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Glycerol was the first cryoprotectant identified for preserving chicken sperm and remains widely used to mitigate cryoinjury during freezing. However, despite its protective properties, glycerol is known to adversely affect hen's fertility following artificial insemination. As early as the 1950s, researchers reported a decrease in fertility at glycerol concentrations exceeding 2 %, which has since been established as the upper safe limit in poultry reproduction. Nevertheless, inconsistent fertility outcomes at this concentration have been documented in both published and unpublished studies, indicating that additional factors may influence the results. We hypothesized that the total volume of glycerol delivered into the hen's oviduct, influenced by sperm concentration and insemination volume, may also impact fertility. To investigate this, we inseminated hens with semen containing a constant 2 % glycerol concentration, but with different sperm concentrations at 2,000, 1,500, and 1,000 × 10⁶ cells/mL, resulting in the delivery of 1, 1.34, and 2 μL of glycerol per insemination, respectively, with each dose containing 100 × 10⁶ sperm cells. Fertility rates progressively declined as the volume of glycerol increased. A 45.7 % reduction in fertility was observed with 1 μL of glycerol, followed by an additional 10.2 % decrease at 1.34 μL. No further decline was noted with 2 μL. In vitro analyses showed that 2 % glycerol significantly impaired sperm motility after 60 min of incubation at 41 °C, while membrane and mitochondrial integrity remained unaffected. No adverse effects were observed during semen preparation at 4 °C. Our findings collectively challenge the exclusive emphasis on glycerol concentration and demonstrate that the total volume of glycerol introduced during insemination significantly impacts fertility. This study reveals for the first time that both glycerol concentration and the volume delivered into the female reproductive tract influence fertilization outcomes. These insights prompt a reexamination of cryoprotectant usage and offer guidance for improving artificial insemination protocols in poultry.