Conventional Slow Freezing Research Articles

Effect of supercooling accompanying the freezing process on ice crystals and the quality of frozen strawberry tissue

Degradation of agricultural product quality occurs hugely during the freezing process, since ice crystal formation with volume expansion and increased osmotic pressure between cell membranes damages their microstructure. Recently, it is studied a freezing accompanied supercooled state for the entire food product, which induces finer and more homogenous ice crystals and improved the quality of soy bean gels after thawing. In this study, texture, drip loss, and microstructural changes were measured in frozen-thawed strawberries prepared by freezing with supercooling. In addition, ice crystals were observed directly using synchrotron X-ray computed tomography in supercooled frozen strawberries. The results showed, although supercooled freezing has the potential to form finer ice crystals in strawberry tissues, the quality of frozen strawberries was not improved compared with conventional slow freezing. It assumed that a higher and momently ice nucleation caused by supercooling was promoted a drastic water migration and huge degradation on strawberry cells by osmotic pressure in the frozen strawberries.

101 Assessment of semen traits in servals (Leptailurus serval) and Canada lynx (Lynx canadensis)

Servals and Canada lynx are managed by species survival plans in North American zoos, but current populations are not sustainable. Increased knowledge of their reproductive biology would benefit breeding management and development of assisted reproductive techniques. The aims of our study were to (1) evaluate effectiveness of urethral catheterization and electroejaculation (EEJ) for semen collection; (2) characterise basal seminal traits; and (3) compare effectiveness of semen cryopreservation methods. Semen was collected from 6 servals and 9 Canada lynx via a urinary catheter (3.5 Fr×22 cm, inserted 15cm into the urethra), followed by EEJ under dexmedetomidine-ketamine anaesthesia. To assess the effect of seasonality on lynx seminal traits, semen was collected before (late January), during (mid-February to mid-March), and after (early April) the peak breeding season. Serval and lynx semen were frozen by conventional slow freezing (i.e. in 0.25-mL straws cooled to 4°C for 2h and frozen in LN vapor) in a soy lecithin-based (SOY) or egg yolk-based (TEY) extender with 4% glycerol and by ultra-rapid freezing (URF; direct pelleting into LN at ≈104°C/min) in SOY medium with 0.2M sucrose. To evaluate post-thaw sperm function in servals, heterologous IVF of domestic cat oocytes was performed, with cleavage rate assessed at 48h post-insemination. Data were analysed by one-way or repeated-measures ANOVA. Data are mean±standard deviation. Sperm recovery by urethral catheterization was negligible in both species, but EEJ allowed sperm collection in all males. Lynx seminal traits were similar during breeding and nonbreeding seasons. Testicular volume (4.81±1.17cm3) and sperm quality (13±11×106 sperm/ejaculate; 49±14% motility; 29±12% normal morphology; 74±13% acrosome integrity) were consistent with previous findings in the lynx genus. Post-thaw sperm quality in lynx has not yet been evaluated. In servals, testes volume was 6.56±2.11cm3 with good sperm quality for most males (46±36×106 sperm/ejaculate; 75±20% motility; 56±36% normal morphology; 84±7% acrosome integrity). Post-thaw, serval sperm acrosome integrity (31±15, 21±13, 24±13% at 0h for TEY, SOY, and URF, respectively; P>0.05) and motility (40±21% at 0h, 20±11% at 6h for TEY; 24±19% at 0h, 6±4% at 6h for SOY; 21±16% at 0h, 3±2% at 6h for URF; treatment: P>0.05; time: P<0.05; interaction: P>0.05) declined substantially. However, thawed sperm could fertilize domestic cat oocytes with no difference among treatments in cleavage success (53±6, 47±4, or 49±14%; TEY, SOY, and URF, respectively; P>0.05), indicating that standard freezing methods are effective in servals. Our findings provide zoos with valuable information about normative reproductive traits in both species. Supported by IMLS and the Roger & Kathy Gross Post-doctoral Fellowship.

