162 Developmental competence of bovine oocytes matured in defined medium

Routinely, bovine oocytes are matured in medium supplemented with 10% fetal bovine serum (FBS). However, due to the undefined nature of FBS and biosafety concerns, a standardized protein supplementation alternative is needed. We hypothesised that oocytes matured in defined medium supplemented with 5% serum substitute (DM) would have higher developmental competence than those matured in FBS-supplemented medium (Control). In 6 replicates, in vitro-produced embryos were produced during the summer season by aspirating cumulus-oocyte complexes from 2- to 8-mm follicles of abattoir ovaries (LAB-COC), or aspirated from FSH-stimulated Holstein and Jersey donors (OPU-COC, n=20; 53 sessions). The LAB-COC (n=1307) and OPU-COC (n=679) were matured for 23h in control medium or DM supplemented with pyruvate and gonadotropins at 6% CO2 in air, fertilized with semen from 1 of 3 or 15 bulls, respectively, and cultured in SCF2 at 38.5°C, 6% CO2, 5% O2 and 89% N2. On Day 7.5 post-fertilization, blastocyst rates were evaluated, and embryos (n=4/replicate) from each group of LAB-COC were stained with 1µg mL−1 Nile Red for lipid quantification, 5 µM Cell Rox® Green (Thermo Fisher Scientific, Waltham, MA, USA) for reactive oxygen species (ROS) assessment and 300 nM Mitotracker Red CMX-Rosamine (Thermo Fisher Scientific) for mitochondrial polarity. Images were obtained with confocal microscopy and fluorescent intensity (AFU) was measured by Image J software (National Institutes of Health, Bethesda, MD, USA) adjusted per cell number. Data were analysed by one-way ANOVA using SAS software (SAS Institute Inc., Cary, NC, USA). Results (Table 1) indicate similar cleavage and blastocyst rates and mitochondrial polarity for embryos from LAB-COC in Control v. DM (P>0.05). However, ROS and lipid content were higher in Control than in DM (P<0.05). For OPU-COC, DM resulted in similar cleavage and blastocyst rates and embryos produced per OPU session compared with Control (P>0.05). Results suggest that maturing oocytes (LAB and OPU) in DM maintain developmental competence, and the resulting embryos had lower lipid content and ROS than those matured in Control medium. Cryopreservation studies and evaluation of pregnancy rates are ongoing. Table 1.Developmental competence of oocytes matured in control or defined medium with 5% serum substitute (DM)

Plasma Rich in Growth Factors Induces Cell Proliferation, Migration, Differentiation, and Cell Survival of Adipose-Derived Stem Cells

Adipose-derived stem cells (ASCs) are a promising therapeutic alternative for tissue repair in various clinical applications. However, restrictive cell survival, differential tissue integration, and undirected cell differentiation after transplantation in a hostile microenvironment are complications that require refinement. Plasma rich in growth factors (PRGF) from platelet-rich plasma favors human and canine ASC survival, proliferation, and delaying human ASC senescence and autophagocytosis in comparison with serum-containing cultures. In addition, canine and human-derived ASCs efficiently differentiate into osteocytes, adipocytes, or chondrocytes in the presence of PRGF. PRGF treatment induces phosphorylation of AKT preventing ASC death induced by lethal concentrations of hydrogen peroxide. Indeed, AKT inhibition abolished the PRGF apoptosis prevention in ASC exposed to 100 μM of hydrogen peroxide. Here, we show that canine ASCs respond to PRGF stimulus similarly to the human cells regarding cell survival and differentiation postulating the use of dogs as a suitable translational model. Overall, PRGF would be employed as a serum substitute for mesenchymal stem cell amplification to improve cell differentiation and as a preconditioning agent to prevent oxidative cell death.