In vitro exposure of bull sperm cells to DMSA-coated maghemite nanoparticles does not affect cell functionality or structure

Figures & data

Table 1. Volumes of SWS, MNP-DMSA work solution and SP-TALP + medium used to form each group.

Treatment group	SWS (μL)	MNP-DMSA work solution (μL)	SP TALP + medium (μL)	Final Fe concentration (mg/mL)
CO	100	–	100	0
G0.015	100	25	75	0.015
G0.03	100	50	50	0.03
G0.06	100	100	–	0.06

SWS: semen work solution; MNP-DMSA: maghemite nanoparticles coated with DMSA; SP-TALP: semen dilution medium corrected with 1.5% of BSA.

Figure 1. Motility (A) and progressive motility (B) of bull sperm in the control and treated groups during the incubation period for 240 min. CO – control group; G0.015: Group treated with MNP-DMSA at the concentration of 0.015 mg Fe/mL; G0.03: Group treated with MNP-DMSA at the concentration of 0.03 mg Fe/mL; G0.06: Group treated with MNP-DMSA at the concentration of 0.06 mg Fe/mL.

Table 2. Motility and progressive motility (mean ± SD) of bull sperm in the control and treated groups during the incubation period for 240 min.

	CO		G0.015		G0.03		G0.06
Time (min)	MOT	PMOT	MOT	PMOT	MOT	PMOT	MOT	PMOT
0	73.2 ± 13.1	60.2 ± 15.4	76.4 ± 6.9	64.1 ± 6.0	74.9 ± 7.3	55.1 ± 22.4	76.1 ± 5.2	61.9 ± 17.5
15	73.1 ± 9.1	58.0 ± 8.7	73.3 ± 7.1	59.8 ± 6.7	71.7 ± 7.6	57.4 ± 9.8	79.7 ± 6.1	65.1 ± 7.9
30	65.2 ± 13.0	50.4 ± 13.4	69.7 ± 9.8	55.0 ± 10.0	70.1 ± 10.0	54.6 ± 9.8	77.8 ± 9.2	63.3 ± 8.6
45	64.3 ± 12.4	52.3 ± 10.5	69.4 ± 6.5	54.7 ± 7.5	65.2 ± 9.5	51.2 ± 10.2	73.9 ± 10.3	57.2 ± 10.5
60	61.3 ± 12.3	47.8 ± 13.0	65.9 ± 8.7	53.0 ± 9.6	64.7 ± 8.9	51.8 ± 10.4	70.7 ± 13.2	57.2 ± 14.4
75	63.0 ± 6.8	50.2 ± 7.8	63.1 ± 12.1	49.3 ± 11.3	64.6 ± 12.0	50.5 ± 12.4	65.1 ± 14.5	52.2 ± 13.4
90	58.8 ± 13.2	47.0 ± 13.4	63.7 ± 13.1	51.7 ± 13.5	63.7 ± 10.9*	48.4 ± 11.7	65.1 ± 17.9	50.8 ± 16.9
105	55.2 ± 9.0	41.2 ± 8.7*	60.7 ± 10.3	47.4 ± 12.1*	56.1 ± 13.4*	42.7 ± 11.9	60.6 ± 16.6	45.6 ± 16.6
120	56.0 ± 9.8	39.9 ± 10.1*	55.1 ± 13.0*	38.8 ± 12.8*	55.0 ± 14.38*	38.6 ± 13.3	58.2 ± 16.9*	44.7 ± 15.7
180	40.6 ± 13.1*	26.8 ± 8.6*	40.4 ± 13.0*	30.4 ± 12.3*	37.0 ± 15.7*	25.8 ± 14.3*	45.6 ± 14.6*	30.2 ± 14.0*
240	31.8 ± 15.7*	23.3 ± 14.5*	32.2 ± 12.4*	21.8 ± 11.8*	27.0 ± 15.8*	18.6 ± 15.4*	33.0 ± 15.3*	22.8 ± 16.1*

MOT: motility; PMOT: progressive motility; CO: control group; G0.015: group treated with MNP-DMSA at the concentration of 0.015 mg Fe/mL; G0.03: group treated with MNP-DMSA at the concentration of 0.03 mg Fe/mL; G0.06: group treated with MNP-DMSA at the concentration of 0.06 mg Fe/mL.

* Indicates values significantly different from T0 (p < 0.05).

Figure 2. Qualitative parameters of sperm movement (mean ± SEM) during the incubation period. (A) STR – straightness coefficient (%), (B) LIN – linearity coefficient (%), (C) BCF – frequency of head displacement (Hz) and (D) ALH – lateral head amplitude (μm). *Indicates significant difference from time 0 (p < 0.05).

Figure 3. Velocity parameters of sperm movement (mean ± SEM) during the incubation period. (A) VAP – mean velocity (μm/s), (B) VSL – linear velocity (μm/s) and (C) VCL – curvilinear velocity (μm/s). *Indicates significant difference from time 0 (p < 0.05).

Figure 4. Percentages (media ± SD) of (A) viable sperm cells, (B) non-viable sperm cells and (C) sperm cells with intact membrane and reacted acrosome during the incubation period with or without MNP-DMSA. ^a,bDifferent letters indicate significant difference among times in the same group (p < 0.05). CO – control group; G0.015: Group treated with MNP-DMSA at the concentration of 0.015 mg Fe/mL; G0.03: Group treated with MNP-DMSA at the concentration of 0.03 mg Fe/mL; G0.06: Group treated with MNP-DMSA at the concentration of 0.06 mg Fe/mL.

Figure 5. Transmission electron micrographies of bull spermatozoa after 4 h of incubation. (A) CO – control group, (B) G0.015: Group treated with MNP-DMSA at the concentration of 0.015 mg Fe/mL, (C) G0.03: Group treated with MNP-DMSA at the concentration of 0.03 mg Fe/mL and (D) G0.06: Group treated with MNP-DMSA at the concentration of 0.06 mg Fe/mL. Note the presence of MNP-DMSA clusters close to the head of the sperm cells in all treated groups (arrows). Nanoparticles were not observed inside the sperm cytoplasm. pm: plasmatic membrane, a: acrosome, nu: nucleus, h: head, t: tail.

Figure 6. Transmission electron micrographies of bull spermatozoa after 4 h of incubation. (A) CO – control group, (B) G0.03: Group treated with MNP-DMSA at the concentration of 0.03 mg Fe/mL. Arrows indicate disruption in plasma membrane in both groups. pm: plasmatic membrane, a: acrosome, nu: nucleus.