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Introduction: To assist save endangered species from extinction and to aid in their care, it is crucial to sustaining their reproductive capacity. The fetus's life and growth processes begin at conception and proceed in accordance with a biological clock's timing. With today's understanding through cryobiology, observing scientific ethics problems and interfering with the clock's operation by stopping its biological time is possible. Practically, it is accomplished by storing the cell at -196 °C, or the temperature of liquid nitrogen, where all metabolic activity ceases. Since 200 years ago, germ cell preservation has been a common practice. Since then, there have been many improvements, particularly for at-risk women. Since then, significant progress has been achieved, and several freezing techniques are being used to preserve ovarian tissue, follicles, and oocytes in women who are at risk of infertility. Different approaches have different levels of success. Among preservation techniques, vitrification performs better and is used more frequently. The cellular configuration of the mammalian oocyte is intricate. This cell's constituent parts are particularly sensitive to osmosis and variations in temperature. For instance, the alterations to the cell membrane that occur during maturation, in vitro fertilization, and the differential between the permeability of water and cryoprotectants can all be mentioned. Oocyte freezing results in a variety of impairments, including a reduction in the quality and viability of cells after thawing. Conclusion:Recent studies are looking for ways to enhance freezing procedures and raise the caliber of frozen oocytes. The favorable or negative effects of freezing on the oocyte and its potential, or embryo development, are the subject of this review article.

Context: Although vitrification is commonly used for oocyte cryopreservation, the cryogenic damage results in poor developmental capacity of oocytes after freezing. Nano-cryopreservation is one of the new methods of vitrification developed in recent years. However, the effect of nano-cryopreservation on mature bovine oocytes remains to be elucidated. Aims: This study aimed to verify the effect of using hydroxyapatite (HA) nanoparticles (NPs) on the vitrification of bovine metaphase II (MII)-stage oocytes. Methods: Bovine MII-stage oocytes were exposed to different HA concentrations (0.01%, 0.05%, and 0.10%) in vitrification solution (VS). After IVF (in vitro fertilisation) and IVC (in vitro culture), the toxicity of HA was assessed by cleavage and blastocyst rates. A suitable concentration of HA nanoparticles was selected according to the results of the first experiment. and then vitrification-thawing was measured. The effect of HA on the developmental capacity of oocytes was assessed by oocyte cleavage rate and blastocyst rate. The mitochondrial membrane potential (MMP) and the intracellular reactive oxygen levels (ROS) of oocytes were measured by staining with a fluorescence probe (JC-1) and an ROS kit after nano-cryopreservation. Key results: The addition of 0.05% HA to the VS did not affect the oocyte morphology; the proportion of oocytes developing with normal morphology was 96.72%. In contrast, this proportion it significantly decreased at a concentration of 0.1% HA in VS (91.69%; P < 0.05). The cleavage rates (56.95% vs 51.20%, 50.67%; P < 0.05) of bovine oocytes exposed to 0.05% HA were significantly higher than in the VS group and VS + 0.1% HA group. After the vitrification-thawing, the oocyte cleavage rates (41.07% vs 33.97%; P < 0.05) and blastocyst rates (12.35% vs 7.38%; P < 0.05) were significantly higher in the VS + 0.05% HA group compared to the VS group. At the 0.05% HA concentration, nano-cryopreserved oocytes had significantly higher MMP (1.35 ± 0.24) and significantly lower ROS (1.43 ± 0.05) than the VS group (P < 0.05, mean ± s.e.m.). Conclusions: The addition of 0.05% HA-NPs in VS could promote bovine MII-stage oocytes' developmental ability after vitrification. This effect may be caused partly by increased mitochondrial membrane activity and decreased ROS. Implications: HA-NPs may be a new class of cryoprotective agent suitable as components for oocyte vitrification. Nanotechnology is of great value in the field of medical biology, but the intersection of nanotechnology and cryogenic bioengineering has not been studied enough. HA nanoparticles were added to the cryoprotectant of bovine oocytes in our study. We found that a certain concentration of nanoparticles had a positive effect on vitrification and postfreezing development capacity. Nano-vitrification of oocytes deserves further exploration in both theory and practice. [ABSTRACT FROM AUTHOR]

