*2.3. Effects of OPs on Sperm Parameters of TP-Induced Mice*

The model of TP-induced mice spermatogenesis dysfunction was established, and mice were treated for 4 weeks. The intervention of TP resulted in severe sperm distortion in mice, and the quantity and quality of sperm were significantly lower than those in the control group. It was even difficult to find an intact normal sperm. As shown in Figure 1A, the sperm morphology of TP-treated mice revealed an increase in acrosome abnormalities: fathead, bent neck, short tail, and coiled-in tail. The treatment of OPs significantly increased the number of normal sperms, elevated the sperm count, improved the sperm motility, and decreased the sperm deformity rate (Figure 1B–D). The effects of different doses of OPs showed a dose dependence, and high dose of OPs treatment significantly ameliorated sperm damage induced by TP (*p* < 0.001).

**Figure 1.** Effects of OPs on sperm quality of ICR mice induced by TP. (**A**) Sperm morphology was observed at 200× magnification; OPs -L, OPs -M, and OPs -H are the groups administrated with 100, 200, and 400 mg/kg OPs separately. (**B**), (**C**), and (**D**) indicate the sperm count, motility, and deformity rate of sperm, respectively. The data were expressed as mean ± SEM, n = 10. Compared with the control group, ### *p* < 0.001; compared with the TP group, \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001.

In this research, VE was chosen as a positive control, based on the fact that its metabolite tocopheryl frequently applied for the promotion of reproductive hormone secretion, increasing sperm numbers and motility, preventing male infertility in the clinic. As shown in Figure 1, VE treatment significantly ameliorated sperm damage induced by TP (*p* < 0.01).

#### *2.4. Effects of OPs on Testicular Injury of TP-Induced Mice*

The results revealed that there was no significant difference in the bodyweight of mice among the groups (Figure 2B). As shown in Figure 2C, the testis index of the TP group was significantly lower than that in the control group (*p* < 0.001), while treatment of OPs ameliorated the testicular weight loss compared with the TP group. The structure of testicular tissue and the number of testicular cells play an important role in spermatogenesis and sperm quality. The testicular structures and cells play important roles during spermatogenesis, while an ample array of factors can influence its quality and quantity [29]. Histological analysis on the tissue sections of H&E staining showed that OPs treatment protected testis tissue against the damage caused by TP. As compared with controls, TP-induced mice showed severe vacuolation of germ cells, enlarged intercellular spaces, irregular shape, and atrophied seminiferous tubules with only a few Sertoli cells, spermatogonia, and primary spermatocytes (Figure 2A). VE and high-dose OPs (400 mg/kg) treatment restored morphological abnormalities compared with the TP group; vacuolation of germ cells and spermatocyte was decreased. The size of the seminiferous tubule, the layer of the spermatocytes, and the number of Sertoli cells were preserved by the treatment of VE and high-dose OPs (Figure 2A). The overall structures of the seminiferous tubule in testis were evaluated by Johnsen's scoring method. Compared with the TP group, the middle and high dose of OPs elevated Johnsen's score significantly (*p* < 0.01) (Figure 2D).

**Figure 2.** Effects of OPs on testicular injury of ICR mice induced by TP. (**A**) Histopathology with H&E staining (200× and 400×) of the testicular section in mice after treatment for 28 days; (**B**) body weight of mice was measured every 3 days; (**C**) testis index was measured by the ratio of testicular weight to body weight; (**D**) Johnsen's score in the testicular tissue was determined in each group. The data were expressed as mean ± SEM, n = 10. Compared with the control group, ### *p* < 0.001; compared with the TP group, \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001.
