Marco-Jiménez F, Viudes-de-Castro M P, Balasch S, Mocé E, Silvestre M A, Gomez E A, Vicente J S
Centro de Investigación y Tecnología Animal (CITA), Instituto Valenciano de Investigaciones Agrarias, Segorbe, Spain.
Cryobiology. 2006 Apr;52(2):295-304. doi: 10.1016/j.cryobiol.2006.01.002. Epub 2006 Feb 24.
Two experiments were designed to evaluate the effect of cryopreservation on morphometric characteristics of the goat sperm head. To address this question, we evaluated the size of the sperm head in fresh control cells, post-cooling cells after equilibration with the glycerol preservation solution, and post-thawing cells. Assessment was by automated morphometric sperm head analysis (ASMA) using phase-contrast microscopy without staining. In the first experiment, ASMA was performed on heterospermic pooled samples (fresh, post-cooling after equilibration with the glycerol preservation solution and post-thawing): length, width, area and perimeter were measured. In the second experiment, sperm viability was assessed by Hoechst staining and head morphometry was carried out as before, simultaneously during the cryopreservation process, and the head size was identified for both live and dead spermatozoa. The data were analysed by principal component analysis (PCA). The purpose of PCA is to derive a small number of linear combinations (principal components) from a set of variables (length, width, area and perimeter), that retain as much of the information in the original variables as possible. The main findings that have emerged from this study are that (i) a simple procedure has been developed for measuring spermatozoa heads without staining, which minimises the possibility that sperm head dimensions were influenced by procedural artefacts; (ii) the dimensions of goat sperm heads after cryopreservation in skimmed milk-glucose medium were smaller than in fresh sperm, but this was due to the equilibration phase with the cryoprotectant and not to the cryopreservation process itself; and (iii) dead spermatozoa showed smaller heads than live sperm, consequent upon the loss of membrane function. No differences were observed between post-cooling cells after equilibration with the glycerol preservation solution and post-thawing spermatozoa and only minor osmotic differences between them and fresh sperm were observed.
设计了两个实验来评估冷冻保存对山羊精子头部形态特征的影响。为解决这个问题，我们评估了新鲜对照细胞、用甘油保存溶液平衡后的冷却后细胞以及解冻后细胞的精子头部大小。通过使用相差显微镜且不染色的自动形态计量精子头部分析（ASMA）进行评估。在第一个实验中，对混合精子样本（新鲜的、用甘油保存溶液平衡后的冷却后样本以及解冻后样本）进行ASMA：测量长度、宽度、面积和周长。在第二个实验中，通过Hoechst染色评估精子活力，并如之前那样在冷冻保存过程中同时进行头部形态测量，确定活精子和死精子的头部大小。数据通过主成分分析（PCA）进行分析。PCA的目的是从一组变量（长度、宽度、面积和周长）中得出少量线性组合（主成分），这些主成分尽可能多地保留原始变量中的信息。本研究得出的主要发现是：（i）已开发出一种简单的程序来测量未染色的精子头部，这将精子头部尺寸受操作假象影响的可能性降至最低；（ii）在脱脂牛奶 - 葡萄糖培养基中冷冻保存后，山羊精子头部尺寸小于新鲜精子，但这是由于与冷冻保护剂的平衡阶段，而非冷冻保存过程本身；（iii）由于膜功能丧失，死精子头部比活精子小。在用甘油保存溶液平衡后的冷却后细胞与解冻后精子之间未观察到差异，并且它们与新鲜精子之间仅观察到微小的渗透压差异。
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