Patrick Jennifer L, Elliott Gloria D, Comizzoli Pierre
Department of Mechanical Engineering and Engineering Science, University of North Carolina at Charlotte, Charlotte, NC, USA; Smithsonian Conservation Biology Institute, National Zoological Park, Washington DC, USA.
Department of Mechanical Engineering and Engineering Science, University of North Carolina at Charlotte, Charlotte, NC, USA.
Theriogenology. 2017 Nov;103:36-43. doi: 10.1016/j.theriogenology.2017.07.037. Epub 2017 Jul 26.
Characterizing the resilience of mammalian cells to non-physiological conditions is necessary to develop preservation and long-term storage strategies at low or ambient temperatures. Using the domestic cat model, the objective of the study was to characterize structural integrity (morphology and DNA damage) as well as functional properties (sperm aster formation and embryo formation after sperm injection) of spermatozoa after microwave-assisted drying to a moisture content compatible with storage in a glassy state at supra-zero temperatures. In Experiment 1, cat epididymal spermatozoa were porated with hemolysin and dried (using a commercial microwave oven set to 20% power) in the presence of trehalose for up to 50 min in a low humidity environment (11%) before measuring moisture content and sample temperature. In Experiment 2, morphology and DNA integrity were evaluated in sperm dried for up to 30 min (using the same method as above) versus fresh spermatozoa. In Experiment 3, the functionality of sperm dried for 30 min versus fresh sperm cells was evaluated after injection into oocytes based on sperm aster formation (5 h post-injection) and embryo development in vitro over 7 days. Moisture contents compatible with dry state storage were reached after 30 min of microwave-assisted drying. After rehydration, sperm morphology was not affected and the percentages of cells with damaged DNA (∼6.5%) was similar to the fresh controls. Sperm aster diameters appeared to be generally smaller for dried-rehydrated cells compared to the fresh controls. This observation was consistent with a lower proportion of blastocyst formation after injection with dried spermatozoa (6.5%) compared to fresh spermatozoa (15%). However, the blastocyst quality based on the total blastomere number was not affected by the sperm treatment. This is the first and encouraging report in any species so far demonstrating that spermatozoa can be dried using microwaves without causing irreversible damage to the cellular structure and function.
为了制定低温或环境温度下的保存和长期储存策略，有必要了解哺乳动物细胞对非生理条件的耐受性。本研究以家猫为模型，目的是表征微波辅助干燥至与零上温度下玻璃态储存相容的水分含量后精子的结构完整性（形态和DNA损伤）以及功能特性（精子星状体形成和精子注射后的胚胎形成）。在实验1中，将猫附睾精子用溶血素打孔，并在低湿度环境（11%）中于海藻糖存在的情况下（使用设置为20%功率的商用微波炉）干燥长达50分钟，然后测量水分含量和样品温度。在实验2中，将干燥长达30分钟（使用上述相同方法）的精子与新鲜精子的形态和DNA完整性进行评估。在实验3中，基于精子星状体形成（注射后5小时）和体外7天的胚胎发育，评估注射到卵母细胞中的干燥30分钟的精子与新鲜精子细胞的功能。微波辅助干燥30分钟后达到了与干燥状态储存相容的水分含量。复水后，精子形态未受影响，DNA受损细胞的百分比（约6.5%）与新鲜对照相似。与新鲜对照相比，干燥复水后的细胞精子星状体直径似乎普遍较小。这一观察结果与注射干燥精子后囊胚形成比例（6.5%）低于新鲜精子（15%）一致。然而，基于总卵裂球数的囊胚质量不受精子处理的影响。这是迄今为止在任何物种中的首个令人鼓舞的报告，表明精子可以用微波干燥而不会对细胞结构和功能造成不可逆转的损害。
