卵胞浆内单精子注射(ICSI)在牛中仍然效率低下。一个原因可能在于用精子注射卵母细胞,这些精子没有经历与体内获能和受精能力相关的分子变化。本研究旨在通过采用荧光激活细胞分选(FACS)在注射前根据获能标记选择精子群体,从而提高牛卵胞浆内单精子注射(压电ICSI)的效率。首先,我们评估了用不同的电容感应器将解冻的精子孵育2小时的效果:肝素,甲基-β-环糊精(MβCD),和二丁酰基环AMP(dbcAMP),在基础获能(C)培养基(Sp-TALP)中单独或组合。通过流式细胞术评估精子获能和质量标记,揭示肝素是精子获能变化的最有效诱导剂。It,因此,选择该治疗作为FACS-压电-ICSI的精子预处理.通过FACS选择显示与精子获能i(Ca2+)水平和顶体完整性相关的标志物的高获能水平(肝素-HCL)和低获能水平(肝素-LCL)的两个细胞群,并用于精子注射。用肝素-HCL精子进行ICSI时前核形成明显高于肝素-LCL和对照组(肝素未分类)组(50%,10%,20%,分别)。此外,注射肝素-HCL精子的囊胚率(22.5%)高于肝素-LCL(10%)和对照组(15.2%)。总之,肝素治疗可有效诱导与精子获能相关的变化。肝素-HCL治疗和FACS的组合能够在ICSI之前精确选择获能精子。提高这项技术在牛种中的效率。
Intracytoplasmic sperm injection (ICSI) remains inefficient in cattle. One reason could lie in the injection of oocytes with sperm that have not undergone molecular changes associated with in vivo capacitation and fertilizing ability. This study aimed to enhance the efficiency of bovine intracytoplasmic sperm injection (piezo-ICSI) by employing fluorescent-activated cell sorting (
FACS) to select the sperm population before injection based on capacitation markers. First, we evaluated the effects of incubating thawed sperm for 2 hours with different capacitating inductors: heparin, methyl-beta-cyclodextrin (MβCD), and dibutyryl cyclic AMP (dbcAMP), alone or in combinations in a basal capacitating (C) medium (Sp-TALP). Sperm capacitation and quality markers were evaluated by flow cytometry, revealing heparin as the most effective inducer of sperm capacitation changes. It, therefore, this treatment was chosen as the sperm pretreatment for
FACS-piezo-ICSI. Two cell populations showing high capacitating levels (Heparin-HCL) and low capacitating levels (Heparin-LCL) of the markers associated with sperm capacitation i(Ca2+) levels and acrosome integrity were selected by
FACS and used for sperm injection. Pronuclear formation was significantly higher when ICSI was performed with Heparin-HCL sperm than with Heparin-LCL and the control group (Heparin unsorted) groups (50 %, 10 %, and 20 %, respectively). Furthermore, injecting Heparin-HCL sperm resulted in a higher blastocyst rate (22.5 %) than Heparin-LCL (10 %) and the control group (15.2 %). In conclusion, heparin treatment effectively induced changes associated with sperm capacitation. The combination of Heparin-HCL treatment and
FACS enabled precise selection of capacitated sperm before ICSI, enhancing the efficiency of this technology in the bovine species.