Cryoprotectant Solution, 1.5 m 1,2-Propanediol

2018 ◽  
Vol 2018 (5) ◽  
pp. pdb.rec096289
Keyword(s):  
Cryoprotectant Solution

scholarly journals Addition of sucrose on cryoprotectant solution of vitrification enhances the quality of Dorper sheep embryos produced in vivo

2015 ◽  
Vol 36 (6Supl2) ◽  
pp. 4257
Author(s):  
Alane Pains Oliveira do Monte ◽  
João Bosco Loiola Filho ◽  
Thais Thatiane Dos Santos Souza ◽  
Mayara De Souza Miranda ◽  
Lívia Correia Magalhães ◽  
...  
Keyword(s):  
Embryo Quality ◽  
Control Group ◽  
Chi Square ◽  
Mass Occurrence ◽  
Chi Square Test ◽  
Vitrification Solution ◽  
Cryoprotectant Solution ◽  
Pellucid Zone

<p>This study aimed to evaluate the efficacy of adding sucrose in vitrification solution of ovine embryos produced <em>in vivo</em>. Forty Dorper ewes were selected and superovulated. Immediately prior to the embryo collection by laparotomy, a laparoscopy was performed to verify the superovulatory response. The recovered flushing was followed by embryo evaluation and embryos were divided in two experimental groups where embryos from Control group were submitted to a traditional vitrification protocol and embryos from Sucrose group to a modified vitrification protocol with sucrose. After warming, embryos were again divided regarding cryoprotectant removal (Indirect) or not (Direct). The embryo quality was classified as embryos of degrees I (excellent or good), II (regular), III (poor) and IV (dead or degenerate). It was also verified the homogeneity of mass, occurrence of embryonic mass retraction and rupture of pellucid zone. The results were expressed as percentages and were subjected to Chi-square test with P &lt; 0.05. The embryos vitrified in the presence of sucrose had lower proportions of lower-quality embryos after warming (22.20 vs. 44.50%), higher percentages of homogeneous embryos after warming (63.89 vs. 38.89 %) while concerning other parameters there was no difference between these groups. It can be concluded that the addition of 0.4 M sucrose during vitrification improves the embryo quality.</p>

scholarly journals Cryopreservation of Coffea arabica L. Zygotic Embryos by Vitrification

2016 ◽  
Vol 44 (2) ◽  
pp. 445-451 ◽  
Author(s):  
Rodrigo Therezan de FREITAS ◽  
Renato PAIVA ◽  
Thais Silva SALES ◽  
Diogo Pedrosa Corrêa da SILVA ◽  
Michele Valquíria dos REIS ◽  
...  
Keyword(s):  
Coffea Arabica ◽  
Seed Storage ◽  
Anatomical Study ◽  
Embryo Cryopreservation ◽  
Zygotic Embryos ◽  
Two Temperatures ◽  
Vitrification Solution ◽  
Cryoprotectant Solution ◽  
Coffea Arabica L ◽  
Coffee Seed

As a consequence of the difficulty in conventional coffee seed storage, biotechnological alternatives such as cryopreservation have been investigated. The objective of this study was to develop a protocol for the cryopreservation of Coffea arabica L. (cv. ‘Catuaí Vermelho’ - IAC 144) zygotic embryos by vitrification. For the cryopreservation study, the embryos were immersed in Plant Vitrification Solution 2 at different times (0, 10, 25, 50, 100, and 250 min) and two temperatures (0 and 25 °C). Subsequently, the best thawing time was determined in a water bath (1, 3, 5 minutes or directly in Recovery Solution). An anatomical study was conducted on non-stored and stored embryos, with or without the use of Plant Vitrification Solution 2. The immersion in cryoprotectant solution for 100 min at 0 °C allows embryo cryopreservation. Embryos can be directly thawed in Recovery Solution after storage in liquid nitrogen. It was observed that Plant Vitrification Solution 2 reduced internal water content in the cells, allowing subsequent embryo growth resumption.

