scholarly journals 87ONE-STEP VERSUS TWO-STEP VITRIFICATION AND IN-STRAW DILUTION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 165
Author(s):  
L.F. Campos-Chillon ◽  
D.J. Walker ◽  
J.F. De La Torre-Sanchez
Keyword(s):  
Least Square ◽  
Bovine Embryos ◽  
Step Method ◽  
Slow Cooling ◽  
Standard Procedures ◽  
Step Procedure ◽  
Main Effect ◽  
One Step ◽  
The One

Slow-cooling techniques are widely used in cryopreservation of bovine embryos. We have previously developed a simple, two-step vitrification technique for direct transfer in the field; however, simplification to one-step vitrification would be attractive. Therefore, factorial combinations of two techniques (one-step and two-step) and two post-thaw temperatures until culture (24 and 37°C) were studied. Blastocysts (n=220) sired by two bulls were obtained in vitro in four replicates. Briefly, oocytes were aspirated from 2–8-mm follicles of ovaries obtained at a slaughterhouse, matured, fertilized and cultured in vitro with standard procedures using chemically defined media (CDM1/2 or G1/2). Two-step embryos were transferred in 1μL into 1mL of V1-CDM (5M ethylene glycol (EG) in HEPES-buffered holding medium (HCDM2)), and one-step embryos into a 7-μL droplet of V2-CDM (7M EG, 0.5M galactose and 18% w/v Ficoll 70 in HCDM2) for 3min at 24°C. Next, embryos for the two-step method were moved in 1μL into a 7μL droplet of V2-CDM at 24°C. Droplets containing embryos (one or two-step) were loaded into 0.25-mL straws preloaded with a 1-cm column of D-HCDM (0.5M galactose in HCDM2), then 0.5cm air, and then 7cm of D-HCDM followed by 0.5cm air. The column containing the embryos (0.5cm (7μL)) was followed with 0.5cm air and 1cm of D-HCDM. Straws were heat-sealed and plunged vertically, sealed end first, into liquid nitrogen just covering the embryo, and the rest of the straw was then slowly immersed. The time from loading to plunging was 40–50s. Straws were thawed in air (24°C) for 10s and then in water horizontally at 37°C until ice disappeared. Straws were gently shaken to mix the columns; then, after 5min at 24 or 37°C, embryos were expelled and cultured in CDM2+5% FCS. Re-expansion and hatching rates were evaluated 48 h post thaw. Data (Table 1) were calculated as a percentage of non-vitrified controls for respective replicates (control means: re-expansion 87%; hatching 74%) and analyzed by ANOVA. There were no main effects of post-thawing temperature (P>0.1), indicating that, after thawing, embryos can be kept at room or body temperature. Also, main effect means for re-expansion and hatching for one-step or two-step addition of cryoprotectant were similar (P>0.1), but there was a tendency for higher survival for the two-step procedure. Further refinements of the one-step technique including EG concentrations, embryological stages and equilibration times should be studied. Table 1 Main effect means (least-square means±SEM) of vitrified embryos (% of non-vitrified controls)

Ultrastructure of bovine in vitro-produced blastocysts cryopreserved by vitrification

Zygote ◽  
1998 ◽  
Vol 6 (1) ◽  
pp. 17-26 ◽  
Author(s):  
Shigeki Ohboshi ◽  
Noboru Fujihara ◽  
Tatsuyuki Yoshida ◽  
Hiroshi Tomagane
Keyword(s):  
Ethylene Glycol ◽  
Cellular Damage ◽  
Bovine Embryos ◽  
Step Procedure ◽  
Direct Exposure ◽  
Subsequent Exposure ◽  
One Step ◽  
The One ◽  
Serious Injuries

SummaryThe objective of this study was to examine ultrastructural aspects of bovine in vitro-produced blastocysts associated with cryopreservation by vitrification. Morphologically good embryos were used and treated with ethylene-glycol-based vitrification solution (VS). The untreated embryos had conventional fine structure. The post-warming embryos treated with direct exposure to VS (one-step procedure) showed cellular damage structurally by cryopreservation, which included loss of microvilli, disruption of the plasma membrane, mitochondrial changes and swelling of the endoplasmic reticulum. However, nuclei and junctional regions seemed to be resistant to cryoinjury. In contrast, the post-warming embryos pre-equilibra ted with 10% ethylene glycol for 5 min and subsequent exposure to VS (two-step procedure) showed less damage than those treated by the one-step procedure. Post-warming embryos treated by the two-step procedure were cultured in vitro for 18 h. Some embryos survived and their structures re-formed to the former state, while other embryos showed serious injuries and could not reconstitute the blastocoele. Three post-warming embryos treated by the two-step procedure that survived after in vitro culture were transferred to three recipients and one of these resulted in pregnancy. These results indicate that cryopreservation by vitrification can damage membranous structures of the cells of bovine embryos, the extent and nature of this damage being dependent on the vitrification procedure.

