Evaluating the Efficacy of Rosemary Extract (Rosmarinus officinalis) as a Natural Antiparasitic Agent in Livestock: A Sustainable Approach to Enhance Animal Health

Evaluating the Efficacy of Rosemary Extract (Rosmarinus officinalis) as a Natural Antiparasitic Agent in Livestock: A Sustainable Approach to Enhance Animal Health | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluating the Efficacy of Rosemary Extract (Rosmarinus officinalis) as a Natural Antiparasitic Agent in Livestock: A Sustainable Approach to Enhance Animal Health Aimal Ali, Asma Akbar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8692058/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Livestock ectoparasites (ticks, mites, lice, fleas, and bed bugs) reduce productivity and increase control costs; resistance to synthetic agents is rising. To evaluate the in vitro antiparasitic efficacy of ethanolic Rosmarinus officinalis (rosemary) extract against major cattle ectoparasites and to compare killing speed across concentrations. Rosemary leaves were shade-dried, powdered, extracted in ethanol, concentrated by rotary evaporation, and tested in Petri-dish bioassays. Five ectoparasite groups collected from naturally infested cattle were exposed to three extract preparations (low, high, and concentrate/undiluted, ensuring consistency with your protocol). Time-to-death (min) and mortality (%) were recorded. One-way ANOVA with Tukey’s HSD was used (α = 0.05). The extract showed a clear dose-response. The high-concentration formulation (reported as 2 g extract in 6 mL ethanol) produced complete mortality within ~ 5–10 minutes across taxa; mites were most sensitive (~ 5 min) and lice were least sensitive (~ 10 min). Treatment effects were significant (ANOVA, p < 0.001). Ethanolic rosemary extract demonstrates rapid, broad-spectrum in-vitro antiparasitic activity and could support integrated pest management strategies. This study provides a standardized, side-by-side comparison of killing speed across multiple cattle ectoparasite taxa using the same extraction approach and endpoint (time-to-death). Antimicrobial Resistance (AMR) Antiparasitic efficacy Green veterinary pharmacology Integrated Pest Management (IPM) Livestock ectoparasites Phytochemicals Rosmarinus officinalis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction There are around 1.5 billion head of cattle worldwide, making the cattle industry an important part of food systems worldwide. Data-Driven Reality Economic losses due to stunted development and milk production, damaged hides, disease transmission, and higher veterinary and control expenses are enormous as a result of parasitic and ectoparasitic infestations of this size. A significant fraction of the world's cattle are at risk of exposure to ticks and tick-borne illnesses, which is said to cost the cattle industry an estimated $ 20–30 billion annually (Makwarela et al., 2023 ). Ticks, mites, lice, and fleas are not only annoying, but they may also spread a variety of zoonotic diseases that are harmful to humans and animals (Ruiz et al., 2025 ). The worldwide cattle business is nevertheless confronted with the widespread and economically devastating problem of parasitic diseases. Reduced weight increase, decreased milk production, and, in extreme circumstances, substantial mortality within herds are all symptoms of these illnesses, which are a major cause of lost productivity (Strydom et al., 2025 ). Ticks, mites, lice, and fleas are not only annoying, but they may also spread a variety of zoonotic diseases that are harmful to humans and animals (Ruiz et al., 2025 ). The infestation rates are often linked to poor physical condition, which causes a vicious circle of physiological stress and higher veterinary expense which have severe economic ramifications (Nizamov, 2025 ). When parasites disrupt the equilibrium of the host immune system, the animal redirects its metabolic resources from growth and reproduction to defense. This isn't only an issue for vets; controlling these pests is crucial to global food security and the future of farming (Brown, Shine, & Rollins, 2025 ). The increasing threat of Antimicrobial Resistance (AMR) has created a significant challenge for the veterinary sector regarding the efficacy of traditional chemical treatments (Tang, Millar, & Moore, 2023 ). Parasitic populations have developed resistance mechanisms more rapidly owing to the misuse of synthetic antibiotics and anthelmintics since the mid-20th century (Guerrero-Encinas et al., 2025 ). An evolutionary "arms race" has rendered several traditional pharmacological therapies ineffective, resulting in chemical residues contaminating animal products and persisting in the environment, while the targeted pests remain unregulated (Lobanovska & Pilla, 2017 ). It is now important to strategically switch to other therapies to keep macrocyclic lactones working. As synthetic drugs based on single molecules are readily resistant to evolution, there is increasing demand in the industry to create alternatives that are more complex and physiologically robust, capable of evading the adaptation mechanisms of modern parasites (Lifschitz et al., 2024 ). Due to these issues, phytochemicals, which are natural compounds derived from plants, have emerged as a novel and intriguing area of investigation in veterinary pharmacology (Ogbuagu et al., 2022 ). Phytochemicals could be a useful substitute for antibiotics since bacteria have a hard time adjusting to them because their chemical profiles are so diverse (Wiles, Pearson, & Beddoe, 2025 ). The World Health Organization claims that a lot of people throughout the world are using plant-based medicines right now, and this trend is starting to spread to animal husbandry as well (Calixto, 2019 ). Rosemary, or Rosmarinus officinalis , is one of these herbs that stands out because it contains so many bioactive chemicals. Carnosic acid and rosmarinic acid are two of these chemicals that are particularly excellent in fighting germs and free radicals (Aziz et al., 2022 ). These plant extracts are commonly used for two things: to help ruminants' digestion and to lessen the amount of methane they release. This is not the same as chemicals that are created in a lab. Rosemary is more than just a pesticide; it is also a full tool in the area of "green veterinary pharmacology” (Kholif et al., 2017 ). Even though parasite control in cattle is routine, there is still a clear need for options that are effective while also being safer for animals and the environment. Although rosemary is known to contain bioactive compounds, there is limited controlled evidence that measures its direct killing effect and, in particular, the time-to-death of different cattle ectoparasites under standardized conditions. Therefore, this study aimed to evaluate the in vitro antiparasitic activity of ethanol-extracted Rosmarinus officinalis and identify the concentration that produces the fastest and most reliable mortality. Specifically, this work tested the extract against key ectoparasite groups in a controlled setting, compared mortality and time-to-death across low, high, and pure extract treatments, and assessed whether the response followed a clear dose–response pattern. The novelty of this study lies in providing a direct, side-by-side comparison across multiple parasite groups using a single extraction method and the same measurable endpoints, which makes the findings easier to interpret and apply. However, because the study was conducted in vitro, it cannot fully reflect real farm conditions where factors such as the animal’s skin and hair, parasite behavior, environmental exposure, product stability, and animal safety may influence outcomes; therefore, formulation work and field trials are needed before routine on-animal use can be recommended. Overall, this study offers a practical foundation for developing rosemary-based approaches that could support more sustainable ectoparasite management and reduce reliance on synthetic products. 2. Methodology 2.1 Study Design and Preparation of Extracts The efficacy of rosemary leaves as a natural ectoparasiticide was evaluated using an experimental in vitro bioassay approach. Fresh leaves of rosemary ( Rosmarinus officinalis L. ) were collected from Khyber nursery, KPK, Peshawar, Pakistan, washed with clean water, and shade-dried to reduce thermal loss of volatile constituents. The dried leaves were mechanically ground into a fine powder using a grinder and stored in airtight containers until extraction. Ethanol was used as the extraction solvent. The crude rosemary extract obtained after solvent removal is shown in Fig. 1 . A weighed amount of plant powder was macerated in ethanol (2000 mL) for 10 days, then filtered through filter paper. The filtrate was concentrated under reduced pressure using a rotary evaporator to remove ethanol and obtain the crude extract. Concentration of the ethanolic filtrate using a rotary evaporator is illustrated in Fig. 2 . Three treatment preparations were produced for testing concentration-dependent effects: Low concentration (diluted), High concentration (2 g crude extract in 6 mL ethanol ), and Undiluted concentrate (15 g crude extract), as shown in Fig. 3 . These preparations were evaluated against the target ectoparasites under controlled exposure conditions, recording mortality and time-to-death as outcome measures. 2.2 Parasite Collection and Bioassay Bedbugs ( Cimex ), ticks ( Rhipicephalus ), mites ( Psoroptes ), lice ( Linognathus ), and fleas ( Ctenocephalides ) were among the five ectoparasite species that were retrieved from naturally affected cattle. After identifying the parasites, they were divided into groups for treatment. In the bioassay, the parasites were exposed to the extract in a controlled environment using petri plates. Death was officially reported after all bodily responses had been exhausted, and the "time-to-death" was calculated in minutes from the time of exposure. 