Open Veterinary Journal, (2025), Vol. 15(7): 3177-3184

Research Article

10.5455/OVJ.2025.v15.i7.28

Functional assessment of kidneys and liver functions in lambs treated with zinc oxide nanoparticles

Raad Mahmood Hussein Al-Zubaidi¹, Ahmad Hanash Al-Zuhairi¹, Marah Salim Hameed³ and Ali Ibrahim Ali AL-Ezzy^4*

¹Department of Medicine, College of Veterinary Medicine, University of Diyala, Iraq

²Department of Physiology, College of Veterinary Medicine, University of Diyala, Iraq

³Department of Pathology, College of Veterinary Medicine, University of Diyala, Iraq

*Corresponding Author: Ali Ibrahim Ali AL-Ezzy. Department of Pathology, College of Veterinary Medicine, University of Diyala, Iraq. Email: alizziibrahim [at] gmail.com

Submitted: 18/02/2025 Revised: 07/06/2025 Accepted: 14/06/2025 Published: 31/07/2025

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ABSTRACT

Background: Zinc oxide nanoparticles (ZnO NPs) have low toxicity and are utilized extensively in medicine. The properties of ZnO NPs include the promotion of growth, the modulation of the immune system, and the regulation of animal reproduction.

Aim: An evaluation of the renal and hepatic function of lambs subjected to ZnO NP treatment.

Methods: Fifteen apparently healthy local breed lambs, aged between 7 and 9 months, were utilized in the present study. The lambs were divided randomly into three equal groups. The control group (group I) received water and glycerol only, the treatment group (group II) received 3.5 mg/kg of ZnO NPs orally, and the third group (group III) received 7 mg/kg of ZnO NPs orally once daily for a period of 90 days. A comprehensive biochemical analysis encompasses a range of essential parameters, including aspartate aminotransferase (AST); alanine aminotransferase; alkaline phosphatase (ALP); serum creatinine; blood urea; and total serum bilirubin (TSB).

Results: A significant increase in TSB was observed among the control group (I) in comparison with the initial baseline. Furthermore, a significant increase in TSB. was noted in group I when it was incorporated with groups II and III at the end of the study. A significant increase in serum AST and ALP was observed among groups I, II, and III at the study’s conclusion in comparison with the initial baseline. However, no significant changes were detected between these groups. Conversely, serum ALP values exhibited a significant decrease at the study’s conclusion in comparison with the baseline. There was a substantial increase in serum urea and creatinine levels at the end of the study in all groups when compared with the baseline.

Conclusion: Due to the different results of toxicological studies on experimental animals. Current recommends carrying out more studies to clarify the toxic effects, in order to install the appropriate doses for animals to determine the desired benefit of nano-zinc to reach logical conclusions.

Keywords: Kidney, Liver, Function, Lambs, Zinc oxide nanoparticles

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Introduction

It is imperative to acknowledge the pivotal function of trace elements in sustaining optimal cell metabolism, thereby ensuring animal health and productivity (Bhalakiya et al., 2019). A deficiency in these elements has been demonstrated to induce perturbations in animal health and productivity. Consequently, the provision of trace elements as mineral supplements is essential to circumvent such deficiencies (Bhalakiya et al., 2019; Palomares, 2022).

Nanotechnology encompasses the production, manipulation, and utilization of materials at the nanoscale level (Kavitha et al., 2013). Nanoparticles (NPs) are particles with a size ranging from 1 to 100 nanometers, and their use is particularly significant in the fields of medicinal chemistry, atomic physics, and other related disciplines (Jeevanandam et al., 2018).

Nanomedicine involves the use of NPs as therapeutic targets, including drug delivery, with a particular emphasis on the central nervous system (Guo et al., 2023). The application of nanomedicine in imaging, diagnostics, and treatments for tissue damage has also been demonstrated (Wan et al., 2025). Zinc oxide NPs (ZnO NPs) have low toxicity and are utilized as antibacterial, anti-inflammatory, and anticancer agent, for drug transport, wound healing, and bioimaging (Xiong, 2013; Zhang and Xiong, 2015; Mishra et al., 2017). This chemical aids animal growth, immune system modulation, and reproduction.

All doses of ZnO NPs have various effects on animal performance. However, in the absence of any indirect contamination of the environment, ZnO NPs are capable of replacing conventional zinc sources at smaller doses, with superior results (Rahman et al., 2022). Consequently, further research is necessary to ascertain the definite bioavailability of ZnO NP in animals (Partha and Somu, 2016).

