Pettitt M J, Buhr M M
Department of Animal and Poultry Science, University of Guelph, Ontario, Canada.
J Androl. 1998 Nov-Dec;19(6):736-46.
To determine how the individual components of extenders affected boar sperm function and membrane structure and to test a new surfactant's cryoprotective ability, boar sperm were cryopreserved in straws in BF5 extender plus or minus egg yolk plus or minus glycerol plus or minus a surfactant (Orvus ES Paste [OEP] or various concentrations of Pluronic F-127). After thawing, sperm function and fluidity of the isolated head plasma membrane (HPM) were determined. Total motility and adenosine triphosphate content (a measure of viability) were superior postthaw in sperm extended in egg yolk plus glycerol (P < 0.05); neither surfactant improved function. Egg yolk plus any other ingredients improved normal acrosome morphology, whereas a combined measure of motility and normal acrosome morphology was better in the presence of 0.33% OEP or 0.1% Pluronic F-127 (P < 0.05 vs. controls). Head plasma membrane was isolated from freshly collected spermatozoa and spermatozoa cryopreserved in the various extenders. Membrane fluidity was monitored with the probes cis-parinaric acid (cPNA), transparinaric acid (tPNA), and 1,6-diphenyl-1 ,3,5-hexatriene (DPH). The cPNA and the DPH monitor the fluidity of gel and liquid-crystalline areas of the membrane, whereas the tPNA preferentially monitors the gel-phase domains of the membrane. Additionally, DPH monitors the hydrophobic core of the bilayer. In the HPM from fresh sperm, the fluidity of each domain changed over time in a manner unique to that domain, and the behavior of the DPH domain varied among boars. The fluidity dynamics of each domain responded uniquely to cryopreservation. The cPNA domain was unaffected, the tPNA domain was altered by four of the eight extenders, and all extenders affected the fluidity of the DPH domain. Membrane structure was significantly correlated with cell function for sperm cryopreserved in extenders that preserved viability and motility. Sperm cryopreserved in egg yolk plus glycerol plus either OEP or 0.1% Pluronic F-127 functioned best when the bulk domains were less fluid initially and the gel domain solidified more slowly. Therefore, the behavior of domains in the HPM of boar spermatozoa is affected by cryopreservation and is related to the postthaw function of boar sperm cryopreserved in different extenders.
为了确定稀释液的各个成分如何影响公猪精子功能和膜结构，并测试一种新型表面活性剂的冷冻保护能力，将公猪精子置于含有或不含蛋黄、甘油以及含有或不含一种表面活性剂（奥尔沃斯ES糊剂[OEP]或不同浓度的普朗尼克F - 127）的BF5稀释液中，在细管中进行冷冻保存。解冻后，测定精子功能以及分离出的头部质膜（HPM）的流动性。在添加蛋黄和甘油的稀释液中冷冻保存的精子解冻后的总活力和三磷酸腺苷含量（活力指标）更高（P < 0.05）；两种表面活性剂均未改善精子功能。蛋黄加任何其他成分可改善顶体正常形态，而在添加0.33% OEP或0.1%普朗尼克F - 127时，活力和顶体正常形态的综合指标更好（与对照组相比，P < 0.05）。从新鲜采集的精子以及在各种稀释液中冷冻保存的精子中分离出头部质膜。使用顺式-副视黄酸（cPNA）、视黄酸（tPNA）和1,6 - 二苯基-1,3,5 - 己三烯（DPH）探针监测膜流动性。cPNA和DPH监测膜的凝胶态和液晶态区域的流动性，而tPNA优先监测膜的凝胶相区域。此外，DPH监测双层膜的疏水核心。在新鲜精子的HPM中，每个区域的流动性随时间变化，其变化方式因区域而异，并且DPH区域的行为在不同公猪之间也有所不同。每个区域的流动性动力学对冷冻保存的反应独特。cPNA区域未受影响，tPNA区域在八种稀释液中的四种中发生改变，并且所有稀释液均影响DPH区域的流动性。对于在能保持活力和运动性的稀释液中冷冻保存的精子，膜结构与细胞功能显著相关。当总体区域最初流动性较小且凝胶区域固化较慢时，在添加蛋黄、甘油以及OEP或0.1%普朗尼克F - 127的稀释液中冷冻保存的精子功能最佳。因此，公猪精子HPM中各区域的行为受冷冻保存影响，并且与在不同稀释液中冷冻保存的公猪精子解冻后的功能相关。
