Nitrogen Tank Research Articles

장기 저장 후 느티만가닥버섯(Hypsizygus marmoreus) 균주의 균사 생존력과 재배 특성에 대한 연구

본 연구는 다양한 조건에서 장기 보존한 느티만가닥버섯 균주의 균사 생존력과 재배성능 검증을 수행하였다. 균사 생존력은 장기 보존된 균주를 9, 21, 33, 45개월 단위로 해동하여 PDA 평판배지에 접종한 다음 1, 2, 3, 4, 5차 계대배양에 따른 균사 생장길이를 측정하여 검증하였다. 액체질소에 보존한 균사를 해동 직후 실시한 1차 계대배양에서는 동결보호제로써 Glycerol 10%를 단용으로 처리한 실험구에서 균사생장이 저조한 결과를 나타내었으나, 5차 계대배양에서는 버섯 균사의 생장력이 다른 실험구와 동일한 수준으로 회복되어, 저장방법에 따른 균사의 생존력에는 큰 영향이 없는것으로 판단된다. 그러나 느티만가닥버섯을 액체 질소에 장기간 보존하고자 할 때는 동결보호제로 사용되는 Glycerol 10%에 Trehalose 5%를 첨가하여 보존해야 균사의 생존력을 유지하는데 도움이 된다. 장기 저장에 따른 느티만가닥버섯 균주의 재배 성능 시험에서는 <TEX>$4^{\circ}C$</TEX> 사면배지에 보호제로써 mineral oil을 사용하는 것이 사면배지 자체로 보존하는 것에 비해 수확량이 감소하는 결과를 나타내므로 장기 저장시에 mineral oil을 사용하는 것은 지양해야 할 것으로 판단된다. Mycelial viability and cultivation characteristics of strains of Hypsizygus marmoreus after long-term storage were investigated. The experimental conditions were the storage at <TEX>$4^{\circ}C$</TEX> using a slant culture technique with or without mineral oil, and in liquid nitrogen tank in the presence of 10% glycerol or 10% glycerol with 5% trehalose. The myceila of four strains of H. marmoreus were thawed at 9, 21, 33, and 45 months after beginning of the storage, and then the growth of the mycelia was measured on a PDA plate with serial transfers to new plate for the recovery. The mycelial growth data after 45 months showed that the mycelia were mostly viable but not fully active particularly when they were stored in liquid nitrogen with 10% glycerol. The growth activity could be fully recovered after second transfer to new PDA plate. Cultivation of mushroom fruiting body using the recovered mycelia also demonstrated that the storage methods employed in this work were applicable for the long-term storage of H. marmoreus.

Development and testing of a zero stitch MLI blanket using plastic pins for space use

New types of MLI blanket have been developed to achieve high thermal performance while maintaining production and assembly workability equivalent to the conventional type. Tag-pins, which are widely used in commercial applications to hook price tags to products, are used to fix the films in place and the pin material is changed to polyetheretherketone (PEEK) for use in space. Thermal performance is measured by using a boil-off calorimeter, in which a rectangular liquid nitrogen tank is used to evaluate the degradation at the bending corner and joint of the blanket. Zero-stitch- and multi-blanket-type MLIs show significantly improved thermal performance (ɛeff is smaller than 0.0050 at room temperature) despite having the same fastener interface as traditional blankets, while the venting design and number of tag-pins are confirmed as appropriate in a depressurization test.

