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This thesis presents the development of a digital microfluidic system to achieve automated sample preparation for the vitrification of mammalian embryos for clinical in vitro fertilization (IVF) applications. This platform included micro devices fabrication, an imaging system, a high voltage control system, and a LabVIEW interface. Individual micro droplets manipulated on the digital microfluidic device were used as micro-vessels to transport a single embryo through a complete vitrification procedure. The device showed cell survival and development rates of 77% and 90%, respectively, which are comparable to the control groups that were manually processed. Technical advantages of this approach, compared to manual operation and channel-based microfluidic vitrification, include automated operation, cryoprotectant concentration gradient generation, and feasibility of loading and retrieval of embryos.

The overall objective of this thesis was to investigate the vitrification of bovine cumulus oocyte complexes (COCs) as a tool for conservation of female genetics. Specifically, the first objective was to compare in vitro maturation rates (i.e. end point Metaphase II (MII) rate) of bovine COCs following vitrification using two different equilibration times (0 vs 10 min) in vitrification solution 1 (VS1) and two different cryodevices (cryotop vs 0.25 mL straw). The MII rate was higher in the non-vitrified control group than in vitrified groups (61 vs 16%, P<0.001). In the vitrified groups, equilibration time in VS1 [TCM-199 + 7.5% ethylene glycol (EG) + 7.5% dimethyl sulfoxide (DMSO) + 20% calf serum (CS)] did not affect MII rate (P=0.964); however, MII rate was higher in COCs vitrified on cryotops than in straws (23 vs 9%, P=0.007).<p> The second objective of this thesis was to compare cleavage and subsequent developmental competence of bovine COCs vitrified using different vitrification solutions (fresh vs frozen) and different equilibration times in VS1 (0 vs 5 min). Immature bovine COCs were vitrified on cryotops using vitrification solutions either prepared fresh or frozen/thawed, and two equilibration times in VS1 (0 vs 5 min). Cleavage and blastocyst production rates were higher (P<0.001) in the non-vitrified control group than vitrified groups (cleavage rate 93 vs 42% and blastocyst rate 31 vs 0.4%). The cleavage rate of COCs vitrified in frozen/thawed solutions with 5 min equilibration was higher (P=0.05) than in other treatment groups. However, the blastocyst rate did not differ (P=0.993) among vitrified groups.<p> We hypothesized that the nuclear stage of bovine COCs at the time of vitrification will affect post-warming in vitro maturation (IVM), cleavage and embryo development. Firstly, a preliminary study was conducted to validate our in vitro maturation system. The COCs were at germinal vesicle (GV, 89%), germinal vesicle breakdown (GVBD, 47%), metaphase I (MI, 90%), and metaphase II (MII, 84%) stages at 0, 6, 12 and 22 h of IVM respectively. In a subsequent study, bovine COCs were vitrified after 0, 6, 12 and 22 h of IVM by loading on cryotops and plunging in liquid nitrogen. Following 1 min in warming solution (TCM-199 + 17% sucrose + 20% CS), COCs were placed in IVM medium to complete 22 h of IVM and nuclear stages were evaluated using lamin A/C-DAPI staining. The nuclear maturation (MII) rates of COCs were 23, 23, 35 and 89% (P<0.001) in the 0, 6, 12 and 22 h IVM groups, respectively. In the final nuclear stage experiment, cleavage and embryo development was determined after vitrification of COCs after 0, 12 and 22 h of IVM. The cleavage and blastocyst rates were higher (P<0.001) in the non-vitrified (control) group than vitrified groups (73 vs 15% and 22 vs 0.3%, respectively). The cleavage rates (14% in 0 h, 17% in 12 h and 14% in 22 h groups; P=0.825) and blastocyst rates (0% in 0 and 22 h and 1% in 12 h groups) did not differ. Results from this study indicated that a greater proportion of COCs progressed to MII if vitrification occurred at MI rather than at GV and GVBD stages. However, the nuclear status of vitrified COCs appeared to have no effect on fertilization or subsequent embryo development.<p> A final set of experiments were designed to determine if the exposure of bovine COCs to cryoprotectant solutions and different warming time intervals affects their ability to become fertilized, cleave and develop into blastocysts. In both experiments, the cleavage and blastocyst rates in the vitrified group were lower (P<0.001) than in the non-vitrified control groups VS1 group, and in the VS1 + vitrification solution 2 (VS2; TCM-199 + 15% EG + 15% DMSO + 20% CS) group. However, the cleavage and blastocyst rates did not differ (P>0.05) in control, VS1 and VS1+VS2 groups. In the second experiment, there was no difference in cleavage rates between 1 and 5 min intervals in the warming solution. In conclusion, cryotop can be used as a preferred cryodevice for vitrification of bovine COCs. Vitrification solutions (VS1 and VS2) and the warming solution can be prepared and stored in the freezer (-20 °C) for convenience. The pre-equilibration of bovine COCs in VS1 had no effect on the nuclear maturation of vitrified/warmed COCs, but 5 min pre-equilibration in VS1 improved cleavage rates. The nuclear stage of COCs at the time of vitrification and cryoprotectant exposure appeared to have no effect on subsequent cleavage. Furthermore, there was no difference in cleavage and blastocyst rates following vitrification and 1 or 5 min warming time intervals. When compared to non-vitrified controls, the vitrification of immature bovine COCs resulted in reduced nuclear maturation, cleavage and embryo development rates. Further studies are needed to elucidate the causes of the poor embryo development in vitrified-warmed bovine COCs at genomic, proteomics and metabolomics levels.

