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Influence of vitrification on mouse metaphase II oocyte spindle dynamics and chromatin alignment

GOMES, Claudia Messias; SILVA, Cristine Ane Silva E.; ACEVEDO, Nicole; BARACAT, Edmund; SERAFINI, Paulo; SMITH, Gary D.
Fonte: ELSEVIER SCIENCE INC Publicador: ELSEVIER SCIENCE INC
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
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Objective: To evaluate influences of vitrification and warming of metaphase II (MII) mouse oocytes on survival, spindle dynamics. spindle morphology, and chromatin alignment on metaphase plates. Design: Experimental animal Study. Setting: University animal laboratory. Animal(s): Eight-week-old B6D2F1 mice. Intervention(s): Denuded MII oocytes were used fresh (control), exposed to vitrification/warming solutions (Sol Expos), or vitrified and warmed (Vitr). Main Outcome Measure(s): Oocyte recovery and survival after warming and the influence of solution exposure and cryopreservation on spindle dynamics and chromatin alignment. Result(s): Cryopreservation of two or 10 oocytes per straw resulted in recovery (100% +/- 0% and 95% +/- 4%, respectively; mean SE) and survival (95% 2% and 98% 2%, respectively). Immediately after warming (Vitr), significantly fewer oocytes assessed with immunocytochemistry contained spindles, compared with control and Sol Expos. When oocytes were placed into a 3 degrees 7C environment for 2 hours after exposure or warming, the ability to recognize spindles by immunocytochemistry was not significantly different between groups. Using live-cell time-lapse imaging with LC-Polscope, similar time-dependent spindle formation dynamics were observed. At 2 hours after collection or treatment...

Prospective randomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification

SMITH, Gary D.; SERAFINI, Paulo C.; FIORAVANTI, Joyce; YADID, Isaac; COSLOVSKY, Marcio; HASSUN, Pericles; ALEGRETTI, Jose Roberto; MOTTA, Eduardo L.
Fonte: ELSEVIER SCIENCE INC Publicador: ELSEVIER SCIENCE INC
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
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Objective: To compare cryopreservation of mature human oocytes with slow-rate freezing and vitrification and determine which is most efficient at establishing a pregnancy. Design: Prospective randomized. Setting: Academically affiliated, private fertility center. Patient(s): Consenting patients with concerns about embryo cryopreservation and more than nine mature oocytes at retrieval were randomized to slow-rate freezing or vitrification of supernumerary (more than nine) oocytes. Intervention(s): Oocytes were frozen or vitrified, and upon request oocytes were thawed or warmed, respectively. Main Outcome Measure(s): Oocyte survival, fertilization, embryo development, and clinical pregnancy. Result(s): Patient use has resulted in 30 thaws and 48 warmings. Women`s age at time of cryopreservation was similar. Oocyte survival was significantly higher following vitrification/warming (81%) compared with freezing/thawing (67%). Fertilization was more successful in oocytes vitrified/warmed compared with frozen/thawed. Fertilized oocytes from vitrification/warming had significantly better cleavage rates (84%) compared with freezing/thawing (71%) and resulted in embryos with significantly better morphology. Although similar numbers of embryos were transferred...

Bovine oocyte vitrification: Effect of ethylene glycol concentrations and meiotic stages

MAGNUSSON, Vanessa; FEITOSA, Weber Beringui; GOISSIS, Marcelo Demarchi; YAMADA, Claudia; TAVARES, Liliam Mara Trevisan; ORTIZ, Mayra Elena; ASSUMPCAO, D`Avila; VISINTIN, Jose Antonio
Fonte: ELSEVIER SCIENCE BV Publicador: ELSEVIER SCIENCE BV
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
37.63%
Success in oocyte cryopreservation is limited and several factors as cryoprotectant type or concentration and stage of oocyte meiotic maturation are involved. The aim of the present study was to evaluate the effect of maturation stage and ethylene glycol (EG) concentration on survival of bovine oocytes after vitrification. In experiment 1, kinetics of oocyte in vitro maturation (IVM) was evaluated. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII) oocytes were found predominantly at 0, 0-10, 10-14, and 18-24 h of INK respectively. In experiment 2, in vitro embryo development after in vitro fertilization (IVF) of oocytes exposed to equilibrium (ES) and vitrification solution VS-1 (EG 30%), or VS-2 (EG 40%) at 0, 12 or 18 It of IVM was evaluated. Only blastocyst rate from oocytes vitrified in SV-2 after 18 h of IVM was different from control oocytes. Hatched blastocyst rates from oocytes vitrified in VS-1 after 12 and 18 h, and SV-2 after 18 h of IVM were different from unvitrified oocytes. In experiment 3, embryo development was examined after IVF of oocytes vitrified using VS-I or VS-2 at 0, 12 or 18 h of IVM. Rates of blastocyst development after vitrification of oocytes in VS-1 at each time interval were similar. However...

