Conventional Freezing Research Articles

Optimal cutting temperature medium embedding and cryostat sectioning are valid for cardiac myofilament function assessment.

To maximize data obtainment from valuable cardiac tissue, we hypothesized that myocardium fixed in optimal cutting temperature (OCT) medium for histology could also be used to investigate the function of myofilament proteins in situ. We compared tissue prepared via conventional liquid nitrogen (LN) snap freezing with tissue fixed in OCT and then sectioned in fiber-parallel orientation. We found that actin-myosin Ca2+ sensitivity, activation rate by Ca2+, cooperativity along the thin filament, as well as cross-bridge cycling rate were unaffected by OCT storage and could reliably be interpreted after sectioning. Absolute values in maximum force generation per cross-sectional area, as well as passive strain, are difficult to investigate after sectioning, as myofibrillar continuity along the preparation cannot be guaranteed. We have shown that myocardial tissue stored in OCT and sectioned before analysis is available for functional analysis, a valuable means of maximizing usage of precious cardiac biopsies.NEW & NOTEWORTHY Myocardial tissue in optimal cutting temperature (OCT) fixation and cryostat sectioning was tested as a means of storing and preparing tissue for myofilament function analysis in relation to conventional liquid nitrogen freezing and dissection. Actomyosin interaction, Ca2+ force activation, and passive compliance were tested. The study concluded that OCT storage and cryostat sectioning do not interfere with the actomyosin cross-bridge dynamics or Ca2+ activation but that absolute tension values suffer and may not be investigated by this method.

The recent state of cryopreservation techniques for ex-situ gene conservation and breeding purposes in small ruminants: A review

The viewpoint of the recent cryopreservation techniques (CT) suggests the use of a reduced volume of cryopreservation solution, high concentration of cryoprotectants and ultra-rapid cooling and warming rates help to reduce cryo-injury and maximize the viability of the preserved animal genetic resources (AnGR). The CT had now become widely accepted as one of the best methods of choice for the ex-situ conservation of AnGR due to its high success rate recorded and no-invasive nature as compared to the conventional slow rate freezing (CSRF). Rapid advances and wide acceptability of the use of assisted reproductive technologies (ART’s) particularly artificial insemination (AI) in animal breeding had resulted in a greater loss of a large number of good quality genes in virtually almost all the native breeds of animals across the globe. Small ruminant (SR) animals are not an exception in such present predicaments situation of erosion and dilution of the valuable AnGR among the native breeds. As a result of this, 148 and 16 breeds of sheep and goats respectively have already become extinct in Europe and the Caucasus. In view of the aforementioned situation, the present review aimed at exploring some of the current states of development, roles played and potentials of CT in the conservation of SR genes and genome for the immediate and future breeding purposes for sustainable development. It basically covers; animal genetic resource, the need to conserve AnGR, tools for ex situ in vitro conservation of AnGR and recent developments in breeding and cryopreservation of SR AnGR.
 Cryopreservation is playing a pivotal role in ex-situ gene conservation of AnGR. Decline in genetic diversity among SR breed population was high in Europe and the Caucasus. There is therefore, need for improvent on current stringent measures on conservation of AnGR in this region of the world.

Cryopreservation of donkey embryos: Comparison of embryo survival rate after in vitro culture between conventional freezing and vitrification