The overall objective of this thesis was to investigate the vitrification of bovine cumulus oocyte complexes (COCs) as a tool for conservation of female genetics. Specifically, the first objective was to compare in vitro maturation rates (i.e. end point Metaphase II (MII) rate) of bovine COCs following vitrification using two different equilibration times (0 vs 10 min) in vitrification solution 1 (VS1) and two different cryodevices (cryotop vs 0.25 mL straw). The MII rate was higher in the non-vitrified control group than in vitrified groups (61 vs 16%, P<0.001). In the vitrified groups, equilibration time in VS1 [TCM-199 + 7.5% ethylene glycol (EG) + 7.5% dimethyl sulfoxide (DMSO) + 20% calf serum (CS)] did not affect MII rate (P=0.964); however, MII rate was higher in COCs vitrified on cryotops than in straws (23 vs 9%, P=0.007).<p> The second objective of this thesis was to compare cleavage and subsequent developmental competence of bovine COCs vitrified using different vitrification solutions (fresh vs frozen) and different equilibration times in VS1 (0 vs 5 min). Immature bovine COCs were vitrified on cryotops using vitrification solutions either prepared fresh or frozen/thawed, and two equilibration times in VS1 (0 vs 5 min). Cleavage and blastocyst production rates were higher (P<0.001) in the non-vitrified control group than vitrified groups (cleavage rate 93 vs 42% and blastocyst rate 31 vs 0.4%). The cleavage rate of COCs vitrified in frozen/thawed solutions with 5 min equilibration was higher (P=0.05) than in other treatment groups. However, the blastocyst rate did not differ (P=0.993) among vitrified groups.<p> We hypothesized that the nuclear stage of bovine COCs at the time of vitrification will affect post-warming in vitro maturation (IVM), cleavage and embryo development. Firstly, a preliminary study was conducted to validate our in vitro maturation system. The COCs were at germinal vesicle (GV, 89%), germinal vesicle breakdown (GVBD, 47%), metaphase I (MI, 90%), and metaphase II (MII, 84%) stages at 0, 6, 12 and 22 h of IVM respectively. In a subsequent study, bovine COCs were vitrified after 0, 6, 12 and 22 h of IVM by loading on cryotops and plunging in liquid nitrogen. Following 1 min in warming solution (TCM-199 + 17% sucrose + 20% CS), COCs were placed in IVM medium to complete 22 h of IVM and nuclear stages were evaluated using lamin A/C-DAPI staining. The nuclear maturation (MII) rates of COCs were 23, 23, 35 and 89% (P<0.001) in the 0, 6, 12 and 22 h IVM groups, respectively. In the final nuclear stage experiment, cleavage and embryo development was determined after vitrification of COCs after 0, 12 and 22 h of IVM. The cleavage and blastocyst rates were higher (P<0.001) in the non-vitrified (control) group than vitrified groups (73 vs 15% and 22 vs 0.3%, respectively). The cleavage rates (14% in 0 h, 17% in 12 h and 14% in 22 h groups; P=0.825) and blastocyst rates (0% in 0 and 22 h and 1% in 12 h groups) did not differ. Results from this study indicated that a greater proportion of COCs progressed to MII if vitrification occurred at MI rather than at GV and GVBD stages. However, the nuclear status of vitrified COCs appeared to have no effect on fertilization or subsequent embryo development.<p> A final set of experiments were designed to determine if the exposure of bovine COCs to cryoprotectant solutions and different warming time intervals affects their ability to become fertilized, cleave and develop into blastocysts. In both experiments, the cleavage and blastocyst rates in the vitrified group were lower (P<0.001) than in the non-vitrified control groups VS1 group, and in the VS1 + vitrification solution 2 (VS2; TCM-199 + 15% EG + 15% DMSO + 20% CS) group. However, the cleavage and blastocyst rates did not differ (P>0.05) in control, VS1 and VS1+VS2 groups. In the second experiment, there was no difference in cleavage rates between 1 and 5 min intervals in the warming solution. In conclusion, cryotop can be used as a preferred cryodevice for vitrification of bovine COCs. Vitrification solutions (VS1 and VS2) and the warming solution can be prepared and stored in the freezer (-20 °C) for convenience. The pre-equilibration of bovine COCs in VS1 had no effect on the nuclear maturation of