Injuries in pacu embryos (Piaractus mesopotamicus) after freezing and thawing

Zygote ◽  
2012 ◽  
Vol 22 (1) ◽  
pp. 25-31 ◽  
Author(s):  
Patrícia Ribeiro Neves ◽  
Ricardo Pereira Ribeiro ◽  
Danilo Pedro Streit ◽  
Maria Raquel M. Natali ◽  
Darci Carlos Fornari ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Sucrose Solution ◽  
Optical Microscope ◽  
Freezing Process ◽  
Control Group ◽  
Embryo Morphology ◽  
Freezing And Thawing ◽  
Yolk Syncytial Layer ◽  
Piaractus Mesopotamicus ◽  
Intracellular Ice Formation

SummaryAlthough the sperm cryopreservation of freshwater and marine teleosts has been feasible for years, the cryopreservation of some fish embryos still remains elusive. Thus, the objective of this experiment was to analyze the embryo morphology after freezing and thawing 40 embryos of Piaractus mesopotamicus immersed into methanol and ethylene glycol, both at 7, 10 and 13% plus 0.1 M sucrose for 10 min. Soon after thawing, three embryos were treated with historesin, stained with hematoxylin–eosin and analyzed under an optical microscope. From every treatment, one palette containing embryos was thawed and incubated, but none of the eggs hatched. Samples containing two embryos were immersed into 10% methanol or 10% ethylene glycol both in association with sucrose, and embryos immersed into only water or sucrose solution were frozen, processed and analyzed using scanning electron microscopy (SEM). In both cases, the control group was immersed into only water. Although the embryos had the chorion, vitello, yolk syncytial layer and blastoderm, all of them were found altered under the optical microscope and by SEM. The chorion was irregular and injured; there was no individuality in the yolk granules; the yolk syncytial layer had an irregular shape, thickness and size; the blastoderm showed injuries in the nucleus shape and sometimes was absent; the blastoderm was located in atypical areas and absent in some embryos. In conclusion, no treatment was effective in preserving the embryos, and none of the embryos avoided injury from intracellular ice formation. These morphological injuries during the freezing process made the P. mesopotamicus embryos unfeasible for hatching.

scholarly journals 114EFFECT OF CYTOCHALASIN B TREATMENT ON VITRIFICATION OF CAPRINE PARTHENOGENIC BLASTOCYSTS

2004 ◽  
Vol 16 (2) ◽  
pp. 179
Author(s):  
S. Nims ◽  
D. Melican ◽  
T. Jellerette ◽  
R. Butler ◽  
W. Gavin
Keyword(s):  
Ethylene Glycol ◽  
Cytochalasin B ◽  
Culture Media ◽  
Sucrose Solution ◽  
Transgenic Animals ◽  
Freezing Process ◽  
Control Group ◽  
System Protection ◽  
Experimental Group

