OBJECTIVE: To monitor time variability of early embryo cleavage by continual monitoring system (time-lapse). To evaluate the impact of patient age for the embryonic growth. To compare pregnancy rate of the time-lapse selected embryos with embryos after ordinary/standard cultivation. DESIGN: Case-control study. SETTING: Centre of assisted reproduction Sanus, Pardubice; PPCHC s.r.o. Hradec Kralove. METHODS: Development of 213 embryos from 44 females was monitored by PrimoVision (time-lapse) system with time frequency of recording 1 image in 12 minutes. The data were evaluated in two groups: infertile patients 35 years (group 35) and control 32 years (group 32). From the collected recordings, time of the first (t2) and of the second (t3) cell cleavage and the time interval between t2 and t3 (cc2) were determined. Symmetrical cellular division to even number of daughter cells, early cleavage and the attainment of blastocyst stage have become the major selection criteria for the embryo transfer. RESULTS: The following average values of studied parameters were found: Group 35: t2 = 27.0 hours, t3 = 38.7 hours, cc2 = 11.7 hours Group 32: t2 = 27.1 hours, t3 = 39.0 hours, cc2 = 11.9 hours, with no significant differences between both groups. Likewise, no significant difference was observed in mean variability of embryo cleavage timing in an individual patient and control: Group 35: t2 = 4.5 hours, t3 = 5.7 hours, Group 32: t2 = 4.5 hours, t3 = 5.1 hours. No relation was observed between the patient age and t2, t3 and cc2 times when evaluated by regression curve (p = 0.60, p = 0.81, p = 0.57). However, embryos of early cleavage have remained in cc2 period significantly shorter period of time compared to embryos of slower cleavage(p = 0.0001). Pregnancy rate at time-lapse selected embryos reached 55.0% while embryos from standard cultivation only 47%. CONCLUSION: The impact of patient age to the cleavage dynamic has not been proved. Relation between the first cell cleavage time and embryo persistence in this stage (cc2) was observed. We may hence recommend cc2 time as convenient parameter at embryo selection for embryo transfer in the centres of assisted reproduction. Our study has shown that embryo selection with time-lapse system (PrimoVision) enhances the success rate of treatment of aging patients.
- MeSH
- blastocysta MeSH
- časosběrné zobrazování metody MeSH
- dospělí MeSH
- lidé MeSH
- přenos embrya metody MeSH
- retrospektivní studie MeSH
- stadium rýhování vajíčka fyziologie MeSH
- těhotenství MeSH
- úhrn těhotenství na počet žen v reprodukčním věku MeSH
- ženská infertilita diagnóza terapie MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Identification of genes that are important for normal preimplantation development is essential for understanding the basics of early mammalian embryogenesis. In our previous study, we have shown that CENPF (mitosin) is differentially expressed during preimplantation development of bovine embryos. CENPF is a centromere-kinetochore complex protein that plays a crucial role in the cell division of somatic cells. To our best knowledge, no study has yet been done on either bovine model, or oocytes and preimplantation embryos. In this study, we focused on the fate of bovine embryos after injection of CENPF double-stranded RNA (dsRNA) into the zygotes. An average decrease of CENPF mRNA abundance by 94.9% or more and an extensive decline in immunofluorescence staining intensity was detected relative to controls. There was no disparity between individual groups in the developmental competence before the 8-cell stage. However, the developmental competence rapidly decreased then and only 28.1% of CENPF dsRNA injected 8-cell embryos were able to develop further (uninjected control: 71.8%; green fluorescent protein dsRNA injected control: 72.0%). In conclusion, these results show that depletion of CENPF mRNA in preimplantation bovine embryos leads to dramatic decrease of developmental competence after embryonic genome activation.
- MeSH
- blastocysta cytologie metabolismus fyziologie MeSH
- buněčné dělení genetika fyziologie MeSH
- chromozomální proteiny, nehistonové antagonisté a inhibitory genetika metabolismus MeSH
- embryonální vývoj genetika fyziologie MeSH
- fertilizace in vitro veterinární MeSH
- kontrolní body buněčného cyklu genetika fyziologie MeSH
- kultivované buňky MeSH
- mikrofilamentové proteiny antagonisté a inhibitory genetika metabolismus MeSH
- skot * embryologie genetika fyziologie MeSH
- stadium rýhování vajíčka metabolismus fyziologie MeSH
- umlčování genů fyziologie MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- skot * embryologie genetika fyziologie MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- centromere protein F MeSH Prohlížeč
- chromozomální proteiny, nehistonové MeSH
- mikrofilamentové proteiny MeSH
The aim of this work was to characterize oocyte fertilization and embryo cleavage in nine AI bulls to find parameters suitable for prediction of in vitro fertility. According to the d8 blastocysts rate, they were categorized as high, medium and low productive (HP, MP and LP, mean: 25.4, 21.0 and 13.6% respectively) bulls. For these categories, oocyte penetration and fertilization efficiency were assessed at 6 and 18 hours post insemination (hpi), respectively. Some presumptive zygotes were cultured and cleaved and fast-cleaved embryo rates were checked at 44 hpi. The penetration rate was significantly higher for HP bulls than for MP and LP bulls (67.9 versus 50.3 and 33.1%; p<0.01). The syngamy rate was significantly higher for HP bulls than for MP and LP bulls (21.4 versus 10.2 and 5.7%; p<0.05). Conversely, no significant differences in fertilization rates were found among HP, MP and LP bulls. The cleavage rate was significantly higher for HP than LP bulls (82.4 versus 74.4%; p<0.01). The fast cleavage rate was significantly higher for both HP and MP bulls, as compared with LP bulls (82.1 and 84.7 versus 73.5%; p<0.01). A strong correlation was found between blastocyst production and penetration (r=0.803), syngamy (r=0.826), cleavage (r=0.635) and fast cleavage (r=0.709). In conclusion, all the evaluated parameters showed a predictive value, the most significant being early penetration and syngamy.
