The polysaccharides from Inonotus obliquus were extracted using reagents such as petroleum ether, 70% methanol, anhydrous ethanol, ether, and acetone, and stored in our laboratory (polysaccharide content: 84%). ART and DHA (purity: 98%) were purchased from Shanghai YuanYe Biotechnology Co., Ltd. High-glucose Dulbecco’s modified Eagle’s medium (DMEM) medium and dimethyl sulfoxide were obtained from Sigma. Fetal bovine serum (FBS) was supplied by Tianjin Haoyang Biological Products Technology Co., Ltd. Phosphate-buffered saline (PBS) powder, trypsin, and modified a PAP staining kit (EA50) were provided by Solarbio. Penicillin and streptomycin were obtained from Nanjing Novozan Biotechnology Co., Ltd. Absolute ethanol, methanol, glutaraldehyde (domestic analytical and pure grade), and Annexin V apoptosis detection kit were purchased from Sangon Biotech (Shanghai) Co., Ltd. The malondialdehyde (MDA) assay and acid phosphatase (ACP) assay kits were procured from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Mouse immunoglobulin G1 (IgG1), IgG2a, IgE, interferon-γ (IFN-γ), interleukin (IL)-4, and nitric oxide (NO) ELISA kits were supplied by Shanghai Langton Biotechnology Co., Ltd. Mouse IL-6 and tumor necrosis factor-α (TNF-α) ELISA kits were provided by Jiangsu enzyme immunoassay Industrial Co., Ltd. The RNA extraction kit was obtained from Promega (Beijing) Biotechnology Co., Ltd. Genomic cDNA first strand synthesis premix reagent and the FastKing one-step reverse transcription-fluorescence quantification kit (SYBR Green) were obtained from Tiangen Biotech (Beijing) Co., Ltd. The mouse anti-sperm antibody (AsAb) ELISA kit was provided by Jingmei Biotechnology Co., Ltd.

A bovine strain of N. caninum was isolated from bovine fetal brain tissue and cultured in the Laboratory of Preventive Veterinary Medicine, Yanbian University, Yanji, China. Vero cells were also maintained in the Laboratory of Preventive Veterinary Medicine, Yanbian University. N. caninum was propagated in Vero cells, and the cells were then cultured in DMEM. Subsequently, 8% heat-inactivated FBS, 100 mg/mL penicillin, and 10 mg/mL streptomycin were added to the cells, and the cells were then incubated at 37 °C in a 5% CO2 environment. Infected Vero cells were washed with ice-cold PBS to purify tachyzoites. Subsequently, first, filter four times using a 27-gage needle, and then filter through a 5-micron filter. The number of parasite cells was determined using a blood cell counting plate.

Male BALB/c mice (age: 8 weeks; weight: approximately 20 g) were housed under controlled temperature (25 ± 1 °C) and humidity (50% ± 5%) with free access to food and water. After 1 week of adaptation to housing conditions, all animals were used for the experiment. The study protocol strictly followed the recommendations provided in the Guide for the Care and Use of Laboratory Animals, Yanbian University. A total of 125 mice were randomly selected, and all mice were randomly assigned to five groups: blank control group, infected model group, IOPs-treated group, ART-treated group, and DHA-treated group, with 25 mice in each group. The male BALB/c mouse model of N. caninum infection was established according to the method of Hang Li (19). The blank control group received an intraperitoneal injection of 0.1 mL of PBS solution, while the remaining four groups were injected with 0.1 mL of a Neospora caninum/PBS suspension (with a Neospora caninum concentration of 1 × 10⁶/mL). Starting from the 7th day after worm infection, the drug was administered by gavage, which was considered as the first day of administration. The dosage was 0.1 mL/10 g, and the drug was continuously administered by gavage for 7 days. The dosage for each group was 200 mg/kg, and the blank control group and model group were given normal saline. Five male mice in each group were sacrificed in batches on the 7th, 14th, 21st, 35th, and 42nd days after treatment administration (19). In accordance with the 3R principles and under a protocol approved by the local Ethical Review Board, mice were euthanized by CO2 asphyxiation using a precision flow meter to ensure a gradual displacement of chamber air. This was immediately followed by cervical dislocation to ensure death prior to tissue collection. After sacrificing the mice, the brains, livers, spleens, testes, and epididymides were dissected and collected for subsequent experiments. Figure 1 is a brief flowchart of the animal experiment part in this study.

