Abstract #213

# 213
A. R. Higginbotham*1, C. M. Owen1, M. Barceló-Fimbres2, L. F. Campos-Chillon1, 1Department of Animal Sciences, California Polytechnic State University, San Luis Obispo, CA, USA;, 2AniCell Biotech LLC, Chandler, AZ, USA.

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 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 mml-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 mml-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)
MediumOocytesCleavage %Blastocyst %Nile Red AFUMitochondria AFU
 SOF46477.0 ± 1.2c18.9 ± 1.4a391.7 ± 23.6a5190 ± 375
 SCF151181.3 ± 1.2d30.6 ± 1.4b274.6 ± 21.8b5674 ± 317
MediumSurvival %Apoptotic cells %
 SOF42.7 ± 5.4A22.1 ± 5.3
 SCF178.3 ± 5.4B22.1 ± 4.5
Freezing additive
 L-AA69.2 ± 5.4C26.3 ± 5.3
 No L-AA51.8 ± 5.4D17.9 ± 4.6
Values with different lowercase superscripts in same column differ (a,bP < 0.01, c,dP < 0.1). Values with different uppercase superscripts in same row differ (A,BP < 0.01, C,DP < 0.05).