Extracellular vesicles (EVs) regulate multiple physiological processes. Seminal plasma contains numerous EVs that may deliver functional molecules such as small RNAs (sRNAs) to the sperm. However, the RNA profiles in the boar seminal plasma extracellular vesicles (SP-EVs) and its function have not been characterized. The aim of this study was to characterize the functions and sRNA profiles in the boar SP-EVs using deep sequencing technology. Briefly, boar SP-EVs were isolated by differential ultracentrifugation and confirmed with a transmission electron microscope (TEM), nanoparticle tracking analysis (NTA), and Western blot. The isolated boar SP-EVs contained numerous and diverse sRNA families, including microRNAs (miRNAs, 9.45% of the total reads), PIWI-interacting RNAs (piRNAs, 15.25% of the total reads), messenger RNA fragments (mRNA, 25.30% of the total reads), and tRNA-derived small RNAs (tsRNA, 0.01% of the total reads). A total of 288 known miRNAs, 37 novel miRNA, and 19,749 piRNAs were identified in boar SP-EVs. The identified ssc-miR-21-5p may confer negative effects on sperm fertility based on a dual-luciferase reporter experiment. This study therefore provides an effective method to isolate SP-EVs and characterizes the sRNA profile.

细胞外囊泡（EVs）调节多种生理过程。精浆中含有大量的电动汽车，可以将诸如小RNA（sRNA）的功能分子传递至精子。然而，公猪精浆细胞外囊泡（SP-EVs）中的RNA谱及其功能尚未被表征。这项研究的目的是使用深度测序技术表征公猪SP-EV中的功能和sRNA谱。简而言之，通过差异超速离心分离公猪SP-EV，并通过透射电子显微镜（TEM），纳米颗粒跟踪分析（NTA）和Western blot进行确认。分离的公猪SP-EV包含许多不同的sRNA家族，包括microRNA（miRNA，占总读数的9.45％），与PIWI相互作用的RNA（piRNA，占总读数的15.25％），信使RNA片段（mRNA，25。总读数的30％）和tRNA衍生的小RNA（tsRNA，总读数的0.01％）。在公猪SP-EV中共鉴定了288个已知的miRNA，37个新的miRNA和19,749个piRNA。根据双荧光素酶报告基因实验，鉴定出的ssc-miR-21-5p可能对精子的繁殖能力产生负面影响。因此，这项研究提供了一种分离SP-EV并表征sRNA谱的有效方法。