2.3 Statistical Analysis All statistical analyses were conducted using SPSS Statistics and Graphpad Prism. For each ectoparasite group, the outcomes were summarized per replicate as time-to-death (minutes) or mortality (%) and expressed as mean ± SD (or mean ± SE, as appropriate). Differences among the three rosemary extract concentrations (low, high, and pure/undiluted) were evaluated using a one-way analysis of variance (ANOVA). Before ANOVA, assumptions were assessed by inspecting residual plots, applying the Shapiro–Wilk test for normality, and Levene’s test for homogeneity of variances. Where ANOVA indicated a significant treatment effect, Tukey’s HSD post hoc test was used for pairwise comparisons between concentrations while controlling the family-wise error rate. Statistical significance was set at p < 0.05, corresponding p-values, and post hoc adjusted p-values for pairwise differences. 3. Results 3.1 Ectoparasite Mortality All of the ectoparasite species that were examined died off when the Rosmarinus officinalis extract was applied. The results were directly related to the level of concentration. High Concentration (2 g + 6 mL ethanol) was the most efficient solution, eliminating all species within 5 to 10 minutes. The low concentration formulations and pure extract, in contrast, needed longer exposure times and were only partially effective. Time-to-death outcomes for the high-concentration rosemary extract are summarized in Table 1 and visualized in Figure 4 . 3.2 Comparative Efficacy Mites exhibited the highest degree of physiological sensitivity to the extract among the assessed species. They only got treatment for five minutes before they died. Lice were the most durable of all the species, since they could live for up to 10 minutes before dying entirely. This variety shows that various species have developed distinct ways to resist, which are likely linked to changes in the thickness of their exoskeletons or the ways they break down toxins in their bodies. Species-specific differences and key post-exposure behavioral observations are presented in Table 2 , with comparative distributions shown in Figure 5 . 3.3 Statistical Significance The One-Way ANOVA showed very significant variations in death rates across the various concentration groups (p < 0.001)21. ANOVA results are summarized in Table 3 . Post hoc comparisons and statistical interpretation are provided in Table 4 . 4. Discussion This study provides compelling empirical evidence for the strong in vitro antiparasitic activity of Rosmarinus officinalis (rosemary) extract against common livestock ectoparasites (Table 5) . The findings demonstrate that the ethanol-based rosemary extract acts as a fast-acting, broad-spectrum natural acaricide, achieving complete mortality within 5-10 minutes across tested species. These results align closely with earlier work by Razzaq et al. (2024), who reported significant antiparasitic efficacy of essential oils such as neem, tea tree, and rosemary against ectoparasitic infestations in cattle. However, unlike Razzaq et al. (2024), who assessed hydro-distilled essential oils qualitatively, the present study provides quantitative time-to-death data, establishing rosemary extract’s superior potency and rapid action. The concentration-dependent effects observed in this study confirm that solvent extraction and formulation play crucial roles in optimizing efficacy. The high-concentration ethanol extract (2 g/6 mL) outperformed both pure and diluted forms, indicating that ethanol enhances the solubility and penetration of active phytochemicals such as carnosic acid and rosmarinic acid. These compounds are known to disrupt neural and mitochondrial functions in arthropods, leading to rapid paralysis and death (Senanayake, 2018). Similar patterns of solvent-dependent efficacy were noted by Razzaq et al. (2024), who observed that essential oil formulations exhibited higher lethality when combined with polar solvents that improved diffusion and surface adherence. Together, these findings underscore the importance of optimizing extraction polarity for achieving high bioactivity in botanical acaricides. Comparison with Gajarmal et al. (2025) further situates this work within the broader framework of ethnoveterinary medicine. Gajarmal and colleagues conducted a comprehensive review of 86 medicinal plant species used traditionally for parasitic control in livestock, identifying phytochemical groups such as flavonoids, tannins, and essential oils as the main bioactive agents. Our results empirically validate this ethnobotanical foundation, providing laboratory confirmation that rosemary, a Lamiaceae family member highlighted in ethnoveterinary records, possesses measurable antiparasitic efficacy through its phenolic diterpenes. This strengthens the link between traditional herbal practices and modern pharmacological validation, bridging the gap between ethnoveterinary knowledge and experimental parasitology. From a mechanistic standpoint, Gajarmal et al. (2025) categorized antiparasitic herbs under Ayurvedic “Krimighna Mahakashaya” groups, associated with internal and external parasite control. The current study substantiates that classification by demonstrating that R.officinalis induces neuro-mitochondrial disruption leading to rapid ectoparasite mortality. Thus, rosemary can be viewed as a modern biochemical representative of this ancient medicinal category, aligning traditional pharmacognosy with contemporary bioassay evidence. When compared with Ahmed et al. (2023), who investigated the anti-ectoparasitic efficacy of methanolic extracts from Calpurnia aurea , Eucalyptus globulus , and Croton macrostachyus on sheep and goat ticks, several notable distinctions emerge. Ahmed et al. achieved up to 80% mortality after 24 hours of exposure, whereas the current rosemary extract caused complete death within 5–10 minutes, highlighting its exceptional speed and potency. The superior performance of rosemary may be attributed to its high phenolic content and solvent-specific extraction efficiency, as ethanol allows better penetration through the arthropod cuticle compared to methanol (Ahmed et al., 2023). Furthermore, while Ahmed et al. (2023) focused solely on ticks, this study demonstrated efficacy against five different ectoparasite taxa, including mites, lice, fleas, and bed bugs, thereby broadening the antiparasitic spectrum and enhancing practical applicability in multi-species infestations. Mechanistic parallels across these studies reveal that plant-derived compounds generally act through multiple synergistic biochemical pathways, including inhibition of chitin synthesis, interference with respiratory enzymes, and disruption of neural signaling. The polyphenolic compounds in rosemary appear to act primarily via oxidative stress and mitochondrial dysfunction, a mechanism supported by observations of rapid paralysis and immobilization in mites and fleas during exposure (Kafle & Chung, 2025). This mode of action differs from that of single-target synthetic acaricides, such as pyrethroids or organophosphates, which often induce resistance due to their narrow molecular targets (Lifschitz et al., 2024). As noted by Guerrero-Encinas et al. (2025), the chemically diverse nature of phytochemicals presents a “multi-target resistance barrier,” reducing the likelihood of adaptive evolution among ectoparasites, a key advantage for long-term Integrated Pest Management (IPM). The interspecific variation observed with mites being the most susceptible and lice the most resilient reflects differential cuticular permeability and detoxification capacities among ectoparasite taxa. This observation parallels the findings of Ahmed et al. (2023), who reported species-specific mortality responses among tick species exposed to different plant extracts. The consistency of such results across independent studies reinforces the conclusion that structural and physiological parasite traits modulate susceptibility to botanical acaricides, underscoring the need for species-tailored concentration thresholds in practical applications. The broader implications of these findings are significant for sustainable livestock management. As highlighted by Gajarmal et al. (2025) and Razzaq et al. (2024), integrating plant-based acaricides like rosemary into IPM frameworks could reduce reliance on synthetic chemicals, thereby mitigating environmental contamination, preventing resistance buildup, and improving animal welfare. Moreover, rosemary’s dual role as a feed additive with digestive and antioxidant benefits Farghaly and Abdullah (2021); Kholif et al. (2017)Kholif et al. (2017) enhances its value as a holistic veterinary solution, aligning with the principles of “green veterinary pharmacology.” Finally, while this study establishes strong in vitro efficacy, further in vivo validation is warranted. Future research should focus on evaluating rosemary extract’s persistence, dermal safety, and field effectiveness in live animals, as well as exploring synergistic formulations with other botanicals such as neem or eucalyptus. Such approaches could yield eco-safe, multi-component acaricides capable of sustaining long-term parasite control without compromising productivity or animal health. Conclusion This study shows that an ethanol extract of Rosmarinus officinalis can kill the tested cattle ectoparasites quickly under laboratory conditions, and that the effect increases with increasing concentration. The high-concentration formulation (2 g extract in 6 mL ethanol) achieved complete mortality across all tested groups within about 5–10 minutes, with mites dying the fastest (around 5 minutes) and lice taking the