Liquid Nitrogen Vapor Research Articles

Cryopreservation of raspberry cultivar accessions bred in Russia from the VIR <i>in vitro</i> collection

Cryobanks use plant cryocollections for long-term preservation of crops which cannot be preserved in seed collections. These are vegetatively propagated crops, accessions of species which form either a small amount of seeds, or recalcitrant seeds. Shoot tips (apexes) of in vitro plants are used for cryopreservation for most berry crops, therefore maintenance of in vitro collections is very important. The VIR in vitro collection includes 150 accessions of Rubus L. species, 85 of them are raspberry cultivars, 59 of which were bred in Russia. These cultivars reflect a wide ecogeographic diversity. Among them, there are raspberry cultivars created at the end of the 19th – first half of the 20th centuries, including cultivars bred by I.V. Michurin and by the pioneer of northern horticulture V.V. Spirin. More than half of national raspberry varieties (33) are listed in the State Register for Selection Achievements Admitted for Usage. Raspberry cultivars from Russian breeding programs have a very limited representation in foreign genebanks. The first aim of the present work was cryopreservation of mostly folk and old Russian raspberry cultivars received by VIR from 1925 till 1950 and their transfer into the cryobank. The second aim of the work was to monitor post-cryogenic regeneration of raspberry cultivars transferred to the cryobank earlier. A modified protocol of the droplet vitrification method by “DV-biotech” was used for cryopreservation of shoot tips of in vitro plants of 10 raspberry cultivars (7 of which are folk and old Russian ones) from the VIR in vitro collection. Post-cryogenic regeneration was evaluated for 17 raspberry cultivars preserved in the cryobank from one to five years. Ten raspberry cultivars (900 apexes) with an average mean post-cryoregenic regeneration value of 38.2±3.0% determined in control tests, were placed in the cryobank for long-term storage. A statistically significant effect of the genotype on the viability of explants after cryopreservation was noted, while the post-cryogenic regeneration was genotype insensitive. Additionally, levels of post-cryogenic regeneration were evaluated for 17 raspberry cultivars (296 apexes) preserved in the cryobank from one to five years. Post-cryogenic regeneration within the 20-70% range was displayed by four raspberry cultivars preserved in the cryobank for one year, and for 8 cultivars conserved there from three to five years post-cryogenic regeneration was within the 10-50% range. According to the results of monitoring, regeneration displayed by 12 raspberry cultivars was within the 10-70% range, which can be considered as a reliable rate of apex preservation in liquid nitrogen vapors in the VIR cryobank. Monitoring of the post-cryogenic regeneration of the raspberry accessions preserved in the VIR cryobank and cryopreservation of new raspberry cultivars will be continued.

Freezing Protocol Optimization for Iberian Red Deer (Cervus elaphus hispanicus) Epididymal Sperm under Field Conditions.

Simple SummaryThe germplasm banks of wild species, such as Iberian red deer, are not widespread, mainly due to the difficulties of collecting and cryopreserving reproductive cells. Optimal freezing protocols under field conditions could be a breakthrough for these species. In this study, epididymal sperm was evaluated using two methods of sperm storage during refrigeration (tube and straw); four equilibration periods (0, 30, 60, and 120 min); and four methods of freezing (cryopreservation in liquid nitrogen vapors in a tank (control) or box, freezing in dry ice, or freezing over a metallic plate). The results showed that samples stored in straws during refrigeration produced less apoptotic spermatozoa and more viable spermatozoa withactive mitochondria. A long equilibration period (120 min) yielded a higher percentage of acrosomal integrity. Moreover, there was no difference in sperm quality between freezing in liquid nitrogen vapors in a tank or box. However, a worse quality was obtained when the samples were cryopreserved in dry ice or over a metallic plate compared to the control. Creating germplasm banks of wild species, such as the Iberian red Deer (Cervus elaphus hispanicus) can be challenging. One of the main difficulties is the obtention and cryopreservation of good-quality reproductive cells when the spermatozoa are obtained from epididymides after death. To avoid a loss of seminal quality during transport, developing alternative methods for cooling and freezing sperm samples under field conditions is necessary. The objective of this study was to evaluate the effects of different durations of equilibrium and different techniques of cooling and freezing on Iberian red deer epididymal sperm quality after thawing to optimize the processing conditions in this species. Three experiments were carried out: (I) evaluation of refrigeration in straws or tubes of 15 mL; (II) study of equilibration period (0, 30, 60, or 120 min); and (III) comparison of four freezing techniques (liquid nitrogen vapor in a tank (C), liquid nitrogen vapor in a polystyrene box (B), dry ice (DY), and placing straws on a solid metallic plate floating on the surface of liquid nitrogen (MP)). For all experiments, sperm motility and kinematic parameters, acrosomal integrity, sperm viability, mitochondrial membrane potential, and DNA integrity were evaluated after thawing. All statistical analyses were performed by GLM-ANOVA analysis. Samples refrigerated in straws showed higher values (p ≤ 0.05) for mitochondrial activity and lower values (p ≤ 0.05) for apoptotic cells. Moreover, the acrosome integrity showed significant differences (p ≤ 0.05) between 0 and 120 min, but not between 30 and 60 min, of equilibration. Finally, no significant differences were found between freezing in liquid nitrogen vapors in a tank or in a box, although there was a low quality after thawing when the samples were cryopreserved in dry ice or by placing straws on a solid metallic plate floating on the surface of liquid nitrogen. In conclusion, under field conditions, it would be possible to refrigerate the sperm samples by storing them in straws with a 120 min equilibration period and freezing them in liquid nitrogen vapors in a tank or box.

