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. 2026 Jun 17;34:100742. doi: 10.1016/j.vas.2026.100742

Effect of novel trace elements on liver and kidney functions, immunity, antioxidant indices and caecal microbiota of growing rabbits

Amr El-Nile a, Mohamed Hafez b, Mohamed I Hassan a, Saber S Hassan c, Mohamed Rashad b, Abdallah E Mohamed c, Alexander I Popov d, Mahmoud Alagawany e, Sobhy MA Sallam f,⁎
PMCID: PMC13333364  PMID: 42440484

Abstract

This study aimed to examine the impact of supplementing water with Novel Trace Elements Mixture (NTEM) on the caecal microbiota and immunological and antioxidant indices of growing rabbits. The study utilized 72 weaning male V-line rabbits that were seven weeks old. The study randomly assigned rabbits into four treatment groups, each containing 18 rabbits with six replicates. The rabbits were randomly assigned to four treatment groups: a control group receiving unsupplemented drinking water and three experimental groups receiving drinking water supplemented with NTEM at levels of 0.5, 1.0, or 1.5 mL/L, respectively. The NTEM stock solution consisted of 5.0 mL of 1.84% aqueous Fe-EDTA, 0.29 g of ZnSO4•7H2O, 1.7 g of MnSO4•H2O, 0.8 g of CuSO4, and 1.8 g of H3BO3 dissolved in 1 L of deionized water. Addition of NTEM at 1 and 1.5 ml/L increased the alkaline phosphatase, while decreased alanine aminotransferase compared to control. Rabbits' drinking water with NTEM led to an increase in serum total antioxidant capacity. Furthermore, a reduction in total bacterial count was observed by adding NTEM levels in drinking water. NTEM at 1.5 ml/L in drinking water reduced E. coli count compared to control and other groups. In conclusion, adding NTEM to drinking water of rabbits showed notable benefits on some parameters of liver and kidney functions, antioxidant and immunity status, as well as the cecal bacterial count especially at levels 1 and 1.5 ml/ L.

Keywords: Animal performance, Caecal microbiota, Immunological indices, Mineral mix, V-line

1. Introduction

The breeding of rabbits is one of the most profitable business ventures, particularly in recent years, as rabbits are known for their abundant production and quick rate of growth compared to other animals (El-Kholy et al., 2025). Therefore, it is essential to take this sector into account when making any major improvements to rabbit production. Accordingly, the importance of rabbits on a national scale may help to address the issue of animal protein shortages (Abou-Saleh et al., 2022). To achieve this, it will also be necessary to provide crucial feed and feed additives. Feed supplements have been utilized to enhance the performance and productivity of animals (El-Nile et al., 2023). Minerals are commonly categorized into macroelements, including Ca, P, K, Na, Cl, and Mg, and trace elements, which comprise Cu, Zn, Mn, B, Fe, Se, Mo, I, and Cr, based on their respective amounts (Council, 2001).

Trace elements are important for many metal-dependent enzymes and protein functions and are essential dietary components for life (Krämer et al., 2007). Generally, trace elements increase productive performance and immune competence (Terpiłowska & Siwicki, 2011). As a result, the lack of important trace elements in the feed and water reduces productivity, and some animals exhibit microelementosis symptoms (Luchin & Darmohray, 2016). Therefore, it is essential to include these mineral components in animal diets to preserve their health and production (Alagawany et al., 2021; Elnesr et al., 2024).

Trace metals, such as copper (Cu) and zinc (Zn), are essential for the functioning of many metal-binding proteins and metalloenzymes in rabbits (Abdelnour et al., 2021). These proteins and enzymes play critical roles in various processes including, immune system function, nutrient metabolism, and overall health (Attia et al., 2016). Cu, Zn, and manganese (Mn) are necessary for maintaining the antioxidant system (Wall, Tran et al., 2016). Zinc, in particular, is a crucial trace element that is redox inactive and serves as a cofactor for antioxidant enzymes, stabilizes sulfhydryl groups in cell membranes to protect against oxidative damage, regulates glutathione metabolism, and strongly induces the production of metallothionein. Metallothionein is essential for sequestering reactive oxygen species generated under stress (Marreiro et al., 2017). On the other hand, Mn has a role in the operation of several organ systems; it is essential for healthy immunological function, blood sugar and cellular energy balance, reproduction, digestion, and bone formation. Also, Mn supports the body's defenses against free radicals. Further, manganese and vitamin K work together to maintain hemostasis and blood coagulation (Aschner & Aschner, 2005).