longest (up to 10 minutes). The statistical results also supported these differences, as the concentrations produced significantly different outcomes (ANOVA, p < 0.001), and the highest concentration performed best in post hoc comparisons. What makes this work novel is that it compares several livestock-relevant ectoparasites side by side using the same extraction method and the same measurable endpoints, especially “time to death,” which gives more practical information than mortality alone. To move from lab evidence to real use, future studies should test rosemary-based preparations in animals and under farm conditions, confirm safety and any potential skin-irritation risk, measure how long the effects last on hair and skin, and determine the most effective delivery method (spray, dip, or pour-on). Researchers should also standardize the extract by measuring key bioactive compounds, checking formulation stability and shelf-life, and exploring whether combining rosemary with other botanicals improves consistency and reduces the need for synthetic products within integrated pest management programs. Declarations Author Contribution Statement Aimal Ali: Writing – original draft, writing – review & editing, Visualization, Resources. Asma Akbar: Data curation, formal analysis, and Supervision. Declaration of competing interest The authors declare no conflicts of interest. Data availability Data will be made available on request. Acknowledgment We are grateful to all the authors for their equal contributions to this manuscript writing and presentation. Declaration of generative AI and AI-assisted technologies in the writing process The figures were created using PowerPoint Presentation. Funding Sources This review paper manuscript did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References Aziz, E., Batool, R., Akhtar, W., Shahzad, T., Malik, A., Shah, M. A., Iqbal, S., Rauf, A., Zengin, G., & Bouyahya, A. (2022). Rosemary species: a review of phytochemicals, bioactivities and industrial applications. South African Journal of Botany , 151 , 3-18. Brown, G. P., Shine, R., & Rollins, L. A. (2025). Does a biological invasion modify host immune responses to parasite infection? Royal Society Open Science , 12 (1), 240669. Calixto, J. B. (2019). The role of natural products in modern drug discovery. Anais da Academia Brasileira de Ciências , 91 (Suppl 3), e20190105. Guerrero-Encinas, I., González-González, J. N., García-Utrera, C. K., Aguilar-Tóala, J. E., & Quihui-Cota, L. (2025). Effects of Yucca Genus Plants on Enteropathogenic Bacteria and Parasites. Revista Brasileira de Farmacognosia , 1-16. Kholif, A., Matloup, O., Morsy, T., Abdo, M., Elella, A. A., Anele, U., & Swanson, K. (2017). Rosemary and lemongrass herbs as phytogenic feed additives to improve efficient feed utilization, manipulate rumen fermentation and elevate milk production of Damascus goats. Livestock science , 204 , 39-46. Lifschitz, A., Nava, S., Miró, V., Canton, C., Alvarez, L., & Lanusse, C. (2024). Macrocyclic lactones and ectoparasites control in livestock: efficacy, drug resistance and therapeutic challenges. International Journal for Parasitology: Drugs and Drug Resistance , 100559. Lobanovska, M., & Pilla, G. (2017). Penicillin’s discovery and antibiotic resistance: lessons for the future? The Yale journal of biology and medicine , 90 (1), 135. Makwarela, T. G., Nyangiwe, N., Masebe, T., Mbizeni, S., Nesengani, L. T., Djikeng, A., & Mapholi, N. O. (2023). Tick Diversity and Distribution of Hard (Ixodidae) Cattle Ticks in South Africa. Microbiology Research , 14 (1), 42-59. https://www.mdpi.com/2036-7481/14/1/4 Nizamov, N. (2025). EFFECTS OF AGE, SEX, AND BODY CONDITION ON ECTOPARASITIC INSECT INFESTATION IN DOMESTIC GOATS. Trakia Journal of Sciences , 23 (1), 7-7. Ogbuagu, O. O., Mbata, A. O., Balogun, O. D., Oladapo, O., Ojo, O. O., & Muonde, M. (2022). Novel phytochemicals in traditional medicine: Isolation and pharmacological profiling of bioactive compounds. International Journal of Medical and All Body Health Research , 3 (1), 63-71. Ruiz, M., Alonso, R. J., Rospide, M., Acosta, D. B., Cavia, R., & Sanchez, J. P. (2025). Diversity and eco‐epidemiology of ectoparasites and Rickettsia spp. associated with the opossums Didelphis albiventris Lund in livestock farms from Argentinian Pampas region. Medical and Veterinary Entomology . Strydom, T., Lavan, R. P., Torres, S., & Pinilla, J. C. (2025). The economic impact of endo-and ectoparasites in dairy cattle. Parasites & Vectors , 18 (1), 495. Tang, K. W. K., Millar, B. C., & Moore, J. E. (2023). Antimicrobial resistance (AMR). British journal of biomedical science , 80 , 11387. Wiles, D., Pearson, J. S., & Beddoe, T. (2025). Harnessing Plant-Derived Terpenoids for Novel Approaches in Combating Bacterial and Parasite Infections in Veterinary and Agricultural Settings. Current Microbiology , 82 (4), 134. https://doi.org/10.1007/s00284-025-04113-4 Ahmed, F. E., Shitu, A., Teshome, Z., Yihune, E., Bitew, M., & Fenta, H. M. (2023). Anti-ectoparasite activity of medicinal herbal plant in terms of reducing ectoparasites effect on sheep and goat skins. Journal of the American Leather Chemists Association , 118 (11), 462-473. Aziz, E., Batool, R., Akhtar, W., Shahzad, T., Malik, A., Shah, M. A., Iqbal, S., Rauf, A., Zengin, G., & Bouyahya, A. (2022). Rosemary species: a review of phytochemicals, bioactivities and industrial applications. South African Journal of Botany , 151 , 3-18. Brown, G. P., Shine, R., & Rollins, L. A. (2025). Does a biological invasion modify host immune responses to parasite infection? Royal Society Open Science , 12 (1), 240669. Calixto, J. B. (2019). The role of natural products in modern drug discovery. Anais da Academia Brasileira de Ciências , 91 (Suppl 3), e20190105. Farghaly, M., & Abdullah, M. (2021). Effect of dietary oregano, rosemary and peppermint as feed additives on nutrients digestibility, rumen fermentation and performance of fattening sheep. Egyptian Journal of Nutrition and Feeds , 24 (3), 365-376. Gajarmal, A., Baheti, S., Patekar, R., Mane, S., Sagar, R., & Rath, S. K. (2025). Bridging India's ethnobotanical traditions and Ayurveda: Exploring galactagogue plants in livestock and human care. Ethnobotany Research and Applications , 31 , 1-35. Guerrero-Encinas, I., Gonzalez-Gonzalez, J. N., Garcia-Utrera, C. K., Aguilar-Toala, J. E., & Quihui-Cota, L. (2025). Effects of Yucca genus plants on enteropathogenic bacteria and parasites. Revista Brasileira de Farmacognosia , 1-16. Kafle, L., & Chung, A.-Y. (2025). Contact toxicity and repellency of lemongrass, spearmint, rosemary oils and their major bioactive compounds on destroyer ants (Trichomyrmex destructor) under laboratory conditions. Discover Applied Sciences , 7 (4), 1-15. Kholif, A., Matloup, O., Morsy, T., Abdo, M., Elella, A. A., Anele, U., & Swanson, K. (2017). Rosemary and lemongrass herbs as phytogenic feed additives to improve efficient feed utilization, manipulate rumen fermentation and elevate milk production of Damascus goats. Livestock science , 204 , 39-46. Lifschitz, A., Nava, S., Miró, V., Canton, C., Alvarez, L., & Lanusse, C. (2024). Macrocyclic lactones and ectoparasites control in livestock: efficacy, drug resistance and therapeutic challenges. International Journal for Parasitology: Drugs and Drug Resistance , 100559. Lobanovska, M., & Pilla, G. (2017). Penicillin’s discovery and antibiotic resistance: lessons for the future? The Yale journal of biology and medicine , 90 (1), 135. Makwarela, T. G., Nyangiwe, N., Masebe, T., Mbizeni, S., Nesengani, L. T., Djikeng, A., & Mapholi, N. O. (2023). Tick Diversity and Distribution of Hard (Ixodidae) Cattle Ticks in South Africa. Microbiology Research , 14 (1), 42-59. https://www.mdpi.com/2036-7481/14/1/4 Nizamov, N. (2025). EFFECTS OF AGE, SEX, AND BODY CONDITION ON ECTOPARASITIC INSECT INFESTATION IN DOMESTIC GOATS. Trakia Journal of Sciences , 23 (1), 7-7. Ogbuagu, O. O., Mbata, A. O., Balogun, O. D., Oladapo, O., Ojo, O. O., & Muonde, M. (2022). Novel phytochemicals in traditional medicine: Isolation and pharmacological profiling of bioactive compounds. International Journal of Medical and All Body Health Research , 3 (1), 63-71. Razzaq, M. A., Imran, M., Khan, M. K., Azeem, A., Raza, M. H., Afzal, M. A., Bilal, H., Rahman, M., Ullah, H., & Abbas, B. (2024). Essential oils as alternative treatments for common parasitic infections in animals. Complementary and alternative medicine: essential oils. Faisalabad, Pakistan: Unique Scientific Publishers , 276-282. Ruiz, M., Alonso, R. J., Rospide, M., Acosta, D. B., Cavia, R., & Sanchez, J. P. (2025). Diversity and eco‐epidemiology of ectoparasites and Rickettsia spp. associated with the opossums Didelphis albiventris Lund in livestock farms from Argentinian Pampas region. Medical and Veterinary Entomology . Senanayake, S. N. (2018). Rosemary extract as a natural source of bioactive compounds. Journal of Food Bioactives , 2 , 51–57-51–57. Strydom, T., Lavan, R. P., Torres, S., & Pinilla, J. C. (2025). The economic impact of endo-and ectoparasites in dairy cattle. Parasites & Vectors , 18 (1), 495. Tang, K. W. K., Millar, B. C., & Moore, J. E. (2023). Antimicrobial resistance (AMR). British journal of biomedical science , 80 , 11387. Wiles, D., Pearson, J. S., & Beddoe, T. (2025). Harnessing Plant-Derived Terpenoids for Novel Approaches in Combating Bacterial and Parasite Infections in Veterinary and Agricultural Settings. Current Microbiology , 82 (4), 134. https://doi.org/10.1007/s00284-025-04113-4 Tables Table 1. Time-to-Death Analysis for High-Concentration Rosemary Extract Ectoparasite Species Time to 100% Mortality Susceptibility Level Mites ( Acari ) 5 Minutes Highest Fleas ( Siphonaptera ) 6–7 Minutes High Bed Bugs ( Cimex ) 7–8 Minutes Moderate Ticks ( Ixodida ) 8–9 Minutes Moderate