Production of human recombinant proteins in the milk of transgenic animals has been shown to be a viable production system. Protection of the animal genetics involved is paramount. Vitrification of embryos is a simple, time-efficient way of preserving an animal’s genetics without the formation of damaging ice crystals during the freezing process. Cytochalasin B has been shown to increase the viability of porcine blastocysts by reducing damage to microfilaments and other cytoskeletal components. These experiments utilized caprine parthenogenic blastocysts as a model to compare the viability of parthenotes treated with or without cytochalasin B prior to and during vitrification. Abattoir oocytes were in vitro-matured in M199 with 10% goat serum containing FSH, LH and gentamycin for 18 to 21h. Parthenogenic blastocysts were produced by treating in vitro matured abattoir oocytes with ionomycin for 5min (5μM) and with 6-dMAP (3mM) for 3h followed by culturing in SOF+0.8% BSA for 7 to 8 days at 38°C with 6% O2, 5% CO2, and 89% N2 in a modular incubator chamber. The experimental group was treated with cytochalasin B (5μg/mL)in the culture media for 30 to 45min prior to and thereafter throughout the vitrification process. All blastocysts (both the experimental group and the control group) were washed through two ovum culture media (OCM) droplets for 5min each. The blastocysts were incubated in vitrification solutions 1 and 2 (10% glycerol in OCM and 10% glycerol+20% ethylene glycol in OCM, respectively) for 5min each, followed by vitrification solution 3 (25% glycerol+25% ethylene glycol in OCM). They were then aspirated immediately into a 0.25cc cryopreservation straw, followed by an air bubble, and then a 0.25M sucrose solution in OCM. The straws were immediately plunged into liquid nitrogen and stored at −196°C. One to four days later, straws were thawed in air for 5s at room temperature, then in 22°C water for 15s. After thawing, the contents of the straw were expelled, mixed, held for 5min, and finally placed in OCM for 5min. Recovered embryos were placed in SOF+20% FBS and incubated at 38°C with 5% CO2 in air overnight. Viability was determined by re-expanding and subsequent hatching of the blastocyst. As shown in Table 1, there were no significant differences between re-expansion and hatching of blastocysts with cytochalasin B treatment compared to blastocysts not treated with cytochalasin B. These results suggest that, unlike porcine embryos (Dobrinsky et al., 2000 Biol Reprod 62, 564–570), cytochalasin B treatment does not improve the post-thaw viability of vitrified caprine parthenogenic blastocysts. Table 1

Sucrose and Facilitated Tucking for Pain Among Neonates Receiving Vaccination, in Puducherry

2017 ◽  
Vol 9 (3) ◽  
Author(s):  
Sujatha S. ◽  
Rebecca Samson ◽  
Christopher Amalraj ◽  
Sundaresan Sundaresan
Keyword(s):  
Sucrose Solution ◽  
Study Groups ◽  
Primary Outcome Measure ◽  
Intradermal Injection ◽  
Control Group ◽  
Intradermal Vaccination ◽  
Term Neonates ◽  
Bcg Vaccination ◽  
Pharmacologic Agents ◽  
Study Intervention

Neglected pain in neonates leads to various ill effects and it can be prevented by using simple and safe non-pharmacological pain relieving measures. Pharmacologic agents are not recommended in neonates for acute pain due toinvasive procedures however, administration of 24% oralsucrose solutionis found to be effective. The objective of this study was to assess the efficacy of 24%oral sucrose in combination with Facilitated tucking during BCG Vaccination through intradermalroute in term neonates which is not done elsewhere. Fifty five healthy term neonates who fulfilled the inclusion criteria such as gestational age above 37 weeks, within 24 hoursof birth age, and neonates delivered only through spontaneous vaginal delivery were included in the study. The study intervention consists of administration of 2 ml of oral 24% sucrose 2 minutes before BCG Vaccination through intradermal route and Facilitated tuckingat the time of vaccination. The primary outcome measure of cumulative NIPS score at 0, 3,5 minuteswas not significant in both the study groups. Whereas there was significant reduction in the level of pain and mean cry time in the neonates of sucrose group. Heart rateand oxygen saturation after intradermal injection also showed significant (p less than 0.001) differenceamong the neonates, who received 24% of oral sucroseand Facilitated tucking than for neonates of control group. Thus oral (24%)sucrose solution given 2 minutes before injection was effective in reducing level of neonatal pain following Intradermal Vaccination. It is a simple, safe and fast acting analgesic and should be considered for minor invasive procedures in term neonates which last for 5-7minutes.

Small-volume vitrification and rapid warming yield high survivals of one-cell rat embryos in cryotubes

2021 ◽  
Author(s):  
Yasuyoshi Fukuda ◽  
Misako Higashiya ◽  
Takahiro Obata ◽  
Keita Basaki ◽  
Megumi Yano ◽  
...  
Keyword(s):  
Cooling Rate ◽  
Small Volume ◽  
Sucrose Solution ◽  
High Concentration ◽  
Rat Embryos ◽  
Low Concentrations ◽  
Intracellular Ice Formation ◽  
Intracellular Ice ◽  
Warming Rate ◽  
Very High