For animal biochemical, metabolic, and physiological processes, boron (B) plays a variety of beneficial roles. It is essential for preserving animal health and avoiding nutritional problems. Recently, studies have focused on identifying the pleiotropic effects of B, which include activating the immune system, working in antioxidant detoxification processes, altering bone metabolism, improving animal performance, and modifying several of bodily functions (Abdelnour et al., 2018; Donoiu et al., 2018; Hunter et al., 2019). There is a strong relationship between iron (Fe) and the immune system, and this is mediated by genes and proteins involved in Fe homeostasis, which play a key role in restricting bacterial access to Fe and thus preventing their growth (Ward et al., 2011). Therefore, the study aimed to investigate the influence of different doses of a novel trace element mixture (NTEM) solution supplementation on liver and kidney function indicators, antioxidant status, immune response, and cecal bacterial count of growing rabbits.

2. Materials and methods

2.1. Ethical statement

The current research was performed at El-Bostan Farm, which is situated within the premises of the Faculty of Agriculture at Damanhour University. Laboratory measurements were carried out at the Arid Lands Cultivation Research Institute, situated in the City of Scientific Research and Technological Applications located in New Borg El-Arab city, Alexandria, Egypt. All practices and investigations of the study were accompanied by approvals of the ethical principles of animal research and approved by Damanhour University's Ethical. Animal Care and Use Committee (Approval No: DUFA-2023–4).

2.2. The preparation of novel trace elements mixture

The solution of trace elements was prepared as follows: 5.0 ml of 1.84% aqueous Fe-EDTA solution, 0.29 g of ZnSO4•7H2O, 1.7 g of MnSO4•H2O, 0.8 g of CuSO4, and 1.8 g of H3BO3 were combined with 1 L of deionized water. The pH of the solution was adjusted to 7.0 using a 10% H3PO4 solution (Hafez et al., 2021). The solution of trace elements was refrigerated at 4 °C until use. The authors have filed a local application for patent protection for the NTEM solution (Patent application ID: EG/P/2023/839, Title "Potential mineral compound that has the ability to enhance growth rates, immunity status, and improve carcass traits for farm animals").

2.3. Animals, experimental design and management

In this experiment, a total of 72 weaning male V-line rabbits were used and aged at seven weeks old. The V-line rabbits used in this study were obtained from the Poultry Research Centre, Faculty of Agriculture, Alexandria University, Egypt. They were acclimatized for one week before starting the experiment during which they were fed on the control diet. The rabbits were randomly allocated into four treatment groups, with each group comprising of six replicates and three rabbits per replicate. The first group was designated as the control group, which did not receive any water supplementation. The second, third, and fourth groups received NTEM at concentrations of 0.5, 1, and 1.5 ml/L, respectively, added to their drinking water twice a week for five weeks. The rabbits were housed individually in wire cages, with dimensions of 35 × 40 × 50 cm for width, height, and length, respectively. Throughout the experimental period, all animal groups were kept in similar administrative and environmental conditions. Throughout the trial, a mean ambient temperature of 32.90 ± 0.95 °C and a humidity level of 59.11 ± 1.02% were documented inside the farm. The rabbits had free access to feed and water. All the groups were fed with the same base diet prepared according to the (NRC, 1977) guidelines, as shown in Table 1.

Table 1.

feed ingredients and calculated chemical composition of the basal diet.