scholarly journals Vitrification of In Vitro Produced Porcine Blastocysts: Influence of Cryoprotectants Toxicity and Embryo Age

2019 ◽  
Vol 47 (1) ◽  
Author(s):  
Lain Uriel Ohlweiler ◽  
Joana Claudia Mezzalira ◽  
Alceu Mezzalira
Keyword(s):  
Cell Number ◽  
Lower Percentage ◽  
Toxicity Tests ◽  
Hatching Rate ◽  
Embryo Viability ◽  
Embryo Survival ◽  
Porcine Embryo ◽  
Negative Role ◽  
Cryoprotectant Solution

Background: Porcine embryos are sensible to all assisted reproduction manipulations, especially the ones that involve cryopreservation. Despite the high cryoprotectant concentrations routinely applied, vitrification is the most effective technique to date. These substances toxicity can also play a negative role in embryo viability. During in vitro porcine embryo production, the speed of development is often unevenly distributed. It is possible that their development speed, affects embryo tolerance to cryoprotectants. This study aimed to evaluate the toxicity of porcine embryos of days 5 or 6 of culture to cryoprotectant agents; as well as to assess embryo survival to vitrification.Material, Methods & Results: Parthenogenetic porcine blastocysts and expanded blastocysts of days 5 and 6 of culture were exposed to toxicity tests (experiments 1 and 2) and vitrification (experiment 3) using different protocols. In the first experiment, three different cryoprotectants were used (Dimethyl sulfoxide - DMSO, Ethylene glycol – EG, and Sucrose - SUC), combined in three different associations (G1: 15% EG + 15% DMSO with 0.5M SUC; G2: 16% EG + 16% DMSO with 0.4M SUC; G3: 18% EG + 18% DMSO with 0.5M SUC). In the fresh Control, embryos of day 6 are more sensible than the ones of day 5, whom showed a lower hatching rate (39.7 vs. 60.8%). After the toxicity (Experiment 1) test, the G1 showed better expansion rates in day 6 (50.0 vs 31.0 and 3.6% for G2 and G3) and higher hatching of day 6 compared to G2 and G3 (23.2, vs. 8.6 and 0.0% for G2 and G3). The fresh non hatched embryos at day 8, derived at day 6, had a lower percentage of cells with cleaved caspase-3 (20.2%) compared with the G1 (30.5%), G2 (31.4%) and G3 (30.5%). The hatched embryos of day 5 from G2 had lower total cell number (TCN) compared with the day 6 hatched embryos, whereas in G1 the TCN was not affected. The second experiment compared EG combined to one of these three extracellular cryoprotectants: Polyvinylpyrrolidone/sucrose/trehalose (respectively groups: PVP, SUC, TRE). The group SUC has raised the best results for day 5 embryos, whereas for day 6 embryos SUC and TRE were both best. The third experiment tested four vitrification protocols, being P1: EG+DMSO+TRE/warming with SUC; P2: EG+DMSO+TRE/warming TRE; P3: EG+TRE/ warming SUC; P4: EG+TRE/warming TRE. The expansion of vitrified day 5 embryos was higher in the P1 (20.0%) in comparison with the other three groups (4.3, 4.3 and 4.4% for P2, P3 and P4, respectively), with no difference for their hatching rates, been it lower comparing to the Control. Day 6 embryos showed no difference in expansion and hatching for the vitrified groups, been them lower than the Control.Discussion: Embryos obtained on day 6 are more sensible than the ones of day 5, fact observed when the embryos were exposed to cryoprotectant solution, as well by the behavior of the no treated Control embryos. The toxicity increases as it does the concentration of intracellular cryoprotectant, where over 16% of the intracellular cryoprotectors already affected the day 6 embryos development. For the day 5 embryos however, 15 or 16% of the intracellular cryoptrotectors, had similar behavior to the embryos. For the extracellular solutions, however, it is variable according the embryos development speed. Indeed, it is necessary to adjust the cryoprotectors to be used to cryopreserve porcine in vitro produced embryos obtained at days 5 and 6 of culture.

