Vitamin E Delivery Systems Increase Resistance to Oxidative Stress in Red Deer Sperm Cells: Hydrogel and Nanoemulsion Carriers

Abstract

Oxidative stress has become a major concern in the field of spermatology, and one of the possible solutions to this acute problem would be the use of antioxidant protection; however, more studies are required in this field, as highly contradictory results regarding the addition of antioxidants have been obtained. Vitamin E is a powerful biological antioxidant, but its low stability and high hydrophobicity limit its application in spermatology, making the use of organic solvents necessary, which renders spermatozoa practically motionless. Keeping this in mind, we propose the use of hydrogels (HVEs) and nanoemulsions (NVEs), alone or in combination, as carriers for the controlled release of vitamin E, thus, improving its solubility and stability and preventing oxidative stress in sperm cells. Cryopreserved sperm from six stags was thawed and extended to 30 × 10⁶ sperm/mL in Bovine Gamete Medium (BGM). Once aliquoted, the samples were incubated as follows: control, free vitamin E (1 mM), NVEs (9 mM), HVEs (1 mM), and the combination of HVEs and NVEs (H + N), with or without induced oxidative stress (100 µM Fe²⁺/ascorbate). The different treatments were analyzed after 0, 2, 5, and 24 h of incubation at 37 °C. Motility (CASA^®), viability (YO-PRO-1/IP), mitochondrial membrane potential (Mitotracker Deep Red 633), lipid peroxidation (C₁₁ BODIPY 581/591), intracellular reactive oxygen species production (CM-H₂DCFDA), and DNA status (SCSA^®) were assessed. Our results show that the deleterious effects of exogenous oxidative stress were prevented by the vitamin E-loaded carriers proposed, while the kinematic sperm parameters (p ˂ 0.05) and sperm viability were always preserved. Moreover, the vitamin E formulations maintained and preserved mitochondrial activity, prevented sperm lipid peroxidation, and decreased reactive oxygen species (ROS) production (p ˂ 0.05) under oxidative stress conditions. Vitamin E formulations were significantly different as regards the free vitamin E samples (p < 0.001), whose sperm kinematic parameters drastically decreased. This is the first time that vitamin E has been formulated as hydrogels. This new formulation could be highly relevant for sperm physiology preservation, signifying an excellent approach against sperm oxidative damage.

Keywords: antioxidant; hydrogel; nanoemulsions; sperm oxidative stress; vitamin E.

Figure 1

Design of the transwell system. (a) Gelation scheme on the plate well; (b) Sperm sample incubation into the transwell insert to avoid the mixture with the hydrogel; (c) Scheme of vitamin E and sperm dynamics across the polycarbonate membrane.

Scheme 1

Nanoemulsion and hydrogel-based vitamin E carrier composition and release mechanism.

Figure 2

Stability and toxicity of the carriers:(a) Storage stability of NVEs in PBS and BGM. Data are stated as mean ± SEM from at least three independent experiments; (b) image of the HVEs using test tube inversion method; (c) differences between control and unloaded hydrogel; (c) vitamin E formulation effects on ROS production and lipid peroxidation. The results show MIF (mean intensity fluorescence). Plots represent the interaction of different treatments × 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).

Figure 3

In vitro release profiles: (a) NVEs in PBS and BGM at 37 °C; (b) HVEs in PBS at 37 °C.

Figure 4

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

Figure 5

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

Figure 6

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

Figure 7

Vitamin E formulation effects on sperm viability. Plots (a,b) represent the triple interaction of different vitamin E treatments × oxidative treatment × incubation time for the flow cytometry analysis of sperm viability. The various lowercase Latin letters (p < 0.05) indicate significant differences between vitamin E treatments, while the different Greek letters (p < 0.05) compare each treatment at the same time with a different oxidative stress status.

Figure 8

Vitamin E formulation effects on mitochondrial activity. Plots represent the triple interaction of different vitamin E treatments × oxidative treatment × incubation time for the flow cytometry analysis of mitochondrial activity (YOPRO1−/Mitotracker deep red+). The various lowercase Latin letters (p < 0.05) indicate significant differences between vitamin E treatments, while the different Greek letters (p < 0.05) compare each treatment at the same time with a different oxidative stress status.

Figure 9

Vitamin E formulation effects on ROS production (a) and lipid peroxidation (b). The results show MFI (mean fluorescence intensity). Plots represent the triple interaction of different vitamin E treatments × 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 vitamin E treatments, while the different Greek letters (p < 0.05) compare each treatment at the same time with a different oxidative stress status.

Figure 10

Vitamin E formulation effects on DNA. Plots (a,b) represent the triple interaction of different vitamin E treatments × 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 vitamin E treatments, while the different Greek letters (p < 0.05) compare each treatment at the same time with a different oxidative stress status.