Lice ( Phthiraptera ) 10 Minutes Lowest Table 2. Time-to-Death Analysis and Key Observations for High-Concentration Rosemary Extract Ectoparasites species Time to 100% Mortality Key Behavioral Observations Post-Exposure Mites (Acari) 5 Minutes Rapid paralysis (< 2min), no visible movement thereafter. Fleas (Siphonaptera) 6-7 Minutes Quick loss of jumping reflex, followed by immobilization. Bedbugs (Cimex) 7-8 Minutes Initial agitation and twitching progressing to sluggish movement. Ticks (Ixodida) 8-9 Minutes Slow withdrawal of legs, gradual loss of attachment ability. Lice (Phthiraptera) 10 Minutes Prolonged period of crawling activity before eventual immobilization. Table 3. Summary of ANOVA Results for Mortality Rates Source of Variation Statistical Result Interpretation Concentration Effect p < 0.001 Highly Significant Error/Residuals -- -- Table 4. Statistical Analysis and Interpretation Analysis component Statistical result Interpretation Concentration effect P<0.001 Highly significant difference in mortality across concentration groups. High vs Low Concentration (Tukey's test) P<0.01 High concentration (2g/6ml) is significantly more effective than low. High v Pure Extract P<0.01 Diluted high-concentration extract outperformed pure extract. Species Susceptibility Variation Observed (mites most, lice least susceptible) Physiological differences (cuticle thickness, detox mechanisms) affect time-to-death. Table 5. Study design Component Description Study type In vitro bioassay evaluating Rosmarinus officinalis extract as an antiparasitic. Experimental station Zoology Lab, Shaheed Benazir Bhutto Women University, Peshawar, Pakistan. Plant Material Rosmarinus officinalis leaves collected from Khyber nursery, KPK, Peshawar; shade-dried and powdered. Extraction Method Powdered leaves extracted in ethanol; solvent removed using a rotary evaporator. Test Species Bed bugs ( Cimex ), ticks ( Rhipicephalus ), mites ( Psoroptes ), lice ( Linognathus ), fleas ( Ctenocephalides ). Exposure Method Parasites were placed in Petri dishes with extract treatments. Treatments (independent variable) T1: Undiluted; T2: Low concentration, (diluted); T3: High concentration Replication R = (3), T = (5), n = (5). Outcomes Mortality and time-to-death (minutes) as primary endpoints. Statistical Analysis One-way ANOVA with Tukey’s post hoc and Levene’s tests for assumptions. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8692058","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":592645513,"identity":"75dd3761-688e-46fd-a017-3818d74a6875","order_by":0,"name":"Aimal Ali","email":"","orcid":"","institution":"Shaheed Benazir Bhutto Women University","correspondingAuthor":false,"prefix":"","firstName":"Aimal","middleName":"","lastName":"Ali","suffix":""},{"id":592645514,"identity":"07f06042-8edb-4193-b64d-604c8b144094","order_by":1,"name":"Asma Akbar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYNCCAwcYGNgb2JghvASQCDFaeA6QrEUigUgt/P2nEz/8OHNH3nzm82ePC9sOM/Cz5xgwF5zBrUXiRu5myZ4bzwzn3M4xN54J1CLZ88aAecYNPG66wbtBgufDYcYZ0jls0rzbDjMY3ADawvMBtw7582c3//zz4bD9DMnjz8Ba7AlpMTiQu02a58bhxBkSDGYQWyRAWvA4zPBG7jZrmTPPkmfwgPzyL51H4syzgsMz8HhfDuiwm2+O3bGdwX782eOCM9Zy/O3JGx8XHMPjfXTAAyIOk6ABCphJ1zIKRsEoGAXDGAAAwSBcvax42BIAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0007-5524-2890","institution":"Shaheed Benazir Bhutto Women University","correspondingAuthor":true,"prefix":"","firstName":"Asma","middleName":"","lastName":"Akbar","suffix":""}],"badges":[],"createdAt":"2026-01-25 11:26:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8692058/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8692058/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103504072,"identity":"c76a4e37-cb24-4894-a910-26c9cecf0d30","added_by":"auto","created_at":"2026-02-26 13:16:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":516183,"visible":true,"origin":"","legend":"\u003cp\u003eRosemary Extract\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8692058/v1/138cb17ca1f6dd770a10bd07.png"},{"id":103082602,"identity":"bb93d632-a75c-45af-b883-b35d5883f0c7","added_by":"auto","created_at":"2026-02-20 14:57:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":758054,"visible":true,"origin":"","legend":"\u003cp\u003eRosemary Run in Rotary\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8692058/v1/d3472c4e90b2bf90ea173c0e.png"},{"id":103082603,"identity":"2d897d42-4513-4a34-b2ef-175a2bf58f03","added_by":"auto","created_at":"2026-02-20 14:57:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":624288,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent Concentrations of Rosemary\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8692058/v1/bc1b1c75d2b6eff4fa4ff8ba.png"},{"id":103504183,"identity":"65770e5f-7579-4165-91f5-ff2b757eb580","added_by":"auto","created_at":"2026-02-26 13:18:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":139646,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of Ectoparasite Mortality - Bar chart comparing mortality times across species\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8692058/v1/afc2421f27c99027ef143d83.png"},{"id":103082605,"identity":"28ae053c-9f27-4a23-9e5c-fb5bb5606e10","added_by":"auto","created_at":"2026-02-20 14:57:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":145947,"visible":true,"origin":"","legend":"\u003cp\u003eComparative Efficacy Between Ectoparasite Species - Box plot showing significant differences\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8692058/v1/832e31aeef258411f937bb81.png"},{"id":105566137,"identity":"69397a97-009d-4851-bb7e-d0b76fc6f322","added_by":"auto","created_at":"2026-03-27 12:55:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3840410,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8692058/v1/cecb28d2-bf4e-4257-8dc2-3411f5b6dd30.pdf"}],"financialInterests":"","formattedTitle":"Evaluating the Efficacy of Rosemary Extract (Rosmarinus officinalis) as a Natural Antiparasitic Agent in Livestock: A Sustainable Approach to Enhance Animal Health","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThere are around 1.5\u0026nbsp;billion head of cattle worldwide, making the cattle industry an important part of food systems worldwide. Data-Driven Reality Economic losses due to stunted development and milk production, damaged hides, disease transmission, and higher veterinary and control expenses are enormous as a result of parasitic and ectoparasitic infestations of this size. A significant fraction of the world's cattle are at risk of exposure to ticks and tick-borne illnesses, which is said to cost the cattle industry an estimated \u003cspan\u003e$\u003c/span\u003e20\u0026ndash;30\u0026nbsp;billion annually (Makwarela et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Ticks, mites, lice, and fleas are not only annoying, but they may also spread a variety of zoonotic diseases that are harmful to humans and animals (Ruiz et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The worldwide cattle business is nevertheless confronted with the widespread and economically devastating problem of parasitic diseases. Reduced weight increase, decreased milk production, and, in extreme circumstances, substantial mortality within herds are all symptoms of these illnesses, which are a major cause of lost productivity (Strydom et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Ticks, mites, lice, and fleas are not only annoying, but they may also spread a variety of zoonotic diseases that are harmful to humans and animals (Ruiz et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The infestation rates are often linked to poor physical condition, which causes a vicious circle of physiological stress and higher veterinary expense which have severe economic ramifications (Nizamov, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhen parasites disrupt the equilibrium of the host immune system, the animal redirects its metabolic resources from growth and reproduction to defense. This isn't only an issue for vets; controlling these pests is crucial to global food security and the future of farming (Brown, Shine, \u0026amp; Rollins, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The increasing threat of Antimicrobial Resistance (AMR) has created a significant challenge for the veterinary sector regarding the efficacy of traditional chemical treatments (Tang, Millar, \u0026amp; Moore, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Parasitic populations have developed resistance mechanisms more rapidly owing to the misuse of synthetic antibiotics and anthelmintics since the mid-20th century (Guerrero-Encinas et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). An evolutionary \"arms race\" has rendered several traditional pharmacological therapies ineffective, resulting in chemical residues contaminating animal products and persisting in the environment, while the targeted pests remain unregulated (Lobanovska \u0026amp; Pilla, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is now important to strategically switch to other therapies to keep macrocyclic lactones working. As synthetic drugs based on single molecules are readily resistant to evolution, there is increasing demand in the industry to create alternatives that are more complex and physiologically robust, capable of evading the adaptation mechanisms of modern parasites (Lifschitz et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Due to these issues, phytochemicals, which are natural compounds derived from plants, have emerged as a novel and intriguing area of investigation in veterinary pharmacology (Ogbuagu et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Phytochemicals could be a useful substitute for antibiotics since bacteria have a hard time adjusting to them because their chemical profiles are so diverse (Wiles, Pearson, \u0026amp; Beddoe, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The World Health Organization claims that a lot of people throughout the world are using plant-based medicines right now, and this trend is starting to spread to animal husbandry as well (Calixto, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Rosemary, or \u003cem\u003eRosmarinus officinalis\u003c/em\u003e, is one of these herbs that stands out because it contains so many bioactive chemicals. Carnosic acid and rosmarinic acid are two of these chemicals that are particularly excellent in fighting germs and free radicals (Aziz et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These plant extracts are commonly used for two things: to help ruminants' digestion and to lessen the amount of methane they release. This is not the same as chemicals that are created in a lab. Rosemary is more than just a pesticide; it is also a full tool in the area of \"green veterinary pharmacology\u0026rdquo; (Kholif et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEven though parasite control in cattle is routine, there is still a clear need for options that are effective while also being safer for animals and the environment. Although rosemary is known to contain bioactive compounds, there is limited controlled evidence that measures its direct killing effect and, in particular, the time-to-death of different cattle ectoparasites under standardized conditions. Therefore, this study aimed to evaluate the in vitro antiparasitic activity of ethanol-extracted \u003cem\u003eRosmarinus officinalis\u003c/em\u003e and identify the concentration that produces the fastest and most reliable mortality. Specifically, this work tested the extract against key ectoparasite groups in a controlled setting, compared mortality and time-to-death across low, high, and pure extract treatments, and assessed whether the response followed a clear dose\u0026ndash;response pattern. The novelty of this study lies in providing a direct, side-by-side comparison across multiple parasite groups using a single extraction method and the same measurable endpoints, which makes the findings easier to interpret and apply. However, because the study was conducted in vitro, it cannot fully reflect real farm conditions where factors such as the animal\u0026rsquo;s skin and hair, parasite behavior, environmental exposure, product stability, and animal safety may influence outcomes; therefore, formulation work and field trials are needed before routine on-animal use can be recommended. Overall, this study offers a practical foundation for developing rosemary-based approaches that could support more sustainable ectoparasite management and reduce reliance on synthetic products.\u003c/p\u003e"},{"header":"2. Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Study Design and Preparation of Extracts\u003c/h2\u003e \u003cp\u003eThe efficacy of rosemary leaves as a natural ectoparasiticide was evaluated using an experimental in vitro bioassay approach. Fresh leaves of rosemary (\u003cem\u003eRosmarinus officinalis L.\u003c/em\u003e) were collected from Khyber nursery, KPK, Peshawar, Pakistan, washed with clean water, and shade-dried to reduce thermal loss of volatile constituents. The dried leaves were mechanically ground into a fine powder using a grinder and stored in airtight containers until extraction. Ethanol was used as the extraction solvent. The crude rosemary extract obtained after solvent removal is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. A weighed amount of plant powder was macerated in ethanol (2000 mL) for 10 days, then filtered through filter paper. The filtrate was concentrated under reduced pressure using a rotary evaporator to remove ethanol and obtain the crude extract. Concentration of the ethanolic filtrate using a rotary evaporator is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Three treatment preparations were produced for testing concentration-dependent effects: Low concentration (diluted), High concentration (2 g crude extract in 6 mL ethanol\u003cem\u003e), and\u003c/em\u003e Undiluted concentrate (15 g crude extract), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. These preparations were evaluated against the target ectoparasites under controlled exposure conditions, recording \u003cem\u003emortality\u003c/em\u003e and time-to-death as outcome measures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Parasite Collection and Bioassay\u003c/h2\u003e \u003cp\u003eBedbugs (\u003cem\u003eCimex\u003c/em\u003e), ticks (\u003cem\u003eRhipicephalus\u003c/em\u003e), mites (\u003cem\u003ePsoroptes\u003c/em\u003e), lice (\u003cem\u003eLinognathus\u003c/em\u003e), and fleas (\u003cem\u003eCtenocephalides\u003c/em\u003e) were among the five ectoparasite species that were retrieved from naturally affected cattle. After identifying the parasites, they were divided into groups for treatment. In the bioassay, the parasites were exposed to the extract in a controlled environment using petri plates. Death was officially reported after all bodily responses had been exhausted, and the \"time-to-death\" was calculated in minutes from the time of exposure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were conducted using SPSS Statistics and Graphpad Prism. For each ectoparasite group, the outcomes were summarized per replicate as time-to-death (minutes) or mortality (%) and expressed as \u003cem\u003emean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/em\u003e (or mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE, as appropriate). Differences among the three rosemary extract concentrations (low, high, and pure/undiluted) were evaluated using a one-way analysis of variance (ANOVA). Before ANOVA, assumptions were assessed by inspecting residual plots, applying the Shapiro\u0026ndash;Wilk test for normality, and \u003cem\u003eLevene\u0026rsquo;s test\u003c/em\u003e for homogeneity of variances. Where ANOVA indicated a significant treatment effect, Tukey\u0026rsquo;s HSD post hoc test was used for pairwise comparisons between concentrations while controlling the family-wise error rate. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, corresponding p-values, and post hoc adjusted p-values for pairwise differences.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Ectoparasite Mortality\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll of the ectoparasite species that were examined died off when the \u003cem\u003eRosmarinus officinalis\u003c/em\u003e extract was applied. The results were directly related to the level of concentration. High Concentration (2 g + 6 mL ethanol) was the most efficient solution, eliminating all species within 5 to 10 minutes. The low concentration formulations and pure extract, in contrast, needed longer exposure times and were only partially effective. Time-to-death outcomes for the high-concentration rosemary extract are summarized in \u003cstrong\u003eTable 1\u003c/strong\u003e and visualized in \u003cstrong\u003eFigure 4\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Comparative Efficacy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMites exhibited the highest degree of physiological sensitivity to the extract among the assessed species. They only got treatment for five minutes before they died. Lice were the most durable of all the species, since they could live for up to 10 minutes before dying entirely. This variety shows that various species have developed distinct ways to resist, which are likely linked to changes in the thickness of their exoskeletons or the ways they break down toxins in their bodies. Species-specific differences and key post-exposure behavioral observations are presented in \u003cstrong\u003eTable 2\u003c/strong\u003e, with comparative distributions shown in \u003cstrong\u003eFigure 5\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Statistical Significance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe One-Way ANOVA showed very significant variations in death rates across the various concentration groups (p \u0026lt; 0.001)21. ANOVA results are summarized in \u003cstrong\u003eTable 3\u003c/strong\u003e. Post hoc comparisons and statistical interpretation are provided in \u003cstrong\u003eTable 4\u003c/strong\u003e.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study provides compelling empirical evidence for the strong in vitro antiparasitic activity of \u003cem\u003eRosmarinus officinalis\u003c/em\u003e (rosemary) extract against common livestock ectoparasites \u003cstrong\u003e(Table 5)\u003c/strong\u003e. The findings demonstrate that the ethanol-based rosemary extract acts as a fast-acting, broad-spectrum natural acaricide, achieving complete mortality within 5-10 minutes across tested species. These results align closely with earlier work by Razzaq et al. (2024), who reported significant antiparasitic efficacy of essential oils such as neem, tea tree, and rosemary against ectoparasitic infestations in cattle. However, unlike Razzaq et al. (2024), who assessed hydro-distilled essential oils qualitatively, the present study provides quantitative time-to-death data, establishing rosemary extract\u0026rsquo;s superior potency and rapid action.\u003c/p\u003e\n\u003cp\u003eThe concentration-dependent effects observed in this study confirm that solvent extraction and formulation play crucial roles in optimizing efficacy. The high-concentration ethanol extract (2 g/6 mL) outperformed both pure and diluted forms, indicating that ethanol enhances the solubility and penetration of active phytochemicals such as carnosic acid and rosmarinic acid. These compounds are known to disrupt neural and mitochondrial functions in arthropods, leading to rapid paralysis and death (Senanayake, 2018). Similar patterns of solvent-dependent efficacy were noted by Razzaq et al. (2024), who observed that essential oil formulations exhibited higher lethality when combined with polar solvents that improved diffusion and surface adherence. Together, these findings underscore the importance of optimizing extraction polarity for achieving high bioactivity in botanical acaricides.