Effect of Caffeine Added to the Activating Solution on Sperm Motility of Fresh and Thawed Semen of Pacu,Piaractus mesopotamicus, and Curimba,Prochilodus lineatus

AbstractThe aim was to evaluate the effect of different concentrations of caffeine added in activating solution over sperm motility in fresh and thawed semen of pacu,Piaractus mesopotamicus, and curimba,Prochilodus lineatus. The activating solutions were prepared with sodium bicarbonate solution of 0.76% (NaHCO3) and caffeine was added at concentrations of 2.5, 5.0, 10.0, and 20.0mM. As control, a solution ofNaHCO30.76 without caffeine was used. Eight males of pacu and 20 males of curimba were used. Aliquots of 200 μL of semen were diluted in 800μLextender solution (DMSO10% andBTS5%), placed in 0.5mLstraws and cryopreserved for 7 d in a liquid nitrogen tank. There was a linear increase in sperm motility for fresh semen of pacu, and for curimba fresh and thawed semen (P&lt; 0.05), due to the increase in the concentration of caffeine. There was a quadratic response for duration of motility for thawed semen of pacu and for fresh semen of curimba (P&lt; 0.05), respectively. These results indicate that addition of caffeine in the activator solution can improve sperm motility parameters, however, is dependent on the species and concentration used.

The sperm’s tale

The sperm’s tale

Performance of Variable Energy Cyclotron Centre superconducting cyclotron liquid nitrogen distribution system

The liquid nitrogen distribution at Variable Energy Cyclotron Centre, Kolkata, India K500 superconducting cyclotron uses parallel branches to cool the thermal shield of helium vessel housing the superconducting coil and the cryopanels. Liquid nitrogen is supplied to the thermal shields from a pressurised liquid nitrogen dewar. Direct measurement of flow is quite difficult and seldom used in an operational cryogenic system. The total flow and heat load of the liquid nitrogen system was estimated indirectly by continuous measurement of level in the liquid nitrogen tanks. A mathematical model was developed to evaluate liquid nitrogen flow in the parallel branches. The model was used to generate flow distribution for different settings and the total flow was compared with measured data.

The maintenance record of the KSTAR helium refrigeration system

Korea Superconducting Tokamak Advanced Research (KSTAR) has a helium refrigeration system (HRS) with the cooling capacity of 9 kW at 4.5 K. Main cold components are composed of 300 tons of superconducting (SC) magnets, main cryostat thermal shields, and SC current feeder system. The HRS comprises six gas storage tanks, a liquid nitrogen tank, the room temperature compression sector, the cold box (C/B), the 1st stage helium distribution box (DB#1), the PLC base local control system interconnected to central control tower and so on. Between HRS and cold components, there's another distribution box (DB#2) nearby the KSTAR device. The entire KSTAR device was constructed in 2007 and has been operated since 2008. This paper will present the maintenance result of the KSTAR HRS during the campaign and discuss the operation record and maintenance history of the KSTAR HRS.

15 THERMORESISTANCE SPERM TEST FOR EPIDIDYMAL STALLION SPERM FROZEN WITH TWO CRYOPROTECTANTS

The Peruvian Paso Horse has become a flagship product of Peru, for which it is very important to work on promoting and preserving their genetic material through semen. The objective of this study was to evaluate the thermoresistance of epididymal sperm of stallion frozen with 2 cryoprotectants (dimethylformamide and glycerol). The testes with the epididymides (n = 16) of 8 adult stallions in good health condition were obtained in the slaughter house and transport at isothermal conditions (30°C). Spermatozoa from the epididymis were recovered and incubated with lactated ringer's solution (Hartman solution) for 30 min, centrifuged at 2000 RPM for 15 min, and resuspended with an extender based on sucrose (7%) and egg yolk (3%) with different levels of cryoprotectants: dimethylformamide-DMF (T4%), glycerol-GLI (4%), and a combination of dimethylformamide-DMF (2%) + glycerol-GLI (2%) with an equilibrium time of 20 min at 5°C. The final dilution was 160 million motile spermatozoa placed on 0.5-mL straws. These straws were kept over liquid nitrogen fumes for 15 min over a tripod stand. After cryopreservation, the straws were stored in liquid nitrogen tanks (–196°C). The thawing process of the straws was initiated at 75°C for 7 s, immediately followed by an evaluation of sperm motility at 4 times 0, 30, 60, and 90 min in which sperm was incubated on water bath at 37°C (thermoresistance test). The data was evaluated (ANOVA) with the SAS 9.12 program (SAS Institute Inc., Cary, NC, USA) and, for the media, Tukey's test was carried out. The results showed an effect of the treatment on the motility of the frozen-thawed semen; dimethylformamide, the cryoprotectant, had the best performance. Table 1.Motility of frozen-thawed stallion epididymal sperm (thermoresistance sperm test)1

