Department of Animal Reproduction and Veterinary Radiology, FMVZ, São Paulo State University, Botucatu, São Paulo, Brazil.
Theriogenology. 2013 Oct 15;80(7):730-7. doi: 10.1016/j.theriogenology.2013.06.010. Epub 2013 Aug 8.
Cryopreservation of spermatozoa is a pivotal tool in assisted reproduction, and studies aiming to establish optimal freezing/thawing protocols are essential to enhance sperm survival. The objectives of the present study were to (1) compare the cryoprotective efficiency of three different glycerol concentrations (3%, 5%, and 7%) on the basis of post-thaw sperm quality and (2) investigate whether the incidence of morphologically abnormal sperm in fresh samples is related to cryodamage sensitivity. Semen was collected from six tomcats using an artificial vagina (total 18 ejaculates). Each ejaculate was diluted using Tris-egg yolk-based extender (TEY), evaluated, equally divided into three aliquots, and rediluted using TEY with and without glycerol to achieve final concentrations of 3%, 5%, and 7%. Samples were loaded into 0.25 mL straws, equilibrated for 60 minutes at 5 °C, frozen, and then thawed at 46 °C for 12 seconds. Fresh and frozen-thawed samples were evaluated for sperm motion parameters (computer-assisted sperm analysis), plasma membrane integrity (PMI; propidium iodide and carboxyfluorescein diacetate), and DNA integrity (acridine orange). Plasma and acrosomal membrane integrity were assessed by flow cytometry (propidium iodide and fluorescein isothiocyanate-conjugated pea (Pisum sativum) agglutinin) immediately after thawing. Sperm motion parameters were also evaluated at 30 and 60 minutes of postincubation. For all treatment groups, cryopreservation significantly impaired the PMI and sperm motion parameters, except for straightness and amplitude of lateral head displacement. DNA integrity showed a slight reduction (P < 0.05) when 3% glycerol was used. The percentage of total motility, progressive motility, and rapid spermatozoa were significantly lower immediately after thawing and up to 60 minutes of incubation for the 3% glycerol group when compared with 5% and 7%. No difference (P > 0.05) was found for PMI, acrosome integrity, and DNA integrity among post-thaw groups. However, higher (P < 0.05) incidence of viable cells with reacted acrosome and dead cells with intact acrosome were observed with 7% and 3% glycerol, respectively. Percentage of morphologically abnormal spermatozoa in fresh sample was positively correlated with PMI only in the 3% glycerol group and negatively correlated with sperm motility in the 5% and 7% groups. In conclusion, the final concentration of 5% glycerol offered better cryoprotective effect for ejaculated cat sperm, and the relationship found between prefreezing sperm morphology and post-thaw sperm quality showed to be dependent on final glycerol concentration.
精子的冷冻保存是辅助生殖的重要工具，因此研究建立最佳的冷冻/解冻方案对于提高精子存活率至关重要。本研究的目的是：（1）比较三种不同甘油浓度（3%、5%和 7%）对解冻后精子质量的冷冻保护效率；（2）研究新鲜样本中形态异常精子的发生率是否与冷冻损伤敏感性有关。使用人工阴道从 6 只公猫中采集精液（共 18 次射精）。每次射精都用 Tris-卵黄基础稀释液（TEY）稀释，评估后将每份精液等分为三份，并用含甘油（终浓度分别为 3%、5%和 7%）的 TEY 重新稀释。样品装入 0.25 mL 细管中，在 5°C 下平衡 60 分钟，冷冻，然后在 46°C 下解冻 12 秒。新鲜和冷冻-解冻样品的精子运动参数（计算机辅助精子分析）、质膜完整性（PMI；碘化丙啶和羧基荧光素二乙酸酯）和 DNA 完整性（吖啶橙）进行评估。解冻后立即通过流式细胞术（碘化丙啶和豌豆凝集素（Pisum sativum）异硫氰酸荧光素结合）评估血浆和顶体膜的完整性。解冻后立即和孵育 30 和 60 分钟评估精子运动参数。对于所有处理组，冷冻保存均显著降低了 PMI 和精子运动参数，但直线性和侧向头部位移振幅除外。当使用 3%甘油时，DNA 完整性略有下降（P<0.05）。与 5%和 7%甘油组相比，3%甘油组的总活力、前向活力和快速精子百分比在解冻后立即和孵育 60 分钟时显著降低。解冻后组间的 PMI、顶体完整性和 DNA 完整性无差异（P>0.05）。然而，分别用 7%和 3%甘油处理时，反应性顶体的活细胞和完整顶体的死细胞的发生率较高（P<0.05）。新鲜样本中形态异常精子的百分比与 PMI 仅在 3%甘油组呈正相关，与 5%和 7%组的精子活力呈负相关。总之，终浓度为 5%的甘油对猫射出精液具有更好的冷冻保护效果，并且发现的冷冻前精子形态与解冻后精子质量之间的关系取决于终浓度的甘油。
