Niosomal nanocarriers loaded with artemisinin has potential to enhance the functional characteristics of cryo-preserved equine spermatozoa.

Niosomal nanocarriers loaded with artemisinin improve the quality and function of frozen-thawed equine sperm by protecting them from damage caused by the freezing process. This study demonstrated enhanced sperm motility, viability, and reduced oxidative stress when using these nanocarriers compared to conventional artemisinin or controls.

Background and Purpose

• Cryopreservation is vital for preserving genetic material in animals but often damages cells due to oxidative stress during freezing and thawing.
• Equine spermatozoa are particularly sensitive to cryo-oxidative damage, leading to reduced viability and fertility post-thaw.
• The study aimed to investigate whether artemisinin, a natural antioxidant, when loaded into niosomal nanocarriers (NN-Art), could better protect sperm cells during cryopreservation compared to free artemisinin or no supplementation.

Methods and Experimental Design

• Equine sperm samples were cryopreserved using a diluent medium either supplemented with:
  • NN-Art (niosomal nanocarriers containing artemisinin),
  • Free artemisinin (Art), or
  • No supplementation (control).
• Post-thaw sperm quality was evaluated by multiple assays:
  • Motility and Kinematics: Computer-assisted sperm analysis (CASA) to measure total motility, progressive motility, and velocity.
  • Plasma Membrane Integrity: Hypoosmotic swelling test (HOS) to assess functional membrane status.
  • Viability: Eosin-nigrosin staining distinguishing live versus dead sperm.
  • Morphology: Hancock’s solution used to identify abnormal sperm shapes.
  • Lipid Peroxidation: Thiobarbituric acid reactive substances (TBARS) assay to evaluate oxidative damage to sperm membranes.
  • Flow Cytometry: Measurement of mitochondrial membrane potential, reactive oxygen species (ROS) levels, sperm chromatin integrity, and apoptotic-like changes.

Key Findings

• Sperm Motility: Addition of NN-Art significantly improved total motility, progressive motility, and straight line velocity compared to both free artemisinin and control groups.
• Viability and Membrane Function: NN-Art led to higher sperm viability and better plasma membrane functionality post-thaw.
• Sperm Morphology: Lower proportions of abnormal sperm morphology were observed with NN-Art supplementation.
• Lipid Peroxidation: NN-Art significantly reduced lipid peroxidation, indicating decreased oxidative membrane damage.
• Mitochondrial Function and ROS Levels: Flow cytometry showed increased mitochondrial membrane potential and decreased ROS levels in sperm treated with NN-Art, suggesting improved energy status and reduced oxidative stress.
• Apoptotic-like Changes: NN-Art decreased late apoptotic sperm percentages, indicating a protective effect against cryo-induced cell death.

Interpretation and Implications

• Niosomal delivery of artemisinin enhances its antioxidant capacity, providing superior protection for equine sperm during the freeze-thaw process.
• The improved motility and membrane integrity suggest that NN-Art helps maintain sperm functionality critical for fertilization potential.
• Reduction in ROS and apoptosis indicates mitigation of cryo-oxidative stress, which is a major cause of sperm quality loss during cryopreservation.
• This nanoformulation approach could be a promising strategy for improving cryopreservation outcomes in equine breeding and potentially other species.
• Further studies may explore in vivo fertility outcomes and optimize dosage for clinical use.