Modification of cellular membranes conveys cryoprotection to cells during rapid, non-equilibrium cryopreservation.

Rapid cooling and re-warming has been shown promising to cryopreserve living cells, which cannot be preserved by conventional slow freezing methods. However, success is limited by the cytotoxicity of highly concentrated cryoprotective agents. Recent results have shown that cryoprotective agents do not need to suppress intracellular ice crystals completely to allow for survival after cryopreservation. Cryoprotective agents like DMSO or ethylene glycol can also lead to a tolerance of cells towards intracellular ice. It is however unclear by which mechanism this tolerance is achieved. These substances are also known to modulate properties of cellular membranes. It is shown here that cryoprotective DMSO and ethylene glycol have a clear influence on the mobility of lipids in the plasma membrane of HeLa cells. To isolate changes of the properties of plasma membranes from effects on ice formation, the membrane properties were modulated in absence of cryoprotective agents. This was achieved by changing their sterol content. In cells with elevated sterol content, an immobile lipid fraction was present, similar to cells treated with DMSO and ethylene glycol. These cells showed also significantly increased plasma membrane integrity after rapid freezing and thawing in the absence of classical cryoprotective agents. However, their intracellular lysosomes, which cannot be enriched with sterols, still got ruptured. These results clearly indicate that a modulation of membrane properties can convey cryoprotection. Upon slow cooling, elevated sterol content had actually an adverse effect on the plasma membranes, which shows that this effect is specific for rapid, non-equilibrium cooling processes. Unraveling this alternative mode of action of cryoprotection should help in the directed design of novel cryoprotective agents, which might be less cytotoxic than classical, empirically-found cryoprotective agents.

Effect of cooling rate on sperm quality of cryopreserved Andalusian donkey spermatozoa

The aim of this study was to evaluate the effect of different cooling rates on post-thaw quality of cryopreserved donkey spermatozoa. Eighteen ejaculates from six adult Andalusian donkeys (three ejaculates per donkey) were collected using an artificial vagina. Pooled semen samples (two ejaculates per pool) were divided into three aliquots, and frozen in Gent freezing extender using three different cryopreservation protocols (P): P1 (conventional slow freezing, as control): semen pre-cooled in an Equitainer for 2 h and frozen in liquid nitrogen (LN2) vapour; P2 (controlled pre-freeze cooling rate): semen pre-cooled at a controlled rate for 73 min and frozen in LN2 vapour; and P3 (rapid freezing) semen frozen immediately in LN2 vapour. After thawing at 37 °C for 30 s, semen samples were assessed for motility, morphology, acrosome and plasma membrane integrity; spermatozoa were also tested for DNA integrity. Significant (P < 0.01) differences were found between the cryopreservation protocols for all sperm parameters evaluated, except for DNA integrity. Semen samples frozen using P2 showed significantly (P < 0.01) higher values for sperm motility, morphology, sperm membrane integrity, and acrosome integrity. On the contrary, P3 reduced sperm motility (P < 0.01) and increased the percentage of spermatozoa with damaged plasma membrane (P < 0.001). In our study, we demonstrated that the sperm of Andalusian donkey is particularly sensitive to the cooling rate used before freezing. Furthermore, Andalusian donkey semen can be successfully cryopreserved using controlled cooling rates combined with freezing in LN2 vapour.

Slow and ultra-rapid freezing protocols for cryopreserving mouflon (Ovis musimon) and fallow deer (Dama dama) epididymal sperm