Vitrificação de embriões Mus domesticus domesticus contidos em volumes diferentes de 9,0 m de etileno glicol.; Vitrification of mus domesticus domesticus embryos exposed to differents volumes of 9.0 m ethylene glycol solution

Assaf, Sabrina Silveira
Fonte: Universidade Federal do Rio Grande do Sul Publicador: Universidade Federal do Rio Grande do Sul
Tipo: Dissertação Formato: application/pdf
POR
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Os experimentos tiveram como objetivo determinar a taxa de eclosão dos embriões vitrificados em volumes diferentes de 9,0 M de etileno glicol. Simultaneamente, testou-se dois procedimentos de estocagem dos fios de teflon, denominados caixa de aço inoxidável e globete/raque. No experimento I, os 881 embriões coletados foram distribuídos em 4 tratamentos: tratamento 1 (T1= controle): 307 embriões foram cultivados in vitro em meio PBSm, acrescido de 0,4% de BSA; tratamento 2 (T2): 292 embriões foram expostos à solução de glicerol 10% acrescida de 0,4% de BSA, envasados em palhetas de 0,25 mL e submetidos ao congelamento pelo método rápido em Biocool; tratamento 3 (T3): 138 embriões foram expostos durante 2 minutos à solução de desidratação (10% de EG + 6% BSA em PBSm) e então transferidos para a solução de vitrificação (50% de EG + 6% de BSA em PBSm), onde permaneceram por 30 segundos e foram colocados em volume de 1 μL no interior de um fio de teflon, medindo 0,4 mm de diâmetro, 2,0 cm de comprimento e 0,05 mm de espessura. Os fios foram acondicionados em uma caixa de aço inoxidável para serem armazenados em nitrogênio líquido; tratamento 4 (T4): 144 embriões foram expostos à solução de desidratação (10% de EG + 6% BSA em PBSm) e após 2 minutos...

Vitrificação de embriões Mus domesticus domesticus envasados em palheta convencional dotada de haste metálica.; Mus domesticus domesticus embryo vitrification using original straws containing a metallic stick

Costa, Alexandre Aiquel Vaz
Fonte: Universidade Federal do Rio Grande do Sul Publicador: Universidade Federal do Rio Grande do Sul
Tipo: Dissertação Formato: application/pdf
POR
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O objetivo deste experimento foi determinar a sobrevivência de blastocistos Mus domesticus domesticus vitrificado em palhetas na presença de uma haste metálica .Blastocistos Mus domesticus domesticus coletados no quarto dia após a fecundação, foram avaliados morfologicamente e divididos aleatoriamente em três grupos. Os embriões do grupo controle foram transferidos para gotas de meio KSOM e cultivados in vitro por 48 horas. Os embriões selecionados para criopreservação foram expostos a uma solução crioprotetora constituída por PBSm + de 10% etileno-glicol + 10% propileno-glicol, para promover uma desidratação das células embrionárias. Após foram transferidos para a solução de vitrificação que continha 20% etilenoglicol + 20% propilenoglicol diluídos em PBSm, e mantidos durante 25 segundos, sendo imediatamente imersos em nitrogênio submetido à vácuo. As taxas de sobrevivência embrionária revelaram uma maior eficiência da técnica com a inserção da peça metálica (56,21% - 86/153) em relação ao método convencional (18,84% - 26/138). Concluímos assim que a presença da peça metálica em contato com a amostra propiciou maior taxa de sobrevivência dos embriões submetidos à vitrificação envasados em palhetas de 0...

