Repurposing advanced DMEM/F-12 cell culture medium to mitigate glycerol toxicity in poultry semen

1. Glycerol is the standard cryoprotectant for avian sperm, yet adding 2% reduces chicken fertility by half. Sperm co-cultured with sperm storage tubules in advanced DMEM/F-12 (adDF12) medium decreased glycerol damage. The following study tested whether adDF12 itself protects sperm from glycerol toxicity compared to conventional Lake PC (LPC) extender.2. Pooled rooster semen was diluted in either LPC or adDF12 with 2% glycerol. Fertility was assessed via artificial insemination (100 × 10⁶ sperm per hen, n = 10 per group). Sperm motility, membrane integrity and mitochondrial activity were evaluated at 4°C and 41°C using computer-assisted analysis and flow cytometry.3. Using adDF12 achieved 56.6% fertility versus 36.3% with LPC, a 55.9% improvement (p = 0.012). This protection operated through enhanced sperm motility at 41°C, total motility increased from 55.2% to 64.9% (p = 0.044) and progressive motility increased from 19.5% to 33.2%. No differences were seen at 4°C. Membrane integrity and mitochondrial activity remained unchanged across treatments.4. The selective motility enhancement seen without mitochondrial changes suggested that adDF12 supported glycolytic rather than oxidative metabolism. Using adDF12 provided glucose (17.5 mM), pyruvate (0.5 mM) and 21 amino acids, substrates largely absent in LPC, which likely enabled flexible ATP generation under glycerol stress.5. These findings established adDF12 as a potential post-thaw resuspension medium for cryopreserved avian semen, directly supporting genetic conservation programmes.