ABSTRACT
Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the 2 or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student´s test (P 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were simi
Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre- and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the fi rst third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may infl uence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells.Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then
ABSTRACT
Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the 2 or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student´s test (P 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were simi
Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre- and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the fi rst third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may infl uence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells.Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then
ABSTRACT
With the increasing use of artificial insemination techniques, little importance has been given to seminal plasma, since it is substituted by other diluters. However, natural sexual intercourse would not work without seminal plasma as diluter for spermatozoa in the epididimus. The study of seminal plasma brings important information for the research on diluters, which are made to substitute the forme. Thus, if the seminal plasma composition were known, it would be easier to preserve semen samples, specially those of species that present cryopreservation problems. Beside, knowledge on "fertility markers" that may be present in seminal plasma would be of great importance in the identification of animals of high reproductive value.
A partir da introdução de técnicas de inseminação artificial, pouca importância tem sido dada ao plasma seminal, já que este é substituído por outros compostos diluidores. Contudo, o processo de monta natural, dificilmente poderia funcionar sem o plasma seminal como diluente para os espermatozóides concentrados no epidídimo. O estudo deste fluído desperta interesse, visto que, os diluidores seminais atuais, tentam de alguma forma substituí-lo. Desta maneira, se sua composição for totalmente revelada, seria mais fácil o processo de preservação do sêmen, principalmente naquelas espécies, onde hoje, se tem dificuldade em criopreservá-lo. Além disto, o conhecimento de "marcadores" de fertilidade, presentes no plasma seminal, seria de grande valia na identificação de animais de alto valor reprodutivo.
ABSTRACT
With the increasing use of artificial insemination techniques, little importance has been given to seminal plasma, since it is substituted by other diluters. However, natural sexual intercourse would not work without seminal plasma as diluter for spermatozoa in the epididimus. The study of seminal plasma brings important information for the research on diluters, which are made to substitute the forme. Thus, if the seminal plasma composition were known, it would be easier to preserve semen samples, specially those of species that present cryopreservation problems. Beside, knowledge on "fertility markers" that may be present in seminal plasma would be of great importance in the identification of animals of high reproductive value.
A partir da introdução de técnicas de inseminação artificial, pouca importância tem sido dada ao plasma seminal, já que este é substituído por outros compostos diluidores. Contudo, o processo de monta natural, dificilmente poderia funcionar sem o plasma seminal como diluente para os espermatozóides concentrados no epidídimo. O estudo deste fluído desperta interesse, visto que, os diluidores seminais atuais, tentam de alguma forma substituí-lo. Desta maneira, se sua composição for totalmente revelada, seria mais fácil o processo de preservação do sêmen, principalmente naquelas espécies, onde hoje, se tem dificuldade em criopreservá-lo. Além disto, o conhecimento de "marcadores" de fertilidade, presentes no plasma seminal, seria de grande valia na identificação de animais de alto valor reprodutivo.