Synthesis of green zinc-oxide nanoparticles and its dose-dependent beneficial effect on spermatozoa during preservation: sperm functional integrity, fertility and antimicrobial activity.

Introduction: The development of an effective extender is important for semen preservation and the artificial insemination (AI) industry. This study demonstrates the beneficial effect of zinc oxide nanoparticles (ZnO-NPs) as an additive to semen extenders to improve semen quality, fertility, and antibacterial activity during liquid preservation in a boar model. Methods: Initially, to find out the safe concentration of ZnO-NPs in sperm cells, a wide range of ZnO-NP concentrations (0, 5, 10, 50, 100, 500, and 1,000 µM) were co-incubated with sperm at 37°C for a cytotoxic study. These NP concentrations were compared to their salt control zinc acetate (ZA) at the same concentrations and to a control group. The effect of the different concentrations of ZnO-NPs on sperm motility, membrane integrity, mitochondrial membrane potential (MMP), and apoptosis was assessed. Accordingly, the non-toxic dose was selected and supplemented in MODENA extender to determine its beneficial effect on the boar semen parameters mentioned and the lipid peroxidation (LPO) levels during liquid preservation at 16°C for 6 days. The non-cytotoxic dosage was subsequently chosen for AI, fertility investigations, and the evaluation of the antibacterial efficacy of ZnO-NPs during preservation hours. An antibacterial study of ZnO-NPs and its salt control at doses of 10 µM and 50 µM was carried out by the colony forming unit (CFU) method. Results and discussion: The cytotoxic study revealed that 5, 10, and 50 µM of ZnO-NPs are safe. Consequently, semen preserved in the MODENA extender, incorporating the non-toxic dose, exhibited 10 and 50 µM ZnO-NPs as the optimal concentrations for beneficial outcomes during liquid preservation at 16°C. ZnO-NPs of 10 µM concentration resulted in a significantly (p < 0.05) improved conception rate of 86.95% compared to the control of 73.13%. ZnO-NPs of 10 and 50 µM concentrations exhibit potent antimicrobial action by reducing the number of colonies formed with days of preservation in comparison to the negative control. The investigation concluded that the incorporation of 10 µM ZnO-NPs led to enhancements in sperm motility, membrane integrity, and MMP, attributed to a reduction in the malondialdehyde (MDA) levels. This improvement was accompanied by a concurrent increase in fertility rates, including farrowing rate and litter size, during the liquid preservation process. Furthermore, ZnO-NPs exhibited an antimicrobial effect, resulting in decreased bacterial growth while preserving boar semen at 16°C for 6 days. These findings suggest that ZnO-NPs could serve as a viable alternative to antibiotics, potentially mitigating antibiotic resistance concerns within the food chain.

Association between levels of insulin-like growth factor-1 in serum and seminal plasma with fresh and frozen-thawed semen characteristics in Beetal bucks.

The aim of this study was to investigate the relationship between the levels of insulin-like growth factor-1 (IGF-1) in serum and seminal plasma and the characteristics of semen in Beetal bucks (Capra hircus). A total of 12 adult Beetal bucks were involved in the study, with each buck providing six ejaculates collected using a standard artificial vagina (n = 72 total). Only qualified semen samples (volume of 0.7 mL, a mass motility rating of 3+ or higher on a 0-+ scale, and individual progressive motility of 80% or more) divided into three fractions were processed for estimation of IGF-1 and other seminal parameters like motility, viability, acrosome integrity, sperm abnormality and superoxide dismutase (SOD) activity. The first and second fraction were diluted and extended with Optixcell extender (1:15 ratio). The first ejaculate fraction was processed for studying fresh semen parameters and the second fraction was cryopreserved for evaluating frozen semen parameters. French mini straws (0.25 mL) were used for semen filling, and polyvinyl alcohol powder of different colours was used for sealing the extended semen. The third fraction of each ejaculate was centrifuged at room temperature (1100 × g for 7 min) to separate the seminal plasma. Additionally, blood samples were taken from each buck on the same day as semen collection, resulting in a total of 36 blood samples. The results revealed a significant positive correlation (r = .4243; p < .05) between the concentration of IGF-1 in both serum and seminal plasma of the Beetal bucks. Furthermore, the concentration of IGF-1 in serum showed significant positive correlations with sperm viability (r = .554; p < .05), acrosome integrity (r = .527; p < .05), post-thaw sperm motility (r = .407; p < .01), post-thaw sperm viability (r = .426; p < .01) and post-thaw acrosome integrity (r = .333; p < .05). However, it had a significant negative correlation with SOD activity in fresh semen (r = -0.458; p < .01). Moreover, the concentration of IGF-1 in seminal plasma demonstrated significant positive correlations with individual progressive motility (r = .341; p < .05), sperm viability (r = .527; p < .05), acrosome integrity (r = .539; p < .05), sperm plasma membrane integrity (r = .464; p < .05), post-thaw sperm motility (r = .644; p < .01), post-thaw sperm viability (r = .643; p < .01), post-thaw acrosome integrity (r = .487; p < .01) and post-thaw sperm plasma membrane integrity (r = .521; p < .01). Additionally, it showed a significant negative correlation with SOD activity in both fresh semen (r = -0.714; p < .01) and frozen semen (r = -0.558; p < .01) of Beetal bucks. Based on these findings, IGF-1 in seminal plasma can be considered as a potential biomarker for the selection of bucks for breeding purposes.