Context: Live birth rates are lower for cryopreserved oocytes than for fresh IVF cycles, indicating a need for improved methodologies. Aims: The aim of this study was to determine if high pressure freezing (HPF) could improve both ultrastructural preservation and cryopreserved oocyte quality when compared to conventional fixation and vitrification methods. Methods: Sheep oocytes and embryos were prepared by HPF or vitrification, with or without cryoprotectants. Frozen oocytes were prepared for transmission electron microscopy or warmed, in vitro fertilised and the recovery and cleavage rates recorded. Key results: Blastocyst rates were similar between fresh, HPF and vitrified embryos. HPF oocytes had improved ultrastructure compared to conventional fixation or vitrification, but had poorer survival and cleavage rates compared to vitrified oocytes. Freeze-substitution of cryopreserved oocytes and transmission electron microscopy demonstrated disruption of the oocyte ultrastructure in the presence of cryoprotectants. Conclusions: Superior preservation of ultrastructure was observed in HPF oocytes compared to vitrification or conventional fixation methods. In the presence of CP, both embryos and oocytes could survive HPF and warming but oocytes had reduced development. Implications: The HPF method has potential to be developed and lead to improved oocyte and embryo cryopreservation and outcomes for assisted reproduction. While freezing oocytes (eggs) for use in human fertility treatments is becoming more common, pregnancy and live birth rates are lower for frozen oocytes than fresh ones. We have shown that sheep oocytes frozen with a high pressure freezing method survive and have improved structural preservation than conventional vitrification and fixation methods. This high pressure freezing method may be further developed to improve preservation of frozen oocytes and subsequent live births. [ABSTRACT FROM AUTHOR]

Context: The vitrification of oocytes is important for the conservation of animals, and the effect of vitrification on methylation patterns of bovine oocytes remains unclear. Aims: This article aims to investigate the effect of vitrification on the DNA methylation patterns on vitrified GV oocytes and their in vitro derived MII oocytes. Methods: 5-MeC staining and single-cell whole genome bisulphite sequencing (SC-WGBS) were utilised to analyse fresh GV oocytes (F_GV group), MII oocytes (F_MII group), vitrified GV oocytes (V_GV group) and their in vitro derived MII oocytes (V_MII group). Key results: Results of both 5-MeC staining and SC-WGBS showed that no significant difference was found between the F_GV group and the V_GV group, while the methylation level of the V_MII group was significantly lower than that of the F_MII group. Moreover, supplementation of 2 μM resveratrol (Res) in IVM medium significantly improved maturation and development ability of vitrified GV oocytes by restoring their DNA methylation levels. Conclusion: In conclusion, vitrification of bovine GV oocytes significantly decreased the DNA methylation level of their in vitro derived MII oocytes, and 2 μM Res improved their development ability by restoring DNA methylation level. Implications: Our results provide an efficient approach to improve the maturation and fertilisation ability of vitrified GV oocytes. 5-MeC and SC-WGBS were utilised to detect the DNA methylation patterns of vitrified bovine GV oocytes and their in vitro derived MII oocytes. Results showed that the methylation level of the V_GV group was similar to that of the F_GV group, while the methylation level of the V_MII group was significantly lower than that of the F_MII group. Moreover, supplementation of 2 μM Res in IVM medium significantly improved maturation and development ability of vitrified GV oocytes by restoring their DNA methylation levels. [ABSTRACT FROM AUTHOR]