The objective of the present study is to evaluate the effect of ZnO NPs treatment of lambs on renal and hepatic functions, weight gain.

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Material and Methods

Ethical consideration

Ethical Approval No.UOD-VMD-7 was granted by the ethical review committee of the Department of Internal Medicine at the College of Veterinary Medicine, University of Diyala, in Iraq.

The area and the population

According to the local ethical legislations for University of Diyala, 15 apparently healthy local breed male lambs, with age range 7–9 months, living in the farm of Veterinary Medicine College, University of Diyala, Baqubah city, Diyala Governorate, 33°45’34.71”N; 44°36’23.97”E, Northeast were included (Al-Ezzy et al., 2016; Bak et al., 2024).

Prior to the trial, biochemical testing of liver and kidney function was performed for all animals, along with an internal parasite investigation. Ivermectin and Oxyclozanid were administered to the lambs as preventative treatments at prescribed dosages of 0.2 and 15 mg/kg B.W., respectively, given twice at intervals of 2 weeks.

According to the study by (Constable et al., 2017), a clinical evaluation of the animals’ mucous membranes, appetite, behaviors, and physical condition was carried out over the course of 2 weeks. Until the end of the experiment, lambs were given 500 g of barley grain per head per day along with straw, green feed, and water.

Feed quantity and composition

The feed quantity is 3% of the body weight, with two meals of concentrated feed, one in the morning and the other in the evening, and roughage (hay) is given freely. The result of the analysis of the feed sample for the zinc appeared the concentration:1.83 mg/100 g, according to (A.O.A.C., 2023).

The components, energy content (kcal/kg dry matter) and crude protein percentage (%) of the feed used in the experiment were determined according to (Thiex et al., 2002), listed in Table 1.

ZnO NPs analysis

Scanning electron microscope

Morphological specifications and size of ZnO NPs were defined according to (Shamhari et al., 2018) by using a scanning electron microscope (SEM) (Shemadzu, Japan, Nano LAB-MOST, SEM MAG:50.0 KX, SEM HV: 20.0 KV) in the Ministry of Science and Technology.

The result of SEM showed a specific morphology and size of ZnO, and the diameters of ZnO NPs varied between 5–50 nm (Fig. 1).

Fig. 1. SEM image of ZnO NPs, show small particles which observed to be bit agglomerated, particles, with diameters between 5–50 nm.

Table 1. Components, energy content (kcal/kg dry matter) and crude protein percentage (%) of the feed used in the experiment.

Fig. 2. XRD patterns of ZnO NPs.

X-ray diffraction

The average particle size (PS) and phase detection of particles were evaluated by X-ray diffractions (XRDs) pattern using X’pert Pro diffract meter (Shemadzu, Japan) that was used to affirm the crystal phases and size, in the University of Technology, Nanotechnology. It was carried out using X-ray diffractometer with Cu- Kα crystal radiation (λ=1.541 A°) scanning at a speed of (10º deg/min) for (2 theta) range of 20º–80º. The diffraction peaks were identified by comparison with (00-036-1451) card/Variable Slit Intensity. The full widths at half maximum in the XRD was used to determine the crystallite size by Scherer’s equation. The strain values η, and the values of dislocation densities δ, were calculated by (Zak et al., 2012).

XRD pattern of ZnO NPs is shown in (Fig. 2), based on the XRD pattern. The pattern shows that all the diffraction peaks are indexed to the hexagonal phase of pure ZnO NPs, ZnO NPs diffraction peaks appearing at scattering angles (2θ)=31.88°, 34.52°, 36.39°, 47.64°, 56.70°, 62.95, 66.48, 68.03, 69.16, and 77.05. The strongest three peaks in 36.39, 31.88, and 34.52, respectively.

Particle size

PS was determined according to the method as described by (Shamhari et al., 2018), by used Brookhaven instruments Corp. (90Plus Particle Sizing Software Ve 5.34), laser in the University of Technology, Nanotechnology. Diluted suspensions are prepared, in the range of 0.0001%–1.0% v/v, using appropriate moisturizing and/or dispersing agents, if necessary. A small ultrasound is sometimes helpful in breaking up loose clumps. Small sample volumes are used as 50 µl. The sample is refundable. U-shaped, disposable, polystyrene cells are used to suspend aqueous and ethanol solutions. It takes a few minutes for the sample and the cell to balance with the effectively controlled temperature environment inside Nano Brook (Iso, 2009).

PS also performed to further confirm the size of ZnO NPs was shown in (Fig. 3), and the mean diameter was 36.5 nm.

