Tsai Ming-Yang, Hung Hsi-Wen, Chen Yu-Hsin, Chen Pin-Chun, Liao Chih-Chiu, Xu Hui-Yi, Lin Hsiu-Lien Herbie
Livestock Management Division, TLRI, MOA, 71246 Tainan, Taiwan.
Animal Nutrition Division, TLRI, MOA, 71246 Tainan, Taiwan.
Poult Sci. 2025 Sep 11;104(11):105840. doi: 10.1016/j.psj.2025.105840.
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 Nevertheless, inconsistent fertility outcomes at this concentration have been documented in both published and unpublished studies, indicating that additional factors may influence the results.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.
甘油是最早被确定用于保存鸡精子的冷冻保护剂，至今仍被广泛用于减轻冷冻过程中的冷冻损伤。然而，尽管甘油具有保护作用，但已知它会对人工授精后母鸡的生育能力产生不利影响。早在20世纪50年代，研究人员就报告说，甘油浓度超过2%时生育能力会下降，此后这一浓度被确定为家禽繁殖的安全上限。然而，已发表和未发表的研究都记录了在此浓度下生育结果不一致的情况，这表明可能有其他因素影响结果。我们推测，受精子浓度和授精量影响，输送到母鸡输卵管中的甘油总量也可能影响生育能力。为了对此进行研究，我们用含有恒定2%甘油浓度但精子浓度不同（分别为2000、1500和1000×10⁶个细胞/毫升）的精液对母鸡进行授精，每次授精分别输送1、1.34和2微升甘油，每剂含有100×10⁶个精子细胞。随着甘油量的增加，受精率逐渐下降。观察到1微升甘油时受精率降低了45.7%，1.34微升时又额外降低了10.2%。2微升时未观察到进一步下降。体外分析表明，在41℃孵育60分钟后，2%的甘油会显著损害精子活力，而膜和线粒体完整性未受影响。在4℃精液制备过程中未观察到不良影响。我们的研究结果共同挑战了对甘油浓度的唯一强调，并表明授精过程中引入的甘油总量会显著影响生育能力。这项研究首次揭示，甘油浓度和输送到雌性生殖道中的量都会影响受精结果。这些见解促使人们重新审视冷冻保护剂的使用，并为改进家禽人工授精方案提供指导。
