Ultrarapid Freezing Research Articles

Ultra-Rapid Freezing Using Droplets Immersed into Liquid Nitrogen in Bull Sperm: Evaluation of Two Cryoprotective Disaccharides and Two Warming Temperatures.

The present study analyzes the effects of different disaccharide concentrations and two thawing temperatures on the characteristics of ultrarapid frozen (URF) bovine sperm, compared with conventional slow-frozen (CF) sperm. For URF sperm, samples were diluted in media comprising 2% bovine serum albumin (BSA) and various nonpermeable cryoprotectants. Five groups were compared: control (without cryoprotectant), sucrose 0.15 M, sucrose 0.3 M, trehalose 0.15 M, and trehalose 0.3 M. In addition, the influence of warming temperatures, 37°C and 65°C, was analyzed. The aspect of different diluents (by drops) immersed in liquid nitrogen was also evaluated. Sperm quality was assessed by measuring motility, viability, acrosome status, and membrane lipid peroxidation (LPO). Moreover, the cryoresistance rate (CR) was determined. The drops immersed in liquid nitrogen showed that crystallization occurred, but not vitrification. CF sperm exhibited significantly higher scores for total motility (TM) and progressive motility (PM), viability, and acrosome integrity, in contrast with URF samples. Cryoprotectants for URF sperm showed a significant (p ≤ 0.05) influence on the TM and PM, viability, acrosome integrity, and CR, but not on LPO. Sperm viability was reduced after ultrarapid freezing, and the control samples were observed to have significantly lower values than those treated with disaccharides. Samples supplemented with 0.3 M sucrose exhibited higher LPO when they were thawed at 37°C. In short, a limited number of spermatozoa were able to maintain their motility and other functional attributes after ultrarapid freezing, but disaccharides showed a moderate protective effect. Samples with trehalose and sucrose at 0.15 and 0.3 M, respectively, showed higher sperm quality than samples containing only BSA. In sum, the function of spermatozoa was moderately maintained when disaccharides were used for ultrarapid freezing, although motility was significantly reduced. In addition, thawing temperatures did not modify the sperm values, suggesting that the easier procedure, that is, 37°C for 30 seconds, can be used.

Ultra-Rapid Freezing Preserves Morphofunctional Integrity and Fertilizing Ability of Epididymal Cat Spermatozoa.

Vitrification and ultra-rapid freezing, which are more commonly used for oocytes and embryos, have recently been applied to spermatozoa in an attempt to make semen cryopreservation in field conditions easier compared to conventional freezing. It is well-known that in case of unexpected death of rare and wild animals, preserving epididymal spermatozoa from isolated testicles represents a great chance of salvaging male germplasm for future use in assisted reproductive technologies. The aim of this study was to evaluate the morphofunctional integrity of cat epididymal spermatozoa ultra-rapid frozen in pellets or straws with two different extenders [E1 (Tris buffer with 20% egg yolk and 0.25 M sucrose) or E2 (Ham's F10 with 1% bovine serum albumin and 0.4 M sucrose)] and to test whether spermatozoa preserved by the best combination were able to fertilize oocytes and produce embryos in vitro by intracytoplasmic sperm injection (ICSI) of in vitro matured cat oocytes. The results showed that E1 and E2 in straw or pellet were comparable (at warming, about 30% normal morphology, 45% intact membranes, and 20% intact acrosomes), except for post-warming motility that was better maintained along time by E1 pellet (21.7 ± 7.4% at warming and 3.6 ± 2.9% after 6 h). Such spermatozoa could fertilize conspecific oocytes and support embryonic development (cleavage 35.5%) as well as frozen control spermatozoa (cleavage 54.29%, p = 0.22). In conclusion, cat epididymal spermatozoa better maintained their morphofunctional features after ultra-rapid freezing with E1 and could successfully produce embryos in vitro after ICSI. This underscores their usefulness as cryobanked material for fertility and biodiversity preservation purposes.