206 Effect of L-Ascorbic Acid, Polydatin, and In-Straw Rehydration of Slow-Frozen In Vitro-Produced Bovine Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 243
Author(s):  
C. M. Dinndorf ◽  
C. M. Owen ◽  
M. Barceló-Fimbres ◽  
L. F. Campos-Chillon
Keyword(s):  
Reactive Oxygen Species ◽  
Ascorbic Acid ◽  
Oxygen Species ◽  
Bovine Embryos ◽  
Freezing Medium ◽  
High Lipid ◽  
Standard Procedures ◽  
Frozen Embryos ◽  
Main Effect

In vitro-produced (IVP) bovine embryos have poor cryotolerance due to high lipid content and reactive oxygen species levels that hinder post-thaw survival. We hypothesised that in-straw rehydration of slow-frozen embryos with sucrose and the addition of the antioxidant polydatin and l-ascorbic acid would increase post-thaw survival. The IVP embryos (n = 116) were generated in 7 replicates by aspirating oocytes from 2- to 8-mm follicles of abattoir ovaries, matured for 23 h, fertilized with semen from 1 of 3 bulls using standard procedures, and cultured in SCF1 medium for 7 days (Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130). Stage 7 embryos were slow-frozen using 1 of 4 protocols in a 2 × 2 factorial design: embryos were equilibrated in conventional slow-freezing media for 20 min [1.5 M ethylene glycol (EG) and 0.5 M sucrose] with 1 mm l-ascorbic acid or 1 μM polydatin, and loaded in the straw adjacent to columns of freezing medium or 0.75 M EG and 0.6 M sucrose and then seeded at −6°C, cooled at 0.5°C min−1, and plunged at –32°C. Embryos were thawed in air for 10 s followed by 30 s in 32 to 35°C water bath. Once straw columns were disrupted, embryos were allowed to equilibrate for 5 min. Subsequently, embryos were washed and placed back in culture and re-expansion was assessed at 24 and 48 h. Data (Table 1) were analysed by ANOVA with means separated by Tukey’s HSD. Results indicate that there was no main effect between the 2 antioxidants or the use of rehydration columns (P < 0.05); however, there was higher (P < 0.05) re-expansion for embryos frozen with polydatin and with rehydration column than embryos frozen with l-ascorbic acid and no rehydration column. This suggests that polydatin coupled with in-straw rehydration (0.75 M EG and 0.6 M sucrose) may improve post-thaw survival of IVP bovine embryos. Table 1.Post-thaw re-expansion rates of embryos exposed to antioxidants and in-straw rehydration (± SEM)

106 THE WARMING PROCEDURE: A FIRST STEP FOR IMPROVING THE NONSURGICAL DEEP INTRAUTERINE TRANSFER OF SOPS-VITRIFIED PORCINE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 165
Author(s):  
J. Gomis ◽  
C. Cuello ◽  
J. Sanchez-Osorio ◽  
M. A. Gil ◽  
I. Parrilla ◽  
...  
Keyword(s):  
Reproductive Performance ◽  
Petri Dish ◽  
Control Group ◽  
Hatching Rate ◽  
Embryo Viability ◽  
Step Method ◽  
One Step ◽  
The One

