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Simple Summary  Artificial insemination with extended semen is a reproductive biotechnological method promising quick genetic improvement and benefits for farmers. Semen collection in farm animals, including rams, is a procedure that takes place under unsterile conditions. Apart from certain bacteria, which are pathogenic and may be transmitted through semen to the female genital tract, causing clinical diseases, semen from healthy donors may also be contaminated during processing of the ejaculate. Consequently, ejaculate contamination with bacteria is inevitable. Antibiotics are a main constituent of semen extenders to avoid growth of bacteria present in ram semen and, subsequently, enhance reproductive performance. During the last decades, however, there is an increasing scientific interest about alternative agents to the conventional antibiotics, such as nanoparticles, to overcome bacterial resistance, which is a serious problem for human and veterinary medicine. Therefore, the aim of this study was to examine the effect of iron oxide and silver nanoparticles’ supplementation on bacterial proliferation control during ram semen processing, as well as their impact on sperm quality and functionality variables.    Abstract  The aim of the study was to investigate the effect of iron oxide (Fe) and silver (Ag) nanoparticles (NPs) on ram semen. A skim milk extender without antibiotics was used as a diluent of 21 ejaculates (8 rams; 2–3 ejaculates/ram). The groups of control (C; semen without NPs), Fe NPs (3.072 mg Fe 3 O 4 /mL semen), and Ag NPs (2.048 mg Ag-Fe/mL semen) were incubated (15 °C; 30 min), and then a magnetic field was used for NPs’ removal. Standard microbiological procedures were performed for all groups. Post-treated samples were stored (15 °C) for 24 h, and sperm variables (kinetics by computer assisted sperm analysis (CASA); viability; morphology; HOST; DNA integrity) were evaluated at 6 and 24 h. Semen data were analyzed by a mixed model for repeated measures and microbiological data with Student’s  t -test for paired samples. At 6 h of storage, VCL and rapid movement-spermatozoa, and at 24 h, total/progressive motility and amplitude of lateral head displacement (ALH) were significantly decreased in group Ag compared to control. In group Fe, progressive/rapid movement-spermatozoa were significantly lower compared to control after 24 h of storage. Only in group Ag was a significant reduction of total bacterial count revealed. In conclusion, the examined Fe NPs demonstrated slight antibacterial effect, while the examined Ag NPs provided higher antibacterial properties accompanied by cytotoxicity.

Toxic and Microbiological Effects of Iron Oxide and Silver Nanoparticles as Additives on Extended Ram Semen