In vitro-produced (IVP) embryos experience poor cryotolerance due to metabolic changes during in vitro culture causing increased lipid accumulation and apoptosis post-thaw. We hypothesised that embryos cultured in a novel SOF for conventional freezing media (SCF1), dehydrated, and allowed longer equilibration before conventional slow freezing would increase post-thaw survival and decrease apoptosis. IVP embryos were produced in 9 replicates by oocytes (n = 3172) aspirated from abattoir ovaries, matured for 23 h, fertilized with semen from 1 of 4 bulls, and cultured in conventional SOF media or SCF1 in 38.5°C in 5% O₂, 5% CO₂, and 90% N₂. Stage 7 blastocysts were stained with 1 µg/mL Nile Red for lipid content and 300 nm Mitotracker Red CMX-Rosamine for mitochondrial polarity. Remaining blastocysts were slow-frozen by 1 of 4 protocols: 2-min dehydration in 0 or 0.6 m sucrose in holding media before equilibration (10 or 20 min) in conventional freezing media (1.5 m ethylene glycol and 0.5 m sucrose in holding media). Embryos were thawed and assessed for re-expansion at 48 h and surviving embryos were stained with 4’6-diamidino-2-phenylindole (DAPI) and a TUNEL assay to determine apoptosis. Ten images per embryo were acquired by confocal microscopy using a 5-µm step size at 40× magnification. Fluorescence of Nile Red and Mitotracker was measured by IMAGE PRO software, 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 one-way ANOVA and means separated by Tukey’s HSD. Post-thaw survival and apoptotic levels (Table 1) were analysed as a factorial 2 (SOF and SCF1) × 2 (0 and 0.6 m sucrose) × 2 (10 and 20 min), and means separated by Tukey’s HSD. No interactions occurred between factors so they were dropped from the model and only main effects are shown. Results indicate that SCF1 increased blastocyst rate, mitochondrial polarity, and post-thaw survival and decreased lipid content and post-thaw apoptosis (P < 0.01). A 20-min equilibration time decreased apoptosis (P < 0.01) and tended to increase post-thaw survival (P < 0.1), suggesting that cryotolerance is improved in embryos cultured in SCF1 and equilibrated for 20 min. Table 1. Effect of media on development, lipid content and mitochondrial polarity (top) and of media, equilibration and dehydration on post-thaw survival and apoptosis (bottom)

Medium	Oocytes	Cleavage %	Blastocyst %	Nile Red AFU	Mitochondria AFU
SOF	1391	88.2 ± 1.4	22.8 ± 1.6a	395.5 ± 14.1a	2845.6 ± 126a
SCF1	1781	86.9 ± 1.4	35.0 ± 1.6b	346.0 ± 14.1b	4011.7 ± 126b
Medium	Survival %		Apoptotic cells %
SOF	49.6 ± 3.5A		31.6 ± 2.3A
SCF1	82.3 ± 3.5B		20.4 ± 1.7B
Equilibration time
10 min	62.1 ± 3.5C		30.6 ± 2.0A
20 min	69.8 ± 3.5D		21.4 ± 2.0B
Dehydration
Sucrose	66.6 ± 3.5		24.9 ± 2.0
No sucrose	65.4 ± 3.5		27.1 ± 2.0

Values with different superscript in same column differ (^a,bP < 0.01). Values with different superscript in same row differ (^A,BP < 0.01, ^C,DP < 0.1).