Demi-embryos were successfully produced by bisection of ICR mouse embryos at preimplantation stages. They were microsurgically bisected using a microsurgical blade attached to a micromanipulator after pretreatment with 0.5 % pronase in PBS for two minutes or not. Embryos with softened zona pellucida were more easily bisected and less damaged than intact embryos. The highest success rate in bisection has been achieved by selecting blastocysts (94.1 % in success rate with intact blastocysts and 100 % in success rate with zona softened blastocysts). Demi-embryos without zona pellucida were cultured in D-PBS or M-16 medium at 37deg C, 5 % CO2 in air for 72 hours for 2-cell stage embryos, 48 hours for 4-to 8-cell stage embryos, 24 hours for morula stage embryos and 6-12 hours for blastocyst stage embryos. For the in vitro culture of 2-cell stage embryos, 100 micro M 2Na-EDTA was added to the media. M-16 medium was better for the in vitro development of mouse embryos than PBS, and PBS is not considered to be suitable for long-term culture of embryos, especially at early stage of cleavage. In M-16 medium, developing rate of demi-embryos of which pair underwent development to form eublastocysts was 15.8 % at 2-cell stage, 16.8 % at 4-cell stage, 38 % at 8-cell stage, 89.6 % at morula stage and 94.4 % at blastocyst stage, respectively. The more rapid and efficient production of demi-embryos and higher viability after bisection can be expected by softening zona pellucida with pronase and by selecting morulae or blastocysts rather than embryos at early stage of cleavage.

O objetivo do presente estudo foi avaliar se diferentes formas de cultivo interferem no efeito do óxido nítrico (NO) sobre a maturação e a integridade da membrana plasmática do complexo cumulus-oócito de bovinos. Para tanto, realizou-se cultivo em gotas sob óleo mineral ou em placas de quatro poços com a adição de diferentes concentrações de nitroprussiato de sódio (SNP, doador de óxido nítrico). Não foi observada diferença (P &gt; 0,05) entre as formas de cultivo quando se avaliou a integridade de membrana plasmática e a expansão das células do cumulus (CC). Contudo, os oócitos dos grupos controle e os cultivados na presença de 10-3 M de SNP, ambos cultivados em placa, apresentaram maior porcentagem de membrana íntegra do que os mesmos tratamentos cultivados em óleo mineral (P &lt; 0,05). Observou-se que a adição de 10-3 M de SNP diminuiu o grau de expansão das CC e de integridade da membrana plasmática dos oócitos, tanto no cultivo em gota sob óleo quanto em placa, diferindo dos outros grupos (P &lt; 0,05). Semelhante à expansão, a forma de cultivo não interferiu na extrusão do primeiro corpúsculo polar, sendo que a adição de 10-3 M de SNP inibiu a extrusão em ambos os sistemas (P &lt; 0,05). Houve um efeito dose-resposta na concentração de NO no meio de maturação em ambos os tipos de cultivo (P &lt; 0,05), sendo que esta foi maior no meio de cultivo sob óleo, exceção feita quando se adicionou 10-3 M de SNP, tratamento no qual não houve diferença nos tipos de cultivo empregados. Estes dados mostram que o sistema de cultivo não interferiu na ação do NO na maturação in vitro de COC bovinos, mas interfere na integridade da membrana plasmática do oócito. | The aim of the present study was to evaluate the influence of different forms of in vitro culture on the nitric oxide action in maturation and membrane integrity on bovine cumulus-oocyte complex. No significant effect was observed between different forms of culture (mineral oil vs plate; P &gt; 0.05), as much for membrane integrity as for expansion of the CC. However, it was observed that oocytes of the groups control and 10-3 M of SNP, cultivated in plate, had presented greater percentage of cell with maintenance of membrane integrity than same treatments cultivated in drop. The addition of 10-3 M of SNP showed an inhibitory effect on the expansion and membrane integrity of CC and oocytes in both, culture in drops under oil and plate (P < 0.05). The culture form did not intervene with the extrusion of the first polar corpuscle and the addition of 10-3 M of SNP inhibited this extrusion in the both systems (P < 0.05). There was a dose-response effect on the concentration of NO in the maturation medium in both types of cultivation (P < 0,05), and this was higher in the culture medium under oil, except when added 10-3 M of SNP, treatment in which there was no difference in the types of cultivation employed. (P<0.05). These data demonstrate that the culture system did not intervene with the action of the NO in the maturation in vitro of bovine COC, but intervened with the integrity of the plasmatic membrane of the oocyte.