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Semen cryopreservation is a well-established procedure used in veterinary assisted reproduction technology applications. We investigated damaging effects of cryopreservation on the structural and ultrastructural characteristics of bull sperm induced at different temperatures and steps during standard cryopreservation procedure using transmission (TEM) and scanning electron microscopy. We also examined the effect of cryopreservation on sperm DNA and chromatin integrity. Five healthy, fertile Friesian bulls were used, and the ejaculates were obtained using an artificial vagina method. The semen samples were pooled and diluted in a tris-yolk fructose (TYF) for a final concentration of 80 × 106 spermatozoa/ml. The semen samples were packed in straws (0.25 ml), and stored in liquid nitrogen (−196°C). Samples were evaluated before dilution, just after dilution (at 37°C), at 2 h and 4 h during equilibration, and after thawing (37°C for 30 s in water bath). In association with step-wise decline in motility and viability, our results showed that the plasma membrane surrounding the sperm head was the most vulnerable structure to cryo-damage with various degrees of swelling, undulation, or loss affecting about 50% of the total sperm population after equilibration and freezing. Typical acrosome reaction was limited to 10% of the spermatozoa after freezing. We also observed increased number of mitochondria with distorted cristae (15%). Chromatin damage was significantly increased by cryopreservation as evident by TEM (9%). This was mainly due to DNA breaks as confirmed by Sperm Chromatin Structure Assay (SCSA) (8.4%) whereas the chromatin structure was less affected as evaluated microscopically by toluidine blue staining. We concluded that, using standard cryopreservation protocol, the most pronounced damage induced by cryopreservation is observed in the plasma membrane. Further improvement of cryopreservation protocols should thus be targeted at reducing plasma membrane damage. Acrosomal, mitochondrial and chromatin damage are also evident but appear to be within acceptable limits as discussed.

There are several obstacles in camel semen cryopreservation; such as increasing semen viscosity and the reduction in motile spermatozoa after ejaculation. Epididymal spermatozoa offer an efficient alternative to overcome these problems and are well-suited for artificial insemination in camels. In the current study, we compared the effects of supplementation with vitamin C, E, inorganic trace elements of selenium (Na2SeO3) and zinc (ZnSO4), and zinc and selenium nanoparticles (ZnONPs and SeNPs, respectively) on the cryopreservation of dromedary camel epididymal spermatozoa. When the SHOTOR extender was supplemented with ZnONPs and SeNPs; the sperm showed increased progressive motility; vitality; and membrane integrity after cooling at 5 °C for 2 h; when compared to the control and vitamin-supplemented groups. Moreover, the ZnONPs and SeNPs supplementation improved the progressive motility, vitality, sperm membrane integrity, ultrastructural morphology, and decreased apoptosis when frozen and thawed. SeNPs significantly increased reduced glutathione (GSH), superoxide dismutase (SOD), and decreased lipid peroxide malondialdehyde (MDA) levels. The advantageous effects of the trace elements were potentiated by reduction into a nano-sized particle, which could increase bioavailability and reduce the undesired liberation of toxic concentrations. We recommend the inclusion of SeNPs or ZnONPs to SHOTOR extenders to improve the cryotolerance of camel epididymal spermatozoa.

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