# The effects of polysaccharides from Inonotus obliquus, artemisinin, and dihydroartemisinin on the reproductive system of male mice infected with Neospora caninum

## TL;DR

This study shows that Inonotus obliquus polysaccharides and dihydroartemisinin help protect male mice from reproductive damage caused by Neospora caninum infection.

## Contribution

The study introduces the protective effects of IOPs and DHA against Neospora caninum-induced reproductive damage in mice.

## Key findings

- IOPs and DHA significantly improved sperm quality and reduced deformity rates in infected mice.
- All three treatments reduced testicular and epididymal tissue damage and improved organ indices.
- IOPs and DHA showed antioxidant, immunomodulatory, and antiapoptotic effects in infected mice.

## Abstract

The present study investigated the protective effects of Inonotus obliquus polysaccharides (IOPs), artemisinin, and dihydroartemisinin (DHA) on Neospora caninum-induced damage of the reproductive system in male BALB/c mice. This study conducted in vivo resistance experiments against Neosporidium using three drugs: polysaccharides from Betula brownii, artemisinin, and dihydroartemisinin. On the basis of establishing a male mouse animal model of new sporidiosis, after gavage administration, the reproductive organ index of male mice was measured at 7d, 14d, 21d, 35d, and 42d, respectively. HE staining and transmission electron microscopy were used to observe the pathological changes of testicular and epididymal tissues. The improved Pap staining method was used to analyze sperm quality, flow cytometry was used to detect apoptosis of spermatogenic cells, MDA and ACP activities were measured, and ELISA was used to detect immunoglobulin IgG1, IgG2a, IgE, and cytokine IFN-γ, IL-4, IL-6, TNF-α. The qPCR method was used to detect the expression of apoptotic genes Bax, Bcl-2, Caspase-3, P53, as well as sperm related genes C-kit, Plzf, Sycp3, Stra8, Dnajb13, Mrto4, and Ipo11, as well as the levels of NO and AsAb. The results showed that IOPs and DHA exhibited significant anti-neosporal activity. Compared to infected mice, IOPs-treated mice showed significantly increased sperm density (p < 0.05) and sperm motility (p < 0.05), while DHA-treated mice exhibited a remarkably reduced sperm deformity rate (p < 0.05). Compared with the model group, the sperm motility in the ART-treated mice was significantl upregulated (p < 0.01). Histopathological analysis revealed that all three treatments ameliorated testicular and epididymal tissue damage, reduced mitochondrial vacuolization, and improved organ indices. Biochemical assays showed a reduced level of malondialdehyde and a high level of acid phosphatase activity in the testicular tissue of treated mice. Immunological assays confirmed decreased levels of immunoglobulin G1 (IgG1), IgG2a, interleukin (IL)-4, IL-6, tumor necrosis-α factor nitric oxide, and anti-sperm antibodies in the treatment groups. Gene expression analysis indicated that IOPs significantly downregulated the expression of caspase-3, p53, and Dnajb13 and upregulated expression of SYCP3 and Stra8. There was no significant difference in the ART group (p < 0.05). DHA markedly reduced Dnajb13 expression but enhanced SYCP3, Stra8, and Ipo11 expression. These findings suggest that ART has no obvious therapeutic effect on male mice infected with Neospora caninum, IOPs and DHA can effectively mitigate N. caninum induced Spermatogenesis block, spermatozoon maturation impairment, and spermatozoon structural defects in male mice antioxidant, immunomodulatory, and antiapoptotic effects and could be considered promising candidates for anti-neosporal therapy.