90 Lipid droplet accumulation in oocytes and embryos can be reduced by decreasing the use of fetal calf serum during in vitro maturation, without compromising subsequent blastocyst rates

Lipid accumulation during in vitro embryo production (IVEP) has been associated with lower embryo developmental potential and cryotolerance, particularly in Zebu breeds. One of the possible causes of this accumulation is the use of fetal calf serum (FCS) during IVEP. The aim of this study was to evaluate the effect of the reduction of FCS concentrations during in vitro maturation (IVM) of Nelore (Bos indicus) cumulus–oocyte complexes (COC) on the accumulation of lipid droplets in matured oocytes and in embryos. The COC used were recovered from slaughterhouse ovaries and those presenting homogeneous cytoplasm and three or more layers of cumulus cells (i.e. scored as grade I) were randomly allocated into two experimental groups, which underwent IVM in the presence of low (1%, n = 172) or high (10%, n = 166) FCS concentrations. Immature COC (n = 16) were used as a negative control for IVM endpoints. The IVM was performed in TCM 199 added with pyruvate, amikacin, human chorionic gonadotrophin, oestradiol, insulin, β-mercaptoethanol and FSH, in an incubator at 38.5°C and 5% CO₂. After 22 h of IVM, part of the COC (n = 19 for 1% FCS and n = 20 for 10% FCS) was denuded and fixed for lipid droplets evaluation. Semen from a single Angus sire was used for IVF. In vitro embryo culture (IVC) was performed in synthetic oviductal fluid (SOF) with 3% FCS, at low oxygen tension (5% CO₂ and 5% O₂) and 38.5°C. At Day 7 of IVC, blastocyst rates were accessed and part of the expanded blastocysts (n = 11 for 1% FCS and n = 20 for 10% FCS) fixed for lipid analysis. The oocytes or blastocysts sampled from each group were stained with Bodipy 493/503 (Molecular Probes) and evaluated by confocal microscopy with laser dissection (LSM Leica Sp8). Lipid quantification was determined by the ration between lipid droplet area in the cytoplasm and total oocyte or embryo cell area. Data were analysed using the GLIMMIX procedure of the SAS (SAS Institute). The cleavage rate (Day 2) was greater in the 10% FCS compared to the 1% FCS (80.0% vs 68.6%; P = 0.0208). The blastocyst rate (Day 7), however, was similar between 10% FCS and 1% FCS (41.0% vs 40.3%, respectively; P = 0.8254). Immature oocytes presented lesser lipid droplets than those IVM either with 1% FCS or 10% FCS (13.1%^a vs 16.9%^b and 19.9%^b, respectively, P = 0.0487). Expanded blastocysts produced from oocytes matured in 1% FCS presented lesser lipid droplets than those from 10% FCS (12.5% vs 17.6%; P = 0.0495). In summary, the reduction of FCS during IVM of Nelore breed oocytes decreased the accumulation of lipid droplets in both oocytes and embryos, without affecting blastocyst rates.

This study was supported by FIVX Apoyar Biotech, Norte Embryo, CAPES, DPG UnB, and FAPDF.