The testes and epididymides were dissected and removed. Subsequently, the adipose tissue and connective tissue were removed from these organs, the organs were weighed, and the reproductive organ index was calculated. The testes or epididymides indices were calculated as the weight of testis or epididymis tissue of each male mice/weight of each male mice (mg/g). Take 0.5 mL of 0.9% saline and place it in a 37 °C water bath. Separate the intact epididymis, place one side of the mouse’s epididymis in preheated physiological saline, and cut it into small pieces. Use a pipette to repeatedly blow and mix the tissue suspension gently for 15–20 times. Place the mixed tissue suspension in a 37 °C constant temperature water bath and wait for the sperm to swim out naturally. Let it sit quietly for 10 min. After preparing the sperm suspension, first observe the sperm motility using a microscope (with a sperm count greater than 200 and at least 5 fields observed). Then, calculate the sperm density using a hemocytometer. Finally, observe the sperm malformation rate through the modified Papanicolaou staining method. The evaluation is conducted within 30 min after sample preparation at 37 °C (20).

On the 7th, 14th, 21st, 35th, and 42nd days after administration, testicular and epididymal tissues were collected from mice in each group. The tissues were immersed in 10% formalin solution, followed by preparation of paraffin sections (with a thickness of 5 μm) (21), and finally stained with hematoxylin and eosin (H&E).

After the mice were sampled, the tissue blocks were fixed in 4% glutaraldehyde and stored at 4 °C for 1 week. After 1 week, remove it and rinse it three times with PBS buffer for 30 min each time. Fix 1% osmium acid in a refrigerator at 4 °C for 2 h. After fixation, rinse with distilled water 3 times for 15 min each time. Dehydrate using gradient alcohol, acetone, and epichlorohydrin, then pre-soak with a mixture of epichlorohydrin and embedding agent for 1 h, and finally soak overnight with embedding agent. Aggregation is carried out through gradient temperature and ultra-thin slicing is performed. Stain with uranyl acetate and lead citrate, and observe and record under transmission electron microscopy. The transmission electron microscope is sourced from Shanghai Zhujin Analytical Instrument Co., Ltd.

An Annexin V-FITC/propidium iodide (PI) kit was used to determine cell apoptosis by FCM analysis. Mice testicular tissue was fragmented, and the tissue fragments were filtered through a 300-mesh filter cloth. The obtained cells were resuspended in 1 × binding buffer, and the cell density was adjusted to the appropriate value. Next, Annexin V-FITC was added to the cells, mixed well, and incubated in the dark at room temperature for 10 min. The cells were then washed with 1 × binding buffer and centrifuged, and the supernatant was discarded. The cell layer pellet was resuspended in 1 × binding buffer, 10 μL PI was added, and the cell suspension was immediately placed in the flow cytometer for apoptosis detection. All the experimental steps above were conducted in accordance with the instructions provided in the reagent kit manual.

Briefly, 0.1 g of the testicular tissue sample was homogenized in 0.9 mL of ice-cold normal saline. The homogenate was centrifuged at 10000 g and 4 °C for 10 min, and the resulting supernatant was used for determining the MDA level and ACP activity. These assays were conducted using the substrate provided by Nanjing Jiancheng Bioengineering Institute in accordance with the manufacturer’s protocol. The optical density was measured using a microplate reader (Bio-Rad), and the MDA and ACP levels were then calculated. All the experimental steps above were conducted in accordance with the instructions provided in the reagent kit manual.

Blood samples were collected from mice eyeballs on 0, 7, 14, 21, 35, and 42 days after treatment administration, and serum was obtained (centrifugation at 1500 g for 15 min at 4 °C). Serum levels of the immunoglobulin subclasses IgG1, IgG2a, and IgE and the cytokines IFN-γ, IL-4, IL-6, and TNF-α were detected by following the procedures mentioned in the assay kit instructions. All the experimental steps above were conducted in accordance with the instructions provided in the reagent kit manual.

