Nanoplastics internalization impairs mitochondrial activity in equine sperm

Nanoplastics (NP) accumulation in biological tissues and their adverse effects on fertility through inflammatory and oxidative stress responses have recently been described as consequences of global plastic pollution. However, little is known about the impact of NP on gametes. This study aimed to assess the internalization of NP and their effects on mature equine spermatozoa. Frozen-thawed ejaculates from five stallions were divided into untreated control (CTR) and samples supplemented with different concentrations (10, 50, 100 and 200 μg/mL) of 30 nm polystyrene NP. At baseline (T0), and after 1 (T1) and 3 h (T3) of incubation at 38 °C, sperm viability, mitochondrial activity, and intracellular ROS were evaluated by flow cytometry, while sperm motility was assessed using a CASA system. NP internalization was analyzed by confocal microscopy and flow cytometry using fluorescent NP. Results showed that NP were internalized by live spermatozoa, accumulating in the post-acrosomal and/or the mid piece region. NP exposure led to reduced sperm viability (CTR T1: 50.7 ± 14.0 % vs 200 μg/mL T1: 36.6 ± 11.8 %, p<0.05), decreased mitochondrial activity (CTR T1: 35.4 ± 15.8 % vs 200 μg/mL T1: 16.3 ± 14.0 %, p<0.001), and increase proportions of live sperm with high intracellular O₂^-· levels (CTR T1: 39.0 ± 10.1 % vs 200 μg/mL T1: 47.3 ± 11.0 %, p<0.05). These results allow us to conclude that equine sperm quality may be compromised by nanoplastics internalization, which pre-eminently impairs mitochondrial activity. This research furnishes some bases for further studies on the potential implications of NP exposure for fertility.