Influence of Prolactin Secretion Changes on Sperm Head Size and Freezability in Ibex and Mouflon.

Aim: This work examined the influence of induced changes in prolactin (PRL) secretion on sperm cryoresistance of ibex and the mouflon. Materials and Methods: PRL secretion was modified in a first experiment by the use of bromocriptine (BCR, dopamine agonist) during the non-breeding season, and in a second experiment by the use of sulpiride (SLP, dopamine D2-receptor antagonist) during the rutting season. Slow and ultra-rapid freezing protocols were used to cryopreserve sperm samples. Results: BCR decreased blood plasma PRL concentrations, whereas SLP increased them. Cryoresistance ratios (CRs) for curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP) in BCR-treated mouflons were lower than in controls using slow-freezing (p < 0.05), while CRs of motility and morphologically normal sperm of BCR-treated mouflons were greater than controls with ultra-rapid freezing (p < 0.05). BCR increased the head sperm dimensions in ibexes (p < 0.001); conversely, BCR decreased the head dimensions in mouflons (p < 0.001). CR-motility, CR-amplitude of lateral head displacement (ALH), CR-viability, and CR-acrosome integrity in SLP-treated mouflons were lower than in controls with slow-freezing (p < 0.01); CR-viability and CR-acrosome were lower than controls with ultra-rapid freezing (p < 0.05). In ibexes, CR-ALH was lower for SLP-treated (p < 0.05). SLP treatment increased head dimensions in ibexes (p < 0.001) but did not affect the sperm head of mouflons. Conclusion: Our findings show that high levels of blood plasma PRL negatively affect the cryoresistance of ibex and mouflon sperm.

Spermatological characteristics and effects of cryopreservation in Lebranche mullet spermatozoa (Mugil liza Valenciennes, 1836): First report of ultra-rapid freezing

The present study investigated the spermatological characteristics of raw semen of Lebranche mullet (Mugil liza), namely pH, and sperm density, and motility; and subsequently evaluated the effects of different times of exposure to cryoprotectants, and the application of an ultra-rapid freezing protocol, on sperm motility and plasma membrane integrity. Semen samples were analyzed undiluted (control) and diluted 1:50 v/v in CF-HBSS + 10% Dimethyl sulfoxide + 30% Ethylene glycol + 94.58 gL−1 Trehalose dehydrate (n = 15). Two treatments – diluted semen samples in cryoprotective medium without ultra-rapid freezing (T1), and diluted semen in cryoprotective medium with ultra-rapid freezing (T2) – were evaluated at 0, 2, 4, 6 and 8 min. The frozen samples were thawed at 37ºC for 60 s. The spermatological characteristics recorded for the semen were: pH: 7.57 ± 0.21; sperm density: 30.4 ± 2.9 × 109 sperm mL−1; motility: 82 ± 4.9%. Sperm motility presented differences after 2 min exposure to cryoprotectants (70.0 ± 2.7%) and ultra-rapid freezing (66.5 ± 5.8%) compared to the control group (98.5 ± 1.9% and 98.5 ± 2.1%, respectively; p < 0.05). On the other hand, the plasma membrane integrity of the spermatozoa after 2 min exposure to cryoprotectants (64.0 ± 8.6%) and ultra-rapid freezing (62.5 ± 5.2%) presented no differences compared to the control group (69.5 ± 3.9% and 70.0 ± 3.5%, respectively p > 0.05); however, differences were observed in the parameters evaluated after longer exposure and cryopreservation times. This is the first report evaluating the effects of different times of exposure to cryoprotectants and direct ultra-rapid freezing in liquid nitrogen on Mugil liza sperm. Our results demonstrated the protocol of sperm ultra-freezing is safe within a time´s window of 2 min of exposure to cryoprotectants, after which a toxicity effect on sperm can be observed.