Expressão gênica das células do cumulus oophorus de bovinos após vitrificação; Gene expression of bovine cumulus oophorus cells after vitrification

Arend, Felipe Lohmann; Vieira, Arnaldo Diniz; Cesaro, Matheus Pedrotti de; Mezzalira, Alceu; Oliveira, Alexandre Tavares Duarte de; Lopes, Rui Fernando Felix
Fonte: Universidade Federal do Rio Grande do Sul Publicador: Universidade Federal do Rio Grande do Sul
Tipo: Artigo de Revista Científica Formato: application/pdf
POR
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Um dos desafios da criobiologia continua sendo o desenvolvimento de um método que proporcione a manutenção da viabilidade após a criopreservação de oócitos imaturos na espécie bovina. A vitrificação tem sido a metodologia que proporciona resultados de sobrevivência após a criopreservação mais promissores para complexos cumulus-oócito (CCOs) imaturos bovinos. Entretanto, a ação dos crioprotetores sobre as células do cumulus oophorus, no que diz respeito à regulação da expressão de genes importantes nesta fase, ainda é pouco compreendida. O objetivo do trabalho foi avaliar a expressão gênica das proteínas ácido hialurônico sintase 2 (HAS2), link protein (HAPLN1), conexina 43 (GJA1) e proteína de choque térmico HSP70-1 (HSP70-1) em células do cumulus oophorus de oócitos imaturos bovinos submetidos a exposição e/ou vitrificação na solução crioprotetora (SV) composta por 20% de etileno glicol (EG) + 20% dimetil sulfóxido (DMSO) + 0,5M de sacarose. Os CCOs foram selecionados e distribuídos em 4 grupos experimentais: G1, CCOs não submetidos a maturação in vitro (MIV); G2, CCOs submetidos à MIV; G3, CCOs maturados após a exposição à SV; e G4, CCOs maturados após a vitrificação com a SV. A MIV foi realizada em TCM 199...

In vitro and in vivo survival of mouse morulas and blastocysts following vitrification in 45% glycerol; Sobrevivência in vitro e in vivo de mórulas e blastocistos murinos vitrificados em meio contendo 45% de glicerol

Bertolini, Marcelo; Lange, Mateus da Costa; Rodrigues, José Luiz Rigo
Fonte: Universidade Federal do Rio Grande do Sul Publicador: Universidade Federal do Rio Grande do Sul
Tipo: Artigo de Revista Científica Formato: application/pdf
ENG
Relevância na Pesquisa
37.58%
A toxidez do agente crioprotetor é um dos aspectos mais críticos no sucesso da vitrificação de embriões mamíferos, variando conforme a concentração, o tempo e a temperatura de exposição. Além disto, embriões de diferentes espécies ou estádios de desenvolvimento podem tolerar diferentes níveis de exposição a agentes crioprotetores. Este estudo visou avaliar a sobrevivência in vitro e in vivo de embriões Mus musculus após a vitrificação em diferentes concentrações e tempos de exposição ao crioprotetor previamente à vitrificação. No Experimento 1, mórulas compactas, blastocistos e blastocistos expandidos foram expostos a 10% de glicerol por 10 min (grupo 1) ou 25% de glicerol por 10, 5 ou 2,5 min (grupos 2, 3 e 4) previamente à exposição à solução de vitrificação contendo 45% de glicerol por 1 min, a 20°C, antes da imersão em nitrogênio líquido. A descongelação ocorreu em banhomaria a 20°C por 20 seg, e a diluição do crioprotetor foi realizada em 1 M de sacarose por 10 min. Após cultivo in vitro por 1 e 48 h, os embriões foram avaliados morfologicamente. A sobrevivência in vitro de mórulas compactas e blastocistos não foi afetada pelas concentrações e tempos de equilíbrio em glicerol previamente à vitrificação. No entanto...

Embryos refrozen-thawed by vitrification lead to live births: Case report

Mauri, Ana L.; Petersen, Claudia G.; Oliveira, João B.A.; Baruffi, Ricardo L.R.; Al-Hasani, Saffa; Franco Jr., José G.
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 93-97
ENG
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Objective: To describe two successful cases of utilizing refrozen blastocysts by vitrification derived from supernumerary embryos. Design: Case report. Setting: Private fertility clinic. Subjects: Two infertility couple. Interventions: Refrozen blastocysts by vitrification derived from supernumerary embryos. Main outcome measures: Obstetric and pediatric results. Results: Two pregnancies obtained from refrozen-warmed blastocysts led to two healthy babies being born without clinical or genetic problems. Conclusion: These case reports support the notion of safely repeating cryopreservation. However, despite these favorable results, there is still a need for prospective controlled studies on the obstetric and neonatal repercussions of refreezing and of vitrification in particular. © 2010 Middle East Fertility Society.

