[version 2; peer review: 2 approved, 2 approved with reservations]
No competing interests were disclosed.
While ivermectin is a common antiparasitic drug used in veterinary medicine, the effects of repeated administration at therapeutic doses have not been well studied. Of particular interest is the impact of ivermectin on electrolyte balance, blood biochemistry, and oxidation-related markers in rabbits, as these animals are known to be sensitive to drug-related metabolic disturbances and side effects.
A total of 10 clinically healthy adult male rabbits (1.5-2 kg) were randomly assigned to a control group with no treatment or to a treatment group receiving ivermectin subcutaneously at a dose of 0.25 mg/kg once weekly for 30 days. The animals were housed in the physiological laboratory of the University of Fallujah under controlled environmental conditions. At the end of the experiment, blood samples were collected, and serum was analyzed for electrolytes, biochemical and oxidative markers, and liver enzyme activity using established laboratory methods.
Repeated administration of ivermectin caused a statistically significant increase in potassium, sodium, chloride, and phosphate concentrations (P≤0.05) compared with baseline, while calcium and magnesium levels remained unchanged. Glucose, total protein, creatinine, cholesterol, and triglyceride levels increased statistically significantly (P≤0.05) compared with the control group, while albumin concentration decreased. Oxidative stress indicators showed increased levels of malondialdehyde (MDA) and decreased levels of reduced glutathione (GSH), as well as superoxide dismutase (SOD) activity. Increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity were also noted.
Repeated administration of ivermectin at therapeutic doses at short intervals causes disturbance in blood electrolyte balance, biochemical profile, and antioxidant system in rabbits, and may also negatively impact liver and kidney functions. These results indicate the need for cautions use of the drug, preferably limited to single administration at long intervals, to minimize the risk of side effects.
The revised version of the manuscript includes substantial improvements in response to the reviewers’ comments. The Introduction was extensively rewritten to provide a more focused background on ivermectin use in rabbits, including its physiological and biochemical effects, pharmacological mechanisms, and existing knowledge gaps related to repeated administration. The study objectives were also clarified and aligned more closely with the title and experimental design. The Materials and Methods section was reorganized using clear subheadings and expanded to include detailed descriptions of the experimental animals, housing conditions, treatment protocol, sample collection procedures, biochemical analyses, and statistical methods. Additional methodological details, including the number of animals per group and serum processing procedures, were added for clarity and reproducibility. The Results section was revised to improve data presentation and statistical reporting. The Discussion section was substantially expanded and restructured to provide deeper scientific interpretation of the findings in relation to oxidative stress, electrolyte imbalance, liver enzyme alterations, and metabolic disturbances associated with repeated ivermectin exposure. Comparisons with previous studies in other animal species and relevant physiological mechanisms were also incorporated. Furthermore, the entire manuscript underwent comprehensive language editing and scientific revision to improve clarity, consistency, and accuracy.
Ivermectin is a semisynthetic derivative of a vermectin, a naturally occurring macrocyclic lactone produced by the bacterium
Despite its proven efficacy and extensive concerns have been raised regarding potential adverse effects in treated animals. Ivermectin exhibits a variety of pharmacokinetic properties, including high lipid solubility, long half-life and extensive tissue distribution, especially in the liver and adipose tissue (
Several studies have shown that avermectins (ivermectin, abamectin, doramectin and eprinomectin) may cause a variety of biochemical, hormonal and histopathological changes in animals exposed to these substances (
Ivermectin is given routinely to rabbits to prevent and treat gastrointestinal parasitic infestations and mange (
Ten clinically healthy adult male rabbits (Oryctolagus cuniculus) weighing 1.5 to 2 kg (aged 10 to 12 months) were included in the study. The animals were maintained in the physiology laboratory of the University of Fallujah under controlled climate conditions: 23 ± 3 °C, 12-hour light and dark periods, and a relative humidity of 50-60%.Food was fed daily using a commercial balanced diet and water was freely available. Before the initiation of the experimental procedures to minimize handling stress and achieve full acclimatization to the laboratory environment. to minimize handling stress and achieve full acclimatization to the laboratory environment.