Effect of physical and thermal pretreatments on enzymatic activity in the production of microporous cassava starch

Cassava starch is modified to increase porosity and lacerations that are limited when only enzymatic treatments are used. This study proposes to improve enzymatic activity of α-amylase and amyloglucosidase on the polymer chains of cassava starch by implementing physical and thermal pretreatments below the gelatinization temperature and before the hydrolytic process. The pretreatments increased the biocatalytic action of the enzymes, causing significant changes in the morphology of the granules, and superficial lacerations were found in samples of starches pretreated with ultrasound (UTS) or annealing and ultra-rapid freezing (ANN-C). At the structural level, the modified starches revealed substantial changes as the infrared spectra reflected a displacement of the absorption bands in the region from 900 to 1100 cm-1. This is associated with an alteration and reorganization of the amorphous and crystalline zones of the granules and is consistent with a decrease in amylose content (from 19.53% to 17.64%) and an increase in the crystallinity index. The thermal behavior of the starches was also modified by increasing the peak temperature (from 68.22°C to 75.38°C) and reducing the gelatinization enthalpy (from 19.34 to 15.79 J/g). UTS and ANN-C pretreatments significantly improved the mesoporous and hydrophilic properties of the modified cassava starches.

Cryopreservation of dog epididymal spermatozoa by conventional freezing or ultra-rapid freezing with nonpermeable cryoprotectant

This study was aimed to assess the effectiveness of two methods for cryopreservation of dog epididymal spermatozoa, one by conventional freezing (CF) with shortening both equilibration and cooling times, and the other by ultra-rapid freezing (URF) with nonpermeable cryoprotectant. Sixty epididymides were recovered from thirty orchiectomized adult dogs and the sperm samples were retrieved by retrograde flushing using TCG-EY (tris, citric acid, glucose + 20% egg yolk) extender and then 20 pools were conformed. Each pool was divided into 2 aliquots and then cryopreserved by CF and URF methods respectively. The CF method maintained the cooled-pool samples for 2h (1h without and 1h with 5% glycerol) and then were frozen by liquid nitrogen (LN2) vapors for 2 min. The URF method cryopreserved the cooled-pool samples using TCG-EY+250 mM sucrose, equilibrating during 30 min (5 °C) and submerging 30-μL drops directly in LN2. The results showed that the URF method produced a lower percentage of total and progressive motilities and acrosome integrity (P < 0.05) than the CF method. However, the kinetic variables (curvilinear and straight-line velocities, straightness, linearity, wobble, amplitude of lateral head displacement, and beat-cross frequency) and plasma membrane integrity did not differ (P > 0.05) between both cryopreservation methods. Unlike the URF method, the width, area and perimeter of sperm head were reduced after the CF method (P < 0.05). In conclusion, despite the low motility achieved after the ultra-rapid freezing method, the similar values of kinetic, viability and head morphometric dimensions to those obtained after conventional freezing, suggest that ultra-rapid freezing with sucrose may be a useful alternative for the cryopreservation of canine epididymal sperm.

Low-Diffusion Fricke Gel Dosimeters with Core-Shell Structure Based on Spatial Confinement.

The diffusion of ferric ions is an important challenge to limit the application of Fricke gel dosimeters in accurate three-dimensional dose verification of modern radiotherapy. In this work, low-diffusion Fricke gel dosimeters, with a core-shell structure based on spatial confinement, were constructed by utilizing microdroplet ultrarapid freezing and coating technology. Polydimethylsiloxane (PDMS), with its excellent hydrophobicity, was coated on the surface of the pellets. The concentration gradient of the ferric ion was realized through shielding half of a Co-60 photon beam field size, and ion diffusion was measured by both ultraviolet-visible spectrophotometry and magnetic resonance imaging. No diffusion occurred between the core-shell pellets, even at 96 h after irradiation, and the diffusion length at the irradiation boundary was limited to the diameter (2–3 mm) of the pellets. Furthermore, Monte Carlo calculations were conducted to study dosimetric properties of the core-shell dosimeter, which indicated that a PDMS shell hardly affected the performance of the dosimeter.

