblong stage), were placed over a 2.5 cm loop of nylon filament. In this procedure, where intracellular cryoprotectant permeation is not required, blastomeres were exposed to cryoprotectants for a maximum of 25 sec prior vitrification. The assayed cryoprotectants (ethylene glycol, propylene glycol, dimethyl sulphoxide, glycerol and methanol) are all frequently used in fish embryo and blastomere cryopreservation. Methanol was finally rejected because of the excessive concentration required for the vitrification (15M). All other cryoprotectants were prepared (individually) at 5 M in Hanks' buffered salt solution (sigma) plus 20% FBS (vitrification solutions: vs). After direct thawing in Hanks' buffered salt solution plus 20% FBS, acceptable survival rates were obtained with ethylene glycol: 82.8%, propylene glycol: 87.7%, dimethyl sulphoxide: 93.4%, and glycerol: 73.9% (p < 0.05). Dimethyl sulphoxide showed the highest blastomere survival rate and allowed the rescue of as much as 20% of the total blastomeres from each zebrafish blastula embryo.     

Optimization of cryopreservation of stem cells cultured as neurospheres: comparison between vitrification, slow-cooling and rapid cooling freezing protocols

We compared cryopreservation of mammalian neural stem cells (NSCs) cultured as neurospheres by slow-cooling (1 C/min) in 10% (v/v) DMSO and cryopreservation by immersion into liquid nitrogen in ethylene glycol (EG)-sucrose solutions that support vitrification (40% (v/v) EG, 0.6 M sucrose) or that do not (37% v/v) EG, 0.6 M sucrose and 30% (v/v) EG, 0.6 M sucrose); the concentration of penetrating cryoprotectant in the last two solutions was lowered with the intention to reduce their toxicity towards NSCs. To protect against contamination a straw-in-straw technique was employed. Vitrification offered the best combination of preservation of structural integrity of neurospheres, cell viability (>96%), multipotency and karyotype. Rapid cooling in 37% (v/v) EG, 0.6 M sucrose afforded good viability but did not preserve structural integrity. Rapid cooling in 30% (v/v) EG, 0.6 M sucrose additionally reduced cell viability to 77%. Slow-cooling reduced cell viability to 65% and damaged the neurospheres. This study suggests that, in contrast to freezing, vitrification has immense potential for the cryopreservation of stem cells cultured as neurospheres or in other structured cultures.     

Effects of cryoprotectant agents and equilibration methods on developmental competence of porcine oocytes

Chemical toxicity of cryoprotectants to in vitro developmental competence of porcine oocytes was examined. In vitro-matured oocytes were exposed to 40 percent ethylene glycol (EG), glycerol (GLY), or 1,2-propanediol (PD), fertilized with spermatozoa, and cultured for 8 days. Compared to treatment with other cryoprotectants, exposure to EG resulted in the development of significantly more blastocysts, but the rate was significantly lower than that of non-exposed control oocytes. In vitro-matured oocytes were also equilibrated in 40 percent EG by 3 multi-step methods, after which their developmental competence was evaluated. The rate of blastocyst development was higher in the 4-step method than in the 2- and 3-step methods of equilibrium. These results indicate that cryoprotectants and equilibration methods affect the developmental competence of porcine oocytes and that EG may be a superior cryoprotectant for vitrification of these cells.     

The effects of different cryoprotectants and the temperature of addition on the survival of red deer epididymal spermatozoa

With the aim of finding an ideal cryoprotectant in a suitable concentration for red deer epididymal spermatozoa conservation, we evaluated the effects of four most commonly used cryoprotectants (CPAs), Glycerol (G), Ethylene glycol (EG), Propylene glycol (PG), and Dimethyl sulfoxide (DMSO), on the sperm survival. Besides, the effects of two temperatures of CPA addition--22 degrees C (ambient temperature) and 5 degrees C--on sperm quality were also tested. For each temperature tested, sperm samples were evaluated after 0, 15, 30 and 60 min of spermatozoa exposition to CPAs. Thus, sperm quality was in vitro judged by microscopic assessments of individual sperm motility (SMI), and of plasma membrane (Viability) and acrosome (NAR) integrities. Overall, DMSO showed the highest toxicity for red deer epididymal spermatozoa, and glycerol the lowest. Thus, at 60 min of incubation SMI results showed that the toxicity to red deer epididymal spermatozoa of the four CPAs are in the following sequence: G approximately = EG approximately = PG < DMSO ('less than' symbol means P < 0.05, and approximate symbol means P = 0.08). Furthermore, our results also showed a differential response of acrosome membrane to temperature of CPAs addition. Regardless of the CPA used, statistically significant variations (P < 0.05) were found between the two temperatures of addition of CPAs for acrosome integrity, the best being 22 degrees C (NAR = 83.8% vs. 69.8%). These data indicate that sperm quality of red deer epididymal spermatozoa, in addition to be affected by the cryoprotectant, can also be influenced by the temperature at which CPAs are added prior to freezing.     

