Sperm-mediated gene transfer: effect on bovine in vitro embryo production.

The technique of sperm-mediated gene transfer (SMGT) can be used to delivery exogenous DNA into the oocyte. However, it has low repeatability and produces inconsistent results. In order to optimize this technique, it is necessary to study the mechanism by which DNA enters the sperm cell and integrates in the sperm genome. Furthermore, studies must focus in the maintenance of sperm cell viability and function. The aim of this study was to evaluate different SMGT protocols of sperm electroporation or capacitation (CaI) aiming to maintain sperm viability in the production of bovine embryos in vitro. Frozen-thawed semen was divided in two experimental groups (electroporation or CaI) and one control group (non-treated cells). For the electroporation method, five different voltages (100, 500, 750, 1000 or 1500 V) with 25 µF capacitance were used. For CaI treatment, combinations of two CaI concentrations (250 nM or 500 nM), two incubation periods of sperm cells with CaI (1 or 5 min) and two incubation periods that mimicked time of sperm cell interaction with exogenous DNA molecules (1 or 2 h) were evaluated. According to our data, electroporation and CaI treatments do not prevent sperm penetration and oocyte fertilization and can be an alternative method to achieve satisfactory DNA delivery in SMGT protocols.

Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos.

The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows®. Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

Treatment of in vitro-Matured Bovine Oocytes With Tauroursodeoxycholic Acid Modulates the Oxidative Stress Signaling Pathway.

In several species, oocyte and embryo competence are improved by the addition of endoplasmic reticulum (ER) stress inhibitors to in vitro maturation (IVM) medium and/or in vitro culture (IVC) medium. This study aimed to evaluate the effects of three concentrations of tauroursodeoxycholic acid (TUDCA; 50, 200, and 1,000 µM), a chemical chaperone for relieving ER stress, during IVM of bovine cumulus-oocyte complexes (COCs) for 24 h. Treated oocytes were analyzed for nuclear maturation, reactive oxygen species (ROS) production, mitochondrial activity, and abundance of target transcripts. In addition, the number of pronuclei in oocytes was evaluated after 18-20 h of insemination, and the rates of blastocyst and hatched blastocyst formation were evaluated after 7 and 8/9 days of culture, respectively. We further evaluated the transcript abundance of embryonic quality markers. Our findings showed that supplementation of IVM medium with 200 µM of TUDCA decreased ROS production and increased abundance of transcripts related to antioxidant activity in oocytes (CAT, GPX1, and HMOX1) and embryos (GPX1 and PRDX3). Interestingly, high concentration of TUDCA (1,000 µM) was toxic to oocytes, reducing the nuclear maturation rate, decreasing mitochondrial activity, and increasing the abundance of ER stress (HSPA5) and cellular apoptosis (CASP3 and CD40) related transcripts. The results of this study suggest that treatment with 200 µM of TUDCA is associated with a greater resistance to oxidative stress and indirectly with ER stress relief in bovine oocytes.