Nuclear maturation and embryo development of porcine oocytes vitrified by cryotop: effect of different stages of in vitro maturation.

The present study was designed to evaluate the viability, meiotic competence and subsequent development of porcine oocytes vitrified using the cryotop method at different stages of in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were cultured in IVM medium supplemented with 1mM dibutyryl cAMP (dbcAMP) for 22 h and then for an additional 22 h without dbcAMP in the medium. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), anaphase I/telophase I (AI/TI) and metaphase II (MII) were found to occur predominantly at 0-22, 26, 32, 38 and 44 h of IVM, respectively. Oocytes were exposed to cryoprotectant (CPA) or vitrified after different durations of IVM (0, 22, 26, 32, 38 and 44 h). After CPA exposure and vitrification, surviving oocytes that were treated before completion of the 44 h maturation period were placed back into IVM medium for the remaining maturation period, and matured oocytes were incubated for 2h. CPA treatment did not affect the viability of oocytes matured for 26, 32, 38 or 44 h, but significantly decreased survival rate of oocytes matured for 0 or 22 h. CPA treatment had no effect on the ability of surviving oocytes to develop to the MII stage regardless of the stage during IVM; however, blastocyst formation following PA was severely lower (P<0.05) than that in the control. At 2h post-warming, the survival rates of oocytes vitrified at 26, 32, 38 and 44 h of IVM were similar but were higher (P<0.05) than those of oocytes vitrified at 0 or 22 h of IVM. The MII rates of surviving oocytes vitrified at 0 and 38 h of IVM did not differ from the control and were higher (P<0.05) than those of oocytes vitrified at 22, 26 or 32 h of IVM. After parthenogenetic activation (PA), both cleavage and blastocyst rates of vitrified oocytes matured for 22, 26, 32, 38 and 44 h did not differ, but all were lower (P<0.05) than those matured 0 h. In conclusion, our data indicate that survival, nuclear maturation and subsequent development of porcine oocytes may be affected by their stage of maturation at the time of vitrification; a higher percentage of blastocyst formation can be obtained from GV oocytes vitrified before the onset of maturation.

Wu G ，Jia B ，Mo X ，Liu C ，Fu X ，Zhu S ，Hou Y ... -  《-》

Reduced competence of immature and mature oocytes vitrified by Cryotop method: assessment by in vitro fertilization and parthenogenetic activation in a bovine model.

Bulgarelli DL ，Vireque AA ，Pitangui-Molina CP ，Silva-de-Sá MF ，de Sá Rosa-E-Silva ACJ ... -  《-》

Bovine oocyte vitrification: effect of ethylene glycol concentrations and meiotic stages.

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 18-24h 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, after vitrification in VS-2, blastocyst rates were less at 18 h than 0 h. Both cleavage rates and blastocyst rates were significantly less in all vitrification groups when compared to control group and only control oocytes hatched. In conclusion, both EG concentration and stage of meiotic maturation affect the developmental potential of oocytes after vitrification.

Magnusson V ，Feitosa WB ，Goissis MD ，Yamada C ，Tavares LM ，D'Avila Assumpção ME ，Visintin JA ... -  《ANIMAL REPRODUCTION SCIENCE》

Comparison of cytoskeletal integrity, fertilization and developmental competence of oocytes vitrified before or after in vitro maturation in a porcine model.

Aim of the study was to investigate the effect of vitrification on viability, cytoskeletal integrity and in vitro developmental competence after in vitro fertilization (IVF) of oocytes vitrified before or after in vitro maturation (IVM) using a pig model. Oocytes from abattoir-derived porcine ovaries were vitrified at either the germinal vesicle (GV) or metaphase II (MII) stage by modified solid surface vitrification (SSV). Oocyte viability was evaluated by stereomicroscopic observation whereas their nuclear stage and morphology of microtubules and F-actin were observed by confocal microscopy after immunostaining. Fertilization was assessed by orcein staining. The survival rate after vitrification was higher for MII-stage than for GV-stage oocytes. However, the ability of surviving oocytes to reach the MII stage after vitrification at the GV stage (GV-vitrified oocytes) was similar to that of control oocytes. Furthermore, after IVM, GV-vitrified oocytes had better spindle and F-actin integrity than oocytes vitrified at the MII stage (MII-vitrified oocytes). In accordance with this result, GV-vitrified oocytes had better ability to extrude the second polar body and support male pronucleus formation after in vitro fertilization (IVF), in comparison to MII-vitrified oocytes. Fertilization rates did not differ among groups. Finally, the ability of GV-vitrified oocytes to develop into embryos was superior to that of MII-vitrified oocytes. However, both vitrified groups showed reduced blastocyst development compared with the control group. In conclusion vitrification of porcine oocytes at the GV stage is advantageous in conferring better cytoskeletal organization and competence to develop to the blastocyst stage in comparison with vitrification at the MII stage.

Egerszegi I ，Somfai T ，Nakai M ，Tanihara F ，Noguchi J ，Kaneko H ，Nagai T ，Rátky J ，Kikuchi K ... -  《-》

Effect of cumulus morphology and maturation stage on the cryopreservability of equine oocytes.

Tharasanit T ，Colleoni S ，Lazzari G ，Colenbrander B ，Galli C ，Stout TA ... -  《REPRODUCTION》