Fraser L, Strzezek J
Department of Animal Biochemistry and Biotechnology, Faculty of Animal Bioengineering, Warmia and Mazury University, Olsztyn, Poland.
Folia Histochem Cytobiol. 2004;42(1):49-55.
The comet assay, under neutral conditions, allows the assessment of DNA integrity influenced by sperm ageing, which is manifested in DNA double-strand breaks. Here, we attempted to use a modified neutral comet assay test (single-cell gel electrophoresis), to our knowledge for the first time, to assess DNA integrity of boar spermatozoa during liquid storage for 96 h at 5 degrees C and 16 degrees C. In this comet assay protocol we used 2% beta-mercaptoethanol prior to the lysis procedure, to aid in removing nuclear proteins. Ejaculates from 3 boars (designated A, C and G) were diluted with a standard semen extender, Kortowo-3 (K-3), which was supplemented with lipoprotein fractions extracted from hen egg yolk (LPFh) or ostrich egg yolk (LPFo). Irrespective of the extender type, the percentage of comet-detected spermatozoa with damaged DNA increased gradually during prolonged storage at 5 degrees C and 16 degrees C. Spermatozoa stored in K-3 extender exhibited elevated levels of DNA damage at both storage temperatures. Significant differences in DNA damage among the boars were more pronounced during storage in LPF-based extenders at 5 degrees C: spermatozoa of boars A and G were less susceptible to DNA damage. The percent of tail DNA in comets was lower in LPF-based extenders, and there were individual variations among the boars. We observed that changes in DNA integrity were dependent on the extender type and storage temperature. A higher level of DNA instability was observed in K-3 extended semen compared with K-3/LPFh or K-3/LPFo extended semen during storage at 5 degrees C. No significant difference in the level of DNA damage between K-3/LPFh and K-3/LPFo was observed. It seems that a long-term storage can affect genomic integrity of boar spermatozoa. The modified neutral comet assay can be used to detect low levels of DNA damage in boar spermatozoa during liquid preservation. Therefore, screening for sperm DNA damage may be used as an additional test of sperm function that can have diagnostic value in practice.
中性条件下的彗星试验可用于评估受精子老化影响的DNA完整性，精子老化表现为DNA双链断裂。在此，我们首次尝试使用改良的中性彗星试验（单细胞凝胶电泳）来评估公猪精子在5℃和16℃下液体保存96小时期间的DNA完整性。在该彗星试验方案中，我们在裂解程序之前使用2%的β-巯基乙醇，以帮助去除核蛋白。从3头公猪（分别为A、C和G）采集的精液用标准精液稀释剂Kortowo-3（K-3）进行稀释，该稀释剂添加了从鸡卵黄（LPFh）或鸵鸟卵黄（LPFo）中提取的脂蛋白组分。无论稀释剂类型如何，在5℃和16℃长期保存期间，检测到DNA受损的彗星精子百分比逐渐增加。保存在K-3稀释剂中的精子在两个储存温度下均表现出较高水平的DNA损伤。在5℃下基于LPF的稀释剂中储存期间，公猪之间DNA损伤的显著差异更为明显：公猪A和G的精子对DNA损伤的敏感性较低。基于LPF的稀释剂中彗星尾巴DNA的百分比更低，并且公猪之间存在个体差异。我们观察到DNA完整性的变化取决于稀释剂类型和储存温度。在5℃储存期间，与K-3/LPFh或K-3/LPFo稀释的精液相比，K-3稀释的精液中观察到更高水平的DNA不稳定性。在K-3/LPFh和K-3/LPFo之间未观察到DNA损伤水平的显著差异。长期储存似乎会影响公猪精子的基因组完整性。改良的中性彗星试验可用于检测公猪精子液体保存期间低水平的DNA损伤。因此，筛选精子DNA损伤可作为精子功能的一项附加检测，在实践中可能具有诊断价值。