Background Cell banking of initial outgrowths from newly derived human embryonic stem cells (hESCs) requires an efficient freezing method. Vitrification is used for the preservation of gametes and early embryos in assisted reproduction techniques (ART). Moreover, vitrification was applied for cryopreservation of hESCs using open pulled straws. Objective To derive and characterize new hESC lines and then use Cryotech and Cryowin tools for their vitrification. Materials and Methods Human ESC lines were generated in a microdrop culture system using mouse embryonic fibroblasts (MEFs) as the feeder layer; this was later scaled up using both MEFs and Yazd human foreskin fibroblasts batch 8 (YhFF#8). To bank the cell lines, master cell banks of 100 Cryotech and Cryowin tools were produced for each individual cell line using the vitrification method; flasks of hESC lines were also cryopreserved using a conventional slow-freezing method. Results The pluripotency of cell lines was assessed by their expression of pluripotency-associated genes (OCT4/POU5F1, NANOG, and SOX2) and markers such as SSEA4, TRA-1-60, and TRA-2-49. Their in vitro capacity to differentiate into germ layers and germ cells using embryoid body (EB) formation and monolayer culture was assessed by screening the expression of differentiation-associated genes. The chromosomal constitution of each hESC line was assessed by G-banding karyotyping. Conclusion Cryotech and Cryowin tools used to vitrify new hESCs at an early stage of derivation is an efficient method of preserving hESCs.

Conjugated linoleic acid (CLA) might be able to improve the cryotolerance of in vitro-produced (IVP) embryos. The effect of two CLA isomers on the cryotolerance of bovine IVP embryos, as well as that of the stage of embryonic development and the method used for cryopreservation was evaluated by three experiments. In Experiment 1, oocytes (n = 3,917) were fertilized in vitro and cultured with 0, 50, 100, or 200 ?M trans-10, cis-12 (t10, c12 CLA). In Experiment 2, fertilized oocytes (n = 2,131) were cultured with 100 ?M t10, c12 or cis-9, trans-11 (c9, t11 CLA), or a combination of both isomers. The embryos were vitrified at the blastocyst (BL) or the expanded blastocyst (EB) stage. In Experiment 3, oocytes (n = 1,720) were fertilized and cultured with or without 100 ?M t10, c12 CLA, and the blastocysts were vitrified or frozen. Blastocyst development rate as well as the rates of re-expansion and hatching after thawing was recorded. Moreover, the mean cell number and mRNA expression of acetyl-CoA carboxylase (ACC1) and stearoyl-CoA desaturase (SCD1) as well as fatty acid synthase (FASN) multienzyme complex were determined. In Experiment 1, the highest concentration of t10, c12 CLA that did not reduce blastocyst development rate was 100 ?M. In Experiment 2, the rates of re-expansion and hatching among the EBs obtained through IVP after supplementation with t10, c12 CLA (73.1% and 57.7%), with c9, t11 CLA (80.0% and 68.6%), with the combination (78.3% and 52.2%), and with the control group (85.4% and 58.3%) were similar. At the BL stage, the rates of re-expansion and hatching were lower than those at the EB stage, and CLA combination allowed a hatching rate (8.0%) lower than that observed in the control group (40.0%). In Experiment 3, the hatching rates for vitrified EBs (vitrified control; 67.4%) and vitrified CLA EBs (65.8%) were higher than those obtained for frozen EBs, exposed (13.3%) or not exposed (28.6%) to CLA. In addition, in Experiment 3, the hatching rate was higher at the EB stage in vitrified groups, while the rates of BL and EB were similar in frozen groups, thus proving that vitrification was more efficient than freezing for IVP bovine embryos. In Experiment 3, CLA isomer t10, C12 did not influence the embryonic cell number or mRNA expression of ACC1 and SCD1 enzymes, but decreased the mRNA expression of FASN. In conclusion, 100 ?M CLA did not affect subsequent embryonic development. However, neither CLA isomer improved the cryotolerance of IVP bovine embryos.