Serum samples were collected from mice on days 0, 7, 14, 21, 35, and 42 after administration, and the levels of AsAb and NO were detected using different ELISA kits according to the manufacturer’s instructions. All the experimental steps above were conducted in accordance with the instructions provided in the reagent kit manual.

In the ELISA experiment, the blood volume of each mouse was (1,100 ± 200) μL, and the serum volume after standing and separation was (450 ± 25) μL, which was detected using a microplate reader. The required mouse materials, dosage, and absorbance for detecting each indicator in 2.7, 2.8, and 2.9 are shown in Table 1.

Total RNA was isolated from the testes of mice in each group by using the RNA extraction kit in accordance with the manufacturer’s instructions. According to the instructions of the kit, the amount of RNA used is 50 ng-2 μg, the total reaction volume is 20 μL, and cDNA reverse transcription is performed under the following conditions: 42 °C for 15 min and 95 °C for 3 min. The obtained cDNA was stored at −20 °C for subsequent analysis. The following mouse gene sequences from GenBank were used for the analysis: Bax, Caspase-3, Bcl-2, p53, c-kit, PlZF, SYCP3, Stra8, Dnajb13, Mrto4, Ipo11, and β-action, and the primer sequences and Genbank accession numbers for all genes are listed in Table 2. The corresponding specific primers were designed using Oligo primer analysis software version 7 and synthesized by Kumei Biological Co., Ltd. Quantitative PCR (qPCR) was performed under the following reaction conditions: 95 °C for 10 min; 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 32 s; and final extension at 55 °C for 40 s, with a temperature change rate of 1 °C/s; the total reaction volume is 50 μL. The fluorescence quantitative PCR instrument is sourced from Analytikjena.

The experimental data were analyzed using SPSS 19.0 and expressed as mean ± standard deviation (⁻X ± SD). Shapiro Wilk test and Levene test were used, respectively, to check the normality and homogeneity of variance of the data. Meet the normal distribution. Analyze the data differences within and between groups using one-way ANOVA and two-way ANOVA. p < 0.05 is statistically significant.

The blank control group showed only a small number of abnormal sperm (Figure 2A); in contrast, the infected model group had a large number of abnormal sperm (Figure 2B). Among the three treatment groups, the ART-treated group had the largest number of abnormal sperm (Figure 2D), followed by the DHA-treated group (Figure 2E), while the IOPs-treated group had the smallest number of abnormal sperm (Figure 2C). The sperm deformity rate of each group is shown in Figure 3B.

The sperm motility of the blank control group, model group, Polyporus obliquus polysaccharide group, artemisinin group, and dihydroartemisinin group was (90.67 ± 3.050)%, (42.09 ± 8.760)%, (73.95 ± 3.330)%, (53.60 ± 7.190)%, and (48.35 ± 6.100)%, respectively. The sperm malformation rates of the blank control group, model group, Polyporus obliquus polysaccharide group, artemisinin group, and dihydroartemisinin group were (8.650 ± 1.330)%, (45.65 ± 4.500)%, (27.40 ± 1.223)%, (42.67 ± 2.560)%, and (33.33 ± 1.750)%, respectively. The sperm densities of the blank control group, model group, Polyporus obliquus polysaccharide group, artemisinin group, and dihydroartemisinin group were (12.65 ± 2.450) × 10⁶/mL, (5.05 ± 1.750) × 10⁶/mL, (7.65 ± 0.600) × 10⁶/mL, (6.70 ± 1.350) × 10⁶/mL, and (7.52 ± 0.5965) × 10⁶/mL, respectively.

The following results were obtained after statistical analysis.

