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In Vitro Effects of Lipopolysaccharide on Rabbit Sperm: Toll-like Receptor 4 Expression, Motility, and Oxidative
Alda Quattrone1, Nour Elhouda Fehri1, Stella Agradi2
1Department of Veterinary Medicine and Animal Sciences, University of Milan, Via dell'Università 6, 26900 Lodi, Italy.
Abstract:
Lipopolysaccharide (LPS)-induced inflammation impairs sperm function; however, its impact on ejaculated rabbit sperm remains unexplored. This dose-response study aims to determine the LPS concentration that negatively affects sperm motility in vitro, while also providing the first identification of TLR4 localization on rabbit spermatozoa. Additionally, it evaluates malondialdehyde (MDA) levels in seminal plasma as an indicator of oxidative stress. Sperm motility was analyzed using computer-assisted sperm analysis (CASA) after incubation with increasing LPS concentrations (0, 50, 100, 200, 400, 600, and 800 µg/mL) at multiple time points (0, 1, 2, and 4 h). LPS doses ≥ 400 µg/mL significantly reduced progressive and non-progressive motility, as well as curvilinear velocity (all p < 0.001), while increasing the proportion of static spermatozoa (p < 0.05). Receiver operating characteristic (ROC) analysis identified 300 µg/mL as the threshold dose for motility decline. Immunofluorescence revealed TLR4 localization in the midpiece of sperm tails, with weak labeling in control samples and a marked increase after 4 h of incubation with 400 μg/mL LPS. MDA levels were assessed using the thiobarbituric acid reactive substances (TBARS) assay with a colorimetric kit, showing no significant effect of LPS treatment. No correlation was found between MDA and other semen parameters. ccThese findings identify TLR4 on rabbit sperm for the first time and establish a threshold LPS dose for future in vitro studies.
Insights
Lipopolysaccharide (LPS) at doses of 400 µg/mL and higher impairs rabbit sperm motility. This study also identified Toll-like receptor 4 (TLR4) on rabbit sperm, establishing a threshold LPS dose for future research.
Area of Science:
- Reproductive Biology
- Immunology
- Toxicology
Background:
- Inflammation induced by lipopolysaccharide (LPS) is known to affect sperm function.
- The specific impact of LPS on ejaculated rabbit sperm and the presence of Toll-like receptor 4 (TLR4) on these cells remain uninvestigated.
Purpose of the Study:
- To determine the LPS concentration that negatively impacts in vitro sperm motility in rabbits.
- To identify TLR4 localization on rabbit spermatozoa.
- To evaluate seminal plasma malondialdehyde (MDA) levels as an indicator of oxidative stress.
Main Methods:
- Sperm motility was assessed using computer-assisted sperm analysis (CASA) after incubation with varying LPS concentrations (0–800 µg/mL) over 4 hours.
- Toll-like receptor 4 (TLR4) localization was determined using immunofluorescence.
- Malondialdehyde (MDA) levels were measured via the thiobarbituric acid reactive substances (TBARS) assay.
Main Results:
- LPS doses of 400 µg/mL and above significantly reduced sperm motility parameters (progressive, non-progressive, and curvilinear velocity) and increased static spermatozoa.
- Receiver operating characteristic (ROC) analysis indicated a threshold LPS dose of 300 µg/mL for motility decline.
- Immunofluorescence confirmed TLR4 presence in the midpiece of rabbit sperm, with increased labeling after LPS exposure. MDA levels were not significantly affected by LPS treatment.
Conclusions:
- This study is the first to identify TLR4 on rabbit spermatozoa.
- A threshold LPS dose of 300 µg/mL was established for impairing rabbit sperm motility in vitro.
- LPS-induced inflammation does not appear to increase oxidative stress in rabbit seminal plasma, as indicated by MDA levels.