\u003c/p\u003e\n\u003cp\u003eComparison with Gajarmal et al. (2025) further situates this work within the broader framework of ethnoveterinary medicine. Gajarmal and colleagues conducted a comprehensive review of 86 medicinal plant species used traditionally for parasitic control in livestock, identifying phytochemical groups such as flavonoids, tannins, and essential oils as the main bioactive agents. Our results empirically validate this ethnobotanical foundation, providing laboratory confirmation that rosemary, a \u003cem\u003eLamiaceae\u003c/em\u003e family member highlighted in ethnoveterinary records, possesses measurable antiparasitic efficacy through its phenolic diterpenes. This strengthens the link between traditional herbal practices and modern pharmacological validation, bridging the gap between ethnoveterinary knowledge and experimental parasitology.\u003c/p\u003e\n\u003cp\u003eFrom a mechanistic standpoint, Gajarmal et al. (2025) categorized antiparasitic herbs under Ayurvedic \u0026ldquo;Krimighna Mahakashaya\u0026rdquo; groups, associated with internal and external parasite control. The current study substantiates that classification by demonstrating that \u003cem\u003eR.officinalis\u003c/em\u003e induces neuro-mitochondrial disruption leading to rapid ectoparasite mortality. Thus, rosemary can be viewed as a modern biochemical representative of this ancient medicinal category, aligning traditional pharmacognosy with contemporary bioassay evidence.\u003c/p\u003e\n\u003cp\u003eWhen compared with Ahmed et al. (2023), who investigated the anti-ectoparasitic efficacy of methanolic extracts from \u003cem\u003eCalpurnia aurea\u003c/em\u003e, \u003cem\u003eEucalyptus globulus\u003c/em\u003e, and \u003cem\u003eCroton macrostachyus\u003c/em\u003e on sheep and goat ticks, several notable distinctions emerge. Ahmed et al. achieved up to 80% mortality after 24 hours of exposure, whereas the current rosemary extract caused complete death within 5\u0026ndash;10 minutes, highlighting its exceptional speed and potency. The superior performance of rosemary may be attributed to its high phenolic content and solvent-specific extraction efficiency, as ethanol allows better penetration through the arthropod cuticle compared to methanol (Ahmed et al., 2023). Furthermore, while Ahmed et al. (2023) focused solely on ticks, this study demonstrated efficacy against five different ectoparasite taxa, including mites, lice, fleas, and bed bugs, thereby broadening the antiparasitic spectrum and enhancing practical applicability in multi-species infestations.\u003c/p\u003e\n\u003cp\u003eMechanistic parallels across these studies reveal that plant-derived compounds generally act through multiple synergistic biochemical pathways, including inhibition of chitin synthesis, interference with respiratory enzymes, and disruption of neural signaling. The polyphenolic compounds in rosemary appear to act primarily via oxidative stress and mitochondrial dysfunction, a mechanism supported by observations of rapid paralysis and immobilization in mites and fleas during exposure (Kafle \u0026amp; Chung, 2025). This mode of action differs from that of single-target synthetic acaricides, such as pyrethroids or organophosphates, which often induce resistance due to their narrow molecular targets (Lifschitz et al., 2024). As noted by Guerrero-Encinas et al. (2025), the chemically diverse nature of phytochemicals presents a \u0026ldquo;multi-target resistance barrier,\u0026rdquo; reducing the likelihood of adaptive evolution among ectoparasites, a key advantage for long-term Integrated Pest Management (IPM).\u003c/p\u003e\n\u003cp\u003eThe interspecific variation observed with mites being the most susceptible and lice the most resilient reflects differential cuticular permeability and detoxification capacities among ectoparasite taxa. This observation parallels the findings of Ahmed et al. (2023), who reported species-specific mortality responses among tick species exposed to different plant extracts. The consistency of such results across independent studies reinforces the conclusion that structural and physiological parasite traits modulate susceptibility to botanical acaricides, underscoring the need for species-tailored concentration thresholds in practical applications.\u003c/p\u003e\n\u003cp\u003eThe broader implications of these findings are significant for sustainable livestock management. As highlighted by Gajarmal et al. (2025) and Razzaq et al. (2024), integrating plant-based acaricides like rosemary into IPM frameworks could reduce reliance on synthetic chemicals, thereby mitigating environmental contamination, preventing resistance buildup, and improving animal welfare. Moreover, rosemary\u0026rsquo;s dual role as a feed additive with digestive and antioxidant benefits Farghaly and Abdullah (2021); Kholif et al. (2017)Kholif et al. (2017) enhances its value as a holistic veterinary solution, aligning with the principles of \u0026ldquo;green veterinary pharmacology.\u0026rdquo;\u003c/p\u003e\n\u003cp\u003eFinally, while this study establishes strong in vitro efficacy, further in vivo validation is warranted. Future research should focus on evaluating rosemary extract\u0026rsquo;s persistence, dermal safety, and field effectiveness in live animals, as well as exploring synergistic formulations with other botanicals such as neem or eucalyptus. Such approaches could yield eco-safe, multi-component acaricides capable of sustaining long-term parasite control without compromising productivity or animal health.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study shows that an ethanol extract of \u003cem\u003eRosmarinus officinalis\u003c/em\u003e can kill the tested cattle ectoparasites quickly under laboratory conditions, and that the effect increases with increasing concentration. The high-concentration formulation (2 g extract in 6 mL ethanol) achieved complete mortality across all tested groups within about 5\u0026ndash;10 minutes, with mites dying the fastest (around 5 minutes) and lice taking the longest (up to 10 minutes). The statistical results also supported these differences, as the concentrations produced significantly different outcomes (ANOVA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and the highest concentration performed best in post hoc comparisons. What makes this work novel is that it compares several livestock-relevant ectoparasites side by side using the same extraction method and the same measurable endpoints, especially \u0026ldquo;time to death,\u0026rdquo; which gives more practical information than mortality alone. To move from lab evidence to real use, future studies should test rosemary-based preparations in animals and under farm conditions, confirm safety and any potential skin-irritation risk, measure how long the effects last on hair and skin, and determine the most effective delivery method (spray, dip, or pour-on). Researchers should also standardize the extract by measuring key bioactive compounds, checking formulation stability and shelf-life, and exploring whether combining rosemary with other botanicals improves consistency and reduces the need for synthetic products within integrated pest management programs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAimal Ali:\u003c/strong\u003e Writing \u0026ndash; original draft, writing \u0026ndash; review \u0026amp; editing, Visualization, Resources. \u003cstrong\u003eAsma Akbar:\u003c/strong\u003e Data curation, formal analysis, and Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to all the authors for their equal contributions to this manuscript writing and presentation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI and AI-assisted technologies in the writing process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe figures were created using PowerPoint Presentation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis review paper manuscript did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAziz, E., Batool, R., Akhtar, W., Shahzad, T., Malik, A., Shah, M. A., Iqbal, S., Rauf, A., Zengin, G., \u0026amp; Bouyahya, A. (2022). Rosemary species: a review of phytochemicals, bioactivities and industrial applications. \u003cem\u003eSouth African Journal of Botany\u003c/em\u003e,\u003cem\u003e 151\u003c/em\u003e, 3-18. \u003c/li\u003e\n\u003cli\u003eBrown, G. P., Shine, R., \u0026amp; Rollins, L. A. (2025). Does a biological invasion modify host immune responses to parasite infection? \u003cem\u003eRoyal Society Open Science\u003c/em\u003e,\u003cem\u003e 12\u003c/em\u003e(1), 240669. \u003c/li\u003e\n\u003cli\u003eCalixto, J. B. (2019). The role of natural products in modern drug discovery. \u003cem\u003eAnais da Academia Brasileira de Ci\u0026ecirc;ncias\u003c/em\u003e,\u003cem\u003e 91\u003c/em\u003e(Suppl 3), e20190105. \u003c/li\u003e\n\u003cli\u003eGuerrero-Encinas, I., Gonz\u0026aacute;lez-Gonz\u0026aacute;lez, J. N., Garc\u0026iacute;a-Utrera, C. K., Aguilar-T\u0026oacute;ala, J. E., \u0026amp; Quihui-Cota, L. (2025). Effects of Yucca Genus Plants on Enteropathogenic Bacteria and Parasites. \u003cem\u003eRevista Brasileira de Farmacognosia\u003c/em\u003e, 1-16. \u003c/li\u003e\n\u003cli\u003eKholif, A., Matloup, O., Morsy, T., Abdo, M., Elella, A. A., Anele, U., \u0026amp; Swanson, K. (2017). Rosemary and lemongrass herbs as phytogenic feed additives to improve efficient feed utilization, manipulate rumen fermentation and elevate milk production of Damascus goats. \u003cem\u003eLivestock science\u003c/em\u003e,\u003cem\u003e 