Cryopreservation of Giraffe Epidydimal Spermatozoa Using Different Extenders and Cryoprotectants.

Simple SummaryGiraffe numbers have been plummeting over the last 30 years by 30–40%. As such, efforts to manage in situ and ex situ populations are increasing. Assisted reproduction techniques such as sperm cryopreservation can help preserve the genetic diversity of giraffe subspecies or enhance genetic exchange between populations. However, to date, the post-thaw motility of recovered sperm has been variable. In this study, spermatozoa were collected from the epididymides of seven giraffes to investigate whether an alternative cryoprotectant could improve sperm motility following conventional cryopreservation. For this, we compared the motility and viability of sperm prior to and after freezing in three different extenders: a commercial equine extender (BotuCrio®; Nidacon, Moedal, Schweden), a commercial bovine extender (Steridyl, Minitube, Tiefenbach, Germany), and an in-house “made” bovine egg yolk extender (TEY). Each was further supplemented with either glycerol or a mix of glycerol and methylformamide cryoprotectants. The results show that spermatozoa frozen with a mix of two cryoprotectants had significantly higher post-thaw motility compared to glycerol alone. Specifically, spermatozoa frozen in TEY and a mix of cryoprotectants achieved post-thaw sperm motility of 57 ± 3%. These results might serve as a blueprint for an improved protocol for giraffe sperm cryopreservation. Giraffe numbers have plummeted over the last 30 years by 30–40%. Thus, their conservation status has been raised from least concern to vulnerable. Efforts to manage in situ and ex situ populations are increasing. Assisted reproduction techniques (ART) such as sperm cryopreservation could help preserve the genetic diversity of giraffe subspecies and, when used for artificial inseminations, enhance genetic exchange between isolated populations. However, to date, the post-thaw motility of recovered sperm has been low and inconsistent. In this study, epididymal sperm collected from the testes of giraffes (n = 7) was frozen in three different extenders, namely, BotuCrio, Steridyl, and test egg yolk (TEY), each supplemented with one of two different cryoprotectants (5% glycerol or a mix of 1% glycerol and 4% methylformamide) and frozen over liquid nitrogen vapor. Across all three extenders, sperm showed significantly better post-thaw results when frozen with a mix of glycerol and methylformamide compared with glycerol alone. Sperm frozen with TEY and a mix of glycerol and methylformamide achieved superior post-thaw total and progressive sperm motility of 57 ± 3% and 45 ± 3%, respectively. These results show the benefit of using alternative cryoprotectants for freezing giraffe spermatozoa and could aid in the application of ARTs for giraffe subspecies or the closely related endangered Okapi.

Evaluation of Semen Quality of Jeju Black Cattle (JBC) to Select Bulls Optimal for Breeding and Establish Freezing Conditions Suitable for JBC Sperm.