This study examines the effectiveness of two methods for cryopreserving post-mortem epididymal sperm - conventional slow freezing employing a short equilibration time with glycerol, and ultra-rapid freezing - from the wild ruminant species Ovis musimon (mouflon) and Dama dama (fallow deer). A Tris-citric acid-glucose (TCG) + 12% egg yolk-based medium was used for the conventional slow freezing of the fallow deer sperm, whereas a Tes-Tris-glucose (TEST) + 6% egg yolk-based medium was used for the mouflon sperm. Glycerol was added to a final concentration of 5% to both media. The same diluents were used for ultra-rapid freezing but replacing the glycerol with 100 mM of sucrose. Sperm variables (motility, viability, acrosome integrity, membrane integrity, and morphological abnormalities) were analyzed before and after cryopreservation. Although values were generally better after the thawing of the conventionally cryopreserved sperm, total sperm motility (38.40 ± 4.44% in mouflon and 31.25 ± 3.37% in fallow deer) and total live sperm (47.19 ± 5.18% in mouflon and 43.13 ± 2.43% in fallow deer) were acceptable for the ultra-rapidly cooled sperm. Independent of the cryopreservation method, membrane integrity, acrosome integrity and the percentages of dead sperm and sperms with a damaged acrosome were better for the cryopreserved mouflon sperm than the fallow deer sperm (P < 0.05). Despite exerting a more harmful effect on sperm variables than conventional freezing, ultra-rapid freezing may be a useful alternative for the cryopreservation of these species' epididymal sperm in the field, as this simple technique does not require sophisticated equipment and expertise.

Effect of Different Straw Volumes and Thawing Rates on Post-Thaw Quality and Fertilization Ability of Cryopreserved Common Carp (Cyprinus carpio) Sperm

Cryopreservation of sperm cells is an essential process applied for long-term conservation of aquatic genetic resources. The goal of this research was to determine effect different of straws volumes and thawing rates on the post-thaw quality and fertilization ability of cryopreserved common carp ( Cyprinus carpio ) sperm. In this study, semen was cryopreserved according to conventional slow freezing protocol. For this aim, the cryosolution contained 75 mM NaCl, 70 mM KCl, 2 mM CaCl 2 , 1 mM MgSO 4 and 20 mM Tris (pH: 8) supplemented with 10% MeOH. Following equilibration at +4°C for 10 min, semen was packed into 0.25, 0.5 and 1.5 mL straws and frozen in liquid nitrogen vapour (for 10 min at -120ºC) and finally stored in liquid nitrogen (-196ºC) tank. Thawing of cryopreserved semen process was performed at 30ºC for 10, 20 and 30 seconds in a water bath. Fertilization was performed using ratio of 1x10 5 spermatozoa/egg. The highest fertility (68.4±2.5%) was determined with cryopreserved sperm packed in 1.5 mL straws that thawed at 30ºC for 30 s. According to the results of this research, sperm cryopreserved with ionic extender containing 10% methanol and packed in 1.5 mL straws are suitable to achieve high fertilization of common carp eggs.

Cryopreservation: Vitrification and Controlled Rate Cooling.

Cryopreservation is the application of low temperatures to preserve the structural and functional integrity of cells and tissues. Conventional cooling protocols allow ice to form and solute concentrations to rise during the cryopreservation process. The damage caused by the rise in solute concentration can be mitigated by the use of compounds known as cryoprotectants. Such compounds protect cells from the consequences of slow cooling injury, allowing them to be cooled at cooling rates which avoid the lethal effects of intracellular ice. An alternative to conventional cooling is vitrification. Vitrification methods incorporate cryoprotectants at sufficiently high concentrations to prevent ice crystallization so that the system forms an amorphous glass thus avoiding the damaging effects caused by conventional slow cooling. However, vitrification too can impose damaging consequences on cells as the cryoprotectant concentrations required to vitrify cells at lower cooling rates are potentially, and often, harmful. While these concentrations can be lowered to nontoxic levels, if the cells are ultra-rapidly cooled, the resulting metastable system can lead to damage through devitrification and growth of ice during subsequent storage and rewarming if not appropriately handled.The commercial and clinical application of stem cells requires robust and reproducible cryopreservation protocols and appropriate long-term, low-temperature storage conditions to provide reliable master and working cell banks. Though current Good Manufacturing Practice (cGMP) compliant methods for the derivation and banking of clinical grade pluripotent stem cells exist and stem cell lines suitable for clinical applications are available, current cryopreservation protocols, whether for vitrification or conventional slow freezing, remain suboptimal. Apart from the resultant loss of valuable product that suboptimal cryopreservation engenders, there is a danger that such processes will impose a selective pressure on the cells selecting out a nonrepresentative, freeze-resistant subpopulation. Optimizing this process requires knowledge of the fundamental processes that occur during the freezing of cellular systems, the mechanisms of damage and methods for avoiding them. This chapter draws together the knowledge of cryopreservation gained in other systems with the current state-of-the-art for embryonic and induced pluripotent stem cell preservation in an attempt to provide the background for future attempts to optimize cryopreservation protocols.

