171 Microplastics are present in bull epididymal sperm and polystyrene bead affects bovine sperm inducing oxidative stress on embryos

The alarming increase in global infertility rates has coincided with the pervasive accumulation of microplastics (MPs) resulting from the poor management of plastic waste. This concerning trend is particularly troubling because only 10% of male infertility cases can be attributed to identifiable causes, leaving a significant knowledge gap in our understanding of their underlying factors. To bridge this critical gap, it is important to explore the connection between the accumulation of MPs and the observed decline in male fertility. Therefore, this study aimed to investigate not only the presence of MPs in epididymal sperm (ES) but also their potential effects on sperm function and embryo development. To detect MPs in ES, bovine ES was collected after slaughter (n = 3) and digested in KOH 10% (24 h, 250 rpm, 60°C), followed by NaClO 7.5% (24 h, 250 rpm, 60°C), filtered in a 0.45-µm pore membrane, digested in HNO₃ 20% (24 h, 250 rpm, 40°C), and filtered again. Microplastics were analysed using Raman spectrometry. Next, thawed bovine sperm were incubated with three different concentrations of a mixture of 1, 0.5, and 0.3 µm polystyrene (PS) beads: (1) 0.7 μg mL⁻¹, blood concentration of PS in cows (bPS, van der Veen et al. 2022 “Plastic particles in livestock feed, milk, meat and blood”); (2) 0.37 μg mL⁻¹, concentration of total MPs in ES (esMP); and (3) 0.026 μg mL⁻¹, concentration of PS in ES (esPS). A control group without beads (CT) was included. The sperm were incubated at 38.5°C, 5% CO₂, and 95% O₂, aliquots were taken at 0, 0.5, 1, 2, 3, and 4 h, checked for motility (n = 4), stained for oxidative stress (CellRox™, n = 4) and acrosome integrity (PNA-Alexa Fluor™ 647, n = 4), fixed with 4% paraformaldehyde, spread in a SuperFrost slide, washed, and analysed. Sperm from all groups were exposed to MPs for 1 h, washed to remove MPs, and used for IVF (n = 2). Cleavage and blastocyst rates were determined, and blastocysts were checked for oxidative stress (CellRox™) and apoptosis (Caspase3). Image analysis was done in ImageJ, and data were analysed using a generalized linear mixed model, and a Tukey post hoc in R. Epididymal sperm presented a mean of 72.5 particles/mL, with silicon, cellulose acetate, and polypropylene being the most abundant. The average particle length was 17.0 ± 27.1 μm, and width 8.3 ± 9.8 μm. All sperm samples incubated with PS exhibited reduced motility compared with the CT at 0.5 h (P < 0.05 for all). However, PS exposure did not affect acrosome or induced oxidative stress and had no effect on cleavage and blastocyst rates. Nonetheless, an increase in oxidative stress was observed in embryos from sperm exposed to bPS compared with CT (P = 0.01) and esPS (P = 0.04). Similarly, bPS tended to increase apoptosis when compared with CT (P = 0.06). By employing realistic exposure concentrations, this research sought to shed light on the comprehensive impact of MPs on bovine sperm and the quality of resulting embryos, providing the first evidence of MPs in bovine epididymal sperm and demonstrating the detrimental effect of PS MPs on sperm motility, as well as their potential to increase oxidative stress in embryos.