Simple SummaryJeju black cattle, a type of native Korean cattle characterized by black fur covering the entire body, inhabit Jeju Special Self-Governing Province, a World Natural Heritage Site. Although this breed was state designated as a natural monument in 2013 due to its characteristics and genetic traits, it is on the verge of extinction and thus there is a need to preserve this breed and further improve its traits. Therefore, we evaluated sperm motility, vitality, and morphology, which have long been considered good predictors of fertility in the absence of female infertility factors. Our findings showed that the semen of the JBC-A bull was superior to the semen of four other JBC bulls. Due to the aging of the population of JBC breeding bulls, strategies should be devised to improve sperm production in vivo.To optimize the reproduction of Jeju black cattle (JBC), freezing conditions for sperm were established and sperm motility, vitality, morphology, and fertility were evaluated to select the optimal bull for breeding. Semen samples from five JBC bulls were individually mixed with freezing medium at a final concentration of 1 × 108 sperm/mL and frozen in liquid nitrogen vapor at a height of 3 or 7 cm (referred to as 3 cm sperm and 7 cm sperm, respectively). When the freezing conditions were compared, the motility of 7 cm sperm was significantly higher than that of 3 cm sperm for the JBC-A bull. The motility, curvilinear velocity, straight-line velocity, and average path velocity of fresh and frozen–thawed sperm were the highest for the JBC-A bull. The vitalities of fresh and frozen–thawed sperm were the highest for the JBC-A/E and JBC-A bulls, respectively. The percentage of normal cells in fresh sperm was the highest for the JBC-D bull. The rates of the normal formation of two pronuclei and total sperm penetration were the highest in zygotes fertilized with sperm from the JBC-A bull. The sperm from the JBC-A bull had superior qualities and are thus the most appropriate choice for the preservation and reproduction of these endangered cattle.

A comprehensive report of long-term stability data for a range ATMPs: A need to develop guidelines for safe and harmonized stability studies

Background aimsAdvanced therapy medicinal products (ATMPs) are novel drugs based on genes, cells or tissues developed to treat many different diseases. Stability studies of each new ATMP need to be performed to define its shelf life and guarantee efficacy and safety upon infusion, and these are presently based on guidelines originally drafted for standard pharmaceutical drugs, which have properties and are stored in conditions quite different from cell products. The aim of this report is to provide evidence-based information for stability studies on ATMPs that will facilitate the interlaboratory harmonization of practices in this area. MethodsWe have collected and analyzed the results of stability studies on 19 different cell-based experimental ATMPs, produced by five authorized cell factories forming the Lombardy “Plagencell network” for use in 36 approved phase I/II clinical trials; most were cryopreserved and stored in liquid nitrogen vapors for 1 to 13 years. ResultsThe cell attributes collected in stability studies included cell viability, immunophenotype and potency assays, in particular immunosuppression, cytotoxicity, cytokine release and proliferation/differentiation capacity. Microbiological attributes including sterility, endotoxin levels and mycoplasma contamination were also analyzed. All drug products (DPs), cryopreserved in various excipients containing 10% DMSO and in different primary containers, were very stable long term at <–150°C and did not show any tendency for diminished viability or efficacy for up to 13.5 years. ConclusionsOur data indicate that new guidelines for stability studies, specific for ATMPs and based on risk analyses, should be drafted to harmonize practices, significantly reduce the costs of stability studies without diminishing safety. Some specific suggestions are presented in the discussion.

Semen collection, evaluation, and cryopreservation in the bonobo (Pan paniscus)

BackgroundCaptive breeding of bonobos (Pan paniscus) has proven to be successful, but maintaining genetic diversity remains a challenge. Cryopreservation of semen is an important potential tool to maintain genetic diversity by preserving current genetic material for future use, as well as facilitating the transport and exchange of genetic material. This study aimed to develop a protocol for semen collection and cryopreservation in the bonobo. Semen was collected from four healthy adult bonobos under general anesthesia during management translocation procedures. Semen collection utilizing urethral catheterization was not successful (n = 1), however, all males (n = 4) responded well to rectal probe electro-ejaculation. Immediately after collection, ejaculates were evaluated for color and admixtures, volume, motility, and concentration. Eosin-Nigrosin staining was prepared to evaluate morphology and viability. Ejaculates were split into two equal volumes and cryopreserved in two different extenders, using a one-step and a two-step approach. Ejaculates were gradually cooled to 4 °C in two hours, subsequently stored in liquid nitrogen vapor for twenty minutes (0.25 ml straws), and finally dropped into liquid nitrogen.ResultsPre-freeze evaluation showed thick, white samples with an average ejaculate volume of 450 µl (100-1000 µl), total motility of 59% (40–80%), viability of 69% (38–85%) and 58% (46–72%) normal spermatozoa. Mainly head (22%) and tail (19%) defects were detected on the Eosin-Nigrosin stain. Ejaculates were highly concentrated, nevertheless, due to the coagulum that caused high viscosity and non-homogenous fractions, only estimations of concentration could be made (1000 million/ml). After 24 h of storage, the post-thaw evaluation showed a loss of quality with an average post-thaw total motility of 15% (5–25%) using the one-step freezing medium, and 19% (5–30%) using the two-step medium. Average post-thaw viability was 15% (4–24%) and 21% (15–29%), respectively.ConclusionsThis report on ejaculates from bonobos obtained by rectal probe electro-ejaculation shows that semen parameters of this species are not completely similar to those of its sibling species, the chimpanzee. Further studies are necessary to develop an optimal protocol for the processing and cryopreservation of bonobo spermatozoa.