93 BLASTOCYST BISECTION TO MULTIPLY BIOPSIED AND VITRIFIED BOVINE EMBRYOS

Dairy cattle breeding schemes increasingly integrate embryo-based genomic selection to accelerate genetic gain. In contrast to the single offspring produced with conventional animal-based genomic selection, multifactorial IVF between elite parents increases genotypes for selection. Genetically superior embryos are identified from biopsies, and only those with the desired genotypes are transferred. To manage the logistics of such schemes, and enable seasonally born progeny, the cryo-preservation of embryos after biopsy and before embryo transfer is critical. Here, we compare 2 methods of cryo-preserving biopsied Day 7 blastocysts and report results from bisecting blastocysts to increase the number of selected embryos for transfer. Abattoir-sourced oocytes were matured in vitro and fertilized with sperm from a single sire. Embryos were cultured for 7 days in a modified Synthetic Oviduct Fluid medium. Approximately 15 cells were biopsied from the mural trophectoderm of grade 1 and 2 blastocysts in Embryo Hold medium minus BSA, using a micro-surgical blade (Bioniche Animal Health, Athens, GA, USA). Following biopsy, each blastocyst was cultured in Embryo Hold with 3 mg mL−1 BSA for ~2 h at 38.5°C to allow for re-expansion. In Experiment 1, embryos were randomly assigned to 1 of 2 cryo-preservation treatments: conventional slow freezing or the Cryologic vitrification method (CVM). Slow freezing entailed freezing in 1.5 M ethylene glycol and 0.1 M sucrose. The CVM involved a 2-step vitrification protocol, with 15% of both ethylene glycol and dimethyl sulphoxide in the final solution comprising Embryo Hold, 20% FCS, 1 M sucrose, and 0.1 mM Ficoll (GE Healthcare). Selected embryos were thawed/warmed and transferred in pairs to the uterine horn ipsilateral to the corpus luteum of each synchronized recipient heifer. In Experiment 2, each biopsied blastocyst was individually vitrified using CVM. Following warming, blastocysts were bisected into approximately equal halves. After ~2 h recovery, pairs of demi-embryos were transferred to recipients categorized with either normal (&gt;2.5, &lt;7 ng mL−1) or low (≥2, &lt;2.5 ng mL−1) plasma progesterone concentrations on Day 5 after oestrus. Embryo survival in both experiments was monitored by ultrasonography of fetal heartbeats up to Day 65 of gestation. Statistical significance was determined using Fisher’s exact test. In Experiment 1, embryo survival on Day 65 was significantly greater with CVM than slow freezing (25/54 = 46% v. 9/54 = 17%; P = 0.002). In Experiment 2, there was no significant difference in the number of fetuses as a percentage of original blastocysts, regardless of normal versus low progesterone levels (13/22 = 59% v. 4/9 = 44%, respectively). In conclusion, vitrification is superior for cryo-preserving biopsied blastocysts, possibly reducing cryo-damage compared with conventional slow freezing, and achieves rates of in vivo development similar to fresh IVF embryos. Embryo bisection potentially provides only a modest increase in the probability of generating a calf from each valuable, genomically selected embryo. Improving embryo competency and other methods of multiplication may maximize this likelihood.