Comparison of the Conventional and Ultra-Rapid Freezing Techniques in Gabon Bucks Throughout the Year.

The aim of this work was to compare the effectiveness of ultra-rapid freezing (UF) and conventional slow freezing (CF) to cryopreserve buck sperm throughout the year. During 1 year, semen from 10 adult Gabon bucks was collected by electroejaculation every 2 weeks. Before and after freezing, samples were selected by density gradient centrifugation, and after sperm selection, the sample was divided into two aliquots. One aliquot was CF with an extender based on Tris, citric acid, and glucose (TCG) +6% yolk +5% glycerol, and maintained at 5°C for 3 hours of equilibration before freezing. The other aliquot was frozen using an UF method with an extender based on TCG +6% yolk +100 mM sucrose, and maintained at 5°C for 30 minutes. The evaluations included the percentages of motile sperm, sperm with progressive motility, quality of sperm motility, and the percentages of sperm with functional membrane, live sperm, sperm with morphoabnormalities, and sperm with intact acrosome. The percentage of sperm with intact acrosome was higher using the conventional freezing method (p < 0.05). After thawing and at pre- and postselection stages, the quality of motility, and the percentages of motile sperm, progressive motile sperm, sperm with functional membrane, and with intact acrosome were greater using CF than UF (p < 0.005). Conventional freezing was more effective than UF to cryopreserve sperm from Gabon bucks, at least in our experimental conditions. Most differences in favor of CF were observed in the quality of motility, and the percentages of motile sperm, progressive motile sperm, sperm with functional membrane, and with intact acrosome during long periods of the year, or even remained throughout it.

Influence of circulating testosterone concentration on sperm cryoresistance: The ibex as an experimental model

Recent studies have noted that the circulating testosterone concentration may affect the ability of spermatozoa to survive cryopreservation. However, few attempts to confirm such a relationship have been made. Wild ruminant species have very marked seasonal changes in their reproductive function and strong annual changes in their plasma testosterone concentration. The present work examines the influence of induced changes in testosterone secretion on sperm variables following conventional slow freezing and ultra-rapid freezing, using the Iberian ibex as an experimental model. In a first experiment, testosterone levels were reduced in the middle of the rutting season (December) using the antiandrogen cyproterone acetate (CA). In a second experiment, testosterone levels were increased at the end of the rutting season (January) via the use of the androgen testosterone propionate (TP). During December, the testosterone concentration was found to be higher in the blood and seminal plasma of untreated males than in those of CA-treated males (p<0.001 and p<0.05, respectively). Compared with controls, the TP-treated animals had higher blood plasma testosterone concentrations but lower seminal plasma testosterone concentrations during January (p<0.01 and p<0.001, respectively). The seminal vesicles of the TP-treated males were larger than those of untreated males (p<0.05). When CA was administered, sperm viability improved compared with controls (p<0.05), irrespective of the freezing protocol followed. For the ultra-rapid freezing procedure, the cryoresistance ratio for motility decreased when TP was administered (p<0.05). The values for fresh sperm morphometric variables decreased during the 50days after the end of CA treatment (p<0.001) and increased over the same time after the end of TP treatment (p<0.001). The circulating testosterone concentration appears to influence sperm cryoresistance. This may explain the seasonal changes seen in sperm freezability in some species, independent of fresh sperm quality.