scholarly journals Spermatic abnormalities of piracanjuba Brycon orbignyanus (Valenciennes, 1849) after cryopreservation

2011 ◽  
Vol 71 (3) ◽  
pp. 693-699 ◽  
Author(s):  
JM. Galo ◽  
DP. Streit-Junior ◽  
RN. Sirol ◽  
RP. Ribeiro ◽  
M. Digmayer ◽  
...  
Keyword(s):  
Dimethyl Sulfoxide ◽  
Liquid Nitrogen ◽  
Distilled Water ◽  
Reproductive Characteristics ◽  
Fish Sperm ◽  
Before And After ◽  
Cryoprotectant Solution ◽  
Fresh Semen

The objective of this research was to verify the presence of spermatic abnormalities on semen of Brycon orbignyanus after cryopreservation. Semen was collected from ten four-year-old males who presented secondary reproductive characteristics for migrating fish. Sperm was evaluated for motility, vigor and spermatic morphology before and after cryopreservation. A cryoprotectant solution was made of 20 mL of yolk egg, 5.0 g of glucose and dimethyl sulfoxide diluted in distilled water (10 mL: 90 mL). The diluted semen (1:3, semen:solution) was submitted to nitrogen steam for 24 hours and then to liquid nitrogen (-196 ºC) for 60 days. Cryopreservation decreased the percentage of normal spermatozoa from 62.20% to 54.60%. Consequently, the percentage of spermatozoa with secondary abnormalities increased from 8.50% to 15.00%. However, there was no difference in primary abnormalities. Both spermatic motility and vigor were decreased in cryopreserved semen compared with fresh semen. In conclusion, cryopreservation of semen of B. orbignyanus increased the percentage of secondary abnormalities and decreased the spermatic motility and vigor.

Survival of Macrobrachium amazonicum embryos submitted to cooling

Zygote ◽  
2017 ◽  
Vol 25 (3) ◽  
pp. 288-295 ◽  
Author(s):  
Arthur Vinícius Lourenço Ferreira ◽  
Moisés Fernandes Martins ◽  
Míriam Luzia Nogueira Martins de Sousa ◽  
Aldeney Andrade Soares Filho ◽  
Célia Maria de Souza Sampaio
Keyword(s):  
Survival Rate ◽  
Storage Time ◽  
Survival Rates ◽  
Negative Control ◽  
Control Groups ◽  
Treatment Groups ◽  
Significant Difference ◽  
Macrobrachium Amazonicum ◽  
Cryoprotectant Solution ◽  
Positive Controls

SummaryCooling techniques have several applications for reproduction in aquaculture. However, few studies have sought to create protocols for cooling and cryopreservation of Macrobrachium amazonicum embryos. Thus, the objective of this work was to verify the survival of M. amazonicum embryos and the correlation between embryonic volume and mortality of M. amazonicum embryos after cooling. Embryo pools were collected from three females and divided into two treatment groups: dimethyl sulfoxide (DMSO) 3% and ethylene glycol (EG) 0.5%, both of them associated with 2 M sucrose. Positive and negative control groups consisted of seawater 10%. Aliquots of 10 µg of embryos were placed in Falcon® tubes containing a cryoprotectant solution and submitted directly to the test temperature of 2°C for 2 and 6 h of cooling. Further analysis of survival and embryonic volume were performed under a stereoscopic microscope. Data were subjected to analysis of variance (ANOVA), and means were compared using the Tukey test at 5%. The highest embryonic survival rate was observed after the shortest storage time for both the DMSO 3% and the 0.5% EG groups, with survival rates of 84.8 ± 3.9 and 79.7 ± 2.8%, respectively. There was a reduction in survival after 24 h, with the DMSO 3% group presenting a survival rate of 71.7 ± 6.6%, and the EG 0.5% group, 66 ± 6.9%. Survival showed a statistically significant difference when compared with the positive controls after 2 h and 24 h of cooling, with 99 ± 0.5% and 95.8 ± 1.5% survival rates, respectively. There was no significant statistical difference in the embryonic volume, but it was possible to observe a change in the appearance of the embryos, from a translucent coloration to an opaque white or brownish coloration, after 24 h in incubators. Thus, it can be concluded that survival is inversely proportional to storage time and that, although there was no change in the embryonic volume after cooling, a change in the appearance of embryos could be observed.

