89 EFFECT OF MEDIA, METABOLIC REGULATORS, AND STAGE OF DEVELOPMENT ON LIPID CONTENT AND MITOCHONDRIAL POLARITY OF IN VITRO-PRODUCED HOLSTEIN EMBRYOS

Abstract

Current bovine embryo culture methods result in accumulation of lipids and reactive oxygen species, possibly due to sub-optimal metabolic regulation. These effects decrease the cryopreservation survival and implantation potential of in vitro-produced (IVP) embryos. Forskolin has been shown to decrease lipid accumulation, and vitamin K₂ (Vit K₂) is thought to decrease oxidative stress from in vitro conditions. The aims of this study were (1) to assess lipid content of embryos cultured with or without forskolin and Vit K₂ in both continuous and sequential SOF-based medium, and (2) to examine individual and combined effects of forskolin and Vit K₂ on mitochondrial polarity. For Experiment 1, a 2 × 2 × 2 factorial design was used to compare culture systems (continuous v. 3-step sequential), additives (no additive v. Vit K₂ (0.5 mM at Day 3) plus forskolin (10 µM at Day 5), and blastocyst stage [6 (early) v. 7 (late)] on overall lipid content. For Experiment 2, mitochondrial polarity of stage 7 blastocysts was analysed from the following groups: no additive, Vit K₂ (0.5 mM at Day 3), forskolin (10 µM at Day 5), and Vit K₂ plus forskolin. IVP embryos (n = 199, Experiment 1; n = 45, Experiment 2) were produced by standard procedures and cultured at 38.5°C in 5% O₂, 5% CO₂, and 90% N₂. For Experiment 1, embryos were stained with 1 μg mL^–1 Nile Red, and two images per embryo were taken along the equatorial plane at 40× magnification. For Experiment 2, embryos were stained with 300 nM MitoTracker Red CMX-Rosamine, and 10 images per embryo were acquired by confocal microscopy with a 5-μm step size at 40× magnification. For both experiments, fluorescence intensity (FI) of each image was measured by Image PRO software with embryo controlled for and background fluorescence corrected. Data (Table 1) were analysed by ANOVA and means were compared by Tukey’s HSD. In Experiment 1, embryos cultured with forskolin and Vit K₂ showed decreased lipid content in both the early and late stage (P < 0.05), with no effect from culture system (P > 0.05). In Experiment 2, forskolin and Vit K₂ individually increased mitochondrial polarity (P < 0.05), but had no combined effect (P > 0.05). In conclusion, these data suggest that while a combination of forskolin and Vit K₂ as media additives reduces lipid accumulation, the interaction between these metabolic regulators may negate their individual effects on mitochondrial polarity.

Table 1.  Fluorescence intensity of Nile Red and MitoTracker Red dyes between treatment groups