Vitrification of immature feline oocytes with a commercial kit for bovine embryo vitrification

Apparicio, M.; Ruggeri, E.; Luvoni, G. C.
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 240-244
ENG
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The aim of this study was to evaluate the suitability of a commercial kit for bovine embryo vitrification for cryopreserving cat oocytes and to evaluate comparatively the effects of its use with slow freezing procedure on cryotolerance in terms of morphology and oocyte resumption of meiosis. Germinal vesicle stage oocytes isolated from cat ovaries were either vitrified (n=72) using a vitrification kit for bovine embryo or slow frozen (n=69) by exposing oocyte to ethylene glycol solution before being transferred to a programmable embryo freezer. After thawing and warming, oocytes were cultured for 48h and then were examined for meiosis resumption using bisbenzimide fluorescent staining (Hoechst 33342). Fresh immature oocytes (n=92) were used as the control group. The proportion of oocytes recovered in a morphologically normal state after thawing/warming was significantly higher in frozen oocytes (94.5%) than in the vitrified ones (75%, p<0.01). Morphological integrity after culture was similar in vitrified (73.6%) and slow frozen oocytes (76.8%); however, only 37.5% of the morphologically normal oocytes resumed meiosis after vitrification compared to 60.9% of those submitted to slow freezing procedure (p<0.01). Fresh oocytes showed higher morphological integrity (91.3%) and meiosis resumption rates (82.6%...

Histological study of rat ovaries cryopreserved by vitrification or slow freezing and reimplanted in the early or late postmenopausal stage

Silva,João Marcos de Meneses e; Pinheiro,Luiz Gonzaga Porto; Leite,José Alberto Dias; Melo,Lígia Helena Ferreira; Lunardi,Franciele Osmarini; Barbosa Filho,Rômulo Cesar Costa; Mendonça,Cindy Vitalino
Fonte: Sociedade Brasileira para o Desenvolvimento da Pesquisa em Cirurgia Publicador: Sociedade Brasileira para o Desenvolvimento da Pesquisa em Cirurgia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/05/2014 EN
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PURPOSE: To compare two rat ovary cryopreservation techniques (vitrification vs. slow freezing) and two postmenopausal stages (early vs. late) with regard to graft take. METHODS: Thirty-three Wistar rats were submitted to bilateral oophorectomy. One ovary was submitted to histological analysis while the other was cryopreserved by slow freezing or vitrification. The cryopreserved ovary was thawed and reimplanted in the greater omentum one week (early menopause) or one month (late menopause) after oophorectomy. One month after ovary reimplantation, the graft take was evaluated macroscopically and histologically. RESULTS: Six of the animals were used ascontrols and seven died. The histological findings of 20 animals included atretic follicles (n=4), primordial follicles (n=2), and corpus luteum with primordial follicles (n=3). No ovarian tissue was found in 11 animals. Vitrification resulted in a higher graft take rate than slow freezing (50% vs. 38.5%), but the difference was not statistically significant. However, the graft take rate was 9.3 times higher in the early than in the late postmenopausal stage (61.5% vs. 14.3%) (p=0.043). CONCLUSION: Vitrification was superior to slow freezing as ovarian cryopreservation technique...

Simplified EM Grid Vitrification Is a Convenient and Efficient Method for Mouse Mature Oocyte Cryopreservation

Kim, Seok Hyun; Ku, Seung-Yup; Sung, Ki Cheong; Kang, Moon Joo; Kim, Sung Ah; Kim, Hee Sun; Oh, Sun Kyung; Jee, Byung Chul; Suh, Chang Suk; Choi, Young Min; Kim, Jung Gu; Moon, Shin Yong
Fonte: Yonsei University College of Medicine Publicador: Yonsei University College of Medicine
Tipo: Artigo de Revista Científica
EN
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This study was performed to evaluate the efficiency of simplified EM grid vitrification, skipping the step of removing the cryoprotectant (5.5 M EG + 1.0 M sucrose) droplet on the grid after loading oocytes, compared to conventional cryopreservation protocols for mouse mature oocytes. Firstly, the recovery, survival, fertilization and hatching rates of simplified EM grid vitrification were compared with those of the slow freezing method using 1.5 M DMSO. Then, conventional EM grid vitrification was compared with simplified EM grid vitrification. Simplified EM grid vitrification showed higher survival, fertilization and hatching rates than those of the slow freezing method (85.6% vs. 63.2%; 51.0% vs. 22.3%; 38.7% vs. 12.5%, p < 0.01, respectively). Moreover, simplified EM grid vitrification showed higher recovery, survival and fertilization rates than those of conventional EM grid vitrification (100% vs. 95.0%, p = 0.024; 90.0% vs. 78.9%, p = 0.033; 56.7% vs. 38.7%, p = 0.021, respectively). Hatching rate tended to be higher for simplified EM grid vitrification compared to conventional EM grid vitrification (41.1% vs. 24.1%). In conclusion, simplified EM grid vitrification is a convenient and efficient method for cryopreservation of mouse mature oocytes...

