86 SIMPLE METHOD FOR EMULSIFICATION OF LIPOPHILIC NUTRIENTS THAT AFFECT PRE-IMPLANTATION DEVELOPMENT OF BOVINE EMBRYOS IN VITRO

Abstract

In order to investigate the effects of bioactive lipophilic nutrients on mammalian pre-implantation embryos in vitro, amphipathic vehicles are commonly used to dissolve the lipophilic substances into culture media. However, easy emulsification of these nutrients would facilitate medium preparation. We report here a simple method for emulsification of lipophilic nutrients that affect bovine pre-implantation embryonic development in vitro. We investigated the effects of emulsified oleic acid (OA) or a mixture of antioxidative vitamins – vitamin E (VE) and β-carotene (BC). Polyglyceryl-10 laurate (P10L) was used as an emulsifier and was dissolved in sterile water at 5.05% (wt/wt) in glass vials. One percent (wt/wt) of OA or a mixture of VE (α-tocopherol) and BC (VE : BC = 1000 : 1 wt/wt) was added into the vial and mixed by using a magnetic stirrer. After first exhibiting white turbidity, the solution became transparent and stabilised, indicating stable emulsification. The emulsified OA and VE+BC were designated as emOA and emVEBC, respectively. Cumulus-enclosed oocytes obtained from abattoir bovine ovaries were in vitro-matured (IVM) for 22 h in modified synthetic oviduct fluid (mSOF) supplemented with 10% (vol/vol) fetal calf serum and 0.2 IU mL^–1 FSH. After IVM, the oocytes were subjected to IVF with Percoll gradient-selected sperm from a single bull in an mSOF-based medium for 20 h. After IVF, presumptive zygotes were freed from the cumulus cells and cultured in mSOF. On Day 3 (IVF = Day 0), embryos that had developed to the 8-cell stage or more (≥8-cell) were subsequently cultured in medium supplemented with 0.05% (vol/vol) of emOA or emVEBC. Blastocyst development from ≥8-cell embryos was assessed on Day 8. In the case of no-additive control and emVEBC, the hatching rate was also assessed on Day 10. All the cultures were performed at 38.5°C under 5% CO₂, 5% O₂, and 90% N₂ and replicated 4 times with ~18 embryos per group per replicate. The development data were statistically analysed by the general linear model. The blastocyst rate in the emOA group (36.4%) was significantly (P < 0.05) lower than that in the no-additive control (54.1%). The blastocyst rate in the emVEBC group (53.9%) was similar to that in the control; however, the hatching rate was significantly higher in the emVEBC group (22.6%) than in the control (9.2%). These data suggest that emulsification of lipophilic nutrients with P10L is an easy method to allow their addition into culture media for investigating their favourable (e.g. antioxidative vitamins) or inhibitory (e.g. OA) effects on pre-implantation development in vitro.