Optimizing electrical activation of porcine oocytes by adjusting pre- and post-activation mannitol exposure times.

Modifying electrical activation conditions have been used to improve in vitro embryo production and development in pigs. However, there is insufficient information about correlations of porcine embryo development with oocyte pre- and post-activation conditions. The purpose of this study was to compare the developmental rates of porcine oocytes subjected to different mannitol exposure times, either pre- or post-electrical activation, and to elucidate the reason for the optimal mannitol exposure time. Mannitol exposure times around activation were adjusted as 0, 1, 2 or 3 min. Blastocyst development were checked on day 7. Exposure of oocytes to mannitol for 1 or 2 min before electrical activation produced significantly higher blastocyst rates than exposure for 0 or 3 min. There was no significant difference in blastocyst rates when activated oocytes were exposed to mannitol for 0, 1, 2 or 3 min after electrical activation. While exposure of oocytes to mannitol for 1 min pre- and 3 min post-activation showed significantly higher blastocyst development than 0 min pre- and 0 min post-activation. It also showed higher maintenance of normal oocyte morphology than exposure for 0 min pre- and 0 min post-activation. In conclusion, exposure of oocytes to mannitol for 1 min pre- and 3 min post-activation seems to be optimal for producing higher in vitro blastocyst development of porcine parthenogenetic embryos. The higher blastocyst development is correlated with higher maintenance of normal morphology in oocytes exposed to mannitol for 1 min pre- and 3 min post-activation.

Effect of roscovitine-treated donor cells on development of porcine cloned embryos.

Synchronization of the donor cell cycle is an important factor for successful animal cloning by nuclear transfer. To improve the efficiency of porcine cloning, in the present report, we evaluated effects of contact inhibition, serum starvation and roscovitine treatment of donor cells on in vitro and in vivo developmental potency of cloned porcine embryos. Fibroblasts derived from a porcine foetus at day 30 of gestation were isolated and cultured to 70% confluency. Then, cells were either cultured to 100% confluency for contact inhibition, or cultured in 0.5% serum for 72 h for serum starvation or with 15 µM roscovitine for 24 h. Cells were most effectively synchronized at G0/G1 in the serum starvation group (87.5%) compared with the contact inhibition and roscovitine treatment groups (76.3% and 79.9% respectively p < 0.05). However, after somatic cell nuclear transfer followed by in vitro culture, the serum starvation group showed a significantly lower blastocyst formation rate (5.6%) compared with the contact inhibition and roscovitine treatment groups (11.6% and 20.0% respectively). Differential expression of apoptosis-related genes and the level of apoptosis in each treatment group explain the variation in developmental competence among the groups. Significantly higher level of apoptosis was observed in the serum starvation group. On the other hand, the roscovitine treatment group shows the lowest level of apoptosis and the best in vitro development among the groups. Cloned embryos derived from roscovitine-treated donor cells were transferred to surrogate pigs. Three healthy live piglets were produced. In conclusion, we suggest that roscovitine treatment of donor cells improves development of cloned porcine embryos and can raise the efficiency of cloned piglet production.

Influence of ovulation status, seasonality and embryo transfer method on development of cloned porcine embryos.

To improve pig cloning efficiency, the present study evaluated the effect of ovulation status, seasonality and embryo transfer (ET) method on in vivo development of cloned porcine embryos. Cloned embryos were transferred to surrogate mothers on the same day of somatic cell nuclear transfer. In pre-ovulation stage (PO), pregnancy rate (PR) and delivery rate (DR) were 36.3% and 9.4%, respectively. In post-ovulation stage, 22.7% PR and 2.1% DR were recorded (both PR and DR are significantly higher in PO). When ET was performed during winter (December-February), spring (March-May), summer (June-August) and autumn (September-November), the PRs were 13.4%, 37.3%, 24.6% and 51.0%, while DRs were 0%, 12.7%, 4.3% and 7.8%, respectively. The highest PRs were recorded in autumn groups. However, DRs were significantly lower in autumn (7.8%) group compared with spring (12.7%) group. The PR was the lowest and no piglets were born in winter group, which might be because of the effect of low temperature during ET. To overcome the low PR in winter group, 0.25 ml straws were used for ET to minimize exposure time of embryos to ambient temperature. The straw ET group showed significantly higher PR in the winter group (23. 9%) compared with the conventional catheter-loading group (7.7%). We suggest that using PO recipient and ET in spring is the best condition for pig cloning. In addition, alternative method to reduce cold shock during ET in winter is necessary.

Effect of dimethyl sulfoxide on cell cycle synchronization of goldfish caudal fin derived fibroblasts cells.

Several studies have previously been conducted regarding cell cycle synchronization in mammalian somatic cells. However, limited work has been performed on the control of cell cycle stages in the somatic cells of fish. The aim of this study was to determine the cell cycle arresting effects of several dimethyl sulfoxide (DMSO) concentrations for different times on different cell cycle stages of goldfish caudal fin-derived fibroblasts. Results demonstrated that the cycling cells or control group (68.29%) yields significantly higher (p < 0.05) arrest in G0/G1 phase compared with the group treated for 24 h with different concentrations (0.5%, 1.0% or 1.5%) of DMSO (64.88%, 65.70%, 64.22% respectively). The cell cycle synchronization in the treatment of cells with 1.0% DMSO at 48 h (81.14%) was significantly higher than that in the groups treated for 24 h (76.82%) and the control group (77.90%). Observations showed that treatment of DMSO resulted in an increase in the proportion of cells at G0/G1 phase for 48 h of culture. However, high levels of apoptotic cells can be detected after 48 h of culture treated with 1% concentration of DMSO.