We previously reported successful nonsurgical deep intrauterine embryo transfer (ET) of fresh in vivo–derived porcine embryos. However, several trials from our laboratory demonstrated that when this procedure was used in combination with vitrified/warmed (VW) embryos, its efficiency was very low. Recently, we have shown that the one-step warming method in syringe, which was used in the earlier trials, compromises the in vitro embryo viability. The aim of this study was to confirm the negative effect of the direct warming in syringe and to evaluate the effect of alternative warming procedures on the in vivo development of VW embryos after nonsurgical ET. In Experiment 1, morulae and early blastocysts were collected on Days 5 to 6 (Day 0: onset of oestrus) and assigned to one of the following groups: 1) syringe group: vitrified embryos (n = 88) were warmed by the one-step method directly in a 1-mL syringe containing 300 μL of warming medium, which was connected to the ET catheter and then transferred to recipients (n = 6); 2) dish group: vitrified embryos (n = 194) were warmed with one-step warming method in a Petri dish containing 1 mL of warming medium, loaded into a Tom Cat catheter and transferred to recipients (n = 13); and 3) control group: fresh embryos (n = 129) were loaded in a 1-mL syringe in 100 μL of transfer medium and transferred to 9 recipients. An average of 15 embryos were transferred to each recipient on Day 4 or 5. Embryos were surgically recovered 24 h after ET. Data were analysed by ANOVA. The embryo recovery rate was similar among groups (range: 70.7 ± 4.8% to 77.2 ± 6.5%). The embryo survival (ES) and the hatching rate (HR) from the control group (94.0 ± 2.1% and 33.4 ± 7.6%, respectively) were higher (P ≤ 0.05) than those from the dish group (80.4 ± 4.6% and 14.5 ± 4.1%, respectively). All embryos from the syringe group were degenerated. Some viable recovered embryos (n = 135) were cultured for 48 h to evaluate their subsequent in vitro development. No differences were observed in ES between the control and the dish group (100.0 ± 0.0% vs 98.9 ± 1.0%). The HR in the control group (71.5 ± 2.1%) was higher (P ≤ 0.01) than that of the dish group (42.7 ± 6.1%). In experiment 2 we evaluated the reproductive performance of naturally cycling recipients after nonsurgical ET of vitrified embryos warmed with the one-step method in a Petri dish. An average of 35 VW morulae and blastocysts were transferred to each recipient (n = 10) on Days 4 to 6. Four recipients returned to oestrus at Days 21 to 22. The remaining 6 recipients were diagnosed pregnant by ultrasonography on Day 26. At Days 50 to 55, 5 recipients remained pregnant. In conclusion, the one-step in syringe warming method for vitrified porcine embryos had a completely adverse effect on the vivo viability, whereas nonsurgical ET of embryos warmed in a Petri dish allowed us to obtain promising reproductive performance. Supported by MICINN (AGL2009-12091) and SENECA (GERM 04543/07).

scholarly journals Efficient Cryopreservation of Populus tremula by In Vitro-Grown Axillary Buds and Genetic Stability of Recovered Plants

Plants ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 77
Author(s):  
Elena O. Vidyagina ◽  
Nikolay N. Kharchenko ◽  
Konstantin A. Shestibratov
Keyword(s):  
Survival Rate ◽  
Axillary Buds ◽  
Long Term Storage ◽  
Average Survival ◽  
Transgenic Lines ◽  
Ssr Loci ◽  
One Step ◽  
The One

Axillary buds of in vitro microshoots were successfully frozen at –196 °C by the one-step freezing method using the protective vitrification solution 2 (PVS2). Microshoots were taken from 11 transgenic lines and three wild type lines. Influence of different explant pretreatments were analyzed from the point of their influence towards recovery after cryopreservation. It was found out that the use of axillary buds as explants after removal of the apical one increases recovery on average by 8%. The cultivation on growth medium of higher density insignificantly raises the regenerants survival rate. Pretreatment of the osmotic fluid (OF) shows the greatest influence on the survival rate. It leads to the increase in survival rate by 20%. The cryopreservation technology providing regenerants average survival rate of 83% was developed. It was based on the experimental results obtained with explant pretreatment. Incubation time in liquid nitrogen did not affect the explants survival rate after thawing. After six months cryostorage of samples their genetic variability was analyzed. Six variable simple sequence repeat (SSR) loci were used to analyze genotype variability after the freezing-thawing procedure. The microsatellite analysis showed the genetic status identity of plants after cryopreservation and of the original genotypes. The presence of the recombinant gene in the transgenic lines after cryostorage were confirmed so as the interclonal variation in the growth rate under greenhouse conditions. The developed technique is recommended for long-term storage of various breeding and genetically modified lines of aspen plants, as it provides a high percentage of explants survival with no changes in genotype.