Treatment by ZnO NPs

Three groups of five male lambs each were randomly selected. As the control group, group I received only glycerol and water as treatment. Group III received oral treatment with 7 mg/kg ZnO NPs once daily for 90 days, while group II received oral treatment with 3.5 mg/kg ZnO NPs.

According to (Al-Zubaedi et al., 2020), equal amounts of water and glycerol were combined, and then ZnO NPs were included to the mixture (as a stabilizing agent for ZnO NPs at room temperature). Each treatment group’s appropriate concentration was determined from the prepared mixture. Only a solution of water and glycerol was administered to the control group. Each animal’s dosage was then determined based on its weight, group, and dose. After that, the animals received oral treatment once a day using a disposable syringe.

Fig. 3. PS of ZnO NPs, average diameter 36.5 nm.

The amount of zinc in lambs feed and administration doses

Table 2 illustrates the amount of zinc in lambs feed compared with the administration doses.

Blood sampling

Blood samples (5 ml) were drawn from the jugular vein and allowed to gently flow along the inside of a sterile, desiccated tube, as described by (Hameed and Al-Ezzy, 2019). After that, the samples were allowed to clot at room temperature in a slanting orientation for as long as one typical Earth revolution on its own axis. Following that, the samples underwent a 5-minute centrifugation process at a rate of 3,000 revolutions per minute. An automatic pipette was then used to extract the clear sera, which were then placed into previously labeled Eppendorf tubes. After that, the tubes were kept at −20°C until they were examined (Hameed and Al-Ezzy, 2024).

Table 2. The amount of zinc in lambs feed and administrative doses.

Biochemical study

The estimation of the following parameters was conducted: aspartate aminotransferase (AST); alanine aminotransferase (ALT); alkaline phosphatase (ALP); serum creatinine; blood urea (BU); and total serum bilirubin (TSB). The estimation of these parameters was conducted according to (Hameed et al., 2024; Hameed and Al-Ezzy, 2024).

Table 3. Show liver function parameters of control and treated groups.

Statistical analysis

All data were denoted as means ± SE. One-ANOVA in SPSS was used for analysis (Hameed et al., 2020). Different letters were used to indicate significant differences at (p < 0.05) (Hussein et al., 2025).

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Results

The results did not show any abnormal clinical signs on the treated animals compared to the control animals, and all the clinical vital signs of the animals were normal. As shown in Table 3, the results of liver function in current study refer to that TSB significant increase in control group (I) at end of study in comparison with zero day, while, in treated groups (II, III) no significant changes at end of study in comparison with zero day, all so, in the between groups significant increase in group I incorporation with group II and III at end of study. The serum values of AST and ALP significantly increased in groups I, II, and III at the end of the study in comparison with zero day, but no significant changes between groups. The serum values of ALP showed significant decreased at end of study in comparison with zero day, While, between groups a significant increase in group I and II incorporation with group III at end of study.

The results of serum value for BU showed no significant changes at end of study in comparison with zero day in all groups. The value of serum creatinine showed no significant changes at end of study in comparison with zero day (Table 3).

The results of serum value for BU showed no significant changes at end of study in comparison with zero day in all groups. The value of serum creatinine showed no significant changes at the end of the study in comparison with zero day (Table 4).

Table 4. Show kidney function parameters of control and treated groups.

Table 5. Shows the effect of adding ZnO NPs to the feed on the weights of lambs in the experiment.

As shown in Table 5, the results showed no significant differences (p ≤ 0.05) in lamb weight between the treatment groups and the control group from the beginning to the end of the experiment.

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Discussion

Zinc is one of the most important trace elements abundant in the animal body, and there are no stores for it in the body (Kosik-Bogacka and Łanocha-Arendarczyk, 2019). Zinc is involved the synthesis of many enzymes and hormones, and is important for the physiological functions of the body, such include alcohol dehydrogenase, ALP, lactate dehydrogenase and superoxide dismutase (Stiles et al., 2024). Zinc is one of the components of free radical scavengers, which is formed as a result of various physiological processes. All these results support and agree with the results of the current study, which proves the role of zinc in activating antioxidant enzymes and ridding the body of the danger of free radicals (Zhao et al., 2014), and also has a role in protecting membranes from bacterial endotoxins (Wei et al., 2022). Zinc is added continuously in ruminant foods for normal functions and to support the daily requirements. The use of nanotechnology to produce zinc NPs represents a potential alternative to organic and inorganic zinc sources (Ali et al., 2018). Studies on the use of zinc NPs have shown better results compared to conventional zinc sources and with less (Swain et al., 2016; Al Jabri et al., 2022).