Abstract To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to achieve this is to convert the water inside the cells into a non-crystalline glass. It is currently believed that to accomplish this vitrification, the cells must be suspended in a very high concentration (20–40%) of a glass-inducing solute, and subsequently cooled very rapidly. Herein, we report that this belief is erroneous with respect to the vitrification of one-cell rat embryos. In the present study, one-cell rat embryos were vitrified with 5 μL of EFS10 (a mixture of 10% ethylene glycol, 27% Ficoll, and 0.45 M sucrose) in cryotubes at a moderate cooling rate, and warmed at various rates. Survival was assessed according to the ability of the cells to develop into blastocysts and to develop to term. When embryos were vitrified at a 2,613 °C/min cooling rate and thawed by adding 1 mL of sucrose solution (0.3 M, 50 °C) at a warming rate of 18,467 °C/min, 58.1 ± 3.5% of the EFS10-vitrified embryos developed into blastocysts, and 50.0 ± 4.7% developed to term. These rates were similar to those of non-treated intact embryos. Using a conventional cryotube, we achieved developmental capabilities in one-cell rat embryos by rapid warming that were comparable to those of intact embryos, even using low concentrations (10%) of cell-permeating cryoprotectant and at low cooling rates.

Human oocyte vitrification with corona radiata, in autologous follicular fluid supplemented with ethylene glycol, preserves conventional IVF potential: birth of four healthy babies

2014 ◽  
Vol 26 (7) ◽  
pp. 1001 ◽  
Author(s):  
Xian-Hong Tong ◽  
Li-Min Wu ◽  
Ren-Tao Jin ◽  
Hong-Bing Luan ◽  
Yu-Sheng Liu
Keyword(s):  
Ethylene Glycol ◽  
Embryo Transfer ◽  
Follicular Fluid ◽  
Control Group ◽  
Human Oocyte ◽  
Oocyte Vitrification ◽  
Human Oocytes ◽  
Corona Radiata ◽  
Fertilisation Rate ◽  
Significant Difference

The aim of this study was to examine the effects of vitrification with autologous follicular fluid (AFF) supplemented with ethylene glycol (EG) and sucrose on human oocytes with corona radiata. A total of 182 human oocytes with corona radiata from fifteen infertile patients were vitrified using either equilibration solutions (ES) and vitrification solution (VS) consisting of AFF, EG and sucrose (AFF group, n = 67) or commercial ES and VS (control group, n = 115). All oocytes were thawed in the next cycle, with surviving oocytes being inseminated by conventional IVF. The clinical outcome of vitrified–warmed oocytes by both vitrification methods was analysed retrospectively. In the AFF group, six patients received embryo transfer, with three couples taking four healthy babies home. In the control group, nine patients received embryo transfer, with four couples taking five healthy babies home. There was no significant difference in the survival rate (91.0 vs 92.2%), two pronuclei (2PN) fertilisation rate (73.8 vs 73.6%), cleavage rate (100 vs 100%), top-quality embryo rate (62.2 vs 59.2%), clinical pregnancy rate (50.0 vs 44.4%), implantation rate (33.3 vs 25%) or take-home baby rate (50.0 vs 44.4%) between the AFF group and the control group, respectively. These results show that AFF supplemented with EG and sucrose is an efficient, cost-effective cryoprotectant for human oocyte cryopreservation. A corona radiata on vitrified–warmed oocytes retains the oocytes’ fertilisation capability in conventional IVF.

Abstract 2419: Left Ventricle Function Preservation by a Novel Synthetic Hydrogel Injection in Myocardial Infarction Rabbit

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Xiaoyan Li ◽  
Xuejun Jliang ◽  
Tao Wang ◽  
Taol Lin ◽  
Congxin Huang ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Heart Failure ◽  
Coronary Artery ◽  
Left Ventricle ◽  
Ethylene Glycol ◽  
Control Group ◽  
Coronary Artery Ligation ◽  
Poly Ethylene Glycol ◽  
Infarcted Myocardium ◽  
Poly Ethylene