A Simple and Efficient Method to Cryopreserve Human Ejaculated and Testicular Spermatozoa in -80°C Freezer.

Human autologous sperm freezing involves ejaculated sperm, and testicular or epididymal puncture sperm freezing, and autologous sperm freezing is widely used in assisted reproductive technology. In previous studies, researchers have tried to cryopreserve sperm from mammals (rats, dogs, etc.) using a −80°C freezer and have achieved success. It is common to use liquid nitrogen vapor rapid freezing to cryopreserve human autologous sperm. However, the operation of this cooling method is complicated, and the temperature drop is unstable. In this study, we compared the quality of human ejaculation and testicular sperm after liquid nitrogen vapor rapid freezing and −80°C freezing for the first time. By analyzing sperm quality parameters of 93 ejaculated sperm and 10 testicular sperm after liquid nitrogen vapor rapid freezing and −80°C freezing, we found reactive oxygen species (ROS) of sperm of the −80°C freezer was significantly lower than liquid nitrogen vapor rapid freezing. Regression analysis showed that progressive motility, ROS, and DNA fragmentation index (DFI) in post-thaw spermatozoa were correlated with sperm progressive motility, ROS, and DFI before freezing. For the freezing method, the −80°C freezer was positively correlated with the sperm progressive motility. Among the factors of freezing time, long-term freezing was negatively correlated with sperm progressive motility and ROS. Although freezing directly at −80°C freezer had a slower temperature drop than liquid nitrogen vapor rapid freezing over the same period, the curves of the temperature drop were similar, and slight differences in the freezing point were observed. Furthermore, there were no statistically significant differences between the two methods for freezing testicular sperm. The method of direct −80°C freezing could be considered a simplified alternative to vapor freezing for short-term human sperm storage. It could be used for cryopreservation of autologous sperm (especially testicular sperm) by in vitro fertilization centers.Clinical Trial Registration: (website), identifier (ChiCTR2100050190).

Successful short-term sperm cryopreservation in brown-marbled grouper (Epinephelus fuscoguttatus) with the utility of ultra-freezer (-80°C).

Production of cryopreserved semen in fish generally requires liquid nitrogen (LN), which is not always easily available in remote areas. To reduce reliance on LN, the aim of the present study was to evaluate whether electric freezer could be a feasible LN-free alternative to cryopreserve brown-marbled grouper sperm. After loading, semen straws were put directly into freezers (-30 or -80°C) for freezing and then transferred to LN for storage. Compared with the conventional LN vapour freezing (straws were put horizontally 3cm above the surface of LN), there was a significant reduction in all tested post-thaw sperm quality parameters in samples frozen at -30°C for 10min, including kinetic parameters (total motility: 85.0% vs. 48.6%), viability (84.7% versus 51.7%), high mitochondrial membrane potential (86.4% vs. 63.7%), ATP content (106.9nM/109 cells vs. 72.9nM/109 cells) and hatching rate (86.3% vs. 45.7%), accompanied with an increasing lipid peroxidation level (MDA content: 11.9nM/109 cells vs. 4.9nM/109 cells). In contrast, frozen with -80 °C ultra-freezer (10min or 12hr) produced similar sperm quality parameters to those using LN, except that temporary storage (12hr) at -80°C yielded lower average path velocity. In conclusion, this study confirmed that -80°C ultra-freezer is an effective alternative to LN for sperm freezing in brown-marbled grouper.