Embryo cryopreservation ensures that genetic biodiversity is preserved over time. This study evaluates the survival of donkey embryos subjected to slow freezing and vitrification after thawing and in vitro culture. Seven-day-old in vivo produced donkey embryos were subjected to slow freezing (SF, N = 14) or vitrification (VIT, N = 22). After one year of cryopreservation, embryos were warmed, washed and placed in incubation for in vitro culture (IVC). In order to assess the embryo viability, the quality grade and developmental stage were recorded after thawing and after 24 and 48 h of IVC. Eleven embryos (SF = 4 and VIT = 7) were incubated under a time-lapse camera, for up to 68 h, in order to determine the area and growth. The survival rate was not influenced by the procedure but by the developmental stage: after 48 h of IVC blastocyst survival rate (1/8, 12.5%) was significantly lower compared to both morulas (8/12, 66.7%) and early blastocysts (11/16, 68.7%) (P < 0.05). Embryo diameter class at recovery did not significantly influence the survival rate. In terms of the embryos that were judged to be alive after 48 h of IVC, quality grade 1 was observed in 7/8 (88%) and 4/12 (33%) of the SF and VIT embryos, respectively (P < 0.05). After time-lapse analysis, the IVC embryo area as well as growth percentage were statistically higher in the SF than the VIT embryos (P < 0.05). In conclusion, no difference in survival rates was found between the two cryopreservation procedures, although embryo quality was more negatively affected by vitrification.

Microwave assisted freezing part 2: Impact of microwave energy and duty cycle on ice crystal size distribution

Among innovative freezing processes, electromagnetic wave assistance during freezing has received great attention in recent years. The literature provides interesting studies indicating that smaller ice crystals may be obtained during freezing with the assistance of microwaves. In the companion paper, we designed a laboratory scale prototype to perform experiments of this type in well controlled conditions at 2.45 GHz. In the current study, we performed freezing experiments using this prototype and developed a protocol to measure ice crystal sizes using X-ray micro-tomography. The methodology was first validated by measuring ice crystal distribution in unidirectional conventional freezing. Then several microwave assisted freezing modalities were studied. Experiments were conducted with pulsed and continuously applied microwaves. The impact of microwave irradiation time was studied at a constant incident microwave power, and then at constant energy supplied to the system. We observed a significant reduction of up to 25% in ice crystal size when microwave assistance was used during freezing with both pulsed and continuously applied microwaves. These observations tend to disconfirm the hypothesis in the literature according to which ice crystal size reduction is due to temperature oscillations. The only remaining hypothesis in the literature is that ice crystal size reduction may be due to the perturbation of the H-bond network in water, which could be a precursor to crystalline structure. It appeared that at constant incident microwave power, the increase in irradiation time tended to decrease ice crystal size. No significant difference in ice crystal size reduction was observed for a constant energy supplied to the system. The amount of energy supplied by microwaves seems to be a factor influencing ice crystal size.Industrial relevanceMicrowave assisted freezing is an emerging process which permits reducing ice crystal size, and thus improving frozen product quality. The measurement of ice crystals in samples frozen in controlled conditions of both freezing and microwave irradiation allows studying the main parameters that induce the reduction of their size. Better understanding of the relevant factors influencing nucleation and/or crystal growth can help to optimize the process.

Effects of Immersion Freezing on Ice Crystal Formation and the Protein Properties of Snakehead (Channa argus).

This study aimed to evaluate the effect of immersion freezing (IF) at different temperatures on ice crystal formation and protein properties in fish muscle. Snakehead blocks were frozen by IF at −20, −30, and −40 °C, and conventional air freezing (AF) at −20 °C. The size of ice crystals in the frozen samples was evaluated using Image J software. Changes in protein properties were analyzed by Fourier transform infrared spectroscopy (FT-IR) and differential scanning calorimetry (DSC). Snakehead blocks frozen using IF contained smaller ice crystals and better microstructures, especially at lower temperatures. The mean cross-sectional areas of ice crystals formed in the frozen samples were 308.8, 142.4, and 86.5 μm2 for IF treatments at −20, −30, and −40 °C, respectively, and 939.6 μm2 for the AF treatment. The FT-IR results show that protein aggregation in the frozen fish blocks was manifested by a decrease in α-helices connected to the increased random coil fraction. The DSC results show that samples prepared by IF had a higher denaturation enthalpy (∆H) and denaturation maximum temperature (Tmax) than those prepared by AF. These results confirm that IF generated a larger number of smaller ice crystals, which is conducive to food preservation.