204\u003c/em\u003e, 39-46. \u003c/li\u003e\n\u003cli\u003eLifschitz, A., Nava, S., Mir\u0026oacute;, V., Canton, C., Alvarez, L., \u0026amp; Lanusse, C. (2024). Macrocyclic lactones and ectoparasites control in livestock: efficacy, drug resistance and therapeutic challenges. \u003cem\u003eInternational Journal for Parasitology: Drugs and Drug Resistance\u003c/em\u003e, 100559. \u003c/li\u003e\n\u003cli\u003eLobanovska, M., \u0026amp; Pilla, G. (2017). Penicillin\u0026rsquo;s discovery and antibiotic resistance: lessons for the future? \u003cem\u003eThe Yale journal of biology and medicine\u003c/em\u003e,\u003cem\u003e 90\u003c/em\u003e(1), 135. \u003c/li\u003e\n\u003cli\u003eMakwarela, T. G., Nyangiwe, N., Masebe, T., Mbizeni, S., Nesengani, L. T., Djikeng, A., \u0026amp; Mapholi, N. O. (2023). Tick Diversity and Distribution of Hard (Ixodidae) Cattle Ticks in South Africa. \u003cem\u003eMicrobiology Research\u003c/em\u003e,\u003cem\u003e 14\u003c/em\u003e(1), 42-59. https://www.mdpi.com/2036-7481/14/1/4 \u003c/li\u003e\n\u003cli\u003eNizamov, N. (2025). EFFECTS OF AGE, SEX, AND BODY CONDITION ON ECTOPARASITIC INSECT INFESTATION IN DOMESTIC GOATS. \u003cem\u003eTrakia Journal of Sciences\u003c/em\u003e,\u003cem\u003e 23\u003c/em\u003e(1), 7-7. \u003c/li\u003e\n\u003cli\u003eOgbuagu, O. O., Mbata, A. O., Balogun, O. D., Oladapo, O., Ojo, O. O., \u0026amp; Muonde, M. (2022). Novel phytochemicals in traditional medicine: Isolation and pharmacological profiling of bioactive compounds. \u003cem\u003eInternational Journal of Medical and All Body Health Research\u003c/em\u003e,\u003cem\u003e 3\u003c/em\u003e(1), 63-71. \u003c/li\u003e\n\u003cli\u003eRuiz, M., Alonso, R. J., Rospide, M., Acosta, D. B., Cavia, R., \u0026amp; Sanchez, J. P. (2025). Diversity and eco‐epidemiology of ectoparasites and Rickettsia spp. associated with the opossums Didelphis albiventris Lund in livestock farms from Argentinian Pampas region. \u003cem\u003eMedical and Veterinary Entomology\u003c/em\u003e. \u003c/li\u003e\n\u003cli\u003eStrydom, T., Lavan, R. P., Torres, S., \u0026amp; Pinilla, J. C. (2025). The economic impact of endo-and ectoparasites in dairy cattle. \u003cem\u003eParasites \u0026amp; Vectors\u003c/em\u003e,\u003cem\u003e 18\u003c/em\u003e(1), 495. \u003c/li\u003e\n\u003cli\u003eTang, K. W. K., Millar, B. C., \u0026amp; Moore, J. E. (2023). Antimicrobial resistance (AMR). \u003cem\u003eBritish journal of biomedical science\u003c/em\u003e,\u003cem\u003e 80\u003c/em\u003e, 11387. \u003c/li\u003e\n\u003cli\u003eWiles, D., Pearson, J. S., \u0026amp; Beddoe, T. (2025). Harnessing Plant-Derived Terpenoids for Novel Approaches in Combating Bacterial and Parasite Infections in Veterinary and Agricultural Settings. \u003cem\u003eCurrent Microbiology\u003c/em\u003e,\u003cem\u003e 82\u003c/em\u003e(4), 134. https://doi.org/10.1007/s00284-025-04113-4 \u003c/li\u003e\n\u003cli\u003eAhmed, F. E., Shitu, A., Teshome, Z., Yihune, E., Bitew, M., \u0026amp; Fenta, H. M. (2023). Anti-ectoparasite activity of medicinal herbal plant in terms of reducing ectoparasites effect on sheep and goat skins. \u003cem\u003eJournal of the American Leather Chemists Association\u003c/em\u003e,\u003cem\u003e 118\u003c/em\u003e(11), 462-473. \u003c/li\u003e\n\u003cli\u003eAziz, E., Batool, R., Akhtar, W., Shahzad, T., Malik, A., Shah, M. A., Iqbal, S., Rauf, A., Zengin, G., \u0026amp; Bouyahya, A. (2022). Rosemary species: a review of phytochemicals, bioactivities and industrial applications. \u003cem\u003eSouth African Journal of Botany\u003c/em\u003e,\u003cem\u003e 151\u003c/em\u003e, 3-18. \u003c/li\u003e\n\u003cli\u003eBrown, G. P., Shine, R., \u0026amp; Rollins, L. A. (2025). Does a biological invasion modify host immune responses to parasite infection? \u003cem\u003eRoyal Society Open Science\u003c/em\u003e,\u003cem\u003e 12\u003c/em\u003e(1), 240669. \u003c/li\u003e\n\u003cli\u003eCalixto, J. B. (2019). The role of natural products in modern drug discovery. \u003cem\u003eAnais da Academia Brasileira de Ci\u0026ecirc;ncias\u003c/em\u003e,\u003cem\u003e 91\u003c/em\u003e(Suppl 3), e20190105. \u003c/li\u003e\n\u003cli\u003eFarghaly, M., \u0026amp; Abdullah, M. (2021). Effect of dietary oregano, rosemary and peppermint as feed additives on nutrients digestibility, rumen fermentation and performance of fattening sheep. \u003cem\u003eEgyptian Journal of Nutrition and Feeds\u003c/em\u003e,\u003cem\u003e 24\u003c/em\u003e(3), 365-376. \u003c/li\u003e\n\u003cli\u003eGajarmal, A., Baheti, S., Patekar, R., Mane, S., Sagar, R., \u0026amp; Rath, S. K. (2025). Bridging India\u0026apos;s ethnobotanical traditions and Ayurveda: Exploring galactagogue plants in livestock and human care. \u003cem\u003eEthnobotany Research and Applications\u003c/em\u003e,\u003cem\u003e 31\u003c/em\u003e, 1-35. \u003c/li\u003e\n\u003cli\u003eGuerrero-Encinas, I., Gonzalez-Gonzalez, J. N., Garcia-Utrera, C. K., Aguilar-Toala, J. E., \u0026amp; Quihui-Cota, L. (2025). Effects of Yucca genus plants on enteropathogenic bacteria and parasites. \u003cem\u003eRevista Brasileira de Farmacognosia\u003c/em\u003e, 1-16. \u003c/li\u003e\n\u003cli\u003eKafle, L., \u0026amp; Chung, A.-Y. (2025). Contact toxicity and repellency of lemongrass, spearmint, rosemary oils and their major bioactive compounds on destroyer ants (Trichomyrmex destructor) under laboratory conditions. \u003cem\u003eDiscover Applied Sciences\u003c/em\u003e,\u003cem\u003e 7\u003c/em\u003e(4), 1-15. \u003c/li\u003e\n\u003cli\u003eKholif, A., Matloup, O., Morsy, T., Abdo, M., Elella, A. A., Anele, U., \u0026amp; Swanson, K. (2017). Rosemary and lemongrass herbs as phytogenic feed additives to improve efficient feed utilization, manipulate rumen fermentation and elevate milk production of Damascus goats. \u003cem\u003eLivestock science\u003c/em\u003e,\u003cem\u003e 204\u003c/em\u003e, 39-46. \u003c/li\u003e\n\u003cli\u003eLifschitz, A., Nava, S., Mir\u0026oacute;, V., Canton, C., Alvarez, L., \u0026amp; Lanusse, C. (2024). Macrocyclic lactones and ectoparasites control in livestock: efficacy, drug resistance and therapeutic challenges. \u003cem\u003eInternational Journal for Parasitology: Drugs and Drug Resistance\u003c/em\u003e, 100559. \u003c/li\u003e\n\u003cli\u003eLobanovska, M., \u0026amp; Pilla, G. (2017). Penicillin\u0026rsquo;s discovery and antibiotic resistance: lessons for the future? \u003cem\u003eThe Yale journal of biology and medicine\u003c/em\u003e,\u003cem\u003e 90\u003c/em\u003e(1), 135. \u003c/li\u003e\n\u003cli\u003eMakwarela, T. G., Nyangiwe, N., Masebe, T., Mbizeni, S., Nesengani, L. T., Djikeng, A., \u0026amp; Mapholi, N. O. (2023). Tick Diversity and Distribution of Hard (Ixodidae) Cattle Ticks in South Africa. \u003cem\u003eMicrobiology Research\u003c/em\u003e,\u003cem\u003e 14\u003c/em\u003e(1), 42-59. https://www.mdpi.com/2036-7481/14/1/4 \u003c/li\u003e\n\u003cli\u003eNizamov, N. (2025). EFFECTS OF AGE, SEX, AND BODY CONDITION ON ECTOPARASITIC INSECT INFESTATION IN DOMESTIC GOATS. \u003cem\u003eTrakia Journal of Sciences\u003c/em\u003e,\u003cem\u003e 23\u003c/em\u003e(1), 7-7. \u003c/li\u003e\n\u003cli\u003eOgbuagu, O. O., Mbata, A. O., Balogun, O. D., Oladapo, O., Ojo, O. O., \u0026amp; Muonde, M. (2022). Novel phytochemicals in traditional medicine: Isolation and pharmacological profiling of bioactive compounds. \u003cem\u003eInternational Journal of Medical and All Body Health Research\u003c/em\u003e,\u003cem\u003e 3\u003c/em\u003e(1), 63-71. \u003c/li\u003e\n\u003cli\u003eRazzaq, M. A., Imran, M., Khan, M. K., Azeem, A., Raza, M. H., Afzal, M. A., Bilal, H., Rahman, M., Ullah, H., \u0026amp; Abbas, B. (2024). Essential oils as alternative treatments for common parasitic infections in animals. \u003cem\u003eComplementary and alternative medicine: essential oils. Faisalabad, Pakistan: Unique Scientific Publishers\u003c/em\u003e, 276-282. \u003c/li\u003e\n\u003cli\u003eRuiz, M., Alonso, R. J., Rospide, M., Acosta, D. B., Cavia, R., \u0026amp; Sanchez, J. P. (2025). Diversity and eco‐epidemiology of ectoparasites and Rickettsia spp. associated with the opossums Didelphis albiventris Lund in livestock farms from Argentinian Pampas region. \u003cem\u003eMedical and Veterinary Entomology\u003c/em\u003e. \u003c/li\u003e\n\u003cli\u003eSenanayake, S. N. (2018). Rosemary extract as a natural source of bioactive compounds. \u003cem\u003eJournal of Food Bioactives\u003c/em\u003e,\u003cem\u003e 2\u003c/em\u003e, 51\u0026ndash;57-51\u0026ndash;57. \u003c/li\u003e\n\u003cli\u003eStrydom, T., Lavan, R. P., Torres, S., \u0026amp; Pinilla, J. C. (2025). The economic impact of endo-and ectoparasites in dairy cattle. \u003cem\u003eParasites \u0026amp; Vectors\u003c/em\u003e,\u003cem\u003e 18\u003c/em\u003e(1), 495. \u003c/li\u003e\n\u003cli\u003eTang, K. W. K., Millar, B. C., \u0026amp; Moore, J. E. (2023). Antimicrobial resistance (AMR). \u003cem\u003eBritish journal of biomedical science\u003c/em\u003e,\u003cem\u003e 80\u003c/em\u003e, 11387. \u003c/li\u003e\n\u003cli\u003eWiles, D., Pearson, J. S., \u0026amp; Beddoe, T. (2025). Harnessing Plant-Derived Terpenoids for Novel Approaches in Combating Bacterial and Parasite Infections in Veterinary and Agricultural Settings. \u003cem\u003eCurrent Microbiology\u003c/em\u003e,\u003cem\u003e 82\u003c/em\u003e(4), 134. https://doi.org/10.1007/s00284-025-04113-4 \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eTime-to-Death Analysis for High-Concentration Rosemary Extract\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEctoparasite Species\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime to 100% Mortality\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSusceptibility Level\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMites (\u003cem\u003eAcari\u003c/em\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e5 