Protective effects of canthaxanthin-loaded seminal exosomes on the quality of human spermatozoa during cryopreservation

Sperm cryopreservation is a common strategy for preserving male fertility; however, it may cause detrimental alterations in the structure and biological role of spermatozoa. The objective of this study is to mitigate the damages caused by cryopreservation through the use of canthaxanthin-loaded seminal exosomes (CAN-loaded EXO). To achieve this, Exosomes were isolated from the human seminal fluid through serial ultracentrifugation and characterized using different methods. Canthaxanthin was loaded into exosomes using the sonication method and incubated with spermatozoa for 1 h. Then the semen was mixed with CryoSperm medium and transferred into straws. The straws were initially placed in nitrogen vapor and then submerged in liquid nitrogen. After one month, the samples were thawed, and sperm parameters, lipid peroxidation, total antioxidant capacity (TAC), reactive oxygen species (ROS) levels, sperm DNA fragmentation (SDF), and sperm apoptosis were assessed. The results demonstrated the successful isolation of exosomes with a spherical morphology, a particle size of 104.5 nm, and a zeta potential of -12.32 mV. Canthaxanthin was loaded into exosomes with an encapsulation efficiency of 63.7 %. Motility and vitality of spermatozoa were improved in the presence of CANloaded EXO compared to the control group (p < 0.05). Incubation of spermatozoa with CAN-loaded EXO enhanced TAC levels and decreased ROS levels, lipid peroxidation, SDF, and apoptosis compared to the control group (p < 0.05). However, CAN-loaded EXO had no significant effect on the morphology of spermatozoa. These findings suggest that exosomes have the potential to serve as a novel drug delivery platform for protecting spermatozoa from cryodamage while enhancing the bioavailability of antioxidants.