A total of 40, aged 8 weeks, sex mature male Sprague Dawley (SD) rats weighting 280 ± 10g were obtained from the East General Hospital. Animals were maintained at temperature (24 ± 2°C), relative humidity (60% ± 5%) with a 12 hour light: dark cycle and with access to food and water ad libitum during the course of the study. This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of East Region Military Command General Hospital (Date20220307/No.2022JLHSXJDWLS-0021).
Chemicals and Experimental design
Lipopolysaccharide (LPS, L2630-10MG) was purchased from Sigma-Aldrich, Co., (USA). Lycopene was obtained from the Nanjing Yuanjian Biotechnology Co., LTD (Nanjing, China).
After 2 weeks of acclimation, the experimental rats were divided into four groups (per n = 10): First group was blank control. Second group was control-received vehicle (CRV, olive oil, 5 ml/kg/d, 4weeks). Third group was treated with LPS (5mg/Kg, dissolved in 0.9% Sodium chloride injection, i.p.). And fourth group received LPS (5mg/Kg, dissolved in 0.9%Sodium chloride injection, i.p) and Lycopene (5mg/kg/d, dissolved in olive oil, i.g, 4weeks).
Epididymal sperm analysis
Detection of sperm parameters was performed according to the WHO laboratory manual for the Examination and processing of human semen (WHO, 2010). The right cauda epididymis were cut centrally and put in plates containing warm (37°C) 0.9%Sodium chloride injection (Chimin Health Management Co., LTD.). Sperm counting was performed using a Neubauer haemocytometer. The semen specimens were evaluated on dishes under a light microscope for sperm motility and sperm concentration. Sperm motility and count were completed by one researcher.
The testicular tissue was preserved in 4% paraformaldehyde over 24 hours. The samples were dehydrated and embedded in paraffin and serially sliced, and they were prepared for hematoxylinand eosin staining. Then, 5-µm sections were cut from the paraffin- embedded tissues, stained with haematoxylin and eosin (HE), After staining, the sections were dehydrated in alcohol, cleared in xylene, and preserved finally. The pictures were examined by light microscope.
Measurement of cytokines
Serum, testis and epididymis tissue were used for the estimation the level of the following cytokines: interleukin1alpha (IL-1α, Cat num: ELK1148), interleukin1beta (IL-1β, Cat num: ELK1272), tumor necrosis factor alpha (TNF-α, Cat num: ELK1396), monocyte chemotactic pretein1(MCP-1, Cat num: ELK5504) and interleukin6(IL-6, Cat num: ELK1158) . Those cytokines were detected in serum, testis and epididymis tissue with commercial kits (ELK Biotechnology CO., LTD, Wuhan, Hubei, China) according to the manufacturer’s instructions.
Testis Antioxidants Biomarkers
Catalase (CAT, A007-1), glutathione peroxidase (GSH-Px, A005-1), and superoxide dismutase (SOD, A001-1) were detected in testis homogenate with commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) according to the manufacturer’s instructions.
Statistical analysis was performed using statistical software SPSS 26.0. Data are presented as the mean ± SD. One-way analysis of variance (ANOVA) was used to determine differences between the normally distributed data. Kruskal–Wallis test was employed to compare non-normally distributed variables. Dunnett´s T3 was employed to compare non-equal variances variables. P < 0.05 was considered to be statistically significant.