After acclimation, rabbits were randomly allocated to one of two groups of five animals: a control group (no treatment) and an ivermectin-treated group (Ivermac-10 ®, ADWIA Pharmaceuticals, Egypt) that received ivermectin at a dose of 0.25 mg/kg body weight by subcutaneous injection once weekly for 4 weeks. Prior to blood collection, animals were sedated with xylazine (6 mg per kg body weight, intramuscular) (
All animal experimentation, including blood sampling or anaesthetic, have been carried out according to the ethical guidelines and recommendations of the American Veterinary Medical Association (AVMA) for proper care and treatment of experimental animals (
| Parameters | Control | Ivermectin | P-value | |
|---|---|---|---|---|
| Glucose mg/dl | 52 ± 3.0 B | 75.6 ± 1.65 A | 0.00021 | |
| Total protein g/dl | 8.26 ± 0.17 B | 11.2 ± 0.55 A | 0.00082 | |
| Albumin g/dl | 5.3 ± 0.3 A | 3.4 ± 0.2 B | 0.00039 | |
| Creatinine mg/dl | 1.93 ± 0.25 B | 3.1 ± 0.29 A | 0.0116 | |
| Uric acid mg/dl | 8.3 ± 0.2 A | 8.24 ± 0.16 A | 0.73 | |
| Cholesterol mg/dl | 44.4 ± 1.8 B | 69.8 ± 1.39 A | 6.7 × 10 −6 | |
| Triglyceride mg/dl | 46.2 ± 1.5 B | 72.4 ± 1.4 A | 2.8 × 10 −7 | |
-Number of animals in each group (5), Data reflected as Mean ± Standard Error (SE).
-The different capital letters indicate significant changes at (P ≤ 0.05) group variation.
Ivermectin substantially increased (P ≤ 0.05) the potassium, sodium, chloride, and phosphate ion levels in blood serum compared to the control. Ivermectin did not affect serum calcium or magnesium concentrations (
| Parameters | Control | Ivermectin | P-value | |
|---|---|---|---|---|
| K + (mmol/L) | 5.24 ± 0.23 B | 6.54 ± 0.2 A | 0.0035 | |
| Na + (mmol/L) | 143.4 ± 0.74 B | 158.4 ± 1.29 A | 0.0023 | |
| Cl - (mmol/L) | 112 ± 0.83 B | 117.4 ± 1.1 A | 0.0042 | |
| Phosphate (U/L) | 3.74 ± 0.27 B | 8.9 ± 0.63 A | 0.00031 | |
| Mg +2 (mg/dl) | 2.23 ± 0.04 A | 2.3 ± 0.11 A | 0.32 | |
| Ca +2 (mg/dl) | 20.1 ± .73 A | 21 ± 1.17 A | 0.58 | |
-Number of animals in each group (5), Data reflected as Mean ± Standard Error (SE).
-The different capital letters indicate significant changes at (P ≤ 0.05) group variation.
The effects of ivermectin treatment on the values of some serum biochemicals are shown in (
| Parameters | Control | Ivermectin | P-value | |
|---|---|---|---|---|
| MDA (μmol/l) | 8.11 ± 0.31 B | 16.14 ± 0.8 A | 1.2 × 10 −7 | |
| GSH (μmol/l) | 14.2 ± 0.94 A | 8.4 ± 1.8 B | 0.0003 | |
| SOD (U/ml) | 12 ± 0.83 A | 7.4 ± 1.1 B | 0.0062 | |
| AST (IU/l) | 33.4 ± 0.49 B | 38.2 ± 0.57 A | 4.6 × 10 −5 | |
| ALT (IU/l) | 14 ± 0.86 B | 21.5 ± 0.93 A | 3.5 × 10 −5 | |
-Number of animals in each group (5), Data reflected as Mean ± Standard Error (SE).
-The different capital letters indicate significant changes at (P ≤ 0.05) variation between groups.
Following repeated administration of ivermectin to rabbits, biochemical changes were observed may indicating liver and kidney dysfunction. Significant increases in serum sodium, potassium, chloride, and phosphate levels (
Significant increases in serum glucose, creatinine, triglycerides, cholesterol and total protein after repeated ivermectin administration (
In addition, ALT, AST and MDA levels increased significantly with a significant decrease in GSH and SOD activity (
The combination of prolonged oxidative stress and decreased liver and kidney function may contribute to progressive metabolic dysregulation. Electrolyte imbalance may reflect altered renal excretion or redistribution, including sodium, potassium, chloride and phosphate ions. These alterations may be associated with metabolic disturbances involving hepatic and renal function are due to hepatic injury, reduced albumin synthesis and impaired glucose and lipid metabolism, all of which are due to long-term oxidative stress. This oxidative imbalance may be related to repeated ivermectin exposure, followed by excessive formation of ROS and eventual exhaustion of antioxidant protection. This pattern is consistent with previous studies in mammals of oxidative and metabolic stress induced by ivermectin (
In summary, this study demonstrates biochemical alterations associated with repeated ivermectin exposure in rabbits. Repeated ivermectin exposure may be associated with oxidative stress and possible mitochondrial, which may contribute to biochemical disturbances, altered renal function, and electrolyte imbalance. These findings suggest that even therapeutic or prolonged exposure may be associated with concurrent hepatic and renal functional disturbances.