vitrified/warmed COCs, but 5 min pre-equilibration in VS1 improved cleavage rates. The nuclear stage of COCs at the time of vitrification and cryoprotectant exposure appeared to have no effect on subsequent cleavage. Furthermore, there was no difference in cleavage and blastocyst rates following vitrification and 1 or 5 min warming time intervals. When compared to non-vitrified controls, the vitrification of immature bovine COCs resulted in reduced nuclear maturation, cleavage and embryo development rates. Further studies are needed to elucidate the causes of the poor embryo development in vitrified-warmed bovine COCs at genomic, proteomics and metabolomics levels.

La cryoconservation du tissu ovarien (CTO) permet aujourd'hui de préserver la fertilité des femmes et jeunes filles devant subir un traitement potentiellement gonadotoxique ou souffrant de pathologie à l'origine d'une insuffisance ovarienne prématurée. En vue d'optimiser les procédures de CTO, l'objectif de notre travail a porté d'une part sur la validation d'un protocole original de congélation lente du tissu ovarien applicable en thérapeutique et d'autre part, sur le développement d'un protocole de vitrification. L'analyse statique du tissu ovarien congelé selon notre protocole de congélation lente montre des résultats similaires en comparaison à ceux obtenus avec le tissu frais en termes de qualité des follicules et de l'endothélium vasculaire ovarien. Néanmoins, le stroma semble plus sensible aux effets délétères de la congélation. L'analyse fonctionnelle montre que le tissu ovarien congelé/décongelé présente une folliculogenèse active après 12 jours de culture in vitro. Les mesures des concentrations en oestradiol dans les milieux de culture et l'étude immunohistochimique du facteur de prolifération PCNA (proliferating cell nuclear antigen) témoignent en effet de l'activité fonctionnelle des follicules décongelés en culture. Dans un second temps, nous avons développé un protocole de vitrification du tissu ovarien. Pour évaluer son efficacité, nous avons réalisé une analyse statique du tissu ovarien vitrifié selon ce protocole versus notre protocole de congélation lente validé. Les résultats obtenus ne montrent aucune différence significative entre les deux méthodes, en termes de préservation de la morphologie et de l'intégrité nucléaire des follicules et du stroma ovarien. En conclusion, notre protocole de congélation lente préserve la qualité des différents compartiments constituant le tissu ovarien et la fonctionnalité de ce tissu. Ces données viennent compléter des travaux préliminaires de l'équipe et permettent de valider ce protocole envisageable maintenant en thérapeutique. La procédure de vitrification développée présente une efficacité similaire à la procédure de congélation lente en termes de préservation de la qualité des follicules et du stroma ovarien. Néanmoins, il serait nécessaire de poursuivre l'étude de l'efficacité de ce protocole par une analyse fonctionnelle. / No abstract available

<p>Crypreservation of oocytes is recently being considered to be a valid choice in infertility treatments.Low survival and fertilization rates due to inefficient slow freeze protocols have been the outcome ofmany previous studies done in the field. However, introduction of the vitrification technique and itsapplication in reproductive medicine and to some extent new improved slow freeze protocols haveshown that oocytes can be cryopreserved with successful outcome.</p><p>In this project the survival rate of oocytes after vitrification with MediCult Vitrification andWarming Media has been studied. Also, a comparison of the carriers Cryoloop (an open system) andCryopette (a closed system) has been performed.</p><p>A total of 43 oocytes were vitrified and warmed according to MediCult's protocol, of which 21oocytes with Cryoloop and 22 with Cryopette. The cells were post-thaw incubated in a physiologicalenvironment for 24h. During that time the morphology and viability were observed and noted after 2h,over night and after 24h. No significant difference in survival rates of the oocytes' was observed usingthe two carriers, and the total survival rate of the oocytes was 83.7%. This indicates thatcryopreservation of oocytes is a valid choice in fertility treatments and that the new product Cryopettecan be used clinically with just as good results as the well established Cryoloop technique. This merits further studies on the applicability of vitrification on oocytes and the Cryopette technique.</p>