Cryopreservation effects on canine sperm morphometric variables and ultrastructure: Comparison between vitrification and conventional freezing

Semen cryopreservation is an increasingly demanded technique in canids, particularly in order to preserve and spread high genetic value material. Sperm vitrification may represent an interesting alternative to costly and time consuming conventional freezing. The objective of this study was to evaluate the effect of sperm vitrification on sperm morphometry and ultrastructure compared to conventional freezing. Pools of nine beagle dogs were both frozen and vitrified. Computerized morphological parameters (length, wide, area and perimeter) and sperm ultrastructure, using scanning and transmission microscopy, were analysed in both fresh and in thawed/warmed samples. There were no differences (p > 0.05) between post-thaw and fresh morphometric variables of the sperm heads. However, cluster analysis revealed that sperm-heads turned out to be smaller after thawing (p < 0.05) in two of the four subpopulations. Vitrification-warming process led to an overall increase in sperm-head size. Furthermore, the sperm head size increased after warming in two subpopulations (p < 0.05). In conclusion, the variations in the sperm head area depended on the cryopreservation procedure (conventional freezing or vitrification). Conventional freezing tended to decrease the head dimensions, at least in some subpopulations, and vitrification led to an overall increase in the sperm head size. Decondensation of chromatin and plasma membrane blebbing in the head region was observed by transmission electron microscopy in several vitrified sperm, which might explain the increase of head dimensions detected by CASA-Morph system.

Control of Ice Nucleation for Subzero Food Preservation

Freezing processes have long been employed for the preservation of foods, providing minimum nutrition loss with a long shelf life period. Freezing plays an essential role in ensuring the safety of food products in all regions of the world. Nonetheless, slow freezing rates and frequently freezing/thawing lead to permanent physicochemical changes, damage of structure, degradation of nutrition values, and color changes through the formation of large ice crystals in food matrix during the cold storage. The size of ice crystals is highly related to the duration of phase transition and degree of supercooling. This paper reviews that the degree of supercooling and nucleation temperature can be controlled by positive or negative pressure and vibration of the dipole and dipole rotation water molecule techniques. Controlling nucleation temperature and suppression of ice crystals in the food matrix could not be achieved by current freezing methods such as air blast, contact, and immersion freezing in the food industry. These present freezing methods are especially focused on increasing the heat transfer rate in foods. Rapid freezing technology may depend on the size and shape of food. However, the size of ice crystals and the suppression of nucleation could be achieved by alternative freezing technologies to overcome the drawbacks of current freezing technologies in the food industry. Conventional freezing technologies can be replaced by emerging freezing techniques, ultrasound irradiation, high pressure, electric field, magnetic field, and microwave-assisted freezing to control the properties of the nucleation and degree of supercooling in the food industry.

Paper-Based Cell Cryopreservation.

The continuous development of simple and practical cell cryopreservation methods is of great importance to a variety of sectors, especially when considering the efficient short- and long-term storage of cells and their transportation. Although the overall success of such methods has been increased in recent years, there is still need for a unified platform that is highly suitable for efficient cryogenic storage of cells in addition to their easy-to-manage retrieval. Here, a paper-based cell cryopreservation method as an alternative to conventional cryopreservation methods is presented. The method is space-saving, cost-effective, simple and easy to manage, and requires no additional fine-tuning to conventional freezing and thawing procedures to yield comparable recovery of viable cells. It is shown that treating papers with fibronectin solution enhances the release of viable cells post thawing as compared to untreated paper platforms. Additionally, upon release, the remaining cells within the paper lead to the formation and growth of spheroid-like structures. Moreover, it is demonstrated that the developed method works with paper-based 3D cultures, where preformed 3D cultures can be efficiently cryopreserved.