Establishment of semen collection technique using electroejaculator and semen cryopreservation of Javan leopard (Panthera pardus melas Cuvier, 1809).

Background and Aim:The Javan leopard (Panthera pardus melas Cuvier, 1809) is a subspecies of Panthera pardus spp., spread across the African and Asian regions. Information on reproductive aspects is crucial for wild animals, including the Javan leopard. In this study, we aimed to developelectroejaculator (EE) techniques and evaluate cryopreservation success in Javan leopard semen.Materials and Methods:The semen of four adult Javan leopards was collected once a week using EE. Placement of the EE probe in the rectum was performed after ultrasound imaging (ultrasonography) to determine the prostate body location. The semen obtained was then evaluated macroscopically and microscopically. Three Javan leopards were used for cryopreservation. The ejaculate was divided into two parts [i.e., one part diluted with AndroMed® (Minitüb, Tiefenbach, Germany) and the other part with Steridyl®(Minitüb, Tiefenbach, Germany)] at a 1:1 ratio immediately after collection and evaluation. The semen was then packed in a 0.25 mL MiniStraw® (Minitüb, Tiefenbach, Germany) then equilibrated at 4°C for 2 h. After equilibration, the straw was then frozen in liquid nitrogen vapor. Frozen semen was then stored in containers until further evaluation.Results:The results showed that ejaculation response occurred at all levels of stimulation, while erections did not always occur. The fastest ejaculation and erection occurred at the fourth voltage. The macroscopic evaluation showed that the semen volume was 0.80±0.26 mL, cloudy white, pH 7.44±0.14, and with watery semen consistency. The microscopic evaluation showed that the sperm motility was 66.98±0.39%, with sperm viability of 75.6±1.79%. Sperm concentration was 62.17±46.95×106 mL−1 with a total concentration of 42.14±23.51×106 cells. Normal sperm morphology is only 40.72±6.26%.Conclusion:This study concluded that the development of a semen collection technique using an EE preceded by imaging of the EE probe location using ultrasound was effective for the ejaculation of Javan leopards. The characteristics of the semen of the Javan leopard showed moderate semen volume, sperm motility, and viability. Javan leopard showed low sperm concentration and normal sperm morphology.

액체질소에 의한 산소결핍 질식사

Asphyxia typically occurs due to oxygen deficiency in confined spaces. A male researcher died in a minor car accident. He had loaded a container filled with liquid nitrogen in his car the day before the accident. Clinical analysis of blood showed that blood alcohol concentration(BAC), carboxyhemoglobin (CO-Hb), drugs and toxins were negative. During the car investigation, it was found that there was frost around the lid of the liquid nitrogen container with a trace amount of liquid nitrogen left. To determine the cause of death, the simulation test was performed by reconstructing the situation of liquid nitrogen leaking in a confined space. The results showed that liquid nitrogen in the container was continuously evaporated and reduced the oxygen concentration in the car. After 20 min the state of oxygen deficiency was reached and then the oxygen concentration was cut down to 6.5 % within 10 hours. It was concluded that the cause of death was oxygen-deficient circumstance resulted from the evaporation of liquid nitrogen in the car. In order to determine the cause of death, it is necessary to perform a simulation test considering all potential factors that may affect the accident.

Banking of AT-MSC and its Influence on Their Application to Clinical Procedures.

Processing of MSCs to obtain a therapeutic product consists of two main steps: 1) the in vitro expansion of the cells until an appropriate number of them is obtained, and 2) freezing and storage of the expanded cells. The last step is critical and must be optimized so that after thawing the cells retain all their physiological properties including the secretory function. In this paper, we evaluated physiological parameters of AT-MSC’s after a full cycle of their processing, particularly freezing and storing at the liquid nitrogen vapor temperature. Based on the recovered proliferative and secretory capacities of the thawed cells, we have designed the optimal technique for processing of MSCs for clinical applications. In our work, we tried to select the best DMSO-based cryoprotectant mixture on the base of post thawing fully retain their properties. We have demonstrated the effectiveness of the use of DMSO in various configurations of the constituent cryoprotective fluids. We have also shown that AT-MSCs that show control levels in most standard tests (viability, shape, culture behaviour, and proliferative properties) after thawing, may show transient variations in some important physiological properties, such as the level of secreted growth factors. Obtained results let us to indicate how to optimize the AT-MSC preparation process for clinical applications. We suggest that before their clinical application the cells should be cultured for at least one passage to recover their physiological stability and thus assure their optimal therapeutic potential.