Minutes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003eHighest\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFleas (\u003cem\u003eSiphonaptera\u003c/em\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e6\u0026ndash;7 Minutes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBed Bugs (\u003cem\u003eCimex\u003c/em\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e7\u0026ndash;8 Minutes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003eModerate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTicks (\u003cem\u003eIxodida\u003c/em\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e8\u0026ndash;9 Minutes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003eModerate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLice (\u003cem\u003ePhthiraptera\u003c/em\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e10 Minutes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003eLowest\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch2\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e \u0026nbsp;\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eTime-to-Death Analysis and Key Observations for High-Concentration Rosemary Extract\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEctoparasites species\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime to 100% Mortality\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eKey Behavioral Observations Post-Exposure\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMites (Acari)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e5 Minutes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eRapid paralysis (\u0026lt; 2min), no visible movement thereafter.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFleas (Siphonaptera)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e6-7 Minutes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eQuick loss of jumping reflex, followed by immobilization.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBedbugs (Cimex)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e7-8 Minutes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eInitial agitation and twitching progressing to sluggish movement.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTicks (Ixodida)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e8-9 Minutes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eSlow withdrawal of legs, gradual loss of attachment ability.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLice (Phthiraptera)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e10 Minutes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eProlonged period of crawling activity before eventual immobilization.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u0026nbsp;\u003c/strong\u003eSummary of ANOVA Results for Mortality Rates\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSource of Variation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStatistical Result\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInterpretation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003eConcentration Effect\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40px;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003eHighly Significant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003eError/Residuals\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.\u0026nbsp;\u003c/strong\u003eStatistical Analysis and Interpretation\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnalysis component\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStatistical result\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInterpretation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConcentration effect\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eP\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eHighly significant difference in mortality across concentration groups.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHigh vs Low Concentration (Tukey\u0026apos;s test)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eP\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eHigh concentration (2g/6ml) is significantly more effective than low.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHigh v Pure Extract\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eP\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eDiluted high-concentration extract outperformed pure extract.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpecies Susceptibility Variation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eObserved (mites most, lice least susceptible)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003ePhysiological differences (cuticle thickness, detox mechanisms) affect time-to-death.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5.\u0026nbsp;\u003c/strong\u003eStudy design\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eComponent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDescription\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStudy type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003eIn vitro bioassay evaluating \u003cem\u003eRosmarinus officinalis\u003c/em\u003e extract as an antiparasitic.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eExperimental station\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003eZoology Lab, Shaheed Benazir Bhutto Women University, Peshawar, Pakistan.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlant Material\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cem\u003eRosmarinus officinalis\u003c/em\u003e leaves collected from Khyber nursery, KPK, Peshawar; shade-dried and powdered.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eExtraction Method\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003ePowdered leaves extracted in ethanol; solvent removed using a rotary evaporator.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTest Species\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003eBed bugs (\u003cem\u003eCimex\u003c/em\u003e), ticks (\u003cem\u003eRhipicephalus\u003c/em\u003e), mites (\u003cem\u003ePsoroptes\u003c/em\u003e), lice (\u003cem\u003eLinognathus\u003c/em\u003e), fleas (\u003cem\u003eCtenocephalides\u003c/em\u003e).\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eExposure Method\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003eParasites were placed in Petri dishes with extract treatments.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatments (independent variable)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003eT1: Undiluted; T2: Low concentration, (diluted); T3: High concentration\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReplication\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eR =\u003c/strong\u003e (3), \u003cstrong\u003eT =\u003c/strong\u003e (5), \u003cstrong\u003en =\u003c/strong\u003e (5).\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOutcomes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003eMortality and time-to-death (minutes) as primary endpoints.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003eOne-way ANOVA with Tukey\u0026rsquo;s post hoc and Levene\u0026rsquo;s tests for assumptions.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antimicrobial Resistance (AMR), Antiparasitic efficacy, Green veterinary pharmacology, Integrated Pest Management (IPM), Livestock ectoparasites, Phytochemicals, Rosmarinus officinalis","lastPublishedDoi":"10.21203/rs.3.rs-8692058/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8692058/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLivestock ectoparasites (ticks, mites, lice, fleas, and bed bugs) reduce productivity and increase control costs; resistance to synthetic agents is rising. To evaluate the in vitro antiparasitic efficacy of ethanolic Rosmarinus officinalis (rosemary) extract against major cattle ectoparasites and to compare killing speed across concentrations. Rosemary leaves were shade-dried, powdered, extracted in ethanol, concentrated by rotary evaporation, and tested in Petri-dish bioassays. Five ectoparasite groups collected from naturally infested cattle were exposed to three extract preparations (low, high, and concentrate/undiluted, ensuring consistency with your protocol). Time-to-death (min) and mortality (%) were recorded. One-way ANOVA with Tukey\u0026rsquo;s HSD was used (α\u0026thinsp;=\u0026thinsp;0.05). The extract showed a clear dose-response. The high-concentration formulation (reported as 2 g extract in 6 mL ethanol) produced complete mortality within ~\u0026thinsp;5\u0026ndash;10 minutes across taxa; mites were most sensitive (~\u0026thinsp;5 min) and lice were least sensitive (~\u0026thinsp;10 min). Treatment effects were significant (ANOVA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Ethanolic rosemary extract demonstrates rapid, broad-spectrum in-vitro antiparasitic activity and could support integrated pest management strategies. This study provides a standardized, side-by-side comparison of killing speed across multiple cattle ectoparasite taxa using the same extraction approach and endpoint (time-to-death).\u003c/p\u003e","manuscriptTitle":"Evaluating the Efficacy of Rosemary Extract (Rosmarinus officinalis) as a Natural Antiparasitic Agent in Livestock: A Sustainable Approach to Enhance Animal Health","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-20 14:57:09","doi":"10.21203/rs.3.rs-8692058/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b7fd0757-25b6-43b4-876f-414b7d122d49","owner":[],"postedDate":"February 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-26T17:49:41+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-20 14:57:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8692058","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8692058","identity":"rs-8692058","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}