Cryoprotectants protect medaka (Oryzias latipes) embryos from chilling injury

This study was conducted to investigate the effect of six cryoprotectants (dimethyl sulfoxide (DMSO), glycerol (Gly), methanol (MeOH), ethylene glycol (EG), 1,2-propylene glycol (PG) and N,N-dimethylformamide (DMF) on the survival of medaka (Oryzias lapites) embryos at low temperatures (0 and -5C). Firstly, the embryos at 8 to 16-cell stages were exposed to different concentrations (1 to 4 mol per L) of DMSO, Gly, MeOH, EG, PG and DMF for 40min at 26C. After removal of the cryoprotectants (CPAs), the embryo survivals were assessed by their development into live fries following 9 day of culture. The results showed that the higher concentration of the CPA, the lower survival of the embryos; and that the toxicity of the six CPAs to medaka embryos is in the order of PG < MeOH = DMSO < Gly < EG < DMF (P < 0.05). Secondly, based on the results obtained above, embryos at 8 to 16-cell stages or other stages were exposed to 2 mol per L of PG, MeOH or DMSO for up to 180 min at 0C and up to 80 min at -5C respectively. The 8 to 16-cell embryos treated with MeOH at low temperatures showed highest survival. Thirdly, when embryos at different stages were treated with 2 mol per L of MeOH at -5C for 60 min, 16-somite stage embryos showed highest survival, followed by 4-somite, neurula, 50 percent epiboly, blastula, 32-cell and 8 to 16-cell embryos. These results demonstrated that PG had the lowest toxicity to medaka embryos among the six permeable CPAs at 26C, whereas MeOH showed highest cryoprotective efficiency under chilling conditions and chilling injury decreased gradually with the development of medaka embryos.     

Cryopreservation of umbilical cord blood-derived mesenchymal stem cells without dimethyl sulfoxide

Cryopreservation of umbilical cord blood-derived mesenchymal stem cells (UCB-derived MSCs) is crucial step for its clinical applications in cell transplantation therapy. In the cryopreservation of MSCs, dimethyl sulfoxide has been widely used as a cryoprotectant (CPA). However, it has been proved that DMSO has toxic side effects to human body. In this study, DMSO-free CPA solutions which contained ethylene glycol (EG), 1, 2-propylene glycol (PG) and sucrose as basic CPAs, supplemented with polyvinyl alcohol (PVA) as an additive, were developed for the cryopreservation of UCB-derived MSCs. The cryopreservation of UCB-derived MSCs was achieved by vitrification via plunging into liquid nitrogen and by programmed freezing via an optical-DSC system respectively. The viability of thawed UCB-derived MSCs was tested by trypan blue exclusion assay. Results showed that the viability of thawed UCB-derived MSCs was enhanced from 71.2% to 95.4% in the presence of PVA for vitrification, but only < 10% to 45% of viability was found for programmed freezing. These results indicate that PVA exerts a beneficial effect on the cryopreservation of UCB-derived MSCs and suggest the vitrification in combination with the dimethyl sulfoxide free CPA solutions supplemented with PVA would be an efficient protocol for the cryopreservation of UCB-derived MSCs.     