Cryogenic wicking of liquid nitrogen in the metallic screens with different weave densities

Liquid wicking in porous media has been applied to the management and transportation of propellant in microgravity. The flow and heat transfer characteristics during wicking of cryogenic fluid in the metallic screens play significant roles in determining the wicking performance. However, the experimental investigations of cryogenic wicking are relatively rare due to experimental difficulties. In the present study, the experimental visualization of wicking of liquid nitrogen is carried out, where five typical Dutch Twill Weaves (DTW) with different weave densities are employed as the test samples. The evolutions of the wicking front during cryogenic wicking are experimentally traced. Moreover, the cryogenic wicking in the DTW has been numerically investigated by considering the thermal non-equilibrium equation in the porous media, which yields a good agreement between the experimental and numerical results due to better accounting the evaporation of liquid nitrogen resulted from the heat conduction of the DTW. As an example, the wicking height of liquid nitrogen in the DTW 200 × 1400 reaches a maximum wicking height of about 0.0851 m, which is improved by about 36.4% ∼ 75.0% compared to the results in the literature. Furthermore, the effect of the weave densities of the DTWs on the flow and heat transfer characteristics are investigated, which provides useful information for the screen channel liquid acquisition device of cryogenic propellant.

Numerical simulation of controlled precision cryosurgery using argon Joule–Thomson and liquid nitrogen evaporation cryoprobes

Cryoablation is the most commonly used type of cryosurgery. Today, this treatment is insufficiently automated and therefore less accurate than alternative technologies that significantly limits and gradually reduces the scope of its application. However, from low temperature engineering point of view cryosurgery has the untapped potential of the improving its accuracy. In this paper three major methodological problems of cryosurgical equipment and procedure improving are considered: the impact on target isotherm location (1) of the soft biotissue moisture content solidification latent heat variation, (2) of the cryoprobe constructions and operating at maximum cooling power including the fluid cryogen features and the accuracy of boundary condition replacing a cryoprobe and (3) target isotherm fixation approach by modifying the cryoprobe operating mode. The numerical results showed that the target isotherm location depends insignificantly on different moisture content of soft biotissue, then it is not a limiting factor for cryoexposure prediction. Also numerical results showed that the theoretically minimum temperature of cryoprobe surface significantly overestimates their movement, this leads to a significant difference between the experimental and simulation results. The typical shape and movement of cryonecrosis isotherm at refined maximum cryoprobe cooling power for two typical constructions of cryoprobes and two cryogens are presented. The novel algorithm of modifying the cryoprobe operation mode for controlled precision cryosurgery is proposed and validated. All this complements the previously obtained data and shows the prospects for performing precision cryosurgery. The total average uncertainty of target isotherm location during the studied preliminary predicting stage of temperature field movement (virtual cryoablation) is estimated no more than ±0.41 mm. This information is expected to be useful for improving the quality of cryosurgery planning algorithms (e.g. for tumor treatment).

A modeling study of spatial and temporal frost growth on the edge of windward fins for a tube-finned heat exchanger

Frosting on the surface of tube-finned heat exchangers is a serious and common problem in the fields of air source heat pump and vaporization of liquid nitrogen. To reveal the frosting characteristics, a frosting model on the edge of windward fins was developed. Based on the previously reported experimental visualization results, the model was simplified by regarding the flat surface of the edge of windward fins to a half-cylindric surface. The predicted frost thickness and frost mass according to the proposed frosting model agreed well with the previously reported experimental data. The numerical results showed that frost layer on the edge of a windward fin grew the fastest along the opposite airflow direction and gradually slower with the increasing θ, angular position relating to the center axis of fin, showing a streamlined profile. The frost thickness, density, and surface temperature all decreased by 31.9%, 19.0%, and 2.2 °C, respectively, while θ increased from 0° to 90° at the end of a 3,600 s frosting period. Additionally, the average water vapor mass flux, total, sensible, and latent heat flux decreased by 17.6%, 17.9%, 18.1%, and 17.6%, respectively. The frosting surface area, frosting rate, total, sensible, and latent heat transfer rates increased by 141.0 times, 119.6 times, 119.0 times, 118.6 times, and 119.8 times, respectively. This study is meaningful for the accurate control of defrosting and thus energy saving.