Morphological evaluation of Prochilodus lineatus embryos after vitrification–thawing in high‐osmolarity cryoprotectant solution

2018 ◽  
Vol 53 (6) ◽  
pp. 1353-1358
Author(s):  
Raphael da Silva Costa ◽  
Caio de Souza Capuzzo ◽  
Cristiele da Silva Ribeiro ◽  
Rosicleire Verissimo‐Silveira ◽  
Diógenes Henrique de Siqueira‐Silva ◽  
...  
Keyword(s):  
Prochilodus Lineatus ◽  
High Osmolarity ◽  
Morphological Evaluation ◽  
Cryoprotectant Solution

25 Cryopreservation of horse testicular tissue as a model for rhinoceros

2019 ◽  
Vol 31 (1) ◽  
pp. 138 ◽  
Author(s):  
M. C. Gómez ◽  
A. Alrashed ◽  
C.-Y. Su ◽  
B. Durrant
Keyword(s):  
Basement Membrane ◽  
Dimethyl Sulfoxide ◽  
Structural Integrity ◽  
Membrane Damage ◽  
Genetic Material ◽  
Testicular Tissue ◽  
Live Cells ◽  
Seminiferous Tubules ◽  
Positive Interaction ◽  
Cryoprotectant Solution

Cryopreservation of testicular tissue (TT) allows retention of valuable genetic material that can be used for conservation of endangered species, such as the northern white rhinoceros (NWR; Ceratotherium simum cottoni). Previously, we found that cryopreservation of NWR TT with a slow controlled cooling rate (CR) method induced morphological alterations in the seminiferous tubules (ST). However, the relative influence of CR, type of medium, and condition of TT from the aged NWR male on TT integrity was not clear. Due to the limited availability of rhinoceros TT, we used the horse as a model for optimization of TT cryopreservation. We evaluated the effect of (1) cryoprotectant solution [PBS (PBS +1.5M dimethyl sulfoxide) v. DMEM (DMEM/F12+10.0% fetal bovine serum+0.05M sucrose+1.5M dimethyl sulfoxide)] and (2) CR [CR1 (−2.0°C min−1 from 0°C to −4.0°C, −15°C min−1 to −12°C, and −0.3°C min−1 to −40°C in a programmable freezer) v. CR2 (same as CR1 but cooled to −8°C and held for 5min before cooling to −40°C) v. CR3 (−1.0°C min−1 from 0°C to −80°C in a CoolCell® freezing device; Corning, Corning, NY, USA)] on the structural integrity of ST from a 2-year-old horse (n=20 ST), cell viability, and expression of spermatogonial stem cells (SSC; GFRα1, and GRP125) and pluripotent markers (SSEA-4, SSEA-1, and OCT-4) in spermatogonial cells isolated from TT frozen with the above treatments (n=3). We found a positive interaction between CR and cryoprotectant solution on structural integrity of fixed and stained TT after freezing in PBS and CR2 that resulted in lower detachment of epithelium cells from the basement membrane (score±standard error of the mean; 0.50±0.1) than that of TT frozen in PBS and CR1 and CR3 (1.00±0.1 and 1.80±0.1, respectively; P&lt;0.001) or in DMEM and CR1 (1.25±0.1), CR2 (1.35±0.1), and CR3 (1.40±0.1; P&lt;0.01) and in lower incidence of basement membrane damage (0.75±0.1) than that of TT frozen in PBS and CR1 (1.17±0.07) and CR3 (1.16±0.07) or in DMEM and CR1 (1.10±0.1), CR2 (1.15±0.1), or CR3 (1.45±0.1; P&lt;0.01). A lower rate of pyknosis was observed in TT frozen with PBS (1.15±0.06) than in TT frozen in DMEM (1.43±0.06; P&lt;0.001). Overall, integrity of ST was improved when TT was frozen in PBS at CR2 having similar percentages of ST with intact epithelium (60%) and basement membrane (35%) as that of refrigerated TT (45 and 50%, respectively) but different from that of TT frozen with PBS at CR1 (10 and 15%, respectively; P&lt;0.05). Flow cytometry analysis of spermatogonial cells revealed that the percentages of live cells from TT frozen in PBS (CR1: 61.5±7.4%; CR2: 59.7±4.8%; CR3: 51.5±4.1%) or DMEM (CR1: 66.2±6.0%; CR2: 59.8±6.0%; CR3: 58.9±6.9%), and expression of SSC and pluripotent markers was similar among all freezing treatments. However, the percentages of live cells from frozen-thawed TT were lower than those of cells isolated from refrigerated TT (80.6±2.2%; P&lt;0.001). Overall, our results showed that (1) structural integrity of horse ST was better maintained when TT was frozen in PBS at CR2 and (2) SSC can be isolated from frozen-thawed TT with a similar relative frequency to that of refrigerated TT.