The effects of acetyl-l-carnitine (ALC) supplementation during IVM on subsequently vitrified buffalo oocytes were evaluated, followed by determination of the mitochondrial DNA copy number, measurement of mitochondrial membrane potential (MMP) and identification of the lipid profile of oocyte membranes as markers of oocyte quality after vitrification. Supplementation with ALC during IVM significantly improved the rates of oocyte cleavage and morula and blastocyst formation, and increased MMP after vitrification compared with unsupplemented vitrified oocytes (P < 0.05). Using a bidirectional orthogonal projection to latent structures discriminant analysis based on positive ion matrix-assisted laser desorption ionisation time-of-flight mass spectrometry data, five phospholipid ions (m/z 728.7 (phosphatidylcholine (PC) 32:3), 746.9 (PC 32:5), 760.6 (PC 34:1), 768.8 (PC P-36:3) and 782.6 (PC 36:4); P < 0.05) were identified as significantly more abundant in fresh oocytes than in unsupplemented vitrified oocytes. Meanwhile, three phospholipid ions (m/z 734.6 (PC 32:0), 760.6 (PC 34:1), and 782.6 (PC 36:4); P < 0.05) were more abundant in ALC-supplemented vitrified oocytes than in unsupplemented vitrified oocytes. Therefore, supplementation with ALC during IVM may improve buffalo oocyte quality after vitrification by enhancing mitochondrial function and altering the phospholipid composition of vitrified oocyte membranes. This is the first report that acetyl-l-carnitine (ALC) supplementation during IVM affects phospholipids profiles of buffalo oocytes after vitrification. Supplementation with ALC has beneficial effects on mitochondrial function and alters the phospholipid composition of vitrified oocyte membranes. ALC improves the quality of mature buffalo oocytes and increases the success of embryo development after vitrification. [ABSTRACT FROM AUTHOR]

Cryopreservation of gametes and embryos has played a critical role in successful assisted reproductive technologies in rodents, domestic farm species, endangered species and humans. With improved success, and changing needs, the utility of gamete or embryo cryopreservation has escalated. In this review we address some of the foundational history of mammalian cryobiology, species-specific utilities, fundamental understandings of cryoprotectant agents and their use in slow-rate freezing and vitrification, and expand on the recent success and uses of oocyte vitrification and warming. In the area of female gamete cryopreservation, emphasis will be placed on not just cell survival, but also perceived and measured affects of cryopreservation on intracellular structures and functions that affect subsequent completion of meiosis with chromatin segregation fidelity, normal fertilisation and embryonic developmental competence. We compare and contrast data from cow, mouse and humans with a focus on using species-comparative developmental biology to guide future studies for improving methodologies for all species. The application of the relatively new technology microfluidics is discussed in relation to moving gradually (i.e. changing the solution over cells in an automated fashion) compared with the stepwise manual movement of cells through changing solution currently used. This use of microfluidics to change the way cells are exposed to cryoprotectant agents can provide new insights into the effects of osmotic stress and cellular strain rates previously unappreciated, precise methods of computational and biological data acquisition and appreciation of morphometric changes to cellular structure in response to different osmotic stresses and strain rates achieved with varying cryoprotectant exposures. Collectively, these devices and methodologies provide a means of achieving incremental improvement of oocyte and zygote cryopreservation with normalised and improved developmental competence. Finally, we look to the past and the future to acknowledge the accomplishment of leaders in the field of mammalian gamete and embryo cryobiology, their inspirational works, their tireless dissemination of information and the potential of new technologies in bioengineering to improve the efficiency and safety of gamete and embryo cryopreservation. [ABSTRACT FROM AUTHOR]

An alarming proportion of Australia's unique plant biodiversity is under siege from a variety of environmental threats. Options for in situ conservation are becoming increasingly compromised as encroaching land use, climate change and introduced diseases are highly likely to erode sanctuaries regardless of best intentions. Ex situ conservation is currently limited to botanic garden living collections and seed banking, with in vitro and cryopreservation technologies still being developed to address ex situ conservation of species not amenable to conventional storage. Cryopreservation (storage in liquid nitrogen) has been used successfully for long-term biosecure storage of shoot tips of several species of threatened Australian plants. We present a case for building on this research and fostering further development and utilisation of cryopreservation as the best means of capturing critical germplasm collections of Australian species with special storage requirements (e.g. recalcitrant-seeded taxa and species with short-lived seeds) that currently cannot be preserved effectively by other means. This review highlights the major issues in cryopreservation that can limit survival including ice crystal damage and desiccation, toxicity of cryoprotective agents, membrane damage, oxidative stress and mitochondrial function. Progress in understanding and mitigating these stresses is vital for advancing cryopreservation for conservation purposes. Cryopreservation is currently the only viable alternative for long-term biosecure germplasm storage of species that cannot be seed-banked effectively. A wide range of plant tissues and organs can be cryopreserved. However, there is wide variation in cryo-capability among species, progress has been made in developing a more holistic understanding of the stresses that cryopreservation imposes on plant germplasm, and in developing novel cryopreservation protocols to counteract them. [ABSTRACT FROM AUTHOR]