Vitrifica????o de o??citos imaturos de eq??inos : caracter??sticas morfol??gicas ultra-estruturais e matura????o nuclear in vitro; Vitrification of immature equine oocytes: ultra structural morphologic characteristics and nuclear in vitro maturation

CURCIO, Bruna da Rosa
Fonte: Universidade Federal de Pelotas; Biotecnologia; Programa de P??s-Gradua????o em Biotecnologia; UFPel; BR Publicador: Universidade Federal de Pelotas; Biotecnologia; Programa de P??s-Gradua????o em Biotecnologia; UFPel; BR
Tipo: Tese de Doutorado Formato: application/pdf
POR
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The aim of the study was to investigate: 1) the ultrastructural morphologic characteristics in equine oocytes subjected to different times of exposure to cryoprotectant solutions and 2) the effect of initial cumulus morphology and cryoprotectants in the nuclear in vitro maturation (IVM) of the vitrified immature equine oocytes. The oocytes were obtained from ovaries from a slaughterhouse. In the first study 30 oocytes where divided in three groups: Control group (G1, n=10); Group 2 (G2, n=10), the oocytes were vitrified for exposure to VS-1 for 3min and VS-2 for 1min; Group 3 (G3, n=10), exposure to VS1 for 1.5min and VS-2 for 30sec. The oocytes were vitrified in open-pulledstraws (OPS). The ultrastructural characteristics where observed using a transmission electron microscope. The oocytes were classified as: I) oocytes morphologically normal; II) oocytes which presented intermediate damage but had completed organelles, and III) oocytes with severe morphological abnormalities. In the second study, compact (Ccp; n=248) and expanded (Cex; n=264) cumulus oocyte complexes were divided in three groups: Control, Treatment-1 (T1 - Ethylene glycol-EG + Dimetyl sulfoxide-DMSO + SIB) and Treatment-2 (T2 - Formamide + EG + DMSO + Polyvinylpyrrolidone + SIB). The control group was immediately matured in vitro...

Efeito dos m??todos de vitrifica????o, OPS e SSV, com adi????o de bloqueador sint??tico de gelo, sobre a viabilidade de o??citos de camundongos e bovinos; Effect of the vitrification methods OPS and SSV, with inclusion of a synthetic ice blocker, on the viability of mice and bovine oocytes

SANTOS, Elisa Caroline da Silva
Fonte: Universidade Federal de Pelotas; Biotecnologia; Programa de P??s-Gradua????o em Biotecnologia; UFPel; BR Publicador: Universidade Federal de Pelotas; Biotecnologia; Programa de P??s-Gradua????o em Biotecnologia; UFPel; BR
Tipo: Dissertação Formato: application/pdf
POR
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Oocyte vitrification is a valuable tool for preservation of genetic material. This study compared the effects of vitrification in OPS and SSV, with addition of SupercoolTM X- 1000 (copolymer), on the viability of mature murine oocytes and immature bovine oocytes. Oocytes were vitrified in OPS and SSV, with addition of 0.1%, 1.0% copolymer and without copolymer, besides a control group with no vitrification. Murine oocytes were evaluated for membrane viability, in the first experiment, and for cleavage rate, in the second experiment. Results were superior with the concentration of 0.1% copolymer, for both methods, in the first experiment. In the second experiment, the SSV method without copolymer presented lower cleavage rate (9.2%) than the control group (26.6%). In the third experiment, bovine oocytes were vitrified and evaluated for maturation and membrane viability, but the results were numerically inferior than those for the control group, for both methods. Those results indicate that both vitrification methods can be used with inclusion of 0.1% of copolymer, for mature murine oocytes, considering membrane viability, but the SSV method without copolymer should not be used due to its low cleavage rate. However, the procedures tested in this study are not recommended for cryopreservation of bovine oocytes.; A vitrifica????o de o??citos ?? uma metodologia valiosa para a conserva????o de material gen??tico. Este trabalho comparou o efeito dos m??todos de vitrifica????o OPS e SSV...

