Curcumin-loaded niosomal nanocarriers offer a promising approach to improve quality characteristics, apoptotic gene expression, and flow cytometry assessments of stallion spermatozoa after thawing.

Overview

• This study investigates the use of curcumin-loaded niosomal nanocarriers (CurLNN) to improve the quality and function of stallion spermatozoa after thawing from cryopreservation.
• The research focuses on how CurLNN affects sperm motility, apoptosis-related gene expression, oxidative stress indicators, and cellular integrity measures.

Background

• Cryopreservation is a widely used technique for preserving spermatozoa, but the freezing and thawing process often leads to oxidative damage that reduces sperm quality and fertility potential.
• Oxidative stress during cryopreservation can cause membrane damage, DNA fragmentation, impaired mitochondrial function, and apoptosis-like changes in sperm cells.
• Antioxidants are explored as additives to cryopreservation media to protect sperm from oxidative damage and improve post-thaw viability and functionality.
• Curcumin is a natural antioxidant with potential to reduce oxidative stress, but its use is limited by poor bioavailability; loading curcumin into niosomal nanocarriers (CurLNN) can enhance delivery and effectiveness.

Objectives

• To evaluate the antioxidant effects of curcumin alone (Cur) and curcumin-loaded niosomal nanocarriers (CurLNN) on stallion sperm post-thaw characteristics.
• To analyze the impact on sperm motility parameters, membrane functionality, oxidative stress markers, and apoptosis-related gene expression in cryopreserved stallion spermatozoa.
• To assess the efficacy of CurLNN compared to free curcumin in improving sperm quality after thawing.

Methodology

• Twenty-five stallion ejaculates were collected and diluted in INRA96 freezing medium supplemented either with 20 μM curcumin or 20 μM CurLNN.
• The samples were cryopreserved and later thawed for analyses.
• Sperm motility was assessed using parameters such as total motility, progressive motility, average path velocity (VAP), straight line velocity (VSL), and straightness (STR).
• Plasma membrane functionality was evaluated to determine membrane integrity.
• The level of sperm abnormalities was recorded.
• Viability was measured alongside malondialdehyde (MDA) levels, a marker of lipid peroxidation and oxidative damage.
• Flow cytometry was employed to analyze mitochondrial membrane potential, reactive oxygen species (ROS) production, apoptotic-like changes, and chromatin integrity.
• Expression of apoptotic regulatory genes was quantified, focusing on Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic) genes.

Key Findings

• Sperm treated with both Cur and CurLNN showed improved total and progressive motility and superior kinematic parameters (VAP, VSL, STR), with CurLNN showing greater enhancement.
• Plasma membrane functionality improved significantly, indicating better membrane integrity after thawing.
• The rate of sperm abnormality decreased significantly with both treatments.
• Viability of sperm cells increased, especially with CurLNN.
• MDA levels were reduced, indicating lowered lipid peroxidation and oxidative stress.
• Flow cytometry results demonstrated enhanced mitochondrial membrane potential and reduced ROS production, suggesting improved mitochondrial function and reduced oxidative damage.
• The proportion of live sperm increased, whereas late apoptotic sperm decreased post-thaw when treated with Cur and CurLNN.
• Bcl-2 (anti-apoptotic) gene expression increased with both treatments, supporting enhanced cell survival.
• CurLNN caused a significant reduction in Bax (pro-apoptotic) gene expression, indicating decreased apoptosis.

Conclusions and Implications

• The encapsulation of curcumin into niosomal nanocarriers (CurLNN) enhanced its antioxidant properties, resulting in better protection of stallion spermatozoa during cryopreservation.
• CurLNN improved multiple aspects of sperm quality post-thaw, including motility, membrane integrity, oxidative stress reduction, and gene expression related to apoptosis.
• This approach shows promise for improving the outcomes of sperm cryopreservation by minimizing oxidative damage and cellular apoptosis.
• Such improvements in sperm quality are crucial for assisted reproduction and breeding programs in equine species.
• Future work may further optimize these nanocarrier systems and explore their application across other species and cryopreservation protocols.