Routine Automated Processing of Cord Blood Units Using the Sepax Cell Processing System.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3646-3646
Author(s):  
Safa Karandish ◽  
Nery Berrios ◽  
Sufira Kiran ◽  
Charisse Ayuste ◽  
Toby Hamblin ◽  
...  
Keyword(s):  
Cord Blood ◽  
Processing Time ◽  
Processing System ◽  
Buffy Coat ◽  
Bar Code ◽  
Cell Processing ◽  
Wide Range ◽  
Significant Difference ◽  
Centrifugation Step ◽  
Cryoprotectant Solution

Abstract We recently incorporated the use of the automated Sepax Cell Processing System (Biosafe SA, Switzerland) for red cell and volume reduction of cord blood units (CBU) before cryopreservation. Now that we have been routinely using this new technique in the laboratory for about six months, we decided to compare the results of this method with our standard manual processing method (Rubinstein, et al, PNAS1995,; 92: 10119–22). For both methods, hespan is added to the cells at final concentration of 20% (v/v). With the Sepax system, after addition of hespan, the cell bag is connected to the Sepax tubing set with the final freeze bag pre-attached to the set. After completion of the automated procedure, buffy coat is collected in the final freeze bag. Cryoprotectant solution is then added directly to the freeze bag. In the manual method, buffy coat and white cell rich plasma layer is collected after first centrifugation step and the white cells are separated from the plasma after the second centrifugation step. Cryoprotectant solution is then added to the cells before transfer to the final freeze bag for cryopreservation. The following are summary of results for each method: Table 1 Manual (n=1160) Sepax (n=311) Pre-Processing volume (ml) 107±30 114±28 Pre-Processing TNC (×10e6) 1196±577 1315±519 TNC Recovery (%) 80±8 83±8 TNC Viability (%) (7-AAD) 97±3 98±3 Total CD34 (×10e6) 4.3±4 4.9±3.6 Total DFU (×10e6) 70±0.9 61.5±20 Post Processing RBC Volume (ml) 9±2 7.3±2 Total Processing Time (including Setup) 60 minutes 30 minutes It is important to note that there was not a significant difference in TNC Recovery over a wide range of Pre-Processing Volume (66–206ml) or Pre-Processing TNC (440 – 3559×106). Since the Sepax device is an automated procedure, issues could arise (i.e. short term loss of electrical power) that would require us to reprocess the CBU before cryopreservation. The Sepax system allows for recovery and reprocessing of the cell using the ‘Purge mode’. We used 5 CBU units to evaluate TNC recovery and viability after purging the cells once and reprocessing. Table 2 TNC Recovery (%) TNC Viability (%) First Buffy Coat 85±5 98±0.9 Post Purge and reprocessing 76±5 98±0.9 Although the TNC recovery was lower after the second procedure, it was still within acceptable limits and the viability of the cells had not changed. These data demonstrates that both methods are equivalent with respect to cell recovery. However, the Sepax System substantially reduces processing time and hands-on operator intervention. Additionally system provides, closed-system processing, bar code reading capability and run data print-out suitable for GMP manufacturing settings.

