Gilmore J A, Du J, Tao J, Peter A T, Critser J K
Cryobiology Research Institute, Methodist Hospital of Indiana, Inc., Indianapolis 46202, USA.
J Reprod Fertil. 1996 May;107(1):87-95. doi: 10.1530/jrf.0.1070087.
A series of six experiments was conducted to determine the fundamental cryobiological properties of boar spermatozoa to develop optimal approaches for cryopreserving this important cell type. In the first experiments, boar spermatozoa samples were diluted in various osmolalities of experimental solutions (185-900 mOsmol kg-1) to provide hypo-, iso-, and hyperosmotic conditions. Equilibrium cell volumes (Expts 1 and 2) were measured after exposure for 3 min and the change in cell volume was measured over time using an electronic particle counter (Expt 3). The isosmotic cell volume was found to be 26.3 +/- 0.39 microns 3 (mean +/- SEM; n = 5). Over this range of osmolalities, boar spermatozoa behaved as linear osmometers (a linear volume versus 1/osm plot, r2 = 0.99) with an osmotically inactive cell fraction of 67.4 +/- 4.5%. The rate of water permeability (Lp) was determined to be 1.03 +/- 0.05 microns min-1 atm-1, which was consistent within and among donors (P > 0.130). A second series of experiments was performed to determine the effect of temperature and osmolality on boar sperm motility (Expt 4), and the effect of osmolality on the integrity of the sperm plasma membrane and its temperature dependence. Plasma membrane integrity was measured before and after boar spermatozoa were returned to an isosmolality (Expt 6). Motility was not affected at 30 degrees C, relative to that at room temperature, but was significantly decreased (P < 0.05) at 8 degrees C and 0 degree C (yielding a relative reduction to 85% and 35% of original motility, respectively; n = 6). Sperm motility was not significantly decreased (P > 0.05) until the osmolality reached 210 mOsmol kg-1, at which time motility began to decrease from 95% to 10% of the original value at 90 mOsmol kg-1. The integrity of the plasma membrane of boar spermatozoa was found to be dependent on temperature, donor and osmolality, decreasing significantly (P < 0.05) below room temperature, and below 185 mOsmol kg-1 (P < 0.05). There was no significant difference (P > 0.10) in the integrity of the plasma membrane of the samples before and after returning to 290 mOsmol kg-1, indicating that osmotic damage occurs during the initial change from isosmotic to hyposmotic media. These osmotic characteristics could be used to determine optimal conditions for cryopreservation of boar spermatozoa.
进行了一系列六个实验，以确定公猪精子的基本低温生物学特性，从而开发出冷冻保存这种重要细胞类型的最佳方法。在第一个实验中，将公猪精子样本稀释于各种渗透压的实验溶液（185 - 900 mOsmol kg⁻¹）中，以提供低渗、等渗和高渗条件。暴露3分钟后测量平衡细胞体积（实验1和2），并使用电子粒子计数器随时间测量细胞体积变化（实验3）。发现等渗细胞体积为26.3 ± 0.39立方微米（平均值 ± 标准误；n = 5）。在这个渗透压范围内，公猪精子表现为线性渗透计（体积与1/渗透压的线性图，r² = 0.99），渗透压不活跃细胞比例为67.4 ± 4.5%。水渗透率（Lp）测定为1.03 ± 0.05微米 分钟⁻¹ 大气压⁻¹，在不同供体之间保持一致（P > 0.130）。进行了第二系列实验，以确定温度和渗透压对公猪精子活力的影响（实验4），以及渗透压对精子质膜完整性及其温度依赖性的影响。在公猪精子恢复到等渗状态前后测量质膜完整性（实验6）。相对于室温，30℃时活力不受影响，但在8℃和0℃时显著降低（P < 0.05）（分别降至原始活力的85%和35%；n = 6）。直到渗透压达到210 mOsmol kg⁻¹时精子活力才显著降低（P > 0.05），此时活力开始从95%降至90 mOsmol kg⁻¹时原始值的10%。发现公猪精子质膜的完整性取决于温度、供体和渗透压，在室温以下以及低于185 mOsmol kg⁻¹时显著降低（P < 0.05）。回到290 mOsmol kg⁻¹后，样本质膜完整性前后无显著差异（P > 0.10），表明在从等渗介质初始转变为低渗介质期间发生了渗透损伤。这些渗透特性可用于确定公猪精子冷冻保存的最佳条件。
