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Effect of vitrification procedure on chromosomal status of embryos achieved from vitrified and fresh oocytes  [PDF]
Javier I García, Luis Noriega-Portella, Luis Noriega-Hoces
Health (Health) , 2011, DOI: 10.4236/health.2011.37077
Abstract: Background: In order to assess the chromosomal status in embryos obtained from vitrified and fresh donated oocytes, preimplantational genetic diagnostic (PGD) was performed after biopsy of one blastomere at day 3. METHODS: A total of 249 oocytes were obtained from 23 oocyte donors, 80 oocytes were used in the vitrified group and 151 oocytes were used in the fresh group. Nine chromosomes (13, 15, 16, 17, 18, 21, 22, X and Y) were investigated by fluorescence in situ hybridization (FISH) analysis in 56 and 121 embryos from vitrified and fresh group respectively. Fertilization, cleavage rate, embryo quality and chromosomal abnormality rate were compared between groups evaluated. Results: Vitrified oocytes showed a survival rate of 97.5%. There was no significant difference in the fertilization rate (82.7% and 91.4%), Day 2 cleavage rate (90.3% and 87.7%) or blastocyst formation rate (31.1% and 44.6%) for the vitrified and fresh groups respectively. Chromosomal abnormality rate (66.1% versus 71.9%), percentage of abnormal blastocysts (61.1% versus 64.8%) and percentage of abnormalities for each analyzed chromosome were similar for the vitrified group compared with the control group. Conclusions: The rates of chromosomal abnormalities in embryos from vitrified oocytes are similar to those published previously; and comparable to those observed in embryos from fresh oocytes. These results confirm that the developmental competence and chromosomal status of embryos obtained from vitrified oocytes is not affected by the vitrification procedure, and they preserve the potential to be fertilized and to develop in to blastocyst stage similar to embryos from fresh oocytes.
Cryopreservation of Seeds and Embryos of Jatropha curcas L.  [PDF]
Julián Andrés Prada, María Elena Aguilar, Ana Abdelnour-Esquivel, Florent Engelmann
American Journal of Plant Sciences (AJPS) , 2015, DOI: 10.4236/ajps.2015.61020
Abstract: Jatropha curcas is a species with a variety of uses. It is grown primarily for oil for biodiesel, but also has agronomic and medicinal applications. Two methods were evaluated for cryopreservation of seeds and zygotic embryos of J. curcas: desiccation followed by rapid immersion of seeds and embryos in liquid nitrogen (LN, -196°C), and vitrification of zygotic embryos. Prior to cryo-preservation, seeds were manually scarified and the moisture content (MC) of seeds and embryos was determined. Explants were disinfected after cryopreservation. Seed germination after LN exposure was 100%. Plantlet development was better in sand substrate than that in vitro. Survival of zygotic embryos after cryopreservation was also 100%, without significant differences between treatments. Optimal development (100%) and plantlet length (51.77 mm) were observed with embryos dried for 60 min to 9.4% MC under laminar flow prior to cryopreservation. Zygotic embryos subjected to the vitrification procedure did not withstand LN exposure. Survival data for non-cryopreserved embryos after each step of the vitrification procedure provided information about embryo tolerance to cryoprotectants.
Efecto del número de embriones por pajuela sobre la viabilidad morfológica post vitrificación en embriones murinos obtenidos in vivo
Cabrera,Pedro; Fernández,Adriana; Díaz,Thaís; Bastidas,Pedro; Molina,Magaly; Bethencourt,Angélica; Vivas,Isis; Reyes,Yuraima; Sifontes,Freddy;
Agronomía Tropical , 2006,
Abstract: the technique of cryopreservation of embryos by vitrification has acquiredgreat relevance world-wide because of the easiness of the process, the low costof the equipment and the high levels of viability of the embryos after vitrification.to determine the effect of the number of embryos per straw on the morphologicalappearance, we used four different numbers of embryos per straw: 1, 2, 4 and6, obtaining no effects (p>0.05) of the number of embryos on the rate of embryorecovery after vitrification, or on the rate of normal embryos. however, thebest results were shown when 4 embryos per straw were used.