The objectives of the current study were to evaluate the effects of supplemental nano-selenium (NSe) and nano-zinc oxide (NZn-O) particles during in vitro maturation (IVM) on DNA damage of cumulus cells, glutathione (GSH) concentration in bovine oocytes, subsequent embryo development and re-expansion rate of vitrified warmed blastocysts. The current study was conducted on bovine ovaries obtained from a local abattoir and transported to the laboratory in sterile phosphate buffer saline with antibiotics at 37°C, within 1 h after slaughter. Ovaries were pooled, regardless of stage of the oestrous cycle of the donor. Only cumulus-intact complexes with evenly granulated cytoplasm were selected for IVM. Experimental design included the following: Experiment 1 studied the effect of addition of 1.0 µg/mL NSe or NZn-O to IVM medium on DNA damage of cumulus cells; Experiment 2 evaluated the effects of NSe or NZn-O on intracellular glutathione in oocytes and cumulus cells; in Experiment 3, the development of oocytes matured in IVM medium supplemented with 1.0 µg/mL NSe or NZn-O was investigated; and in Experiment 4, the effects of adding 1.0 µg/mL NSe and NZn-O to in vitro fertilisation media on vitrified oocytes and embryos were investigated. The DNA damage in cumulus cells decreased with supplemental NSe and NZn-O at concentration of 1 µg/mL in the IVM medium (180.2 ± 21.4, 55.8 ± 4.3 and 56.6 ± 3.9 for the control and NSe and NZn-O groups respectively). Total GSH concentrations increased following supplementation with 1 µg/mL NSe and 1 µg/mL NZn-O, compared with the control group. Re-expansion rate of vitrified warmed blastocysts in experimental media containing NSe and NZn-O with ethylene glycol was higher than that of the control. In conclusion, providing NSe and NZn-O during oocyte maturation significantly increased both intracellular GSH concentration and DNA integrity of cumulus cells. Optimal embryo development was partially dependent on the presence of NSe and NZn-O during IVM. NSe and NZn-O during oocyte maturation act as a good cryoprotective agents of vitrified, warmed blastocysts. The present study evaluated the effects of supplemental nano-selenium (NSe) and nano-zinc oxide (NZn-O) particles during in vitro maturation (IVM) on DNA damage of cumulus cells, glutathione concentration in bovine oocytes, subsequent embryo development and the re-expansion rate of vitrified warmed blastocysts. Optimal embryo development was partially dependent on the presence of NSe and NZn-O during IVM. NSe and NZn-O during oocyte maturation act as good cryoprotective agents of vitrified, warmed blastocysts. [ABSTRACT FROM AUTHOR]