Testicular index: The blank control and infected model groups showed significant decrease in the testicular index on the 7th, 14th, 21st, 35th, and 42nd days after treatment (p < 0.01). Specifically, the infected model and IOPs-treated groups showed significant increase in the testicular index on the 7th, 14th, 21st, 35th, and 42nd days after treatment (p < 0.01). The infected model and ART-treated groups showed a significant increase in the testicular index on the 7th day after treatment (p < 0.05), which further increased on the 14th, 21st, 35th, and 42nd days after treatment (p < 0.01). The infected model and DHA groups showed no significant difference in the testicular index on the 7th day after treatment (p > 0.05), but which increased significantly on the 14th, 21st, 35th, and 42nd days after treatment (p < 0.01; Figure 4A).

In the blank control group, the testicular index of mice showed no significant trend from day 7 to day 42. In the model group, the testicular index exhibited a pattern of initial decrease followed by an increase, with notable fluctuations during the first 21 days—declining from (3.649 ± 0.054) mg/g to (3.432 ± 0.049) mg/g, and then rising to (3.622 ± 0.028) mg/g by day 42. In the Inonotus obliquus polysaccharide group, the testicular index demonstrated an initial increase followed by a decrease, also with clear fluctuations in the first 21 days—rising from (3.803 ± 0.043) mg/g to (3.868 ± 0.114) mg/g, then dropping to (3.793 ± 0.030) mg/g by day 42. In the artemisinin group, a similar initial increase followed by a decrease was observed, with marked fluctuations within the first 14 days—increasing from (3.797 ± 0.025) mg/g to (3.908 ± 0.034) mg/g, before declining to (3.834 ± 0.039) mg/g at day 42. In contrast, the dihydroartemisinin group displayed a slight but steady increasing trend, with the testicular index gradually rising from (3.760 ± 0.037) mg/g on day 7 to (3.809 ± 0.016) mg/g by day 42.

Epididymis index: The blank control and infected model groups showed significant decrease in the epididymis index on the 7th, 14th, 21st, 35th, and 42nd days after treatment (p < 0.01). Moreover, all three treatment groups when comparison with infected group showed highly significant increase in on the 7th, 14th, 21st, 35th, and 42nd days after treatment (p < 0.01; Figure 4B).

In the blank control group, the epididymal index of mice showed no significant trend from day 7 to day 42. The model group exhibited an initial decrease followed by an increase, with notable fluctuations during the first 21 days—declining from (1.883 ± 0.047) mg/g to (1.583 ± 0.064) mg/g, before rising to (1.703 ± 0.064) mg/g by day 42. The Inonotus obliquus polysaccharide group demonstrated an increase followed by a decrease. Marked fluctuations were observed in the first 14 days, with values rising from (2.098 ± 0.037) mg/g to (2.197 ± 0.089) mg/g. From day 21 onward, the index declined, reaching (1.946 ± 0.031) mg/g by day 42. The artemisinin group showed a similar pattern of an initial rise and subsequent fall. Prominent fluctuations occurred within the first 14 days, increasing from (2.145 ± 0.010) mg/g to (2.221 ± 0.032) mg/g. Starting from day 21, the values decreased, falling to (2.049 ± 0.030) mg/g at the end of the treatment period (day 42). In contrast, the dihydroartemisinin group displayed no significant trend in the epididymal index from day 7 to day 42.

As shown in Figure 8, the blank control and infected model groups exhibited a highly significant difference in the level of spermatogenic cell apoptosis (p < 0.01). Similarly, all three treatment groups and the infected model group showed a highly significant difference in the level of spermatogenic cell apoptosis (p < 0.01).

The spermatogenic cell apoptosis rates showed significant differences among the experimental groups. As expected, the model group exhibited a markedly increased apoptosis rate of (18.330 ± 0.300)%, compared to (6.867 ± 0.125)% in the blank control group. Among the treatment groups, the Inonotus obliquus polysaccharide group demonstrated the lowest rate at (9.390 ± 0.100)%, followed by the dihydroartemisinin group at (12.500 ± 0.200)% and the artemisinin group at (14.400 ± 0.150)%, all of which were lower than that of the model group.