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.

Experimental study on the migration and expansion of liquid nitrogen in loose media with different dip angles

ABSTRACT The goaf is the main disaster area for natural fires in mines, and liquid nitrogen has become an essential medium for fire prevention in goafs due to its excellent cooling and inerting properties.At present, there is no in-depth research on the transport and vaporization of liquid nitrogen after injection into the goaf, and it is impossible to quantitatively evaluate the cooling inerting effect after liquid nitrogen injection. To address the above problems, this project proposes to carry out the following research: to study the effects of different inclination angles on the transport and vaporization of liquid nitrogen and temperature distribution in the loose medium in confined space by monitoring the temperature field changes during the pressure injection of liquid nitrogen in the loose medium in confined space.The results show that the temperature field distribution is very uneven after liquid nitrogen is injected into the loose medium in the restricted space.It is found that the vaporization rate of liquid nitrogen decreases with the increase of diffusion time and diffusion area, and the instantaneous vaporization rate of liquid nitrogen decreases with the larger inclination angle under the same injection position. Compared with the horizontal state, the maximum vaporization rate decreased 84.79% when the maximum inclination angle was 15°, which indicates that the vaporization rate was greatly affected by the inclination angle of the surface. In the vertical direction of the experimental box, the cooling area of liquid nitrogen in the experimental box is mainly concentrated within 0.05 m height from the bottom of the experimental box, and the changes of the “cooling restricted zone” after liquid nitrogen injection into the goaf are further explained during the experiment.It is found that the “cooling restricted zone” mainly exists in the upper part of the goaf, under the inclined condition, and the area increases with the increase of the inclination angle, so the increase of the angle is not conducive to the effective cooling effect of liquid nitrogen.The research results of the project will help to improve the theory of liquid nitrogen fire prevention in mines and provide theoretical support for the fire prevention technology of liquid nitrogen direct injection in goafs.

Changes in Structure and Corrosion Resistance of Cryogenically Treated WC-Co Cemented Carbides

The aim of this study was to evaluate changes in the corrosion resistance and microstructure of deeply cryogenic treated (DCT) cemented carbides (CC). Changes in corrosion resistance were determined by potentiodynamic testing in artificial mine water. Microstructural changes were characterized using X-ray diffraction analysis. Four sorts of WC-Co cemented carbides with different WC grain sizes and volume fractions of cobalt binder were used for the experiment. Specimens were divided into two groups. The first group of specimens was deeply cryogenic treated (DCT) at -186°C in dry liquid nitrogen vapor. The second group of specimens was tempered at 450°C. The results of the experiments show that DCT increased the amount of ε-Co in all the types of CC compared to the tempered group of specimens. This microstructural change and a change in the micro-stresses in the surfaces of the specimens contributed to increasing the corrosion resistance of CC after DCT.

Cell Viability Assessment Using Fluorescence Vital Dyes and Confocal Microscopy in Evaluating Freezing and Thawing Protocols Used in Cryopreservation of Allogeneic Venous Grafts.

The authors present their contribution to the improvement of methods suitable for the detection of the freezing and thawing damage of cells of cryopreserved venous grafts used for lower limb revascularization procedures. They studied the post-thaw viability of cells of the wall of cryopreserved venous grafts (CVG) immediately after thawing and after 24 and 48 h culture at +37 °C in two groups of six CVG selected randomly for slow thawing in the refrigerator and rapid thawing in a water bath at +37 °C. The grafts were collected from multi-organ and tissue brain-dead donors, cryopreserved, and stored in a liquid nitrogen vapor phase for five years. The viability was assessed from tissue slices obtained by perpendicular and longitudinal cuts of the thawed graft samples using in situ staining with fluorescence vital dyes. The mean and median immediate post-thaw viability values above 70% were found in using both thawing protocols and both types of cutting. The statistically significant decline in viability after the 48-h culture was observed only when using the slow thawing protocol and perpendicular cutting. The possible explanation might be the “solution effect damage” during slow thawing, which caused a gentle reduction in the graft cellularity. The possible influence of this phenomenon on the immunogenicity of CVG should be the subject of further investigations.

