Vitamin E Lipid-Based Nanodevices as a Tool for Ovine Sperm Protection against Oxidative Stress: Impact on Sperm Motility

Abstract

The advent of nanotechnology in the field of animal reproduction has led to the development of safer and more efficient therapies. The use of nanotechnology allows us to avoid the detrimental effects of certain traditional antioxidants, such as Vitamin E. Its hydrophobic nature makes mandatory the use of organic solvents, which are toxic to sperm cells. This study aims to evaluate the efficiency of vitamin E nanoemulsions (NE) on ram (Ovis aries) spermatozoa. For this purpose, the effect of three NE concentrations (6, 12, and 24 mM) were assessed on sperm of 10 mature rams of the Manchega breed. Sperm samples were collected by artificial vagina, pooled, and diluted in Bovine Gamete Medium. The samples were stored at 37 °C and assessed at 0, 4, 8, and 24 h under oxidative stress conditions (100 µM Fe²⁺/ascorbate). Motility (CASA), viability (YO-PRO/IP), acrosomal integrity (PNA-FITC/IP), mitochondrial membrane potential (Mitotracker Deep Red 633), lipoperoxidation (C₁₁ BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (SCSA^®®) were assessed. A linear mixed-effects models were used to analyze the effects of time, NE, and oxidant (fixed factors) on sperm parameters, and a random effect on the male was also included in the model with Tukey's post hoc test. Protection of ram spermatozoa with NE resulted in a more vigorous motility under oxidative stress conditions with respect Control and Free vitamin E, while preventing the deleterious effects of oxidative stress coming from the production of free radicals and lipid peroxidation. These results ascertain the high relevance of the use of delivery systems for sperm physiology preservation in the context of assisted reproduction techniques.

Keywords: drug liberation; nanoemulsions; nanotechnology; sperm oxidative stress; vitamin e.

Figure 1

Vitamin E nanoemulsions on confocal microscopy. Ram spermatozoa stained with the fluorescent combination of Hoechst and DiD oil under a confocal laser scanning microscope. (a) Nucleus of sperm head stained by Hoechst 33342. (b) Vitamin E nanoemulsions labelled with DiD (DiIC₁₈(5). (c) Spermatozoa marked with the fluorescent combination.

Figure 2

Vitamin E nanoemulsions effects on total and progressive sperm motility. Plots show the triple interaction of different treatments of vitamin E × oxidative treatment × incubation time for CASA-derived variables. The various lowercase Latin letters (p < 0.05) indicate significant differences between treatments of vitamin E, while asterisks (p < 0.05) compare each treatment at the same time with the different oxidative stress status.

Figure 3

Vitamin E nanoemulsions effects on velocity sperm parameters. Plots show the triple interaction of different treatments of vitamin E × oxidative treatment × incubation time for CASA-derived variables. The various lowercase Latin letters (p < 0.05) indicate significant differences between treatments of vitamin E, while asterisks (p < 0.05) compare each treatment at the same time with the different oxidative stress status.

Figure 4

Vitamin E nanoemulsions effects on sperm viability. Plots show the triple interaction of different treatments of Vitamin E × oxidative treatment × incubation time for the flow cytometry analysis of sperm viability. The various lowercase Latin letters (p < 0.05) indicate significant differences between treatments of vitamin E.

Figure 5

Vitamin E nanoemulsions effects on acrosome integrity. Plots show the triple interaction of different treatments of Vitamin E × oxidative treatment × incubation time for the flow cytometry analysis of mitochondrial activity (PNA−/PI−). The various lowercase Latin letters (p < 0.05) indicate significant differences between treatments of vitamin E, while different Greek letters (p < 0.05) compare each treatment at the same time with the different oxidative stress status.

Figure 6

Vitamin E nanoemulsions effects on mitochondrial activity. Plots show the triple interaction of different treatments of Vitamin E × oxidative treatment × incubation time for the flow cytometry analysis of mitochondrial activity (YO-PRO-1−/Mitotracker deep red+). The various lowercase Latin letters (p < 0.05) indicate significant differences between treatments of vitamin E.

Figure 7

Vitamin E nanoemulsions effects on ROS production and lipid peroxidation. Plots show the triple interaction of different treatments of Vitamin E × oxidative treatment × incubation time for the flow cytometry analysis of reactive oxygen species (median fluorescence of H2DCFDA in PI− sperm) and lipid peroxidation (median green fluorescence of BODIPY C11). The various lowercase Latin letters (p < 0.05) indicate significant differences between treatments of vitamin E, while asterisks (p < 0.05) compare each treatment at the same time with the different oxidative stress status.

Figure 8

Vitamin E nanoemulsions effects on DNA. Plots represent the triple interaction of different treatments of Vitamin E × oxidative treatment × incubation time for the flow cytometry analysis of DNA Fragmentation Index and High DNA Stainability (median fluorescence of sperm assessed by SCSA^®®). The various lowercase Latin letters (p < 0.05) indicate significant differences between treatments of vitamin E, while asterisks (p < 0.05) compare each treatment at the same time with the different oxidative stress status.