Metastable–solid phase diagrams derived from polymorphic solidification kinetics

Nonequilibrium processes during solidification can lead to kinetic stabilization of metastable crystal phases. A general framework for predicting the solidification conditions that lead to metastable-phase growth is developed and applied to a model face-centered cubic (fcc) metal that undergoes phase transitions to the body-centered cubic (bcc) as well as the hexagonal close-packed phases at high temperatures and pressures. Large-scale molecular dynamics simulations of ultrarapid freezing show that bcc nucleates and grows well outside of the region of its thermodynamic stability. An extensive study of crystal-liquid equilibria confirms that at any given pressure, there is a multitude of metastable solid phases that can coexist with the liquid phase. We define for every crystal phase, a solid cluster in liquid (SCL) basin, which contains all solid clusters of that phase coexisting with the liquid. A rigorous methodology is developed that allows for practical calculations of nucleation rates into arbitrary SCL basins from the undercooled melt. It is demonstrated that at large undercoolings, phase selections made during the nucleation stage can be undone by kinetic instabilities amid the growth stage. On these bases, a solidification-kinetic phase diagram is drawn for the model fcc system that delimits the conditions for macroscopic grains of metastable bcc phase to grow from the melt. We conclude with a study of unconventional interfacial kinetics at special interfaces, which can bring about heterogeneous multiphase crystal growth. A first-order interfacial phase transformation accompanied by a growth-mode transition is examined.

Slow and ultra-rapid freezing protocols for cryopreserving roe deer (Capreolus capreolus) epididymal sperm collected at different times of year

The roe deer is a monoestrous species with a very short rutting season. The present work reports the most suitable period for collecting epididymal sperm and describes the effect of two cooling rates on the post-thaw quality of sperm. Testes were collected 24–48 h after death. Samples of sperm flushed from the epididymis were subjected to either (1) dilution in a Tris-citric acid-glucose-egg yolk-based medium with glycerol, and slow freezing in straws, or (2) dilution in the same extender but replacing the glycerol with 100 mM of sucrose, and ultra-rapid freezing in pellets. Sperm motility, acrosome and membrane integrity, morphometry and morphological abnormalities were analysed before and after cryopreservation. Spermatogenic activity was investigated via histological examination of testis sections. Several testes collected between April, May and September showed no spermatogenic activity. All those collected in June–August showed spermatogenic activity. No significant difference was detected in the cryoresistance ratios associated with the conventional slow freezing, between sperm collected during the pre-rutting (April–May) and rutting (June–August) periods. No significant differences were seen between the slow-frozen-thawed and the ultra-rapid-frozen-thawed sperm in terms of percentage of viable sperm or the percentage of sperm with morphological abnormalities. Slow freezing returned significantly better (P<0.05) values for post-thaw acrosome integrity (43.3% vs. 25.0%) and straight-line velocity (19 μm/s vs. 4 μm/s). For both freezing methods, sperm heads were smaller post-thawing than pre-freezing (P<0.001). In conclusion, both the pre-rutting and rutting season are suitable periods for freezing roe deer sperm. Ultra-rapid freezing did not provide suitable results.

Facile synthesis of perovskite phosphors and nanocrystals using laundry detergent by ultra-rapid freezing for light-emitting diodes application

Confined metal halide perovskites (MHP) phosphors and nanocrystals have attracted increasing research attentions due to their excellent optoelectronic properties. However, the large-scale and eco-friendly preparation of such materials are still facing great challenges due to their intrinsic ionic characteristics. Herein, we demonstrate an ultra-rapid freezing strategy for preparing perovskite phosphors and nanocrystals in water using laundry detergent as the surfactant. In this work, high-quality CsPbX3 (X = Cl, Br) and MAPbBr3 (MA = CH3NH3+) phosphors and their nanocrystals have been rapidly synthetized at ultra-low temperature (−196 °C). Intriguingly, the production of perovskite phosphors can be easily scaled up to gram level. The obtained CsPbBr3 nanorods present narrow emission width and high photoluminescence quantum yield of 75% for highly pure green light emission. Furthermore, photoluminescence-based light-emitting diodes (PL-LEDs) with CsPbBr3 nanorods as phosphors have realized the intrinsic green emission at 520 nm and superior luminance of 32930 cd∙m−2. This work provides a low-cost and green method for large-scale preparation of quality perovskite phosphors and nanocrystals for practical applications in LEDs, printing industry and anti-counterfeiting recognition fields.