Vitrificación de ovocitos bovinos y su uso en el desarrollo partenogenético de embriones
Ruiz,J; Correa,JE; Martínez,M;
Archivos de medicina veterinaria , 2010, DOI: 10.4067/S0301-732X2010000100011
Abstract: the aim of this study was to evaluate vitrification effects on the viability of chemically activated oocytes in order to produce parthenogenetic bovine embryos. bovine oocytes retrieved from ovaries obtained in a slaughterhouse were matured in vitro for 20-22 hours and then assigned to the following groups: i (n=76): vitrified/thawed oocytes, ii (n=119): exposed oocytes to cryoprotectans without vitrification and iii (n=142): control oocytes. bovine oocytes were vitrified in microdrops on a precooled aluminum foil floating in liquid nitrogen, using an equilibrium solution with 4% ethylene glycol and a vitrification solution with 35% ethylene glycol, 5% polyvinyl-pyrrolidone and 0,4 m trehalose. the vitrified microdrops were stored in liquid nitrogen and were thawed after 1-3 days of storage. the oocytes of the 3 groups were parthenogenetically activated by 4-min exposure to 5 μm ca ionomycin at room temperature followed through 5 hours incubation in 6-dimethylaminopurine at 38.5 oc in a 5% co2 in humidified atmosphere. embryos were cultivated on msof medium during 8-9 days. the rates of oocytes survival were 55.1% and 93.7% to vitrified/thawed (i) and exposed (ii) oocytes respectively. the rates of cleavage were 55.3%, 72.3% and 74.6%; and development to blastocysts were 7.1%, 17.4% and 21.7% in groups i, ii and iii respectively. these results demonstrate that the oocyte vitrification technique has been set up in our laboratory and parthenogenetic bovine embryos can be produced from such as vitrified/thawed oocytes.
Vitrificacion de blastocitos bovinos producidos in vitro con el método Open Pulled Straw (OPS): Primer reporte*
Silva,M. E.; Berland,M. A;
Archivos de medicina veterinaria , 2004, DOI: 10.4067/S0301-732X2004000100009
Abstract: the purpose of this work is to present preliminary produced in vitro using the open pulled straw (ops) method. open pulled straws were made by pulling 0.25 ml inner and outer diameter were reduced to half of their original and deposited individually in vitrification solutions 1 and dimethyl - sulfoxide (dmso), for 1 minute and 20 μl containing the embryo pulled into the straw by capillarity into liquid nitrogen. eighty three blastocysts were recovered after thawing. fifthy four percent of these were re- expanded or hatched after 24 hours of in vitro culture. after 72 hours of in vitro culture 29% of the blastocysts matured. even though our results are slightly lower to those reported in the literature, they show that it is possible to successfully vitrify bovine blastocysts produced in vitro using the ops method
Vitrifica??o de ovócitos imaturos de bovinos utilizando etilenoglicol associado à trehalose e polivinilpirrolidona
Souza, M.R.;Costa, E.P.;Torres, C.A.A.;Guimar?es, J.D.;Fagundes, L.M.;
Arquivo Brasileiro de Medicina Veterinária e Zootecnia , 2003, DOI: 10.1590/S0102-09352003000500011
Abstract: this study aimed to evaluate the effects of vitrification procedure of immature bovine oocytes using ethylene glycol (eg) associated with trehalose and polivinylpyrrolidone on the percentage of recovered oocytes with normal morphology and nuclear maturation, fecundation and cleavage rates for in vitro cultivated embryos. ovary oocytes of slaughtered cows were randomly allotted to three treatments (t): ti - oocytes neither undenuded nor vitrified, tii - vitrified oocytes with cumulus oophorus, tiii - undenuded vitrified oocytes. the percentage of recovered oocytes and oocytes with normal morphology after vitrification was different for tii and tiii (92.2 and 72.6%, 79.0 and 63.3% for tii and tiii, respectively). all normal oocytes were cultivated at 38.5oc in atmosphere with 5% co2 for 24 hours. after culture, the oocytes were fecundated and the embryos were cultivated in vitro for seven days. the nuclear maturation, fecundation and cleavage rates for ti, tii and tiii were different (83.9, 70.0 and 44.0%, 17.5, 23.7 and 5.1%, 0.0, 0.0 and 0.0% for ti, tii and tiii, respectively). morulas and blastocysts were obtained only for ti (21.4%). these results indicate that the protocol used for vitrification procedure is not recommended for cryopreservation of immature bovine oocytes.
Vitrificación como técnica de crioconservación de embriones bovinos
Archivos de medicina veterinaria , 2002, DOI: 10.4067/S0301-732X2002000200002
Abstract: over the last 40 years cells and embryos of mammals have been preserved, at room temperature, refrigeration temperature and by different cryoconservation methods. these techniques have evolved into more simple, practical and less expensive freezing procedures. one of these is vitrification, a process of solidification that uses a highly concentrated solution which does not crystallize during freezing; its viscosity increases with the descent of the temperature until the formation of an amorphous solid state similar to glass. for this reason, exposure and freezing rates should be quick enough to avoid toxicity and the formation of intracellular ice, which can cause embryonic damage. in order for the embryos to support the osmotic shock, they should be equilibriated with a less concentrated crioprotectant solution before being exposed to the vitrificant solution for their later freezing. several vitrification procedures have been published about embryo preservation, using different crioprotectants, concentration, volume, addition method, temperatures, exposition time, freezing rate, thawing procedure and dilution, to maintain the function, normal structure and viability of the embryo. these techniques have also been experimented with in vitro and in vivo produced embryos, at different developmental stages. this paper aims to review the present bovine embryo cryopreservation methods, particulary vitrification, as well as to mention the latest procedures and progress so that new researchers may have an updated literature review to start their works.
