211 EFFECT OF AMNIOTIC FLUID DERIVED STEM CELL-CONDITIONED MEDIUM ON DEVELOPMENT AND POST-THAW SURVIVAL OF IN VITRO-PRODUCED HOLSTEIN EMBRYOS

Abstract

In vitro-produced (IVP) embryos have altered metabolism with lower blastocyst rates and higher lipid accumulation compared with IVP embryos. Culturing embryos with amniotic fluid derived stem cell-conditioned media (AFS) may help mimic in vivo conditions and improve embryo development. We hypothesised that culturing embryos with AFS will improve in vitro culture conditions and developmental competence. The IVP embryos were produced in 5 replicates by aspirating oocytes (n = 1469) of 2 to 8 mm from abattoir ovaries. Oocytes were matured for 23 h, fertilized with semen from 1 of 2 bulls, and cultured in a novel SOF medium (SOF for conventional freezing media) in 38.5°C in 5% O_2, 5% CO₂, and 90% N₂. Media was supplemented with either 0 (control), 5, or 10% AFS. Stage 7 blastocysts were stained with 1 µg mL⁻¹ of Nile Red and 300 nM Mitotracker Red CMX-Rosamine (Molecular Probes Inc., Eugene, OR, USA) to measure lipid content and mitochondrial polarity, respectively. Ten images per stained embryo were acquired by confocal microscopy using a 5 µM step size at 40× magnification. Remaining blastocysts were slow frozen using 2 min of dehydration in 0.6 M sucrose before equilibration for 10 min in a conventional freezing medium (1.5 M ethylene glycol and 0.5 M sucrose). Embryos were thawed and re-expansion was assessed at 24 and 48 h. Data were analysed by general linear model using a one-way ANOVA with means separated by Tukey’s HSD. Results (Table 1) indicated no difference in development, lipid content, mitochondrial polarity, or post-thaw survival (P > 0.01), suggesting that growth factors present in AFS did not improve culture conditions.

Table 1.  Main effect of AFS on embryo developmental competence and post-thaw survival rates