Crypreservation of oocytes is recently being considered to be a valid choice in infertility treatments.Low survival and fertilization rates due to inefficient slow freeze protocols have been the outcome ofmany previous studies done in the field. However, introduction of the vitrification technique and itsapplication in reproductive medicine and to some extent new improved slow freeze protocols haveshown that oocytes can be cryopreserved with successful outcome. In this project the survival rate of oocytes after vitrification with MediCult Vitrification andWarming Media has been studied. Also, a comparison of the carriers Cryoloop (an open system) andCryopette (a closed system) has been performed. A total of 43 oocytes were vitrified and warmed according to MediCult's protocol, of which 21oocytes with Cryoloop and 22 with Cryopette. The cells were post-thaw incubated in a physiologicalenvironment for 24h. During that time the morphology and viability were observed and noted after 2h,over night and after 24h. No significant difference in survival rates of the oocytes' was observed usingthe two carriers, and the total survival rate of the oocytes was 83.7%. This indicates thatcryopreservation of oocytes is a valid choice in fertility treatments and that the new product Cryopettecan be used clinically with just as good results as the well established Cryoloop technique. This merits further studies on the applicability of vitrification on oocytes and the Cryopette technique.

This Technology in Industry Fellowship (TIF) funded Masters Project was structured around Computational Fluid Dynamics (CFD) modelling for Lemar Environmental Ltd (Lemar). This study is a component of a larger programme that is being undertaken by Lemar concerning the vitrification process. The modelling has built on an initial model developed by CSIRO for Lemar and has been carried out under the direction of Canterbury University. The modelling involved computer simulations and detailed comparisons of the gas flow for both high and low swirl vanes, in both the steady state and transient modes. The output of this activity; velocity profiles (tangential and axial), vorticity, as well as particle tracking (in steady state mode only) were compared to literature and evaluated for both scenarios. As the study was restricted to the gas flow in transient mode, no recommendations and extrapolated modifications to the burner geometry and plant equipment can be made as they have to be verified by the particle motion within the gas flow. The steady state particle simulations obtained through this project, did not provide sufficient evidence to conclude that particles attach to the outer wall and only demonstrated the influences that the high and low swirl had on the particles. Further investigations of transient particle tracking would provide an overall interpretation as to whether or not the dried sludge particles bounced or stuck to the viscous slag layer and a commentary as to their movement in the chamber. Lemar's strategic vitrification programme is still active and the resulting redesign process is nearing completion and modifications to the plant are expected to be finalised by January 2008. Following extensive testing by Lemar it is understood that they would be looking to seek venture capital in order to progress the project to the market. In order for the final stage of the sewage sludge vitrification plant project to commence, Lemar has been in consultation with subject matter experts in the field, as well as undertaking trials on the plant, computer modelling and research into both the technical and international marketing prospects for the combustion technology. The detailed analysis and research undertaken through the CFD modelling conducted for this Project, recommends that Lemar conducts further CFD modelling to investigate transient particle tracking before any plant or geometry modifications are proposed and undertaken in order to optimise the ash capture which is a key output of the vitrification process.

