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开发一种基于改性聚二甲基硅氧烷装置的微流控系统,用于选择牛附睾精子。

Development of a microfluidic system structured on a modified polydimethylsiloxane device for the selection of bovine epididymal spermatozoa.

机构信息

Laboratory of Reproductive Technologies and Innovations in Animal Models, Federal Rural University of Semi-Arid, Mossoró, RN, Brazil.

Laboratory of Animal Germplasm Conservation, Federal Rural University of Semi-Arid, Mossoró, RN, Brazil.

出版信息

Reprod Toxicol. 2022 Jun;110:1-8. doi: 10.1016/j.reprotox.2022.03.005. Epub 2022 Mar 16.

Abstract

Microfluidic systems are on the rise in several studies that evaluate reproductive cells. However, the material used for manufacturing can still be considered relatively expensive. The objective of this study was to develop a new microfluidic device, using a modified polydimethylsiloxane ((PDMS) Silpuran®), test its viability and carry out a selection of bovine sperm. Sperm was collected from epididymis (n = 10) and evaluated at different incubation times (60 min, 120 min, 180 min) to assess polydimethylsiloxane toxicity, where a tube was used as a control and the microfluidic device as treatment. An additional ten epididymis were used for the sperm selection test, which utilized four types of solutions: in vitro maturation medium (IVM) with and without oocyte, progesterone and saline solution (SS). The Percoll gradient was used as a control and the microfluidic device as treatment. The kinetic parameters of sperm were evaluated using the computer-assisted semen analysis (CASA). Morphology was performed with Bengal Rose, the integrity, and viability of the sperm using the hypoosmotic test and fluorescent microscopy probes, respectively. Mann-Whitney test was used in the first experiment, Kruskall-Wallis variance analysis tests with post hoc and Student-Newman-Keuls used in the second experiment. Regarding the non-toxic effects, most motility parameters demonstrated the superiority of the microfluidic device compared to the control. In the second experiment, the sperm showed equivalence between the microfluidic device and the Percoll gradient Silpuran® PDMS was not toxic to the cells and can be efficient for selecting bovine sperm, achieving better results in a medium for IVM with or without oocytes.

摘要

微流控系统在评估生殖细胞的多项研究中兴起。然而,用于制造的材料仍被认为相对昂贵。本研究的目的是开发一种新的微流控装置,使用改良的聚二甲基硅氧烷((PDMS) Silpuran®),测试其可行性并进行牛精子的选择。从附睾收集精子(n=10),并在不同孵育时间(60 min、120 min、180 min)下进行评估,以评估聚二甲基硅氧烷的毒性,其中将试管作为对照,微流控装置作为处理。另外 10 个附睾用于精子选择试验,使用四种溶液:有和没有卵母细胞的体外成熟培养基(IVM)、孕酮和生理盐水(SS)。Percoll 梯度用作对照,微流控装置作为处理。使用计算机辅助精液分析(CASA)评估精子的动力学参数。用孟加拉玫瑰进行形态学评估,用低渗试验和荧光显微镜探针分别评估精子的完整性和活力。在第一个实验中使用 Mann-Whitney 检验,在第二个实验中使用 Kruskall-Wallis 方差分析检验,并用事后和 Student-Newman-Keuls 检验。关于无毒作用,大多数运动参数表明微流控装置优于对照。在第二个实验中,精子在微流控装置和 Percoll 梯度之间表现出等效性。Silpuran® PDMS 对细胞没有毒性,可有效用于选择牛精子,在有或没有卵母细胞的 IVM 培养基中取得更好的效果。

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