The blank control and model groups showed a highly significant increase (p < 0.01) in MDA levels on the 7th, 14th, 21st, 35th, and 42nd test days. The MDA level of the infected model group and those of the IOPs-treated and ART-treated groups showed significant reduction on the 7th, 14th, 21st, 35th, and 42nd test days (p < 0.01). Although the MDA levels of the DHA-treated and infected model groups showed no significant difference on the 7th test day (p > 0.05), significant reduction were noted on the 14th, 21st, 35th, and 42nd test days (p < 0.01; Figure 9A).

The MDA concentrations in testicular tissue exhibited distinct temporal patterns across the experimental groups. In the blank control group, MDA levels showed a minor transient increase followed by a return to baseline, rising from (4.622 ± 0.392) nmol/mg to (5.281 ± 0.448) nmol/mg before declining to (4.633 ± 0.393) nmol/mg. In contrast, the model group displayed a progressive increase in MDA concentration, from (11.670 ± 0.214) nmol/mg on day 7 to (16.910 ± 0.160) nmol/mg by day 42. The Inonotus obliquus polysaccharide group demonstrated a more complex trend: MDA levels initially decreased from (11.670 ± 0.214) nmol/mg on day 7 to (5.613 ± 0.368) nmol/mg by day 14, then increased to (13.450 ± 0.666) nmol/mg on day 35, before finally falling to (6.044 ± 0.837) nmol/mg on day 42. The artemisinin group exhibited an initial increase followed by a decrease. After noticeable fluctuations during the first 35 days—rising from (5.992 ± 1.246) nmol/mg to (10.040 ± 0.163) nmol/mg—the concentration declined to (6.820 ± 0.134) nmol/mg by day 42. Similarly, the dihydroartemisinin group also showed a pattern of decrease, increase, and subsequent decrease, with marked early fluctuations. Levels dropped from (11.05 ± 0.281) nmol/mg on day 7 to (5.280 ± 0.197) nmol/mg by day 14, gradually rose to (14.26 ± 1.571) nmol/mg on day 35, and finally decreased to (5.164 ± 0.023) nmol/mg by the end of the treatment period (day 42).

ACP activity in testicular tissue showed highly significant reduction between the blank control and infected model groups on the 7th, 14th, 21st, 35th, and 42nd test days (p < 0.01). Although the infected model and IOPs-treated groups showed no significant difference in ACP activity on the 7th test day (p > 0.05), significant increase were noted on the 14th, 21st, 35th, and 42nd test days (p < 0.01). The ACP activity showed a significant increase between the infected model and ART-treated groups on the 7th, 14th, 21st, 35th, and 42nd test days (p < 0.05 for the 35th day and p < 0.01 for the remainder). Significant increase in ACP activity between the infected model and DHA-treated groups were noted on the 7th, 14th, 21st, 35th, and 42nd test days (p < 0.01; Figure 9B).

The concentration of acid phosphatase (ACP) in testicular tissue showed distinct temporal patterns across the experimental groups. In the blank control group, ACP levels initially decreased, then increased, and finally stabilized, with notable fluctuations observed within the first 14 days. Specifically, the concentration declined from (412.91 ± 0.890) U/L to (359.60 ± 1.196) U/L, subsequently rose to (385.8 ± 13.76) U/L, and eventually decreased to (366.0 ± 0.382) U/L before plateauing. In the model group, ACP concentration followed a pattern of initial decrease, subsequent increase, and final decline. From day 7 (200.03 ± 6.412) U/L, it dropped to (183.0 ± 9.652) U/L by day 14, increased to (305.9 ± 3.201) U/L on day 21, and finally decreased to (274.4 ± 6.834) U/L. The Inonotus obliquus polysaccharide group exhibited a gradual increase in ACP concentration, rising from (213.64 ± 0.701) U/L to (344.4 ± 0.154) U/L over the experimental period. In the artemisinin group, ACP levels showed an initial rise followed by a decline, with considerable fluctuations during the first 21 days. The concentration increased from (232.00 ± 12.440) U/L to (379.03 ± 6.248) U/L and later decreased to (337.12 ± 3.716) U/L. Similarly, the dihydroartemisinin group displayed an initial increase followed by a decrease, with marked fluctuations in the first 21 days. ACP levels rose from (222.33 ± 1.544) U/L to (336.90 ± 12.97) U/L and subsequently stabilized.