Ingredients (kg/ton) Chemical composition (g/kg)
Yellow corn 100.0 Dry matter 903.2
Barley 130.0 Organic matter 804.8
Molasses 30.0 Crude protein 172.4
Clover hay 395.0 Crude fibre 134.6
Wheat bran 150.0 Ether extract 28.00
Soybean meal 175.0 Nitrogen-free extract 569.8
Dicalcium phosphate 8.0 Ash 95.20
Limestone 5.0 digestible energy (kcal/kg) 2464
Sodium chloride 3.0
Vitamin and minerals mixture* 3.0
DL-methionine 1.0
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⁎

Provides per kg of diet: Vit.A,1200 IU; Vit.D3, 2500 IU; Vit. E, 10 mg; Vit. K3, 3 mg; Vit.B1, 1 mg; Vit.B2, 4 mg; Pantothenic acid, 10 mg; Nicotinic acid, 20 mg; Folic acid, 1 mg; Biotin, 0.05 mg; Niacin, 40 mg; Vit.B6, 3 mg; Vit. B12, 20 mg; Choline Chloride, 400 mg; Mn, 62 mg; Fe,44 mg; Zn, 56 mg; I, 1 mg; Cu, 5 mg and Se, 0.01 mg.

2.4. Sampling

At the end of the experiment, six rabbits per group were individually weighed and anesthetized by intramuscular injection of xylazine (5 mg/kg body weight) and ketamine (35 mg/kg body weight). Humane euthanasia was then performed in accordance with internationally accepted guidelines for the care and use of laboratory animals (M2.2.1 Parenteral Injection according to Underwood and Anthony (2020). Blood samples were collected aseptically into sterile tubes for further analysis. The blood was collected in sterile tubes, with each animal receiving a separate tube for serum metabolite analysis. The tubes were left to coagulate at room temperature for 30 min and then separated into serum and cellular components through centrifugation at 3500 rpm for 15 min. After collection, the serum samples were securely stored at −20 °C until they were analyzed.

2.4.1. Liver and kidney functions indicators

Commercially available kits based on calorimetric measurements were used to determine all blood biochemical variables. The levels of several serum components, including alkaline phosphatase (ALP, U/L), aspartate aminotransferase (AST, U/L), alanine aminotransferase (ALT, U/L), urea (mg/dl), and creatinine (mg/dl), were analyzed using commercially available kits from Diamond Diagnostics (located at 23 El-Montazah St. Heliopolis, Cairo, Egypt). The analysis was performed spectrophotometrically.

2.4.2. Antioxidant status, oxidative stress and immune response

The total antioxidant capacity (TAC) was measured in mg/dl using the method outlined in Koracevic et al. (2001), while the malondialdehyde (MDA) concentration was determined in mmol/dl using the method described in Festa and Thiele (2011). The researchers assessed the animal's immune response by utilizing two measures - phagocytic activity (PA, %) and phagocytic index (PI, %). The methods used to determine these indicators were similar to the ones described in previous studies (Kawahara et al., 1991). The levels of immunoglobulins (IgG and IgM) were determined using a validated ELISA-based procedure according to Akiba et al. (1982).

2.5. Caecum microbial count

The American Public Health Association method (Marshall, 1992) was used to determine the total number of bacteria in the caecum, while the numbers of Escherichia coli, Clostridium Spp., and Lactobacillus were determined using the method outlined in Mackie and McCartney (1953). Colony forming unit (CFU) incubation was employed, and the samples were incubated at 30 °C for 2–7 days.

2.6. Statistical analysis

The researchers used the Generalized Linear Model (GLM) technique in the Statistical Package for Social Sciences (SPSS®) software to conduct their statistical analysis. The methodology for this technique was previously described in the literature. (SPSS, 2011). One-way analysis of variance was conducted using the formula Yij = µ + τj + εij, where µ represents the general mean, τ represents the treatment effect, and ε represents the experimental random error. Treatment means were compared using Duncan’s multiple range test (Duncan, 1955).

3. Results

3.1. Liver and kidney functions

Table 2 and Fig. 1 present the impact of water supplementation of NTEM on the liver and kidney function of growing rabbits. The results indicated that all liver and kidney parameters were significantly influenced by the addition of NTEM to the drinking water of growing rabbits, except for AST. The supplementation of NTEM at levels of 1 and 1.5 ml/L in the drinking water increased (p < 0.05) the ALP concentration compared to the group that received 0.5 ml/L and the control group. On the other hand, the addition of NTEM decreased (p < 0.05) the concentrations of ALT, and the lowest serum ALT value was observed in the group that received 1 ml/L of NTEM. Moreover, serum urea concentration was decreased (p < 0.05) with the addition of NTEM at levels of 1 and 1.5 ml/L, while serum creatinine concentration was decreased (p < 0.05) in the groups that received NTEM levels compared to the control group.