INNOVATIVE ULTRA-RAPID VITRIFICATION METHOD FOR FIVE OR FEWER TESTICULAR SPERMATOZOA FROM NON-OBSTRUCTIVE AZOOSPERMIC MEN AFTER MICROSURGICAL TESTICULAR SPERM EXTRACTION (MICRO-TESE)

Clinical outcomes of ICSI when using only five or fewer testicular spermatozoa after cryopreservation have been unsuccessful and are considered to be inferior to those using testicular fresh spermatozoa from Micro-TESE. A possible cause of these poor results has been the lack of a successful freezing technique. In these cases, repeated Micro-TESE and simultaneous oocyte pick up has been the only available treatment. We developed an innovative ultra-rapid vitrification method for five or fewer testicular spermatozoa from non-obstructive azoospermic men after Micro-TESE. This study included 113 non-obstructive azoospermic men using Micro-TESE, it was conducted retrospectively at St. Mother Clinic between 2011 and 2018. Each motile spermatozoon was carefully aspirated tail first into the pipette, put into a 2-μl microdroplet media of the vitrification medium near the tip of the Cryotop (Kitazato Corporation, Tokyo, Japan) submerged in liquid nitrogen vapor for 2 min and then immediately plunged into liquid nitrogen. The vitrified spermatozoa were warmed by dipping them into a droplet media. Successfully recovered motile sperm were selected and used for ICSI. Number of patients, transfer cycles and collected sperms were 113, 192 and 560. Mean age of patients and their wives were 32.0±3.7y and 28.4±5.8y. Clinical pregnancy rate, miscarriage rate, live birth rate and number of live offspring were 24.0% (46/192), 19.6% (9/46), 19.3% (37/192) and 37 (Male: Female = 17: 20). Sperm recovery rate and survival rate were 90.3% (506/560) and 70.4% (356/506). Fertilization rate and mean number of transferred embryos were 51.6% (99/192) and 1.73 (1-2). Mean gestational weeks and mean body weight at birth were 39.23±5.27w and 2852.31±314.28g. No congenital anomalies were observed in any of the babies. This method for cryopreserving five or fewer spermatozoa from non-obstructive azoospermic men showed a recovery rate above 90% and a survival rate of about 70% which is the highest data ever been reported. The clinical pregnancy rate was 24.0% and 37 healthy babies were born.

Linking in‐situ and ex‐situ populations of threatened amphibians through genome banking

AbstractGenetic variation of the endangered Puerto Rican crested toad (PRCT; Peltophryne lemur) has dwindled over time in both wild and captive populations, leading to long‐term sustainability issues for the recovery program. To address this challenge, we propose that PRCTs can be used as a model species to show how in‐situ and ex‐situ populations can be linked through sperm biobanking and gamete transfer, expanding genetic variability of both populations. Male toads (n = 10) in Guayanilla, Puerto Rico were administered human chorionic‐gonadotropin (hCG) and luteinizing‐hormone‐releasing‐hormone analog (GnRHa) to stimulate spermiation. Sperm was collected noninvasively from 9/10 males, cryopreserved in 10% trehalose with either 10% N,N‐dimethylformamide (DMFA) or 10% dimethyl sulfoxide (DMSO), and transported in liquid nitrogen vapor to the National Amphibian Genome Resource Bank at Mississippi State University. Ultrasonography was used to identify females (n = 3) with mature oocytes for GnRHa‐induced oviposition of eggs for in‐vitro fertilization (IVF). Post‐thaw sperm motility was 28% and 25% for sperm cryopreserved with DMFA or DMSO, respectively. Of the 9,672 eggs used for IVF, 4% (n = 306/6,981) were fertilized with frozen–thawed sperm, compared with 20% (n = 525/2691) fertilized with fresh sperm controls. Overall, 46 toadlets were produced from frozen–thawed sperm. After 4.5 months of headstarting, 14 juvenile toads produced from various genetic crosses using frozen–thawed sperm were released to new sites in the wild, introducing unique genetic representation and new founder lines. After 1.5 years, 24 adult toads produced using frozen–thawed sperm continue to thrive in the captive collection, and one of these males has now produced an F2 generation of offspring with 5,085 tadpoles released to the wild. This transformational study is the first to produce reproductively viable adult PRCTs using cryopreserved sperm from hormonally‐induced wild males and captive females, introducing innovative methods that link in‐situ and ex‐situ populations of endangered amphibians to revolutionize genetic management.