Research and Application of the Local Differential Freezing Technology in Deep Alluvium

Aiming at the complicated engineering conditions of the auxiliary shaft repair in the Banji coal mine, it was proposed to seal the water around the shaft lining by differential control freezing technology using double rows of holes. The outer row of holes is completely frozen, and the inner row of holes is local differential frozen according to the degree of destruction of the shaft lining. The local differential freezing pipe was successfully developed according to engineering requirements. Numeral simulations were used to predict the development of the freezing temperature field; the results showed that the inward expansion range of the frozen wall formed by the inner row freezing holes was effectively limited and the temperature drop rate of the shaft lining was significantly reduced after the local differential freezing technique was adopted. The on‐site monitoring data showed that the temperature of the limited freezing layer was about 5°C higher than that of the conventional freezing layer. During the drainage work and the construction of the new shaft lining, the thickness and average temperature of the frozen wall remained stable, indicating that the implementation of the local differential freezing technology achieved the expected results. Further analysis showed that when the temperature of the limited freezing part of freezing pipes in the inner row was controlled within the range of −15 to −10°C, not only could the frozen wall reach the design thickness and strength but the frost heaving pressure on the existing shaft lining could be effectively eliminated.

Effect of cryoprotectant-free vitrification versus conventional freezing on human testicular sperm motility: a prospective comparative study

This study aimed to demonstrate the effect of conventional freezing versus cryoprotectant-free vitrification on the recovery of testicular sperm motility. Testicular samples were obtained from 50 patients with azoospermia for testicular biopsy ± potential sperm storage. We retrieved 100 spermatozoa from each patient divided equally into two straws. They were frozen using conventional freezing as a control group and cryoprotectant-free vitrification in micro-capillary system using open–pulled straws. Seven days later, cryopreserved straws were thawed and assessed in duplicate. The mean sperm motility between the original spermatozoa sample and the post warming sample was reduced after conventional freezing compared to cryoprotectant-free vitrification (4.48 ± 2.09% versus 3.25 ± 1.92%, p < 0.001; 4.48 ± 2.09% vs 3.68 ± 1.93%, p < 0.001, respectively). There was a significant difference between the two methods regarding the mean sperm motility after warming (3.38 ± 1.86% versus 3.76 ± 1.88%, p = 0.015). The mean recovery percent of testicular sperm motility from the original sperm sample was lower (p = 0.02) after conventional freezing compared to cryoprotectant-free vitrification (78.4 ± 28.17% versus 85.37 ± 23.63%). Overall, the rate of post-thaw recovery of human testicular sperm motility improved using cryoprotectant-free vitrification compared to conventional freezing.

Cryopreservation induces higher oxidative stress levels in Bos indicus embryos compared with Bos taurus

Freezing and thawing of Bos indicus embryos affect their quality for embryo transfer. The objective of this study was to compare the levels of reactive oxygen species between Bos indicus and Bos taurus embryos produced in vivo, before and after conventional freezing, as well as to analyze damage caused by apoptosis and lipid peroxidation. Bos indicus has higher levels of reactive oxygen species than Bos taurus embryos, both fresh (14.32 ± 1.41 auf vs 8.07 ± 1.15 auf (arbitrary units of fluorescence), P < 0.05) and after freezing (20.91 ± 1.21 auf vs 14.39 ± 0.58 auf, P < 0.05). The number of apoptotic nuclei is also significantly higher in Bos indicus embryos than Bos taurus (8.28 ± 0.80 vs 1 ± 0.57, P < 0.05) but highlighting a notable increase after the freeze-thaw process in both subspecies (Bos indicus from 8.28 ± 0.80 to 10.71 ± 0.42, P < 0.05; Bos taurus from 1 ± 0.57 to 5.5 ± 1.15, P < 0.05). Finally, although lipid peroxidation is lower in Bos indicus embryos before freezing in comparison with Bos taurus (2.46 ± 0.14 vs 4.20 ± 0.51), the effect after the freeze-thaw process showed an increase of 4.34 in Bos indicus than Bos taurus embryos (51.45 ± 5.52 auf vs 11.85 ± 2.88 auf, P < 0.05). In conclusion, in comparison with Bos taurus, Bos indicus embryos undergo greater oxidative stress causing increases in the cryopreservation process, promoting major cell damage and lowering embryonic viability.