45 SURVIVAL OF HOLSTEIN IN VITRO-PRODUCED EMBRYOS CULTURED IN NOVEL SYNTHETIC OVIDUCTAL FLUID MEDIA (SCF1) AND DEHYDRATED PRIOR TO CRYOPRESERVATION

In vitro-produced (IVP) embryos experience poor cryotolerance due to metabolic changes during in vitro culture causing increased lipid accumulation and apoptosis post-thaw. We hypothesised that embryos cultured in a novel SOF for conventional freezing media (SCF1), dehydrated, and allowed longer equilibration before conventional slow freezing would increase post-thaw survival and decrease apoptosis. IVP embryos were produced in 9 replicates by oocytes (n = 3172) aspirated from abattoir ovaries, matured for 23 h, fertilized with semen from 1 of 4 bulls, and cultured in conventional SOF media or SCF1 in 38.5°C in 5% O2, 5% CO2, and 90% N2. Stage 7 blastocysts were stained with 1 µg mL−1 Nile Red for lipid content and 300 nM Mitotracker Red CMX-Rosamine for mitochondrial polarity. Remaining blastocysts were slow-frozen by 1 of 4 protocols: 2-min dehydration in 0 or 0.6 M sucrose in holding media before equilibration (10 or 20 min) in conventional freezing media (1.5 M ethylene glycol and 0.5 M sucrose in holding media). Embryos were thawed and assessed for re-expansion at 48 h and surviving embryos were stained with 4′6-diamidino-2-phenylindole (DAPI) and a TUNEL assay to determine apoptosis. Ten images per embryo were acquired by confocal microscopy using a 5-µM step size at 40× magnification. Fluorescence of Nile Red and Mitotracker was measured by IMAGE PRO software, and cells stained for TUNEL were analysed by a cell counter plug-in. Blastocyst rate, Nile Red, and Mitotracker data (Table 1) were analysed by one-way ANOVA and means separated by Tukey’s HSD. Post-thaw survival and apoptotic levels (Table 1) were analysed as a factorial 2 (SOF and SCF1) × 2 (0 and 0.6 M sucrose) × 2 (10 and 20 min), and means separated by Tukey’s HSD. No interactions occurred between factors so they were dropped from the model and only main effects are shown. Results indicate that SCF1 increased blastocyst rate, mitochondrial polarity, and post-thaw survival and decreased lipid content and post-thaw apoptosis (P &lt; 0.01). A 20-min equilibration time decreased apoptosis (P &lt; 0.01) and tended to increase post-thaw survival (P &lt; 0.1), suggesting that cryotolerance is improved in embryos cultured in SCF1 and equilibrated for 20 min. Table 1.Effect of media on development, lipid content and mitochondrial polarity (top) and of media, equilibration and dehydration on post-thaw survival and apoptosis (bottom)

English

Fresh date fruits (Barhi cultivar) at Khalal stage of maturity were frozen by two different methods; individual quick freezing (IQF), and conventional slow freezing (CSF). The frozen dates were stored for 9 months at -20°C and -40°C, through conventional and individual quick freezing methods, respectively. Texture profile analysis parameters (hardness, elasticity, chewiness and resilience), color, and nutrition (enzymes and sugars) properties for fresh and frozen dates were measured after every three months of frozen storage. IQF technique showed better result in quality maintaining of fresh date fruits than CSF. Color and textural parameters of the frozen dates were affected by the freezing method and the frozen storage period. IQF method is superior in preserving the fresh Barhi fruits color values, textural parameters and nutritional aspects compared

Comparison of vitrification and conventional slow freezing for cryopreservation of ovarian tissue with respect to the number of intact primordial follicles: A meta-analysis.