The method of cryovacuum condensation of thin gas films and, in particular, condensation involving the formation of glassy states, provides ample opportunities for precise control and maintenance of the formation conditions of studied samples. This has prompted researchers to formulate and address the question of the relationship between the formation conditions/structure of molecules and the degree of kinetic stability of cryocondensates, including organic glasses. This study focused on thin films of Freon 134a cryovacuum condensates condensed on a cooled metal substrate comprised of gas at deposition temperatures ranging from 16–100 K and gas phase pressures from 10–4 to 10–6 Torr. A comparison between the vibrational spectra of Freon 134a in the gas phase and in the cryocondensed state is provided. The results of IR spectrometric studies of cryovacuum condensates of Freon 134a (2.5 μm thick) in the frequency range 400–4200 cm–1 are presented. Based on the obtained spectra and data on their thermally stimulated transformations, an assumption is made that, in the temperature range 16–60 K, Freon 134a cryocondensates are in an amorphous state with different degrees of amorphization. At the vitrification temperature of 70 K, transition from the amorphous glassy state to the state of the supercooled liquid phase takes place, followed by its crystallization into the state of a plastic crystal. In the temperature range of 78–80 K, the transition of a plastic crystal to a crystalline state with a monoclinic lattice begins. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate isolated bovine preantral follicles after vitrification procedure. Ovaries were obtained at a local slaughterhouse and submitted to follicular isolation. From the obtained suspension, one part was destined to control group. The remaining suspension was divided into two parts, one to the toxicity test and the other one to the vitrification procedure. For vitrification, the follicles were exposed for 1 min to vitrification solution-1, composed of 10% ethylene glycol, 10% DMSO in saline solution (NaCl 0.9%), and then 1 min to vitrification solution-2, composed of 20% ethylene glycol, 20% DMSO in the same saline solution supplemented with 0.5 M sucrose. The follicles were vitrified by use of glass capillaries and remained vitrified for 24 h. After warming, they were transferred to a 0.25 M sucrose solution for 5 min and then transferred to a 0.15 M sucrose solution for another 5 min. For the toxicity test, the follicles were only exposed to cryoprotectants and analised. The preantral follicles were classified as non-viable orviable when they were stained or not stained by trypan blue, respectively. Differences (P0.05) from that found after vitrification, indicating that the reduction of percentage of viable preantral follicles was due to a toxic effect of cryoprotectants and not to the vitrification procedure itself.

This study was conducted to produce buffalo embryos in vitro from stepwise vitrified immature oocytes. Cumulus oocyte complexes (COCs) were obtained from the ovaries of slaughtered buffalo and were collected from the local abattoir. Selected COCs were exposed to a vitrification solution consisting of 40% ethylene glycol (EG) plus 0.3 M trehalose and 20% polyvinyl pyrrolidone (PVP) for 1 min and loaded in 0.25 ml plastic mini-straws containing 100 µl of 10% sucrose. The loaded cryostraws were cryopreserved by stepwise vitrification and were stored in liquid nitrogen for 4 to 6 months. Data analysis revealed a high percentage of post-thawing morphologically normal immature oocytes (80.7%) with a low percentage of damaged oocytes. There were no significant differences in the maturation (82.1%), cleavage (47.6%) and buffalo embryo development (15.4%) produced by the stepwise vitrified immature oocytes in comparison to the three observations in fresh oocytes (88.3%, 50.4% and 19.4%, respectively, p

Background: Lysophosphatidic acid (LPA) contributes to follicular activation, oocyte maturation, in vitro fertilization, and embryo implantation., Objective: This study was designed to evaluate the effects of LPA to improve the development of isolated follicles derived from whole mouse cultured vitrified ovaries., Materials and Methods: In this experimental study, first, the 1-wk-old mouse ovaries in the non-vitrified and vitrified groups were cultured in the presence of 20 µM of LPA for 1 wk. Then, their isolated preantral follicles were cultured individually for 12 days in the presence or absence of 40 µM of LPA. The following evaluations were done for the cultured follicles: a viability test using Calcein AM staining, flow cytometry using annexin V/Pi, and analysis of the expression of genes by real-time reverse transcription polymerase chain reaction. The maturation rates of the oocytes were compared among groups and some of the released metaphase II oocytes were subjected to in vitro fertilization., Results: In all LPA treated groups, the rates of survival and follicular development were higher, and the incidence of cell death and expression of pro-apoptotic genes were lower, than in the non-LPA supplemented groups (p = 0.035). There was no significant difference between the vitrified and non-vitrified groups regarding follicular or oocyte development, but the expression of Bad and LPA receptors genes was significantly altered in the vitrified LPA supplemented group in comparison with the non-vitrified LPA supplemented group (p = 0.028)., Conclusion: LPA improved the survival and developmental potential of the isolated follicles. Despite some alterations in the expression of apoptosis-related genes in the vitrified ovaries, LPA had positive effects on the survival and development of these follicles., Competing Interests: The authors declare that there is no conflict of interest., (Copyright © 2022 Abedpour et al.)