N. caninum is an obligate, intracellular parasitic protozoan that causes abortion, stillbirth, and reproductive system damage in pregnant animals, resulting in substantial economic losses to animal husbandry (22, 23). Currently, there is no effective vaccine or drug for controlling neosporosis. The present study found that IOPs, ART, and DHA can effectively alleviate the symptoms of reproductive system damage in male mice with N. caninum infection. Some studies have found that DHA is more stable, less toxic, and has a better antimalarial effect compared to ART (9). Other studies have also shown that IOP has a certain protective effect on mice infected with Neospora caninum (24). In this study, all results collectively indicate that DHA is more effective than ART in treating male mice infected with Neospora caninum, but the difference between DHA and IOP is not significant at present. This requires further verification through more in vivo experiments, such as studies on male mice and their offspring.

The assessment of sperm quality primarily involves the following parameters: sperm viability, teratozoospermia rate (percentage of abnormal forms), sperm concentration, sperm motility, and one-hour survival rate. Sperm quality is considered abnormal and suboptimal if the viability falls below 60%, the teratozoospermia rate exceeds 20%, the concentration is lower than 9.5 × 10⁶/mL, the motility is less than 70%, or the one-hour survival rate is below 60%. Such abnormalities may adversely affect offspring reproduction to varying degrees (25, 26). Therefore, the sperm quality of mice in the infected model group was significantly decreased, which manifested as reduced sperm motility, increased deformity rate, and decreased sperm density; this finding was consistent with the results of previous studies that N. caninum can interfere with male reproductive function (19). Additionally, the sperm quality parameters (sperm motility, deformity rate, and density) of mice in the drug treatment group were significantly improved compared to those in the infected model group (27), with DHA showing the most prominent effect, which may be related to its high-efficiency antiparasitic activity and rapid metabolic characteristics. In this study, on the 35th day of drug administration, a decrease in sperm count was observed in the IOPs-treated groups mice. Combined with subsequent expression experiments of spermatogenesis-related genes, abnormalities in the expression of three genes, namely Plzf, Sycp3, and stra8, were observed on the 35th day of drug administration. However, no existing research can explain why this phenomenon occurs. This may require deeper investigation.

The reproductive organs are crucial components of the body’s reproductive system, with the testis and epididymis being two significant internal reproductive organs in male animals. Previous studies have indicated that Neospora can harm the reproductive system of male animals (19). In the HE staining results, it was observed that the testes and epididymis of mice in the blank control group exhibited normal structures. However, in the model group, abnormal arrangement of spermatogenic cells at various stages, shedding of spermatogenic cells, reduction in sperm count, shortening of epithelial cells in the epididymal ducts, and even rupture of the duct walls were evident. Following treatment with polysaccharides from IOPs, ART, and DHA, the pathological changes in the testes and epididymis tissues showed varying degrees of improvement. The number of sperm in the lumens increased, the number of shed spermatogenic cells decreased, the arrangement of spermatogenic cells became clearer, the number of interstitial cells between the ducts increased, and there was a slight rupture of the duct walls. Transmission electron microscopy results revealed normal morphology and structure of various organelles in the tissues and organs of mice in the blank control group. In contrast, the organelles within the cells of mice in the model group exhibited numerous abnormalities, including extensive vacuolization of mitochondria, reduced numbers of Golgi complexes and ribosomes, uneven distribution of peripheral dense fibers in sperm, shrinkage of nuclear membranes, and unclear boundaries. After treatment with polysaccharides from IOPs, the vacuolization of mitochondria significantly improved, the nuclear membrane remained normal, and malformed sperm were rarely observed. Based on the analysis of these three experimental results, it is confirmed that Neospora can cause damage to various tissues and organs, particularly the testes and epididymis, in male mice. However, polysaccharides from IOPs, ART, and DHAn can all mitigate this damage to varying extents, thereby enhancing the body’s resistance to Neospora.