Myocardial infarction and the subsequent heart failure remain among the world’s prominent health challenges. Other studies have demonstrated that bio-derived materials improve cardiac function after implantation for angiogenic potential. In this study, we hypothesized that injection of biomaterials into infarcted myocardium can preserve left ventricle (LV) function through its prevention of paradoxical systolic bulging. Infarction was induced in rabbit myocardium by coronary artery ligation. In sham-operated rabbits (n = 5), a suture was tied loosely around the left anterior descending coronary artery without ligating it. 7 dayslater, 100μl α-cyclodextrin (CD) solution and 100μl poly (ethylene glycol)-b-polycaprolactone-(dodecanedioic acid)-polycaprolactone-poly (ethylene glycol)(MPEG-PCL-MPEG) solution (n = 7) was injected simultaneously through Duploject applicator into the infarcted myocardium. Solid hydrogel matrix formed by linear MPEG-PCL-MPEG polymer threading into the cavities of the α-cyclodextrin after mixing. Injection of phosphate buffered saline (PBS) served as controls (n = 7). 28 days after the treatments, histological analysis indicated that injection of hydrogel prevented scar expansion and wall thinning compared with group ( P < 0.05) without more microvessel density in infarcted myocardium ( P = 0.70).By echocardiography, LV ejection fraction was significantly greater in the hydrogel group (56.09 ± 8.42%) than the control group (37.26 ± 6.36%, P = 0.001). The LV end-diastolic and end-systolic diameters were 2.07 ± 0.33 cm and 1.74 ± 0.30cm in the control group, respectively. Smaller LV end-diastolic diameter (1.61 ± 0.26cm, P = 0.005) and smaller end-systolic diameter (1.17 ± 0.23cm, P = 0.001) were found in the hydrogel group. These results suggest that α-CD/MPEG-PCL-MPEG hydrogel injection could serve structural and mechanical support of an injured LV replacing some of the functions of the damaged ECM and thus prevented paradoxical motion serves, which may eventually lead to LV remodeling and dilation prevention. Our study should initiate further experimental and clinical studies exploring potential approaches to the treatment of postinfarction heart failure.

Volumetric Changes in Cells During Freezing and Thawing

1980 ◽  
Vol 102 (2) ◽  
pp. 91-97 ◽  
Author(s):  
J. M. Knox ◽  
G. S. Schwartz ◽  
K. R. Diller
Keyword(s):  
Experimental Data ◽  
Image Analysis ◽  
Close Agreement ◽  
Living Cells ◽  
Freezing Process ◽  
Freezing And Thawing ◽  
Image Analysis Technique ◽  
Computerized Image Analysis ◽  
Analysis Technique ◽  
Thawing Process

A thermodynamic model is presented to describe the combined freezing and thawing process for living cells. Continuous changes in the cell volume are predicted according to the thermal protocol imposed on the system. Experimental verification of the model is sought by monitoring continuously the volume of cells as frozen on a cryomicroscope. The volumes of individual cells are measured from sequential photomicrographs by a computerized image analysis technique. The model and experimental data are in quite close agreement for the freezing process, but upon thawing the experimentally measured volumes consistently increased much more rapidly than predicted by the model. The model can be made to conform to the data by accounting for a substantial influx of electrolyte to the cell at subfreezing temperatures.

scholarly journals Effect of an alcoholic diet on dental caries and on Streptococcus of the mutans group: study in rats

2007 ◽  
Vol 21 (2) ◽  
pp. 101-105 ◽  
Author(s):  
Karla Zanini Kantorski ◽  
Daniela Martins de Souza ◽  
Verônica Quispe Yujra ◽  
Juliana Campos Junqueira ◽  
Antonio Olavo Cardoso Jorge ◽  
...  
Keyword(s):  
Dental Caries ◽  
Smooth Surface ◽  
Sucrose Solution ◽  
Control Diet ◽  
Ethanol Solution ◽  
Control Group ◽  
Water Control ◽  
Occlusal Caries ◽  
Occlusal Surfaces ◽  
Significant Difference