Effect of different avian egg yolk types on fertilization ability of cryopreserved common carp (Cyprinus carpio) spermatozoa

In spite of the fact that egg yolk from different avian species has successfully been used as an additive for the cryopreservation of sperm in mammalian species, its efficacy for cryopreserving fish sperm has not previously been tested comparatively. Therefore, the present study was carried out to determine the effect of egg yolks from different avian species, namely domestic chicken (Gallus gallus domesticus), turkey (Meleagris gallopavo) and quail (Coturnix coturnix), on post-thaw motility and fertilization ability of cryopreserved common carp spermatozoa. Egg yolks from chicken, turkey and quail were analysed for moisture, total fat, protein, cholesterol and phospholipid profile. Total fat and cholesterol contents of the turkey egg yolk were higher than chicken and quail egg yolks (p < 0.05). Semen was frozen according to conventional slow freezing procedure. The extender contained 350 mM glucose, 30 mM Tris and 5 % glycerol supplemented with different ratios of avian egg yolk (10, 15 and 20 %). Semen was equilibrated at 4 °C for 15 min and placed into 0.25-ml straws and frozen in liquid nitrogen vapour (for 10 min at −120 °C) and finally stored in liquid nitrogen (−196 °C) tank. The frozen spermatozoa were thawed in a water bath at 35 °C for 30 s. Fertilization was conducted using a ratio of 1 × 105 spermatozoa/egg. Cryopreservation experiments resulted in higher post-thaw motility and fertilization rates. Mean post-thaw motility of cryopreserved spermatozoa was between 45 and 80 %, and fertilization rates, expressed as the percentage of eyed embryos, ranged from 70 to 95 %. In conclusion, the present study showed that turkey and quail egg yolks are suitable alternatives to the chicken egg yolk for the cryopreservation of common carp spermatozoa.

Bone marrow stroma‐mediated resistance to FLT3 inhibitors in FLT3‐ITD AML is mediated by persistent activation of extracellular regulated kinase

A consistent pattern of response has been observed when FMS-like tyrosine kinase 3 (FLT3) tyrosine kinase inhibitors (TKIs) have been used as monotherapy to treat patients with relapsed or refractory FLT3- internal tandem duplication (ITD) acute myeloid leukaemia (AML). Circulating blasts are cleared from the peripheral blood, while bone marrow blasts are either unaffected or are cleared from the marrow at a much slower rate. We used an in vitro model of FLT3-ITD AML blasts co-cultured with normal human bone marrow stromal cells to investigate the basis for this dichotomous response pattern to FLT3 inhibitors. We have found that in blasts on stroma, potent FLT3 inhibition predominantly results in cell cycle arrest rather than apoptosis. The anti-apoptotic effect is mediated through a combination of direct cell-cell contact and soluble factors. The addition of exogenous FLT3 ligand (FL) augments the protection, primarily by shifting the 50% inhibitory concentration for FLT3 inhibition upwards. Cytokine-activated extracellular regulated kinase (ERK), rather than STAT5, appears to be the most important downstream signalling protein mediating the protective effect, and inhibition of MEK significantly abrogates stromal-mediated resistance. These findings explain the phenomenon of peripheral blood versus bone marrow blast responses and suggest that the combination of potent FLT3 inhibition and MEK inhibition is a promising strategy for the treatment of FLT3-ITD AML.