Background:Vitrification is the standard method for cryopreserving human oocytes and embryos, but its effects on ovarian tissue are uncertain. The purpose of this meta-analysis was to compare the proportion of intact primordial follicles in ovarian tissue cryopreserved with vitrification versus slow freezing.Methods:Medline, Cochrane, EMBASE, and Google Scholar databases were searched until November 11, 2014 using combinations of the search terms: ovarian tissue, cryopreservation, vitrification, follicle, follicles. Inclusion criteria were randomized controlled trails, two-arm prospective studies, and retrospective studies in which ovarian tissues were preserved by vitrification or conventional slow freezing. The primary outcome was the proportion of intact primordial follicles.Results:Six studies were included in the meta-analysis. The number of patients ranged from 3 to 20, and age ranged from 20 to 43 years. Total number of morphologically intact follicles ranged from 14 to 2058, among which 6 to 724 were primordial. The pooled odds ratio (OR) showed no significant difference in the proportion of intact primordial follicles after slow freezing or vitrification (OR = 1.228, 95% confidence interval [CI]: 0.769–1.961, P = 0.390). Sensitivity analysis using the leave-one-out approach indicated no considerable changes in the direction and magnitude of the pooled estimates when individual studies were excluded one at a time, indicating good reliability of the current analysis.Conclusions:Vitrification and slow freezing produce equivalent results with respect to intact primordial follicles for the cryopreservation of human ovarian tissue. However, the included studies varied in the cryopreservation protocols used.

Freezing of fresh Barhi dates for quality preservation during frozen storage.

Fresh harvested dates are perishable and there is a need for extending their shelf life while preserving their fresh like quality characteristics. This study evaluates three different freezing methods, namely cryogenic freezing (CF) using liquid nitrogen; individual quick freezing (IQF) and conventional slow freezing (CSF) in preserving the quality and stability of dates during frozen storage. Fresh dates were frozen utilizing the three methods. The produced frozen dates were frozen stored for nine months. The color values, textural parameters, and nutrition qualities were measured for fresh dates before freezing and for the frozen dates every three months during the frozen storage. The frozen dates’ color values were affected by the freezing method and the frozen storage period. There are substantial differences in the quality of the frozen fruits in favor of cryogenic freezing followed by individual quick freezing compared to the conventional slow freezing. The results revealed large disparity among the times of freezing of the three methods. The freezing time accounted to 10 min for CF, and around 80 min for IQF, and 1800 min for CSF method.

Cryogenic freezing of fresh date fruits for quality preservation during frozen storage

Fresh date fruits, especially Barhi cultivar, are favored and widely consumed at the Khalal maturity stage (first color edible stage). These fruits are seasonal and perishable and there is a need for extending their shelf life. This study evaluates two different freezing methods, namely cryogenic freezing using liquid nitrogen and conventional deep freezing on preserving the quality and stability of date fruits (cv. Barhi) at Khalal maturity stage. Fresh date fruits (cv. Barhi) at Khalal stage were frozen utilizing the two methods. The produced frozen dates were stored under frozen storage conditions for nine months (at −20°C and −40°C for the conventional and cryogenic freezing, respectively). Color values, textural properties (hardness, elasticity, chewiness and resilience), and nutrition attributes (enzymes and sugars) for fresh dates before freezing and for the frozen dates were measured every three months during the frozen storage. Color values of the frozen dates were affected by the freezing method and the frozen storage period. There are substantial differences in the quality of the frozen fruits in favor of cryogenic freezing compared to the conventional slow freezing. The results revealed a large disparity between the times of freezing of the two methods. The freezing time accounted to 10min in the cryogenic freezing method, whereas it was 1800min for the conventional slow freezing system.

Seasonal Preservation Success of the Marine Dinoflagellate Coral Symbiont, Symbiodinium sp.

Coral reefs are some of the most diverse and productive ecosystems on the planet, but are threatened by global and local stressors, mandating the need for incorporating ex situ conservation practices. One approach that is highly protective is the development of genome resource banks that preserve the species and its genetic diversity. A critical component of the reef are the endosymbiotic algae, Symbiodinium sp., living within most coral that transfer energy-rich sugars to their hosts. Although Symbiodinium are maintained alive in culture collections around the world, the cryopreservation of these algae to prevent loss and genetic drift is not well-defined. This study examined the quantum yield physiology and freezing protocols that resulted in survival of Symbiodinium at 24 h post-thawing. Only the ultra-rapid procedure called vitrification resulted in success whereas conventional slow freezing protocols did not. We determined that success also depended on using a thin film of agar with embedded Symbiodinium on Cryotops, a process that yielded a post-thaw viability of >50% in extracted and vitrified Symbiodinium from Fungia scutaria, Pocillopora damicornis and Porites compressa. Additionally, there also was a seasonal influence on vitrification success as the best post-thaw survival of F. scutaria occurred in winter and spring compared to summer and fall (P < 0.05). These findings lay the foundation for developing a viable genome resource bank for the world’s Symbiodinium that, in turn, will not only protect this critical element of coral functionality but serve as a resource for understanding the complexities of symbiosis, support selective breeding experiments to develop more thermally resilient strains of coral, and provide a ‘gold-standard’ genomics collection, allowing for full genomic sequencing of unique Symbiodinium strains.