The objective of this study was to evaluate the effects of an alcohol diet on Streptococcus of the mutans group and on dental caries in the oral cavity of rats. Forty animals were divided into 3 groups according to the following liquid diets: 20% ethanol solution (Alcohol Group, AG), 27% sucrose solution (Isocaloric Group, IG), and water (Control Group, CG). After 56 days, samples were collected and plated on Mitis Salivarius Bacitracin agar to assess the number of colony forming units (CFU/mL) of Streptococcus of the mutans group. The animals were sacrificed and the jaws were removed in order to assess the occurrence of dental caries on the smooth and occlusal surfaces using stereomicroscopy. The data were submitted to ANOVA and Tukey test. The average numbers of CFU/mL (10³) were: 8.17 (AG), 9.78 (IG), and 5.63 (CG). There was no significant difference among the groups for the occurrence of occlusal caries. Regarding smooth surface caries, in the upper jaw, the caries number in the IG (1.58) was similar to that in the AG (2.06) and in the CG (1.14), and the number of caries in the AG was higher than in the CG; in the lower jaw there was significant difference among the 3 groups: AG (1.14), IG (2.00) and CG (0.43). The diets with the alcohol and sucrose solutions presented a tendency of increasing the colonization by Streptococcus of the mutans group and of increasing the occurrence of smooth surface dental caries in rat molars when compared to the control diet.

Vitrification of bovine IVF blastocysts in an ethylene glycol/sucrose solution and heat-stable plant-extracted proteins

1997 ◽  
Vol 47 (4) ◽  
pp. 865-879 ◽  
Author(s):  
A.T. Palasz ◽  
H. Gustafsson ◽  
H. Rodriguez-Martinez ◽  
L. Gusta ◽  
B. Larsson ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Sucrose Solution ◽  
Heat Stable

62 EXPOSURE TO ETHYLENE GLYCOL AND DIMETHYL SULFOXIDE CAUSES ACTIVATION AND SPINDLE ANOMALIES IN BUFFALO (BUBALUS BUBALIS) OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 131 ◽  
Author(s):  
M. De Blasi ◽  
E. Mariotti ◽  
M. Rubessa ◽  
S. Di Francesco ◽  
G. Campanile ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Embryo Development ◽  
Sucrose Solution ◽  
Bubalus Bubalis ◽  
Meiotic Spindle ◽  
Blastocyst Development ◽  
Chi Square ◽  
Chi Square Test ◽  
Simple Exposure

Despite the increasing interest, buffalo oocyte cryopreservation is still inefficient, especially in terms of blastocyst development after IVF. The aim of this work was to evaluate chromatin and spindle organization of buffalo in vitro-matured oocytes after vitrification/warming by cryotop and after their simple exposure to cryoprotectants (CP). An overall amount of 251 COC was selected and matured in vitro. In the vitrification group, COC were first exposed to 10% ethylene glycol (EG) + 10% DMSO for 3 min, and then to 20% EG + 20% of DMSO and 0.5 m sucrose, loaded on cryotops, and plunged into liquid nitrogen within 25 s. Oocytes were warmed into a 1.25 m sucrose solution for 1 min and then to decreasing concentrations of sucrose (0.625 m, 0.42 m, and 0.31 m) for 30s each. In order to test CP toxicity, COC were simply exposed to the vitrification and warming solutions. Two hours after warming, oocytes were fixed and immunostained for microtubules using a method previously described (Messinger SM and Albertini DF 1991 J. Cell Sci. 100, 289–298), stained for nuclei with Hoechst, and examined by fluorescence microscopy. Fresh in vitro-matured oocytes were fixed and stained as controls. Data were analyzed by chi-square test; results are shown in Table 1. The percentages of MII oocytes in the control and vitrification groups were greater than in the toxicity group, in which a greater percentage of telophase II stage oocytes were found compared with both the control and vitrification groups, indicating occurrence of activation. Of the MII oocytes, both exposure to CP and vitrification procedures gave greater percentages of oocytes with abnormal spindle and abnormal chromatin configuration compared with the control. An unexpected datum was the evidence of a significant percentage of spontaneously activated oocytes in the toxicity group. We speculate that the lack of activation in the vitrification group may be related to the slowing down of metabolic activity subsequent to thermal shock, and hence, that activation after vitrification may occur later than 2 h post-warming. In conclusion, the simple exposure to CP causes activation of the COC and damage to the cytoskeleton similar to that induced by the whole vitrification protocol. The damages to the meiotic spindle and DNA fragmentation may lead to aneuploidy incompatible with subsequent embryo development and account for the poor embryo development currently recorded in buffalo. Table 1.Chromatin and spindle organization in oocytes vitrified and exposed to cryoprotectants