Zebrafish embryos (Danio rerio) using microinjection

Low membrane permeability is one of the major obstacles to the successful cryopreservation of zebrafish embryos. The aim of the present study was to explore if this could be overcome by yolk modification with different cryoprotectants by micro-injection. Initial investigation of two cryoprotectants, methanol and sucrose, was undertaken to determine their suitability for micro-injection supplementation of the yolk mass. Intact zebrafish embryos at 50% epiboly stage were injected with Hanks' solution, 5.2 M methanol or 1.3 M sucrose yielding approximate final concentrations of 2.0 and 0.5 M of the cryoprotectants within the yolk sac respectively. After micro-manipulation, the embryos were cultured at 28 degree C for three days and their survival assessed at the hatching stage. All micro-manipulations performed in the present study resulted in a significant decrease in embryo survival (P < 0.05). Embryos micro-injected with methanol or sucrose were also subjected to a cooling procedure. They were placed in 3M methanol + 0.5 M sucrose at room temperature for 30 min and then cooled from 20 degree C to 0 degree C at 2 degree/min, from 0 degree C to -7.5 degree/min at 1 degree/min, seeded at -7.5 degree C and held for 10 min, before cooling at 0.3 degree/min to - 20 degree C or until full crystallization in all embryos. The processes of extra- and intracellular crystallization were studied by cryomicroscopy. The temperature of intracellular crystallization did not differ significantly between control and injected embryos. However, it was found that intracellular crystallization did not always happen instantly after extracellular crystallization.     

Comparison of three techniques for cryopreservation and reestablishment of long-term Gentiana tibetica suspension culture

Cryogenic storage of cell suspensions allows long-term maintenance of cultures. The main purpose of the study was to develop a successful cryogenic protocol for 10-year-old embryogenic cell suspensions of G. tibetica. We examined three techniques of freezing: (I) controlled-rate cooling with various cryoprotectants (0.1-0.5 M DMSO, 0.5-1.0 M sucrose, 0.5-1.0 M glycerol, 0.25-1.0 M proline) or preculture with 0.4 M sorbitol and cryoprotectants (0.065-0.1 M DMSO, 0.2-0.8 M proline), (II) vitrification (PVS2) and (III) encapsulation. Cell viability was assessed by the TTC test and biomass increase. After controlled-rate cooling the majority of cells were lethally damaged, with only 3% viability observed. Vitrification and encapsulation approaches were more effective, assuring high levels of post-thaw viability ca. 85% and 7%, respectively. The encapsulation procedure gave faster recovery of the culture suspension than did vitrification, and ensured culture homogeneity and embryogenic competence.     

Studies on cryopreservation of luc gene transfected bluegill sunfish fibroblast cell line

The effect of cryopreservation on the survival of luc gene transfected bluegill sunfish fibroblast (BF-2) cells was investigated. Propane-1,2-diol was found to be the least toxic cryoprotectant when compared with DMSO and methanol. Both propane-1,2-diol and DMSO are effective in protecting cells from freezing damage. Whilst there were no significant differences in cell survival between cryoprotectant concentration (10 or 15%) and culture age used in this study, 7-day old culture appeared to be more resistant to freezing without cryoprotectant when compared with 3- and 14-day old culture. The highest cell survival values obtained with propane-1,2-diol (10%) and DMSO (10%) protection were 96.2 1.2% and 94.0 3.1% respectively. Initial subsequent cell growth after cryopreservation was slower than their non-frozen controls. The survival of transfected BF-2 cells (BF-2/luc1) after cryopreservation were very similar to those obtained with wild type cells being: 94.0 3.1% and 95.2 1.5% respectively with 10% DMSO protection. These results suggested that genetically modified fish cell lines may be equally amenable to cryopreservation as the wild type.     

Studies on the toxicity of dimethyl sulfoxide, ethylene glycol, methanol and glycerol to loach (Misgurnus fossilis) sperm and the effect on subsequent embryo development

The process of sperm cryopreservation consists of several steps: equilibration of sperm in cryoprotectant medium, freezing of sperm to subzero temperatures, low temperature storage and thawing of the sperm suspension. It has been shown that cryopreservation can cause some damage to the genetic material of cells although the mechanism and significance of these changes are still unknown. The aim of this work was to study the effect of cryoprotectant equilibration process on genetic damage of Loach (Misgurnus fossilis) sperm, using embryo survival as an indicator. Decrease in embryo survival after the 20th stage is generally believed to result from the failure in the genome function of embryos. In the first set of the experiments, Loach sperm were equilibrated in cryoprotectants Me2SO, ethylene glycol, methanol and glycerol (0.6, 1.2, 2.5 M) for 60 min at 10 degree C. The effect of cryoprotectant equilibration on sperm was evaluated based on the survival of embryos derived from cryoprotectant treated sperm. Embryo survival was evaluated at the following stages: 7th, 14th, 17th, 20th, 23rd, 26th, 31st, 34th, 35th, 36th and 37th. Cryoprotectants at concentrations greater than 1.2 M had significant effect on the survival of the embryos after the 20th stage. The effect of glycerol was the most significant with 64.8 +/- 2.4% of embryos survival compared to 77.0 +/- 2.4% for control. Me2SO treatment also effects embryo survival significantly. Possible mechanisms of the genetic instability of cryoprotectants are discussed.     