Electrical activation induces reactive oxygen species in porcine embryos.

The objectives were to determine factors affecting generation of reactive oxygen species (ROS) in porcine embryos after electrical activation of oocytes, and the effects of an antioxidant and chemical agent on ROS generation. Greater ROS were induced by electrical activation compared to IVF (mean+/-S.E.M., 14.6+/-0.8 vs. 9.2+/-0.4, P<0.05). Furthermore, ROS generation in embryos after electrical activation was significantly increased by higher intensity and longer duration electrical pulses and by higher exogenous Ca(2+) concentrations. Cleavage rate and blastocyst formation rate were not directly related to the level of ROS. Supplementation of the IVC medium with 0.5mM glutathione (GSH) reduced ROS (9.2+/-0.4 vs. 14.7+/-0.9, P<0.05). Treatment with the chemical activation agent, 6-dimethylaminopurine (6-DMAP) for 3h did not induce further ROS generation in combination with electrical activation, but it improved blastocyst formation rate (53.8+/-1.1 vs. 23.7+/-3.5, P<0.05). We concluded that generation of ROS should be considered for optimizing electrical activation and that supplementing an antioxidant or combining electrical and chemical activation induced lower ROS generation in electrically activated porcine embryos.

Effects of culture conditions and nuclear transfer protocols on blastocyst formation and mRNA expression in pre-implantation porcine embryos.

This study investigated the effects of culture conditions and somatic cell nuclear transfer (SCNT) protocols on in vitro development of porcine SCNT embryos and on expression patterns of genes involved in stress (heat shock protein 70.2, HSP70.2), trophoblastic function (integrin beta1, ITGB1), metabolism (phosphoglycerate kinase 1, PGK1), apoptosis (BAX), and imprinted gene (insulin-like growth factor 2 receptor, IGF2R). In Experiment 1, supplementing modified North Carolina State University (mNCSU) medium with 10% FBS at Day 4 of culture increased SCNT blastocyst formation (22.9 vs. 10.7%, P<0.05), number of inner cell mass cells (13.3+/-4.3 vs. 7.6+/-2.2, P<0.05), and total cells (57.9+/-19.5 vs. 36.3+/-8.2, P<0.05) in cloned blastocysts. In Experiment 2, using culture medium with 10% FBS, 1.0mM calcium in fusion/activation medium (1.0C), and 7.5mug/mL cytochalasin B treatment (0.1C&CB) yielded higher rates (P<0.05) of blastocysts (33.6 and 33.3%, respectively) relative to the control (0.1mM calcium fusion medium, 0.1C; 18.3%). Total cell numbers of blastocysts were increased (P<0.05) in 1.0C (77.4+/-28.9) compared to the control (58.5+/-22.6). In vitro-derived blastocysts had higher expression levels of BAX and lower levels of HSP70.2, IGF2R compared to their in vivo-derived counterparts. Supplementing culture medium with 10% FBS increased relative abundances of BAX mRNA in SCNT blastocysts relative to in vivo-derived blastocysts. The transcript level of ITGB1 in blastocyst from 0.1C&CB was lower than in vivo blastocysts. In conclusion, different culture conditions or SCNT protocols affected in vitro development of SCNT embryos and altered several important genes (BAX, HSP70.2, IGTB1, and IGF2R) compared to conventional in vivo-derived blastocysts.

Influence of oocyte donor and embryo recipient conditions on cloning efficiency in dogs.

To determine factors that affect the efficiency of dog cloning by somatic cell nuclear transfer, the present study was performed to investigate 1) the effects of surgical history (non-operated/operated) and parity (nullipara/multipara) on the recovery of in vivo canine oocytes; 2) the effects of surgical history and parity of recipients on the pregnancy and delivery; and 3) the effects of synchronization state (AA, advanced asynchrony; SY, synchrony; RA, retarded asynchrony) between oocytes donor and recipient on the pregnancy and delivery. Oocyte recovery rate was significantly higher in non-operated dogs compared to operated dogs (93.8 vs. 89.6%, P < 0.05) and not different between nulliparous dogs and multiparous dogs. Delivery rate was also significantly higher in non-operated dogs compared to operated dogs (2.8 vs. 1.0%, P < 0.05) and in nulliparous dogs than multiparous dogs (3.0 vs. 1.7%, P < 0.05). Even though SY showed increased pregnancy and delivery rate (20.0% and 3.0%) compared to AA (15.0% and 2.0%) and RA (0.0% and 0.0%), there was no significant difference. In conclusion, we recommend non-operated dogs as experimental dogs and nulliparous dogs as recipient dogs to increase delivery rate after transfer of somatic cell nuclear transferred embryos, but further study is needed to find out appropriate synchrony status at the transfer.