Cryopreservation of scaly carp (Cyprinus carpio) sperm: effect of different cryoprotectant concentrations on post-thaw motility, fertilization and hatching success of embryos

The aim of the present study was to determine the effect of various cryoprotectants on post-thaw sperm quality and fertilizing capacity of cryopreserved scaly carp (Cyprinus carpio) semen. The present study focused on freezing of scaly carp sperm utilizing a practical and inexpensive protocol for aquaculture. Semen was diluted with Kurokura’s extender composing 3.6 g/l NaCl, 10 g/l KCl, 0.22 g/l CaCl2, 0.08 g/l MgCl2 and 0.2 g/l NaHCO3. The extender contained three different cryoprotectants (DMSO, DMA and egg yolk) at ratios of 5, 10 and 15 %. Semen was placed into 0.25-ml straws and exposed to liquid nitrogen vapor (−120 °C) using an insulated box with an adjustable tray for 10 min and then plunged into liquid nitrogen (−196 °C) tank. The thawing process was performed in a water bath at 40 °C for 10 s. The results indicated that type of cryoprotectants and their concentrations are rather effective in scaly carp sperm cryopreservation on post-thaw sperm quality, while they are very important in order to obtain high fertilization rates. The highest fertilization rate was determined as 96.4 ± 0.15 % with 15 % egg yolk, while the highest hatching rate was determined as 99.3 ± 0.80 with 15 % DMA. In conclusion, the applied cryopreservation method for scaly carp sperm is suitable to fertilize high amounts of eggs.

Comparison of different cooling rates for fibroblast and keratinocyte cryopreservation

Easy, cost-effective and reliable cryopreservation protocols are crucial for the successful and effective application of tissue engineering. Several different protocols are in use, but no comprehensive comparisons across different machine-based and manual methods have been made. Here, we compare the effects of different cooling rates on the post-thaw survival and proliferative capacity of two basic cell lines for skin tissue engineering fibroblasts and keratinocytes, cultured and frozen in suspension or as a monolayer. We demonstrate that effectiveness of cryopreservation cannot be reliably determined immediately after thawing: the results at this stage were not indicative of cell growth in culture 3 days post-thaw. Cryopreservation of fibroblasts in an adherent state greatly diminishes their subsequent growth potential. This was not observed when freezing in suspension. In keratinocytes, however, adherent freezing is as effective as freezing in suspension, which could lead to significant cost and labour savings in a tissue-engineering environment. The 'optimal' cryopreservation protocol depends on cell type and intended use. Where time, ease and cost are dominant factors, the direct freezing into a nitrogen tank (straight freeze) approach remains a viable method. The most effective solution across the board, as measured by viability 3 days post-thaw, was the commonly used, freezing container method. Where machine-controlled cryopreservation is deemed important for tissue-engineering Good Manufacturing Practice, we present results using a portfolio of different cooling rates, identifying the 'optimal' protocol depending on cell type and culture method. Copyright © 2013 John Wiley & Sons, Ltd.

Pressure variations in a cryogenic liquid storage tank subjected to periodic excitations

The pressure change in a partially filled liquid nitrogen tank, subjected to periodic lateral forces, has been investigated experimentally. The cylindrical tank has a radius of R=0.148m and is filled up to 69% of the total volume (43×10−3m3). Six different sloshing conditions were considered, with the wave amplitude b of the first asymmetric mode ranging from b/R=0.12 up to wave breaking conditions of b/R⩾0.54. The tank was pressurized with nitrogen vapor and the pressure at sloshing initiation was in general pi≈250kPa. Pressure drops in the order of 100kPa have been measured and the dependency of these pressure drops on wave amplitude has been determined. The integrated temperature sensors allowed to measure the vapor temperature change and, with a high resolution, the thermal boundary layer in the liquid. The effective diffusion coefficient model of Das and Hopfinger (2009) [2] has been extended and allows calculating the temperature distribution in the thermal boundary layer, as well as the pressure drop as a function of the effective diffusion coefficient. A novel result is that the calculated temperature distribution in the thermal boundary layer deviates from the measured one, relating to a stop in the pressure drop. The sloshing Nusselt number NuS=De/D0 is shown to correlate well with a Reynolds number that contains the wave amplitude.