Zinc oxide and selenium nanoparticles can improve semen quality and heat shock protein expression in cryopreserved goat (Capra hircus) spermatozoa.

BACKGROUND: Reactive oxygen species (ROS) are strongly linked with oxidative stress (OS) generated during the process of sperm cryopreservation. Indeed, cellular damage from ROS has been implicated during sperm cryopreservation which causes deterioration in sperm quality and antioxidant nanoparticles (NPs) have been successful in preventing such damage. The interaction of NPs with sperm cells has been less frequently explored in farm animals. OBJECTIVE: The present study explored the effect of NP supplementation on sperm ultrastructure, potential interaction with sperm membrane (plasma and acrosome membrane), heat shock protein (HSP) gene expression levels and sperm quality in cryopreserved buck semen. MATERIALS AND METHODS: Thirty-two (32) ejaculates were collected from four (4) adult male bucks and then diluted in Tris- citric acid- fructose- egg yolk (TCFY) extender containing the Zinc-oxide (ZnO) and Selenium (Se) NP treatments (T0: Control; TZn: 0.1 mg/mL ZnO NPs and TSe: 1 µg/mL Se NPs) after initial evaluation. Diluted semen was packed in 0.25 mL French mini straws and then stored in liquid nitrogen (LN2). Sperm parameters, lipid peroxidation (LPO) profile, sperm head morphology ultrastructural classification under transmission electron microscope (TEM), potential interaction of NPs with sperm membrane and expression of HSP genes were evaluated in the different treatment groups. RESULTS: We found a significant (p < 0.05) increase in the percentage of spermatozoa with intact plasma membrane, and intact acrosome in the ZnO (0.1 mg/mL) and Se (1 µg/mL) NP supplemented groups in comparison to the frozen control group. TEM assessment revealed no internalization of both ZnO and Se NPs into the sperm structure. Few occasional contacts of ZnO NPs with the sperm membrane and a few agglomerates of Se NPs around the area of damaged membranes were visualized. HSP70 and HSP90 mRNA levels were significantly (p < 0.001) higher in the NP supplemented groups in comparison to the control. HSP70 and HSP90 mRNA levels had a strong positive association with sperm motility and a weak to moderate association with other sperm parameters. CONCLUSIONS: Current findings indicated that ZnO NPs are more potent than Se NPs in ameliorating peroxidative damages during sperm cryopreservation, increases semen quality parameters possibly by increasing the expression levels of HSP genes in buck semen. Furthermore, NP supplementation may have a potential role in preserving sperm head ultrastructure by acting as an antioxidant and reducing OS during various degrees of cellular insults, which needs to be further explored.

In Vitro and In Vivo Studies on the Efficacy of Zinc-Oxide and Selenium Nanoparticle in Cryopreserved Goat (Capra hircus) Spermatozoa.

Different nanoparticles (NPs) are currently being investigated for their potential role as cryoprotectant during semen cryopreservation in several mammalian species. It may be possible to improve semen quality following cryopreservation by supplementation of NPs in the freezing extenders. The present study was carried out in semen collected from four (4) Assam Hill Goat bucks (10 ejaculates per buck) to investigate the effect of supplementing zinc oxide (ZnO) and selenium (Se) NPs in Tris-citric acid-fructose yolk (TCFY) extender on in vitro sperm quality and in vivo fertility rate after freeze-thawing. The size morphology and zeta potential of ZnO and Se NPs were evaluated prior to its incorporation in the freezing extender. Qualified semen samples (> 70% progressive motility) were divided into five (5) aliquots and then diluted in TCFY extender containing ZnO and Se NP supplementation at different concentrations (T0, control; T1, 0.1 mg/mL ZnO NPs; T2, 0.5 mg/mL ZnO NPs; T3, 0.5 µg/mL Se NPs; and T4, 1 µg/mL Se NPs). Diluted semen was packed in 0.25 mL straws and then stored in liquid nitrogen. After thawing, post-thaw in vitro sperm attributes were evaluated. Finally, the effect of NPs on in vivo fertility rate was checked in heat-synched does (n = 70) by artificial insemination (AI) using straws that showed superior results during the in vitro study. Results showed that ZnO and Se NPs were poly-crystalline in nature with particle size below 100 nm (nm). The evaluated post-thaw sperm in vitro attributes were significantly (p < 0.001) higher in T1 in comparison to T0. The antioxidant enzyme activities were significantly (p < 0.001) higher in T1. Lipid peroxidation (LPO) profile was significantly (p < 0.001) lower in T1. Sperm motility and mitochondrial membrane potential (MMP) had a highly significant (r = 0.580, p < 0.05) association in T1. No significant (p > 0.05) differences in pregnancy rates were recorded after AI in the different treatments. In conclusion, extender supplemented with 0.1 mg/mL ZnO NPs improved post-thaw semen quality of goat spermatozoa consequently by increasing activities of endogenous antioxidant enzymes thereby lowering LPO levels. However, improved in vitro outcomes might not correspond to improved field fertility outcomes.