Piglets produced from in vivo blastocysts vitrified using the Cryologic Vitrification Method (solid surface vitrification) and a sealed storage container

Beebe, L.; Bouwman, E.; McIlfatrick, S.; Nottle, M.
Fonte: Elsevier Science Inc Publicador: Elsevier Science Inc
Tipo: Artigo de Revista Científica
Publicado em //2011 EN
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The objective was to develop a simple successful porcine cryopreservation protocol that prevented contact between embryos and liquid nitrogen, avoiding potential contamination risks. In vivo-derived blastocysts were collected surgically from donor pigs, and two porcine embryo vitrification protocols (one used centrifugation to polarize intracytoplasmic lipids, whereas the other did not) were compared using the Cryologic Vitrification Method (CVM), which used solid surface vitrification. The CVM allowed embryos to be vitrified, without any contact between embryos and liquid nitrogen. Both protocols resulted in similar in vitro survival rates (90% and 94%) and cell number (89 ± 5 and 99 ± 5) after 48 h in vitro culture of vitrified and warmed blastocysts. The protocol that did not use centrifugation was selected for continued use. To protect vitrified embryos from contact with liquid nitrogen and potential contamination during storage, a sealed outer container was developed. Use of this sealed outer container did not affect in vitro survival of cryopreserved blastocysts. In vivo blastocysts (n = 151) were collected, vitrified, and stored using the selected protocol and sealed container. These embryos were subsequently warmed and transferred to six recipients; five became pregnant and farrowed a total of 26 piglets. This embryo vitrification method allowed porcine embryos to be successfully vitrified and stored without any contact with liquid nitrogen.; L.F.S. Beebe...

The presence of 1 mM glycine in vitrification solutions protects oocyte mitochondrial homeostasis and improves blastocyst development

Zander, D.; Cashman, K.; Lane, M.
Fonte: Kluwer Academic/Plenum Publ Publicador: Kluwer Academic/Plenum Publ
Tipo: Artigo de Revista Científica
Publicado em //2013 EN
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PURPOSE: Embryos generated from oocytes which have been vitrified have lower blastocyst development rates than embryos generated from fresh oocytes. This is indicative of a level of irreversible damage to the oocyte possibly due to exposure to high cryoprotectant levels and osmotic stress. This study aimed to assess the effects of vitrification on the mitochondria of mature mouse oocytes while also examining the ability of the osmolyte glycine, to maintain cell function after vitrification. METHODS: Oocytes were cryopreserved via vitrification with or without 1 mM Glycine and compared to fresh oocyte controls. Oocytes were assessed for mitochondrial distribution and membrane potential as well as their ability to fertilise. Blastocyst development and gene expression was also examined. RESULTS: Vitrification altered mitochondrial distribution and membrane potential, which did not recover after 2 h of culture. Addition of 1 mM glycine to the vitrification media prevented these perturbations. Furthermore, blastocyst development from oocytes that were vitrified with glycine was significantly higher compared to those vitrified without glycine (83.9 % vs. 76.5 % respectively; p < 0.05) and blastocysts derived from oocytes that were vitrified without glycine had significantly decreased levels of IGF2 and Glut3 compared to control blastocysts however those derived from oocytes vitrified with glycine had comparable levels of these genes compared to fresh controls. CONCLUSION: Addition of 1 mM glycine to the vitrification solutions improved the ability of the oocyte to maintain its mitochondrial physiology and subsequent development and therefore could be considered for routine inclusion in cryopreservation solutions.; Deirdre Zander-Fox & Kara S. Cashman & Michelle Lane