Human sperm vitrification: A scientific report.

The sperm vitrification developed by this group is based on the ultrarapid freezing of a vitrification solution composed of a non-permeable cryoprotectant (saccharides and protein), in which previously selected spermatozoa are resuspended, free of seminal plasma, and then plunged directly into liquid nitrogen. Compared to traditional sperm freezing, vitrification does not cause chemical or physical damage to the intracellular structures and reduces the damage to the plasma membrane because no ice crystals form, thus preserving motility and DNA integrity. This manuscript is a review of the vitrification methodology developed by the authors' research group, including studies showing the application in human reproduction therapy. The authors perform a review of the work initiated more than a decade ago by this research group, on the implementation of sperm vitrification, a more effective technique for cryopreservation of human spermatozoa, discussing the results obtained by other authors and the projection of this technique. The vitrification technique has been developed in selected spermatozoa free of seminal plasma supplemented with saccharides such as sucrose, trehalose, and dextran, together with albumin, providing a high motility rate and protective structures of the cytoskeleton. In patients, it can be used to preserve their fertility for oncological reasons, genetics, inflammatory diseases, or reproductive medicine techniques. The possibility that vitrified spermatozoa can be preserved at temperatures of -80°C can simplify sample storage, optimizing the space and time as well as operator safety. Vitrification techniques have demonstrated the preservation of selected spermatozoa without seminal plasma and with non-permeable cryoprotectants and protein. Currently, it is one of the most effective ways to maintain sperm function and has been used in in vitro fertilization or intrauterine insemination in humans, achieving healthy live births.

Using thin film freezing to minimize excipients in inhalable tacrolimus dry powder formulations.

We investigated the feasibility of preparing high-potency tacrolimus dry powder for inhalation using thin film freezing (TFF). We found that using ultra-rapid freezing can increase drug loading up to 95% while maintaining good aerosol performance. Drug loading affected the specific surface area and moisture sorption of TFF formulations, but it did not affect the chemical stability, physical stability, and dissolution of tacrolimus. Tacrolimus remained amorphous after storage at 40°C/75% RH, and 25°C/60% RH for up to 6months. Lactose functioned as a bulking agent, and it had little to no effect as a stabilizer for amorphous tacrolimus due to a lack of interaction between the drug and excipient. Additionally, the aerosol performance of TFF tacrolimus/lactose (95/5) did not significantly change after six months of storage at 25°C/60% RH. For processing parameters, the solids content and the processing temperature did not affect the aerosol performance of tacrolimus. Furthermore, both low- and high-resistance RS01 showed optimal and consistent aerosol performance over the 1-4kPa pressure drop range. In conclusion, TFF is a suitable technology for producing inhalable powder that contain high drug loading and have less flow rate dependence.

Optimization of Sperm Cryopreservation Protocol for Peregrine Falcon (Falco peregrinus).

Simple SummaryThe process of semen cryopreservation can have multiple advantages in an ex situ conservation program. However, there is a necessity to adapt the protocol to the specificity of each species. With that in mind, we aimed to optimize the sperm freezing/thawing process and study the effect of different cryoprotectants in the peregrine falcon.Sperm cryopreservation is a complex process that needs to be adapted to wild and domestic avian species to ensure proper efficiency. Because of its accessibility, the peregrine falcon may be used as a good model for studying other raptor species. To find the most optimal cryopreservation protocol for peregrine falcon ejaculates, sperm parameters such as motility, viability, DNA fragmentation, acrosome integrity, and mitochondrial activity were analyzed under different conditions by varying the freezing method (slow freezing in straws vs. ultrarapid freezing in pellets), thawing conditions (37 °C for 30 s vs. 5 °C for 1 min), type of cryoprotectant (DMA vs. DMSO), and concentration of DMSO (4% vs. 8%). Results show that slow cryopreservation in straws yielded greater percentages (p < 0.05) of motile spermatozoa (22.5% ± 4.4% vs. 0.0% ± 4.1%), viable spermatozoa with intact acrosomes (84.6% ± 4.3% vs. 77.4% ± 4.3%), and spermatozoa with active mitochondria (41.0% ± 6.7% vs.12.8% ± 6.7%), compared with those obtained by the ultrarapid freezing in pellets. However, no differences were found between different thawing conditions. Moreover, all sperm motility parameters were greater (p < 0.05) when DMSO was used during freezing compared with DMA, although the use of 3% and 8% DMSO produced similar results. In conclusion, these results represent important progress in the study of falcon semen cryopreservation protocol, highlighting the crucial steps of the process and the most suitable conditions.

