de Oliveira Rodrigo Arruda, Scarlet Dragos, Ille Natascha, Aurich Christine
Centre for Artificial Insemination and Embryo Transfer, Vetmeduni, Vienna, Austria.
Obstetrics, Gynaecology, and Andrology, Vetmeduni, Vienna, Austria.
Theriogenology. 2017 Feb;89:289-294. doi: 10.1016/j.theriogenology.2016.11.009. Epub 2016 Nov 14.
A decrease in fertility of equine semen during cooled-storage so far has mainly been attributed to changes in sperm membrane function. In the present study we hypothesized that cooled-storage also changes the sperm DNA methylation level. For this purpose, semen was collected from 10 fertile stallions and processed for cooled-storage at 5 °C. Two final concentrations, 50 × 10 and 100 × 10 cells/mL, were used. Semen was analyzed for total motility, progressive motility, membrane integrity, phosphatidylserine translocation (PST), mitochondrial membrane potential and chromatin condensation, immediately after processing and at 24 h-intervals until 72 h of storage. DNA cytosine methylation was assessed by ELISA after DNA extraction and denaturation. DNA methylation did neither change over time nor was affected by semen concentration. Total motility, progressive motility, membrane integrity, PST, mitochondrial membrane potential and chromatin condensation changed over storage time, but no differences between semen concentrations could be demonstrated. The results demonstrate that cooled-storage of equine semen does not induce changes in sperm DNA cytosine methylation. In cooled-semen of good quality, a concentration of 100 × 10 sperm/mL does not affect semen longevity. It can be concluded that a better fertility of cooled-stored than cryopreserved stallion semen is at least in part a result of only minor influences of cooled-storage on DNA integrity and methylation.
到目前为止，马精液在冷藏过程中生育力的下降主要归因于精子膜功能的变化。在本研究中，我们假设冷藏也会改变精子DNA甲基化水平。为此，从10匹生育能力正常的种马采集精液，并在5℃下进行冷藏处理。使用了两种终浓度，即50×10⁶和100×10⁶个细胞/mL。在处理后以及在冷藏72小时内每隔24小时对精液进行总活力、前向运动力、膜完整性、磷脂酰丝氨酸易位（PST）、线粒体膜电位和染色质凝聚分析。DNA提取和变性后，通过ELISA评估DNA胞嘧啶甲基化。DNA甲基化既不随时间变化，也不受精液浓度影响。总活力、前向运动力、膜完整性、PST、线粒体膜电位和染色质凝聚随冷藏时间变化，但精液浓度之间未显示出差异。结果表明，马精液的冷藏不会诱导精子DNA胞嘧啶甲基化的变化。在优质冷藏精液中，100×10⁶个精子/mL的浓度不会影响精液寿命。可以得出结论，冷藏精液比冷冻保存的种马精液具有更好的生育力，至少部分原因是冷藏对DNA完整性和甲基化的影响较小。
