USDA, Agricultural Research Service, Beltsville, MD 20705, USA.
Poult Sci. 2011 Feb;90(2):435-43. doi: 10.3382/ps.2010-00998.
The carbohydrate-rich zone on the sperm surface is essential for inmunoprotection in the female tract and early gamete interactions. We recently have shown the glycocalyx of chicken sperm to be extensively sialylated and to contain residues of mannose, glucose, galactose, fucose, N-acetyl-galactosamine, N-acetyl-glucosamine, and N-acetyl-lactosamine. Our objective here was to evaluate the effects of 3 different cryopreservation methods on the sperm glycocalyx. Semen from roosters was pooled, diluted, cooled to 5°C, and aliquoted for cryopreservation using 6% dimethylacetamide (DMA), 11% dimethylsulfoxide (DMSO), or 11% glycerol (GOH). For the DMA method, semen was equilibrated for 1 min with cryoprotectant and rapidly frozen by dropping 25-µL aliquots into liquid nitrogen. For the other methods, semen was equilibrated for either 1 min (DMSO) or 20 min (GOH), loaded into straws, and frozen with a programmable freezer. Thawing rates mimicked the freezing rates (e.g., rapid for DMA; moderate for DMSO and GOH). Aliquots of thawed and fresh, unfrozen semen were incubated with 1 of 12 fluorescein isothiocyanate-conjugated lectins and counterstained with propidium iodide, and mean fluorescence intensity (MFI) was assessed by flow cytometry. For each lectin, the MFI of propidium iodide-negative (viable sperm) was compared among the fresh and frozen-thawed treatments (n = 5). For sperm frozen with GOH and DMA, the MFI of most lectins was similar (P > 0.05) to that of fresh sperm, whereas only 5 of 12 lectins were similar between fresh and DMSO-frozen sperm. Sperm from all 3 methods had higher (P < 0.05) MFI for lectins specific for N-acetyl-glucosamine and β-galactose than did fresh sperm. Fewer sperm were damaged (P < 0.001) with GOH than with DMA or DMSO, and membrane integrity was correlated with MFI for 9 of 12 lectins (P < 0.05). These data indicate that surface carbohydrates are altered during cryopreservation, and that cryoprotectant type and freezing-thawing rates affect the degree of modification. Although the glycoconjugates have not yet been identified, it is likely that these cryopreservation-induced changes contribute to the reduced fertility of frozen-thawed chicken semen.
精子表面富含碳水化合物的区域对于女性生殖道中的免疫保护和早期配子相互作用至关重要。我们最近表明，鸡精子的糖萼广泛地被唾液酸化，并含有甘露糖、葡萄糖、半乳糖、岩藻糖、N-乙酰半乳糖胺、N-乙酰葡萄糖胺和 N-乙酰乳糖胺残基。我们的目的是评估 3 种不同的冷冻保存方法对精子糖萼的影响。将公鸡的精液混合、稀释、冷却至 5°C，然后等分用于冷冻保存，使用 6%二甲基乙酰胺 (DMA)、11%二甲基亚砜 (DMSO) 或 11%甘油 (GOH)。对于 DMA 方法，精液用冷冻保护剂平衡 1 分钟，然后通过将 25-µL 等分试样滴入液氮中快速冷冻。对于其他方法，精液用 DMSO 平衡 1 分钟或 GOH 平衡 20 分钟，装入 straws 中，用可编程冷冻机冷冻。解冻速率模拟冷冻速率（例如，DMA 快速；DMSO 和 GOH 中等）。解冻和新鲜未冷冻精液的等分试样与 12 种荧光素异硫氰酸酯结合的凝集素中的 1 种孵育，并与碘化丙啶复染，通过流式细胞术评估平均荧光强度 (MFI)。对于每种凝集素，将新鲜和冷冻解冻处理之间的碘化丙啶阴性（存活精子）的 MFI 进行比较（n = 5）。对于用 GOH 和 DMA 冷冻的精子，大多数凝集素的 MFI 与新鲜精子相似（P > 0.05），而只有 12 种凝集素中的 5 种在新鲜和 DMSO 冷冻的精子之间相似。与新鲜精子相比，来自所有 3 种方法的精子的 MFI 对 N-乙酰葡萄糖胺和 β-半乳糖特异性的凝集素更高（P < 0.05）。与 DMA 或 DMSO 相比，GOH 处理的精子损伤更少（P < 0.001），并且膜完整性与 12 种凝集素中的 9 种的 MFI 相关（P < 0.05）。这些数据表明，冷冻保存过程中表面碳水化合物发生了变化，并且冷冻保护剂类型和冷冻-解冻速率会影响修饰的程度。尽管尚未鉴定出糖缀合物，但很可能这些冷冻保存引起的变化导致冷冻-解冻鸡精液的生育力降低。