Table 2.

Influence of novel trace elements mixture levels on liver functions of growing V-line rabbits at 12 weeks of age.

Item Control NTEM (ml/L)
 SEM P value
0.5 1.0 1.5
Liver functions1
ALP (U/L) 12.26b 10.93b 14.52a 14.59a 0.47 0.005
ALT (IU/L) 33.15a 25.01c 14.63d 29.66b 1.54 <0.001
AST (IU/L) 27.32 29.66 27.92 28.79 0.44 0.269
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The superscript letters "a-d" in the presented data indicate significant differences within each row at a significance level of p-value <0.05.

1

ALP: alkaline phosphatase, ALT: alanine aminotransferase, AST: aspartate aminotransferase.

Fig. 1.

Fig 1 dummy alt text

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Influence of novel trace elements mixture levels on kidney functions of growing V-line rabbits at 12 weeks of age.

3.2. Antioxidant and immune indices

The effects of water supplementation of NTEM on the antioxidant and immune indices of rabbits are presented in Fig. 2 and Table 3. The supplementation of NTEM in the drinking water of growing rabbits resulted in an improvement (p < 0.05) in the overall serum antioxidant capacity (TAC) in comparison to the control group. Whereas, adding the NTEM at levels of 1 and 1.5 ml/L to drinking water reduced (p < 0.05) serum MDA of growing rabbits. Moreover, the level of serum phagocytic activity (PA) was increased (p < 0.05) with the addition of the NTEM at a level of 1.5 ml/L to drinking water. No significant differences were observed in phagocytic index (PI), immunoglobulin M (IgM) and, immunoglobulin G (IgG) between the control and treated groups (Table 3).

Fig. 2.

Fig 2 dummy alt text

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Influence of novel trace elements mixture levels on oxidative status (TAC: Total antioxidant capacity, MDA: malondialdehyde) of growing V-line rabbits at 12 weeks of age.

Table 3.

Influence of novel trace elements mixture levels on immunity status of growing V-line rabbits at 12 weeks of age.

Item Control NTEM (ml/L)
 SEM P value
0.5 1.0 1.5
Immunity status1
PA % 19.65b 20.02ab 20.08ab 21.18a 0.21 0.049
PI % 2.80 2.78 2.78 2.82 0.01 0.369
IgM (mg/dl) 995.71 1002.20 992.61 990.64 3.69 0.413
IgG (mg/dl) 256.26 258.03 262.94 257.31 4.16 0.867
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The superscript letters "a" and "b" in the presented data indicate significant differences within each row at a significance level of p-value <0.05.

1

PA: phagocytic activity, PI: phagocytic index, IgG and IgM: immunoglobulins G and M.

3.3. Cecal microbiota

The effects of water supplementation of NTEM on the caecal bacterial count are shown in Table 4. Results showed significant differences among all treatments on caecal bacterial count. The supplementation of NTEM at 0.5 and 1 ml/L levels to the drinking water of growing rabbits resulted in a reduction (p < 0.05) in total bacterial count (TBC) compared to the control and other groups. The lowest TBC value was observed in the group that received 1 ml/L of NTEM. Meanwhile, NTEM at levels 1 and 1.5 ml/L in rabbit drinking water reduced (p < 0.05) E. coli count. In addition, the Clostridia count was decreased (p < 0.05) with all NTEM levels; While Lactobacillus was increased in all treatment groups compared to the control group.

Table 4.

The impact of novel trace elements mixture levels on the caecal microbiota count (log10cfu/g) in V-line rabbits during their growth stage at 12 weeks old.

Item Control NTEM (ml/L)
 SEM P value
0.5 1.0 1.5
Total Bacterial count 8.75a 7.90b 4.14c 9.17a 0.43 <0.001
E. coli 5.78a 5.23a 3.87b 2.28c 0.33 <0.001
Clostridium 14.48a 3.70b 0.00c 0.00c 1.26 <0.001
Lactobacillus 0.80c 2.33b 5.08a 5.47a 0.46 <0.001
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The superscript letters "a-c" in the presented data indicate significant differences within each row at a significance level of p-value <0.05.

