Magnetic nanopurification of ram semen using Mase V particles for depletion of morbid and DNA fragmented spermatozoa
Abstract
The aim of this study was to enhance the quality of fresh ram semen by purification of spermatozoa using combination of annexin V and lectin (PNA and LCA) coated nanoparticles. Semen samples from two rams were collected regularly during the breeding season using electro-ejaculation. Samples (n=5) with poor quality (decreased sperm motility and viability) were chosen for the nanopurification procedure. Fresh (control) samples and purified (depleted from damaged spermatozoa) samples were analysed by CASA for total and progressive motility. Flow cytometry was used to check presence of apoptotic-like changes (annexin V-FITC) and integrity of acrosome (PNA-AF488 and rhodamine LCA) as well as proportion of dead cells (propidium iodide) in semen samples before and after nanopurification. Transmission electron microscopy (TEM) was performed to assess changes in sperm ultrastructure and interaction with nanoparticles. Purified ram spermatozoa exhibited increased viability (p<0.01) and both motility parameters (p<0.05) and decreased proportion of spermatozoa with damaged acrosome (p<0.05). On the other hand, no significant differences were observed for annexin V and LCA positive spermatozoa between fresh and purified samples. TEM analysis revealed occurrence of spermatozoa with swollen acrosome and damaged plasma membrane as well as abundance of nanoparticles surrounding the cells or bound to the membrane in positively stained spermatozoa. In conclusion, nanopurification process can improve the quality of ram semen, mainly in terms of sperm viability, motility and presence of damaged spermatozoa, which might result in better fertilization ability of insemination doses or even higher cryosurvival rates of stored spermatozoa.
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