213 Effect of Novel Sof Medium and L-Ascorbic Acid During Cryopreservation of in Vitro-Produced Jersey Cattle Embryos

Abstract

Jersey embryos have high lipid content and poor cryotolerance. High lipid and reactive oxygen species concentrations are associated with poor post-thaw survival and increased post-thaw apoptosis. It was hypothesized that culturing embryos in SOF-based medium (SCF1; SOF for conventional freezing will decrease lipid content, and adding l-ascorbic acid (l-AA) to freezing media will increase cryotolerance and decrease post-thaw apoptosis. A 2 × 2 factorial design was used to compare SOF v. SCF1 and additives in freezing media (control v. L-AA). In vitro-produced blastocysts were produced in 5 replicates by aspirating oocytes (n = 975) from 2 to 8 mm follicles of abattoir ovaries, maturing for 23 h, fertilizing with semen from 1 of 2 bulls, and culturing in SOF medium or SCF1 in 38.5°C in 5% O2, 5% CO2, and 90% N2. Randomly selected Day 7 blastocysts were stained with 1 µg mL−1 Nile Red for lipid content and 300 nM Mitotracker Red CMX-Rosamine (Molecular Probes Inc., Eugene, OR, USA) for mitochondrial polarity. Remaining blastocysts were placed in 0.6 M sucrose in holding media for 2 min followed by equilibration in 1.5 M ethylene glycol and 0.5 M sucrose in holding media for 10 min with 0 or 0.1 mM l-AA. Blastocysts were thawed and assessed for re-expansion at 24 and 48 h, then stained with 4′,6-diamidino-2-phenylindole and a TUNEL assay to measure apoptosis. Ten images per stained blastocyst were acquired by confocal microscopy using a 5 µM step size at 40× magnification. Image Pro software was used to measure fluorescence of Nile Red and Mitotracker, and cells stained for TUNEL were analysed by a cell counter plug-in. Blastocyst rate, Nile Red, and Mitotracker data (Table 1) were analysed by 1-way ANOVA and means were separated by Tukey’s HSD. Post-thaw survival and apoptotic levels (Table 1) were analysed as a factorial 2 (SOF and SCF1) by 2 (0 and 0.1 mM l-AA) and means were separated by Tukey’s HSD. Results (Table 1) indicate SCF1 increased blastocyst rate and post-thaw survival and decreased lipid content (P < 0.01) with no effect on mitochondrial polarity. Post-thaw, l-AA (Table 1) increased survival (P < 0.05) but had no effect on apoptosis. The SCF1 medium increases development and lowers lipid content, whereas l-AA may lower reactive oxygen species to increase cryotolerance. Table 1. Effect of media on development, lipid content, and mitochondrial polarity (top part), and of media and l-AA on post-thaw survival and apoptosis (lower part)

Exogenous ascorbic acid enhances vitrification survival of porcine in vitro-developed blastocysts but fails to improve the in vitro embryo production outcomes

“…Supplementation of AsA to cryopreservation media of mouse and bovine embryos had also beneficial effects by reducing peroxidation and increasing cryotolerance [48,49].…”