Trehalose is an agent useful in maintaining the integrity of many biological systems submitted to various stresses. It is also presumed to improve specimen preparation for electron microscopy and to reduce beam damage. Here we study the effect of trehalose on the preparation and observation by cryo-electron microscopy of thin vitrified films of biological suspensions. We observe that trehalose, as compared to sucrose, can indeed reduce electron beam damage to biological particles, as determined from the dose necessary for the onset of bubbling. Surprisingly, we also find that the contrast of biological particles is higher in a vitrified solution of trehalose than in one of sucrose. This effect can be explained if the water evaporation during the specimen preparation is less in the presence of trehalose than with sucrose, but we do not yet understand the underlying reasons since the evaporation properties of both sugars are similar at a macroscopic level. We conclude that trehalose is truly a remarkable substance and that more investigation is needed in order to fully understand its properties, and that the addition of ca. 3–5% trehalose to biological suspensions is a simple and useful method to reduce commonly arising drying artefacts and water evaporation in the thin film vitrification method. [ABSTRACT FROM AUTHOR]

In tissue‐cultured Cynara scolymus L. (globe artichoke) vitrification (hyperhydric transformation) can only be overcome by increasing the concentration of agar. However, with increasing concentrations of Difco Bacto agar (8–15 g/I) the availability of labelled kinetin is decreased. There is some evidence for postulating that cytokinins under inductive conditions of low agar concentration or high matrix potential are evocators of vitrification. Both the brand and concentration of agar also affect the chemical and physical characteristics of a culture medium. Impurities introduced with the agar are responsible for significant differences in the concentration of an element in comparable media with different levels of agar, and are easily detected by conductance. Penetrometer measurements also show large ranges in solidity of media with increasing concentrations, not only within the type of agar, but also for the same concentration within different brands. [ABSTRACT FROM AUTHOR]

Plant cryobiology has primarily emerged from the classical fields of cryobiology and plant stress physiology. Cryopreservation tools are now available to geneticists for germplasm preservation and the field itself is advancing significantly through the use of molecular techniques. Long-term preservation of vegetatively propagated tissues can minimize the risks of long-term maintenance under tissue culture or field conditions. Cells can be successfully cryopreserved when the adverse affects of ice crystal formation are mitigated by the removal of water or procedures to limit ice formation and crystal growth. The addition of cryoprotectant solutions to hydrated cells may improve the survival of microdissected shoot tips or embryonic axes. Recent discoveries in the genetic pathways leading to cold acclimation and freezing tolerance suggest the involvement of key cold-regulated genes in the acquisition of cold tolerance in plant tissues. Model systems of banana and Arabidopsis have revealed the involvement of genes and proteins in the glycolytic and other metabolic pathways, particularly processes involved in dehydration tolerance, osmoprotection, and membrane transport. Furthermore, successful recovery appears to be dependent upon the presence of antioxidant protection from reactive oxygen species. Characterization of specific genes and proteins will lead to significant advances in plant cryobiology research.

Ovarian tissue banking is a feasible strategy for fertility preservation for young women after cancer treatments. Ovarian tissue, after thawing, is used for several options; orthotopic grafting (normal site), autologous heterotopic grafting and collection of ovarian follicles for culture. Recent reports of live birth encouraged clinicians and researchers to apply this technology to premature ovarian failure (POF) resulting from strong cancer therapy. Grafting, however, carries a risk of malignant cell recurrence. For safety, development of a culture method is necessary but optimum culturing conditions for less-developed follicles abundant in the ovary are not well known. In the present article, the current status of ovarian tissue cryopreservation, and in vitro oocyte growth and maturation from the preserved ovaries are reviewed.

Cryopreservation, in vitro maturation, fertilization and culture can be applied to various processes across a wide range of species, that is, for the breeding and reproduction of farm animals, preservation of genetic variants in laboratory animals, and the conservation of wild species. In particular, the storage of oocytes by cryopreservation and IVM following cryopreservation, might become effective alternative assisted reproduction treatments for infertile patients. For example, in a clinical context, these techniques might be important for patients who are at risk of losing their ovarian function because of extirpative therapy, chemotherapy or radiation. Thus, it is important for assisted reproductive technology to improve IVM and cryopreservation techniques. In the present review, we introduce our recent studies on vitrification and IVM of germinal vesicle stage oocytes in animals.