41 Effect of vitrification on DNA integrity of human spermatozoa

The aim of our study was to investigate the effect of vitrification with sucrose on swim-up-prepared human spermatozoa in comparison with standard, conventional manual freezing with permeable cryoprotectants. After informed consent, 35 ejaculates were obtained from 35 patients with normozoospermia who were patients of a fertility clinic. All specimens used for this study had fulfilled the following quality criteria for spermatozoa concentration and motility on IVOS (Hamilton Thorne). Semen analysis was performed according to published guidelines of the World Health Organization (WHO Laboratory Manual for the Examination and Processing of Human Semen, 2010). After swim-up, each sample was centrifuged, resuspended with the basic medium (human tubal fluid + 1% human serum albumin) to achieve a concentration of 5×106 spermatozoa/mL, and finally aliquoted into two equal subsamples. Each of these aliquots was assigned to one of two groups: group 1 included conventionally cryopreserved spermatozoa and group 2 included spermatozoa that were vitrified. For conventional cryopreservation, freezing media (15% (vol/vol) glycerol, 20% (vol/vol) egg yolk) and citrate was added to the washed spermatozoa in a 1:2 ratio. The sperm suspension was aspirated into 0.5-mL straws (CryoBioSystem). Subsequent to the room-temperature incubation for 10min, straws were placed horizontally in the vapour phase for 15min and then submerged into liquid nitrogen. For thawing, cryopreserved straws were immersed in water (23°C) for 5min. For preparation of vitrification solution, the basic medium (human tubal fluid + 1% human serum albumin) was diluted 1:1 with 0.5M sucrose. Immediately after processing, the sperm suspension was diluted in a 1:1 ratio with the vitrification solution to reach a final sucrose concentration of 0.25M. The vitrification and sperm solution (300μL) were aspirated into the straws 0.5mL. Straws were then left at room temperature (20-21°C) for 10min and subsequently submerged horizontally into the liquid nitrogen (Isachenko et al. 2012 J. Androl. 33, 462-468; https://doi.org/10.2164/jandrol.111.013789) and stored similarly to the conventionally cryopreserved straws. To thaw, vitrified straws were immersed in a water bath (42°C) for 20s. The DNA fragmentation was analysed using the APO DIRECT kit (BD PharmingenTM). The cells were stained according to the manufacturer's protocol, followed by flow cytometry analysis CyFlow (Sysmex-Partec). An analysis of variance with a significance of 0.05 for nonparametric statistical analysis to establish differences between groups was used. In our study, no statistically significant differences were observed in the total motility, progressive motility, or velocity parameters of spermatozoa (P&amp;gt;0.05) post-thawing. Also, higher percentages of DNA fragmentation (35.1±8.1% vs. 20.1±6.8%; P&amp;lt;0.05) were found in spermatozoa cryopreserved by means of vitrification with sucrose compared with conventional cryopreservation. Therefore, these methods are comparable and either can be implemented for the storage of spermatozoa to be used for future assisted-reproduction-technology procedures. Vitrification of human spermatozoa provides a simpler, faster, more cost-effective alternative to conventional cryopreservation methods.