Slow freezing and vitrification of mouse morula and early blastocysts

Zander, D.; Lane, M.; Hamilton, H.
Fonte: Kluwer Academic/Plenum Publ Publicador: Kluwer Academic/Plenum Publ
Tipo: Artigo de Revista Científica
Publicado em //2013 EN
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PURPOSE: To assess the relative success of morula and early blastocyst slow freezing and vitrification in regards to survival and implantation rates utilising protocols which could be clinically implemented as a viable alternative to expanded blastocyst stage freezing. METHODS: Mouse morula and early blastocysts were either slow frozen/thawed or vitrified/warmed. Their subsequent survival, blastocyst development and blastocyst cell number and allocation to either the inner cell mass, trophectoderm or epiblast was assessed. In addition blastocysts were also transferred to pseudopregnant recipients and implantation and fetal development was determined. RESULTS: Vitrification of both morula and early blastocysts resulted in significantly higher rates of survival and blastocyst development compared to slow freezing. In addition slow frozen early blastocysts had significantly reduced blastocyst cell number compared to control however vitrified morula and early blasocyts and slow frozen morula had equivocal blastocyst cell numbers. Transfer of blastocysts from both methods of cryopreservation resulted in similar implantation rates however the placentas created from slow frozen early blastocysts were significantly lighter than control (95.5 g ± 5.4 vs. 122.0 g ± 4.2 respectively). CONCLUSIONS: Vitrification resulted in significantly higher rates of morula and early blastocyst survival and blastocyst development compared to slow freezing. In addition this study has validated the use of a closed DMSO free vitrification protocol which could then be investigated for use in the clinical setting as an alternative to expanded blastocyst freezing.; Deirdre Zander-Fox...

Bovine oocyte vitrification: effect of ethylene glycol concentration and meiotic stages

MAGNUSSON, Vanessa; FEITOSA, Weber Beringui; GOISSIS, Marcelo Demarchi; YAMADA, Claudia; TAVARES, Liliam Mara Trevisan; ASSUMPÇÃO, Mayra Elena Ortiz D'Ávila; VISINTIN, José Antonio
Fonte: Amsterdam Publicador: Amsterdam
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
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Success in oocyte cryopreservation is limited and several factors as cryoprotectant type or concentration and stage of oocyte meiotic maturation are involved. The aim of the present study was to evaluate the effect of maturation stage and ethylene glycol (EG) concentration on survival of bovine oocytes after vitrification. In experiment 1, kinetics of oocyte in vitro maturation (IVM) was evaluated. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII) oocytes were found predominantly at 0, 0-10, 10-14, and 18-24 h of IVM, respectively. In experiment 2, in vitro embryo development after in vitro fertilization (IVF) of oocytes exposed to equilibrium (ES) and vitrification solution VS-1 (EG 30%), or VS-2 (EG 40%) at 0, 12 or 18 h of IVM was evaluated. Only blastocyst rate from oocytes vitrified in SV-2 after 18 h of IVM was different from control oocytes. Hatched blastocyst rates from oocytes vitrified in VS-1 after 12 and 18 h, and SV-2 after 18 h of IVM were different from unvitrified oocytes. In experiment 3, embryo development was examined after IVF of oocytes vitrified using VS-1 or VS-2 at 0, 12 or 18 h of IVM. Rates of blastocyst development after vitrification of oocytes in VS-1 at each time interval were similar. However...

Controle da vitrificação do cravo (Dianthus caryophyllus L.) in vitro; Control of carnation vitrification (Dianthus caryophyllus L.) in vitro

Cuzzuol, G.R.F.; Gallo, L.A.; Almeida, M. de; Crocomo, O.J.
Fonte: Universidade de São Paulo. Escola Superior de Agricultura Luiz de Queiroz Publicador: Universidade de São Paulo. Escola Superior de Agricultura Luiz de Queiroz
Tipo: info:eu-repo/semantics/article; info:eu-repo/semantics/publishedVersion; ; ; ; ; Formato: application/pdf
Publicado em 01/12/1995 POR
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37.44%
Baixos níveis de benziladenina (BAP), baixo potencial de água no meio e baixa umidade condicionada por tampas de algodão foram capazes de inibir a vitrificação de cravo (Dianthus caryophyllus L.) cultivado In vitro, mas essas condições implicaram no baixo desenvolvimento das plantas e da taxa de propagação. Elevados níveis de NH4NO3 demonstraram serem altamente promotores da vitrifícação assinalada pelo alto conteúdo de proteína, enquanto relação inversa foi constatada para altos níveis de CaCl2, aos quais seguiu-se aumento na atividade da peroxidase. Os resultados permitiram estabelecer um protocolo para controle da vitrificação do cravo, constituído de 4,0 g/1 de "Gelrite", 0,5 mg/1 de ácido naftalenoacético (ANA), 0,05 mg/1 de BAP, doses normais das soluções salinas do meio MS e vedação do tipo tampas de algodão para cultivo de ápices meristemáticos. Para a fase de multiplicação, este protocolo deve ser alterado para 0,5 mg/1 de BAP, 10,3 mM de NH4NO3 e 12,0 mM de CaCl2.; Low levels of benzyl adenine (BAP), low water potential of the growth medium and low humidity due to cotton covers, inhibited vitrification of carnation (Dianthus caryophyllus L.) in vitro. However, under these conditions, a low development of plants and a decrease in the multiplication ratio...