Made available in DSpace on 2014-06-11T19:29:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-12-17Bitstream added on 2014-06-13T20:59:30Z : No. of bitstreams: 1 paschoal_dm_me_botfmvz.pdf: 1363323 bytes, checksum: f767f2dcabcc3f599b8e36c74c4d096c (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo desta dissertação foi avaliar a viabilidade e a resistência à criopreservação após a realização de lipólise química utilizando-se o forskolin. Embriões Bos taurus indicus (OZ) e Bos taurus indicus X Bos taurus taurus (OMZT) produzidos in vitro, foram comparados em meio de cultivo contendo ou não soro fetal bovino (SFB). Para o experimento1, 1.052 oócitos de graus I e II foram colhidos de ovários de vacas Bos taurus indicus de abatedouro, dividindo em 7 réplicas. Os oócitos foram maturados em TCM 199 acrescido de SFB, Piruvato de Sódio, FSH, LH e Penicilina/Estreptomicina. A fertilização foi realizada com sêmen de touro Nelore (Bos taurus indicus) ou com sêmen de touro Simental (Bos taurus taurus), em meio HTF acrescido de BSA, Piruvato de Sódio, Cafeína, Heparina, Penicilinamina, Hipotaurina, Epinefrina e Penicilina/Estreptomicina. Os prováveis zigotos foram cultivados em meio SOFaa, acrescido de BSA, Piruvato de Sódio, Penicilina/Estreptomicina, suplementado ou não com SFB (Grupo 2,5%SFB e Grupo 0%SFB). No D6 os blastocistos foram novamente divididos em 2 sub-grupos com ou sem adição de 10μM de forskolin (total de 4 grupos - 2,5%SFB, 0%SFB, 2,5%+F e 0%+F). Para o experimento 2 os embriões foram expostos a solução de vitrificação 1 (SV1 = 5M de Etileno Glicol) durante 3 minutos e transferidos para uma gota com 15μL da solução de vitrificação 2 (SV2 = composta por 7M de Etileno Glicol, 0.5M de Galactose e 18% (w/v) de Ficoll 70) sendo, em seguida envasados em palhetas francesas estéreis de 0,25 mL. Para o aquecimento, as palhetas foram mantidas em ar por 8 segundos e depois em água a 37°C por 15 segundos. Como controle dos experimentos, embriões bovinos produzidos in vivo coletados de vacas Nelores foram utilizados. A taxa de clivagem e de produção de blastocisto no experimento 1, não diferiu entre os tratamentos dentro dos grupo... / The main objective of this dissertation was to evaluate the viability and cryotolerance of bovine embryos treated with forskolin to induce chemical lipolysis. In vitro produced Bos taurus indicos (OZ) and Bos taurus indicus X Bos taurus taurus (OMZT) embryos were compared in culture conditions containing or not fetal calf serum (FCS). For Experiment 1, 1.052 grade I and II Bos taurus indicos oocytes were obtained from slaughterhouse ovaries and divided in 7 replicates. The oocytes were matured in TCM 199 supplemented with FCS, Sodium Piruvate, FSH, LH and Penicillin/Streptomycin. Fertilization was performed using semen from one Nelore bull (Bos taurus indicos) and one Simental bull (Bos taurus taurus) in HTF medium supplemented with BSA, Sodium Pyruvate, Caffeine, Heparine, Penicilamine, Hipotaurine, Epinephrine and Penicillin/Streptomycin. The presumptive zygotes were cultured in SOFaa with BSA, Sodium Pyruvate and Penicillin/Streptomycin supplemented or not with FCS (Group 2.5%FCS and Group 0%FCS). On day 6 embryos from both groups were divided in two sub-groups containing or not 10μM of forskolin (total of 4 groups - 2.5%FCS, 0%FCS, 2.5%+F and 0%+F). For Experiment 2 the embryos were exposed to a vitrification solution 1 (SV1 = 5M of Ethylene Glycol) during 3 minutes and transferred to a vitrification solution 2 (SV2 = 7M Ethylene Glycol + 0.5M Glucose + 18% Ficoll). While in the SV2 embryos were packaged in 0.25 mL straws, incubated for 1 minute in Liquid N2 vapor and plunged into Liquid N2. For warming the straws were held in air for 8 seconds and plunged in 37oC water bath for 15 seconds. As a control, in vivo produced embryos collected from Nelore cows were used. The cleavage and blastocyst production rates, in Experiment 1, was not different among groups of OZ or OMZT embryos (P>0.05). However, a significant difference was observed in blastocyst production rates when... (Complete abstract click electronic access below)