Effect of warming temperatures on donkey sperm vitrification in 0.5 mL straws in comparison to conventional freezing

Aim of study: There is little information about vitrification of sperm in large volumes (up to 0.5 mL). This study aimed to develop the vitrification technique in 0.5 mL straws in donkey sperm, evaluating the effect of three warming temperatures.Area of study: Cordoba, Spain.Material and methods: Ejaculates from five donkeys were divided in four groups: one control subjected to conventional slow freezing (C) and three vitrified in 0.5 mL straws and warmed using different protocols (W1: 37ºC/30s, W2: 43ºC/20s and W3: 70ºC/8s+37ºC/52s). Sperm motility, kinematic parameters, plasma membrane and acrosome integrity were evaluated. Conventional freezing resulted in significantly higher values for total (42.7±19.6%), and progressive motility (30.3±16.7%), plasma membrane (49.1±10.4%) and acrosome integrity (39.6±14.5%) respect to vitrification method.Main results: Values after warming ranged between 0.2-2.8% for total motility; 0.2-2.1% for progressive motility; 5.5-20.0% for plasma membrane integrity and 14.5-29.8% for acrosome integrity in all warming protocols after sperm vitrification. However, no differences were found between W3 and C for kinematic parameters; and W3 resulted in significantly higher values for membrane integrity (20.0±11.0%) in comparison to W1 (5.5±3.6%) and W2 (9.3±8.4%).Research highlights: High warming rates seem to be better for donkey sperm vitrification in large volumes; but this methodology is still not an alternative to conventional sperm freezing.

Ejaculated compared with epididymal stallion sperm vitrification

The aim of this study was to evaluate the effect of trehalose and lactose extenders on ejaculated and epididymal stallion sperm vitrification. Ejaculated semen samples were collected from seven fertile stallions, and cauda epididymis samples were collected from ten stallion carcasses after slaughter. Both the ejaculated and the epididymis samples were diluted and vitrified using INRA 96® and bovine serum albumin as well as trehalose or lactose. As a control, ejaculated and epididymal samples were collected and frozen using the conventional method. Vitrification was performed by immersing sperm suspensions directly in LN2. After thawing or devitrification, there was assessment of samples for sperm motility using computer-assisted analysis. Viability was assessed using SYBR-14 and propidium iodide (PI) and acrosome integrity by fluorescein using isothiocyanate combined with peanut agglutinin (FITC-PNA) and PI. Epididymal sperm vitrification with trehalose (EPT) or lactose (EPL) resulted in greater progressive sperm motility than sperm of the control sample (EPC). After post-thaw/devitrification of sperm in the EPT group, sperm motility was greater (P<0.001) compared to that using EPL (50.72 ± 5.09% compared with 34.21 ± 3.02%). The results from assessment of ejaculated sperm samples after undergoing the vitrification process indicated cells were less viable (P<0.001) than the control (EJC) sample. In conclusion, vitrification of epididymal stallion sperm using trehalose might be a beneficial alternative for the long-term storage of sperm samples with great economic value. Spermatozoa from vitrified ejaculates of stallions, however, had lesser motility and viability rates than samples subjected to conventional freezing.

YbMgGaO4: A Triangular‐Lattice Quantum Spin Liquid Candidate

AbstractResonating valence bond ground state, the prototype of a quantum spin liquid (QSL), had been first proposed by P. W. Anderson back in 1973 on the triangular lattice. In such a 2D QSL, spinons created as unpaired spins can propagate by locally rearranging the uncorrelated valence bonds. However, the corresponding candidate materials are still rare to date. YbMgGaO4, first proposed in 2015 may fill this gap: the magnetic rare‐earth Yb3+ ions arrange on the triangular lattice with the perfect R‐3m symmetries and with a large interlayer distance. No conventional spin freezing is observed down to 40 mK without residual magnetic entropy, despite the significant antiferromagnetic coupling of ≈2 K. This report reviews and classifies the relevant experimental and theoretical progress on some (effective) spin‐1/2 triangular antiferromagnets, especially on YbMgGaO4, followed by discussion and outlook on some of the pending issues.