The results of TSB showed significant increase in group I in compression with treated groups, the reason for this may be due to weak immunity to resist pathogens, as indicated by other studies (Zhao et al., 2014; Parashuramulu et al., 2015), where they prove that zinc enhancing immunity. The serum bilirubin level is important as a marker of basic risks to renal function (Kawamoto et al., 2014). Studies have indicated evidence that zinc has antioxidant and anti-inflammatory properties (Guo et al., 2020). It has been found that serum zinc concentrations correlate with inflammatory markers, including TNF, IL-6, and CRP, and zinc plays an important role in the decrease of these inflammatory cytokines in the blood via down-regulation (Jarosz et al., 2017). According to Kapitulnik (2004) and Chen et al. (2020) “Bilirubin is an influential endogenous anti-oxidant, that scavenges peroxyl radicals, also inhibits oxidative stress, and bilirubin has anti-inflammatory, anti-oxidative and immunosuppressive characteristics.”

Kidney function is determined by BU nitrogen (BUN) and creatinine indicators. So if signs of kidney failure appear, a clear change in BUN and creatinine levels, and therefore, the results of current study agree with (Wang et al., 2006) which stated that the use of nano Zn has less effect on kidney function compared to traditional Zn sources and this is likely due to the fact that Nano Zn is less toxic (Swain et al., 2016). But these results do not agree with (Najafzadeh et al., 2013), which found a significant increase in creatinine level in the nano group, and this difference may be due to the difference in the size and dose of nano zinc in addition to the amount of zinc present in the diet before adding the experimental dose of nano zinc.

On the other hand, the potential danger of high levels of zinc NPs in diets is unknown and the toxicological consequences are not sufficiently known. In addition, the majority of toxicological studies were conducted on rodents as in live models (Argmann et al., 2005; Chong et al., 2021). It should be noted that the most important organs targeted by zinc NPs include the liver, spleen, heart, pancreas, and bones when exposed orally (Wang et al., 2008; Fujihara and Nishimoto, 2024). The cytotoxic effects of ZnO NPs were dose- and time-dependent including oxidative stress, cell membrane lipid peroxidation and oxidative DNA damage (Lin et al., 2009; Najafzadeh et al., 2013). According to (Najafzadeh et al., 2013; Rahman et al., 2022) lambs treated with 20 mg/kg ZnO NPs orally for 25 days suffered from slight hepatic toxicity and sever renal damage. Zinc toxicity is related to the concentration of free ions (Kasemets et al., 2009; Kool et al., 2011). But zinc ZnO NPs are likely to remain in NP form for a longer time, and thus are less toxic than inorganic salts such as ZnCl2 (Hooper et al., 2011; Ali et al., 2018).

The results showed no significant differences (p ≤ 0.05) in lamb weight between the treatment groups and the control group from the beginning to the end of the experiment. This result was consistent with what was indicated by (Mohamed et al., 2017), who noted no significant differences in live body weight when using ZnO NPs in dry ewes’ rations. On the other hand, (Zaboli et al., 2013), noted no significant difference in weight gain in Angora goat kids when using conventional zinc. On the other hand, the results of the current study did not agree with what (Hussein, 2020) indicated, as the results of his study indicated that the body weight of Iraqi local goats exposed to experimental zinc deficiency increased significantly in the groups treated with ZnO NP compared to the control group, and this may be attributed to the difference in animals’ response to treatment, especially in animals that already suffer from zinc deficiency.

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Conclusions

Zinc is an essential element in the diet of animals, and at the same time it is mostly excreted into the environment and this leads to direct pollution of the environment. For this reason, solutions in various studies have tended to use zinc NPs in lower doses in livestock feed and to obtain the best results in terms of productivity and health of animals, while reducing environmental pollution damage. In light of the different results of studies on the toxicity of zinc NPs, we recommend that further studies be carried out in this research field.

Limitations of the study and future directions

Current study limited to the male lambs, it’s recommended in future to complete the study on ewe lambs and evaluation of sex effect on bioavailability of Zn. Its recommended, in future to design a study for evaluation of zinc level in the blood and in the feed for estimation of bioavailability of Zn in serum.

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Acknowledgments

The authors deeply appreciated for the efforts of technicians at the college of veterinary medicine, University of Diyala.

Conflict of interest

The authors disclosed that there is no conflict of interest.

Funding

The authors disclosed that they share the total cost of current research project.

Authors’ contributions

Authors disclosed that they were equally contribute in planning, methodology, data collection and analysis, and writing of manuscript.

Data availability

All the required data were included with in the text.

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