Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, Milan, Italy.
Theriogenology. 2011 Jun;75(9):1613-22. doi: 10.1016/j.theriogenology.2011.01.001. Epub 2011 Mar 11.
Semen cryopreservation is fundamental both for the practice of artificial insemination, and for the conservation of genetic resources in cryobanks; nevertheless, there is still not an efficient standard freezing procedure assuring a steady and suitable level of fertility in fowl, and consequently there is no systematic use of frozen semen in the poultry industry. This study examined changes in motility (CASA), cell membrane integrity (Ethidium Bromide (EtBr) exclusion procedure and stress test) and DNA fragmentation (neutral comet assay) in fowl spermatozoa before, during and after cryopreservation and storage at -196 °C. An optimized comet assay for chicken semen was studied and applied to the analyses. Semen collected from 18 Mericanel della Brianza (local Italian breed) male chicken breeders was frozen in pellets and thawed in a water bath at 60 °C. Measurements were performed on fresh semen soon after dilution, after equilibration with 6% dimethylacetamide at 4 °C (processed semen) and after thawing. Sperm DNA damage occurred during cryopreservation of chicken semen and the proportion of spermatozoa with damaged DNA significantly increased from 6.2% in fresh and 6.4% in processed semen to 19.8% in frozen-thawed semen. The proportion of DNA in the comet tail of damaged spermatozoa was also significantly affected by cryopreservation, with an increase found from fresh (26.3%) to frozen-thawed (30.9%) sperm, whereas processed semen (30.1%) didn't show significant differences. The proportion of total membrane damaged spermatozoa (EtBr exclusion procedure) did not increase by 4 °C equilibration time, and greatly and significantly increased by cryopreservation; the values recorded in fresh, processed and frozen semen were 2.9, 5.6, and 66.7% respectively. As regards the proportion of damaged cells in the stress test, all values differed significantly (7.1% fresh semen, 11.7% processed semen, 63.7% frozen semen). Total motility was not affected by equilibration (52.1% fresh semen, 51.9% processed semen), whereas it decreased significantly after cryopreservation (19.8%). These results suggest a low sensitivity of frozen-thawed chicken spermatozoa to DNA fragmentation, therefore it should not be considered as a major cause of sperm injuries during cryopreservation.
精液冷冻保存对于人工授精的实践和低温库中遗传资源的保存都至关重要；然而，目前还没有一种有效的标准冷冻程序可以确保禽类的稳定和合适的生育能力，因此家禽业并没有系统地使用冷冻精液。本研究检查了在冷冻保存和-196°C 储存过程中，鸡精子的运动能力（CASA）、细胞膜完整性（溴化乙锭（EtBr）排除程序和应激测试）和 DNA 碎片化（中性彗星试验）的变化。研究并应用了一种优化的鸡精液彗星试验。从 18 只 Mericanel della Brianza（当地意大利品种）雄性种鸡中采集精液，制成精液冻块并在 60°C 的水浴中解冻。在稀释后立即、在 4°C 用 6%二甲基乙酰胺平衡后（处理精液）以及解冻后，对新鲜精液进行测量。鸡精液冷冻保存过程中发生精子 DNA 损伤，冷冻解冻后精子 DNA 损伤的比例从新鲜精液的 6.2%和处理精液的 6.4%显著增加到 19.8%。彗星尾部受损精子的 DNA 比例也受到冷冻保存的显著影响，发现从新鲜精子（26.3%）到冷冻解冻精子（30.9%）增加，而处理精液（30.1%）则没有显著差异。总膜受损精子的比例（EtBr 排除程序）在 4°C 平衡时间内没有增加，而冷冻保存时则大大增加；新鲜、处理和冷冻精液中的记录值分别为 2.9%、5.6%和 66.7%。关于应激试验中受损细胞的比例，所有值均有显著差异（新鲜精液 7.1%，处理精液 11.7%，冷冻精液 63.7%）。总活力不受平衡的影响（新鲜精液 52.1%，处理精液 51.9%），但冷冻保存后显著下降（19.8%）。这些结果表明，冷冻解冻鸡精子对 DNA 碎片化的敏感性较低，因此不应将其视为冷冻保存过程中精子损伤的主要原因。