A time-lapse evaluation of conventional slow freezing and vitrification of PN-stage embryos

A time-lapse evaluation of conventional slow freezing and vitrification of PN-stage embryos

Validation of microSecure vitrification (μS-VTF) for the effective cryopreservation of human embryos and oocytes

A novel, aseptic closed system vitrification (VTF) technique for the cryopreservation of embryos and oocytes has been developed and clinically validated in this study. It combines the practicality of embryo-containing sterile flexipettes stored safely and securely with 0.3ml CBS™ embryo straws possessing weld seals. The cooling and warming rates of this double container system were determined using a data logger. Upon direct plunging into LN2, the flexipettes cool at an average rate of 1391°C/min, while warming occurs at an average rate of 6233°C/min in a 37°C 0.5M sucrose bath. Direct deposition of the flexipette into a warming bath insured a rapid transition between −100 and −60°C to minimize potentially harmful recrystalization associated with devitrification. In conclusion, the μS-VTF system has exhibited higher (p<0.05) intact survival, implantation and live birth rates than conventional slow freezing methods. The effective embryo transfer of vitrified blastocysts proved similar to or better than fresh embryo transfer outcomes. The sustained clinical use of μS-VTF has justified a change in our infertility practice.Capsule: The microSecure vitrification (μS-VTF) procedure is a low-cost, non-commercial, aseptic, closed system that offers technical simplicity and repeatability, while effectively attaining an estimated 4:1 warming-to-cooling rate ratio, which supports excellent embryo survival and sustained viability.

Vitrification of cleavage stage day 3 embryos results in higher live birth rates than conventional slow freezing: a RCT.

Is the live birth rate (LBR) per embryo thawed/warmed higher when Day 3 cleavage stage embryos are cryopreserved by vitrification compared with slow freezing? The LBR per embryo thawed/warmed was higher after vitrification than after slow freezing on Day 3, based on better embryo survival, quality and availability of embryos in the vitrification group. Post-thawing survival rate of cleavage-stage embryos has been reported to be higher after vitrification than after slow freezing. This RCT was performed in an academic tertiary center between September 2011 and March 2013. If supernumerary embryos were available on Day 3, patients were randomized at the time of cryopreservation using a computerized system to determine a simple allocation to the vitrification group or the slow freezing group and all embryos were frozen with the same technique. The primary outcome of this study was the LBR per embryo thawed/warmed. Power calculation revealed that 184 thawed embryos were needed in each group (β = 0.8, α < 0.05) to test the hypothesis that the LBR per embryo thawed/warmed was significantly higher (16%) after vitrification than after slow freezing (6%). Patients <40 years old undergoing their first oocyte retrieval (OR), with embryo transfer and with supernumerary embryos on Day 3, were randomized. Day 3 embryos with ≥6 cells, <25% fragmentation and morphologically equal blastomeres were cryopreserved by slow freezing (using 1,2-propanediol and 0.1 M sucrose as cryoprotectant) or by closed vitrification using commercially available freezing/vitrification media. Survival was defined as ≥50% cells were intact after thawing. Thawed embryos were further cultured overnight. In total, 307 patients were randomized to slow freezing (155 patients, 480 embryos) or vitrification (152 patients, 495 embryos). By March 2013, 200 embryos were thawed after slow freezing in 95 cycles for 79 patients and 217 embryos were warmed after vitrification in 121 cycles in 90 patients. The LBR per embryo thawed/warmed was significantly higher after vitrification (16.1% (35/217)) than after slow freezing (5.0% (10/200); P < 0.0022; relative risk (RR) 3.23; 95% confidence interval (CI) 1.64-6.35). Similarly, the implantation rate per embryo thawed/warmed was higher after vitrification (20.7% (45/217) than after slow freezing (7.5% (15/200); P = 0.0012; RR 2.76; CI 1.59-4.81). The survival rate was significantly higher after vitrification (84.3% (183/217) than after slow freezing (52.5% (105/200); P < 0.0001). Significantly more embryos were fully intact after vitrification (75.4% (138/183) than after slow freezing (28.6% (30/105); P < 0.0001). The number of transfers was significantly higher after vitrification (90.1% (109/121)) than after slow freezing (73.7% (70/95); P = 0.0024). Survival rates in the slow freezing group were low in this study. Additional RCTs are needed to compare reproductive outcome after vitrification and after slow freezing with 1,2-propanediol and 0.2 M sucrose, since this method has been reported to have better survival than the method used in our study. Our findings are only applicable to the specific slow freezing cryopreservation medium used in our study, and not to any other commercially available media. When compared with slow freezing using 1,2-propanediol and 0.1 M sucrose as cryoprotectant, vitrification of Day 3 cleavage stage embryos resulted in a higher LBR per embryo warmed, and may therefore result into a higher cumulative delivery rate after one oocyte retrieval. None. NCT02013024.