Development of a Prototype for Extra-Virgin Olive Oil Storage with Online Control of Injected Nitrogen

<abstract> <bold><sc>Abstract.</sc></bold> The conservation of oil is a very complex operation because, to maintain the quality level over time, it is necessary to take precautions to avoid increased acidity and the onset of rancidity caused by oxidation, as well as problems related to the anaerobic fermentation of suspended matter in the oil or deposited on the bottom of the storage vessels. This article describes the development of a prototype integrated olive oil storage system. The storage system consists of a nitrogen gas generator, three stainless steel tanks, and a feedback controller for nitrogen concentration and tank temperature. In the first experiment, three tanks were used to test the functionality of the system. The experimental design was based on the creation of a different modified atmosphere in the headspace of each tank using the nitrogen gas generator and feedback controller. In the second experiment, four tanks were used to validate the functionality of the prototype system using a different type of extra-virgin olive oil. The results of this research show that the prototype performed well in terms of its ability to detect and adjust the atmosphere of the tank headspace. In the first experiment, the oxygen percentage in the headspace of the three tanks remained between 0.7% and 0.9%. Chemical analyses of acidity, total phenol content, and peroxide index showed that the controlled headspace made it possible to maintain acidity levels within the range of 0.30% to 0.37%, total phenol content in the range of 247 to 252 ppm (gallic acid equivalent), and peroxide index values in the range of 8.80 to 9.70 meq O₂ per kg of oil during the experiment. The proposed prototype provided effective control and regulation of the oxygen concentration in the headspace of the tanks and can help maintain the chemical characteristics of extra-virgin olive oil over time. The second experiment confirmed that the prototype storage system was able to maintain the chemical characteristics of the stored extra-virgin olive oil.

Facial Rejuvenation With Staged Injections of Cryopreserved Fat and Tissue Cocktail

Facial rejuvenation by autologous fat transfer is common in aesthetic plastic surgery. The main drawback is progressive resorption, requiring repeated harvesting and microfat grafting. The authors present a method for cryopreservation of excess harvested fat and tissue to enable subsequent use of previously harvested excess material. Fat grafts were harvested using a 50-mL syringe and a 3- or 4-mm cannula. A tissue "cocktail" composed of dermis, fascia, and fat was prepared from excised scar tissue, tissue from abdominoplasty, or tissue from reduction mammaplasty. Cocktail specimens were placed in sterile tubes, immersed in a liquid nitrogen tank (-196°C), and stored at -80°C. At 3- to 6-month intervals, repeated cryopreserved fat graft injections were performed. Patients were evaluated by comparing preoperative and postoperative photographs. Between 2000 and 2010, a total of 5199 cryopreserved fat or tissue injections were performed in 2439 consecutive patients (age range, 19-80 years). Nasolabial folds and lips were the most common injection sites. Clinical outcomes were satisfactory, and improved contour was achieved in most patients after repeated injections. Cryopreservation of excess tissue for future injection is promising since repetitive injections are often required after resorption of microfat grafts. In our study, the survival of cryopreserved tissue cocktail or fat was comparable to that of fresh fat grafts and is therefore an effective adjuvant method for facial rejuvenation.

Seed cryopreservation of the native cacti Discocactus zehntneri, Pilosocereus gounellei and Stephanocereus luetzelburgii from Bahia, Brazil