This study was conducted in full compliance with the ethical guidelines approved by the Scientific Research Ethics Committee of the Faculty of Veterinary Medicine, University of Fallujah (Approval No. 6; 13/10/2025),
Underlying data Zenodo: Dataset for the Ivermectin Study in Rabbits (2025).
Data are available under the terms of the
The authors thank the study staff as well as the study participants.
The study investigates an important issue:
It includes a
Electrolytes Biochemical profile Oxidative stress markers Liver enzyme activity The dose used (0.25 mg/kg) is within a
Only
This greatly reduces
Blood samples were taken only at the end of the experiment. Therefore, it is unclear:
When the changes started Whether they were progressive or temporary Whether the effects were reversible The experiment lasted only
This is insufficient to assess
The study suggests possible liver and kidney damage based on ALT, AST, and creatinine levels. However, there is
Significant changes in Na⁺, K⁺, Cl⁻, and phosphate were reported. However, the study provides
Only one dose level was tested.
Is the work clearly and accurately presented and does it cite the current literature?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
Yes
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Is the study design appropriate and is the work technically sound?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
Yes
Reviewer Expertise:
endocrine disorders single-cell technologies
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
General Evaluation
The manuscript investigates the physiological, biochemical, and oxidative stress impacts of repeated therapeutic subcutaneous administrations of ivermectin (0.25 mg/kg} weekly for 4 weeks) in male rabbits. Given the ubiquitous use of ivermectin in veterinary medicine for treating conditions like sarcoptic mange, evaluating subclinical toxicities associated with repeated exposure is highly relevant.
The authors have visibly addressed several structural shortcomings from Version 1 by organizing the methodology with better kit descriptions and clarifying group numbers (n=5). However, critical scientific gaps, contradictions within the text, and reporting vulnerabilities remain in this revised version. Notably, the manuscript still claims to evaluate "physiological" responses, yet it only reports serum biochemical data. Additionally, there are major discrepancies in statistical parameter reporting and questionable physiological baselines that require thorough rectification before approval.
Major Comments
1. Misalignment of Title/Objectives with Results (Missing Physiological Data):
The title and objectives explicitly promise an evaluation of physiological responses. True physiological data typically include clinical signs, body weight changes, feed/water intake, fecal output, or vital signs (temperature, heart rate, respiration rate). The results presented are exclusively biochemical endpoint markers from serum. The authors must either supply the missing clinical/physiological data or remove "Physiological" from the title and text to avoid misleading the reader.
2. Inconsistencies in Methodological Terminology & Blinding:
In the Material and Methods section, the authors state: "Independent two-sample t-test was used to evaluate differences between the occlusion and control groups." The term "occlusion group" is highly confusing contextually. It appears to be a copy-paste or translation error for the "ivermectin-treated group". This must be corrected to maintain professional clarity.
There is no mention of whether sample analysis or data handling was blinded. Given the small sample size (n=5 per group), bias control is crucial.
3. Statistical Reporting and Logical Errors:
4. Biochemical and Physiological Implausibility:
If total protein rises drastically while albumin plummets, it implies an immense, unaddressed spike in the globulin fraction. The authors completely ignore this dynamic in the discussion. What is driving this massive globulin increase? Is it an acute phase inflammatory response, or severe hemoconcentration/dehydration? This needs profound biochemical interpretation.
Minor Comments
1. Typographical and Grammatical Errors:
Material and Methods (Paragraph 1): The sentence fragment "...to minimize handling stress and achieve full acclimatization to the laboratory environment." is accidentally duplicated back-to-back at the end of the paragraph. Please excise the duplicate text.
Introduction (Paragraph 1): Typo in "Its antiparasitic activity is primarily mediatrd through...". Correct "mediatrd" to "mediated".
Discussion (Paragraph 2): Grammatical error in "Reduced serum albumin may impaired hepatic synthetic function...". Change to "Reduced serum albumin may indicate impaired hepatic synthetic function..." or "...may impair...".
2. Incomplete Manufacturer Data:
In the Material and Methods section, the colorimetric kits are cited with manufacturers and cities. Please provide the full formal corporate names, specific catalog numbers if available, and complete locations (City, Country) for all diagnostic test kits to maximize transparency and reproducibility.
3. Units Format Consistency:
Ensure standard scientific formatting is used uniformly across all tables.
Conclusion
This manuscript provides practical insights into the subclinical biochemistry profiles of ivermectin overuse. However, it requires a Moderate-to-Major Revision to eliminate textual errors, reconcile highly unusual reference baselines, and properly align the stated physiological scope with the purely biochemical results presented.
Is the work clearly and accurately presented and does it cite the current literature?
Partly
If applicable, is the statistical analysis and its interpretation appropriate?