4. Discussion

In order to encourage sustainability, supplementation with bioavailable organic trace elements may be a better option because it benefits all parties involved in the feed production chain, up to the end consumer. Additionally, animal diets are supplemented with macro- and micro-elements that increase nutrient utilization. This supplementation is important for proper growth and development, but excessive amounts can lead to imbalances and health issues (Amr et al., 2023).

The enhanced liver function may be ascribed to the safeguarding properties of the trace elements found in the NTEM mixture that support regular liver metabolism. Our findings are in accordance with the study conducted by Elkomy et al. (2015), which demonstrated that rabbits given diets enriched with various amounts of boron showed a gradual decline in the liver aminotransaminase enzymes (AST and ALT) compared to the group fed on a basal diet. The reason behind the decline in AST and ALT enzyme activity caused by boron supplementation may be due to the protective effects of boron on normal liver metabolism. Furthermore, Abdel-Wareth et al. (2022) observed that when ZnO nanoparticles were added to rabbit diets, there was a significant decrease in serum AST and ALT enzyme activities compared to the control group. The decrease in these enzymes indicates a betterment in liver function (Kew, 2000).

In the present study, water supplementation of NTEM levels significantly decreased serum urea and creatinine concentrations. This may be due to the important role of trace elements as enzyme cofactors that improve protein metabolism. Our result is consistent with Helal et al. (2018) who, found that concentrations of total protein, urea and, creatinine were significantly decreased with the addition of Zn and Cu to growing rabbit's diet. Furthermore, Chrastinová et al. (2015) reported that Zn is a component of Zn metalloenzymes, which preserve the structural integrity of proteins, and may be the cause of these improvements in the serum biochemistry of rabbits.

The results obtained from the study showed that supplementing growing rabbits with NTEM levels had a significant impact on the TAC and MDA levels. The NTEM groups had a higher TAC level and lower blood MDA levels compared to the control group. The increased TAC levels in the NTEM groups may be attributed to trace elements, which helps protect against oxidative stress and reduces lipid peroxidation. MDA is a biomarker that indicates overall lipid peroxidation levels, and studies have shown that Zn and B minerals can protect cells from DNA damage and lipid peroxidation, which can lead to various pathological disorders (Elkomy et al., 2015). In addition, Zn may induce the creation of proteins high in sulfhydryl groups, such as metallothionein (a protein rich in cysteine) and glutathione (the primary intracellular non-protein thiol), which could lead to the subsequent reduction in lipid peroxidation (Marreiro et al., 2017). Additionally, Zn can stop protein sulfhydryl groups from oxidation and reduce their reactivity due to its direct binding to the sulfhydryl (Kucková et al., 2021).

On the other side, copper contributes to the antioxidant system by participating in the enzymes ceruloplasmin and Cu-Zn superoxide dismutase (SOD). Superoxide radicals in the cytosol are converted to hydrogen peroxide by the copper-zinc SOD enzyme (Halliwell & Gutteridge, 2015). Antioxidant system, a defense system of the organisms to free radicals, includes the enzymatic and nonenzymatic system (Shen & Song, 2021). The non-enzymatic systems comprise mainly vitamin, cysteine, glutathione (GSH) and mineral element. The enzymatic system consists of antioxidant enzymes, including mainly catalase (CAT), SOD and glutathione peroxidase (GSH-Px). Antioxidant system can catalyze rapidly the superoxide anion (O2-) to produce disproportionation reaction, eliminate superoxide anion, and protect cells of organism from damage of free radicals (Song et al., 2021). Cu supplementation, in the range of growth promotion, can increase activities of GSH-Px, and SOD in serum in cattle, pigs and chickens (Shen et al., 2021). When Cu nutrient is deficient or excessive, lipid peroxidation produces a lot of MDA, consumes a large amount of SOD and GSH-Px, and finally causes a significant decrease of SOD and GSH-Px content, and free radical scavenging capacity decreased (Li et al., 2021). Furthermore, Zn stimulates the production of metallothionein, a protein that binds to metals and may reduce hydroxide radicals, and playing an antioxidant role (Weiss & Spears, 2006).