- MeSH
- blastocysta cytologie metabolismus MeSH
- embryo savčí fyziologie MeSH
- fertilita MeSH
- interakce spermie a vajíčka * MeSH
- kultivované buňky MeSH
- oocyty fyziologie MeSH
- skot MeSH
- spermie fyziologie MeSH
- stadium rýhování vajíčka fyziologie MeSH
- umělá inseminace veterinární MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The pig ovary contains a large number of growing oocytes, which do not mature in vitro and cannot be readily used in various biotechnologies. This study was conducted to determine the possibility of inducing meiotic maturation in growing pig oocytes with an internal diameter of 110 microm, which had developed partial meiotic competence. Most of these oocytes spontaneously stopped maturation at the metaphase I stage (68%); a limited number proceeded to the metaphase II stage (26%). Treatment with calcium ionophore A23187 (50 microM for 5 or 10 min) after 24h in vitro culture overcame the block at the metaphase I stage, and treated growing pig oocytes matured to the metaphase II stage (66%). Oocytes in which maturation had been induced by calcium ionophore were again treated with calcium ionophore. Up to 58% of the treated oocytes were activated. Parthenogenetic development in oocytes treated with ionophore for meiosis induction and activation was very limited. The portion which reached morula stage did not exceed 8% and at most 3% developed to the blastocyst stage.
- MeSH
- calcimycin farmakologie MeSH
- časové faktory MeSH
- ionofory farmakologie MeSH
- meióza účinky léků fyziologie MeSH
- oocyty cytologie účinky léků růst a vývoj MeSH
- partenogeneze účinky léků fyziologie MeSH
- prasata fyziologie MeSH
- stadium rýhování vajíčka účinky léků fyziologie MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- calcimycin MeSH
- ionofory MeSH
The aim of our study was to investigate the parthenogenetic activation of in vitro matured pig oocytes after their combined treatment with calcium ionophore A 23187 and the inhibitor of protein kinases, 6-dimethylaminopurine (6-DMAP) and to study the further embryonic development of oocytes activated using this treatment. The oocytes were exposed to ionophore (10, 25 or 50 microM) for 0.5, 1, 3, 5 or 7 min and then cultured with 6-DMAP (0 or 2 microM) The highest activation rate (up to 88% of the activated eggs reached the pronuclear stage) was observed after combined treatment of the oocytes with 50 microM ionophore and 6-DMAP. The highest rate of embryonic development was observed after treatment with 25 microM ionophore without 6-DMAP, when up to 51% of the eggs developed beyond two-cell stage, 2% of the eggs developed up to the stage of morula and up to 3% of the eggs reached the stage of blastocyst. When 50 microM ionophore was used, the embryonic development of the activated eggs was arrested before the morula and blastocyst stage. After treatment of the activated eggs with 6-DMAP, we did not observe any development beyond the stage of 16 blastomeres. We can conclude that combined treatment with calcium ionophore A 23187 and 6-DMAP increases the activation rate in pig oocytes matured in vitro, but this combined treatment exerts a detrimental effect on further embryonic development of the activated eggs.
- MeSH
- adenin analogy a deriváty farmakologie MeSH
- calcimycin farmakologie MeSH
- časové faktory MeSH
- inhibitory enzymů farmakologie MeSH
- inhibitory proteinkinas MeSH
- ionofory farmakologie MeSH
- kultivované buňky MeSH
- oocyty účinky léků růst a vývoj fyziologie MeSH
- partenogeneze MeSH
- prasata fyziologie MeSH
- stadium rýhování vajíčka účinky léků fyziologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- adenin MeSH
- calcimycin MeSH
- inhibitory enzymů MeSH
- inhibitory proteinkinas MeSH
- ionofory MeSH
- N(6),N(6)-dimethyladenine MeSH Prohlížeč
- MeSH
- blastocysta účinky léků fyziologie ultrastruktura MeSH
- daktinomycin farmakologie MeSH
- fertilizace * MeSH
- krysa rodu Rattus MeSH
- morula účinky léků fyziologie ultrastruktura MeSH
- stadium rýhování vajíčka fyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- daktinomycin MeSH
The non-cellular investments were damaged in only 2.8% of rabbit morulae frozen/ thawed in plastic straws. Similar results (4.8%) were obtained with glass ampoules only when morulae were slowly frozen and slowly thawed in siliconized ampoules. Freezing/thawing by other methods and the use of untreated ampoules resulted in high incidence of embryos with damaged investments (41-61%). Morulae frozen/thawed by a method allowing an almost complete elimination of investment injury displayed high survival when slowly frozen and rapidly thawed (90.7%) while slow freezing and slow thawing decreased survival (74.5%). Morulae subjected to rapid freezing were for the first time successfully stored at--196 degrees C. After rapid thawing 59.6% morulae survived. The mechanism of damage to the non-cellular investments of rabbit embryos is discussed.
