Infection of Trichinella spiralis Affects the Reproductive Capacity of ICR/CD-1 Male Mice by Reducing the Urine Pheromone Contents and Sperm Quality

Abstract

Female mice can discriminate the urinary odors of male mice due to their olfactory acuity. Parasitic infection or subclinical infection can decrease the odor attractiveness of male mice and finally lead to aversion or avoidance responses in odor selection for female mice. Trichinella spiralis is a kind of tissue-parasitizing nematode that causes trichinellosis, a zoonotic parasitic disease that spreads throughout the world. However, the reproductive injury caused by Trichinella spiralis infection was not fully revealed. In this study, we explored the effect of Trichinella spiralis infection on the reproductive capacity in ICR/CD-1 male mice. We identified eight volatile compounds in urine by GC-MS analysis, and the results indicated that the contents of dimethyl sulfone, Z-7-tetradecen-1-ol, 6-Hydroxy-6-methyl-3-heptanone and (S)-2-sec-butyl-4,5-dihydrothiazole were significantly downregulated after parasitic infection, which might lead to the reduction of attractiveness of male mice urine to females. On the other hand, parasitic infection decreased sperm quality and downregulated the expression levels of Herc4, Ipo11, and Mrto4, and these genes were strongly related to spermatogenesis. In summary, this study revealed that the reproductive injury caused by Trichinella spiralis infection in ICR/CD-1 male mice could be associated with a decrease in urine pheromone content and sperm quality.

Keywords: ICR/CD-1 male mice; Trichinella spiralis; reproductive injury; sperm quality; urine pheromone content.

Figure 1

Trichinella spiralis infection decreased growth rate of ICR/CD-1 male mice and induced specific antibody production. (A) The significant differences between the infection group and control group appeared at 30 days post-infection and parasitic infection decreased host growth rate. (B) Parasitic infection induced specific antibody production, the results of absorbance 450 nm were determined by ELISA method and the two groups showed significant differences at 20-, 30- and 40-days post-infection. All data are presented as means ± standard deviation, Two-way ANOVA was used for significance analysis, *** p < 0.001, **** p < 0.0001.

Figure 2

Trichinella spiralis infection decreased the attractiveness of male mouse urine to female mice. (A) At 0-, 10-, 20- and 30-days post-infection, the urine protein levels in infection group were determined and showed significant decreases when compared with the control group. (B) The behavioral test was performed at 10, 20 and 30 days after Trichinella spiralis infection, the two groups showed significant differences at 20- and 30-days post-infection. All data are presented as means ± standard deviation, Two-way ANOVA was used for significance analysis, * p < 0.1; ** p < 0.01, **** p < 0.0001.

Figure 3

The infection of Trichinella spiralis decreased the pheromone content in the male mice urine. (A) The representative gas chromatogram from infection group was presented and the GC peaks were indicated. (B,C) The comparison of the absolute content of Z-7-tetradecen-1-ol (GC Peak 3), Dimethyl sulfone (GC Peak 5), 6-Hydroxy-6-methyl-3-heptanone (GC Peak 6), and (S)-2-sec-butyl-4,5-dihydrothiazole (GC Peak 8) between the infection group and the control group. (D) The comparison of the relative amount of 1-Tetradecanol (GC peak 4), Dimethyl sulfone (GC peak 5), 6-Hydroxy-6-methyl-3-heptanone (GC peak 6), and R,R-3,4-dehydro-exo-brevicomin (GC peak 7) between the infection group and control group. All data are presented as means ± standard deviation, Two-way ANOVA was used for comparison between the two groups, * p < 0.1, ** p < 0.01, **** p < 0.0001.

Figure 4

Trichinella spiralis infection hampered the reproductive capacity of the ICR/CD-1 male mice. (A) Parasitic infection decreased the conception rate of male mice. (B) The average number of offspring in the second births from the infection group was significantly lower than that in the control group. (C) The average body weight of the offspring after birth showed no significant differences between the two groups. (D) After weaning, the offspring body weight showed significant decreases at the second births for the infection group. All data are presented as means ± standard deviation, Two-way ANOVA was used for comparison between the infection group and the control group, * p < 0.1, **** p < 0.0001, ns non-significant.

Figure 5

Trichinella spiralis infection damaged spermatogenesis and decreased the sperm quality in ICR/CD-1male mice. Sperm count (A) and sperm morphology (B) were significantly decreased after parasitic infection, and the sperm deformities (C) were significantly increased. It should be noted that the sperm damage caused by parasitic infection would get worse over time. All data are presented as means ± standard deviation, Two-way ANOVA was used for comparison between the infection group and the control group, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 6

Parasitic infection downregulated the gene expression levels of Herc4 (A), Ipo11 (B) and Mrto4 (C). The gene expression levels were determined by qRT-PCR. Each sample was tested in triplicate, and β-actin was used as the normalization control. All data are presented as means ± standard deviation, Two-way ANOVA was used for comparison between the two groups, * p < 0.1, ** p < 0.01, **** p < 0.0001, ns non-significant.