The native cacti species from Bahia, Discocactus zehntneri, Pilosocereus gounellei and Stephanocereus luetzelburgii , as other members of the Family Cactaceae, have been dramatically affected by illegal traffic, and with the destruction and fragmentation of their habitats. Considering the potential extinction risks for endangered species, and the several applications of these species, some actions on ex situ conservation of cacti are required. Thus, this work aimed to evaluate the physiological quality of seeds from D. zehntneri, P. gounellei and S. luetzelburgii stored in liquid nitrogen (-196°C) for 0, 7 and 30 days. For this purpose, seeds of the three species were transferred to cryovials and then immersed directly in liquid nitrogen for 7 or 30 days. After the storage period, the seeds were removed from the nitrogen tank, and thawed for 1 h at ambient temperature and subsequently chemically sterilized. The seeds were inoculated onto Petri dishes lined with two layers of germitest paper. The germination rate of the three species was not reduced by exposure to liquid nitrogen, as well as D. zehntneri that had the best results for speed and germination rate observed in seeds stored for 30 days. Keywords : Cacti, cryoconservation, ex situ conservation, germination African Journal of Biotechnology Vol. 12(21), pp. 3250-3254

Establishment of Simple and Efficient Methods for Plant Material Harvesting and Storage to Allow DNA Extraction from a Myrtaceae Species with Medicinal Potential

Genomic DNA isolation is an essential procedure to allow many genetic applications and analyses like that using molecular markers. Obtaining good quality DNA samples is the first step to succeed in such analysis and it depends on effective procedures for harvesting and preserving the plant material, and also for DNA extraction. The use of fresh material is ideal for DNA isolation, however, in studies that involve the harvesting of wild plants this is not always possible, since in most cases, populations are distant from the research laboratory. An alternative is freezing the plant material in liquid nitrogen in the field, but this practice is not feasible in many cases, given the difficulty and danger of transporting the liquid nitrogen tank on places of difficult access. In this study, we present four simple, efficient and low cost methodologies that have been successfully tested for a Myrtaceae species with medicinal potential collected at different biomes, Atlantic Rainforest and Brazilian Savanna (Cerrado). Among the main advantages observed are the reduced use of liquid nitrogen, the use of inexpensive materials and ease of transport and storage of samples. The methods presented here can potentially be applied to other species of this botanical family.

84 EFFECT OF A STRESSOR ON CANINE SPERM DNA FRAGMENTATION USING THE SPERM CHROMATIN DISPERSION TEST

Recently, a new procedure for the analysis of sperm DNA fragmentation has been developed for human and different mammalian species (Sperm-Halomax®), based on the sperm chromatin dispersion test (SCDt); however, no studies has been performed specifically on canine frozen–thawed-stressed semen but is there for cooled semen. The aim of this work was to assess the effect of a stressor (24 h in an oven at 38°C) on canine frozen–thawed semen using the SCDt to resemble what happens in the female reproductive tract. For this purpose, ejaculates were collected by digital manipulation from 4 healthy beagle dogs and the sperm-rich fraction of the ejaculates from 3 different dogs was pooled each time. All the pooled semen samples (n = 4) used presented physiological values concerning to routine semen parameters (motility, morphology, and sperm concentration). After evaluation, semen samples were centrifuged and the sperm pellet resuspended to a final concentration of 100 × 106 sperm mL–1 in 2 steps with CaniPRO Freeze (Minitub, Tiefenbach, Germany). Sperm were slowly cooled to 5°C and then loaded into 0.5-mL plastic straws. After that, straws were frozen in liquid-nitrogen vapours for 10 min and stored into a nitrogen tank. Straws were thawed in a water bath (30 s/37°C) and incubated for 24 hours at 38°C before analysis. The sperm DNA fragmentation was assessed in fresh semen and frozen–thawed-stressed samples using the Sperm-Halomax® commercial kit specifically developed for canine semen (Halotech DNA SL, Madrid, Spain) following the manufacturer’s instructions. Slides were stained for green fluorescence staining and 500 sperm per slide were counted using fluorescence microscopy. The sperm DNA fragmentation index (%) was compared between fresh and frozen–thawed-stressed semen samples by ANOVA. Results were expressed as mean ± standard error of the mean. The results obtained showed that subjecting thawed semen to 24 h in an oven at 38°C significantly increased (P &lt; 0.05) DNA fragmentation compared with fresh semen (2.7% ± 0.2 v. 1.4 ± 0.1%). The stress factor was performed to simulate the viability of canine thawed sperm (12–24 h) when a bitch is inseminated with frozen semen. It would be interesting to perform further studies to relate sperm DNA fragmentation and fertility of frozen–thawed canine semen. In conclusion, frozen–thawed-stressed semen samples increased the sperm DNA fragmentation index measured using a SCDt. Further studies are needed to relate sperm DNA fragmentation with fertility rates or cryopreservation success.

