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Abstract：
Cryopreservation results in the destabilization of the sperm plasma membrane, leading to negative side effects such as premature cryocapacitation, apoptosis and the low mitochondrial activity of bovine spermatozoa. Low-density lipoproteins (LDL) and trehalose have been used in seminal freezing to protect the integrity and stability of sperm membranes. Likewise, trehalose can increase the mitochondrial activity of sperm. The objective of this study was to evaluate the membrane stability and mitochondrial activity of bovine sperm after being frozen and treated with LDL sources and trehalose. Ten ejaculates from five bulls were cryopreserved under the treatments, CEY: chicken egg yolk (20% v/v); CCEY: centrifuged CEY (20% v/v); LDL: LDL (8% v/v); T: trehalose (100 mM); and TLDL: T (100 mM) plus LDL (8% v/v). After thawing, membrane stability and mitochondrial membrane potential (ΔΨM) were assessed by flow cytometry through the M-540/Yopro-1 and DiOC6/PI probes. The structural membrane integrity (SMI) was evaluated by fluorescence microscopy using SYBR14/PI dyes. A generalized linear model was adjusted, and the means were compared using the Tukey test. Centrifuged chicken egg yolk and LDL had a higher proportion of non-cryocapacitated non-apoptotic sperm (M-Y-), while CEY and T had the largest populations of cryocapacitated non-apoptotic sperm (M+Y-) and cryocapacitated apoptotic sperm (M+Y+). Centrifuged chicken egg yolk also showed a higher proportion of sperm with high-ΔΨM. Treatments that included egg yolk or purified LDL had a positive effect on SMI. Centrifuged chicken egg yolk has a superior cryoprotective effect on membrane stability and mitochondrial activity of bovine semen over the conventional use of CEY or the individual or simultaneous use of LDL and trehalose.
摘要：
冷冻保存导致精子质膜不稳定，导致不良副作用，如过早冷冻，细胞凋亡和牛精子的低线粒体活性。低密度脂蛋白（LDL）和海藻糖已用于精液冷冻，以保护精子膜的完整性和稳定性。同样，海藻糖可以增加精子的线粒体活性。这项研究的目的是评估冷冻并用LDL来源和海藻糖处理后牛精子的膜稳定性和线粒体活性。在处理下，将五头公牛的十只射精冷冻保存，CEY:鸡蛋黄(20%v/v);CCEY:离心CEY(20%v/v);LDL:LDL(8%v/v);T:海藻糖(100mM);和TLDL:T(100mM)加LDL(8%v/v)。解冻后,通过M-540/Yopro-1和DiOC6/PI探针通过流式细胞术评估膜稳定性和线粒体膜电位(ΔkW)。使用SYBR14/PI染料通过荧光显微镜评估结构膜完整性(SMI)。对广义线性模型进行了调整，并使用Tukey检验比较平均值。离心鸡蛋黄和低密度脂蛋白的非冷冻非凋亡精子（M-Y-）比例较高，而CEY和T的冷冻凋亡非凋亡精子（MY-）和冷冻凋亡精子（MY）的种群最大。离心的鸡蛋蛋黄也显示出较高的精子比例。包括蛋黄或纯化的LDL的处理对SMI具有积极作用。与常规使用CEY或单独使用LDL和海藻糖相比，离心鸡蛋黄对牛精液的膜稳定性和线粒体活性具有优越的冷冻保护作用。