Cryopreservation of adult bovine testicular tissue for spermatogonia enrichment

To develop a procedure for cryopreservation of adult bovine testis tissue, the effects of dimethyl sulphoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), and their concentrations (v/v), as well as different thawing temperatures, on the cell viability of bovine testis tissue after freezing/thawing were examined. The highest testicular cell viabilities came from the media containing DMSO (85.3 ± 1.2 percent), PG (82 ± 1.0 percent) and EG (83.4 ± 1.0 percent) at 10 percent concentration respectively. Using 10 percent DMSO gave significantly higher spermatogonia percentage (61.1 ± 1.2 percent, P < 0.001) than processing with 10 percent PG (54.3 ± 0.6 percent) or 10 percent EG (55 ± 1.8 percent) after differential plating. Thawing in water bath of 37 or 97-100 degree C also provided significantly higher viabilities (85.1 ± 1.0, 85 ± 1.0 percent, P < 0.01, respectively) and spermatogonia percentages (56.6 ± 2.0, 56.6 ± 2.6 percent, P < 0.01, respectively) than that thawing at 4C (23.4 ± 0.8 percent for total viability, 8.97 ± 1.0 percent for spermatogonia percentage). Collectively, 10 percent DMSO and thawing in 37-100 degree C water baths were appropriate for the cryopreservation of bovine testicular tissue and subsequent spermatogonia enrichment.     

Cryopreservation of human ovarian tissue by direct plunging into liquid nitrogen: negative effect of disaccharides in vitrification solution

Given that it has been possible to successfully cryopreserve human ovarian tissue by direct plunging into liquid nitrogen, this study was designed to establish the future direction to be taken in this line of research. Bovine oviductal epithelial fragments (as a tissue model) and large biopsy fragments (approximately 2.0 cubic mm) of human ovarian tissue were used for cryopreservation. Two protocols were tested: with permeable cryoprotectants (dimethyl sulphoxide, propylene glycol, glycerol) + egg yolk + sucrose or trehalose + a synthetic blocker of ice nucleation, Supercool X-1000; and using only permeable cryoprotectants (glycerol and ethylene glycol) + egg yolk + Supercool X-1000. The cryopreserved tissue specimens were subsequently thawed and the cryoprotectants removed by dilution in graded sucrose solutions. Both the dynamic growth and hormonal activity of the ovarian tissue pieces, vitrified using only permeable cryoprotectants, were greater than after vitrification in a mixture of permeable cryoprotectants and sucrose. Unlike the case for other reproductive tissue (spermatozoa, oocytes, embryos), these findings suggest that the cryopreservation of ovarian tissue by direct plunging into liquid nitrogen must be achieved by vitrification using only permeable cryoprotectants and agents that prevention ice formation.     

Implementation of garlic cryopreservation techniques in the national plant germplasm system

The USDA-ARS National Plant Germplasm System (NPGS) maintains more than 200 Allium sativum (garlic) accessions at the Western Regional Plant Introduction Station in Pullman, WA. All accessions must be grown out in the field annually since garlic plants from these accessions do not reliably produce seeds and bulbs do not store well. Shoot tips excised from garlic cloves can be successfully cryopreserved using either Plant Vitrification Solution 2 (PVS2; 15 percent v/v DMSO, 15 percent v/v ethylene glycol, 30 percent v/w glycerol, 0.4 M sucrose) or Plant Vitrification Solution 3 (PVS3; 50 percent v/w sucrose, 50 percent v/w glycerol). We compared regrowth of shoot tips representing diverse garlic germplasm after exposure to either PVS2 or PVS3 during the cryopreservation procedure. At the USDA-ARS National Center for Genetic Resources Preservation, a component of the NPGS, we consider accessions successfully preserved if a minimum of 40 percent of explants exhibit regrowth after liquid nitrogen exposure and at least 60 viable shoot tips remain in long-term storage. Ten of twelve diverse garlic accessions were successfully cryopreserved using either PVS2 or PVS3 as cryoprotectants. Five genotypes had the best post liquid nitrogen regrowth after exposure to PVS2, four genotypes had the best regrowth after exposure to PVS3, and three genotypes performed equally well using either cryoprotectant solution. This project is part of an ongoing program to cryopreserve accessions of NPGS clonal crop collections.     