Medium composition for effective slow freezing of embryonic cell lines derived from marine medaka (Oryzias dancena).

This study was conducted to identify optimal medium composition for freezing Oryzias dancena embryonic cell lines. Different freezing media consisting of various concentration of dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), and trehalose were prepared and long-term cultured embryonic cell line was frozen in each freezing medium by conventional slow freezing program for 7days. Through measurement of viability and growth of post-thaw cells frozen in each freezing medium, it was determined that optimal composition of three components was 10% DMSO, 20% FBS, and 0.1M trehalose. The post-thaw cells frozen in optimal freezing medium showed similar morphology and growth rate with non-frozen cells. Next, this condition was applied to two different sets of experiment; (1) freezing of the same cells during expanded period (57days) and (2) freezing of short-term cultured cells from other batches for 7days. The viability of post-thaw cells was significantly low and comparable in set 1 and 2, respectively, when compared with the result of long term-cultured cells frozen in optimal freezing medium for 7days and similar morphology and growth rate with non-frozen counterparts were detected in the post-thaw cells from both sets. In conclusion, this study first reports the optimal medium composition for freezing O. dancena embryonic cells, which can contribute to fish species preservation as well as improvement of cell-based biotechnology by providing stable cell storage.

Ovarian tissue vitrification is more efficient than slow freezing in protecting oocyte and granulosa cell DNA integrity

Ovarian tissue cryopreservation is the primary treatment modality currently available to women at risk of losing their ovarian function due to cytotoxic therapy. However, the impact of these techniques on the oocyte DNA integrity is not elucidated. Here we have investigated the effect of vitrification and conventional slow freezing of eight week old Swiss albino mouse ovarian tissues on the oocyte and granulosa cell DNA integrity using the comet assay. The intracellular levels of reactive oxygen species in oocytes was measured by 2′,7′-dichlorodihydrofluorescein diacetate fluorescence. The cryopreservation of ovarian tissue by the slow freezing technique resulted in a significantly higher level of DNA fragmentation in oocytes in comparison to vitrification (p < 0.05) whereas DNA fragmentation in granulosa cells was significantly higher than the control (p < 0.01). Further, reactive oxygen species were significantly elevated in oocytes derived from slow freezing when compared to vitrification (p < 0.05). Therefore, we conclude that the ovarian tissue slow freeze-thawing makes the oocyte and granulosa cells more vulnerable to DNA damage whereas vitrification appears to be a safer method than slow freezing for ovarian tissue cryopreservation.

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.