The antioxidant dimethylthiourea improves IVF efficiency and decreases cumulus cell apoptosis in pigs.

Abattoir ovaries, which are the main source of oocytes for reproductive biotechnologies, arrive at the laboratory under ischaemic conditions. Reoxygenation generates reactive oxygen species (ROS) in ischaemic tissues, which could affect oocyte quality. The aim of this study was to evaluate the effect of supplementation of media with dimethylthiourea (DMTU) during the collection and washing of cumulus-oocyte complexes (COC) on ROS levels, COC apoptosis and oocyte nuclear and cytoplasmic maturation. Thus, the collection (TCM-199) and washing (TCM-199 with 10% porcine follicular fluid, sodium pyruvate and antibiotics) media were supplemented with 1 and 10mM DMTU. In the control group, the media were not supplemented with DMTU. Intracellular ROS levels decreased significantly in the DMTU-treated groups (P<0.05). Although no effects on rate of nuclear maturation were observed, DMTU significantly increased sperm penetration rates without increasing polyspermy (P<0.05). The addition of 10mM DMTU to the collection and washing media enhanced IVF efficiency. DMTU did not modify the early or late apoptosis of oocytes. Both concentrations of DMTU significantly increased viability and decreased the apoptosis of cumulus cells (P<0.05). These results suggest that the addition of 1 or 10mM of DMTU to the media during the collection and washing of porcine COCs is useful for decreasing cumulus apoptosis mediated by ROS and for optimising the IVF of porcine oocytes.

Vesicles Cytoplasmic Injection: An Efficient Technique to Produce Porcine Transgene-Expressing Embryos.

The use of vesicles co-incubated with plasmids showed to improve the efficiency of cytoplasmic injection of transgenes in cattle. Here, this technique was tested as a simplified alternative for transgenes delivery in porcine zygotes. To this aim, cytoplasmic injection of the plasmid alone was compared to the injection with plasmids co-incubated with vesicles both in diploid parthenogenic and IVF zygotes. The plasmid pcx-egfp was injected circular (CP) at 3, 30 and 300 ng/µl and linear (LP) at 30 ng/µl. The experimental groups using parthenogenetic zygotes were as follows: CP naked at 3 ng/µl (N = 105), 30 ng/µl (N = 95) and 300 ng/µl (N = 65); Sham (N = 105); control not injected (N = 223); LP naked at 30 ng/µl (N = 78); LP vesicles (N = 115) and Sham vesicles (N = 59). For IVF zygotes: LP naked (N = 44) LP vesicles (N = 94), Sham (N = 59) and control (N = 79). Cleavage, blastocyst and GFP+ rates were analysed by Fisher's test (p < 0.05). The parthenogenic CP naked group showed lower cleavage respect to control (p < 0.05). The highest concentration of plasmids to allow development to blastocyst stage was 30 ng/µl. There were no differences in DNA fragmentation between groups. The parthenogenic LP naked group resulted in high GFP rates (46%) and also allowed the production of GFP blastocysts (33%). The cytoplasmic injection with LP vesicles into parthenogenic zygotes allowed 100% GFP blastocysts. Injected IVF showed higher cleavage rates than control (p < 0.05). In IVF zygotes, only the use of vesicles produced GFP blastocysts. The use of vesicles co-incubated with plasmids improves the transgene expression efficiency for cytoplasmic injection in porcine zygotes and constitutes a simple technique for easy delivery of plasmids.