Proteomic analysis reveals the potential positive effects of Mito-TEMPO on ram sperm motility and fertility during cryopreservation

Abstract

The aim of this study was to investigate the effects of mitochondria-targeted antioxidant Mito-TEMPO on the protein profile of ram sperm during cryopreservation and evaluate the cryoprotective roles of Mito-TEMPO on ram sperm quality and fertilization capacity. Semen collected from 8 Dorper rams was cryopreserved in TCG-egg yolk extender supplemented with various concentrations of Mito-TEMPO (0, 20, 40 and 60 μM). After thawing, sperm characteristics, antioxidant status and the abundance of hexose transporters (GLUT 3 and 8) were analyzed. The cervical artificial insemination (AI) was performed to evaluate the fertilization ability of cryopreserved ram sperm. The alterations of sperm proteomic profile between the control and MT₄₀ groups were determined using iTRAQ-coupled LC-MS. Supplementation with 40 μM of Mito-TEMPO resulted in the highest post-thaw sperm motility and kinematics. Sperm quality, antioxidant capacity and glucose transporter abundance of frozen-thawed ram sperm were elevated in the MT₄₀ group. The inclusion of 40 μM Mito-TEMPO in freezing extender also resulted in the higher pregnancy rate of ewes. A total of 457 proteins including 179 upregulated proteins and 278 downregulated proteins were defied as differentially expressed proteins (DEPs) using fold change (FC) > 1.2 with P < 0.05. Sixty-one DEPs with (FC > 1.5) were dramatically regulated by Mito-TEMPO. These DEPs are mainly involved in sperm motility, energy metabolism and capacitation. Our data suggest that the beneficial effects of Mito-TEMPO on sperm motility and fertility potential of cryopreserved ram semen are achieved by regulating sperm antioxidant capacity and sperm proteins related to energy metabolism and fertility.

Keywords: Antioxidation; Cryopreservation; Mito-TMPO; Proteomics; Ram semen.