A one-step method for direct nonsurgical transfer of frozen-thawed bovine embryos. I. Basic studies

Cryobiology ◽  
1982 ◽  
Vol 19 (6) ◽  
pp. 673-674 ◽  
Author(s):  
S.P. Leibo ◽  
A.W. West ◽  
B. Perry
Keyword(s):  
Bovine Embryos ◽  
Step Method ◽  
One Step ◽  
Basic Studies

scholarly journals An Oligoimide Particle as a Pickering Emulsion Stabilizer

Polymers ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 1071 ◽  
Author(s):  
Yu-Jin Cho ◽  
Dong-Min Kim ◽  
In-Ho Song ◽  
Ju-Young Choi ◽  
Seung-Won Jin ◽  
...  
Keyword(s):  
Particle Concentration ◽  
Pickering Emulsion ◽  
Pickering Emulsions ◽  
Step Method ◽  
Wetting Properties ◽  
Emulsion Droplets ◽  
Step Procedure ◽  
Oil In Water ◽  
One Step ◽  
Oil Emulsions

A pyromellitic dianhydride (PMDA) and 4,4′-oxydianiline (ODA)-based oligoimide (PMDA-ODA) was synthesized by a one-step procedure using water as a solvent. The PMDA-ODA particles showed excellent partial wetting properties and were stably dispersed in both water and oil phases. A stable dispersion was not obtained with comparison PMDA-ODA particles that were synthesized by a conventional two-step method using an organic solvent. Both oil-in-water and water-in-oil Pickering emulsions were prepared using the oligoimide particles synthesized in water, and the size of the emulsion droplet was controlled based on the oligoimide particle concentration. The oligoimide particles were tested to prepare Pickering emulsions using various kinds of oils. The oil-in-water Pickering emulsions were successfully applied to prepare microcapsules of the emulsion droplets. Our new Pickering emulsion stabilizer has the advantages of easy synthesis, no need for surface modification, and the capability of stabilizing both oil-in-water and water-in-oil emulsions.

Influence of amide versus ester linkages on the anticancer properties of the new flavone–biotin conjugates

2019 ◽  
Vol 74 (7-8) ◽  
pp. 193-200
Author(s):  
Monika Stompor ◽  
Marta Świtalska ◽  
Agata Bajek ◽  
Joanna Wietrzyk
Keyword(s):  
Cell Lines ◽  
Amide Bond ◽  
Step Method ◽  
Fluorescent Reporter ◽  
Bifunctional Molecules ◽  
Anticancer Properties ◽  
One Step ◽  
3T3 Cell Lines ◽  
Mcf 7

Abstract Novel biotinylated C-6 substituted flavones were synthesised by a one-step method that connects biotin to 6-hydroxyflavone and 6-aminoflavone by esterification and amidation of hydroxyl and amino groups, respectively. The obtained compounds, 6-O-biotinylflavone and 6-biotinylamidoflavone, are the bifunctional molecules composed of a flavone moiety as a fluorescent reporter and biotin as a cancer-targeting unit. Antiproliferative activity was evaluated using SRB assays in MCF-7, MCF-10A, HepG2, MDA-MB-231, 4T1, and Balb/3T3 cell lines. In vitro evaluation revealed that compounds with biotin moiety displayed better cell selectivity between the cancer and normal cells than the parental substrates. These results indicate that anticancer effect is not related to the position of biotin moiety, but it is related to the presence of ester or amide bond. 6-O-Biotinylflavone was more active than 6-hydroxyflavone against human breast (MDA-MB-231) and liver (HepG2) cancer cells with IC50 (concentration of tested agent that inhibits proliferation of the cell population by 50%) values equal to 78.5 ± 18.8 μM and 133.2 ± 14.2 μM, respectively. Non biotinylated 6-aminoflavone was more active than 6-biotinylamidoflavone against all tested cell lines, with IC50 values between 34.3 ± 9.1 μM (4T1) and 173.86 ± 24.3 μM (MCF-7).

Bias in the one-step method for pooling study results

1990 ◽  
Vol 9 (3) ◽  
pp. 247-252 ◽  
Author(s):  
Sander Greenland ◽  
Alberto Salvan
Keyword(s):  
Step Method ◽  
Study Results ◽  
One Step ◽  
The One

Survival of vitrified in vitro–produced bovine embryos after a one-step warming in-straw cryoprotectant dilution procedure

2015 ◽  
Vol 83 (5) ◽  
pp. 881-890 ◽  
Author(s):  
J.N. Caamaño ◽  
E. Gómez ◽  
B. Trigal ◽  
M. Muñoz ◽  
S. Carrocera ◽  
...  
Keyword(s):  
Bovine Embryos ◽  
One Step
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