Partly
Are all the source data underlying the results available to ensure full reproducibility?
Partly
Is the study design appropriate and is the work technically sound?
Yes
Are the conclusions drawn adequately supported by the results?
No
Are sufficient details of methods and analysis provided to allow replication by others?
Partly
Reviewer Expertise:
Biochemistry
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Peer Review Report
Summary of the Article
The manuscript entitled “Physiological and Biochemical Responses to Repeated Administration of Ivermectin in Rabbits” investigates the physiological and biochemical effects associated with repeated ivermectin administration in rabbits. The study evaluates changes in selected hematological, biochemical, and physiological parameters to assess the potential impact and safety profile of repeated ivermectin exposure. The topic is relevant due to the widespread use of ivermectin in veterinary medicine and the need to better understand its effects following repeated administration.
Overall Assessment
The manuscript addresses an important veterinary and pharmacological topic. The study is generally well designed, and the findings contribute useful information regarding the physiological and biochemical responses of rabbits to repeated ivermectin treatment. The manuscript is clearly written and presents results that may be valuable for researchers and veterinary practitioners.
Major Comments
The study addresses a relevant scientific question and provides data that may contribute to understanding the safety and biological effects of repeated ivermectin administration.
The experimental design appears appropriate for the objectives of the study. However, the authors may further strengthen the manuscript by providing additional discussion comparing their findings with those reported in previous studies.
The discussion section could be expanded to better explain the physiological mechanisms responsible for the observed changes in the measured parameters.
The authors should clearly highlight the practical implications of their findings for veterinary practice and future research.
Minor Comments
Some grammatical and stylistic revisions would improve readability.
The manuscript would benefit from a more detailed description of certain methodological procedures to enhance reproducibility.
Ensure that all abbreviations are defined at their first appearance in the text.
A careful review of formatting and reference style is recommended.
Strengths
The study investigates a topic of practical veterinary importance.
Appropriate physiological and biochemical parameters were evaluated.
The manuscript is generally well organized and easy to follow.
The findings provide useful information regarding repeated ivermectin administration in rabbits.
Weaknesses
The discussion could provide deeper interpretation of the biological significance of the findings.
Additional comparison with recent literature would strengthen the conclusions.
Some methodological details may require further clarification.
Recommendation
Approved
The manuscript is scientifically sound and suitable for indexing after consideration of the minor comments above.
Is the work clearly and accurately presented and does it cite the current literature?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
Yes
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Is the study design appropriate and is the work technically sound?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
Yes
Reviewer Expertise:
animal physiology
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
The manuscript addresses an interesting and relevant topic, particularly considering the widespread use of ivermectin as a broad-spectrum antiparasitic agent in animals. However, the potential effects of continuous or repeated dosing on organismal health remain insufficiently understood, which makes this study potentially valuable.
Despite this, the manuscript requires substantial improvement in terms of clarity, structure, methodological detail, and scientific depth before it can be considered for publication.
The Introduction is currently too general and lacks sufficient focus on the specific research problem. The authors are encouraged to:
Expand the background on the
Provide a more detailed overview of the
Ensure alignment between the
Clearly and explicitly state the
The Methods section lacks clarity and organization and should be substantially revised:
Structure the section using clear subheadings, such as:
The manuscript does not specify the
The description of procedures should be improved for clarity and correctness. For example: “The blood samples were centrifuged at 3000 rpm for 10 minutes to obtain serum, which was subsequently stored at −20 °C until analysis.”
The study claims to evaluate
Ensure that all variables described in the objectives and methods are
Present physiological data (if collected) in a clear and structured manner, including appropriate statistical analysis.
The Discussion requires significant improvement and deeper scientific interpretation:
The section currently focuses mainly on reporting results rather than interpreting them in the context of existing literature. The authors should incorporate and compare their findings with
Provide a structured discussion addressing:
Each variable should be discussed in relation to known physiological mechanisms and potential effects of ivermectin.
The manuscript has potential but requires
Improve the overall quality of scientific writing. Provide missing methodological details. Strengthen the Discussion with critical analysis and literature support. Include additional relevant references to contextualize the findings.
Only after substantial revision and clarification of these aspects can the manuscript be adequately evaluated for indexing.
Reviewer Expertise:
NA
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Dear Oscar Vivanco-Galván,
We sincerely appreciate your valuable comments and constructive suggestions regarding our manuscript. All comments have been carefully considered, and substantial revisions have been made throughout the manuscript to improve its scientific quality, clarity, organization, and methodological accuracy.
In addition, the manuscript has undergone comprehensive language editing and scientific revision to improve clarity and consistency throughout the text.
We are grateful for your insightful comments, which significantly improved the quality of the manuscript.
Sincerely ,
Sabea Khamees Abed