25 Effect of taxol and epothilone B on meiotic spindle stabilization in vitrified bovine oocytes

2021 ◽  
Vol 33 (2) ◽  
pp. 120
Author(s):  
E. Girka ◽  
K. R. Bondioli
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Recovery Period ◽  
Normal Chromosome ◽  
Meiotic Spindle ◽  
Control Group ◽  
Chromosome Arrangement ◽  
Epothilone B ◽  
Significant Difference ◽  
Bovine Oocytes

Vitrification has the potential to be a valuable technique for preservation of bovine oocytes; however, this method often results in abnormal microtubule and chromosome arrangement. The aim of this experiment was to evaluate taxol and epothilone B as meiotic spindle stabilising pretreatments in a vitrification protocol. Bovine oocytes were purchased and matured invitro during shipment. At 18h of maturation, oocytes were divided randomly into control, taxol, and epothilone B treatments (Table 1). All treatments were prepared in invitro maturation (IVM) medium (IVF Biosciences). Partially denuded oocytes were incubated in either control or treatment medium for 15min at 38.5°C before vitrification. Oocytes were incubated in an equilibration solution (10% dimethyl sulfoxide, 10% ethylene glycol) for 5min, transferred to a vitrification solution (20% dimethyl sulfoxide, 20% ethylene glycol, 0.5M sucrose), loaded onto a Cryolock, and plunged into liquid nitrogen within 45s. For warming, a Cryolock was placed directly into a 0.5M sucrose solution and incubated for 3min. Oocytes were transferred to a 0.25M solution for 3min and washed in the basal solution used for vitrification and warming media (Dulbecco’s phosphate-buffered saline, 20% fetal bovine serum). Once warmed, oocytes were transferred to IVM medium for a 4-h recovery period and completely denuded before staining. Staining to evaluate spindle morphology was performed with anti α-tubulin primary antibody and secondary antibody Alexa Fluor 488. Oocytes were also stained with Hoechst to evaluate chromosome arrangement. Both spindle morphology and chromosome arrangement data were analysed using a logistic regression with a binomial response variable (normal/abnormal). Both 0.5μM and 1.0μM Taxol treatments had no effect on either meiotic spindle or chromosome arrangement compared with the control group (P&gt;0.05). The 2.0μM taxol treatment improved chromosome configuration (P&lt;0.05) with no effect on microtubule distribution compared with the control group (P&gt;0.05). All epothilone B treatments resulted in disruption of microtubule distribution and chromosome arrangement compared with control (P&lt;0.001) and resulted in a consistent abnormality hypothesised to be tubulin polymerization. These results indicate that taxol is capable of increasing the occurrence of normal chromosome arrangement in vitrified bovine oocytes and that epothilone B may cause additional harm to the oocyte that is not associated with the metaphase plate. Table 1. Effect of stabilisation agents on meiotic spindle of invitro-matured bovine oocytes Treatment n Normal microtubule distribution (%) Normal chromosome arrangement (%) Control 100 44 47 0.5μM Taxol 104 44 37 1.0μM Taxol 98 43 56 2.0μM Taxol 102 49 62a 0.5μM Epothilone B 103 11b 11b 1.0μM Epothilone B 97 6b 8b 2.0μM Epothilone B 100 2b 1b aP&lt;0.05;. bP&lt;0.001: Different superscripts within a column indicate a significant difference.

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