Orientador: Fernanda da Cruz Landim e Alvarenga / Banca: Maria Denise Lopes / Banca: Claudia Barbosa Fernandes / Resumo: O objetivo desta dissertação foi avaliar a viabilidade e a resistência à criopreservação após a realização de lipólise química utilizando-se o forskolin. Embriões Bos taurus indicus (OZ) e Bos taurus indicus X Bos taurus taurus (OMZT) produzidos in vitro, foram comparados em meio de cultivo contendo ou não soro fetal bovino (SFB). Para o experimento1, 1.052 oócitos de graus I e II foram colhidos de ovários de vacas Bos taurus indicus de abatedouro, dividindo em 7 réplicas. Os oócitos foram maturados em TCM 199 acrescido de SFB, Piruvato de Sódio, FSH, LH e Penicilina/Estreptomicina. A fertilização foi realizada com sêmen de touro Nelore (Bos taurus indicus) ou com sêmen de touro Simental (Bos taurus taurus), em meio HTF acrescido de BSA, Piruvato de Sódio, Cafeína, Heparina, Penicilinamina, Hipotaurina, Epinefrina e Penicilina/Estreptomicina. Os prováveis zigotos foram cultivados em meio SOFaa, acrescido de BSA, Piruvato de Sódio, Penicilina/Estreptomicina, suplementado ou não com SFB (Grupo 2,5%SFB e Grupo 0%SFB). No D6 os blastocistos foram novamente divididos em 2 sub-grupos com ou sem adição de 10μM de forskolin (total de 4 grupos - 2,5%SFB, 0%SFB, 2,5%+F e 0%+F). Para o experimento 2 os embriões foram expostos a solução de vitrificação 1 (SV1 = 5M de Etileno Glicol) durante 3 minutos e transferidos para uma gota com 15μL da solução de vitrificação 2 (SV2 = composta por 7M de Etileno Glicol, 0.5M de Galactose e 18% (w/v) de Ficoll 70) sendo, em seguida envasados em palhetas francesas estéreis de 0,25 mL. Para o aquecimento, as palhetas foram mantidas em ar por 8 segundos e depois em água a 37°C por 15 segundos. Como controle dos experimentos, embriões bovinos produzidos in vivo coletados de vacas Nelores foram utilizados. A taxa de clivagem e de produção de blastocisto no experimento 1, não diferiu entre os tratamentos dentro dos grupo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The main objective of this dissertation was to evaluate the viability and cryotolerance of bovine embryos treated with forskolin to induce chemical lipolysis. In vitro produced Bos taurus indicos (OZ) and Bos taurus indicus X Bos taurus taurus (OMZT) embryos were compared in culture conditions containing or not fetal calf serum (FCS). For Experiment 1, 1.052 grade I and II Bos taurus indicos oocytes were obtained from slaughterhouse ovaries and divided in 7 replicates. The oocytes were matured in TCM 199 supplemented with FCS, Sodium Piruvate, FSH, LH and Penicillin/Streptomycin. Fertilization was performed using semen from one Nelore bull (Bos taurus indicos) and one Simental bull (Bos taurus taurus) in HTF medium supplemented with BSA, Sodium Pyruvate, Caffeine, Heparine, Penicilamine, Hipotaurine, Epinephrine and Penicillin/Streptomycin. The presumptive zygotes were cultured in SOFaa with BSA, Sodium Pyruvate and Penicillin/Streptomycin supplemented or not with FCS (Group 2.5%FCS and Group 0%FCS). On day 6 embryos from both groups were divided in two sub-groups containing or not 10μM of forskolin (total of 4 groups - 2.5%FCS, 0%FCS, 2.5%+F and 0%+F). For Experiment 2 the embryos were exposed to a vitrification solution 1 (SV1 = 5M of Ethylene Glycol) during 3 minutes and transferred to a vitrification solution 2 (SV2 = 7M Ethylene Glycol + 0.5M Glucose + 18% Ficoll). While in the SV2 embryos were packaged in 0.25 mL straws, incubated for 1 minute in Liquid N2 vapor and plunged into Liquid N2. For warming the straws were held in air for 8 seconds and plunged in 37oC water bath for 15 seconds. As a control, in vivo produced embryos collected from Nelore cows were used. The cleavage and blastocyst production rates, in Experiment 1, was not different among groups of OZ or OMZT embryos (P>0.05). However, a significant difference was observed in blastocyst production rates when... (Complete abstract click electronic access below) / Mestre