76 THE EFFECT OF GLYCEROL CONCENTRATIONS IN EGG-YOLK CITRATE EXTENDER ON THE QUALITY OF CRYOPRESERVED NGUNI BULL SEMEN

There are bull shortages in South African poor rural areas. Artificial-insemination technology could play a significant role on breeding emerging farmer’s cattle. The objective of this study was to compare glycerol concentrations (0, 4, 8, or 12%) during freezing of Nguni bull semen to conduct AI in different villages. Semen was collected by electro ejaculator from 2 Nguni bulls of known and proven fertility. Collected semen samples were kept in a thermo flask (37°C) and transported to the laboratories within 10 min after collections. Semen samples were pooled and evaluated by Sperm Class Analyser® and allocated randomly per treatment. Semen was then diluted (1 : 2 v:v) with egg-yolk citrate extender supplemented with either 0% (negative control), 4, 8, and 12% of glycerol concentration or AndroMed® (positive control). Semen samples were equilibrated for 4 h at 5°C. After equilibration period, samples were loaded into 0.5-mL straws and placed horizontally into the controlled rate (–5, –8, –10, –12, –15, –25, –35°C min–1) from 5°C until target temperature of –80°C is reached. The frozen semen straws were stored in a liquid nitrogen tank (–196°C) until thawing. Treatment means were separated using Fisher’s protected t-test least, and data are presented as mean ± SD. There was a significant differences (P &lt; 0.05) between raw total sperm motility (83.3 ± 19.3) and frozen–thawed sperm with either 0% (0.0 ± 0.0), 4% (30.2 ± 13.4), 8% (47.9 ± 12.5), or 12% (61.5 ± 4.7) of glycerol and on AndroMed® (27.7 ± 17.8) group. Regardless of the glycerol concentrations used, the freezing-thawing process reduced (P &lt; 0.05) the Nguni total sperm motility rate compared to uncryopreserved sperm (83.3 ± 19.3). In conclusion, egg-yolk citrate extender supplemented with 12% glycerol yielded a better (P &lt; 0.05) total sperm motility rate (61.5 ± 4.7) as compared with the 0% (0.0 ± 0.0), 4% (30.2 ± 13.4), 8% (47.9 ± 12.5), and AndroMed® (27.7 ± 17.8) group. Further studies are required to test other levels of glycerol concentrations (&gt;12%) on freezing Nguni semen and conducting AI.

2. Roles of intracellular ice formation, vitrification of cell water, and recrystallization of intracellular ice on the survival of mouse embryos and oocytes