In the current study, both NTEM levels (0.5 and 1.0 ml/L) significantly decreased total microbial count compared to the control group. Moreover, all NTEM levels significantly decreased E. coli and Clostridium spp. On the other side, Lactobacillus spp increased significantly with all NTEM treatments. This result may be due to the role of trace elements as antibacterial properties. According to recent research, Symbiotic intestinal microorganisms compete with their host for the use of trace metal elements. Moreover, the metabolic processes of trace metal elements in the host and microorganisms affect the organism's health. Supplementation or the lack of trace metal elements in the host can change the intestinal microbial community structure and function (Ma et al., 2023).

Further, Refaie et al. (2015) reported that dietary Nano-Cu supplementation enhanced the population of Lactobacillus and the overall bacterial count while decreasing the population of E. coli and Clostridium Spp. Contrary to coliforms, which are dangerous, lactobacillus is helpful to hosts in animal intestines (Santos et al., 2006). It is generally recognized that the intestinal microbiota is essential for the host animals' nutritional and immune function (Rehman et al., 2008). Additionally, Gunalan et al. (2012) observed that ZnO nanoparticles are effective against a variety of bacterial and fungal pathogens due to their antibacterial activity and antimicrobial efficiency. Moreover, Sentürk et al. (2024) reported that the administration of B significantly altered the composition of the gut microbiota, resulting in a rise in advantageous species such as Barnesiella and Alistipes. Additionally, there was a decrease in some taxa associated with inflammation and illness, such as Clostridium XIVb and Bilophila. Notable increases in genera like Treponema and Catellicoccus were observed, suggesting the potential of boron compounds to enrich microbial communities with unique metabolic functions. This result might be attributable to the NTEM role in creating a favorable environment for Lactobacillus growth and reducing harmful bacteria.

5. Conclusion

In conclusion, adding the NTEM in rabbit drinking water showed notable benefits on liver and kidney functions, antioxidant and some immunity parameters, as well as the cecal bacterial count. Thus, the NTEM as a mineral mix, environmental friendly prepared, is recommended for using as water supplementation for the growing rabbits especially at levels 1 and 1.5 ml/ L. However, further evaluation and investigation of other parameters are necessary to provide more in-depth information and confirm its benefits for animal production.

Ethics approval and consent to participate

All practices and investigates of the study were accompanied by approvals of the ethical principles of animal research and approved by Damanhour University's Ethical. Animal Care and Use Committee (Approval No: DUFA-2023–4). We obtained informed consent from the owner of the birds involved in the study. All methods were carried out according to relevant guidelines and regulations (Ethics Committee, Damanhour University, Egypt). The study was carried out in compliance with the ARRIVE guidelines.

Consent for publication

Not applicable.

AI Disclosure

The authors used an artificial intelligence–assisted language tool (ChatGPT) solely to improve the clarity, and grammar of the manuscript. The AI tool was not used for data collection, data analysis, interpretation of results, or generation of scientific content. The authors take full responsibility for the content of the manuscript.

Funding

This research received financial support from the Deanship of Graduate Studies and Scientific Research at Qassim University, Saudi Arabia No (QU-APC-2026).

CRediT authorship contribution statement

Amr El-Nile: Conceptualization, Methodology, Investigation, Data curation, Formal analysis, Writing – original draft. Mohamed Hafez: Software, Data curation, Writing – review & editing. Mohamed I. Hassan: Methodology, Investigation, Data curation, Validation, Writing – review & editing. Saber S. Hassan: Methodology, Validation, Supervision, Writing – review & editing. Mohamed Rashad: Formal analysis, Writing – review & editing. Abdallah E. Mohamed: Conceptualization, Validation, Writing – review & editing. Alexander I. Popov: Software, Writing – review & editing. Mahmoud Alagawany: Writing – review & editing, Writing – original draft. Sobhy M.A. Sallam: Software, Funding acquisition, Project administration, Writing – review & editing.

Declaration of competing interest

No potential conflicts of interest declared.

Acknowledgements

This research received financial support from the Deanship of Graduate Studies and Scientific Research at Qassim University, Saudi Arabia No (QU-APC-2026).

Data availability

  • All the data generated or analyzed during this study are included in this published article.

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