Cryopreservation and Culture of Testicular Tissues: An Essential Tool for Biodiversity Preservation.

Systematic cryo-banking of reproductive tissues could enhance reproductive management and ensure sustainability of rare mammalian genotypes. Testicular tissues contain a vast number of germ cells, including at early stages (spermatogonia and spermatocytes), that can potentially develop into viable spermatozoa after grafting or culture in vitro, and the resulting sperm cells then can be used for assisted reproductive techniques. The objective of this review was to describe current advances, limitations, and perspectives related to the use of testicular tissue preservation as a strategy for the conservation of male fertility. Testes can be obtained from mature or prepubertal individuals, immediately postmortem or by orchiectomy, but testicular biopsies could also be an alternative to collect samples from living individuals. Testicular fragments can be then cryopreserved by using slow or ultra-rapid freezing, or even vitrification methods. The composition of cryopreservation media can vary according to species-specific characteristics, especially regarding the cryoprotectant type and concentration. Finally, spermatozoa have been usually obtained after xenografting of testicular fragments into severely immunodeficient mice, while this method still has to be optimized after in vitro culture conditions.

Sequential interval micro-droplet loading in closed hemi-straw carrier system: A convenient and efficient method for ultra-rapid cryopreservation in extreme oligozoospermia

Cryopreservation of human spermatozoa with low concentration while maintaining adequate post-thawing motility remains a major challenge for male fertility preservation. A convenient and efficient ultra-rapid freezing method for small amounts of human spermatozoa in a closed Hemi-Straw carrier system (CHS) was developed. Spermatozoa from 60 healthy men were involved in a parameter refining test and another 15 extreme oligozoospermic specimens were assigned to a verification test. A commercialized sperm freezing medium, Quinn's Advantage® Sperm Freeze medium (glycerol and sucrose as the cryoprotective agent) was used in the study. The results showed that the highest recovery rates would be obtained via the method of 2 μl single droplet sequential interval loading, by placing the straw at 1 cm above the liquid nitrogen (LN2) surface for 60 s during freezing and 2 cm above the LN2 for 2 min during thawing. This method was applied in cryopreservation for the normozoospermic specimens and compared with a conventional slow freezing method. The results were better than those in the control group in the total motility recovery rate (77.8 ± 11.2% vs 56.6 ± 11.9%, P < 0.01), progressive motility recovery rate (77.6 ± 13.2% vs 47.7 ± 14.6%, P < 0.01), 24 h survival index (60.9 ± 13.4% vs 42.1 ± 14.1%, P < 0.01) and the sperm DNA fragment index (4.2 ± 3.7% vs 5.8 ± 3.7%, P = 0.126). This method was applied to the oligozoospermic specimens. Motile spermatozoa could be found in 12 of 15 cases in the ultra-rapid freezing group, while only in 7 cases in control group. The results indicated that this freezing method was simple, convenient and bio-safe for cryopreservation of severe oligozoospermic specimens.