Comparative Study between Slow Conventional Freezing and Cryoprotectant-Free Vitrification of Human Spermatozoa in Large Volume

Background: Cryopreservation is the collection, freezing, and long term storage of sperm, and is a highly effective method of protecting male fertility. Cryopreservation of semen has been widely used as a vital method for fertility preservation of male patients before undergoing chemotherapy, radiotherapy, and/or surgery that may lead to testicular failure or ejaculatory dysfunction. Objective: The aim of this study was to compare the aseptic technology of cryoprotectant-free vitrification of human spermatozoa in large volume, to conventional freezing protocol as regards post thawing motility, vitality and sperm DNA fragmentation. Patients and methods: This study included a total of 20 male patients seeking seminal fluid analysis, attending at the andrology laboratory of a specialized IVF center (ADAM International Hospital for Fertility and Sterility, Giza, Egypt. Patients were presented with the diagnoses of normozoospermia, oligozoospermia (either isolated or combined with asthenozoospermia or teratozoospermia). Results: Motility of (in a large volume (300 µl) in the absence of permeable cryoprotectants displayed significant statistically lower levels as compared to conventional Sperm Freezing. It was shown in different groups at different times (post thawing and 1-hour and 24-hour) of assessment that motility of vitrified spermatozoa decreases in comparison with slow conventional freezing as we go from the baseline. DNA fragmentation of vitrified spermatozoa showed higher levels as compared to conventional slow freezing but there is no significant statistical difference between vitrification and conventional slow freezing in DNA fragmentation. Conclusion: It could be concluded that vitrification technique was quite far away from comparison with slow conventional freezing protocol, and still need for further modifications and wide scale of study to achieve the good results.

Comparative Study between Slow Conventional Freezing and Cryoprotectant-Free Vitrification of Human Spermatozoa in Large Volume

Background: Cryopreservation is the collection, freezing, and long term storage of sperm, and is a highly effective method of protecting male fertility. Cryopreservation of semen has been widely used as a vital method for fertility preservation of male patients before undergoing chemotherapy, radiotherapy, and/or surgery that may lead to testicular failure or ejaculatory dysfunction. Objective: The aim of this study was to compare the aseptic technology of cryoprotectant-free vitrification of human spermatozoa in large volume, to conventional freezing protocol as regards post thawing motility, vitality and sperm DNA fragmentation. Patients and methods: This study included a total of 20 male patients seeking seminal fluid analysis, attending at the andrology laboratory of a specialized IVF center (ADAM International Hospital for Fertility and Sterility, Giza, Egypt. Patients were presented with the diagnoses of normozoospermia, oligozoospermia (either isolated or combined with asthenozoospermia or teratozoospermia). Results: Motility of (in a large volume (300 µl) in the absence of permeable cryoprotectants displayed significant statistically lower levels as compared to conventional Sperm Freezing. It was shown in different groups at different times (post thawing and 1-hour and 24-hour) of assessment that motility of vitrified spermatozoa decreases in comparison with slow conventional freezing as we go from the baseline. DNA fragmentation of vitrified spermatozoa showed higher levels as compared to conventional slow freezing but there is no significant statistical difference between vitrification and conventional slow freezing in DNA fragmentation. Conclusion: It could be concluded that vitrification technique was quite far away from comparison with slow conventional freezing protocol, and still need for further modifications and wide scale of study to achieve the good results.

Catalase incorporation in freezing mixture leads to improved recovery of cryopreserved iPSC lines

Among the various types of stem cells, induced pluripotent stem cells (iPSCs) have gained much attention due to their pluripotent nature. iPSCs help us to understand the processes that regulate pluripotency and specialization. However, in order to use them in various applications in regenerative medicine, their efficient cryopreservation and recovery after the freezing injury is critical. Here we have used an antioxidant catalase, as an additive to the conventional freezing mixture containing 50% FBS and 10% DMSO. The hiPSCs were frozen as aggregates by using a programmable freezer and then stored in liquid nitrogen at −196 °C. It was seen that catalase improved the revival efficiency by reducing the late apoptotic populations and increasing the live cell fraction. Catalase also retained the pluripotent nature of iPSCs in a better way post revival. This improvement could be attributed to reduction of total ROS and apoptosis, which are the two main factors that cause damage during freezing. Our data suggest that catalase could be a useful additive while freezing hiPSCs.