Cryopreservation of spermatozoa of black marlin, Makaira indica (Teleostei: Istiophoridae)

As a first step towards the development of a method for the cryopreservation of black marlin spermatozoa, this study investigated the effect of dimethylsulfoxide (DMSO) concentration and pellet size on post-thaw spermatozoal motility. Spermatozoa were recovered from the spermatic duct of testes retrieved post-mortem from four adult black marlin caught in the Coral Sea spawning grounds of Australia. Undiluted spermatozoa were stored on ice for 4 to 10 hours during transport to shore, then evaluated for motility after activation in seawater (1:10 v:v). Spermatozoa were prepared for cryopreservation in pellets by extension (1:3 v:v) in a defined fish Ringer's solution to give two final DMSO concentrations of 2.5% or 5.0%. Diluted spermatozoa were frozen directly on a dry ice block in pellet sizes of either 0.25 ml or 0.50 ml. Frozen pellets were thawed in a water bath at 40 degrees C for 60 seconds and assessed for post-thaw motility following activation in seawater. Spermatozoa recovered within 50 minutes of death and chilled on ice for 4 to 10 hours showed a mean (+/- SEM) motility immediately following activation of 91.6 +/- 7.9%. 50% of the spermatozoa remained motile for approximately 4 to 5 minutes. Following cryopreservation, mean motility declined significantly across all cryoprotectant and pellet size combinations (P < 0.001) but spermatozoa frozen in 2.5% DMSO showed higher motility than those frozen in 5.0% DMSO (P = 0.014). Pellet size had no effect on post-thaw motility (P = 0.179).     

Influence of exposure to vitrification solutions on 2-cell mouse embryos: I. Intracellular potassium and sodium content

Intracellular concentrations of potassium and sodium in two-cell mouse embryos in G1/S phase after exposure to vitrification solutions containing vitrificant agents (VFAs) ethane-1,2-diol (ethylene glycol, EG), propane-1,2-diol (propylene glycol, PG), or dimethyl sulfoxide (DMSO), and sucrose (S) after incubation in Dulbecco solution were measured by electron probe microanalysis (EPMA) as described earlier (CryoLetters, 2006, 27: 87-98). The 4-step protocol was as followed: 10% VFA for 10 min => 30% VFA + 0.7 M S for 1.5 min ==> 0.5 M S for 10 min ==> 10 min pure Dulbecco's. The cytoplasmic concentration of potassium and sodium in immediately flashed out from the oviduct embryos was in range of 120 +/- 2 mM, with good concordance with the previous data (CryoLetters, 2006, 27:87-98). Exposure in Dulbecco's for 30 min did not alter elemental composition, neither did exposure in PG or DMSO for 1.5 min. In contrast, exposure for 1.5 min in EG dropped the level of potassium to 96 +/- 2 mM while elevating level of cytoplasmic sodium to 136 +/- 3 mM. Further exposure to 30%-EG for 3 min led to a two-fold decrease of both elements (60 +/- 3 mM and 66 +/- 2 mM for K and Na, respectively).     

Cryopreservation of Centaurea ultreiae (Compositae) a critically endangered species from Galicia (Spain)

Ex situ conservation of endangered plants is an important aim in order to preserve biodiversity of flora in threatened ecosystems. Among the biotechnological techniques which can be used, cryopreservation is emerging as a preferred option in many instances. This study describes a cryopreservation technique developed for shoot tips of the endangered species Centaurea ultreiae (Compositae) using a vitrification procedure. Basal medium (BM) for preculture and loading phases consisted of 1/2 MS basal salts with modified vitamins (3 microM thiamine). For preculturing shoot tips, BM with five osmotic treatments were investigated: 0.3 M sucrose +/- 20 microM ABA, 0.6 M glycerol +/- 20 microM ABA and 0.25 M sucrose + 0.25 M glycerol + 10 microM ABA. A loading solution treatment (BM with 2 M glycerol and 0.4 M sucrose) was applied prior to exposure of shoot tips to PVS2 and found to be indispensable to obtaining successful post-LN recovery. Highest (95.5%) regrowth of LN immersed shoot tips was obtained following incubation on BM + 0.3 M sucrose + 20 microM ABA or 0.25 M sucrose + 0.25 M glycerol + 10 microM ABA, with loading treatment and PVS2 exposure for 20 minutes at 0 degree C. Keywords: cryopreservation, encapsulation, endangered species, ex situ conservation, vitrification.