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    Dose- and size-dependent effects of Fe3O4 nanoparticles on bull sperm cryopreservation
    (Academic Press Inc Elsevier Science, 2026) Ozturk, Ali Erdem; Dadi, Seyma; Bodu, Mustafa; Korkmaz, Oya; Atay, Yunus Emre; Sariozkan, Serpil; Ocsoy, Ismail
    Fe3O4 nanoparticles (Fe3O4 NPs) are widely used in health sciences, including reproductive biotechnology. In reproductive systems, they have been applied in sperm cryopreservation, nano-purification, drug delivery, and toxicity assessments. However, most studies have evaluated Fe3O4 NPs at only a single size and dose, even though their biological effects may vary with size-dependent characteristics. Therefore, this study aimed to investigate how magnetic Fe3O4 NPs of different sizes and concentrations influence the cryopreservation of bull sperm. Initially Fe3O4 NPs with three sizes (similar to 6, similar to 20, and similar to 80 nm) were synthesized, and two concentrations (10 and 50 mu g/mL) were added to diluted semen samples, supplemented with a commercial extender and cryopreserved. The findings showed that 6 nm Fe3O4 NPs maintained total and progressive motility at levels similar to the control group at both doses, whereas a reduction in both motility parameters was observed only in the Fe20-10 group. No statistically significant differences were found in kinematic parameters, acrosomal, or plasma membrane integrity. Conversely, the 50 mu g/mL doses of the 20 and 80 nm groups decreased mitochondrial membrane potential (MMP). Chromatin condensation improved in all experimental groups compared to the control, which correlates with reduced DNA damage across nanoparticle (NP) treatments. Zeta potential measurements demonstrated that surface charge varied with particle size, and microscopic evaluations revealed the accumulation of positively charged Fe3O4 NPs in the head region. Overall, this study showed that Fe3O4 NPs exhibit distinct size-dependent characteristics, underscoring the importance of preliminary trials to determine the optimal NP size for spermatological applications.
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    Evaluation of AFP III-Conjugated Fe3O4 Nanoparticles on Bull Sperm Cryopreservation
    (Wiley, 2025) Ozturk, Ali Erdem; Dadi, Seyma; Bodu, Mustafa; Korkmaz, Oya; Atay, Yunus Emre; Sariozkan, Serpil; Ocsoy, Ismail
    Antifreeze proteins are naturally occurring proteins that are known for their ability to inhibit ice formation. Although effective in nature, they have failed to produce the expected results in cryobiological studies. However, the lack of a significant effect when used alone has raised the following question: Could conjugation with Fe3O4 nanoparticles (Fe3O4 NPs), which are frequently used in drug delivery studies, enable closer interaction with spermatozoa and provide cumulative protection? To address this question, bull spermatozoa were used. First, Fe3O4 NPs (similar to 6 nm) and AFP III-conjugated Fe3O4 NPs (AFP III@Fe3O4 NPs) were synthesised and characterised by dynamic light scattering, zeta potential analysis, and FTIR spectroscopy to confirm the conjugation. Then, sperm samples collected from five different Holstein bulls for five replications were diluted with AndroMed extender and divided into five groups: control, Fe3O4 NPs only, AFP III only (0.1 and 0.5 mu g/mL), and AFP III@Fe3O4 NPs (0.042 and 0.075 mu g/mL). After cryopreservation, spermatological parameters and DNA condensation were analysed. The AFP III@Fe3O4 NPs (0.075 mu g/mL) exhibited significantly higher motility and DNA integrity compared to the other treatment groups (p < 0.05). However, no statistically significant differences were observed in mitochondrial membrane potential, membrane, and acrosomal integrity. Analyses of motility and DNA damage supported the protective effect of the AFP III@Fe3O4 NPs 0.075 mu g/mL formulation. Thus, we have demonstrated the applicability of AFP III@Fe3O4 NPs, for the first time, in bull sperm cryopreservation and have introduced a novel nano-biotechnological approach for improving the success of assisted reproductive technologies in livestock.
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    Size-dependent cryoprotective effects of curcumin nanoparticles on DNA integrity and motility in cryopreserved Angora buck sperm
    (Frontiers Media Sa, 2026) Ozturk, Ali Erdem; Sariozkan, Serpil; Bodu, Mustafa; Atay, Yunus Emre; Sahin, Derya; Korkmaz, Oya; Ocsoy, Ismail
    Curcumin is a potent antioxidant and anti-inflammatory agent derived from the Curcuma longa plant. However, its low water solubility and limited bioavailability restrict its effectiveness in cryopreservation applications. In this study, curcumin nanoparticles (CNp) were produced in two different sizes (20 and 80 nm) to increase bioavailability, and their effects on the freezing of Angora buck sperm were investigated. Semen was collected a total of six times from five Angora bucks (aged 2-3 years). The collected semen was pooled in equal volumes, diluted with a Tris-based egg yolk diluent, and distributed among the experimental groups. Experimental groups set as: control, CNp 20-1 (20 nm CNp at 1 mu g/ml), CNp 20-5 (20 nm CNp at 5 mu g/ml), CNp 80-1 (80 nm CNp at 1 mu g/ml), and CNp 80-5 (80 nm CNp at 5 mu g/ml), and were drawn into 0.25 ml straws and frozen. After thawing, motility, kinematic parameters, plasma membrane and acrosomal integrity (PMAI), mitochondrial membrane potential (MMP), and DNA integrity analyses were performed. The highest motility value was observed in the CNp 20-1 group (p < 0.05), but the groups containing 80 nm CNp reduced MMP (p < 0.01). There was no statistical difference between groups in PMAI. DNA integrity remained within acceptable limits in all groups and was better preserved compared to the control group (p < 0.05). In addition to these findings, dynamic light scattering (DLS) analyses revealed that CNps exhibited increased water-absorbing capacity and aggregation. The hydrodynamic diameters of 20 and 80 nm CNps were found to be 223.3 and 289.3 nm, respectively. The findings indicate that the size of curcumin nanoparticles significantly affects aggregation behavior, motility, and DNA integrity parameters. These results suggest that curcumin nanoparticles with a 20 nm size may offer a more effective strategy for the cryopreservation of Angora buck sperm.