A Preliminary Study On The Aggregation Of Porcine For Making Chimeras

Chimera is an individual with a double chromosome complement which is integrated by two or more embyos or cells with different hereditary. The formation of chimera can be achieved by two means, one is blastocyst injection, the other one is aggregation, the latter method is comparatively easier while has lower success rate. The low aggregation rate ascribes to the difficulty in acquiring embyos, as the foundation for aggregation is comparatively synchronous development of embyos. Futhermore, the technique and manipulation of each tache in embyos aggregation are exigent, and there are many influencing factors such as the remove of zone pellucide, conditions for aggregation, culture medium for aggregated embyos, culture environment and so on. In recent years, as the stem cell technology developing rapidly and it's applied potential constantly appearing, the embryonic stem cells with organic consistency obtained by stem cell nuclear transfer have become an critical trench to resolve numerous medicinal problems. Therefore, the aggregation of embyos from different individuals of the same breed has become a required process to that trench. However, the low embyos aggregation rate seriously limit the development of this technique. This experiment did systemic investigation on the critical teches of aggregation such as the remove of zona pellucide, concentration for aggregation, development of aggregated embyos, systems for aggregation, innovation of culture method and so on. The effects of these teches on the different individual embyos aggregation of the same breed were studied. The objective of this experiment is to find fitting processes for the making of porcine chimera to improve the development rate of aggregated embyos and prolong the developmental stage of aggregated embyos. 1, research on removing of zone pellucideThe test adopted Acid Tyrode, 0.25% pronase, 0.3% pronase, 0.5 % pronase to remove zone pellucide of 8-cell embryo. Then through observing the percentage of obtained naked embryo, the aggregation rate and aggregated embryos' development rate, the effects of four kinds of liquid on the aggregation result were estimated. The results showed: the group treated with 0.3% pronase got the highest naked embryo rate (14/17, 82.4%) (P<0.05), while the group treated with Acid Tyrode had the lowest rate (8.5/16, 53.1%) (P<0.05). The aggregation rates of the 0.25% pronase treated group (9/13, 69.2%) and the 0.3% pronase treated group (10/14, 71.4%) were significant higher than those of the Acid Tyrode treated group (5/8.5, 53.1%) and 0.50% pronase treated group (7/11, 63.6%) (P<0.05) .The development rate of aggregated embyos of the 0.25% pronase treated group (6/9, 66.7%) and the 0.3% pronase treated group (7/10, 70.0%) were higher than those of Acid Tyrode treated group (1/5, 20.0%) and the 0.5% pronase treated group(3/7, 42.9%) (P<0.05) The group treated with leaded the lowest development rate (P<0.05). 0.30% pronase was consequently the bestchoice to remove zone pellucide, for it could lead the highest naked oocytes rate, aggragation rate, and development rate of aggragated embryos.2, Study on the effects of various concentrations on embyos aggregation.Three different concentrations of PHA (0.03%,0.06%,0.12%) were adopted in this experiment to investigate the relation between the effiency and the concentration of PHA, the aggregation effiency was estimated by aggregation rate and development rate of aggregated embyos. The results indicated that along with the concentration of PHA increased, the aggregation rate of embyos also increased (P<0.05) ,their aggregation rates were 11/17 (64.7%), 12/16 (75.0%), 14/18 (77.8%) , respectively.The development rate of aggregated embyos of 0.06%PHA treated group (9/12,75.0%) was significantly higher than those of 0.03%PHA treated group (7/11,63.6%) and 0.12%PHA treated group (9/14,64.3%) (P<0.05) . As a result, 0.06%PHA is an optimal concentration in embyos aggregation for higher aggregation rate and development rate of aggregated embyos was obstained with that concentration.3, research on different stages of embyos used for aggregation.The aggregation rate and development rate of aggregated embryos were used as indexs to confirm the effects of different stages of embyos on aggregation efficiency. The results demonstrated that the aggregation rate(14/17, 82.4%) and the development rate of aggregated embyos(10/14, 71.4%) of 8-cell with 8-cell embryos were significantly higher than those of 4-cell with 8-cell embryos(aggregation rate: 18/25, 72.0%, development rate of aggregated embyos: 11/18, 61.1%) and 8-cell with morula(aggregation rate: 13/19, 68.4%, development rate of aggregated embyos: 8/13, 61.5%)(P<0.05).4, study on different aggregation systems.The aggregation rate and development rate of aggregated embryos were used as indexs to compare the effects of PHA aggregation and hollowness aggregation on embyos aggregation efficiency. The results indicated that there was no difference on aggregation rate between the two systems 76.9%(10/13) VS73.3 (11/15) (P>0.05) . The development rate of aggregated embryos of self-made hollowness system (8/11, 72.7%) was significantly higher than that of 0.06%PHA aggregation (7/10, 70.0%), but there was no significant difference between them (P>0.05) . Therefore, hollowness aggregation could be used for embyos aggregation.5, study on diverse culture methods of aggregated embryos.Two culture methods were compared according as the development rate of aggregated embryos. The results showed that the aggregated embryos cultured in hollowness (11/16, 68.8%) had appreciably higher development rate than that of these cultured in culture dish (9/13, 69.2%) ,but there was no significant difference between them (P>0.05) .