Methods for partial denudment of maturated bovine oocytes submitted to vitrification; METODOLOGIAS DE DESNUDAMENTO PARCIAL DE OÓCITOS BOVINOS MATURADOS E SUBMETIDOS À VITRIFICAÇÃO

MEZZALIRA, A.; CUCCO, D.C.; BUNN, S.; CRUZ, F.B.; WERLICH, D.E.; VIEIRA, A.D.; SANTOS, R.M.
Fonte: UFPR Publicador: UFPR
Tipo: info:eu-repo/semantics/article; info:eu-repo/semantics/publishedVersion; Artigo Avaliado pelos Pares Formato: application/pdf
Publicado em 27/01/2006 POR
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37.52%
Different methods of partial denudation of maturated bovine oocytes were evaluated for in vitro embryo production and vitrification procedures, in three experiments. First the effect of partial denudation by successive pipetting was evaluated. In the second experiment, the denudation by pipetting was compared with the use of hyaluronidase, and finally, the effect of both methods was evaluated in regard to the vitrification of oocytes. Oocytes were submitted to treatments after 22 hours of maturation in TCM 199 medium. For the vitrification, they were firstly exposed for 30 seconds to an equilibrium solution and for 20 seconds to a vitrification solution (20% EG + 20% DMSO + 0.3M SUC), loaded in open pulled straws (OPS) and plunged into liquid nitrogen. The rewarming of the oocytes suspensions was carried out by plunged them in decreasing sucrose concentrations. Oocytes of all treatments were submitted to an additional 2 hours maturation period, followed by fertilization. Presumptive zygotes were cultured in SOFaaci medium. In the first experiment the denudation resulted in a decreased blastocyst rate (28.7% to 20.5% – P; Diferentes metodologias de desnudamento parcial de oócitos bovinos maturados foram avaliadas na produção in vitro de embriões e na vitrificação...

Stepwise vitrification of in vitro produced buffalo Blastocysts

Abd-Allah,S.M.
Fonte: Archivos de Zootecnia Publicador: Archivos de Zootecnia
Tipo: info:eu-repo/semantics/article; journal article; info:eu-repo/semantics/publishedVersion Formato: text/html; application/pdf
Publicado em 01/09/2011 ENG
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27.72%
The purpose of this study was to evaluate the use of a stepwise vitrification as a method for cryopreservation of in vitro-produced (IVP) buffalo blastocysts and to compare the results with post-thaw survival rate of buffalo blastocysts frozen by stepwise vitrification with those frozen by conventional vitrification (one step method). Selected IVP blastocysts were exposed to a vitrification solution consisting of 40% ethylene glycol (EG) plus 0.3 M trehalose and 20% polyvinyl pyrrolidone (PVP) for 1 min and loaded in 0.25 ml plastic mini straws containing 100 μl of 10% sucrose. The loaded cryostraws were cryopreserved by either the stepwise vitrification or one step vitrification and stored in liquid nitrogen for one month. After thawing and removal of cryoprotectants, embryos exhibiting intact zona pellucida and uniform blastomeres were considered suitable for in vitro culture. Of the embryos cryopreserved by stepwise and one step vitrification, 100 and 60%, respectively, recovered embryos post-thawing. Similarly 95.4 and 71.1% of embryos cryopreserved by stepwise and one step vitrification were exhibiting good embryos post-thawing. Post-thaw blastocysts were serially washed in tissue culture medium 199 (TCM-199) for 5 min in both cases. They were then cultured in TCM-199 supplemented with 10% fetal calf serum for 2448 h. Development to hatched blastocyst stage was considered the initial indicator of success of cryopreservation of embryos. The rates of blastocyst re-expansion and hatching of stepwise vitrified blastocysts (66 and 55%...