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.     

Therapeutic efficacy of zeta-cypermethrin pour-on for the treatment of biting and sucking lice in cattle under field conditions

OBJECTIVE: To assess the efficacy of zeta-cypermethrin pour-on to control cattle lice. DESIGN: Five field trials in south-eastern Australia. PROCEDURE: Zeta-cypermethrin pour-on, deltamethrin pour-on and pour-on vehicle were applied to groups of 10 cattle. Lice were counted before treatment and 14, 28, 42 and 56 days after treatment. RESULTS: Zeta-cypermethrin pour-on given at 2.5 mg/kg was equivalent to, or marginally more effective than a deltamethrin pour-on at 0.75 mg/kg. It eliminated B bovis and H eurysternus and gave good control of L vituli and S capillatus. Zeta-cypermethrin at 1 mg/kg gave good control of B bovis and H eurysternus but was not satisfactory against L vituli and S capillatus. CONCLUSION: Zeta-cypermethrin pour-on, given at 2.5 mg/kg, is an effective treatment for cattle lice control. Zeta-cypermethrin, and other synthetic pyrethroid pour-ons, are the treatment of choice to control B bovis.     

Mechanistic target of rapamycin is activated in bovine granulosa cells after LH surge but is not essential for ovulation

The LH surge induces functional and morphological changes in granulosa cells. Mechanistic target of rapamycin (mTOR) is an integrator of signalling pathways in multiple cell types. We hypothesized that mTOR kinase activity integrates and modulates molecular pathways induced by LH in granulosa cells during the preovulatory period. Cows were ovariectomized and granulosa cells collected at 0, 3, 6, 12 and 24 hr after GnRH injection. While RHEB mRNA levels increased at 3 and 6 hr, returning to basal levels by 12 hr after GnRH treatment, RHOA mRNA levels increased at 6 hr and remained high thereafter. Western blot analyses revealed increased S6K phosphorylation at 3 and 6 hr after GnRH injection. Similarly, mRNA levels of ERK1/2, STAR and EGR‐1 were higher 3 hr after GnRH treatment. Rapamycin treatment inhibited mTOR activity and increased AKT activity, but did not alter ERK1/2 phosphorylation and EGR1 protein levels in cultured bovine granulosa cells. Rapamycin also inhibited LH‐induced increase in EREG mRNA abundance in granulosa cells in vitro. However, intrafollicular injection of rapamycin did not suppress ovulation. These findings suggest that mTOR is involved in the control of EREG expression in cattle, which may be triggered by LH surge stimulating RHEB and S6K activity.