58 MULTI-THERMAL GRADIENT FREEZING ALLOWS THE CRYOPRESERVATION OF SHEEP WHOLE OVARIES WITH THE SAME EFFICIENCY OF OVARIAN FRAGMENTS

2013 ◽  
Vol 25 (1) ◽  
pp. 176
Author(s):  
T. A. L. Brevini ◽  
S. Maffei ◽  
G. Pennarossa ◽  
A. Arav ◽  
F. Gandolfi
Keyword(s):  
Cooling Rate ◽  
Nude Mice ◽  
Thermal Gradient ◽  
Ovarian Tissue ◽  
Sample Volume ◽  
Ovarian Tissue Cryopreservation ◽  
Cortical Region ◽  
Cryoprotectant Solution ◽  
Whole Ovary ◽  
Whole Ovaries

Ovarian tissue cryobanking is proposed as an effective option for preserving female fertility in cancer patients. At present 2 options are available: cryopreservation of ovarian cortical fragments or of the whole ovary. The use of whole ovary reduces ischemic insult. However, the larger the sample volume, the more difficult it is to introduce the cryoprotective agents and to ensure an adequate cooling rate that minimizes tissue damage. For this reason, we used the multi-thermal gradient method, based on running the sample through a temperature gradient. This allows a homogeneous cooling rate through the whole sample independently from its volume. The aim of the study was to determine whether multi-thermal gradient freezing allows a substantial reduction of the damages induced by cryopreservation of large samples by comparing the viability of cortical fragments versus whole ovaries after thawing and grafting in nude mice. Sheep ovaries were collected at the local abattoir and randomly divided into 3 groups: A) ovaries frozen as cortical fragments, B) ovaries frozen as whole organs, and C) fresh ovaries immediately processed for further analysis (control). Ovarian fragments (10 × 5 × 1 mm) were sliced from the cortical region and immersed into cryoprotectant solution (Leibovitz L-15 medium, 10% FCS, and 1.5 M dimethyl sulfoxide), while whole ovaries were perfused with the same solution. Samples were placed into glass freezing tubes 16 mm in diameter filled with cryoprotectant solution. Samples were frozen with the multi-thermal gradient freezing apparatus (Core Dynamics, Ness Ziona, Israel) progressing along the thermal gradient at a rate of 0.01 mm s–1, resulting in a cooling rate of 0.3°C min–1. Two weeks later, samples were thawed by plunging the tubes into a 37°C water bath with gentle shaking. Whole ovaries were perfused with 10 mL of HEPES-Talp medium, 0.5 M sucrose, and 10 IU mL–1 of heparin and their cortical region was cut into fragments. These fragments and those derived from group A were rehydrated in L-15 medium with decreasing sucrose concentrations. Fragments (2 × 2 × 1 mm) were xenografted in the dorsal region of 6 nude mice for each group. Mice were killed after 8 weeks and grafts were collected for analysis. Cryopreserved samples were compared with each other and fresh controls (group C). Morphologically normal follicles at primordial, primary, and secondary stages were visible in all samples. Cell proliferation was assessed measuring Ki-67 mRNA and counting immunohistochemically positive cells. The FSH receptor and GDF9 gene expression were used to evaluate tissue viability. No significant differences for any of these parameters were measured amongst the groups. We conclude that directional freezing is an effective method for ovarian tissue cryopreservation independently from the sample volume, thus overriding the limitations usually associated with whole-organ banking. Supported by AIRC IG 10376 and by Carraresi Foundation.

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