Strategies to Minimize Various Stress-Related Freeze-Thaw Damages During Conventional Cryopreservation of Mammalian Spermatozoa.

The aim of the article is to report a review on different sperm cryopreservation techniques, various stress-related freeze-thaw damages altering sperm structure and function during conventional cryopreservation, and strategies to minimize these stresses. Sperm cryopreservation has allowed indefinite storage and successful transportation of valuable germplasm from proven sites at distant locations, for genetic upgradation through implementation of reproductive techniques, such as artificial insemination. Different techniques for sperm cryopreservation have been proposed such as conventional freezing techniques, directional freezing, and sperm vitrification. Drawbacks related to conventional freezing methods, such as heterogeneous ice nucleation and repeated freeze-thaw cycles at the ice front that disrupts and kill sperm cells, led to the emergence of the directional freezing technique. Sperm vitrification is advantageous as there is no ice crystal-induced physical damages to sperm. However, sperm vitrification has less applicability as encouraging results are only reported in human, dog, and cat. In spite of several drawbacks, conventional freezing techniques are still most widely used for sperm cryopreservation. Spermatozoa experience stresses in the form of cold shock, osmotic stress, and mainly oxidative stress during conventional cryopreservation ultimately reduces the sperm viability and fertility. Several attempts have been made in the past to minimize all these stresses individually or in combination. Membrane fluidity was increased to prevent the cold shock and cryocapacitation-like changes by the addition of cholesterol to the membrane. Antifreeze proteins were added in semen extender to minimize freeze-thaw damages due to heterogeneous ice nucleation and ice recrystallization. Oxidative stress was reduced either by neutralizing reactive oxygen species (ROS) through enzymatic, nonenzymatic, plant-based antioxidants or reductants; or by minimizing the level of sources like the semen radiation exposure, leucocytes, and dead and defective spermatozoa, which lead to ROS production during the semen cryopreservation process. A novel approach of minimizing oxidative stress was to reduce the oxygen tension in sperm microenvironment that is, extender by partial deoxygenation process, as a number of literatures pointed out direct link of O2 with ROS production. When compared with other strategies, partial deoxygenation of semen extender with N2 gassing is found as a cost-effective, comparatively easy and a potential approach to large-scale frozen semen production.

Conventional freezing vs. cryoprotectant-free vitrification of epididymal (MESA) and testicular (TESE) spermatozoa: Three live births

Data of cryoprotectant-free vitrification of human testicular and epididymal spermatozoa are limited. The aim of this investigation was to compare two aseptic technologies of TESE (testicular) and MESA (epididymal) spermatozoa cryopreservation: standard conventional freezing with the use of cryoprotectants and cryoprotectant-free vitrification. Sperm motility, capacitation-like changes, acrosome reaction and the mitochondrial membrane potential of frozen (5% glycerol, −10 °C/min) and vitrified (Human Tubal Fluid + 1% Human Serum Albumin+0.25 M sucrose, plunging into liquid nitrogen of capillaries with spermatozoa isolated from liquid nitrogen (aseptic method) were compared. The quality of the cryoprotectant-free vitrified MESA- and TESE-spermatozoa was higher than that of spermatozoa conventionally frozen with permeable cryoprotectants. Intracellular sperm injection (ICSI) was performed with vitrified spermatozoa. We report the birth of three healthy babies from two women following ICSI with motile MESA- and TESE-spermatozoa vitrified without cryoprotectants. This is the first report of full-term pregnancies and babies born after ICSI with epididymal and testicular spermatozoa vitrified without cryoprotectants. In conclusion, cryoprotectant-free vitrification can be successfully applied for the cryopreservation of motile TESE- and MESA-spermatozoa.