Trehalose-Based Polyethers for Cryopreservation and Three-Dimensional Cell Scaffolds

The capability to slow ice growth and recrystallization is compulsory in the cryopreservation of cells and tissues to avoid injuries associated with the physical and chemical responses of freezing and thawing. Cryoprotective agents (CPAs) have been used to restrain cryoinjury and improve cell survival, but some of these compounds pose greater risks for the clinical application of cryopreserved cells due to their inherent toxicity. Trehalose is known for its unique physicochemical properties and its interaction with the phospholipids of the plasma membrane, which can reduce cell osmotic stress and stabilized the cryopreserved cells. Nonetheless, there has been a shortage of relevant studies on the synthesis of trehalose-based CPAs. We hereby report the synthesis and evaluation of a trehalose-based polymer and hydrogel and its use as a cryoprotectant and three-dimensional (3D) cell scaffold for cell encapsulation and organoid production. In vitro cytotoxicity studies with the trehalose-based polymers (poly(Tre-ECH)) demonstrated biocompatibility up to 100 mg/mL. High post-thaw cell membrane integrity and post-thaw cell plating efficiencies were achieved after 24 h of incubation with skin fibroblast, HeLa (cervical), and PC3 (prostate) cancer cell lines under both controlled-rate and ultrarapid freezing protocols. Differential scanning calorimetry and a splat cooling assay for the determination of ice recrystallization inhibition activity corroborated the unique properties of these trehalose-based polyethers as cryoprotectants. Furthermore, the ability to form hydrogels as 3D cell scaffolds encourages the use of these novel polymers in the development of cell organoids and cryopreservation platforms.

Development of an Efficient ‘One-Step Freezing’ Cryopreservation Protocol for a Georgian Provenance of Chestnut (Castanea sativa Mill.) Zygotic Embryos

Experiments were performed to determine the influence of various dehydration and vitrification treatment times on the ‘one-step freezing’ cryopreservation of embryonic axes (EAs), composed of zygotic embryos and cotyledon residuals, from mature seeds of a Georgian provenance of chestnut (Castanea sativa Mill.). Dehydration was carried out in laminar flow hood from 1 to 5 h, and vitrification experiments were carried out by immersion of EAs in PVS2 vitrification solution up to 120 min, both followed by direct immersion in liquid nitrogen. Both systems resulted in inducing specimen tolerance to ultra-rapid freezing, although to a different extent. Full germination of cryo-stored EAs after 5 h of dehydration (reducing moisture content from initial 66% to 21%) has been increased from 0% to 66.7%. A pre-treatment of EAs in PVS2 vitrification solution for 30 min produced fully developed plantlets at a rate of 55.6% in post-cryopreservation. Plantlet regrowth from cryopreservation was faster in EAs that underwent the dehydration/‘one-step freezing’ procedure. All the plantlet from cryopreserved EAs could be easily acclimatized, producing healthy potted plants. Finally, the TTC test showed to be useful for a fast evaluation of specimen survival after thawing and, as a consequence, to speed up the development of optimized cryo-protocols.&#x0D; &#x0D; *********&#x0D; In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 4, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue.&#x0D; *********

Cryopreservation and transfer of pig embryos

Recent advances in cryopreservation and non-surgical transfer of pig embryos have made embryo transfer in pigs a commercially viable technology especially for the international transfer of valuable genetic material. Early research demonstrated that early stage pig embryos were highly sensitive to temperatures below 15°C and this sensitivity decreased with development to peri-hatching blastocysts which is accompanied by a reduction in lipid content. Removal of the lipid prior to freezing was found to improve the freezability and resulted in the birth of piglets. It was shown that by polarising the lipid material out of the blastomeres and using ultra rapid freezing (vitrification) the early stage zona pellucida intact embryo would also survive freezing and result in the birth of piglets. Other factors that have contributed to successful cryopreservation of the early stage pig embryo include the use of cytoskeletal stabilising agents, specific cryoprotectants, increasing cooling rates using open pulled straws or micro droplets and assisted hatching. The present review discusses the relative importance of each of these factors. The development of non-surgical transfer technology and its importance in the application of embryo transfer in the pig industry is also reviewed.