N. caninum infection can induce oxidative stress, leading to a high MDA level and decreased ACP activity in testicular tissue, which suggests exacerbated lipid peroxidation and impaired Sertoli cell function (28–30). NO is a highly reactive gaseous molecule produced by macrophages and formed in liver cells, duodenal cells, and vascular endothelial cells. Studies abroad have shown that high concentrations of NO can inhibit the secretion of testosterone in the body, thereby suppressing sperm production. AsAb is an antibody produced by the male animal’s body itself. It can hinder the normal sperm production, cause sperm agglutination and immobilization, interfere with sperm energy acquisition, thereby reducing sperm survival rate and vitality, leading to infertility symptoms in male animals (31, 32). The results of this study show that the NO level in the serum of mice in the model group is significantly higher than that in the blank control group. However, after administration of three drugs, namely IOP, ART, and DHA, the NO level in the serum gradually returns to normal with time. Compared to the blank control group, the AsAb concentration in the serum of mice in the model group increases and reaches its peak on day 35 of the experiment, indicating that Neospora can cause testicular tissue damage. Furthermore, IOP, ART, and DHA can improve this result; after administration of these drugs, the AsAb concentration decreases relatively. This suggests that Neospora can increase the concentration of AsAb, thereby hindering normal sperm production, reducing sperm motility and vitality, and causing reproductive disorders in male mice.

IgG antibody is considered a key indicator of the humoral immune response triggered by Neospora infection. Neospora infection elicits a Th1-type immune response, and fluctuations in cytokine levels are regarded as a crucial feature of cellular immunity, particularly factors like pro-inflammatory cytokines IL-4 and TNF-α, which are regarded as decisive factors in combating Neospora infection. IFN-γ is primarily produced by activated Th cells and NK cells, while IL-6 stimulates the growth and activation of B cells, leading to antibody production and inhibiting Th1-type immune responses and IFN-γ production. IgE is associated with type I allergic reactions (33–35). The serum levels of proinflammatory factors (TNF-α, IFN-γ, and IL-6) and immunoglobulins (IgG1, IgG2a, and IgE) increased in the infected model group, while the inflammatory response was significantly attenuated after drug treatment, indicating that these three drugs may inhibit excessive inflammatory response by regulating Th1/Th2 immune balance.

qPCR and FCM analysis demonstrated that N. caninum infection can upregulate the expression of proapoptotic genes (Bax, caspase-3, and p53) and inhibit the expression of antiapoptotic genes (Bcl-2), resulting in excessive apoptosis of spermatogenic cells (36, 37). Drug treatment restored the expression of apoptosis-related genes, with IOPs and DHA showing the optimum effect. Dnajb13 is a member of type II heat shock protein 40 family (Hsp40). In testicular tissue structure, Dnajb13 is localized in the cytoplasm of developing sperm cells and mature sperm tail and plays a key role in normal spermatogenesis. Additionally, Mrto4 is abundantly expressed in testis tissue, and it can regulate polyadenylation and deadenylation of some specific mRNAs during spermatogenesis. Ipo11 influences the expression of TSLC1/IGSF4, which enables spermatogenic cells to develop and mature faster (38). During the spermatogenesis process, the expressed Mrto4 and Ipo11 proteins bind to RNA and regulate this process, which is particularly important for maintaining male reproductive function. The expression of key spermatogenesis-related genes (c-kit, PLZF, SYCP3, and Stra8) and spermatogenesis function-related genes (Mrto4 and Ipo11) was downregulated in the infected model group, and their expression levels were restored following drug treatment; this finding confirmed that these three drugs can improve male reproductive function by regulating spermatogenesis-related pathways.

The present study systematically evaluated the protective effects of IOPs, ART, and DHA on the reproductive system of male mice with N. caninum infection for the first time and confirmed that these three drugs can improve reproductive function by reducing oxidative damage, regulating immune response, inhibiting apoptosis, and promoting the expression of spermatogenesis-related genes. DHA showed the most significant inhibitory effect among the three drugs, and its administration schedule can be further optimized in the future to enhance its clinical application potential. This study provides an important theoretical basis for developing anti-neosporosis drugs.