Although sperm were successfully cryopreserved in 1950, another 22 years elapsed before the successful preservation of a mammalian embryo. In 1963, I had carried out physical–chemical analyses indicating that if cells were cooled too rapidly, they would undergo lethal intracellular ice formation (IIF), and defined what is meant by “too rapidly” for a particular cell. To avoid IIF, the cell had to dehydrate during cooling, and the required rate of cooling depended chiefly on the permeability of a particular cell to water and on the temperature coefficient of that permeability. Between 1968 and 1972, Stanley Leibo and I turned our attention to the survival of yeast, mouse marrow stem cells, and Chinese Hamster tissue culture cells as functions of cooling rate and temperature. We found that plots of survival vs. cooling rate formed an inverted “U”, with high survivals at a medium rate, and lower survivals after very slow and very rapid cooling. We proposed a “Two-Factor” hypothesis which stated that the right hand arm of the inverted U was a result of IIF at high cooling rates. Interestingly, the optimum cooling rate for maximum survival varied over a wide range for the different cells. This was chiefly because the cells differed widely in water permeability and the temperature coefficient of that permeability. In fact if the known values of these parameters were used to generate the theoretical curves, the predicted relation between IIF and cooling rate agreed quite closely with the observed drop in survival vs. cooling rate. In 1971 David Whittingham reported the successful cryopreservation of mouse 8-cell embryos cooled at 50 °C/min in PVP. Leibo and I were excited but perplexed about these results – perplexed because our computations indicated that mouse embryos would undergo lethal IIF when cooled at that rate. We calculated that the cooling rate would have to be about 1 °C/min.. That turned out to be the case. With that change and the substitution of Me 2 SO for PVP, David, Stanley and I obtained very good in vitro and in vivo survival of 8-cell embryos cooled to −196 °C (or −269 °C). The ability to cryopreserve mouse embryos quickly had important applied consequences and important fundamental consequences. Perhaps, the most important direct application was that it permitted the long-term maintenance of thousands of mutant lines of mice in the form of preimplantation embryos in a few liquid nitrogen tanks. The indirect applications stemmed from the fact that essentially the same procedures worked for early embryos of over 20 other mammals, including humans. In the last decade, clinical interest has been emphasizing the cryopreservation of human oocytes. They have proved more difficult to freeze and so interest has shifted somewhat to cryopreserving them by vitrification. As discussed, one way to avoid lethal IIF is to cool cells slowly enough so that the freezable water in the cell flows out of the cell and freezes externally. An alternative approach is to avoid intracellular ice by vitrifying cellular water. Vitrification involves converting water into a non-crystalline or amorphous glass. To achieve this conversion, it has been the strong belief that cells have to be in solutions containing a very high concentration of glass-inducing solutes and have to be cooled very rapidly. In recent work, however, we have found that neither of these is true for mouse oocytes. The important factor is not the cooling rate – it is the warming rate, and the warming rate has to be very high. Furthermore, if the warming rate is very high, one can cut in half the concentration of solutes in the vitrification solution and still obtain high survivals.

117IN - Biomarker Development Needs Tissue Repositories: No Honey without Bees

ABSTRACT Pathologists have traditionally archived the tissues on which they have established the diagnoses for their patients. Like many other professional societies, the Swiss Society for Pathology has recommended that paraffin blocks be retained for a minimum of 20 years (40 years for pediatric tumors). In addition to paraffin blocks, fresh tumor and non-tumorous tissues can be frozen rapidly and kept for many years in freezers at -80°C or in nitrogen tanks. The research value of these archived tissues depends largely on the disposition of clinical information, on patient survival, and – ideally – on data concerning tumor response to treatment (chemotherapy, targeted treatments). With this information, combined with the modern tools of molecular pathology, notably deep sequencing, novel cancer markers with prognostic and/or predicitve values can be detected in historic cohorts and verified in independent historic cohorts, before ultimately being tested prospectively in “new” cancer patients (the “honey”). Certainly, this can only be successful, (i) if the material is adequately stored and quality-controlled (is there truely invasive carcinoma in the tissue block ?), (ii) if the logistics for clinical data management is professionally organized, and (iii) if there exists an understanding and a consent of the patients whose tissues are to be used for research. The first two tasks lay in the hands of professionals who run the bank (pathologists, technicians, data managers), and have to be compensated for their work (the “bees”). The third task needs to be solved at the level of the general population. The last few years have seen many fruitful discussions and conferences dedicated to establishing a consensus between the general population (patients) and the scientific community (researchers). A recent survey among 1600 French- and German-speaking Swiss citizens has shown that the population is highly- and quite unconditionally - in favor of biobanking for research, provided that patients are properly informed and that their (written) consent is asked. Disclosure The author has declared no conflicts of interest.