Antioxidants and Oxidants in Boar Spermatozoa and Their Surrounding Environment Are Associated with AMPK Activation during Liquid Storage

Abstract

Activation of the AMP-activated protein kinase (AMPK) has been demonstrated to be beneficial for boar sperm quality and functionality, while the underlying mechanism of AMPK activation of boar spermatozoa remains obscure. This study aimed to explore the effect of antioxidants and oxidants in boar spermatozoa and their surrounding fluid (SF) on the activation of AMPK during the liquid storage. Ejaculates from Duroc boars, routinely used for semen production, were collected and diluted to a final concentration of 25 × 106/mL. In experiment 1, twenty-five semen samples from eighteen boars were stored at 17 °C for 7 days. In experiment 2, three pooled semen samples created from nine ejaculates of nine boars were used, and each sample was treated with 0, 0.1, 0.2, and 0.4 μM/L H2O2 and stored at 17 °C for 3 h. Sperm quality and functionality, antioxidants and oxidants in boar spermatozoa and SF, the intracellular AMP/ATP ratio, and the expression levels of the phosphorylated AMPK (Thr172) were determined. Sperm quality significantly decreased with storage time in terms of viability (p < 0.05). Antioxidant and oxidant levels were markedly affected with storage time, with a decline in the SF total antioxidant capacity (TAC) (p < 0.05), SF malondialdehyde (MDA) (p < 0.05), and the sperm’s total oxidant status (TOS), as well as a fluctuation in sperm superoxidase dismutase-like (SOD-like) activity (p < 0.05). The intracellular AMP/ATP ratio increased (p < 0.05) on day 4 and subsequently decreased to its lowest value on days 6 and 7 (p < 0.05). The phosphorylated AMPK levels increased from day 2 to day 7 (p < 0.05). Correlation analyses indicate that sperm quality during liquid storage was correlated to antioxidants and oxidants in spermatozoa and SF (p < 0.05), which were correlated to the phosphorylation of sperm AMPK (p < 0.05). Treatment with H2O2 induced damages in sperm quality (p < 0.05), a decline in antioxidant levels (SF TAC, p < 0.05; sperm SOD-like activity, p < 0.01), an increase in oxidant levels (SF MDA, p < 0.05; intracellular ROS production, p < 0.05), a higher AMP/ATP ratio (p < 0.05), and phosphorylated AMPK levels (p < 0.05) in comparison with the control. The results suggest that antioxidants and oxidants in boar spermatozoa and SF are involved in AMPK activation during liquid storage.

Post-Thaw Storage Temperature Influenced Boar Sperm Quality and Lifespan through Apoptosis and Lipid Peroxidation

“…Antioxidant and oxidant levels in boar sperm and the surrounding fluid have been demonstrated to be associated with boar sperm quality and functionality during liquid storage through influencing the phosphorylation of AMP-activated protein kinase (AMPK) [23].…”

“…The mitochondrial membrane potential was determined using Mitotracker Deep Red 633 staining following a procedure described by Li et al (2023) [23]. Briefly, cytometric tubes containing a 100 µL sperm sample (25 × 10 6 sperm/mL) was loaded with 3 µL of H-42 (0.05 mg/mL in PBS), 2 µL of PI (0.5 mg/mL in PBS), and 5 µL of Mitotracker (0.2 µM of PBS in a stock solution of 1 mM in DMSO) and incubated at 37 • C for 15 min.…”

“…A microplate reader (PowerWave XS; Bio-Tek Instruments, Winooski, VT, USA) was utilized to determine the contents or activities of antioxidants and oxidants in boar SPZ and SN, as previously described by Li et al (2023) [23]. SN samples were measured directly.…”

Advancements in Genetic Biomarkers and Exogenous Antioxidant Supplementation for Safeguarding Mammalian Cells against Heat-Induced Oxidative Stress and Apoptosis

“…It is well established that AMPK signaling plays a key role in the tight junctions (TJs) and cell proliferation of testis Sertoli cells [ 143 , 144 , 145 , 146 , 147 , 148 ]. In addition, Ni et al [ 147 ] highlighted that Sertoli cells play a key role in lactate supply, maintenance of cell junctions, and support for germ cells’ mitosis and meiosis.…”

“…Once this balance is disrupted, the microenvironment of the testis and the quality of sperm will be affected. When α1AMPK was conditionally knocked out in mouse SCs, the mutant mice still showed an abnormal phenotype, including thin-head spermatozoa, reduced expression of junctional proteins (β-catenin, vimentin, occludin, and ZO-1), and deregulation of energy homeostasis [ 143 , 145 , 147 , 148 ]. Consistently, a study has documented that curcumin (natural antioxidant and anti-inflammatory compound) supplementation rescues porcine Sertoli cell impairment and TJs by inhibiting the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome through the AMPK/SIRT3/SOD2/mtROS signaling pathway [ 150 ].…”