Caracteriza??o isozimática e atividade de peroxidase em folhas de plantas hiperídrica, intermediária e normal de Bidens pilosa L. mantidas in vitro
Oliveira, José Emílio Zanzirolani de;Amaral, Cláudio Lúcio Fernandes;Casali, Vicente Wagner Dias;
Ciência e Agrotecnologia , 2008, DOI: 10.1590/S1413-70542008000100004
Abstract: activity of peroxidase (ec and isozymes analysis of a bidens pilosa clone maintained in vitro culture were characterized in hyperhydric, intermediary and normal plants. electrophorese in starch gels (12%) of six isozymes systems was tested, polymorphisms in peroxidase and acid phosphatase (ec were detected. there was absence of polymorphism in phosphoglucoisomerase (ec, phosphoglucomutase (ec, glutamate oxaloacetate transaminase (ec and malate dehydrogenase (ec comparing the activity of peroxidase enzyme, it was higher in hyperhydric and intermediary plants in relation to normal ones. enzymatic variability is a potential tool as hyperhydricity marker in plants grown in vitro.
Viabilidade e fertiliza??o in vitro de oócitos bovinos após vitrifica??o
Galbinski, Sérgio;Bos-Mikich, Adriana;Ferrari, Arnaldo Nicola;
Revista Brasileira de Ginecologia e Obstetrícia , 2003, DOI: 10.1590/S0100-72032003000800003
Abstract: purpose: to verify vitrification techniques using 6 m dmso to cryopreserve in vitro matured bovine oocytes, and to assess the effects of the time of exposure to vitrification solutions (vs). methods: dilutions of vs were prepared from the stock vs (vs 100%) consisting of 6 m dmso to give 25 and 65% dmso solutions. bovine oocytes were in vitro matured for 18-22 h. matured oocytes were placed first into 25% vs, at room temperature for 5 min, then transferred to 65% vs, before being pipetted into the 100% vs in plastic straws. three experimental groups were formed: in the first group, time of pipetting through 65% vs and loading the straw took up to 60 s, in the second group it did not exceed 30 s. for thawing, straws were held in air for 10 s and then in a water bath for 10 s. the contents of each straw were expelled in sucrose solution and held for 5 min. in the third experimental group, oocytes went through all vs, but were not vitrified. all retrieved oocytes were inseminated. for control, fresh, in vitro matured oocytes were inseminated. results: after vitrification, 69.1 and 59.8% of the oocytes were retrieved from the 30 s and 60 s groups, respectively, and 93 and 89% of these oocytes appeared morphologically normal 24 h after insemination, respectively. in the group of oocytes exposed without vitrification, 75.6% were retrieved and 84.7% were morphologically viable, 24 h after insemination. no fertilization was observed in the experimental groups. among controls, 65.4% were fertilized. conclusions: the vitrification technique using 6 m dmso is not a feasible approach to cryopreserve in vitro matured bovine oocytes. decreasing the time of exposure to vs did not overcome deleterious effects of the procedure on the fertilizability of oocytes. improvements in the technique are needed to protect the zona pellucida and oolemma.
Vitrifica??o de ovócitos desnudados ou n?o e previamente maturados in vitro
Fagundes, Letícia Martins;Costa, Eduardo Paulino da;Torres, Ciro Alexandre Alves;Amaral Filha, Wald'ma Sobrinho;Silva, Trícia Osório da;Gioso, Marilú Martins;
Revista Brasileira de Zootecnia , 2004, DOI: 10.1590/S1516-35982004000500004
Abstract: this study aimed at the evaluation of the effects from cryopreservation of bovine oocytes in vitro matured, by using ethylene glycol (eg) associated to trehalose and polyvinylpyrrolidone (pvp), of ovary oocytes of slaughtered cows, randomly assigned to three treatments. treatment 0 (t0 - control): oocytes that were desnuded and not vitrified. treatment 1 (t1): cryopreservation of in vitro matured oocytes with cumulus oophorus. tratamento 2 (t2): cryopreservation of in vitro matured desnuded oocytes. the percentage of recovered oocytes after cryopreservation and with normal morphology was different for vitrified oocytes (94.7 and 76.8%; 69.5 and 48.85% for t1 and t2, respectively). the main changes ultrastructural in vitrificated oocytes were prematurely released of cortical granules. later, all normal oocytes were fecundated and cultivated at 38.5oc in atmosphere with 5% co2 for seven days. the fecundation and cleavage rates for treatments were different (56.2, 41.7 and 12.5%; 36.3, 0.0 and 0.0%, for t0, t1 and t2, respectively). morulas and blastocysts were obtained only in t0 (34.5%). these results indicate that, the used protocols, for vitrification procedure is not indicated for cryopreservation of matured bovine oocytes.
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