Correa J R, Heersche G, Zavos P M
Department of Animal Sciences, University of Kentucky, Lexington, KY 40546, USA.
Theriogenology. 1997 Feb;47(3):715-21. doi: 10.1016/s0093-691x(97)00029-0.
The objective of this study was to assess the sperm membrane integrity and permeability of frozen-thawed bovine spermatozoa, processed at varying temperatures during and after thawing, by exposing the spermatozoa to standardized hypoosmotic conditions. The hypoosmotic swelling (HOS) test was employed to measure changes in sperm membrane functional status and permeability. Frozen specimens (from 5 bulls) were thawed at 37h degrees C for 10 sec and transferred to a water bath at 37 (Aliquot 1), 21 (Aliquot 2) or 5 degrees C (Aliquot 3) to complete thawing (1 to 2 min). The specimens were maintained and processed at these temperatures for additional 5 to 10 min. Specimens were slowly diluted 1:1 (v/v) and washed with Ham's F-10 media containing 3% (w/v) BSA. The HOS test was performed by adding 0.1 ml of the sperm specimen to 1.0 ml of a 100 mOsm/L HOS diluent. The following treatments were performed: 1) Aliquot 1 (control), specimens were incubated in HOS solutions at 37 degrees C for 5 min; 2) Aliquot 2, specimens were incubated in HOS solutions at 21 or 37 degrees C for 5 min; and 3) Aliquot 3, specimens were incubated in HOS solutions at 5 or 37 degrees C for 5 min. Samples were obtained from the sperm specimen-HOS diluent mixtures at 1 min intervals (during the 5 min incubation period), fixed and assessed for sperm swelling patterns. The sperm response to the HOS test for specimens processed at temperatures below 37 degrees C was higher when samples were incubated in HOS diluents at 37 degrees C. This finding indicates that the potential for sperm swelling (measurement of sperm membrane functional status) can be maintained when spermatozoa are processed at temperatures below 37 degrees C. The highest response to the HOS test was observed in spermatozoa processed at 21 degrees C and incubated in a HOS solution at 37 degrees C. The response to the HOS test was superior to the one observed in specimens maintained and processed at 37 degrees C throughout. Thawing of spermatozoa at 37 degrees C, followed by processing at 21 degrees C seems to reduce the negative effects associated with osmotic shock and results in the preservation of the sperm membrane functional status during the in vitro handling of frozen-thawed bovine spermatozoa.
本研究的目的是通过将牛精子置于标准化的低渗条件下，评估冻融后在解冻期间及解冻后不同温度下处理的牛精子的细胞膜完整性和通透性。采用低渗肿胀（HOS）试验来测量精子膜功能状态和通透性的变化。将冷冻标本（来自5头公牛）在37℃解冻10秒，然后转移至37℃（样品1）、21℃（样品2）或5℃（样品3）的水浴中完成解冻（1至2分钟）。标本在这些温度下保持并处理5至10分钟。标本缓慢按1:1（v/v）稀释，并用含3%（w/v）牛血清白蛋白的哈姆F-10培养基洗涤。通过将0.1 ml精子标本加入1.0 ml 100 mOsm/L的低渗肿胀稀释液中进行HOS试验。进行了以下处理：1）样品1（对照），标本在37℃的低渗肿胀溶液中孵育5分钟；2）样品2，标本在21℃或37℃的低渗肿胀溶液中孵育5分钟；3）样品3，标本在5℃或37℃的低渗肿胀溶液中孵育5分钟。在5分钟孵育期间，每隔1分钟从精子标本-低渗肿胀稀释液混合物中取样，固定并评估精子肿胀模式。当在37℃的低渗肿胀稀释液中孵育样品时，在低于37℃温度下处理的标本对HOS试验的精子反应更高。这一发现表明，当精子在低于37℃的温度下处理时，精子肿胀的可能性（精子膜功能状态的测量）可以维持。在21℃处理并在37℃的低渗肿胀溶液中孵育的精子中观察到对HOS试验的最高反应。对HOS试验的反应优于在整个过程中在37℃保持和处理的标本中观察到的反应。在37℃解冻精子，然后在21℃处理似乎可以减少与渗透休克相关的负面影响，并在冻融牛精子的体外处理过程中保持精子膜功能状态。
